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Free access

BD Wyse, SL Linas and TJ Thekkumkara

GH/growth factors have been shown to increase angiotensin type 1 receptor expression. In the present study we determined the cis-acting regulatory region controlling GH-induced transcription of the human type-1 angiotensin receptor (hAT(1)). In human proximal tubule cells transfected with a chloramphenicol acetyl transferase (CAT) reporter plasmid under the control of the hAT(1) promoter, GH induced CAT activity. Serial deletions of the hAT(1) promoter region indicated that an area between -314 bp and -70 bp upstream of the 5'-end of the cDNA sequence was essential for this activation to occur. Although sequence analysis identified putative multiple nuclear protein binding sites in this region, we determined that a 12 bp sequence (5'-GAGAGGGAGGAG-3', GAGA box) located between -161 bp and -149 bp was important for GH-mediated activation. Using mobility shift assays we demonstrated increased DNA binding activity to the labeled GAGA box in nuclear extracts treated with GH, suggesting this sequence is a GH response element. Southwestern analysis identified an 18 kDa GAGA box-binding protein (GAGA-BP). GH-induced activity of the GAGA-BP occurred within 2.5 min and reached a maximum at 5 min. Activation did not require de novo protein synthesis. Removal of the GAGA box abolished GH-induced transcription as well as basal transcription of the hAT(1) gene. Additional studies demonstrated that epidermal growth factor, platelet-derived growth factor and insulin activate the GAGA-BP, suggesting these growth factors can also regulate the transcription of the hAT(1) gene through the GAGA box. Our data show that the GAGA-BP acts as a trans-acting factor binding to the cis-acting regulatory element in the hAT(1) promoter, which is necessary for the basal and growth factor(s)-mediated transcriptional activation of the hAT(1) gene.

Free access

Lai Jin, Qichun Zhang, Rui Guo, Lina Wang, Juejin Wang, Rong Wan, Rongjian Zhang, Youhua Xu and Shengnan Li

Urocortin (Ucn), a corticotropin-releasing factor (CRF)-related neuropeptide binding both CRF type 1 receptor (CRFR1) and CRFR2, has recently been found in prostate cancer. However, no report has yet been known to elucidate the roles of Ucn in prostate cancer via the two receptors. In this study, the expression of both CRFR1 and CRFR2 in the mouse prostate cancer cell line RM-1 were detected and cellular apoptosis was monitored in the presence of CRF or Ucn2, the CRFR1- and CRFR2-selective agonist respectively. CRF promoted apoptosis while Ucn2 exerted the opposite effect. CRF reduced Bcl-2 expression, induced Bax expression, and hyperpolarized the mitochondrial membrane potential to activate caspase-9. On the contrary, Ucn2 increased Bcl-2 expression and decreased Bax expression, in which phosphorylation of Akt and cyclic AMP response element-binding (CREB) was involved. Pretreatment with phosphatidylinositide 3-kinase/Akt inhibitor (LY-294002) prior to Ucn2 led to downregulation of CREB phosphorylation and hence reduced Bcl-2 expression. These effects of CRF and Ucn2 were abolished by antalarmin (Anta) and antisauvagine-30, the CRFR1- and CRFR2-selective antagonist respectively. In LNCaP cell line, similar effects on cell apoptosis by CRF and Ucn2 were observed. In summary, our results demonstrated CRFR1 and CRFR2 expression in prostate cancer and indicated the opposite apoptotic roles of the two different CRFRs. These data may contribute to uncovering the pathophysiological function of endogenous Ucn in prostate tumorigenesis and progression.

Free access

Bruria Funkenstein, Ekaterina Krol, Elena Esterin and Yong-soo Kim

Myostatin (MSTN), a negative regulator of muscle growth and a member of the transforming growth factor-β superfamily, can bind the two activin type 2 receptors (ACVR2). It has been previously shown that WT mice injected with ACVR2B extracellular domain (ACVR2B-ECD) had higher muscle mass. Likewise, fish larvae immersed in Pichia pastoris culture supernatant, containing goldfish Acvr2b-ECD, showed enhanced larval growth. However, it is not clear whether fish Mstn1 and Mstn2 signal through the same receptor and whether fish express more than one acvr2b gene. In the current study, three cDNAs encoding acvr2b (saacvr2b-1, saacvr2b-2a, and saacvr2b-2b) were cloned from gilthead sea bream. All three contain the short extracellular binding domain, a short transmembrane region, and a conserved catalytic domain of serine/threonine protein kinase. Bioinformatics analysis provided evidence for the existence of two acvr2b genes (acvr2b-1 and acvr2b-2) in several other fish species as well, probably as a result of gene or genome duplication. The two isoforms differ in their amino acid sequences. The direct inhibitory effect of Acvr2b-ECD on Mstn activity was tested in vitro. The saAcvr2b-1-ECD was expressed in the yeast P. pastoris. Evidence is provided for N-glycosylation of Acvr2b-1-ECD. The affinity-purified Acvr2b-1-ECD inhibited recombinant mouse/rat/human mature MSTN activity when determined in vitro using the CAGA-luciferase assay in A204 cells. A lower inhibitory activity was obtained when unprocessed purified, furin-digested, and activated saMstn1 was used. Results of this study demonstrate for the first time the existence of two acvr2b genes in fish. In addition, the study shows that bioactive fish Acvr2b-ECD can be produced from P. pastoris.

Free access

Lai Jin, Chuanhua Li, Rong Li, Zongxing Sun, Xianjun Fang and Shengnan Li

Peripheral corticotropin-releasing hormone receptors (CRHRs) are G protein-coupled receptors that play different roles depending on tissue types. Previously, we discovered the mechanism of CRHR-mediated apoptosis of mouse prostate cancer cell line (RM-1) to be a change of Bcl-2:Bax ratio, and CRH was found to inhibit transforming growth factor β migration of breast cancer cells via CRHRs. In the present study, we investigated cytosolic calcium-dependent phospholipase A2 (cPLA2) bridging CRHR activations and Bcl-2:Bax ratio and the effect of CRHR activation on cell migration. Silencing of cPLA2 attenuated a CRHR1 agonist, CRH-induced apoptosis, and the decrease of the Bcl-2:Bax ratio, whereas silencing of cPLA2 aggravated CRHR2 agonist, Urocortin 2 (Ucn2)-inhibited apoptosis, and the increase of the Bcl-2:Bax ratio. CRH in a time- and concentration-dependent manner increased cPLA2 expression mainly through interleukin 1β (IL1β) upregulation. Ucn2 decreased cPLA2 expression through neither tumor necrosis factor α nor IL1β. CRH-suppressed decay of cPLA2 mRNA and Ucn2 merely suppressed its production. Overexpression of CRHR1 or CRHR2 in HEK293 cells correspondingly upregulated or downregulated cPLA2 expression after CRH or Ucn2 stimulation respectively. In addition, both CRH and Ucn2 induced migration of RM-1 cells. Our observation not only established a relationship between CRHRs and cell migration but also for the first time, to our knowledge, demonstrated that cPLA2 participates in CRHR1-induced apoptosis and CRHR2-inhibited apoptosis.

Free access

L Badinga, S Song, RC Simmen, JB Clarke, DR Clemmons and FA Simmen

The coexpression of IGF (-I and -II) peptides, corresponding receptors, and IGF binding proteins (IGFBPs) in uterine endometrium suggests that a significant component of IGF action in this tissue is via autocrine or paracrine pathways, or both. The present study examined whether IGF-II and a major uterine-expressed IGF-II binding protein, IGFBP-2, modulate endometrial epithelial cell mitogenesis. Serum-deprived porcine endometrial glandular epithelial (GE) cells of early pregnancy were treated with various concentrations of IGFs, recombinant porcine (rp) IGFBP-2, or both, and examined for changes in cellular mitogenesis by incorporation of [(3)H]thymidine into DNA. Recombinant human (rh) IGF-II stimulated DNA synthesis in a dose-dependent manner. Human [Leu(27)]-IGF-II, an analog with selective affinity for the IGF-II (type II) receptor, increased thymidine uptake by twofold compared with untreated GE cells. When added in combination with an equimolar concentration of rhIGF-I, [Leu(27)]-IGF-II or rhIGF-II stimulated thymidine incorporation to a greater extent than did rhIGF-I alone. Ligand blot analysis of GE cell conditioned medium revealed the presence of four IGFBPs with molecular masses of 48, 31, 23, and 15 kDa. Physiological concentrations of rpIGFBP-2 (nM range) increased both basal and IGF-induced DNA synthesis in GE cells. At equimolar concentrations, Des(1-6)IGF-II (an IGF-II analog with much reduced affinity for IGFBPs) and rpIGFBP-2 had additive effects on GE cell mitogenesis, suggesting that the IGFBP-2 modulation of uterine cell growth may involve both IGF-dependent and IGF-independent pathways. Our results demonstrate the complex interplay of IGF system components in uterine endometrial epithelial growth regulation in vitro, identify IGF-II and IGFBP-2 as locally coexpressed uterine epithelial cell mitogens, and suggest the presence of a functional signaling pathway by which IGF-II stimulates epithelial cell proliferation via the type II receptor.

Free access

Katherine A Robinson, Jonathan W Brock and Maria G Buse

Thioredoxin-interacting protein (Txnip) is a metabolic regulator, which modulates insulin sensitivity and likely plays a role in type 2 diabetes. We studied the regulation of Txnip in 3T3-L1 adipocytes. Cells were incubated under different conditions and Txnip was measured by immunoblotting. We confirmed that high glucose markedly increases Txnip expression by promoting transcription. Insulin decreases Txnip protein levels. Rapamycin under most conditions decreased Txnip, suggesting that mTOR complex-1 is involved. The acute effects of insulin are mainly posttranscriptional; insulin (100 nM) accelerates Txnip degradation more than tenfold. This effect is cell type specific. It works in adipocytes, preadipocytes and in L6 myotubes but not in HepG2 or in HEK 293 cells or in a pancreatic β-cell line. The ubiquitin/proteasome pathway is involved. Degradation of Txnip occurred within 15 min in the presence of 3 nM insulin and overnight with 0.6 nM insulin. Proteasomal Txnip degradation is not mediated by a cysteine protease or an anti-calpain enzyme. Okadaic acid (OKA), an inhibitor of phosphoprotein phosphatases (pp), markedly reduced Txnip protein and stimulated its further decrease by insulin. The latter occurred after incubation with 1 or 1000 nM OKA, suggesting that insulin enhances the phosphorylation of a pp2A substrate. Incubation with 0.1 μM Wortmannin, a PI3 kinase inhibitor, increased Txnip protein twofold and significantly inhibited its insulin-induced decrease. Thus, while OKA mimics the effect of insulin, Wortmannin opposes it. In summary, insulin stimulates Txnip degradation by a PI3 kinase-dependent mechanism, which activates the ubiquitin/proteasome pathway and likely serves to mitigate insulin resistance.

Free access

CW Hay and K Docherty

Cell engineering or gene therapy may represent an alternative to current methods of treating diabetes mellitus. Cells could be engineered to secrete insulin ex vivo for transplantation or the insulin gene could be administered directly by injection into muscle. A problem has been that non-neuroendocrine cells lack the endoproteases (PC3/1 and PC2) that are responsible for the processing of proinsulin to insulin. This can be surmounted by engineering the paired basic amino acid processing sites within proinsulin to sites that would be recognized by the ubiquitously expressed protease, furin. However, in every study to date, the expression of the furin-cleavable construct was greatly reduced relative to that of the unmodified proinsulin construct. We investigated possible causes for this, including mRNA stability, the presence of additional CpG islands, and the amino acid substitutions within furin-cleavable proinsulin. Several furin-cleavable rat proinsulin I cDNAs were engineered and used to transfect human HEK293, rat L6 and mouse C(2)C(12) cell lines. The stability of wild-type and furin-cleavable proinsulin mRNA in transfected C(2)C(12) cells was measured by RT-PCR. Comparison of the decay rates in the presence of actinomycin D showed no significant difference between the two species of mRNA. A furin-cleavable proinsulin cDNA was created to contain the same distribution of CpG islands as wild-type proinsulin. Comparison of insulin-like immunoreactivity in all three cell lines transfected with either this construct or a widely used furin-cleavable proinsulin containing additional CpG islands showed that the presence of the extra CpG islands had no effect. Studies to examine amino acid substitutions used to create furin consensus sequences showed that the addition of basic residues at the C-peptide/A-chain junction was responsible for the reduced production of furin-cleavable proinsulin. Using this information, we engineered a cDNA for furin-cleavable rat proinsulin I that was efficiently processed to mature insulin and expressed at the same level as wild-type proinsulin.

Free access

E Anastasi, C Santangelo, A Bulotta, F Dotta, B Argenti, C Mincione, A Gulino, M Maroder, R Perfetti and U Di Mario

The elucidation of mechanisms regulating the regeneration and survival of pancreatic beta cells has fundamental implications in the cell therapy of type 1 diabetes. The present study had the following three aims: 1. to investigate whether pancreatic ductal epithelial cells can be induced to differentiate into insulin-producing cells by exposing them to hepatocyte growth factor (HGF); 2. to characterize some of the molecular events leading to their differentiation toward a beta-cell-like phenotype; 3. to evaluate the susceptibility of newly differentiated insulin-secreting cells to cytokine-induced apoptosis, a mechanism of beta-cell destruction occurring in type 1 diabetes. We demonstrated that HGF-treated rat pancreatic ductal cell line (ARIP) cells acquired the capability to transcribe the insulin gene and translate its counterpart protein. HGF-treated cells also exhibited a glucose-dependent capability to secrete insulin into the cultured medium. Expression analysis of some of the genes regulating pancreatic beta-cell differentiation revealed a time-dependent transcription of neurogenin-3 and Neuro-D in response to HGF. Finally, we determined the susceptibility to proinflammatory cytokine (PTh1)-induced apoptosis by incubating HGF-treated and untreated ARIP cells with a cocktail of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). Such treatment induced apoptotic death, as determined by the TUNEL technique, in about 40% of HGF-treated, insulin-secreting ARIP cells, while untreated ARIP cells were resistant to PTh1-induced apoptosis. In conclusion, we showed that HGF promotes the differentiation of ARIP cells into pancreatic beta-cell-like cells, and that the differentiation toward an insulin-secreting phenotype is associated with the appearance of susceptibility to cytokine-induced apoptosis.

Free access

Cynthia S Ritter and Alex J Brown

Vitamin D compounds regulate PTH at the transcriptional level, presumably via binding to the vitamin D receptor (VDR), but the exact mechanism is presently unclear. We recently reported that the several vitamin D prohormones with low VDR affinity suppressed PTH, even when their activation was inhibited, raising the possibility that their actions may be VDR independent. To test this hypothesis, we developed a novel organ culture that allowed the assessment of activities of the prohormones on PTH release from wild-type and VDR-null thyroparathyroid explants. The cultures remained viable with respect to PTH release for at least 2 weeks. Full suppression of PTH by the native vitamin D hormone, 1α,25-dihydroxyvitamin D3 [1α,25 (OH)2D3], required 2 days, consistent with a transcriptional mechanism, and was reversible, indicating that reduced PTH was not attributable to cell death. Inhibition of PTH release by 1α,25 (OH)2D3 and two prohormones, 25-hydroxyvitamin D3 and 1α-hydroxyvitamin D2, was observed in explants from wild-type mice but not in those from VDR-null mice. These findings 1) are the first direct demonstration of the role of the VDR in regulation of PTH by 1α,25(OH)2D3, 2) confirm that the suppressive actions of the vitamin D prohormones are mediated by the VDR, and 3) introduce a novel organ culture model that allows the ex vivo study of the function of parathyroid glands from transgenic animals.

Free access

Qi Zhang, Jing Liu, Jia Liu, Wenhui Huang, Limin Tian, Jinxing Quan, Yunfang Wang and Ruilan Niu

Non-alcoholic fatty liver disease is associated with hepatic microangiopathy and liver inflammation caused by type 2 diabetes mellitus. Oxidised LDL (oxLDL) is involved in proinflammatory and cytotoxic events in various microcirculatory systems. The lectin-like oxLDL receptor 1 (LOX1) plays a crucial role in oxLDL-induced pathological transformation. However, the underlying mechanism of oxLDL's effects on liver microcirculation disturbances remains unclear. In this study, we investigated the effects of oxLDL on LOX1 (OLR1) expression and function, as well as on the fenestration features of human liver sinusoidal endothelial cells (HLSECs) in vitro. Primary HLSECs were obtained and cultured. The cells were treated with various concentrations of oxLDL (25, 50, 100 and 200 μg/ml), and the cytotoxicity and expression of LOX1 were examined. Furthermore, LOX1 knockdown was performed using siRNA technology, and the changes in intracellular reactive oxygen species (ROS), NFκB, p65, (p65), endothelin 1 (ET1 (EDN1)), eNOS (NOS3) and caveolin 1 (CAV1) levels were measured. Cells were treated with 100 μg/ml oxLDL, and the fenestra morphology was visualised using scanning electron microscopy. oxLDL significantly increased LOX1 expression at both the mRNA and protein levels in HLSECs in a dose- and time-dependent manner. oxLDL stimulation increased ROS generation and NFκB activation, upregulated ET1 and caveolin 1 expression, downregulated eNOS expression and reduced the fenestra diameter and porosity. All of these oxLDL-mediated effects were inhibited after LOX1 knockdown. These results reveal a mechanism by which oxLDL stimulates the production of LOX1 through the ROS/NFκB signalling pathway and by which LOX1 mediates oxLDL-induced endothelial injury and the defenestration of HLSECs.