Transcription of LIPE gene encoding hormone-sensitive lipase/cholesteryl esterase is regulated by SF-1 in human adrenocortical cells: involvement of protein kinase A signal transduction pathway

in Journal of Molecular Endocrinology
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  • Department of Biochemistry and Molecular Biology, Poznan University of Medical Sciences, 6 Swiecickiego St., 60-781 Poznan, Poland

The study was designed to elucidate the influence of the protein kinase A (PKA) signal transduction pathway on transcription of the LIPE gene encoding hormone-sensitive lipase/cholesteryl esterase (HSL) in H295R cells. HSL is one of the key enzymes involved in steroid hormone synthesis, and ACTH, with mediation of the PKA pathway, increases its activity. However, the mode of regulation of LIPE gene expression by ACTH remains unknown. It was found that stimulation of the PKA pathway by the adenylyl cyclase activator, forskolin, caused a twofold increase in LIPE transcript accompanied by appreciable rise in the protein product of the gene and cortisol output. RNA polymerase II inhibitor abolished, and protein synthesis inhibitor attenuated this effect. Forskolin and PKA catalytic subunit increased transcriptional activity of LIPE promoter A in cells transfected with the luciferase reporter vector. Overexpression of steroidogenic factor-1 (SF-1) increased LIPE promoter activity, while transient silencing of SF-1 expression with specific siRNAs abolished forskolin-stimulated LIPE transcription. It is concluded that ACTH via the PKA pathway stimulates expression of SF-1, which activates transcription of LIPE presumably by interaction with putative binding sequences within promoter A. A novel mechanism contributing to the long-term effect of ACTH on adrenal steroidogenesis is proposed: ACTH stimulates transcription of SF-1, which interacts with the putative SF-1-binding sequences within the promoter and activates LIPE transcription. An increased level of HSL results in an enhanced supply of cholesterol required for steroid hormone synthesis.

Abstract

The study was designed to elucidate the influence of the protein kinase A (PKA) signal transduction pathway on transcription of the LIPE gene encoding hormone-sensitive lipase/cholesteryl esterase (HSL) in H295R cells. HSL is one of the key enzymes involved in steroid hormone synthesis, and ACTH, with mediation of the PKA pathway, increases its activity. However, the mode of regulation of LIPE gene expression by ACTH remains unknown. It was found that stimulation of the PKA pathway by the adenylyl cyclase activator, forskolin, caused a twofold increase in LIPE transcript accompanied by appreciable rise in the protein product of the gene and cortisol output. RNA polymerase II inhibitor abolished, and protein synthesis inhibitor attenuated this effect. Forskolin and PKA catalytic subunit increased transcriptional activity of LIPE promoter A in cells transfected with the luciferase reporter vector. Overexpression of steroidogenic factor-1 (SF-1) increased LIPE promoter activity, while transient silencing of SF-1 expression with specific siRNAs abolished forskolin-stimulated LIPE transcription. It is concluded that ACTH via the PKA pathway stimulates expression of SF-1, which activates transcription of LIPE presumably by interaction with putative binding sequences within promoter A. A novel mechanism contributing to the long-term effect of ACTH on adrenal steroidogenesis is proposed: ACTH stimulates transcription of SF-1, which interacts with the putative SF-1-binding sequences within the promoter and activates LIPE transcription. An increased level of HSL results in an enhanced supply of cholesterol required for steroid hormone synthesis.

Introduction

Steroid hormone synthesis in the adrenal cortex requires a continuous supply of free cholesterol. However, endogenous synthesis provides only about 20% of the cholesterol required for corticosteroid formation (Kraemer 2007), the main source being plasma lipoproteins, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) (Azhar & Reaven 2002, Connelly & Williams 2004). LDL is taken up by the cells via LDL receptor-mediated endocytosis, and free cholesterol is liberated from cholesteryl esters in lysosomes by ‘acid’ cholesteryl esterase (EC 3.1.1.13). The uptake of HDL cholesterol requires scavenger receptor class B type 1 (SR-B1) and is limited to cholesteryl esters, which are hydrolysed in the cytosol by hormone-sensitive lipase/cholesteryl esterase (HSL) EC 3.1.1.79 (Rodrigueza et al. 1999). In the cytosol, free cholesterol is esterified with activated fatty acids by acyl-CoA cholesterol acyl transferase (ACAT) (EC 2.3.1.26). Cholesteryl esters formed are stored in lipid droplets, which mainly contain cholesteryl esters and phospholipids with some proteins and negligible amounts of free cholesterol and triacylglycerols (Boyd & Trzeciak 1973).

The main source of substrate for steroid hormone synthesis is free cholesterol liberated by HSL from cholesteryl esters stored in lipid droplets (Trzeciak & Boyd 1973). HSL is a fatty acyl hydrolase that cleaves fatty acyl esters of glycerol and cholesterol (Kraemer & Shen 2002). This enzyme plays an important role in the control of energy homoeostasis by releasing free fatty acids from acylglycerols stored in adipocytes for transport and oxidation in energy-requiring tissues (Yeaman 1990). HSL also regulates provision of free cholesterol for steroid hormone synthesis in the adrenal cortex, testis, ovary and placenta. HSL is most active in adipose tissue. In steroidogenic tissues including the adrenal cortex, the enzyme is about three times less active, and its activity in pancreatic β-cells is even lower, while in the heart and macrophages, it is negligible (Khoo et al. 1993, Langfort et al. 1999, Lindvall et al. 2004).

It is well known that both cholesterol release and the amount of steroid hormones produced in the adrenal cortex are regulated by ACTH (Trzeciak & Boyd 1974, Boyd et al. 1975). ACTH binds to a specific receptor coupled with membrane-bound adenylyl cyclase; thus, cAMP is generated and activates the protein kinase A (PKA) signal transduction pathway, which leads to phosphorylation and activation of HSL (Trzeciak & Boyd 1974).

With the exception of the catalytic site, the primary structure of HSL differs from that of other lipases. Within the structure of HSL, two functional domains can be distinguished. The N-terminal domain is responsible for interaction with lipase translocating protein (lipotransin), adipose lipid-binding protein (ALBP) and steroidogenic acute regulator (StAR; Shen et al. 1999, 2003, Syu & Saltiel 1999). The C-terminal domain comprises the substrate-binding site, catalytic triad: Ser424, Asp692 and His723, and the regulatory loop containing several Ser residues which can be phosphorylated by different kinases (Contreras et al. 1996). A particular role is played by Ser563 which is phosphorylated by PKA (Holm 2003). Following PKA-dependent phosphorylation of Ser563, HSL forms a complex with lipotransin which translocates the enzyme to the surface of lipid droplets (Egan et al. 1992, Syu & Saltiel 1999). Owing to the ATPase activity of lipotransin and at the expense of a high-energy bond of ATP, HSL is liberated from the complex with lipotransin in order to exert its catalytic function (Syu & Saltiel 1999). Activation of PKA also results in phosphorylation of perilipins, covering lipid droplets and preventing access of HSL to its substrate (Londos et al. 1995, Servetnick et al. 1995). Upon phosphorylation, perilipins dissociate, and HSL is capable of binding to the surface of lipid droplets and hydrolysing cholesteryl esters. All effects mentioned above are parts of the short-term regulation of steroidogenesis by ACTH and precede the long-term regulation involving stimulation of transcription of genes encoding steroidogenic cytochromes P450 (CYP), 3-β-hydroxysteroid dehydrogenase (HSD3B) and StAR (Orth & Kovacs 1998).

In the adipocyte line 3T3-F442A, stimulation of the PKA pathway results in inhibition of transcription of the LIPE gene, encoding HSL (Plee-Gautier et al. 1996). On the contrary, in Leydig cells, human chorionic gonadotrophin acting through the PKA pathway induces expression of this gene (Kraemer et al. 1993).

To date, however, it has not been shown whether activation of the PKA pathway is accompanied by increased transcription of LIPE in adrenocortical cells. Such finding might constitute a novel, important element in the long-term response to ACTH.

The LIPE gene, located on chromosome 19q13.1, is composed of nine exons plus six additional exons (A, B, C, D, T1 and T2), which are transcribed in different tissues, and the transcription is controlled by tissue-specific promoters (Levitt et al. 1995, Laurin et al. 2000).

In adipose tissue, expression of LIPE is controlled by promoter B located upstream from non-coding exon B and containing exon 1. In the adrenal cortex, however, expression of this gene is driven by promoter A, situated 15 kb upstream from exon 1 (Grober et al. 1997). Promoter A precedes exon A which encodes a translation start codon, and this extends the open reading frame and results in the addition of 43 amino acids to the N-terminal end of HSL. In the testes, promoter T, located more than 15 kb upstream of exon 1, contains two additional exons, T1 and T2. Exon T1 encodes a translation start codon and additional 301 amino acids of the N-terminus of HSL, while transcription of exon T2 results only in extending the 5′-UTR of the transcript (Blaise et al. 1999). Like exon T2, non-coding exons C and D extend the 5′-UTR of the transcripts. The presence of different promoters results in the differential expression of three isoforms of human HSL. In adipocytes, it is mostly the 84 kDa form which is expressed, and in adrenal cortex and pancreatic β-cells, the 89 kDa form is the major one, whereas in the testes, the 120 kDa form predominates.

To date, investigations have been focused on regulation of the LIPE expression in adipose tissue, where the 84 kDa isoform predominates. The main regulators of LIPE expression in the adipose tissue are glucose and glucocorticoids (Slavin et al. 1994, Botion & Green 1999, Smih et al. 2002). However, in the adrenal cortex, the expression of the 89 kDa isoform has not been investigated. Elucidation of the ACTH effect on transcription of LIPE gene, in particular the activity of promoter A, is therefore essential for understanding mechanisms regulating substrate supply for steroid hormone synthesis in the adrenal cortex.

It has been firmly established that steroidogenic factor-1 (SF-1) is involved in the regulation of steroidogenic gene expression by ACTH (Morohashi et al. 1992, Val et al. 2003). SF-1 binds to specific binding sequences in target gene promoters and regulates transcription of a number of genes including CYP, HSD3B and StAR. SF-1 also plays an important role in the differentiation of cells producing steroid hormones.

This study has been designed to determine whether transcription of the LIPE gene encoding HSL is regulated by ACTH via cAMP and the PKA signal transduction pathway in human adrenocortical cells. An attempt has also been made to identify transcription factor(s) responsible for LIPE expression and their target sequences in LIPE promoter A.

Materials and methods

Materials

Forskolin, 8Br-cAMP, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), cycloheximide (CX), insulin, transferrin, sodium selenite mixture (ITS) and antibiotic/antimycotic (ABAM) were purchased from Sigma–Aldrich. Foetal bovine serum (FBS) was obtained from Gibco, and Ultroser G was a product of Pall Corporation (Port Washington, NY, USA).

TRItidy G reagent was purchased from Applichem (Darmstadt, Germany); reverse transcription kit and random hexamers were purchased from Novazym (Poznan, Poland); LC FastStart DNA Master SYBR Green1 and FuGene HD were purchased from Roche Diagnostics and Dual-Luciferase Reporter Assay System was purchased from Promega.

Antibody directed against HSL was obtained from Cell Signalling Technology, Inc. (Danvers, MA, USA), while anti-SF-1 antibody, anti-β-actin antibody and SF-1 siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protein Assay Dye reagent was obtained from Bio-Rad, and the Western Blotting Detection System was supplied by Amersham.

Constructs

The pGL3 vector, containing the Firefly luciferase gene under the control of LIPE promoter A fragments, was donated by Dr C Holm (Lund, Sweden), and the reference vector, harbouring Renilla reniformis gene (pRL-TK), was supplied by Dr J Li (Lyon, France). Expression vector pCHV-SF-1 harbouring the SF-1 gene was presented by Dr K Parker (Dallas, USA), and the pMT-CMVα vector, containing cDNA of the α subunit of PKA, was donated by Dr G S McKnight (Washington DC, USA).

Cell culture and treatments

Human adrenocortical cells (line H295R), purchased from American Type Culture Collection (Manassas, VA, USA), were cultured in combined DMEM/Ham's F-12 1:1 (v/v) containing l-glutamine (2.5 mM), which was obtained from Sigma–Aldrich. The medium was supplemented with 3% FBS, 4% Ultroser G, 1% ITS (1.0 mg/ml insulin, 0.55 mg/ml transferrin and 0.5 μg/ml sodium selenite) and ABAM. Other reagents used for cell culture were purchased from Gibco. Cortisol concentration in the culture medium was estimated by electrochemiluminescence using the Cobas 6000 instrument (Roche Diagnostics).

Incubation of the H295R cells with test substances

After confluence was reached, 24 h before treatment, the medium was substituted with the same medium but it was serum-free, containing ITS and test substances: forskolin, DRB or CX. Incubation was conducted for either 24 or 48 h as indicated. All incubations with test substances were conducted in serum-free medium. After incubation, the cells were washed with PBS, harvested and lysed with modified radioimmunoprecipitation assay buffer.

Isolation of RNA, reverse transcription and amplification of cDNA

RNA was isolated according to the phenol–chloroform method (Chomczynski & Sacchi 1987) using TRItidy reagent (Applichem). The concentration of RNA was measured by spectrophotometry (Eppendorf, Hamburg, Germany), and 1 μg of RNA was transcribed with the use of MMLV reverse transcriptase and random hexamers (Novazym) according to the manufacturer's instructions.

Quantitative analysis of the LIPE and SF-1 transcripts was conducted by real-time PCR (RT-qPCR) with the use of the LightCycler1.0 System (Roche Diagnostics) and specific primers (Table 1). Results were normalised to the mitochondrial ribosomal protein L19 gene (MRPL19), which was used as the reference.

Table 1

Oligonucleotide primers used for RT-qPCR

cDNAPrimers forward (F) and reverse (R)Annealing temperature (°C)Product size (bp)
LIPEF: 5′-GCGGTGGCGAAAAGACAAG-3′62239
R: 5′-GGTCCAGGTCAAAGAGGTG-3′
SF-1F: 5′-ACCCACAGTCGCCACCGT-3′60201
R: 5′-GAGCAGTCCGTAGTGGTAG-3′
MRPL19F: 5′-ACTTTATAATCCTCGGGTC-3′62171
R: 5′-ACTTTCAGCTCATTAACAG-3′

Incubation of the cells with the luciferase reporter vectors and determination of luciferase activity

The cells were transfected with the reporter vector (pGL3) harbouring the Firefly luciferase gene under control of the LIPE promoter A fragments (−30, −60, −343, −1150 and −2150 bp) together with the expression vector harbouring SF-1 gene or pMT-CEVα vector containing the PRKACA gene encoding catalytic subunit α (Cα) of PKA. For transfections, FuGene HD reagent (Roche Diagnostics) was used. After transfection, the cells were incubated for 48 h, harvested and lysed. Firefly luciferase activity in the cell lysates was determined using the Dual Luciferase System and 20/20n Luminometer (Turner BioSystems, Sunnyvale, CA, USA). Transfection efficiency was normalised to the co-transfected reference vector pRL-TK containing the Renilla luciferase gene.

Transfection of the cells containing SF-1 siRNAs and determination of LIPE expression level

Silencing of SF-1 expression was achieved by transfection of H295R cells with the mixture of three siRNAs complementary to the SF-1 transcript (Santa Cruz). Transfection was conducted under optimal conditions, and the FuGene HD reagent was used. The effectiveness of silencing was monitored by western blotting at 24 and 48 h after transfection. SF-1 levels were estimated using anti-SF-1 antibody followed by HRP-conjugated anti-γ-globulin secondary antibody and detection of peroxidase activity. The effect of SF-1 silencing on the level of LIPE transcript was determined by RT-qPCR, while the influence of SF-1 silencing on HSL protein level was determined by western blotting with the use of specific antibody and HRP-conjugated secondary antibody.

Computer software and Internet addresses

Primers were designed using Oligo v.6.71 software, and DNA sequences were obtained from GenBank (http://www.ncbi.nlm.nih.gov/BLAST/, Genew: human gene nomenclature Database http://www.gene.ucl.ac.uk/egi-bin/nomenclature/).

Statistical analysis

Statistical analysis of the results was carried out with the aid of GraphPad InStat v.3.05 (GraphPad Software, La Jolla, CA, USA) and Microsoft Excel 2007. The results are the mean (±s.e.m.) from at least two duplicates from three (or more) separate experiments. To estimate the influence of test substances on the level of transcripts, one-way ANOVA or two-way ANOVA tests were applied. All results were also tested with post-hoc Student–Newman–Keuls test. Significance of the differences between individual samples was tested at the level of *P<0.05, **P<0.01 or ***P<0.001.

Results

Stimulation of the PKA pathway results in induction of LIPE transcription

It is known that ACTH, acting via the PKA pathway, induces steroidogenic gene expression in the adrenal cortex. Therefore, we assumed that LIPE transcription might also be affected. Since H295R cells lack ACTH receptor, we stimulated the PKA pathway with the adenylyl cyclase activator, forskolin, or alternatively with 8Br-cAMP. Upon stimulation of the PKA pathway for 24 h, a two- to threefold increase in the level of the LIPE transcript, accompanied by appreciable increase in the protein product of the gene and cortisol output, was observed (Fig. 1). Although after incubation for 36 or 48 h without or with forskolin the level of the LIPE transcript was about 20% higher than after 24 h (not shown), the twofold increase in the transcript level was insignificant. An inhibitor of RNA polymerase II, DRB, abolished this effect, indicating that stimulation of the LIPE expression took place at the transcriptional level.

Figure 1
Figure 1

Effects of forskolin, 8Br-cAMP and an inhibitor of RNA polymerase II or protein synthesis inhibitor on the level of the LIPE transcript. The H295R cells were incubated for 24 h without (control) or with 25 μM forskolin, 0.1 μM 8Br-cAMP and/or 75 μM 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) or else with 10 μg/ml cycloheximide (CX). Following incubation, total RNA was extracted, and cDNA was obtained by reverse transcription. Specific cDNA was then amplified and quantified by PCR (RT-qPCR). The results are the average of four separate experiments ±s.e.m.; ***P<0.001; **P<0.01. The HSL level, determined by western blotting (fold control is shown in brackets), and cortisol output without forskolin (C) and under forskolin stimulation (F) are shown in the insets.

Citation: Journal of Molecular Endocrinology 46, 1; 10.1677/JME-10-0035

In order to examine whether stimulation of LIPE expression depends on the synthesis of proteins, which might affect transcription of the gene, we incubated the cells with forskolin and/or protein synthesis inhibitor, CX. It was found that CX slightly lowered the basal expression level and significantly alleviated the effect of forskolin on LIPE transcription (Fig. 1), suggesting that newly synthesised protein(s) are required for stimulation of the LIPE transcription.

The state of chromatin condensation, which also might be affected by stimulation of the PKA pathway, was excluded since histone deacetylase inhibitor, trichostatin A, did not influence the stimulatory effect of forskolin on the LIPE expression (not shown).

The mode of regulation of transcriptional activity of LIPE promoter A by PKA

To investigate the regulation of transcriptional activity of promoter A by the PKA pathway, H295R cells were transfected with the vector containing Firefly luciferase gene under control of the −2150 bp fragment of the LIPE promoter and were incubated with forskolin, which significantly increased its transcriptional activity. This suggested that stimulation of the PKA pathway increased the expression of LIPE. As expected, in the cells transfected with the luciferase reporter vector containing the same promoter fragment cloned in an inverted position, very low transcriptional activity and no effect of forskolin were observed (Fig. 2). Overexpression of the catalytic subunit α of PKA (Cα) caused a greater than twofold increase in the transcriptional activity of promoter A (Fig. 2) documenting that the effect of forskolin is mediated by PKA.

Figure 2
Figure 2

Transcriptional activity of the LIPE promoter A as affected by forskolin or catalytic subunit of protein kinase A. The H295R cells were transfected with the pGl3 vector containing a −2150 fragment of the LIPE promoter A in normal or inverted position (inv) directing Firefly luciferase reporter gene expression. The cells were either incubated for 48 h without (control) or with 25 μM forskolin or were co-transfected with the vector expressing catalytic subunit α of protein kinase A (Cα) and were incubated for 48 h. After incubations, luciferase activity was determined, as described in the Materials and methods section. RLU, relative luciferase units. The results are the average of four separate experiments ±s.e.m.; **P<0.01.

Citation: Journal of Molecular Endocrinology 46, 1; 10.1677/JME-10-0035

The PKA pathway regulates transcription of the SF-1 gene

SF-1 is the principal transcription factor involved in the expression of a number of steroidogenic genes whose transcription is controlled by the PKA pathway (Morohashi et al. 1992, Val et al. 2003). Therefore, we presumed that stimulation of the PKA pathway might also affect SF-1 transcription and decided to evaluate the effect of forskolin on the level of SF-1 transcript.

As a result of 24 h incubation of H295R cells with forskolin, a greater than twofold increase in the level of SF-1 transcript was observed (Fig. 3). This was accompanied by more than threefold increase in SF-1 protein (Fig. 3 inset). This suggested that the protein product of this gene might be involved in the induction of LIPE expression.

Figure 3
Figure 3

The influence of forskolin on SF-1 transcript levels. The H295R cells were incubated for 24 h without (control) or with 25 μM forskolin. Following incubation, total RNA was extracted. Specific cDNAs were then amplified and quantified by PCR (RT-qPCR). The results are the average of four separate experiments ±s.e.m.; ***P<0.001. The SF-1 level, determined by western blotting (fold control is shown in brackets), is shown in the inset.

Citation: Journal of Molecular Endocrinology 46, 1; 10.1677/JME-10-0035

SF-1 stimulates transcriptional activity of LIPE promoter A

In order to demonstrate the effect of SF-1 on the activity of promoter A, the cells transfected with the luciferase reporter vectors under control of the promoter fragments ranging from −30 to −2150 bp were co-transfected with the vector expressing SF-1, and the luciferase activity was determined. It was shown that transfection of the same vector into COS cells, in which SF-1 is not expressed, caused accumulation of SF-1 protein detected by western blotting with the use of specific antibody (not shown).

It was found that overexpression of SF-1 in H295R cells resulted in a more than twofold increase in the transcriptional activity of the −2150 and −1150 bp but not the shorter (−343, −100, −60 and −30) fragments of the promoter (Fig. 4). This indicates that SF-1 stimulates the expression of LIPE by interaction with the longer fragments of the promoter. Computer analysis of the DNA sequence of promoter A revealed two putative SF-1-binding sequences localised within the −1400 to −1420 bp region, suggesting direct interaction of SF-1 with the −2150 bp promoter fragment. The interaction of SF-1 with the −1150 bp fragment, which does not contain any SF-1-binding sequences, may be indirect and requires additional protein(s).

Figure 4
Figure 4

Influence of overexpression of SF-1 on the transcriptional activity of the LIPE promoter A. The H295R cells were transfected with the pGl3 vector containing deletion fragments of LIPE promoter A directing Firefly luciferase reporter gene expression (white bars). Parallel samples were co-transfected with the expression vector encoding SF-1 (black bars). The cells were incubated for 48 h, and luciferase activity was measured as described in the Materials and methods section. RLU, relative luciferase units. The results are the average of four separate experiments ±s.e.m.; **P<0.01; *P<0.05.

Citation: Journal of Molecular Endocrinology 46, 1; 10.1677/JME-10-0035

In order to demonstrate that SF-1 is essential for the PKA-mediated LIPE expression, H295R cells were transfected with siRNAs complementary to the SF-1 transcript. Transient silencing of the SF-1 expression resulted in a major decrease in SF-1 protein, while β-actin level was unaffected. The silencing of SF-1 also caused a significant inhibition of forskolin-stimulated LIPE expression and a slight, but insignificant, attenuation of its basal expression (Fig. 5). This clearly shows that SF-1 expression is required for stimulation of LIPE transcription by the PKA pathway. Silencing of SF-1 did not affect basal HSL protein level; forskolin increased HSL protein content, while SF-1 siRNA attenuated the effect of forskolin (Fig. 5 inset). Similarly, cortisol output was stimulated by forskolin, basal output was slightly lowered by SF-1 siRNA, while SF-1 silencing predictably did not influence the effect of forskolin since forskolin activates the PKA pathway, which results in phosphorylation and activation of HSL (not shown).

Figure 5
Figure 5

Effect of forskolin on the LIPE expression in the cells in which SF-1 is silenced. The cells were either left intact (C) or SF-1 was silenced by introducing SF-1 siRNAs. The cells were incubated without or with 25 μM forskolin. After incubation, total RNA was extracted; cDNA was obtained by reverse transcription and was quantified by PCR (black bars). The results are the average of four separate experiments ±s.e.m.; ***P<0.001. SF-1 and β-actin protein content in the cells transfected with SF-1 siRNAs is shown in the inset. In parallel experiments, the level of HSL protein was estimated in these cells by western blotting (inset) and quantified by densitometry (white bars).

Citation: Journal of Molecular Endocrinology 46, 1; 10.1677/JME-10-0035

Discussion

To investigate LIPE transcription, we chose the human adrenal tumour cell line H295R, which has been widely used as a model system (Rainey et al. 1993, 1994, Staels et al. 1993). These cells contain all major steroidogenic enzymes, including HSL, and respond to stimulation of the PKA pathway by increasing steroid hormone output, but they lack the ACTH receptor. Therefore, to investigate the effects of stimulation of the PKA pathway, adenylyl cyclase activator (forskolin) or a cAMP derivative (8Br-cAMP) capable of crossing the cell membrane was used instead.

Stimulation of the PKA pathway by forskolin caused a greater than twofold increase in the amount of the LIPE transcript, quantified by real-time PCR. This was accompanied by an increase in the protein product of the gene and cortisol output. A similar result was obtained when 8Br-cAMP was applied. Since DRB, an inhibitor of RNA polymerase II, abolished the effect of forskolin, we concluded that an increase of LIPE expression took place at a transcriptional level. The inhibitory effect of CX, on the other hand, indicated that stimulation of LIPE transcription depended on de novo synthesis of an unknown protein(s), which might affect expression of this gene.

Moreover, forskolin significantly increased transcriptional activity of the LIPE promoter fragment controlling expression of the luc reporter gene. The effect was specific since it was achieved only when the promoter was cloned into the vector in the proper orientation.

A similar effect was obtained when cells were transfected with the reporter vector containing LIPE promoter together with the vector expressing the catalytic subunit α of PKA, demonstrating that PKA activity is required for the stimulation of LIPE transcription.

Taken together, these observations indicate that ACTH stimulates LIPE expression at a transcriptional level via cAMP and PKA and that a protein(s) synthesised de novo is (are) required for this stimulation.

During the search for a stimulatory protein, CREB was excluded since the LIPE promoter does not contain a cAMP-response element (CRE) (Smale 1997, Lindvall et al. 2004). The best candidate for such a protein was SF-1, a principal stimulator of expression of almost all genes involved in steroid hormone synthesis (Val et al. 2003). We found that stimulation of the PKA pathway led to a significant increase in the expression of SF-1. This suggested that in the adrenal cortex, this pathway activated the expression of SF-1, thus supplying enough SF-1 protein required for stimulation of LIPE transcription.

In order to demonstrate the effect of SF-1 on LIPE transcription, we overexpressed SF-1 in H295R cells transfected with the vector containing luc reporter gene under control of LIPE promoter fragments. This caused a greater than twofold increase in transcriptional activity of the −2150 and −1150 bp fragments but not the shorter ones. Analysis of the LIPE promoter sequence reveals the presence of two putative SF-1-binding sequences localised between −1400 and −1420 bp.

Transfection experiments revealed that SF-1 overexpression also led to an increase in the transcriptional activity of the −1150 bp fragment of LIPE promoter. However, no SF-1-binding sequences were found in the entire fragment. Since activation of both LIPE promoter fragments by overexpression of SF-1 was almost equally strong, we postulate that the shorter fragment was activated by SF-1 indirectly and that other, yet unidentified, protein(s) was (were) required for this effect. We demonstrated that CX attenuated forskolin-induced transcription of LIPE. This suggested that a protein or proteins, synthesised de novo, were required for this effect. We postulate that one of those proteins might be SF-1.

Silencing of SF-1 expression by specific siRNAs abolished the stimulatory effect of forskolin on LIPE expression. This indicates that in transduction of the tropic signal from ACTH through its receptor, cAMP and PKA, SF-1 might play a role as the final regulator that directly or indirectly stimulates LIPE expression.

We conclude that ACTH stimulates expression of LIPE via the PKA signal transduction pathway, the principal regulator being the protein product of SF-1, and that the PKA pathway induces expression of this gene. Thus, we demonstrate the contribution of the LIPE and SF-1 expression to the long-term effect of ACTH on steroid hormone synthesis in the adrenal cortex.

Our results enable us to propose a hypothetical mechanism by which the LIPE transcription is regulated in the adrenal cortex (Fig. 6). Stimulation of the PKA pathway by ACTH leads to an increase in the expression of SF-1 and its protein product, and SF-1 by direct or indirect interaction with LIPE promoter A activates LIPE expression. Although our preliminary observations suggest that SF-1 might directly interact with SF-1-binding sequences localised in promoter A between −1400 and −1420, SF-1 might also contribute to the stimulation of LIPE transcription by indirect interaction with the promoter region −1150 to −343, which does not contain SF-1-binding sequences, but this would require an additional, yet unknown protein(s).

Figure 6
Figure 6

Hypothetical mechanism of regulation of the LIPE transcription by the protein kinase A pathway in the H295R cells. AC, adenylyl cyclase; PKA, protein kinase A; SF-1, steroidogenic factor-1; HSL, hormone-sensitive lipase/cholesterol esterase. Asterisks denote activated forms.

Citation: Journal of Molecular Endocrinology 46, 1; 10.1677/JME-10-0035

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

The present study was supported by a grant no 4131/P01 from the State Committee of Scientific Research.

Acknowledgements

The authors are grateful to Dr C Holm (Lund, Sweden), Dr K Parker (Dallas, USA), Dr G S McKnight (USA) and Dr J Li (Lyon, France) for providing constructs used in the experiments and to Dr G Schoenhals for critical reading of the manuscript.

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  • Connelly MA & Williams DL 2004 SR-BI and HDL cholesteryl ester metabolism. Endocrine Research 30 697703.doi:10.1081/ERC-200043979.

  • Contreras JA, Karlsson M, Osterlund T, Laurell H, Svensson A & Holm C 1996 Hormone-sensitive lipase is structurally related to acetylcholinesterase, bile salt-stimulated lipase, and several fungal lipases. Building of a three-dimensional model for the catalytic domain of hormone-sensitive lipase. Journal of Biological Chemistry 271 3142631430.doi:10.1074/jbc.271.49.31426.

    • Search Google Scholar
    • Export Citation
  • Egan JJ, Greenberg AS, Chang MK, Wek SA, Moos MC Jr & Londos C 1992 Mechanism of hormone-stimulated lipolysis in adipocytes: translocation of hormone-sensitive lipase to the lipid storage droplet. PNAS 89 85378541.doi:10.1073/pnas.89.18.8537.

    • Search Google Scholar
    • Export Citation
  • Grober J, Laurell H, Blaise R, Fabry B, Schaak S, Holm C & Langin D 1997 Characterization of the promoter of human adipocyte hormone-sensitive lipase. Biochemical Journal 328 453461.

    • Search Google Scholar
    • Export Citation
  • Holm C 2003 Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. Biochemical Society Transactions 31 11201124.doi:10.1042/BST0311120.

    • Search Google Scholar
    • Export Citation
  • Khoo JC, Reue K, Steinberg D & Schotz MC 1993 Expression of hormone-sensitive lipase mRNA in macrophages. Journal of Lipid Research 34 19691974.

  • Kraemer FB 2007 Adrenal cholesterol utilization. Molecular and Cellular Endocrinology 265–266 4245.doi:10.1016/j.mce.2006.12.001.

  • Kraemer FB & Shen WJ 2002 Hormone-sensitive lipase: control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis. Journal of Lipid Research 43 15851594.doi:10.1194/jlr.R200009-JLR200.

    • Search Google Scholar
    • Export Citation
  • Kraemer FB, Patel S, Singh-Bist A, Gholami SS, Saedi MS & Sztalryd C 1993 Detection of hormone-sensitive lipase in various tissues. II. Regulation in the rat testis by human chorionic gonadotropin. Journal of Lipid Research 34 609616.

    • Search Google Scholar
    • Export Citation
  • Langfort J, Ploug T, Ihlemann J, Saldo M, Holm C & Galbo H 1999 Expression of hormone-sensitive lipase and its regulation by adrenaline in skeletal muscle. Biochemical Journal 340 459465.doi:10.1042/0264-6021:3400459.

    • Search Google Scholar
    • Export Citation
  • Laurin NN, Wang SP & Mitchell GA 2000 The hormone-sensitive lipase gene is transcribed from at least five alternative first exons in mouse adipose tissue. Mammalian Genome 11 972978.doi:10.1007/s003350010185.

    • Search Google Scholar
    • Export Citation
  • Levitt RC, Liu Z, Nouri N, Meyers DA, Brandriff B & Mohrenweiser HM 1995 Mapping of the gene for hormone sensitive lipase (LIPE) to chromosome 19q13.1→q13.2. Cytogenetics and Cell Genetics 69 211214.doi:10.1159/000133966.

    • Search Google Scholar
    • Export Citation
  • Lindvall H, Nevsten P, Strom K, Wallenberg R, Sundler F, Langin D, Winzell MS & Holm C 2004 A novel hormone-sensitive lipase isoform expressed in pancreatic beta-cells. Journal of Biological Chemistry 279 38283836.doi:10.1074/jbc.M311365200.

    • Search Google Scholar
    • Export Citation
  • Londos C, Brasaemle DL, Gruia-Gray J, Servetnick DA, Schultz CJ, Levin DM & Kimmel AR 1995 Perilipin: unique proteins associated with intracellular neutral lipid droplets in adipocytes and steroidogenic cells. Biochemical Society Transactions 23 611615.

    • Search Google Scholar
    • Export Citation
  • Morohashi K, Honda S, Inomata Y, Handa H & Omura T 1992 A common trans-acting factor, Ad4-binding protein, to the promoters of steroidogenic P-450s. Journal of Biological Chemistry 267 1791317919.

    • Search Google Scholar
    • Export Citation
  • Orth DN & Kovacs WJ 1998 The adrenal cortex. In Williams Textbook of Endocrinology. Eds JD Wilson, JW Foster, HM Kronenberg & PR Larsen. Philadelphia: W.B. Saunders Company.

  • Plee-Gautier E, Grober J, Duplus E, Langin D & Forest C 1996 Inhibition of hormone-sensitive lipase gene expression by cAMP and phorbol esters in 3T3-F442A and BFC-1 adipocytes. Biochemical Journal 318 10571063.

    • Search Google Scholar
    • Export Citation
  • Rainey WE, Bird IM, Sawetawan C, Hanley NA, McCarthy JL, McGee EA, Wester R & Mason JI 1993 Regulation of human adrenal carcinoma cell (NCI-H295) production of C19 steroids. Journal of Clinical Endocrinology and Metabolism 77 731737.doi:10.1210/jc.77.3.731.

    • Search Google Scholar
    • Export Citation
  • Rainey WE, Bird IM & Mason JI 1994 The NCI-H295 cell line: a pluripotent model for human adrenocortical studies. Molecular and Cellular Endocrinology 100 4550.doi:10.1016/0303-7207(94)90277-1.

    • Search Google Scholar
    • Export Citation
  • Rodrigueza WV, Thuahnai ST, Temel RE, Lund-Katz S, Phillips MC & Williams DL 1999 Mechanism of scavenger receptor class B type I-mediated selective uptake of cholesteryl esters from high density lipoprotein to adrenal cells. Journal of Biological Chemistry 274 2034420350.doi:10.1074/jbc.274.29.20344.

    • Search Google Scholar
    • Export Citation
  • Servetnick DA, Brasaemle DL, Gruia-Gray J, Kimmel AR, Wolff J & Londos C 1995 Perilipins are associated with cholesteryl ester droplets in steroidogenic adrenal cortical and Leydig cells. Journal of Biological Chemistry 270 1697016973.doi:10.1074/jbc.270.28.16970.

    • Search Google Scholar
    • Export Citation
  • Shen WJ, Sridhar K, Bernlohr DA & Kraemer FB 1999 Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein. PNAS 96 55285532.doi:10.1073/pnas.96.10.5528.

    • Search Google Scholar
    • Export Citation
  • Shen WJ, Patel S, Natu V, Hong R, Wang J, Azhar S & Kraemer FB 2003 Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein: facilitation of cholesterol transfer in adrenal. Journal of Biological Chemistry 278 4387043876.doi:10.1074/jbc.M303934200.

    • Search Google Scholar
    • Export Citation
  • Slavin BG, Ong JM & Kern PA 1994 Hormonal regulation of hormone-sensitive lipase activity and mRNA levels in isolated rat adipocytes. Journal of Lipid Research 35 15351541.

    • Search Google Scholar
    • Export Citation
  • Smale ST 1997 Transcription initiation from TATA-less promoters within eukaryotic protein-coding genes. Biochimica et Biophysica Acta 1351 7388.doi:10.1016/S0167-4781(96)00206-0.

    • Search Google Scholar
    • Export Citation
  • Smih F, Rouet P, Lucas S, Mairal A, Sengenes C, Lafontan M, Vaulont S, Casado M & Langin D 2002 Transcriptional regulation of adipocyte hormone-sensitive lipase by glucose. Diabetes 51 293300.doi:10.2337/diabetes.51.2.293.

    • Search Google Scholar
    • Export Citation
  • Staels B, Hum DW & Miller WL 1993 Regulation of steroidogenesis in NCI-H295 cells: a cellular model of the human fetal adrenal. Molecular Endocrinology 7 423433.doi:10.1210/me.7.3.423.

    • Search Google Scholar
    • Export Citation
  • Syu LJ & Saltiel AR 1999 Lipotransin: a novel docking protein for hormone-sensitive lipase. Molecular Cell 4 109115.doi:10.1016/S1097-2765(00)80192-6.

    • Search Google Scholar
    • Export Citation
  • Trzeciak WH & Boyd GS 1973 The effect of stress induced by ether anaesthesia on cholesterol content and cholesteryl-esterase activity in rat-adrenal cortex. European Journal of Biochemistry 37 327333.doi:10.1111/j.1432-1033.1973.tb02991.x.

    • Search Google Scholar
    • Export Citation
  • Trzeciak WH & Boyd GS 1974 Activation of cholesteryl esterase in bovine adrenal cortex. European Journal of Biochemistry 46 201207.doi:10.1111/j.1432-1033.1974.tb03612.x.

    • Search Google Scholar
    • Export Citation
  • Val P, Lefrancois-Martinez AM, Veyssiere G & Martinez A 2003 SF-1 a key player in the development and differentiation of steroidogenic tissues. Nuclear Receptor 1 8 doi:10.1186/1478-1336-1-8.

    • Search Google Scholar
    • Export Citation
  • Yeaman SJ 1990 Hormone-sensitive lipase – a multipurpose enzyme in lipid metabolism. Biochimica et Biophysica Acta 1052 128132.doi:10.1016/0167-4889(90)90067-N.

    • Search Google Scholar
    • Export Citation

(N Derebecka-Hołysz is now at Institute of Natural Fibres and Medical Plants, Poznan, Poland)

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      Society for Endocrinology

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    Effects of forskolin, 8Br-cAMP and an inhibitor of RNA polymerase II or protein synthesis inhibitor on the level of the LIPE transcript. The H295R cells were incubated for 24 h without (control) or with 25 μM forskolin, 0.1 μM 8Br-cAMP and/or 75 μM 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) or else with 10 μg/ml cycloheximide (CX). Following incubation, total RNA was extracted, and cDNA was obtained by reverse transcription. Specific cDNA was then amplified and quantified by PCR (RT-qPCR). The results are the average of four separate experiments ±s.e.m.; ***P<0.001; **P<0.01. The HSL level, determined by western blotting (fold control is shown in brackets), and cortisol output without forskolin (C) and under forskolin stimulation (F) are shown in the insets.

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    Transcriptional activity of the LIPE promoter A as affected by forskolin or catalytic subunit of protein kinase A. The H295R cells were transfected with the pGl3 vector containing a −2150 fragment of the LIPE promoter A in normal or inverted position (inv) directing Firefly luciferase reporter gene expression. The cells were either incubated for 48 h without (control) or with 25 μM forskolin or were co-transfected with the vector expressing catalytic subunit α of protein kinase A (Cα) and were incubated for 48 h. After incubations, luciferase activity was determined, as described in the Materials and methods section. RLU, relative luciferase units. The results are the average of four separate experiments ±s.e.m.; **P<0.01.

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    The influence of forskolin on SF-1 transcript levels. The H295R cells were incubated for 24 h without (control) or with 25 μM forskolin. Following incubation, total RNA was extracted. Specific cDNAs were then amplified and quantified by PCR (RT-qPCR). The results are the average of four separate experiments ±s.e.m.; ***P<0.001. The SF-1 level, determined by western blotting (fold control is shown in brackets), is shown in the inset.

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    Influence of overexpression of SF-1 on the transcriptional activity of the LIPE promoter A. The H295R cells were transfected with the pGl3 vector containing deletion fragments of LIPE promoter A directing Firefly luciferase reporter gene expression (white bars). Parallel samples were co-transfected with the expression vector encoding SF-1 (black bars). The cells were incubated for 48 h, and luciferase activity was measured as described in the Materials and methods section. RLU, relative luciferase units. The results are the average of four separate experiments ±s.e.m.; **P<0.01; *P<0.05.

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    Effect of forskolin on the LIPE expression in the cells in which SF-1 is silenced. The cells were either left intact (C) or SF-1 was silenced by introducing SF-1 siRNAs. The cells were incubated without or with 25 μM forskolin. After incubation, total RNA was extracted; cDNA was obtained by reverse transcription and was quantified by PCR (black bars). The results are the average of four separate experiments ±s.e.m.; ***P<0.001. SF-1 and β-actin protein content in the cells transfected with SF-1 siRNAs is shown in the inset. In parallel experiments, the level of HSL protein was estimated in these cells by western blotting (inset) and quantified by densitometry (white bars).

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    Hypothetical mechanism of regulation of the LIPE transcription by the protein kinase A pathway in the H295R cells. AC, adenylyl cyclase; PKA, protein kinase A; SF-1, steroidogenic factor-1; HSL, hormone-sensitive lipase/cholesterol esterase. Asterisks denote activated forms.

  • Azhar S & Reaven E 2002 Scavenger receptor class BI and selective cholesteryl ester uptake: partners in the regulation of steroidogenesis. Molecular and Cellular Endocrinology 195 126.doi:10.1016/S0303-7207(02)00222-8.

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    • Export Citation
  • Blaise R, Grober J, Rouet P, Tavernier G, Daegelen D & Langin D 1999 Testis expression of hormone-sensitive lipase is conferred by a specific promoter that contains four regions binding testicular nuclear proteins. Journal of Biological Chemistry 274 93279334.doi:10.1074/jbc.274.14.9327.

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    • Export Citation
  • Botion LM & Green A 1999 Long-term regulation of lipolysis and hormone-sensitive lipase by insulin and glucose. Diabetes 48 16911697.doi:10.2337/diabetes.48.9.1691.

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    • Export Citation
  • Boyd GS & Trzeciak WH 1973 Cholesterol metabolism in the adrenal cortex: studies on the mode of action of ACTH. Annals of the New York Academy of Sciences 212 361377.doi:10.1111/j.1749-6632.1973.tb47607.x.

    • Search Google Scholar
    • Export Citation
  • Boyd GS, Arthur JR, Beckett GJ, Mason JI & Trzeciak WH 1975 The role of cholesterol and cytochrome P-450 in the cholesterol side chain cleavage reaction in adrenal cortex and corpora lutea. Journal of Steroid Biochemistry 6 427436.doi:10.1016/0022-4731(75)90167-3.

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    • Export Citation
  • Chomczynski P & Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Analytical Biochemistry 162 156159.doi:10.1016/0003-2697(87)90021-2.

    • Search Google Scholar
    • Export Citation
  • Connelly MA & Williams DL 2004 SR-BI and HDL cholesteryl ester metabolism. Endocrine Research 30 697703.doi:10.1081/ERC-200043979.

  • Contreras JA, Karlsson M, Osterlund T, Laurell H, Svensson A & Holm C 1996 Hormone-sensitive lipase is structurally related to acetylcholinesterase, bile salt-stimulated lipase, and several fungal lipases. Building of a three-dimensional model for the catalytic domain of hormone-sensitive lipase. Journal of Biological Chemistry 271 3142631430.doi:10.1074/jbc.271.49.31426.

    • Search Google Scholar
    • Export Citation
  • Egan JJ, Greenberg AS, Chang MK, Wek SA, Moos MC Jr & Londos C 1992 Mechanism of hormone-stimulated lipolysis in adipocytes: translocation of hormone-sensitive lipase to the lipid storage droplet. PNAS 89 85378541.doi:10.1073/pnas.89.18.8537.

    • Search Google Scholar
    • Export Citation
  • Grober J, Laurell H, Blaise R, Fabry B, Schaak S, Holm C & Langin D 1997 Characterization of the promoter of human adipocyte hormone-sensitive lipase. Biochemical Journal 328 453461.

    • Search Google Scholar
    • Export Citation
  • Holm C 2003 Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. Biochemical Society Transactions 31 11201124.doi:10.1042/BST0311120.

    • Search Google Scholar
    • Export Citation
  • Khoo JC, Reue K, Steinberg D & Schotz MC 1993 Expression of hormone-sensitive lipase mRNA in macrophages. Journal of Lipid Research 34 19691974.

  • Kraemer FB 2007 Adrenal cholesterol utilization. Molecular and Cellular Endocrinology 265–266 4245.doi:10.1016/j.mce.2006.12.001.

  • Kraemer FB & Shen WJ 2002 Hormone-sensitive lipase: control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis. Journal of Lipid Research 43 15851594.doi:10.1194/jlr.R200009-JLR200.

    • Search Google Scholar
    • Export Citation
  • Kraemer FB, Patel S, Singh-Bist A, Gholami SS, Saedi MS & Sztalryd C 1993 Detection of hormone-sensitive lipase in various tissues. II. Regulation in the rat testis by human chorionic gonadotropin. Journal of Lipid Research 34 609616.

    • Search Google Scholar
    • Export Citation
  • Langfort J, Ploug T, Ihlemann J, Saldo M, Holm C & Galbo H 1999 Expression of hormone-sensitive lipase and its regulation by adrenaline in skeletal muscle. Biochemical Journal 340 459465.doi:10.1042/0264-6021:3400459.

    • Search Google Scholar
    • Export Citation
  • Laurin NN, Wang SP & Mitchell GA 2000 The hormone-sensitive lipase gene is transcribed from at least five alternative first exons in mouse adipose tissue. Mammalian Genome 11 972978.doi:10.1007/s003350010185.

    • Search Google Scholar
    • Export Citation
  • Levitt RC, Liu Z, Nouri N, Meyers DA, Brandriff B & Mohrenweiser HM 1995 Mapping of the gene for hormone sensitive lipase (LIPE) to chromosome 19q13.1→q13.2. Cytogenetics and Cell Genetics 69 211214.doi:10.1159/000133966.

    • Search Google Scholar
    • Export Citation
  • Lindvall H, Nevsten P, Strom K, Wallenberg R, Sundler F, Langin D, Winzell MS & Holm C 2004 A novel hormone-sensitive lipase isoform expressed in pancreatic beta-cells. Journal of Biological Chemistry 279 38283836.doi:10.1074/jbc.M311365200.

    • Search Google Scholar
    • Export Citation
  • Londos C, Brasaemle DL, Gruia-Gray J, Servetnick DA, Schultz CJ, Levin DM & Kimmel AR 1995 Perilipin: unique proteins associated with intracellular neutral lipid droplets in adipocytes and steroidogenic cells. Biochemical Society Transactions 23 611615.

    • Search Google Scholar
    • Export Citation
  • Morohashi K, Honda S, Inomata Y, Handa H & Omura T 1992 A common trans-acting factor, Ad4-binding protein, to the promoters of steroidogenic P-450s. Journal of Biological Chemistry 267 1791317919.

    • Search Google Scholar
    • Export Citation
  • Orth DN & Kovacs WJ 1998 The adrenal cortex. In Williams Textbook of Endocrinology. Eds JD Wilson, JW Foster, HM Kronenberg & PR Larsen. Philadelphia: W.B. Saunders Company.

  • Plee-Gautier E, Grober J, Duplus E, Langin D & Forest C 1996 Inhibition of hormone-sensitive lipase gene expression by cAMP and phorbol esters in 3T3-F442A and BFC-1 adipocytes. Biochemical Journal 318 10571063.

    • Search Google Scholar
    • Export Citation
  • Rainey WE, Bird IM, Sawetawan C, Hanley NA, McCarthy JL, McGee EA, Wester R & Mason JI 1993 Regulation of human adrenal carcinoma cell (NCI-H295) production of C19 steroids. Journal of Clinical Endocrinology and Metabolism 77 731737.doi:10.1210/jc.77.3.731.

    • Search Google Scholar
    • Export Citation
  • Rainey WE, Bird IM & Mason JI 1994 The NCI-H295 cell line: a pluripotent model for human adrenocortical studies. Molecular and Cellular Endocrinology 100 4550.doi:10.1016/0303-7207(94)90277-1.

    • Search Google Scholar
    • Export Citation
  • Rodrigueza WV, Thuahnai ST, Temel RE, Lund-Katz S, Phillips MC & Williams DL 1999 Mechanism of scavenger receptor class B type I-mediated selective uptake of cholesteryl esters from high density lipoprotein to adrenal cells. Journal of Biological Chemistry 274 2034420350.doi:10.1074/jbc.274.29.20344.

    • Search Google Scholar
    • Export Citation
  • Servetnick DA, Brasaemle DL, Gruia-Gray J, Kimmel AR, Wolff J & Londos C 1995 Perilipins are associated with cholesteryl ester droplets in steroidogenic adrenal cortical and Leydig cells. Journal of Biological Chemistry 270 1697016973.doi:10.1074/jbc.270.28.16970.

    • Search Google Scholar
    • Export Citation
  • Shen WJ, Sridhar K, Bernlohr DA & Kraemer FB 1999 Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein. PNAS 96 55285532.doi:10.1073/pnas.96.10.5528.

    • Search Google Scholar
    • Export Citation
  • Shen WJ, Patel S, Natu V, Hong R, Wang J, Azhar S & Kraemer FB 2003 Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein: facilitation of cholesterol transfer in adrenal. Journal of Biological Chemistry 278 4387043876.doi:10.1074/jbc.M303934200.

    • Search Google Scholar
    • Export Citation
  • Slavin BG, Ong JM & Kern PA 1994 Hormonal regulation of hormone-sensitive lipase activity and mRNA levels in isolated rat adipocytes. Journal of Lipid Research 35 15351541.

    • Search Google Scholar
    • Export Citation
  • Smale ST 1997 Transcription initiation from TATA-less promoters within eukaryotic protein-coding genes. Biochimica et Biophysica Acta 1351 7388.doi:10.1016/S0167-4781(96)00206-0.

    • Search Google Scholar
    • Export Citation
  • Smih F, Rouet P, Lucas S, Mairal A, Sengenes C, Lafontan M, Vaulont S, Casado M & Langin D 2002 Transcriptional regulation of adipocyte hormone-sensitive lipase by glucose. Diabetes 51 293300.doi:10.2337/diabetes.51.2.293.

    • Search Google Scholar
    • Export Citation
  • Staels B, Hum DW & Miller WL 1993 Regulation of steroidogenesis in NCI-H295 cells: a cellular model of the human fetal adrenal. Molecular Endocrinology 7 423433.doi:10.1210/me.7.3.423.

    • Search Google Scholar
    • Export Citation
  • Syu LJ & Saltiel AR 1999 Lipotransin: a novel docking protein for hormone-sensitive lipase. Molecular Cell 4 109115.doi:10.1016/S1097-2765(00)80192-6.

    • Search Google Scholar
    • Export Citation
  • Trzeciak WH & Boyd GS 1973 The effect of stress induced by ether anaesthesia on cholesterol content and cholesteryl-esterase activity in rat-adrenal cortex. European Journal of Biochemistry 37 327333.doi:10.1111/j.1432-1033.1973.tb02991.x.

    • Search Google Scholar
    • Export Citation
  • Trzeciak WH & Boyd GS 1974 Activation of cholesteryl esterase in bovine adrenal cortex. European Journal of Biochemistry 46 201207.doi:10.1111/j.1432-1033.1974.tb03612.x.

    • Search Google Scholar
    • Export Citation
  • Val P, Lefrancois-Martinez AM, Veyssiere G & Martinez A 2003 SF-1 a key player in the development and differentiation of steroidogenic tissues. Nuclear Receptor 1 8 doi:10.1186/1478-1336-1-8.

    • Search Google Scholar
    • Export Citation
  • Yeaman SJ 1990 Hormone-sensitive lipase – a multipurpose enzyme in lipid metabolism. Biochimica et Biophysica Acta 1052 128132.doi:10.1016/0167-4889(90)90067-N.

    • Search Google Scholar
    • Export Citation