p38 MAPK regulates steroidogenesis through transcriptional repression of STAR gene

in Journal of Molecular Endocrinology
Authors:
Syed Kashif ZaidiGeriatric Research, Division of Endocrinology, Division of Gastroenterology and Hepatology, Department of Obstetrics and Gynecology, State Key Laboratory of Cellular Stress Biology, Program in Molecular Medicine, Education and Clinical Center (GRECC-182B), VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304, USA
Geriatric Research, Division of Endocrinology, Division of Gastroenterology and Hepatology, Department of Obstetrics and Gynecology, State Key Laboratory of Cellular Stress Biology, Program in Molecular Medicine, Education and Clinical Center (GRECC-182B), VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304, USA

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Wen-Jun ShenGeriatric Research, Division of Endocrinology, Division of Gastroenterology and Hepatology, Department of Obstetrics and Gynecology, State Key Laboratory of Cellular Stress Biology, Program in Molecular Medicine, Education and Clinical Center (GRECC-182B), VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304, USA
Geriatric Research, Division of Endocrinology, Division of Gastroenterology and Hepatology, Department of Obstetrics and Gynecology, State Key Laboratory of Cellular Stress Biology, Program in Molecular Medicine, Education and Clinical Center (GRECC-182B), VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304, USA

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Stefanie BittnerGeriatric Research, Division of Endocrinology, Division of Gastroenterology and Hepatology, Department of Obstetrics and Gynecology, State Key Laboratory of Cellular Stress Biology, Program in Molecular Medicine, Education and Clinical Center (GRECC-182B), VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304, USA

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Alex BittnerGeriatric Research, Division of Endocrinology, Division of Gastroenterology and Hepatology, Department of Obstetrics and Gynecology, State Key Laboratory of Cellular Stress Biology, Program in Molecular Medicine, Education and Clinical Center (GRECC-182B), VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304, USA

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Mark P McLeanGeriatric Research, Division of Endocrinology, Division of Gastroenterology and Hepatology, Department of Obstetrics and Gynecology, State Key Laboratory of Cellular Stress Biology, Program in Molecular Medicine, Education and Clinical Center (GRECC-182B), VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304, USA

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Jiahuai HanGeriatric Research, Division of Endocrinology, Division of Gastroenterology and Hepatology, Department of Obstetrics and Gynecology, State Key Laboratory of Cellular Stress Biology, Program in Molecular Medicine, Education and Clinical Center (GRECC-182B), VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304, USA

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Roger J DavisGeriatric Research, Division of Endocrinology, Division of Gastroenterology and Hepatology, Department of Obstetrics and Gynecology, State Key Laboratory of Cellular Stress Biology, Program in Molecular Medicine, Education and Clinical Center (GRECC-182B), VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304, USA

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Fredric B KraemerGeriatric Research, Division of Endocrinology, Division of Gastroenterology and Hepatology, Department of Obstetrics and Gynecology, State Key Laboratory of Cellular Stress Biology, Program in Molecular Medicine, Education and Clinical Center (GRECC-182B), VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304, USA
Geriatric Research, Division of Endocrinology, Division of Gastroenterology and Hepatology, Department of Obstetrics and Gynecology, State Key Laboratory of Cellular Stress Biology, Program in Molecular Medicine, Education and Clinical Center (GRECC-182B), VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304, USA

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Salman AzharGeriatric Research, Division of Endocrinology, Division of Gastroenterology and Hepatology, Department of Obstetrics and Gynecology, State Key Laboratory of Cellular Stress Biology, Program in Molecular Medicine, Education and Clinical Center (GRECC-182B), VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304, USA
Geriatric Research, Division of Endocrinology, Division of Gastroenterology and Hepatology, Department of Obstetrics and Gynecology, State Key Laboratory of Cellular Stress Biology, Program in Molecular Medicine, Education and Clinical Center (GRECC-182B), VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304, USA

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Free access

STAR/StarD1, part of a protein complex, mediates the transport of cholesterol from the outer to inner mitochondrial membrane, which is the rate-limiting step for steroidogenesis, and where steroid hormone synthesis begins. Herein, we examined the role of oxidant-sensitive p38 MAPKs in the regulation of STAR gene transcription, using model steroidogenic cell lines. Our data indicate that oxidant activation of p38 MAPK exhibits a negative regulatory role in the induction of functional expression of STAR, as evidenced by enhanced induction of STAR (mRNA/protein) expression and increased steroidogenesis during pharmacological inhibition of p38 MAPK or in cells with increased transient overexpression of a dominant-negative (dn) form of p38 MAPKα or p38 MAPKβ. Studies with rat Star-promoter demonstrated that overexpression of p38 MAPKα-wt, -β, or -γ significantly reduced both basal and cAMP-sensitive promoter activity. In contrast, overexpression of p38 MAPKα-dn, -β, or -γ enhanced the Star promoter activity under basal conditions and in response to cAMP stimulation. Use of various constitutively active and dn constructs and designer knock-out cell lines demonstrated that MKK3 and MKK6, the upstream activators of p38 MAPKs, play a role in p38 MAPKα-mediated inhibition of Star promoter activity. In addition, our studies raised the possibility of CREB being a potential target of the p38 MAPK inhibitory effect on Star promoter activity. Collectively, these data provide novel mechanistic information about how oxidant-sensitive p38 MAPKs, particularly p38 MAPKα, contribute to the negative regulation of Star gene expression and inhibit steroidogenesis.

Abstract

STAR/StarD1, part of a protein complex, mediates the transport of cholesterol from the outer to inner mitochondrial membrane, which is the rate-limiting step for steroidogenesis, and where steroid hormone synthesis begins. Herein, we examined the role of oxidant-sensitive p38 MAPKs in the regulation of STAR gene transcription, using model steroidogenic cell lines. Our data indicate that oxidant activation of p38 MAPK exhibits a negative regulatory role in the induction of functional expression of STAR, as evidenced by enhanced induction of STAR (mRNA/protein) expression and increased steroidogenesis during pharmacological inhibition of p38 MAPK or in cells with increased transient overexpression of a dominant-negative (dn) form of p38 MAPKα or p38 MAPKβ. Studies with rat Star-promoter demonstrated that overexpression of p38 MAPKα-wt, -β, or -γ significantly reduced both basal and cAMP-sensitive promoter activity. In contrast, overexpression of p38 MAPKα-dn, -β, or -γ enhanced the Star promoter activity under basal conditions and in response to cAMP stimulation. Use of various constitutively active and dn constructs and designer knock-out cell lines demonstrated that MKK3 and MKK6, the upstream activators of p38 MAPKs, play a role in p38 MAPKα-mediated inhibition of Star promoter activity. In addition, our studies raised the possibility of CREB being a potential target of the p38 MAPK inhibitory effect on Star promoter activity. Collectively, these data provide novel mechanistic information about how oxidant-sensitive p38 MAPKs, particularly p38 MAPKα, contribute to the negative regulation of Star gene expression and inhibit steroidogenesis.

Introduction

Extensive experimental evidence now indicates that aging in humans and animal models is associated with a general decline in steroid hormone production (Zaidi et al. 2012). Similarly, a number of in vitro studies have clearly shown that isolated adrenocortical cells and testicular Leydig cells (Zaidi et al. 2012) of older rats of several different strains synthesize and secrete less steroid hormone in response to tropic hormone or its second messenger, cAMP, than do cells from younger animals. These changes in steroid hormone production and secretion do not appear to be a function of reduced tropic hormone signaling or a defect in steroid hormone-synthesizing enzymes.

Previous work from this laboratory has shown that an adequate amount of cholesterol is not available to the adrenal (adrenocortical cells) and testis (Leydig cells) in aging rats (Popplewell & Azhar 1987, Liao et al. 1993, Sun et al. 2008, Zaidi et al. 2012) for the initial and rate-limiting step in steroid biosynthesis, i.e. translocation of cholesterol from the outer mitochondrial membrane (OMM) to the P450 side-chain cleavage enzyme (P450scc/Cyp11A1), which is localized in the matrix side of the inner mitochondrial membrane (IMM) and converts cholesterol to pregnenolone, the precursor of all steroid hormones (Stocco & Clark 1996, Stocco 2001, Hu et al. 2010, Miller & Bose 2011). This aging-induced attenuation of cholesterol transport to mitochondria is not due to a loss of cellular cholesterol stores, but results from the downregulation of STAR (Leers-Sucheta et al. 1999, Luo et al. 2001, Wang et al. 2005, Sun et al. 2008) and peripheral-type benzodiazepine receptor or translocator protein (TSPO; Culty et al. 2002, Sun et al. 2008). These components of the steroidogenic machinery (Stocco & Clark 1996, Stocco 2001) probably function in concert (Liu et al. 2006) to facilitate rate-limiting cholesterol transport from the OMM to the IMM (Hu et al. 2010, Miller & Bose 2011). Although various cellular and molecular mechanisms controlling this aging defect have not been definitely identified, excessive reactive oxygen species (ROS) production (Azhar et al. 1995, Cao et al. 2004, Abidi et al. 2008a,b, Zaidi et al. 2012) and ROS-induced oxidative damage to STAR have been suggested as a potential mechanism for impaired cholesterol transport to the IMM for steroid production during aging.

p38 MAPKs are members of the MAPK family that are activated by a variety of environmental stresses and inflammatory cytokines (Roux & Blenis 2004, Coulthard et al. 2009, Cuadrado & Nebreda 2010, Remy et al. 2010, Corrêa & Eales 2012). The four members of the p38 MAPK isoform family are p38 MAPKα (MAPK14), p38 MAPKβ (SAPK2b), p38 MAPKγ (SAPK3, ERK6, or MAPK14), and p38 MAPKδ (SAPK4 or MAPK13) (Han et al. 1994, Lee et al. 1994, Rouse et al. 1994, Coulthard et al. 2009, Cuadrado & Nebreda 2010, Remy et al. 2010). These isoforms differ in their organ/tissue distribution, regulation of kinase activation and subsequent phosphorylation of downstream substrates, kinases or transcription factors, and sensitivity to pharmacological inhibition by pyridinyl imidazole molecules such as SB203580 and SB202190 (Bain et al. 2007, Coulthard et al. 2009, Cuadrado & Nebreda 2010, Remy et al. 2010). Various forms of cellular stress, including oxidative stress, u.v. irradiation, hypoxia, and ischemia, as well as inflammatory cytokines, stimulate the activity of p38 MAPKs (Coulthard et al. 2009, Cuadrado & Nebreda 2010, Remy et al. 2010) by dual phosphorylation of Thr180 and Tyr182 in a Thr–Gly–Tyr motif. This phosphorylation is mediated by upstream MAPKs (MAPKKs), MKK3 and MKK6 (Derijard et al. 1995, Raingeaud et al. 1995, 1996, Han et al. 1996), which are in turn activated by several different and overlapping sets of MAPKKKs or MEKKs (Coulthard et al. 2009, Cuadrado & Nebreda 2010, Remy et al. 2010).

Recently, we have shown that aging leads to increased activation of p38 MAPKα in rat adrenocortical cells (Abidi et al. 2008a). This effect appears to be specific for p38 MAPKα, as the expression of neither ERK1/2 nor JNK1/2 was altered during aging (Abidi et al. 2008a). We also showed that the use of p38 MAPK inhibitors (SB203580 and SB202190) and antioxidants (NAC and MnTMPyP) partially restored the steroidogenesis in adrenal cells from old animals, indicating that the p38 MAPK signaling cascade facilitates the inhibitory actions of excessive oxidative insult on adrenal steroid hormone production caused by aging (Abidi et al. 2008a). These results were further complemented by in vitro studies using a mouse adrenocortical tumor cell line, Y-1, showing that activated p38 MAPKs mediate oxidant (excessive oxidative stress)-induced inhibition of steroid production (Abidi et al. 2008b).

In the present study, we sought to determine molecular mechanisms by which the oxidant-sensitive p38 MAPK inhibits cAMP-induced steroidogenesis. Herein, we show that the inhibitory effect of p38 MAPK on cAMP-induced steroidogenesis in model steroidogenic cell lines is due to a decrease in StAR expression and its promoter activity. Furthermore, expression and functional activity of cAMP response-element binding protein (CREB), a mediator of tropic hormone/cAMP-stimulated STAR gene transcription, is also sensitive to the inhibitory actions of p38 MAPK. The current study describes the molecular events responsible for the p38 MAPK-mediated negative regulation of STAR gene transcription and inhibition of steroidogenesis.

Materials and methods

Reagents and antibodies

N6, 2′-O-dibutyryl cAMP (Bt2cAMP), SB 203580, SB 202190, xanthine, xanthine oxidase (XO), microbial, fatty acid–free BSA, hydrogen peroxide, and monoclonal Anti-FLAG M2 antibody and mouse monoclonal anti-actin antibody were obtained from Sigma–Aldrich. 4-Hydroxy-2-nonenal (HNE) was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). The NEP-PER Nuclear Protein Extraction Kit, and Promega Dual-Luciferase Reporter Kit (E1980) were purchased from Thermo Fisher Scientific (Rockford, IL) and Promega Corporation respectively. Anti-phospho-CREB (Ser133), anti-CREB, pan anti-p38 MAPK, and anti-phospho-p38 MAPK (Thr180/Tyr182) antibodies that detect endogenous levels of total and phosphorylated (activated) forms of p38 MAPKα, β, or γ protein, respectively, were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). IRDye 800CW goat anti-rabbit and IRDye 680LT goat anti-mouse secondary antibodies were purchased from LI-COR Corporate (Lincoln, NE, USA). SYBR Green Master Mix and Lipofectamine 2000 were supplied by Applied Biosystems and Invitrogen (Life Technologies) respectively.

Plasmids

The generation of Rat Star promoter-pGL3 constructs p-1862-luc, p-1413-luc, p-998-luc, p-545-luc, and p-342-luc has been described in an earlier publication (Sandhoff et al. 1998). The mouse Star promoter constructs, pGL3 (p-966-luc; Manna et al. 2002), were kindly supplied by Dr Douglas Stocco (Texas Tech University Health Sciences Center, Lubbock, TX, USA). The pcDNA3-Flag-tagged-p38 MAPKα-wt, p38 MAPKβ-wt, p38 MAPKγ-wt, p38 MAPKδ-wt, p38 MAPKα/AF-dn (dominant negative) p38 MAPKβ/AF-dn, p38 MAPKγ/AF-dn, and p38 MAPKδ/AF-dn constructs used here have been described previously (Pramanik et al. 2003). The dn forms (AF) of human p38 MAPKα, p38 MAPKβ, p38 MAPKγ, and p38 MAPKδ cDNA constructs were created by substituting Thr188 with Ala and Tyr190 with Phe in the TGY dual phosphorylation site. Because AF mutant proteins cannot be phosphorylated, they act as dn proteins and interfere with the functions of their respective p38 MAPKs. The pcDNA3-Flag-tagged MKK3-wt, MKK3 (AA)-dn, MKK3 (EE)-ca (constitutively active), MKK6-wt, MKK6 (AA)-dn, and MKK6 (EE)-ca expression constructs used here were generated as reported previously (Han et al. 1996, Raingeaud et al. 1996); ca MKK3 (EE)-ca and dn MKK3 (AA)-dn were constructed by replacing Ser189 and Thr193 with Glu and Ala residues respectively. Likewise, ca MKK6 (EE)-ca and dn MKK6 (AA)-dn were constructed by replacing Ser207 and Thr211 with Glu and Ala residues respectively. HA-MKK6–p38 MAPKα-ca, HA-MKK6–p38 MAPKγ-ca, HA-MKK6–p38α/AGF-dn, and HA-MKK6–p38γ/AGF-dn fusion constructs were kindly provided by Dr Guan Chen (University of Wisconsin, Madison, WI, USA). RSV-CREB plasmid was purchased from Addgene (Cambridge, MA, USA). CRE-Reporter (luc) and activator protein 1 (AP1) (TPA-response element (TRE)) Reporter (luc) kits were obtained from Qiagen–SA Biosciences (Valencia, CA, USA). Both CRE-Reporter (luc) and AP1 (TRE) Reporter (luc) are inducible reporter constructs that encode a firefly luciferase gene under the control of a basal promoter element (TATA box) joined to tandem six repeats (6×) of CRE and AP1/TRE transcriptional response elements respectively.

Cell culture

Mouse Y-1 adrenocortical tumor cells, mouse MLTC-1 testicular Leydig tumor cells, and human embryonic kidney-293 (HEK-293) cells were originally obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). MLTC-1 cells were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin, 100 units/ml penicillin, and 1% l-glutamine in a humidified atmosphere of 5% CO2 at 37 °C. Y-1 cells were maintained at 37 °C in a humidified 5% CO2 atmosphere in Ham's F-10 medium supplemented with 2.5% FBS, 12.5% horse serum, 100 μg/ml streptomycin, and 100 units/ml penicillin. MKK3/6 wt, MKK3−/−, MKK6−/−, and MKK3/6 double knock-out (DKO) mouse embryonic fibroblasts (MEFs) (Brancho et al. 2003) were maintained in DMEM containing 10% FBS, 100 μg/ml streptomycin, and 100 units/ml penicillin at 37 °C with 5% CO2.

Transfections and luciferase assays

MLTC-1 (or other cell types such as Y1, and MEFs, MKK3/6 wt, MKK3−/−, MKK6−/−, and MKK3/6−/−) cells were seeded at a density of 1×105 cells/well in a 12-well plate containing a cell-type-specific culture medium as described earlier. After overnight incubation, cells were transiently transfected with a rat p-1862 Star–Luc (firefly luciferase) construct (1 μg)±native p38 MAPKα (wt) (1 μg) or dn p38 MAPKα (1 μg) using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen, Life Technologies). A control reporter construct containing Renilla luciferase (Rluc, pRL-TK) (50 ng) was co-transfected for normalization of transfection efficiency. In some cases, MLTC-1 cells were transfected with rat p-1862 Star–Luc (firefly luciferase) construct (1 μg)+pRL-TK (50 ng) and ± p38 MAPKβ-wt (1 μg), ± p38 MAPKβ-dn, ± p38 MAPKγ-wt (1 μg), ± p38 MAPKγ-dn (1 μg),± p38 MAPKδ-wt, ± p38 MAPKδ-dn, ± MKK6–p38 MAPKα-ca, ± MKK6–p38 MAPKα-dn, ± MKK6–p38 MAPKγ-ca, ± MKK6–p38 MAPKγ-dn, ± MKK3-wt,± MKK3-dn (AA), ± MKK3-ca (EE), ± MKK6-wt,± MKK6-dn (AA), or ± MKK6-ca (EE). In each case, total DNA concentration was kept constant to 3 μg with the addition of inert pcDNA3. After 48 h, cells were incubated with or without Bt2cAMP for 5 h, washed, harvested using cell lysis buffer (Promega Corporation), and subsequently assayed for both firefly and Renilla luciferase activities using the Promega Dual-Luciferase Reporter Kit (E1980) and a SpectraMax L luminescence Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) or GloMax-20/20 Single-Tube Luminometer (Promega Corporation). Firefly luciferase activities were normalized to Renilla luciferase reporter activities and shown as -fold induction compared with control (empty vector). The results shown are the average of triplicate determinations with the error bars representing s.e.m. All the experiments were independently repeated at least two to three times.

In other assays, MLTC-1 cells were transfected with CRE-luc (firefly luciferase)+Renilla luciferase (Rluc, pRL-TK), ± CREB, or ± CREB+p38 MAPKα-wt or p38 MAPKα-dn, for 48 h. At the end of incubation, washed dishes were further incubated with or without Bt2cAMP followed by measurement of firefly and Renilla luciferase activities. Likewise, MLTC-1 cells were transfected with AP1/TRE-luc (firefly luciferase)+Renilla luciferase (Rluc, pRL-TK) ± p38 MAPKα-wt or p38 MAPKα-dn and, after 48 h, dishes were further incubated with or without AP1/TRE activator TPA (10 nM) for 30 min. The cell lysates were then quantified for firefly and Renilla luciferase activities.

Transfection of MLTC-1 cells with p38 MAPK constructs and measurement of steroid (progesterone) secretion

MLTC-1 cells were seeded at a density of 1×105 cells/well in a 12-well plate containing RPMI 1640 medium supplemented with 10% FBS, 100 μg/ml streptomycin, 100 units/ml penicillin, and 1% l-glutamine (under 5% CO2 at 37 °C) for 24 h before transfection. The cells were transfected with native wt-p38 MAPKα (1 μg) and/or dn-p38 MAPKα (1 μg) or pcDNA3 (3 μg) using Lipofectamine 2000 as recommended by the manufacturer (Invitrogen, Life Technologies). In each case, the total amount of plasmid (3 μg) was kept the same with the addition of inert pcDNA3. Forty-eight hours after transfection, the medium was replaced with a fresh medium and the cells (triplicate wells) were treated with ± Bt2cAMP (2.5 mM) and incubations continued for an additional 5 h. At the end of incubation, media were aspirated and stored frozen until analyzed for secreted progesterone content by RIA as described earlier (Reaven et al. 1995). The cells were washed once with PBS and then processed for the determination of Star/StarD1 mRNA expression by quantitative RT-PCR as described below.

Quantitative real-time PCR

Total RNA from MLTC-1 and Y-1 cells was isolated by using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA concentration and purity were quantified using a NanoDrop Spectrophotometer (Thermo Scientific). Samples (2 μg) RNA were reverse transcribed to cDNA at 42 °C for 1 h using SuperScript II reverse transcriptase (Invitrogen) and oligo(dT) and random hexamers. Quantitative real-time PCR (qRT-PCR) was carried out using specific primers (Table 1) and amplified using SYBR Select Master Mix for CFX (Applied Biosystems) in an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). The results are presented as relative mRNA expression levels calculated using the formula 2−ΔΔCT, where ΔΔCT=ΔCTtarget−ΔCTreference with 36B4 (60S acidic ribosomal protein P0) as the reference gene.

Table 1

Oligonucleotide sequences used in quantitative real-time PCR (qRT-PCR) analysis

GeneForwardReverse
m-Star/StarD15′-GGATTAAGGCACCAAGCTGT-3′5′-CCAGTTGAGAACCAAGCAGA-3′
m-StarD25′-TGTGCTGAACTCCAGAAACC-3′5′-ATAAAGTCCACTTGGCTGGTC-3′
m-StarD35′-CCTGCTCTTCATCTCCCTTC-3′5′-CTCCTGCTCCAAGTTCTTCC-3′
m-StarD45′-TCCTGTGGTGTGAGTGTTGA-3′5′-TGGCTTGGACTGTCTTTGAG-3′
m-StarD55′-GGGAATCTGTACCGAGGAGA-3′5′-AACTGGCTTTATGCAATCCC-3′
m-StarD65′-CACAGTTCGCACTCCTCTGT-3′5′-CTCTGCAGGAACAAACTCCA-3′
m-StarD75′-ATGTTCGGCGGTATAGTGTG-3′5′-GGATAACCATTTGGGATTCG-3′
m-StarD105′-CTGGAACCTGACCTACAGCA-3′5′-CCATCCGACACTTGATCTTG-3′
m-p38 MAPKα5′-CCCCAGAGATCATGCTGAAT-3′5′-ACAACGTTCTTCCGGTCAAC-3′
m-p38 MAPKβ5′-ATGTAGCGGTGAACGAGGA-3′5′-CCACATATCCGGTCATCTCC-3′
m-p38 MAPKγ5′-GAGACTTGAAGCCTGGCAAC-3′5′-CCGGGTTACCACATATCCTG-3′
m-p38 MAPKδ5′-CGCCACACAGACACTGAGA-3′5′-ATGATGCAACCAACAGACC-3′
m-Cyclophilin5′-TGGAGAGCACCAAGACAGACA-3′5′-TGCCGGAGTCGACAATGAT-3′
m-36B45′-CCTGAAGTGCTCGACATCAC-3′5′-CGATCTGCAGACACACACTG-3′

Western blotting analysis

The cells were washed with PBS and then harvested in RIPA buffer, containing a Complete Protease Inhibitor Cocktail and PhosSTOP phosphatase inhibitor cocktail (both from Roche Diagnostics) directly to plates and scraping into tubes. The cells were briefly sonicated, and centrifuged for 5 min. The protein concentrations of the lysates were measured using the Pierce BCA Protein Assay Reagent Kit (Thermo Fischer Scientific, Rockford, IL, USA). Proteins from cell lysates (20 μg) were resolved on a precast 4–20% gradient SDS–PAGE and electro-transferred onto a Hybond nitrocellulose membrane (Amersham, GE Healthcare, Piscataway, NJ, USA). The membranes were blocked, incubated with anti-p38 MAPK, anti-phospho-p38 MAPK, anti-CREB, anti-phospho-CREB, and mouse anti-β-actin antibodies, washed, and incubated with IRDye 800CW goat anti-rabbit and IRDye 680LT goat anti-mouse secondary antibodies. The membrane blots were then imaged using an ODYSSEY Infrared Imaging System (LI-COR Biosciences). Quantification of bands was made with the analysis software provided with the imaging system.

Statistical analysis

Data are presented as mean±s.e.m. Statistical analyses were performed using ANOVA followed by the Bonferroni's post test using GraphPad Prism Software, Prism 4 (GraphPad Software, Inc., San Diego, CA, USA). A P value of <0.05 was considered statistically significant. All experiments were performed independently at least three times.

Results

Effect of Bt2cAMP stimulation on mRNA levels of Star and Star-related lipid transfer proteins in MLTC-1 and Y-1 cells

The human and mouse genomes each contain 15 genes encoding Star-related lipid transfer (START) domain-containing proteins (STARD; Soccio & Breslow 2003). STAR (STARD1), the prototype of the STARD family of proteins, plays an essential role in moving cholesterol substrate to the IMM of steroidogenic cells of the adrenal gland and gonads, where the first committed step in steroidogenesis occurs (Stocco 2001, Miller & Bose 2011). Herein, we assessed the mRNA levels of Star (StarD1) (subsequently referred to as Star) and other StarD family members under basal conditions and in response to cAMP stimulation in two steroidogenic cell lines, MLTC-1 and Y-1. Total RNA samples were analyzed for Star, StarD2, 3, 4, 5, 6, 7, and 10 mRNA expression by qRT-PCR. The results shown in Fig. 1 indicate that Star mRNA is most abundantly expressed in both MLTC-1 and Y-1 cells and that Bt2cAMP (a cell-permeable analog of cAMP) stimulation further increased its mRNA levels by four- to fivefold in both cell types. Relatively low mRNA expression of StarD4 and StarD7 was also detected in MLTC-1 cells, and Bt2cAMP (2.5 mM) treatment of these cells enhanced StarD4 expression by four- to fivefold (Fig. 1). In Y-1 cells, low, but significant, mRNA expression was noted for StarD4, StarD5, and StarD7, although their levels were not affected by Bt2cAMP stimulation. The higher levels of mRNA expression of STAR protein compared with other STARD proteins in MLTC-1 and Y-1 cells are consistent with its critical role in the regulation of steroidogenesis.

Figure 1
Figure 1

qRT-PCR analysis of StarD1/Star, StarD2, StarD3, StarD4, StarD5, StarD6, StarD7, and StarD10 mRNA levels in mouse Y-1 adrenal cells and mouse Leydig tumor cells, MLTC-1, treated with or without Bt2cAMP (2.5 mM) for 6 h. Groups of RNA samples from Y-1 or MLTC-1 cells were analyzed by qRT-PCR as described in the ‘Materials and methods’ section. The levels of expression of StarD1/Star, StarD2, StarD3, StarD4, StarD5, StarD6, StarD7, and StarD10 mRNAs are shown. Expression of cyclophilin was used for normalization. (A) Y-1 cells and (B) MLTC-1 cells. Each value represents mean±s.e.m. of four separate determinations. ***P<0.0001 basal (control) vs Bt2cAMP.

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-13-0287

Expression of endogenous mRNA levels of p38 MAPK isoforms in MLTC-1 and Y-1 cells

Using qRT-PCR, we next determined the relative mRNA expression levels of each of the four p38 MAPK isoforms in MLTC-1 and Y-1 cells (Fig. 2). p38 MAPKα expression was most abundant in both cell types; only low levels of p38 MAPKβ were detected in Y-1 cells, while its mRNA levels were roughly 40% of the levels of p38 MAPKα in MLTC-1 cells (Fig. 2). Low levels of p38 MAPKγ mRNA were also detected in Y-1 cells as well as in MLTC-1 cells. The expression of p38 MAPKδ, however, was barely detectable in MLTC-1, and Y-1 cells showed only low expression (Fig. 2). These results led us to conclude that p38 MAPKα is the predominant form in both cell types, but significant expression of p38 MAPKβ and p38 MAPKγ was also detected in MLTC-1 and Y-1 cells respectively. Thus, most of the follow-up studies focused on the p38 MAPKα isoform.

Figure 2
Figure 2

QRT-PCR analyses of p38 MAPK isoforms, p38 MAPKα, p38 MAPKβ, p38 MAPKγ, and p38 MAPKδ mRNA levels in cultured Y-1 and MLTC-1 cells. Groups of RNA samples from Y-1 or MLTC-1 cells were analyzed by qRT-PCR as described in the ‘Materials and methods’ section. The levels of expression of p38 MAPKα, p38 MAPKβ, p38 MAPKγ, and p38 MAPKδ mRNAs are shown. Expression of 36B4 was used for normalization. (A) Y-1 cells and (B) MLTC-1 cells. Each value represents mean±s.e.m. of four separate determinations. *P<0.05 p38α vs p38β (MLTC-1) and ***P<0.001 p38α vs p38β (Y-1), p38γ or p38δ.

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-13-0287

Effects of oxidants and Bt2cAMP on Star mRNA expression in steroidogenic cell lines

To examine the effect of oxidants on Star mRNA expression, MLTC-1 and Y-1 cells were pre-exposed to xanthine/XO (X/XO; superoxide anion), hydrogen peroxide, lipid peroxidation product, HNE, or vehicle (control) for 1 h, the dishes washed and treated or not treated a cell membrane-permeable cAMP analog, Bt2cAMP, for 5 h. Total RNA was isolated and Star mRNA measured using qRT-PCR. When cells were treated for 5 h, the results demonstrated that Star mRNA levels, as expected, increased threefold to fourfold following treatment with 2.5 mM Bt2cAMP compared with nontreatment control (Fig. 3). Treatment of cells with a superoxide-generating system (X/XO), hydrogen peroxide, or a lipid peroxidation product, HNE, under basal conditions caused a significant reduction in Star mRNA levels in MLTC-1 and Y-1 cells, ranging from 30 to 80% (Fig. 3). Likewise, pre-exposure of cells to these three oxidants caused a variable, but significant, reduction in Bt2cAMP-stimulated Star mRNA levels (Fig. 3). These results indicate that exposure of steroidogenic cells to different oxidants results in downregulation of StAR gene expression.

Figure 3
Figure 3

Exposure of Y-1 cells (A) and MLTC-1 cells (B) to xanthine/xanthine oxidase (X/XO) generated superoxide anion (O2 −•) or hydrogen peroxide (H2O2) attenuates mRNA expression of Star/StarD1 both under basal conditions and in response to cAMP stimulation. Cultured Y-1 and MLTC-1 cells were exposed to ± 0.1 mM xanthine and 10 mU/ml XO or ± 50 μM hydrogen peroxide (H2O2) for 1 h. At the end of incubation, the dishes were washed and subsequently incubated with or without Bt2cAMP (2.5 mM) for 5 h, followed by isolation of total RNA and the quantification of the Star/StarD1 mRNA levels by qRT-PCR. Each value represents mean±s.e.m. of four separate determinations. ***P<0.0001 basal (control) vs Bt2cAMP.

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-13-0287

Oxidant activation of p38 MAPK negatively affects cAMP transcriptional regulation of Star

The results described above indicate that oxidant exposure of steroidogenic cells results in downregulation of Star mRNA levels. As the p38 MAPK pathway has been implicated in mediating oxidative-stress-induced inhibition of steroid hormone synthesis (Abidi et al. 2008a,b), we investigated the role of p38 MAPK signaling in oxidant modulation of cAMP-mediated transcriptional regulation of STAR protein. MLTC-1 cells were transiently transfected with a construct containing the promoter region (−2862) of rat Star, a control reporter construct containing Renilla luciferase for normalization of transfection efficiency and with or without an expression plasmid encoding FLAG-p38 MAPKα, and, after 48 h, cells were exposed to vehicle (control), superoxide anion (O2 −•), X/XO, the generator of O2 −•), H2O2, or HNE (a lipid peroxidation product) for an additional 60 min. After extensive washing, cells were treated with ± Bt2cAMP (2.5 mM) for 3 h, harvested and cell lysates analyzed for firefly and Renilla luciferase activities or subjected to western blotting analysis for the measurement of total and phosphorylated (activated) forms of p38 MAPKα. As shown in Fig. 4A, short-term exposure of transfected cells to optimal doses of superoxide anion, H2O2, or HNE resulted in p38 MAPKα activation, as indicated by an increased level of phosphorylation at residues Thr180 and Tyr182 (approximately 10–30% (phopsho-p38 MAPK:total p38 MAPK ratios: control=1.00; superoxide anion=1.08; H2O2=1.34; and HNE=1.24)). Basal levels of Star promoter luciferase activity were significantly decreased when cells were co-transfected with a p38 MAPKα construct (Fig. 4B). Basal promoter activity was further decreased when p38 MAPKα-transfected cells were exposed to oxidants: superoxide anion (O2 −•), H2O2, or 4-HNE (Fig. 4B). Likewise, cAMP-stimulated promoter activity was also decreased with the introduction of p38 MAPKα. However, no additional decreases in cAMP-stimulated promoter activity were noted following oxidant exposure of p38 MAPKα-transfected cells (Fig. 4B). These p38 MAPKα overexpression studies indicate that inhibitory actions of oxidants on Star promoter activity are mediated by p38 MAPKα.

Figure 4
Figure 4

Evaluation of the effects of p38 MAPKα (wt) overexpression and the effects of X/XO generated O2 −•, H2O2, and 4-HNE on basal and cAMP-stimulated rat Star promoter (luciferase) activity in MLTC-1 cells. MLTC-1 cells were transiently transfected with a rat p-1862 Star–Luc (firefly luciferase) construct (1 μg)+Renilla luciferase (Rluc, pRL-TK) (50 ng) and ± native p38 MAPKα (wt) (1 μg) using Lipofectamine 2000. After 48 h, dishes were exposed to ± xanthine (0.1 mM)/XO (10 mU/ml), ± 50 μM H2O2, or ± 50 μM 4-hydroxynonenol (4-HNE) for 1 h, washed, incubated with or without Bt2cAMP (2.5 mM) for 5 h, cells were lysed using cell lysis buffer (Promega Corporation) and subsequently assayed for both firefly and Renilla luciferase activities using a Promega Dual-Luciferase Reporter Kit (E1980). In some cases, cell lysates were subjected to SDS–PAGE followed by western blotting analysis and the quantification of the total and the phosphorylated p38 MAPKα bands. (A) Western blots showing the bands stained for either total p38 MAPKα or its phosphorylated form. (B) Rat Star promoter luciferase activity. **P<0.01 basal (control) vs Bt2cAMP and ***P<0.0001 basal (control) vs Bt2cAMP.

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-13-0287

Effects of overexpression of native (wt) and dn p38 MAPK isoforms on Star promoter activity

The above findings indicate that p38 MAPKα exerts a negative effect on both basal and cAMP-stimulated Star promoter activity. To establish p38 MAPK isoform specificity, we next examined the relative efficacy of native (wt) p38 MAPKα, p38 MAPKβ, p38 MAPKγ, and p38 MAPKδ and their respective dn forms on Star promoter activity. MLTC-1 cells were transfected with STAR-reporter luciferase plasmid alone or together with plasmids encoding native or dn FLAG-p38 MAPK isoforms, and luciferase activities were quantified. As before, expression of wt p38 MAPKα-wt reduced the basal promoter activity by approximately 50%, whereas expression of dn p38 MAPKα-dn enhanced the basal promoter activity by about twofold (Fig. 5A). Qualitatively similar results were obtained when Y-1 or HEK-293 cells were substituted for MLTC-1 or rat Star promoter (p-1862-rat StAR) reporter construct was replaced with a mouse Star promoter reporter construct (p960-mouse Star; Manna et al. (2002); data not shown). Results presented in Fig. 5B and C show that overexpression of p38 MAPKβ-wt or p38 MAPKγ-wt also significantly suppressed the transfected rat Star promoter reporter activity both under basal conditions and in response to cAMP stimulation. On the other hand, expression of p38 MAPKβ-dn, similar to p38 MAPKα-dn, enhanced basal as well as cAMP-stimulated promoter activity, but the use of p38 MAPKγ-dn showed no such stimulatory effect. In contrast, expression of p38 MAPKδ-wt showed no inhibitory effect on promoter activity when measured under basal conditions, but significantly decreased cAMP-stimulated promoter activity (Fig. 5D). On the other hand, transient overexpression of p38 MAPKδ-dn variably but significantly enhanced cAMP-stimulated promoter activity (Fig. 5D). These results led us to conclude that p38 MAPKα and to a lesser extent p38 MAPKβ, p38 MAPKγ, and p38 MAPKδ function as negative regulators of Star gene transcription.

Figure 5
Figure 5

Effects of overexpression of wt and dn p38 MAPKα, p38 MAPKβ, p38 MAPKγ, and p38 MAPKδ constructs on Star promoter activity in MLTC-1 cells. The cells were transiently transfected with p-1862 Star–Luc (firefly luciferase) construct plus Renilla luciferase (Rluc, pRL-TK), and empty vector, p38 MAPKα-wt, p38 MAPKα-dn, p38 MAPKβ-wt, p38 MAPKβ-dn, p38 MAPKγ-wt, p38 MAPKγ-dn, p38 MAPKδ-wt, or p38 MAPKδ-dn. Forty-eight hours after transfection, the dishes were washed and subsequently incubated with or without Bt2cAMP (2.5 mM) for 5 h and cells lysed using cell lysis buffer (Promega Corporation) and subsequently assayed for both firefly and Renilla luciferase activities. (A) p38 MAPKα, (B) p38 MAPKβ, (C) p38 MAPKγ, and (D) p38 MAPKδ. Each value represents mean±s.e.m. of four separate determinations. wt (native); dn, dominant negative. *P<0.01 basal (control) vs Bt2cAMP; **P<0.01 basal (control) vs Bt2cAMP; ***P<0.0001 basal (control) vs Bt2cAMP; ††P<0.01 Bt2cAMP p38δ-dn vs Bt2cAMP p38α-wt; and †††P<0.0001 Bt2cAMP control vs Bt2cAMP p38α-wt.

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-13-0287

To further establish the inhibitory action of p38 MAPKα on Star promoter activity, p38 MAPK inhibitor experiments were carried out. To this end, MLTC-1 cells were either transfected with Star promoter alone or Star promoter plus p38 MAPKα-wt construct and then treated with selective inhibitors of p38 MAPK, SB203580 and SB202190 (Bain et al. 2007). Luciferase measurement results indicated that SB203580 or SB202190 treatment resulted in a small increase in basal promoter activity in MLTC-1 cells transfected with Star promoter alone (Table 2). Co-transfection of Star promoter with p38 MAPKα resulted in an efficient reduction in Star promoter activity under basal conditions. This reduction in basal promoter activity was completely restored following treatment of transfected cells with either SB203580 or SB202190 (Table 2). Qualitatively similar results were obtained when luciferase measurements were carried out using extracts from cAMP-treated cells (data not shown).

Table 2

Effects of p38 MAPK inhibitors and overexpression of native/wt and dominant negative (dn) forms of p38 MAPKα either separately or in combination on Star promoter (luciferase) activity in MLTC-1 cells. Mouse Leydig tumor cells MLTC-1 were transiently transfected with rat p-1862 rStAR–luc (firefly luciferase) construct (1 μg)±plasmid-encoding native (wt) FLAG p38 MAPKα (1 μg). A control reporter construct containing Renilla luciferase (Rluc, pRL-TK; 50 ng) was co-transfected for normalization of transfection efficiency. After 48 h, cells were incubated with or without Bt2cAMP (2.5 mM) for 4 h and then with or without SB203580 (10 μM) or SB202190 (10 μM) for an additional hour, harvested and analyzed for firefly and Renilla luciferase activities. Firefly luciferase activities were normalized to Renilla luciferase reporter activities and shown as -fold induction compared with control (empty vector). Each value represents mean±s.e.m. of four separate determinations

AdditionsRelative p-1862 rStAR–luc activity (U)±s.e.m.
SB2035806.56±0.873*
SB2021907.76±0.850*
p38 MAPKα2.96±0.807
SB203580+p38 MAPKα7.18±0.917
SB202190+p38 MAPKα9.42±1.953

*P<0.01 p38 MAPKα vs SB203580 or SB202190 and P<0.01 p38 MAPKα vs SB203580+p38 MAPKα or SB202190+p38 MAPKα.

The results presented in Fig. 6 demonstrate that transient overexpression of p38 MAPKα-wt in MLTC-1 cells significantly decreased the endogenous mRNA levels of STAR protein both under basal conditions and in response to cAMP stimulation. In contrast, transient overexpression of a p38 MAPKα-dn significantly upregulated both basal as well as cAMP-stimulated Star mRNA levels. These results further confirmed that p38 MAPKα negatively regulates the gene transcription of STAR protein.

Figure 6
Figure 6

Effects of transient overexpression of wt and dominant-negative p38 MAPKα on basal and cAMP-stimulated endogenous mRNA levels of Star in MLTC-1 cells. MLTC-1 cells were transiently transfected with empty vector, p38 MAPKα-wt, or p38 MAPKα-dn and, 48 h after transfection, the dishes were washed and subsequently incubated with or without Bt2cAMP (2.5 mM) for 5 h. Following incubation, the dishes were subjected to total RNA isolation and the RNA samples quantified for Star (StarD1) mRNA levels by qRT-PCR. Each value represents mean±s.e.m. of four separate determinations. *P<0.01 Bt2cAMP p38α-dn vs basal (control) Bt2cAMP; ***P<0.01 Bt2cAMP p38α-wt vs basal (control) Bt2cAMP; and †††P<0.001 Bt2cAMP p38α-dn vs Bt2cAMP p38α-wt.

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-13-0287

MKK3 and MKK6 contribute to the p38 MAPK-mediated suppression of Star promoter activity

Two closely related dual-specificity upstream protein kinases, MKK3 and MKK6, phosphorylate and activate p38 MAPK at the activation site Thr–Gly–Tyr (Han et al. 1996, Enslen et al. 1998, Wysk et al. 1999, Coulthard et al. 2009, Cuadrado & Nebreda 2010, Remy et al. 2010). We determined whether MKK3 and MKK6 play a role in p38 MAPK-mediated suppression of Star promoter activity. MLTC-1 cells (which express predominantly p38 MAPKα but also significant amount of p38 MAPKβ, Fig. 2) were co-transfected with a rat Star firefly luciferase reporter construct as described earlier (rat p-1862 StAR–Luc) plus a reporter construct containing Renilla luciferase (Rluc, pRL-TK) with or without a plasmid encoding a ca form of MKK3/MMK6, FLAG-MKK3-ca (MKK3 (EE))/FLAG-MKK6-ca (MKK6 (EE)), or a dn MKK3/MKK6, FLAG-MKK3-dn (MKK (AA))/FLAG-MKK6-dn (MKK (AA)). After 48 h, the cells were treated with or without Bt2cAMP (2.5 mM) for 5 h, harvested, and cell extracts analyzed for dual luciferase activities. As shown in Fig. 7A, overexpression of MKK3-ca significantly reduced both basal and cAMP-stimulated promoter activity. In contrast, transfections with an MKK3-dn construct upregulated both the basal as well as cAMP-stimulated reporter activity. Likewise, overexpression of MKK6-ca led to a significant inhibition of basal and cAMP-stimulated Star promoter activity (Fig. 7A). Similar to the effect of the dn form of MKK3, overexpression of MKK6-dn also enhanced promoter activity both under basal conditions and in response to cAMP stimulation. These results imply that both MKK3 and MKK6 are equally effective and participate in p38 MAPK-mediated repression of Star promoter activity.

Figure 7
Figure 7

Regulation of Star promoter activity in MITC-1 cells in response to transient overexpression of MKK3, MKK6, or MKK6–p38 MAPKα/γ constructs and mouse embryonic fibroblasts (MEFs) lacking MKK3, MKK6, or both. (A) MLTC-1 cells were cotransfected with p-1862 Star–Luc (firefly luciferase) construct (1 μg) plus Renilla luciferase (Rluc, pRL-TK) (50 ng), and empty vector, p38 MAPKα-wt, p38 MAPKα-dn, MKK3-ca, MKK3-dn, MKK6-ca, or MKK6-dn. At the end of 48 h, the dishes were treated with or without Bt2cAMP (2.5 mM) for an additional 5 h and subsequently cell lysates assayed for firefly and Renilla luciferase activities. (B) MLTC-1 cells were transiently transfected with empty vector, p38 MAPKα-wt, p38 MAPKα-dn, MKK6–p38 MAPKα-ca, MKK6–p38 MAPKα-dn, MKK6–p38 MAPKγ-ca, or MKK6–p38 MAPKγ-dn and, after 48 h, further treated with or without Bt2cAMP for 5 h. Cell lysates were assayed for firefly and Renilla luciferase activities as described above. (C) Wt (MKK3/6) MEFs and MEFs deficient in MKK3−/−, MKK6−/−, or both (MKK3−/−/MKK6−/−) were co-transfected with p-1862 StarLuc (firefly luciferase) and Renilla luciferase (Rluc, pRL-TK) constructs for 48 h, stimulated with or without Bt2cAMP for an additional 5 h, followed by determination of firefly and Renilla luciferase activities. (D) MKK3/6 and MKK3−/−/MKK6−/− DKO MEFs were co-transfected with p-1862 Star–Luc (firefly luciferase) and Renilla luciferase (Rluc, pRL-TK) constructs. In a third set MKK3−/−/MKK6−/− DKO MEFs were co-transfected with MKK3-ca+MKK6-ca plus p-1862 Star–Luc (firefly luciferase) and Renilla luciferase (Rluc, pRL-TK) constructs for 48 h followed by treatment with ± Bt2cAMP. Cell lysates were assayed for firefly and Renilla luciferase activities, as described earlier. Each value represents mean±s.e.m. of four separate determinations. ca, constitutively active; dn, dominant negative. All values presented (A) show statistically significant differences between basal vs Bt2cAMP and basal (control) or basal Bt2cAMP vs p38α-wt, MKK3-ca, or MKK6-ca. The values presented in (B) show statistically significant differences between basal vs Bt2cAMP and basal (control) or basal Bt2cAMP vs p38α-wt, MKK6–p38α-ca, or MKK6–p38γ-ca. (C) Basal (control) or basal Bt2cAMP WT3/6 show statistically significant differences from the corresponding Bt2cAMP MKK3−/−, MKK6−/−, or MKK3/6−/− groups. (D) ***P<0.001 wt MKK3/6 Bt2cAMP vs MKK3/6−/− Bt2cAMP; and ††P<0.01 MKK3/6−/− Bt2cAMP vs MKK3/6 plus MKK3-ca+MKK6-ca Bt2cAMP.

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-13-0287

Next we undertook the approach of enzyme–substrate fusion (Robinson et al. 1998, Zheng et al. 1999, Qi et al. 2007) to further examine the MKK–p38 MAPK regulation of Star promoter activity. This approach is based on the fact that p38 MAPK becomes ca when linked to its upstream activator such as MKK6 (Qi et al. 2007). Herein, we used constructs in which p38 MAPKα-ca, p38 MAPKγ-ca, p38α/AGF-dn, or p38γ/AGF-dn (representative p38 MAPKs) were fused in-frame with the activator MKK6 (MKK6 activates all isoforms of p38 MAPK) through a decapeptide linker as reported previously by Qi et al. (2007). MLTC-1 cells were transiently transfected with Star promoter with or without expression vector encoding HA-MKK6–p38 MAPKα-ca, HA-MKK6–p38 MAPKγ-ca, HA-MKK6–p38α/AGF-dn, or HA-MKK6–p38γ/AGF-dn, and after 48 h cell lysates were analyzed for firefly and Renilla luciferase activities. As a positive control, some dishes were also transfected with p38 MAPKα (wt) or p38 MAPKα-dn plasmid DNA. Results presented in Fig. 7B demonstrate that transfection of cells with either MKK6–p38 MAPKα-ca or MKK6–p38 MAPKγ-ca suppressed the expression of Star promoter activity, whereas use of MKK6–p38 MAPKα/AGF-dn or MKK6–p38 MAPKγ/AGF-dn enhanced the promoter activity. As expected, transfection of cells with p38 MAPKα-wt or p38 MAPKα-dn decreased and increased the promoter activity respectively. Thus, p38 MAPKs, including p38 MAPKα and their upstream activators MKK3 and MKK6, work in concert to modulate Star promoter activity.

To more directly define the roles that MKK3 and MKK6 play in p38 negative regulation of the Star promoter activity mediated by MAPKs, we assessed the expression of Star promoter activity in MEFs with targeted disruption of Mkk3 (Map2k3), Mkk6 (Map2k6) or both Mkk3 and Mkk6 genes. MKK3/6 (wt), MKK3−/−, MKK6−/−, and MKK3/6−/− (DKO) MEFs (Brancho et al. 2003) were transfected with a rat Star reporter construct (48 h), treated with or without Bt2cAMP (2.5 mM, 5 h), and, subsequently, cell lysates analyzed for luciferase activities. As shown in Fig. 7C, expression of Star promoter activity was substantially higher (both basal and cAMP stimulated) in single MKK3−/− or MKK6−/− MEFs as compared with wt MEFs that express both MKK3 and MKK6. Interestingly, use of MKK3/6−/− DKO MEFs produced no additive effect, indicating that MKK3 and MKK6 play complementary roles and activate similar p38 MAPKs, such as p38 MAPKα that mediate negative regulation of Star promoter activity. Finally, the results presented in Fig. 7D demonstrate that simultaneous overexpression of MKK3-ca and MKK6-ca in MKK3/6−/− DKO MEFs significantly decreased both basal and c-AMP-stimulated Star promoter luciferase activity as compared with cells transfected with the control DNA.

cAMP-stimulated Star mRNA and progesterone levels in relation to p38 MAPK

The above studies provided strong evidence that p38 MAPKs, particularly p38 MAPKα, function as negative regulators of Star promoter activity. We next evaluated the inhibitory actions of p38 MAPKα on steady-state Star mRNA levels and its relevance to steroidogenesis. Expression of p38 MAPKα-wt significantly decreased both basal and cAMP-induced Star mRNA levels. In contrast, when MLTC-1 cells were transfected with p38 MAPKα-dn, basal and cAMP-stimulated levels were significantly increased compared with levels for the control cells and the cells transfected with p38 MAPKα-wt (data not shown).

We also measured progesterone synthesis and secretion in the culture medium from cells transfected with either p38 MAPKα-wt or p38 MAPKα-dn and subsequently stimulated without or with Bt2cAMP. As shown in Table 3, progesterone accumulation in each case paralleled changes in Star mRNA expression. The nonstimulated MLTC-1 cells transiently expressing p38 MAPKα (wt) decreased progesterone production by approximately 75–80%, whereas expression of p38 MAPKα-dn increased medium progesterone levels by approximately twofold. Likewise, cAMP-stimulated progesterone secretion by p38 MAPKα-wt-expressing cells was reduced by approximately 50% when compared with that seen in control cells. On the other hand, expression of p38 MAPKα-dn resulted in approximately a twofold increase in cAMP-stimulated progesterone production as compared with control cells (Table 3).

Table 3

Effects of overexpression of native/wt and dominant-negative (dn) forms of p38 MAPKα on steroid secretion by MLTC-1 cells. Mouse Leydig tumor cells, MLTC, were transiently transfected with empty vector, plasmid-encoding native (wt) FLAG p38 MAPKα (1 μg), or dn Flag p38 MAPKα (1 μg). After 48 h, cells were treated with or without Bt2cAMP (2.5 mM) for 5 h and subsequently culture medium samples collected and analyzed for progesterone secretion by RIA. Each value represents mean±s.e.m. of four separate determinations

Additionsng Progesterone secreted/105 cells/5 h±s.e.m.
Basal8.53±0.45*
Bt2cAMP (2.5 mM)34.83±3.01
p38 MAPKα-wt2.43±0.41
p38 MAPKα-wt+Bt2cAMP (2.5 mM)17.20±0.33
p38 MAPKα-dn17.84±0.95
p38 MAPKα-dn+Bt2cAMP (2.5 mM)72.33±1.84§

*P<0.05 basal (control) vs p38 MAPKα-wt; P<0.001 Bt2cAMP vs MAPKα-wt; P<0.01 MAPKα-wt vs p38 MAPKα-wt+Bt2cAMP or p38 MAPKα-dn; and §P<0.001 MAPKα-wt vs p38 MAPKα-dn+Bt2cAMP.

Effect of p38 MAPK on CREB-mediated regulation of Star promoter activity

Expression of Star is mainly regulated at the transcriptional level through a cAMP-protein kinase A (PKA)-dependent mechanism (Manna et al. 2009). Herein, we examined the effect of p38 MAPK on CREB-mediated regulation of Star promoter activity. As the CREB-dependent transcriptional response is primarily dependent on CREB phosphorylation (Shaywitz & Greenberg 1999, Johannessen et al. 2004, Altarejos & Montminy 2011), we initially examined the effects of p38 MAPKα-wt and p38 MAPKα-dn on the cAMP/PKA-stimulated phosphorylation (Ser133) of CREB protein in MLTC-1 cells. The results presented in Fig. 8 demonstrate the effects of p38 MAPKα-wt and p38 MAPKα-dn constructs on the levels of the cAMP/PKA-stimulated phosphorylated form of CREB (p-CREB). Transfection of cells with p38 MAPKα-wt increased cAMP-stimulated phospho-CREB levels, and the use of p38 MAPKα-dn resulted in much greater stimulation of its phospho-levels.

Figure 8
Figure 8

Effects of overexpression of p38 MAPKα-wt and p38 MAPKα-dn on the levels of total (t) and phospho-forms of CREB in response to treatment of cells with or without Bt2cAMP. Cells were transfected with either an empty vector, p38 MAPKα-wt, or p38 MAPKα-dn, and, after 48 h, treated with or without Bt2cAMP. The cell lysates were subjected to SDS–PAGE followed by western blotting analysis. The blots were developed using anti-CREB (total) or anti-phospho-CREB (Ser133). (A) Western blot of p-CREB, t-CREB and p-ATF. (B) Quantification of ratios of p-CREB:t-CREB. (C) Quantification of ratios of p-ATF:t-ATF. t, total; p, phosphorylated.

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-13-0287

The results presented in Fig. 8 also demonstrate that phosphorylation of ATF-1, which is 65% identical to CREB in its primary amino acid sequence (Shaywitz & Greenberg 1999), was also increased in response to the overexpression of p38 MAPKα-wt or p38 MAPKα-dn construct. The potential effect of p38 MAPKα on CREB was further studied in MLTC-1 cells using a CRE-reporter (luciferase) construct. The data presented in Fig. 9A show that transient transfection of MLTC-1 cells with CRE, CREB, and p38 MAPKα-wt resulted in a significant reduction in basal CRE-luciferase activity as compared with cells transfected with CRE alone or CRE+CREB. In contrast, transient expression of CRE, CREB, and p38 MAPKα-dn in these cells resulted in an approximately 50% increase in basal CRE-luciferase activity compared with results obtained with CRE or CRE+CREB construct. Results presented in Fig. 9B demonstrate that co-transfection of the cells with p38 MAPKα-wt decreased the Bt2cAMP-induced luciferase activity. Expression of p38 MAPKα-dn construct, however, showed no effect on CRE-luciferase activity in response to cAMP stimulation (Fig. 9B). We also evaluated the effects of overexpression of p38 MAPKα-wt and p38 MAPKα-dn on AP1 (TRE) luciferase activity in MLTC-1 cells. As illustrated in Fig. 9C and D, transient expression of p38 MAPKα-wt or p38 MAPKα-dn, however, showed no effect on AP1 luciferase activity either under basal conditions or in response to TPA stimulation. These results led us to conclude that p38 MAPKα inhibits cAMP-induced Star promoter activity primarily by interfering with the PKA-mediated CREB phosphorylation and its functional expression.

Figure 9
Figure 9

p38 MAPKα-mediated downregulation of CRE (A and B), but not AP1/TRE (C and D), reporter activity in MLTC-1 cells. MLTC-1 cells were transfected with Cre-Luc (firefly luciferase)+Renilla luciferase (Rluc, pRL-TK), with or without CREB, or CREB+p38 MAPKα-wt or p38 MAPKα-dn for 48 h. At the end of the incubation, washed dishes were further incubated with or without Bt2-cAMP followed by measurement of firefly and Renilla luciferase activities. Likewise, cells were transfected with AP1/TRE-luc (firefly luciferase)+Renilla luciferase (Rluc, pRL-TK), with or without p38 MAPKα-wt or p38 MAPKα-dn, and, after 48 h, dishes were further incubated with or without AP1/TRE activator TPA (10 nM) for 30 min. The cell lysates were then quantified for firefly and Renilla luciferase activities. Each value represents mean±s.e.m. of four separate determinations. **P<0.01 CRE + CREB p38α-dn vs CRE + CREB p38α-wt; †††P<0.001 basal (control) vs Bt2cAMP; ‡‡P<0.01 basal (control) vs TPA; and ‡‡‡P<0.001 basal (control) vs TPA.

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-13-0287

Discussion

STAR protein plays a pivotal role in the tropic peptide hormone-regulated acute phase of steroidogenesis, mediating the rate-limiting translocation of cholesterol from the OMM to the IMM, where side-chain cleavage enzyme (P450scc; Cyp11A) carries out the first committed step in steroidogenesis, i.e. conversion of cholesterol to pregnenolone, the precursor of all steroid hormones (Stocco & Clark 1996, Stocco 2001, Hu et al. 2010, Miller & Bose 2011). We and others previously reported that expression of STAR protein is downregulated in both the adrenal gland and testicular Leydig cells of aging rats, and, as a result, adequate amounts of cholesterol are not transported to mitochondria, leading to defective steroidogenesis during aging (Leers-Sucheta et al. 1999, Luo et al. 2005, Wang et al. 2005, Sun et al. 2008). These changes have been linked to excessive oxidative stress and increased ROS production (Azhar et al. 1995, Chen et al. 2001, 2008, Cao et al. 2004, Abidi et al. 2008a,b, Zaidi et al. 2012). The observed increased expression of the active (phospho) form of oxidant-sensitive p38 MAPKα during aging led to the suggestion that this kinase mediates the anti-steroidogenic actions of excessive oxidative stress during aging (Abidi et al. 2008a,b). The current study examined the molecular mechanisms by which oxidant-sensitive p38 MAPK negatively affects Star gene transcription. Our initial qRT-PCR data indicate that Star (StarD1) is most abundantly expressed in both Y-1 and MLTC-1 cells and that Bt2cAMP stimulation further increased its mRNA levels by fourfold to fivefold in both cell types. The expression of StarD2 and StarD3, which belong to the same subgroup as StarD1 as well as a second subgroup of STARD proteins, STARD4, STARD5, and STARD6, was very low in Y-1 and MLTC-1 cells and their expression was not affected by cAMP stimulation of either cell type. These results are at variance with the results reported for another mouse Leydig tumor cell line, MA-10, where stimulation of cells with a cAMP analog was reported to stimulate mRNA expression of StarD4, in addition to a robust induction of StAR/StarD1 (Soccio et al. 2005). Similar to our data, however, the mRNA levels of StarD3 and StarD5 were induced by cAMP stimulation; the levels of StarD2, which is a phosphatidylcholine transfer protein (PCTP/StarD2) and StarD6, whose expression is limited to germ cells of testis (Soccio et al. 2002, Gomes et al. 2006, Bose et al. 2008), were not determined in this model Leydig cell line (Soccio et al. 2005). Interestingly, it has been shown that StarD6 exhibits similar activity to that of Star/StarD1 in inducing steroidogenesis in a cell-free mitochondrial system (Bose et al. 2008). Our qRT-PCR measurements also indicated that p38 MAPKα is the predominant endogenous isoform in both cell lines, although significant expression of p38 MAPKβ was also noted in MLTC-1 cells. In contrast, only low levels of p38 MAPKγ and p38 MAPKδ were detected in both cell types. These results are in agreement with an earlier report from this laboratory (Abidi et al. 2008b), but are at variance with another report showing that p38 MAPKδ is expressed at very high levels in human steroidogenic tissues (Wang et al. 1997). Whether the low expression of p38 MAPKδ noted here in mouse Y-1 adrenocortical tumor cells and mouse MLTC-1 Leydig tumor cells represents a species difference or tumor effect needs to be explored. As p38 MAPKα isoform (mRNA) is highly expressed in both Y-1 and MLTC-1 cells, most of the studies aimed at defining the effects of p38 MAPK on Star/StarD1 gene transcription were carried out with p38 MAPKα. However, we acknowledge that mRNA levels of the four p38 MAPKs in theory may not reflect the actual protein levels. Technical difficulties associated with currently available isoform-specific p38 MAPK antibodies, as well as lack of availability of p38 MAPKδ antibody, prevented us from measuring the protein levels of individual p38 MAPK isoforms.

In accordance with a previous observation that ROS-mediated excessive oxidative stress inhibits steroidogenesis (Margolin et al. 1990, Gatzuli et al. 1991, Endo et al. 1993, Stocco et al. 1993, Kodaman et al. 1994, Musicki et al. 1994, Carlson et al. 1995, Diemer et al. 2003, Abidi et al. 2008b), our data demonstrate that treatment of Y-1 and MLTC-1 cells with oxidants, superoxide anion (O2 −•), H2O2, or a lipid peroxidation product, 4-HNE results in a significant reduction in the levels of Star mRNA both under basal conditions and in response to cAMP stimulation. In addition, we provide evidence that exposure of MLTC-1 cells transiently overexpressing p38 MAPKα to the above mentioned three oxidants resulted in increased phosphorylation (activation) of p38 MAPKα, along with decreased Star promoter activity. The specificity studies demonstrated that p38 MAPKα, p38 MAPKβ, and p38 MAPKγ were highly effective at suppressing Star promoter activity both under basal conditions and in response to cAMP stimulation, although p38 MAPKα was relatively more effective than p38 MAPKβ or p38 MAPKγ. In contrast, expression of dn forms of p38 MAPKα, p38 MAPKβ, or p38 MAPKγ increased the expression of basal as well as cAMP-stimulated promoter activity. Likewise p38 MAPKα/β inhibitors, SB203580 or SB202190, prevented p38 MAPKα-mediated suppression of promoter activity, further confirming that p38 MAPK functions as a negative regulator of Star gene transcription. Although overexpression of p38 MAPKδ-wt construct exhibited no significant effect on basal Star promoter activity, transient overexpression of p38 MAPKδ-dn, however, significantly increased the cAMP-stimulated promoter activity over the values seen with the use of p38 MAPKδ-wt construct. Finally, use of various constructs of upstream activators of p38 MAPKs, MKK3 and MKK6 (Derijard et al. 1995, Raingeaud et al. 1995, 1996, Han et al. 1996), MKK6–p38-MAPKα, and MKK6–p38-MAPKγ fusion constructs (Robinson et al. 1998, Zheng et al. 1999, Qi et al. 2007) and MEFs with targeted disruption of Mkk3, Mkk6, or both Mkk3 and Mkk6 genes (Brancho et al. 2003, Kang et al. 2006) further established that p38 MAPKs, particularly oxidant-sensitive p38 MAPKα, are involved in the negative regulation of Star gene transcription.

Extensive evidence now indicates that the expression of Star is mainly regulated at the transcriptional level through a cAMP-PKA-dependent mechanism (Manna et al. 2009). Transcriptional activation of target genes by the cAMP-PKA signaling pathway is mainly achieved via cAMP-responsive element (CRE)-binding protein (CREB) transcription factor. cAMP-activated PKA catalyzes the phosphorylation of CREB at serine residue 133 (Altarejos & Montminy 2011). Phosphorylation of CREB at Ser133 is required for CREB-induced gene transcription and promotes recruitment of the transcriptional co-activator CREB-binding protein (CBP) and its paralogue p300 (Mayr & Montminy 2001). Although Star promoter lacks a consensus CRE (TGACGTCA), three CRE-like sites, CRE1, CRE2, and CRE3, have been characterized in the proximal region of the Star promoter (Manna et al. 2002), of which the CRE2 site overlaps with an AP1/TRE (5-TGAG/CCTCA-3)-binding site (Manna & Stocco 2007) that can bind both CRE (CREB and its family member CRE modulator (CREM), and ATF1 and also ATF2) and AP1/TRE (Fos/Jun)-binding proteins (Shaywitz & Greenberg 1999, Shaulian & Karin 2002). Our findings demonstrate that transient overexpression of p38 MAPKα-wt increased phospho-CREB (p-Ser133) levels, and the use of p38 MAPKα-dn resulted in a greater increase in phospho-CREB (p-Ser133) levels. Interestingly, neither p38 MAPKα-wt nor p38 MAPKα-dn increased the phosphorylation status of ATF-1, which is 65% identical to CREB in its primary amino acid sequence (Shaywitz & Greenberg 1999). At present, we do not know the precise mechanism(s) by which p38 MAPKα and p38 MAPKα-dn upregulate the cAMP-PKA-mediated Ser133 phosphorylation of CREB. As CREB is also a substrate for the p38 MAPK-regulated MSK kinase (Hauge & Frödin 2006), it is possible that both PKA and MSK simultaneously but variably catalyze CREB phosphorylation and in this process lead to increased CREB phosphorylation. The data obtained with the dn form of the p38 MAPKα raised another possibility that other kinases (e.g. PKC, calmodulin kinases, and glycogen synthase kinase; Impey & Goodman 2001, Johannessen et al. 2004, Johannessen & Moens 2007) that also phosphorylate CREB may cooperate with p38 MAPKα/MSK and interfere with the ability of PKA to catalyze Ser133 phosphorylation. These various possibilities are likely to decrease CREB activity and, consequently, Star gene transcription and its promoter activity.

The potential effect of p38 MAPKα on CREB function was studied in MLTC-1 cells using a CRE-reporter (luciferase) approach. Various measurements led to the conclusion that p38 MAPKα, by interfering with PKA-mediated Ser133 phosphorylation of CREB, inhibits its function. On the other hand, both p38 MAPKα-wt and p38 MAPKα-dn proteins showed no significant effect on AP1 reporter activity. We used AP1 (TRE) as a control, because the CRE and AP1 (TRE) consensus sequences differ by only one nucleotide (CRE, TGACGTCA and AP1/TRE, TGACTCA) (Hai & Curran 1991, Shaulian & Karin 2002) and both recognition sequences can bind ATF/CREB and the Fos/Jun family of proteins, albeit with a variable affinity (Hai & Curran 1991).

In summary, our data indicate that oxidant/ROS exposure of steroidogenic cells leads to simultaneous inhibition and activation of Star mRNA and phospho-p38 MAPK protein levels respectively. In addition, results regarding Star promoter activity reported in this study indicate that p38 MAPKα and to some extent p38 MAPKβ and p38 MAPKγ inhibit Star gene transcription and that upstream kinases, MKK3 and MKK6, play a significant role in this process. Finally, our data provide evidence that p38 MAPKα negatively regulates Star gene transcription by potentially interfering with cAMP-PKA-mediated phosphorylation and the functional expression of the CREB transcription factor.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This research was supported in part by the Department of Veterans Affairs (Office of Research and Development, Medical Research Service) and by grant numbers 2R01HL33881 and 1R01HL92473 from the National Institutes of Health.

Author contribution statement

S K Z, W-J S, F B K, and S A conceived and designed the experiments; S K Z performed the experiments; W-J S, F B K, and S A interpreted the results; S K Z, S B, W-J S, F B K, and S A drafted and edited the manuscript; S B and A B prepared the figures; M P M, J H, and R J D provided critical reagents; and S K Z, W-J S, S B, A B, M P M, J H, R J D, F B K, and S A approved the final version of the manuscript.

References

  • Abidi P, Leers-Sucheta S, Cortez Y, Han J & Azhar S 2008a Evidence that age-related changes in p38 MAP kinase contribute to the decreased steroid production by the adrenocortical cells from old rats. Aging Cell 7 168178. (doi:10.1111/j.1474-9726.2007.00364.x).

    • Search Google Scholar
    • Export Citation
  • Abidi P, Zhang H, Zaidi SM, Shen W-J, Leers-Sucheta S, Cortez Y, Han J & Azhar S 2008b Oxidative stress-induced inhibition of adrenal steroidogenesis requires participation of p38 mitogen-activated protein kinase signaling pathway. Journal of Endocrinology 198 193207. (doi:10.1677/JOE-07-0570).

    • Search Google Scholar
    • Export Citation
  • Altarejos JY & Montminy M 2011 CREB and the CRTC co-activators: sensors for hormonal and metabolic signals. Nature Reviews. Molecular Cell Biology 12 141151. (doi:10.1038/nrm3072).

    • Search Google Scholar
    • Export Citation
  • Azhar S, Cao L & Reaven E 1995 Alteration of the adrenal antioxidant defense system during aging in rats. Journal of Clinical Investigation 96 1414. (doi:10.1172/JCI118177).

    • Search Google Scholar
    • Export Citation
  • Bain J, Plater L, Elliott M, Shpiro N, Hastie C, Mclauchlan H, Klevernic I, Arthur J, Alessi D & Cohen P 2007 The selectivity of protein kinase inhibitors: a further update. Biochemical Journal 408 297315. (doi:10.1042/BJ20070797).

    • Search Google Scholar
    • Export Citation
  • Bose H, Whittal R, Ran Y, Bose M, Baker B & Miller W 2008 StAR-like activity and molten globule behavior of StARD6, a male germ-line protein. Biochemistry 47 22772288. (doi:10.1021/bi701966a).

    • Search Google Scholar
    • Export Citation
  • Brancho D, Tanaka N, Jaeschke A, Ventura J-J, Kelkar N, Tanaka Y, Kyuuma M, Takeshita T, Flavell RA & Davis RJ 2003 Mechanism of p38 MAP kinase activation in vivo. Genes and Development 17 19691978. (doi:10.1101/gad.1107303).

    • Search Google Scholar
    • Export Citation
  • Cao L, Leers-Sucheta S & Azhar S 2004 Aging alters the functional expression of enzymatic and non-enzymatic anti-oxidant defense systems in testicular rat Leydig cells. Journal of Steroid Biochemistry and Molecular Biology 88 6167. (doi:10.1016/j.jsbmb.2003.10.007).

    • Search Google Scholar
    • Export Citation
  • Carlson JC, Sawada M, Boone DL & Stauffer JM 1995 Stimulation of progesterone secretion in dispersed cells of rat corpora lutea by antioxidants. Steroids 60 272276. (doi:10.1016/0039-128X(94)00053-F).

    • Search Google Scholar
    • Export Citation
  • Chen H, Cangello D, Benson S, Folmer J, Zhu H, Trush MA & Zirkin BR 2001 Age-related increase in mitochondrial superoxide generation in the testosterone-producing cells of Brown Norway rat testes: relationship to reduced steroidogenic function? Experimental Gerontology 36 13611373. (doi:10.1016/S0531-5565(01)00118-8).

    • Search Google Scholar
    • Export Citation
  • Chen H, Pechenino AS, Liu J, Beattie MC, Brown TR & Zirkin BR 2008 Effect of glutathione depletion on Leydig cell steroidogenesis in young and old brown Norway rats. Endocrinology 149 26122619. (doi:10.1210/en.2007-1245).

    • Search Google Scholar
    • Export Citation
  • Corrêa SA & Eales KL 2012 The Role of p38 MAPK and its substrates in neuronal plasticity and neurodegenerative disease. Journal of Signal Transduction 2012 649079. (doi:10.1155/2012/649079).

    • Search Google Scholar
    • Export Citation
  • Coulthard LR, White DE, Jones DL, McDermott MF & Burchill SA 2009 p38 MAPK: stress responses from molecular mechanisms to therapeutics. Trends in Molecular Medicine 15 369379. (doi:10.1016/j.molmed.2009.06.005).

    • Search Google Scholar
    • Export Citation
  • Cuadrado A & Nebreda A 2010 Mechanisms and functions of p38 MAPK signalling. Biochemical Journal 429 403417. (doi:10.1042/BJ20100323).

  • Culty M, Luo L, Yao Z-X, Chen H, Papadopoulos V & Zirkin BR 2002 Cholesterol transport, peripheral benzodiazepine receptor, and steroidogenesis in aging Leydig cells. Journal of Andrology 23 439447. (doi:10.1002/j.1939-4640.2002.tb02251.x).

    • Search Google Scholar
    • Export Citation
  • Derijard B, Raingeaud J, Barrett T, Wu I-H, Han J, Ulevitch RJ & Davis RJ 1995 Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267 682685. (doi:10.1126/science.7839144).

    • Search Google Scholar
    • Export Citation
  • Diemer T, Allen JA, Hales KH & Hales DB 2003 Reactive oxygen disrupts mitochondria in MA-10 tumor Leydig cells and inhibits steroidogenic acute regulatory (StAR) protein and steroidogenesis. Endocrinology 144 28822891. (doi:10.1210/en.2002-0090).

    • Search Google Scholar
    • Export Citation
  • Endo T, Aten R, Leykin L & Behrman H 1993 Hydrogen peroxide evokes antisteroidogenic and antigonadotropic actions in human granulosa luteal cells. Journal of Clinical Endocrinology and Metabolism 76 337342. (doi:10.1210/jcem.76.2.7679398).

    • Search Google Scholar
    • Export Citation
  • Enslen H, Raingeaud J & Davis RJ 1998 Selective activation of p38 mitogen-activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6. Journal of Biological Chemistry 273 17411748. (doi:10.1074/jbc.273.3.1741).

    • Search Google Scholar
    • Export Citation
  • Gatzuli E, Aten RF & Behrman HR 1991 Inhibition of gonadotropin action and progesterone synthesis by xanthine oxidase in rat luteal cells. Endocrinology 128 22532258. (doi:10.1210/endo-128-5-2253).

    • Search Google Scholar
    • Export Citation
  • Gomes C, Oh S-D, Kim J-W, Chun S-Y, Lee K, Kwon H-B & Soh J 2006 Expression of the putative sterol binding protein Stard6 gene is male germ cell specific. Biology of Reproduction 72 651658. (doi:10.1095/biolreprod.104.032672).

    • Search Google Scholar
    • Export Citation
  • Hai T & Curran T 1991 Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity. PNAS 88 37203724. (doi:10.1073/pnas.88.9.3720).

    • Search Google Scholar
    • Export Citation
  • Han J, Lee J, Bibbs L & Ulevitch R 1994 A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265 808811. (doi:10.1126/science.7914033).

    • Search Google Scholar
    • Export Citation
  • Han J, Lee J-D, Jiang Y, Li Z, Feng L & Ulevitch RJ 1996 Characterization of the structure and function of a novel MAP kinase kinase (MKK6). Journal of Biological Chemistry 271 28862891. (doi:10.1074/jbc.271.6.2886).

    • Search Google Scholar
    • Export Citation
  • Hauge C & Frödin M 2006 RSK and MSK in MAP kinase signalling. Journal of Cell Science 119 30213023. (doi:10.1242/jcs.02950).

  • Hu J, Zhang Z, Shen W-J & Azhar S 2010 Review: Cellular cholesterol delivery, intracellular processing and utilization for biosynthesis of steroid hormones. Nutrition & Metabolism 7 4772. (doi:10.1186/1743-7075-7-47).

    • Search Google Scholar
    • Export Citation
  • Impey S & Goodman R 2001 CREB signaling – timing is everything. Science's STKE 2001 pe1. (doi:10.1126/stke.2001.82.pe1).

  • Johannessen M & Moens U 2007 Multisite phosphorylation of the cAMP response element-binding protein (CREB) by a diversity of protein kinases. Frontiers in Bioscience 12 18141832. (doi:10.2741/2190).

    • Search Google Scholar
    • Export Citation
  • Johannessen M, Delghandi MP & Moens U 2004 What turns CREB on? Cellular Signalling 16 12111227. (doi:10.1016/j.cellsig.2004.05.001).

  • Kang YJ, Seit-Nebi A, Davis RJ & Han J 2006 Multiple activation mechanisms of p38α mitogen-activated protein kinase. Journal of Biological Chemistry 281 2622526234. (doi:10.1074/jbc.M606800200).

    • Search Google Scholar
    • Export Citation
  • Kodaman P, Aten R & Behrman H 1994 Lipid hydroperoxides evoke antigonadotropic and antisteroidogenic activity in rat luteal cells. Endocrinology 135 27232730. (doi:10.1210/endo.135.6.7988463).

    • Search Google Scholar
    • Export Citation
  • Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ & Keys JR 1994 A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372 73946. (doi:10.1038/372739a0).

    • Search Google Scholar
    • Export Citation
  • Leers-Sucheta S, Stocco DM & Azhar S 1999 Down-regulation of steroidogenic acute regulatory (StAR) protein in rat Leydig cells: implications for regulation of testosterone production during aging. Mechanisms of Ageing and Development 107 197203. (doi:10.1016/S0047-6374(98)00149-3).

    • Search Google Scholar
    • Export Citation
  • Liao C, Reaven E & Azhar S 1993 Age-related decline in the steroidogenic capacity of isolated rat Leydig cells: a defect in cholesterol mobilization and processing. Journal of Steroid Biochemistry and Molecular Biology 46 3947. (doi:10.1016/0960-0760(93)90207-D).

    • Search Google Scholar
    • Export Citation
  • Liu J, Rone MB & Papadopoulos V 2006 Protein–protein interactions mediate mitochondrial cholesterol transport and steroid biosynthesis. Journal of Biological Chemistry 281 3887938893. (doi:10.1074/jbc.M608820200).

    • Search Google Scholar
    • Export Citation
  • Luo L, Chen H & Zirkin B 2001 Leydig cell aging: steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme. Journal of Andrology 22 149156. (doi:10.1002/j.1939-4640.2001.tb02165.x).

    • Search Google Scholar
    • Export Citation
  • Luo L, Chen H & Zirkin BR 2005 Temporal relationships among testosterone production, steroidogenic acute regulatory protein (StAR), and P450 side chain cleavage enzyme (P450scc) during Leydig cell aging. Journal of Andrology 26 2531. (doi:10.1002/j.1939-4640.2005.tb02868.x).

    • Search Google Scholar
    • Export Citation
  • Manna PR & Stocco DM 2007 Crosstalk of CREB and Fos/Jun on a single cis-element: transcriptional repression of the steroidogenic acute regulatory protein gene. Journal of Molecular Endocrinology 39 261277. (doi:10.1677/JME-07-0065).

    • Search Google Scholar
    • Export Citation
  • Manna PR, Dyson MT, Eubank DW, Clark BJ, Lalli E, Sassone-Corsi P, Zeleznik AJ & Stocco DM 2002 Regulation of steroidogenesis and the steroidogenic acute regulatory protein by a member of the cAMP response-element binding protein family. Molecular Endocrinology 16 184199. (doi:10.1210/mend.16.1.0759).

    • Search Google Scholar
    • Export Citation
  • Manna PR, Dyson MT & Stocco DM 2009 Role of basic leucine zipper proteins in transcriptional regulation of the steroidogenic acute regulatory protein gene. Molecular and Cellular Endocrinology 302 111. (doi:10.1016/j.mce.2008.12.009).

    • Search Google Scholar
    • Export Citation
  • Margolin Y, Aten RF & Behrman HR 1990 Antigonadotropic and antisteroidogenic actions of peroxide in rat granulosa cells. Endocrinology 127 245250. (doi:10.1210/endo-127-1-245).

    • Search Google Scholar
    • Export Citation
  • Mayr B & Montminy M 2001 Transcriptional regulation by the phosphorylation-dependent factor CREB. Nature Reviews. Molecular Cell Biology 2 599609. (doi:10.1038/35085068).

    • Search Google Scholar
    • Export Citation
  • Miller WL & Bose HS 2011 Early steps in steroidogenesis: intracellular cholesterol trafficking Thematic Review series: genetics of human lipid diseases. Journal of Lipid Research 52 21112135. (doi:10.1194/jlr.R016675).

    • Search Google Scholar
    • Export Citation
  • Musicki B, Aten R & Behrman H 1994 Inhibition of protein synthesis and hormone-sensitive steroidogenesis in response to hydrogen peroxide in rat luteal cells. Endocrinology 134 588595. (doi:10.1210/endo.134.2.7507829).

    • Search Google Scholar
    • Export Citation
  • Popplewell P & Azhar S 1987 Effects of aging on cholesterol content and cholesterol metabolizing enzymes in the rat adrenal gland. Endocrinology 121 6473. (doi:10.1210/endo-121-1-64).

    • Search Google Scholar
    • Export Citation
  • Pramanik R, Qi X, Borowicz S, Choubey D, Schultz RM, Han J & Chen G 2003 p38 Isoforms have opposite effects on AP-1-dependent transcription through regulation of c-Jun the determinant role of the isoforms in the p38 MAPK signal specificity. Journal of Biological Chemistry 278 48314839. (doi:10.1074/jbc.M207732200).

    • Search Google Scholar
    • Export Citation
  • Qi X, Pohl NM, Loesch M, Hou S, Li R, Qin J-Z, Cuenda A & Chen G 2007 p38α antagonizes p38γ activity through c-Jun-dependent ubiquitin–proteasome pathways in regulating Ras transformation and stress response. Journal of Biological Chemistry 282 3139831408. (doi:10.1074/jbc.M703857200).

    • Search Google Scholar
    • Export Citation
  • Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ & Davis RJ 1995 Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. Journal of Biological Chemistry 270 74207426. (doi:10.1074/jbc.270.13.7420).

    • Search Google Scholar
    • Export Citation
  • Raingeaud J, Whitmarsh AJ, Barrett T, Derijard B & Davis RJ 1996 MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Molecular and Cellular Biology 16 12471255.

    • Search Google Scholar
    • Export Citation
  • Reaven E, Tsai L & Azhar S 1995 Cholesterol uptake by the ‘selective’ pathway of ovarian granulosa cells: early intracellular events. Journal of Lipid Research 36 16021617.

    • Search Google Scholar
    • Export Citation
  • Remy G, Risco AM, Iñesta-Vaquera FA, González-Terán B, Sabio G, Davis RJ & Cuenda A 2010 Differential activation of p38MAPK isoforms by MKK6 and MKK3. Cellular Signalling 22 660667. (doi:10.1016/j.cellsig.2009.11.020).

    • Search Google Scholar
    • Export Citation
  • Robinson MJ, Stippec SA, Goldsmith E, White MA & Cobb MH 1998 A constitutively active and nuclear form of the MAP kinase ERK2 is sufficient for neurite outgrowth and cell transformation. Current Biology 8 11411152. (doi:10.1016/S0960-9822(07)00485-X).

    • Search Google Scholar
    • Export Citation
  • Rouse J, Cohen P, Trigon S, Morange M, Alonso-Llamazares A, Zamanillo D, Hunt T & Nebreda AR 1994 A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78 10271037. (doi:10.1016/0092-8674(94)90277-1).

    • Search Google Scholar
    • Export Citation
  • Roux PP & Blenis J 2004 ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiology and Molecular Biology Reviews 68 320344. (doi:10.1128/MMBR.68.2.320-344.2004).

    • Search Google Scholar
    • Export Citation
  • Sandhoff TW, Hales DB, Hales KH & McLean MP 1998 Transcriptional regulation of the rat steroidogenic acute regulatory protein gene by steroidogenic factor 1. Endocrinology 139 48204831. (doi:10.1210/endo.139.12.6345).

    • Search Google Scholar
    • Export Citation
  • Shaulian E & Karin M 2002 AP-1 as a regulator of cell life and death. Nature Cell Biology 4 E131E136. (doi:10.1038/ncb0502-e131).

  • Shaywitz AJ & Greenberg ME 1999 CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annual Review of Biochemistry 68 821861. (doi:10.1146/annurev.biochem.68.1.821).

    • Search Google Scholar
    • Export Citation
  • Soccio R & Breslow J 2003 StAR-related transfer (START) proteins: mediators of intracellular lipid metabolism. Journal of Biological Chemistry 278 2218322186. (doi:10.1074/jbc.R300003200).

    • Search Google Scholar
    • Export Citation
  • Soccio R, Adams R, Romanowski M, Sehayek E, Burley S & Breslow J 2002 The cholesterol-regulated StarD4 gene encodes a StAR-related lipid transfer protein with two closely related homologues, StarD5 and StarD6. PNAS 99 69436948. (doi:10.1073/pnas.052143799).

    • Search Google Scholar
    • Export Citation
  • Soccio R, Adams R, Maxwell K & Breslow J 2005 Differential gene regulation of StarD4 and StarD5 cholesterol transfer proteins. Activation of StarD4 by sterol regulatory element-binding protein-2 and StarD5 by endoplasmic reticulum stress. Journal of Biological Chemistry 280 1941019418. (doi:10.1074/jbc.M501778200).

    • Search Google Scholar
    • Export Citation
  • Stocco DM 2001 StAR protein and the regulation of steroid hormone biosynthesis. Annual Review of Physiology 63 193213. (doi:10.1146/annurev.physiol.63.1.193).

    • Search Google Scholar
    • Export Citation
  • Stocco D & Clark B 1996 Regulation of the acute production of steroids in steroidogenic cells. Endocrine Reviews 17 221244. (doi:10.1210/edrv-17-3-221).

    • Search Google Scholar
    • Export Citation
  • Stocco D, Wells J & Clark B 1993 The effects of hydrogen peroxide on steroidogenesis in mouse Leydig tumor cells. Endocrinology 133 28272832. (doi:10.1210/endo.133.6.8243310).

    • Search Google Scholar
    • Export Citation
  • Sun Z, Shen W-J, Sucheta S-L & Azhar S 2008 Impact of aging on cholesterol transport protein expression and steroidogenesis in rat testicular Leydig cells. Open Longevity Science 2 7685. (doi:10.2174/1876326X00802010076).

    • Search Google Scholar
    • Export Citation
  • Wang X, Diener K, Manthey C, Wang S, Rosenzweig B, Bray J, Delaney J, Cole C, Chan-Hui P & Mantlo N et al. 1997 Molecular cloning and characterization of a novel p38 mitogen-activated protein kinase. Journal of Biological Chemistry 272 2366823674. (doi:10.1074/jbc.272.38.23668).

    • Search Google Scholar
    • Export Citation
  • Wang X, Shen C, Dyson M, Eimerl S, Orly J, Hutson J & Stocco D 2005 Cyclooxygenase-2 regulation of the age-related decline in testosterone. Endocrinology 146 42024208. (doi:10.1210/en.2005-0298).

    • Search Google Scholar
    • Export Citation
  • Wysk M, Yang DD, Lu H-T, Flavell RA & Davis RJ 1999 Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for tumor necrosis factor-induced cytokine expression. PNAS 96 37633768. (doi:10.1073/pnas.96.7.3763).

    • Search Google Scholar
    • Export Citation
  • Zaidi SK, Shen W-J & Azhar S 2012 Impact of aging on steroid hormone biosynthesis and secretion. Open Longevity Science 6 130. (doi:10.2174/1876326X01206010001).

    • Search Google Scholar
    • Export Citation
  • Zheng C, Xiang J, Hunter T & Lin A 1999 The JNKK2–JNK1 fusion protein acts as a constitutively active c-Jun kinase that stimulates c-Jun transcription activity. Journal of Biological Chemistry 274 2896628971. (doi:10.1074/jbc.274.41.28966).

    • Search Google Scholar
    • Export Citation

S K Zaidi is now at Center of Excellence in Genomic Research, King Abdulaziz University, Kingdom of Saudi Arabia, Jeddah 21589, Saudi Arabia

 

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    qRT-PCR analysis of StarD1/Star, StarD2, StarD3, StarD4, StarD5, StarD6, StarD7, and StarD10 mRNA levels in mouse Y-1 adrenal cells and mouse Leydig tumor cells, MLTC-1, treated with or without Bt2cAMP (2.5 mM) for 6 h. Groups of RNA samples from Y-1 or MLTC-1 cells were analyzed by qRT-PCR as described in the ‘Materials and methods’ section. The levels of expression of StarD1/Star, StarD2, StarD3, StarD4, StarD5, StarD6, StarD7, and StarD10 mRNAs are shown. Expression of cyclophilin was used for normalization. (A) Y-1 cells and (B) MLTC-1 cells. Each value represents mean±s.e.m. of four separate determinations. ***P<0.0001 basal (control) vs Bt2cAMP.

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    QRT-PCR analyses of p38 MAPK isoforms, p38 MAPKα, p38 MAPKβ, p38 MAPKγ, and p38 MAPKδ mRNA levels in cultured Y-1 and MLTC-1 cells. Groups of RNA samples from Y-1 or MLTC-1 cells were analyzed by qRT-PCR as described in the ‘Materials and methods’ section. The levels of expression of p38 MAPKα, p38 MAPKβ, p38 MAPKγ, and p38 MAPKδ mRNAs are shown. Expression of 36B4 was used for normalization. (A) Y-1 cells and (B) MLTC-1 cells. Each value represents mean±s.e.m. of four separate determinations. *P<0.05 p38α vs p38β (MLTC-1) and ***P<0.001 p38α vs p38β (Y-1), p38γ or p38δ.

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    Exposure of Y-1 cells (A) and MLTC-1 cells (B) to xanthine/xanthine oxidase (X/XO) generated superoxide anion (O2 −•) or hydrogen peroxide (H2O2) attenuates mRNA expression of Star/StarD1 both under basal conditions and in response to cAMP stimulation. Cultured Y-1 and MLTC-1 cells were exposed to ± 0.1 mM xanthine and 10 mU/ml XO or ± 50 μM hydrogen peroxide (H2O2) for 1 h. At the end of incubation, the dishes were washed and subsequently incubated with or without Bt2cAMP (2.5 mM) for 5 h, followed by isolation of total RNA and the quantification of the Star/StarD1 mRNA levels by qRT-PCR. Each value represents mean±s.e.m. of four separate determinations. ***P<0.0001 basal (control) vs Bt2cAMP.

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    Evaluation of the effects of p38 MAPKα (wt) overexpression and the effects of X/XO generated O2 −•, H2O2, and 4-HNE on basal and cAMP-stimulated rat Star promoter (luciferase) activity in MLTC-1 cells. MLTC-1 cells were transiently transfected with a rat p-1862 Star–Luc (firefly luciferase) construct (1 μg)+Renilla luciferase (Rluc, pRL-TK) (50 ng) and ± native p38 MAPKα (wt) (1 μg) using Lipofectamine 2000. After 48 h, dishes were exposed to ± xanthine (0.1 mM)/XO (10 mU/ml), ± 50 μM H2O2, or ± 50 μM 4-hydroxynonenol (4-HNE) for 1 h, washed, incubated with or without Bt2cAMP (2.5 mM) for 5 h, cells were lysed using cell lysis buffer (Promega Corporation) and subsequently assayed for both firefly and Renilla luciferase activities using a Promega Dual-Luciferase Reporter Kit (E1980). In some cases, cell lysates were subjected to SDS–PAGE followed by western blotting analysis and the quantification of the total and the phosphorylated p38 MAPKα bands. (A) Western blots showing the bands stained for either total p38 MAPKα or its phosphorylated form. (B) Rat Star promoter luciferase activity. **P<0.01 basal (control) vs Bt2cAMP and ***P<0.0001 basal (control) vs Bt2cAMP.

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    Effects of overexpression of wt and dn p38 MAPKα, p38 MAPKβ, p38 MAPKγ, and p38 MAPKδ constructs on Star promoter activity in MLTC-1 cells. The cells were transiently transfected with p-1862 Star–Luc (firefly luciferase) construct plus Renilla luciferase (Rluc, pRL-TK), and empty vector, p38 MAPKα-wt, p38 MAPKα-dn, p38 MAPKβ-wt, p38 MAPKβ-dn, p38 MAPKγ-wt, p38 MAPKγ-dn, p38 MAPKδ-wt, or p38 MAPKδ-dn. Forty-eight hours after transfection, the dishes were washed and subsequently incubated with or without Bt2cAMP (2.5 mM) for 5 h and cells lysed using cell lysis buffer (Promega Corporation) and subsequently assayed for both firefly and Renilla luciferase activities. (A) p38 MAPKα, (B) p38 MAPKβ, (C) p38 MAPKγ, and (D) p38 MAPKδ. Each value represents mean±s.e.m. of four separate determinations. wt (native); dn, dominant negative. *P<0.01 basal (control) vs Bt2cAMP; **P<0.01 basal (control) vs Bt2cAMP; ***P<0.0001 basal (control) vs Bt2cAMP; ††P<0.01 Bt2cAMP p38δ-dn vs Bt2cAMP p38α-wt; and †††P<0.0001 Bt2cAMP control vs Bt2cAMP p38α-wt.

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    Effects of transient overexpression of wt and dominant-negative p38 MAPKα on basal and cAMP-stimulated endogenous mRNA levels of Star in MLTC-1 cells. MLTC-1 cells were transiently transfected with empty vector, p38 MAPKα-wt, or p38 MAPKα-dn and, 48 h after transfection, the dishes were washed and subsequently incubated with or without Bt2cAMP (2.5 mM) for 5 h. Following incubation, the dishes were subjected to total RNA isolation and the RNA samples quantified for Star (StarD1) mRNA levels by qRT-PCR. Each value represents mean±s.e.m. of four separate determinations. *P<0.01 Bt2cAMP p38α-dn vs basal (control) Bt2cAMP; ***P<0.01 Bt2cAMP p38α-wt vs basal (control) Bt2cAMP; and †††P<0.001 Bt2cAMP p38α-dn vs Bt2cAMP p38α-wt.

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    Regulation of Star promoter activity in MITC-1 cells in response to transient overexpression of MKK3, MKK6, or MKK6–p38 MAPKα/γ constructs and mouse embryonic fibroblasts (MEFs) lacking MKK3, MKK6, or both. (A) MLTC-1 cells were cotransfected with p-1862 Star–Luc (firefly luciferase) construct (1 μg) plus Renilla luciferase (Rluc, pRL-TK) (50 ng), and empty vector, p38 MAPKα-wt, p38 MAPKα-dn, MKK3-ca, MKK3-dn, MKK6-ca, or MKK6-dn. At the end of 48 h, the dishes were treated with or without Bt2cAMP (2.5 mM) for an additional 5 h and subsequently cell lysates assayed for firefly and Renilla luciferase activities. (B) MLTC-1 cells were transiently transfected with empty vector, p38 MAPKα-wt, p38 MAPKα-dn, MKK6–p38 MAPKα-ca, MKK6–p38 MAPKα-dn, MKK6–p38 MAPKγ-ca, or MKK6–p38 MAPKγ-dn and, after 48 h, further treated with or without Bt2cAMP for 5 h. Cell lysates were assayed for firefly and Renilla luciferase activities as described above. (C) Wt (MKK3/6) MEFs and MEFs deficient in MKK3−/−, MKK6−/−, or both (MKK3−/−/MKK6−/−) were co-transfected with p-1862 StarLuc (firefly luciferase) and Renilla luciferase (Rluc, pRL-TK) constructs for 48 h, stimulated with or without Bt2cAMP for an additional 5 h, followed by determination of firefly and Renilla luciferase activities. (D) MKK3/6 and MKK3−/−/MKK6−/− DKO MEFs were co-transfected with p-1862 Star–Luc (firefly luciferase) and Renilla luciferase (Rluc, pRL-TK) constructs. In a third set MKK3−/−/MKK6−/− DKO MEFs were co-transfected with MKK3-ca+MKK6-ca plus p-1862 Star–Luc (firefly luciferase) and Renilla luciferase (Rluc, pRL-TK) constructs for 48 h followed by treatment with ± Bt2cAMP. Cell lysates were assayed for firefly and Renilla luciferase activities, as described earlier. Each value represents mean±s.e.m. of four separate determinations. ca, constitutively active; dn, dominant negative. All values presented (A) show statistically significant differences between basal vs Bt2cAMP and basal (control) or basal Bt2cAMP vs p38α-wt, MKK3-ca, or MKK6-ca. The values presented in (B) show statistically significant differences between basal vs Bt2cAMP and basal (control) or basal Bt2cAMP vs p38α-wt, MKK6–p38α-ca, or MKK6–p38γ-ca. (C) Basal (control) or basal Bt2cAMP WT3/6 show statistically significant differences from the corresponding Bt2cAMP MKK3−/−, MKK6−/−, or MKK3/6−/− groups. (D) ***P<0.001 wt MKK3/6 Bt2cAMP vs MKK3/6−/− Bt2cAMP; and ††P<0.01 MKK3/6−/− Bt2cAMP vs MKK3/6 plus MKK3-ca+MKK6-ca Bt2cAMP.

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    Effects of overexpression of p38 MAPKα-wt and p38 MAPKα-dn on the levels of total (t) and phospho-forms of CREB in response to treatment of cells with or without Bt2cAMP. Cells were transfected with either an empty vector, p38 MAPKα-wt, or p38 MAPKα-dn, and, after 48 h, treated with or without Bt2cAMP. The cell lysates were subjected to SDS–PAGE followed by western blotting analysis. The blots were developed using anti-CREB (total) or anti-phospho-CREB (Ser133). (A) Western blot of p-CREB, t-CREB and p-ATF. (B) Quantification of ratios of p-CREB:t-CREB. (C) Quantification of ratios of p-ATF:t-ATF. t, total; p, phosphorylated.

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    p38 MAPKα-mediated downregulation of CRE (A and B), but not AP1/TRE (C and D), reporter activity in MLTC-1 cells. MLTC-1 cells were transfected with Cre-Luc (firefly luciferase)+Renilla luciferase (Rluc, pRL-TK), with or without CREB, or CREB+p38 MAPKα-wt or p38 MAPKα-dn for 48 h. At the end of the incubation, washed dishes were further incubated with or without Bt2-cAMP followed by measurement of firefly and Renilla luciferase activities. Likewise, cells were transfected with AP1/TRE-luc (firefly luciferase)+Renilla luciferase (Rluc, pRL-TK), with or without p38 MAPKα-wt or p38 MAPKα-dn, and, after 48 h, dishes were further incubated with or without AP1/TRE activator TPA (10 nM) for 30 min. The cell lysates were then quantified for firefly and Renilla luciferase activities. Each value represents mean±s.e.m. of four separate determinations. **P<0.01 CRE + CREB p38α-dn vs CRE + CREB p38α-wt; †††P<0.001 basal (control) vs Bt2cAMP; ‡‡P<0.01 basal (control) vs TPA; and ‡‡‡P<0.001 basal (control) vs TPA.

  • Abidi P, Leers-Sucheta S, Cortez Y, Han J & Azhar S 2008a Evidence that age-related changes in p38 MAP kinase contribute to the decreased steroid production by the adrenocortical cells from old rats. Aging Cell 7 168178. (doi:10.1111/j.1474-9726.2007.00364.x).

    • Search Google Scholar
    • Export Citation
  • Abidi P, Zhang H, Zaidi SM, Shen W-J, Leers-Sucheta S, Cortez Y, Han J & Azhar S 2008b Oxidative stress-induced inhibition of adrenal steroidogenesis requires participation of p38 mitogen-activated protein kinase signaling pathway. Journal of Endocrinology 198 193207. (doi:10.1677/JOE-07-0570).

    • Search Google Scholar
    • Export Citation
  • Altarejos JY & Montminy M 2011 CREB and the CRTC co-activators: sensors for hormonal and metabolic signals. Nature Reviews. Molecular Cell Biology 12 141151. (doi:10.1038/nrm3072).

    • Search Google Scholar
    • Export Citation
  • Azhar S, Cao L & Reaven E 1995 Alteration of the adrenal antioxidant defense system during aging in rats. Journal of Clinical Investigation 96 1414. (doi:10.1172/JCI118177).

    • Search Google Scholar
    • Export Citation
  • Bain J, Plater L, Elliott M, Shpiro N, Hastie C, Mclauchlan H, Klevernic I, Arthur J, Alessi D & Cohen P 2007 The selectivity of protein kinase inhibitors: a further update. Biochemical Journal 408 297315. (doi:10.1042/BJ20070797).

    • Search Google Scholar
    • Export Citation
  • Bose H, Whittal R, Ran Y, Bose M, Baker B & Miller W 2008 StAR-like activity and molten globule behavior of StARD6, a male germ-line protein. Biochemistry 47 22772288. (doi:10.1021/bi701966a).

    • Search Google Scholar
    • Export Citation
  • Brancho D, Tanaka N, Jaeschke A, Ventura J-J, Kelkar N, Tanaka Y, Kyuuma M, Takeshita T, Flavell RA & Davis RJ 2003 Mechanism of p38 MAP kinase activation in vivo. Genes and Development 17 19691978. (doi:10.1101/gad.1107303).

    • Search Google Scholar
    • Export Citation
  • Cao L, Leers-Sucheta S & Azhar S 2004 Aging alters the functional expression of enzymatic and non-enzymatic anti-oxidant defense systems in testicular rat Leydig cells. Journal of Steroid Biochemistry and Molecular Biology 88 6167. (doi:10.1016/j.jsbmb.2003.10.007).

    • Search Google Scholar
    • Export Citation
  • Carlson JC, Sawada M, Boone DL & Stauffer JM 1995 Stimulation of progesterone secretion in dispersed cells of rat corpora lutea by antioxidants. Steroids 60 272276. (doi:10.1016/0039-128X(94)00053-F).

    • Search Google Scholar
    • Export Citation
  • Chen H, Cangello D, Benson S, Folmer J, Zhu H, Trush MA & Zirkin BR 2001 Age-related increase in mitochondrial superoxide generation in the testosterone-producing cells of Brown Norway rat testes: relationship to reduced steroidogenic function? Experimental Gerontology 36 13611373. (doi:10.1016/S0531-5565(01)00118-8).

    • Search Google Scholar
    • Export Citation
  • Chen H, Pechenino AS, Liu J, Beattie MC, Brown TR & Zirkin BR 2008 Effect of glutathione depletion on Leydig cell steroidogenesis in young and old brown Norway rats. Endocrinology 149 26122619. (doi:10.1210/en.2007-1245).

    • Search Google Scholar
    • Export Citation
  • Corrêa SA & Eales KL 2012 The Role of p38 MAPK and its substrates in neuronal plasticity and neurodegenerative disease. Journal of Signal Transduction 2012 649079. (doi:10.1155/2012/649079).

    • Search Google Scholar
    • Export Citation
  • Coulthard LR, White DE, Jones DL, McDermott MF & Burchill SA 2009 p38 MAPK: stress responses from molecular mechanisms to therapeutics. Trends in Molecular Medicine 15 369379. (doi:10.1016/j.molmed.2009.06.005).

    • Search Google Scholar
    • Export Citation
  • Cuadrado A & Nebreda A 2010 Mechanisms and functions of p38 MAPK signalling. Biochemical Journal 429 403417. (doi:10.1042/BJ20100323).

  • Culty M, Luo L, Yao Z-X, Chen H, Papadopoulos V & Zirkin BR 2002 Cholesterol transport, peripheral benzodiazepine receptor, and steroidogenesis in aging Leydig cells. Journal of Andrology 23 439447. (doi:10.1002/j.1939-4640.2002.tb02251.x).

    • Search Google Scholar
    • Export Citation
  • Derijard B, Raingeaud J, Barrett T, Wu I-H, Han J, Ulevitch RJ & Davis RJ 1995 Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267 682685. (doi:10.1126/science.7839144).

    • Search Google Scholar
    • Export Citation
  • Diemer T, Allen JA, Hales KH & Hales DB 2003 Reactive oxygen disrupts mitochondria in MA-10 tumor Leydig cells and inhibits steroidogenic acute regulatory (StAR) protein and steroidogenesis. Endocrinology 144 28822891. (doi:10.1210/en.2002-0090).

    • Search Google Scholar
    • Export Citation
  • Endo T, Aten R, Leykin L & Behrman H 1993 Hydrogen peroxide evokes antisteroidogenic and antigonadotropic actions in human granulosa luteal cells. Journal of Clinical Endocrinology and Metabolism 76 337342. (doi:10.1210/jcem.76.2.7679398).

    • Search Google Scholar
    • Export Citation
  • Enslen H, Raingeaud J & Davis RJ 1998 Selective activation of p38 mitogen-activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6. Journal of Biological Chemistry 273 17411748. (doi:10.1074/jbc.273.3.1741).

    • Search Google Scholar
    • Export Citation
  • Gatzuli E, Aten RF & Behrman HR 1991 Inhibition of gonadotropin action and progesterone synthesis by xanthine oxidase in rat luteal cells. Endocrinology 128 22532258. (doi:10.1210/endo-128-5-2253).

    • Search Google Scholar
    • Export Citation
  • Gomes C, Oh S-D, Kim J-W, Chun S-Y, Lee K, Kwon H-B & Soh J 2006 Expression of the putative sterol binding protein Stard6 gene is male germ cell specific. Biology of Reproduction 72 651658. (doi:10.1095/biolreprod.104.032672).

    • Search Google Scholar
    • Export Citation
  • Hai T & Curran T 1991 Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity. PNAS 88 37203724. (doi:10.1073/pnas.88.9.3720).

    • Search Google Scholar
    • Export Citation
  • Han J, Lee J, Bibbs L & Ulevitch R 1994 A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265 808811. (doi:10.1126/science.7914033).

    • Search Google Scholar
    • Export Citation
  • Han J, Lee J-D, Jiang Y, Li Z, Feng L & Ulevitch RJ 1996 Characterization of the structure and function of a novel MAP kinase kinase (MKK6). Journal of Biological Chemistry 271 28862891. (doi:10.1074/jbc.271.6.2886).

    • Search Google Scholar
    • Export Citation
  • Hauge C & Frödin M 2006 RSK and MSK in MAP kinase signalling. Journal of Cell Science 119 30213023. (doi:10.1242/jcs.02950).

  • Hu J, Zhang Z, Shen W-J & Azhar S 2010 Review: Cellular cholesterol delivery, intracellular processing and utilization for biosynthesis of steroid hormones. Nutrition & Metabolism 7 4772. (doi:10.1186/1743-7075-7-47).

    • Search Google Scholar
    • Export Citation
  • Impey S & Goodman R 2001 CREB signaling – timing is everything. Science's STKE 2001 pe1. (doi:10.1126/stke.2001.82.pe1).

  • Johannessen M & Moens U 2007 Multisite phosphorylation of the cAMP response element-binding protein (CREB) by a diversity of protein kinases. Frontiers in Bioscience 12 18141832. (doi:10.2741/2190).

    • Search Google Scholar
    • Export Citation
  • Johannessen M, Delghandi MP & Moens U 2004 What turns CREB on? Cellular Signalling 16 12111227. (doi:10.1016/j.cellsig.2004.05.001).

  • Kang YJ, Seit-Nebi A, Davis RJ & Han J 2006 Multiple activation mechanisms of p38α mitogen-activated protein kinase. Journal of Biological Chemistry 281 2622526234. (doi:10.1074/jbc.M606800200).

    • Search Google Scholar
    • Export Citation
  • Kodaman P, Aten R & Behrman H 1994 Lipid hydroperoxides evoke antigonadotropic and antisteroidogenic activity in rat luteal cells. Endocrinology 135 27232730. (doi:10.1210/endo.135.6.7988463).

    • Search Google Scholar
    • Export Citation
  • Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ & Keys JR 1994 A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372 73946. (doi:10.1038/372739a0).

    • Search Google Scholar
    • Export Citation
  • Leers-Sucheta S, Stocco DM & Azhar S 1999 Down-regulation of steroidogenic acute regulatory (StAR) protein in rat Leydig cells: implications for regulation of testosterone production during aging. Mechanisms of Ageing and Development 107 197203. (doi:10.1016/S0047-6374(98)00149-3).

    • Search Google Scholar
    • Export Citation
  • Liao C, Reaven E & Azhar S 1993 Age-related decline in the steroidogenic capacity of isolated rat Leydig cells: a defect in cholesterol mobilization and processing. Journal of Steroid Biochemistry and Molecular Biology 46 3947. (doi:10.1016/0960-0760(93)90207-D).

    • Search Google Scholar
    • Export Citation
  • Liu J, Rone MB & Papadopoulos V 2006 Protein–protein interactions mediate mitochondrial cholesterol transport and steroid biosynthesis. Journal of Biological Chemistry 281 3887938893. (doi:10.1074/jbc.M608820200).

    • Search Google Scholar
    • Export Citation
  • Luo L, Chen H & Zirkin B 2001 Leydig cell aging: steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme. Journal of Andrology 22 149156. (doi:10.1002/j.1939-4640.2001.tb02165.x).

    • Search Google Scholar
    • Export Citation
  • Luo L, Chen H & Zirkin BR 2005 Temporal relationships among testosterone production, steroidogenic acute regulatory protein (StAR), and P450 side chain cleavage enzyme (P450scc) during Leydig cell aging. Journal of Andrology 26 2531. (doi:10.1002/j.1939-4640.2005.tb02868.x).

    • Search Google Scholar
    • Export Citation
  • Manna PR & Stocco DM 2007 Crosstalk of CREB and Fos/Jun on a single cis-element: transcriptional repression of the steroidogenic acute regulatory protein gene. Journal of Molecular Endocrinology 39 261277. (doi:10.1677/JME-07-0065).

    • Search Google Scholar
    • Export Citation
  • Manna PR, Dyson MT, Eubank DW, Clark BJ, Lalli E, Sassone-Corsi P, Zeleznik AJ & Stocco DM 2002 Regulation of steroidogenesis and the steroidogenic acute regulatory protein by a member of the cAMP response-element binding protein family. Molecular Endocrinology 16 184199. (doi:10.1210/mend.16.1.0759).

    • Search Google Scholar
    • Export Citation
  • Manna PR, Dyson MT & Stocco DM 2009 Role of basic leucine zipper proteins in transcriptional regulation of the steroidogenic acute regulatory protein gene. Molecular and Cellular Endocrinology 302 111. (doi:10.1016/j.mce.2008.12.009).

    • Search Google Scholar
    • Export Citation
  • Margolin Y, Aten RF & Behrman HR 1990 Antigonadotropic and antisteroidogenic actions of peroxide in rat granulosa cells. Endocrinology 127 245250. (doi:10.1210/endo-127-1-245).

    • Search Google Scholar
    • Export Citation
  • Mayr B & Montminy M 2001 Transcriptional regulation by the phosphorylation-dependent factor CREB. Nature Reviews. Molecular Cell Biology 2 599609. (doi:10.1038/35085068).

    • Search Google Scholar
    • Export Citation
  • Miller WL & Bose HS 2011 Early steps in steroidogenesis: intracellular cholesterol trafficking Thematic Review series: genetics of human lipid diseases. Journal of Lipid Research 52 21112135. (doi:10.1194/jlr.R016675).

    • Search Google Scholar
    • Export Citation
  • Musicki B, Aten R & Behrman H 1994 Inhibition of protein synthesis and hormone-sensitive steroidogenesis in response to hydrogen peroxide in rat luteal cells. Endocrinology 134 588595. (doi:10.1210/endo.134.2.7507829).

    • Search Google Scholar
    • Export Citation
  • Popplewell P & Azhar S 1987 Effects of aging on cholesterol content and cholesterol metabolizing enzymes in the rat adrenal gland. Endocrinology 121 6473. (doi:10.1210/endo-121-1-64).

    • Search Google Scholar
    • Export Citation
  • Pramanik R, Qi X, Borowicz S, Choubey D, Schultz RM, Han J & Chen G 2003 p38 Isoforms have opposite effects on AP-1-dependent transcription through regulation of c-Jun the determinant role of the isoforms in the p38 MAPK signal specificity. Journal of Biological Chemistry 278 48314839. (doi:10.1074/jbc.M207732200).

    • Search Google Scholar
    • Export Citation
  • Qi X, Pohl NM, Loesch M, Hou S, Li R, Qin J-Z, Cuenda A & Chen G 2007 p38α antagonizes p38γ activity through c-Jun-dependent ubiquitin–proteasome pathways in regulating Ras transformation and stress response. Journal of Biological Chemistry 282 3139831408. (doi:10.1074/jbc.M703857200).

    • Search Google Scholar
    • Export Citation
  • Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ & Davis RJ 1995 Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. Journal of Biological Chemistry 270 74207426. (doi:10.1074/jbc.270.13.7420).

    • Search Google Scholar
    • Export Citation
  • Raingeaud J, Whitmarsh AJ, Barrett T, Derijard B & Davis RJ 1996 MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Molecular and Cellular Biology 16 12471255.

    • Search Google Scholar
    • Export Citation
  • Reaven E, Tsai L & Azhar S 1995 Cholesterol uptake by the ‘selective’ pathway of ovarian granulosa cells: early intracellular events. Journal of Lipid Research 36 16021617.

    • Search Google Scholar
    • Export Citation
  • Remy G, Risco AM, Iñesta-Vaquera FA, González-Terán B, Sabio G, Davis RJ & Cuenda A 2010 Differential activation of p38MAPK isoforms by MKK6 and MKK3. Cellular Signalling 22 660667. (doi:10.1016/j.cellsig.2009.11.020).

    • Search Google Scholar
    • Export Citation
  • Robinson MJ, Stippec SA, Goldsmith E, White MA & Cobb MH 1998 A constitutively active and nuclear form of the MAP kinase ERK2 is sufficient for neurite outgrowth and cell transformation. Current Biology 8 11411152. (doi:10.1016/S0960-9822(07)00485-X).

    • Search Google Scholar
    • Export Citation
  • Rouse J, Cohen P, Trigon S, Morange M, Alonso-Llamazares A, Zamanillo D, Hunt T & Nebreda AR 1994 A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78 10271037. (doi:10.1016/0092-8674(94)90277-1).

    • Search Google Scholar
    • Export Citation
  • Roux PP & Blenis J 2004 ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiology and Molecular Biology Reviews 68 320344. (doi:10.1128/MMBR.68.2.320-344.2004).

    • Search Google Scholar
    • Export Citation
  • Sandhoff TW, Hales DB, Hales KH & McLean MP 1998 Transcriptional regulation of the rat steroidogenic acute regulatory protein gene by steroidogenic factor 1. Endocrinology 139 48204831. (doi:10.1210/endo.139.12.6345).

    • Search Google Scholar
    • Export Citation
  • Shaulian E & Karin M 2002 AP-1 as a regulator of cell life and death. Nature Cell Biology 4 E131E136. (doi:10.1038/ncb0502-e131).

  • Shaywitz AJ & Greenberg ME 1999 CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annual Review of Biochemistry 68 821861. (doi:10.1146/annurev.biochem.68.1.821).

    • Search Google Scholar
    • Export Citation
  • Soccio R & Breslow J 2003 StAR-related transfer (START) proteins: mediators of intracellular lipid metabolism. Journal of Biological Chemistry 278 2218322186. (doi:10.1074/jbc.R300003200).

    • Search Google Scholar
    • Export Citation
  • Soccio R, Adams R, Romanowski M, Sehayek E, Burley S & Breslow J 2002 The cholesterol-regulated StarD4 gene encodes a StAR-related lipid transfer protein with two closely related homologues, StarD5 and StarD6. PNAS 99 69436948. (doi:10.1073/pnas.052143799).

    • Search Google Scholar
    • Export Citation
  • Soccio R, Adams R, Maxwell K & Breslow J 2005 Differential gene regulation of StarD4 and StarD5 cholesterol transfer proteins. Activation of StarD4 by sterol regulatory element-binding protein-2 and StarD5 by endoplasmic reticulum stress. Journal of Biological Chemistry 280 1941019418. (doi:10.1074/jbc.M501778200).

    • Search Google Scholar
    • Export Citation
  • Stocco DM 2001 StAR protein and the regulation of steroid hormone biosynthesis. Annual Review of Physiology 63 193213. (doi:10.1146/annurev.physiol.63.1.193).

    • Search Google Scholar
    • Export Citation
  • Stocco D & Clark B 1996 Regulation of the acute production of steroids in steroidogenic cells. Endocrine Reviews 17 221244. (doi:10.1210/edrv-17-3-221).

    • Search Google Scholar
    • Export Citation
  • Stocco D, Wells J & Clark B 1993 The effects of hydrogen peroxide on steroidogenesis in mouse Leydig tumor cells. Endocrinology 133 28272832. (doi:10.1210/endo.133.6.8243310).

    • Search Google Scholar
    • Export Citation
  • Sun Z, Shen W-J, Sucheta S-L & Azhar S 2008 Impact of aging on cholesterol transport protein expression and steroidogenesis in rat testicular Leydig cells. Open Longevity Science 2 7685. (doi:10.2174/1876326X00802010076).

    • Search Google Scholar
    • Export Citation
  • Wang X, Diener K, Manthey C, Wang S, Rosenzweig B, Bray J, Delaney J, Cole C, Chan-Hui P & Mantlo N et al. 1997 Molecular cloning and characterization of a novel p38 mitogen-activated protein kinase. Journal of Biological Chemistry 272 2366823674. (doi:10.1074/jbc.272.38.23668).

    • Search Google Scholar
    • Export Citation
  • Wang X, Shen C, Dyson M, Eimerl S, Orly J, Hutson J & Stocco D 2005 Cyclooxygenase-2 regulation of the age-related decline in testosterone. Endocrinology 146 42024208. (doi:10.1210/en.2005-0298).

    • Search Google Scholar
    • Export Citation
  • Wysk M, Yang DD, Lu H-T, Flavell RA & Davis RJ 1999 Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for tumor necrosis factor-induced cytokine expression. PNAS 96 37633768. (doi:10.1073/pnas.96.7.3763).

    • Search Google Scholar
    • Export Citation
  • Zaidi SK, Shen W-J & Azhar S 2012 Impact of aging on steroid hormone biosynthesis and secretion. Open Longevity Science 6 130. (doi:10.2174/1876326X01206010001).

    • Search Google Scholar
    • Export Citation
  • Zheng C, Xiang J, Hunter T & Lin A 1999 The JNKK2–JNK1 fusion protein acts as a constitutively active c-Jun kinase that stimulates c-Jun transcription activity. Journal of Biological Chemistry 274 2896628971. (doi:10.1074/jbc.274.41.28966).

    • Search Google Scholar
    • Export Citation