[Show abstract][Hide abstract] ABSTRACT: -NADPH oxidase 4 (Nox4) has been implicated in cardiac remodeling, but its precise role in cardiac injury remains controversial. Furthermore, little is known about the downstream effector signaling pathways activated by Nox4-derived ROS in the myocardium. We investigated the role of Nox4 and Nox4 associated signaling pathways in the development of cardiac remodeling.
-Cardiac-specific human Nox4 transgenic mice (c-hNox4Tg) were generated. Four groups of mice were studied: 1) control mice (CTL): littermates that are negative for hNox4 transgene but Cre positive; 2) c-hNox4 Tg mice; 3) angiotensin II (AngII)-infused CTL mice and 4) c-hNox4Tg mice infused with AngII. The c-hNox4Tg mice exhibited approximately 10-fold increase in Nox4 protein expression and 8-fold increase in the production of reactive oxygen species, and manifested cardiac interstitial fibrosis. AngII-infusion to CTL mice increased cardiac Nox4 expression and induced fibrosis and hypertrophy. The Tg mice receiving AngII exhibited more advanced cardiac remodeling and robust elevation in Nox4 expression, indicating that AngII worsens cardiac injury, at least partially by enhancing Nox4 expression. Moreover, hNox4 transgene and/or AngII-infusion induced the expression of cardiac fetal genes and activated the Akt-mTOR and NFκB signaling pathways. Treatment of AngII-infused c-hNox4Tg mice with GKT137831, a Nox4/Nox1 inhibitor, abolished the increase in oxidative stress, suppressed Akt-mTOR and NFκB signaling pathway and attenuated cardiac remodeling.
-Upregulation of Nox4 in the myocardium causes cardiac remodeling through activating Akt-mTOR and NFκB signaling pathways. Inhibition of Nox4 has therapeutic potential to treat cardiac remodeling.
[Show abstract][Hide abstract] ABSTRACT: Exposure of proximal tubular epithelial cells to high glucose contributes to the accumulation of tubulointerstitial and matrix proteins in diabetic nephropathy, but how this occurs is not well understood. We investigated the effect of the signaling molecule tuberin, which modulates the mammalian target of rapamycin pathway, on renal hypertrophy and fibronectin expression. We found that the kidney mass was significantly greater in partially tuberin-deficient (TSC2(+/-) ) diabetic rats than wild-type diabetic rats. Furthermore, TSC2(+/-) rats exhibited significant increases in the basal levels of phospho-tuberin and fibronectin expression in the kidney cortex. Increased levels of phosphorylated tuberin associated with an increase in fibronectin expression in both wild-type and TSC2(+/-) diabetic rats. Treatment with insulin abrogated the diabetes-induced increase in fibronectin expression. In vitro, high glucose enhanced fibronectin expression in TSC2(+/-) primary proximal tubular epithelial cells; both inhibition of Akt and inhibition of the mammalian target of rapamycin could prevent this effect of glucose. In addition, forced expression of tuberin in tuberin-null cells abolished the expression of fibronectin protein. Taken together, these data suggest that tuberin plays a central role in the development of renal hypertrophy and in modulating the production of the matrix protein fibronectin in diabetes.
Journal of the American Society of Nephrology 08/2012; 23(10):1652-62. DOI:10.1681/ASN.2012030285 · 9.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective of this study is to identify and characterize the genetic variants related to the glomerular filtration rate (GFR) linkage on 2q37. Of the positional candidate genes, we selected IRS1 and resequenced its 2-kb promoter region and exons for sequence variants in 32 subjects. A total of 11 single nucleotide polymorphisms (SNPs) were identified. To comprehensively cover the 59-kb-long intron-1, eight additional tagging SNPs were selected from the HapMap. All the 19 SNPs were genotyped by TaqMan Assay in the entire data set (N = 670; 39 families). Association analyses between the SNPs and GFR and type 2 diabetes-related traits were performed using the measured genotype approach. Of the SNPs examined for association, only the Gly(972)Arg variant of IRS1 exhibited a significant association with GFR (P = 0.0006) and serum triglycerides levels (P = 0.003), after accounting for trait-specific covariate effects. Carriers of Arg972 had significantly decreased GFR values. Gly(972)Arg contributed to 26% of the linkage signal on 2q. Expression of IRS1 mutant Arg972 in human mesangial cells significantly reduced the insulin-stimulated phosphorylation of IRS1 and Akt kinase. Taken together, the data provide the first evidence that genetic variation in IRS1 may influence variation in GFR probably through impaired insulin receptor signaling.
[Show abstract][Hide abstract] ABSTRACT: Apoptosis contributes to the development of diabetic nephropathy, but the mechanism by which high glucose (HG) induces apoptosis is not fully understood. Because the tuberin/mTOR pathway can modulate apoptosis, we studied the role of this pathway in apoptosis in type I diabetes and in cultured proximal tubular epithelial (PTE) cells exposed to HG. Compared with control rats, diabetic rats had more apoptotic cells in the kidney cortex. Induction of diabetes also increased phosphorylation of tuberin in association with mTOR activation (measured by p70S6K phosphorylation), inactivation of Bcl-2, increased cytosolic cytochrome c expression, activation of caspase 3, and cleavage of PARP; insulin treatment prevented these changes. In vitro, exposure of PTE cells to HG increased phosphorylation of tuberin and p70S6K, phosphorylation of Bcl-2, expression of cytosolic cytochrome c, and caspase 3 activity. High glucose induced translocation of the caspase substrate YY1 from the cytoplasm to the nucleus and enhanced cleavage of PARP. Pretreatment the cells with the mTOR inhibitor rapamycin reduced the number of apoptotic cells induced by HG and the downstream effects of mTOR activation noted above. Furthermore, gene silencing of tuberin with siRNA decreased cleavage of PARP. These data show that the tuberin/mTOR pathway promotes apoptosis of tubular epithelial cells in diabetes, mediated in part by cleavage of PARP by YY1.
Journal of the American Society of Nephrology 02/2011; 22(2):262-73. DOI:10.1681/ASN.2010040352 · 9.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The tuberous sclerosis complex (TSC) is caused by defects in one of two tumor suppressor genes, TSC-1 or TSC-2. The TSC-2 gene encodes tuberin, a protein involved in the pathogenesis of kidney tumors, both angiomyolipomas and renal cell carcinomas. We investigated a potential role for tuberin in regulating a key DNA repair pathway. Downregulation of tuberin in human renal epithelial cells using siRNA resulted in a marked decrease in the abundance of the 8-oxoG-DNA glycosylase (OGG1). Mouse embryonic fibroblasts deficient in tuberin (TSC2(-/-) and TSC2(+/-)) also had markedly decreased OGG1 mRNA and protein expression, as well as undetectable OGG1 activity accompanied by accumulation of 8-oxodG. Gel shift analyses and chromatin immunoprecipatation identified the transcription factor NF-YA as a regulator of OGG1 activity. The binding of NF-YA to the OGG1 promoter was significantly reduced in TSC2(-/-) compared with TSC2(+/+) cells. Introduction of TSC2 cDNA into the tuberin-deficient cells restored NF-YA and OGG1 expression. Transcriptional activity of the OGG1 promoter was also decreased in tuberin-null cells. In addition, mutation of both CAAT boxes, the sites to which NF-YA binds, completely inhibits OGG1 promoter activity. These data provide the first evidence that tuberin regulates a specific DNA repair enzyme, OGG1. This regulation may be important in the pathogenesis of kidney tumors in patients with TSC.
American journal of physiology. Renal physiology 02/2008; 294(1):F281-90. DOI:10.1152/ajprenal.00370.2007 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Angiotensin II (Ang II) induces protein synthesis and hypertrophy through arachidonic acid (AA)- and redoxdependent activation of the serine-threonine kinase Akt/PKB in mesangial cells (MCs). The role of NAD(P)H oxidase component p22( phox ) was explored in this signaling pathway and in Ang II-induced expression of the extracellular matrix protein fibronectin. Ang II causes activation of Akt/PKB and induces fibronectin protein expression, effects abrogated by phospholipase A(2) inhibition and mimicked by AA. Ang II and AAalso elicited an increase in fibronectin expression that was reduced with a dominant negative mutant of Akt/PKB. Exposure of the cells to hydrogen peroxide stimulates Akt/PKB activity and fibronectin synthesis. The antioxidant N-acetylcysteine abolished Ang II- and AA-induced Akt/PKB activation and fibronectin expression. Western blot analysis revealed high levels of p22( phox ) in MCs. Antisense (AS) but not sense oligonucleotides for p22( phox ) prevented ROS generation in response to Ang II and AA. AS p22( phox ) inhibited Ang II- or AA-induced Akt/PKB as well as protein synthesis and fibronectin expression. These data provide the first evidence, in MCs, of activation by AAof a p22( phox )-based NAD(P)H oxidase and subsequent generation of ROS. Moreover, this pathway mediates the effect of Ang II on Akt/PKB-induced protein synthesis and fibronectin expression.
[Show abstract][Hide abstract] ABSTRACT: Monocyte chemotactic protein-1 (MCP-1) recruits activated phagocytes to the site of tissue injury. Interferon-gamma (IFN-gamma) present in the microenvironment of glomerulus acts on mesangial cells to induce local production of MCP-1. The mechanism by which IFN-gamma stimulates expression of MCP-1 is not clear. We therefore examined the role of PI 3 kinase signaling in regulating the IFN-gamma-induced MCP-1 expression in mesangial cells. Blocking PI 3 kinase activity with Ly294002 attenuated IFN-gamma-induced MCP-1 protein and mRNA expression. IFN-gamma increased Akt kinase activity in a PI 3 kinase-dependent manner. Expression of dominant negative Akt kinase inhibited serine phosphorylation of STAT1alpha, without any effect on its tyrosine phosphorylation, and decreased IFN-gamma-induced expression of MCP-1. These data for the first time indicate a role for PI 3 kinase-dependent Akt kinase in MCP-1 expression. We have recently shown that along with Akt, PKCepsilon is a downstream target of PI 3 kinase in IFN-gamma signaling. Similar to dominant negative Akt kinase, dominant negative PKCepsilon also inhibited serine phosphorylation of STAT1alpha without any effect on tyrosine phosphorylation. Dominant negative PKCepsilon also abrogated MAPK activity, resulting in decrease in IFN-gamma-induced MCP-1 expression. Furthermore, Akt and PKCepsilon are present together in a signaling complex. IFN-gamma had no effect on this complex formation, but did increase PKCepsilon-associated Akt kinase activity. PKCepsilon did not regulate IFN-gamma-induced Akt kinase. Finally, expression of dominant negative Akt kinase blocked IFN-gamma-stimulated MAPK activation. These data provide the first evidence that PI 3 kinase-dependent Akt and PKCepsilon activation independently regulate MAPK activity and serine phosphorylation of STAT1alpha to increase expression of MCP-1.
[Show abstract][Hide abstract] ABSTRACT: Cellular mechanisms responsible for the loss of capillary wall permselectivity in diabetic nephropathy are not well characterized. ZO-1 is a junctional protein involved in the assembly and proper function of a number of tight junctions and is also expressed at the junction of podocytes with the slit diaphragm. We investigated the effect of diabetes and high glucose concentration on the expression of ZO-1 in animal models of both type 1 and 2 diabetes and in rat glomerular epithelial cells. In diabetic animals, immunohistochemistry and Western blotting showed decreased expression of ZO-1 in glomeruli. Immunogold electron microscopy revealed redistribution of ZO-1 from the podocyte membrane to the cytoplasm in the diabetic animals. Exposure of rat glomerular epithelial cells to high glucose resulted in a decrease in the intensity of ZO-1 staining and redistribution of ZO-1 from the membrane to the cytoplasm, changes that are attenuated by blockade of the angiotensin II type 1 receptor. ZO-1 protein expression and serine and tyrosine phosphorylation of ZO-1 were also decreased in cells exposed to high glucose. These findings suggest that alterations in the content and localization of ZO-1 may be relevant to the pathogenesis of proteinuria in diabetes.
[Show abstract][Hide abstract] ABSTRACT: Podocytes or glomerular epithelial cells (GECs) are important targets of the diabetic microenvironment. Podocyte foot process effacement and widening, loss of GECs and hypertrophy are pathological features of this disease. ANG II and oxidative stress are key mediators of renal hypertrophy in diabetes. The cellular mechanisms responsible for GEC hypertrophy in diabetes are incompletely characterized. We investigated the effect of high glucose on protein synthesis and GEC hypertrophy. Exposure of GECs to high glucose dose dependently stimulated [(3)H]leucine incorporation, but not [(3)H]thymidine incorporation. High glucose resulted in the activation of ERK1/2 and Akt/PKB. ERK1/2 pathway inhibitor or the dominant negative mutant of Akt/PKB inhibited high glucose-induced protein synthesis. High glucose elicited a rapid generation of reactive oxygen species (ROS). The stimulatory effect of high glucose on ROS production, ERK1/2, and Akt/PKB activation was prevented by the antioxidants catalase, diphenylene iodonium, and N-acetylcysteine. Exposure of the cells to hydrogen peroxide mimicked the effects of high glucose. In addition, ANG II resulted in the activation of ERK1/2 and Akt/PKB and GEC hypertrophy. Moreover, high glucose and ANG II exhibited additive effects on ERK1/2 and Akt/PKB activation as well as protein synthesis. These additive responses were abolished by treatment of the cells with the antioxidants. These data demonstrate that high glucose stimulates GEC hypertrophy through a ROS-dependent activation of ERK1/2 and Akt/PKB. Enhanced ROS generation accounts for the additive effects of high glucose and ANG II, suggesting that this signaling cascade contributes to GEC injury in diabetes.
American journal of physiology. Renal physiology 04/2006; 290(3):F741-51. DOI:10.1152/ajprenal.00313.2005 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. We investigated the role of the NAD(P)H oxidase Nox4 in generation of reactive oxygen species (ROS), hypertrophy, and fibronectin expression in a rat model of type 1 diabetes induced by streptozotocin. Phosphorothioated antisense (AS) or sense oligonucleotides for Nox4 were administered for 2 weeks with an osmotic minipump 72 h after streptozotocin treatment. Nox4 protein expression was increased in diabetic kidney cortex compared with non-diabetic controls and was down-regulated in AS-treated animals. AS oligonucleotides inhibited NADPH-dependent ROS generation in renal cortical and glomerular homogenates. ROS generation by intact isolated glomeruli from diabetic animals was increased compared with glomeruli isolated from AS-treated animals. AS treatment reduced whole kidney and glomerular hypertrophy. Moreover, the increased expression of fibronectin protein was markedly reduced in renal cortex including glomeruli of AS-treated diabetic rats. Akt/protein kinase B and ERK1/2, two protein kinases critical for cell growth and hypertrophy, were activated in diabetes, and AS treatment almost abolished their activation. In cultured mesangial cells, high glucose increased NADPH oxidase activity and fibronectin expression, effects that were prevented in cells transfected with AS oligonucleotides. These data establish a role for Nox4 as the major source of ROS in the kidneys during early stages of diabetes and establish that Nox4-derived ROS mediate renal hypertrophy and increased fibronectin expression.
[Show abstract][Hide abstract] ABSTRACT: Angiotensin II (Ang II) stimulates hypertrophy of glomerular mesangial cells. The signalling mechanism by which Ang II exerts this effect is not precisely known. Downstream potential targets of Ang II are the extracellular-signal-regulated kinases 1 and 2 (ERK1/ERK2). We demonstrate that Ang II activates ERK1/ERK2 via the AT1 receptor. Arachidonic acid (AA) mimics the action of Ang II on ERK1/ERK2 and phospholipase A2 inhibitors blocked Ang II-induced ERK1/ERK2 activation. The antioxidant N-acetylcysteine as well as the NAD(P)H oxidase inhibitors diphenylene iodonium and phenylarsine oxide abolished both Ang II- and AA-induced ERK1/ERK2 activation. Moreover, dominant-negative Rac1 (N17Rac1) blocks activation of ERK1/ERK2 in response to Ang II and AA, whereas constitutively active Rac1 resulted in an increase in ERK1/ERK2 activity. Antisense oligonucleotides for Nox4 NAD(P)H oxidase significantly reduce activation of ERK1/ERK2 by Ang II and AA. We also show that protein synthesis in response to Ang II and AA is inhibited by N17Rac1 or MEK (mitogen-activated protein kinase/ERK kinase) inhibitor. These results demonstrate that Ang II stimulates ERK1/ERK2 by AA and Nox4-derived reactive oxygen species, suggesting that these molecules act as downstream signal transducers of Ang II in the signalling pathway linking the Ang II receptor AT1 to ERK1/ERK2 activation. This pathway involving AA, Rac1, Nox4, reactive oxygen species and ERK1/ERK2 may play an important role in Ang II-induced mesangial cell hypertrophy.
[Show abstract][Hide abstract] ABSTRACT: ANG II induces protein synthesis through the serine-threonine kinase Akt/protein kinase B (PKB) in mesangial cells (MCs). The mechanism(s) of activation of Akt/PKB particularly by G protein-coupled receptors, however, is not well characterized. We explored the role of the small GTPase Rac1, a component of the phagocyte NADPH oxidase, and the gp91phox homologue Nox4/Renox in this signaling pathway. ANG II causes rapid activation of Rac1, an effect abrogated by phospholipase A2 inhibition and mimicked by arachidonic acid (AA). Northern blot analysis revealed high levels of Nox4 transcript in MCs and transfection with antisense (AS) oligonucleotides for Nox4 markedly decreased NADPH-dependent reactive oxygen species (ROS)-producing activity. Dominant negative Rac1 (N17Rac1) as well as AS Nox4 inhibited ROS generation in response to ANG II and AA, whereas constitutively active Rac1 stimulated ROS formation. Moreover, N17Rac1 blocked stimulation of NADPH oxidase activity by AA. N17Rac1 or AS Nox4 abolished ANG II- or AA-induced activation of the hypertrophic kinase Akt/PKB. In addition, AS Nox4 inhibited ANG II-induced protein synthesis. These data provide the first evidence that activation by AA of a Rac1-regulated, Nox4-based NAD(P)H oxidase and subsequent generation of ROS mediate the effect of ANG II on Akt/PKB activation and protein synthesis in MCs.
American journal of physiology. Renal physiology 09/2003; 285(2):F219-29. DOI:10.1152/ajprenal.00414.2002 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BMP-7, a member of the bone morphogenetic protein subfamily of the TGFbeta-superfamily is highly expressed in the murine kidney. BMP-7 is involved in fetal nephron development and mesenchymal to epithelial cell differentiation. Constitutive BMP-7 expression is found in tubular and glomerular epithelial cells of the adult kidney. BMP-7 may play a role in physiology and pathophysiology of the adult kidney since BMP-7 gene expression in acute renal ischemia is diminished and injection of recombinant BMP-7 into rats with ischemic acute renal failure preserves renal function. In order to investigate the transcriptional regulation of BMP-7, this study was undertaken to clone and characterize the promoter of the murine BMP-7 gene. A 1394 bp sequence of the 5'-flanking region of the BMP-7 gene was isolated and subcloned. No TATA and CAAT box consensus motifs could be identified as shown for promoters of other BMPs. Using in vitro transfection assays, the 5'-flanking region revealed moderate to strong basal promoter activity. PMA increased basal BMP-7 promoter activity. Thus BMP-7 gene transcription might involve at least in part a PKC-dependent pathway. The cloning of a 5'-flanking region of the BMP-7 gene should provide a useful tool for future studies on the transcriptional regulation of BMP-7 gene expression.
[Show abstract][Hide abstract] ABSTRACT: Angiotensin II (Ang II) exerts contractile and trophic effects in glomerular mesangial cells (MCs). One potential downstream target of Ang II is the protein kinase Akt/protein kinase B (PKB). We investigated the effect of Ang II on Akt/PKB activity in MCs. Ang II causes rapid activation of Akt/PKB (5-10 min) but delayed activation of phosphoinositide 3-kinase (PI3-K) (30 min). Activation of Akt/PKB by Ang II was not abrogated by the PI3-K inhibitors or by the introduction of a dominant negative PI3-K, indicating that in MCs, PI3-K is not an upstream mediator of Akt/PKB activation by Ang II. Incubation of MCs with phospholipase A2 inhibitors also blocked Akt/PKB activation by Ang II. AA mimicked the effect of Ang II. Inhibitors of cyclooxygenase-, lipoxyogenase-, and cytochrome P450-dependent metabolism did not influence AA-induced Akt/PKB activation. However, the antioxidants N-acetylcysteine and diphenylene iodonium inhibited both AA- and Ang II-induced Akt/PKB activation. Dominant negative mutant of Akt/PKB or antioxidants, but not the dominant negative form of PI3-K, inhibited Ang II-induced protein synthesis and cell hypertrophy. These data provide the first evidence that Ang II induces protein synthesis and hypertrophy in MCs through AA/redox-dependent pathway and Akt/PKB activation independent of PI3-K.
The FASEB Journal 10/2001; 15(11):1909-20. DOI:10.1096/fj..01-0165com · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stromelysin-1, matrix metalloproteinase-3 (MMP-3), is an important endopeptidase selectively expressed by somatic cells in organ tissues. The renal tubulointerstitium, for example, comprises tubular epithelium and interstitial fibroblasts forming the principal mass of the kidney. We observed that mRNA encoding stromelysin-1 is detectable in murine renal fibroblasts, but not in proximal tubular epithelium. Transcripts measured by RNase protection assay in renal fibroblasts increase following exposure to phorbol ester, and thereafter, activated stromelysin-1 protein can be detected in culture media by Western blotting. A 6.4 Kb genomic clone containing the putative stromelysin-1 promoter was isolated and a relevant 2.1 Kb PstI restriction fragment including 2.1 Kb of the immediate 5'-flanking region was sequenced on both strands. Two transcriptional start sites were identified by primer extension; the major start site corresponded to a previously established position in the rat promoter, and a second undescribed minor transcriptional start site was located 16 bp upstream of the primary site. A HiNF-A chromatin-activating element at -106 bp was found in the early promoter region of pR336 and an active AP-1 site at -72 bp with an Ets/PEA-3 motif at -203 bp was suggested by transient transfection of luciferase minigenes into renal fibroblasts responsive to phorbol ester. This Ets element was identical to a site in the early promoter of the fibroblast-specific gene FSP1. A baseline enhancement in activity of pR336 in fibroblasts was further observed with the addition of 5' flanking sequence out to -1980 bp. This additional region of flanking sequence contains two modular regions: one of multiple PEA-3 elements between -684 bp and -1955 bp and a second region between -1929 bp and -1980 bps containing a second AP-1 site at -1929 bp, a MBF-1/ MEP-1 metal binding site, and a PPAR peroxisome proliferator element at -1950 bp. Our findings implicate a gene structure with expected activity in a mesenchymal phenotype. The PKC-dependent regulation of the stromelysin-1 gene supports the notion that it may be modulated during inflammation or tissue remodeling.
Kidney International 08/1997; 52(1):120-9. DOI:10.1038/ki.1997.311 · 8.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Perlecan, the basement membrane heparan sulfate proteoglycan (HSPG), has been fully cloned from mouse and human tissues. When a cRNA probe of murine perlecan cDNA was employed in RNase protection assay to test whether rat glomerular epithelial cells (GEC) constitutively express perlecan, several bands of hybridization were seen, suggesting that sequences between rat and murine perlecan may not be identical. Using primers based on published cDNA sequences of murine and human perlecan and poly A+ RNA of rat GEC, we synthesized a 497 bp product (RPD-I) by RT-PCR. The deduced aminoacid sequence showed an 85% and 88% homology with domain I of murine and human perlecan, respectively. The three putative sites containing the consensus sequence SGD for attachment of heparan sulfate chains were fully conserved in the rat perlecan as was a site (NFT) for attachment of N-linked oligosaccharide. RPD-I detected a > 9.5 kb transcript of perlecan in RNA of GEC, similar in size to that present in rat glomeruli. Employing a riboprobe synthesized from RPD-I in RNase protection assay we examined whether dbcAMP regulated perlecan expression in the GEC. At 1, 6, 24 and 48 h of incubation, 1 mM dbcAMP caused 43%, 32%, 47% and 40% reduction in mRNA abundance of perlecan, respectively. Immunoprecipitation showed a corresponding reduction of 61%, 70% and 65% in the synthesis of 35SO4 labeled basement membrane HSPG by the GEC following 12, 24 and 48 h of incubation with dbcAMP. Following incubation for 1 and 24 h prostaglandins, PGE1 and PGE2 (1 uM), known activators of glomerular adenylate cyclase, reduced perlecan mRNA abundance to a similar extent as dbcAMP on northern analysis. Our results show that glomerular basement membrane HSPG synthesized by the GEC belongs to the perlecan family. Decrease of GEC perlecan gene expression and synthesis by cAMP and prostaglandins may be of relevance to proteinuric states characterized by activation of these mediators.
[Show abstract][Hide abstract] ABSTRACT: Mesangial cells predominantly express platelet-derived growth factor (PDGF)-A chain mRNA and release PDGF. Mesangial cell PDGF-A chain mRNA abundance is regulated by several agents including phorbol esters. We have recently demonstrated that induction of PDGF-A chain mRNA abundance in response to phorbol 12-myristate 13-acetate is primarily due to gene transcription. We have now analyzed the 5'-flanking region of the PDGF-A chain promoter to identify DNA binding protein(s) which have the potential to regulate PDGF-A chain gene transcription in human mesangial cells. DNase I footprint analysis of the 5'-flanking region of the PDGF-A chain promoter identifies a DNase I protected region at the location -82 to -102 corresponding to the sequence 5'-GGCCCGGAATCCGGGGGAGGC-3'. Therefore, nuclear extracts from human mesangial cells contain a protein, PDGF-A-BP-1, that binds to a DNA sequence (-82 to -102) in the promoter region of the PDGF-A chain gene. Gel mobility shift analysis using labeled oligomer corresponding to the binding site for PDGF-A-BP-1 indicates that PDGF-A-BP-1 is induced by phorbol ester in mesangial cells as well as fat-storing cells (> 20 fold). Egr-1 protein does not bind to labeled PDGF-A-BP-1 oligomer and does not compete with the binding of PDGF-A-BP-1. In addition, SP-1 binding sequence does not compete with the binding sequence of the mesangial cell protein. PDGF-A-BP-1 appears to represent a novel protein which is induced by phorbol ester and thus has the potential for an important role in the transcriptional regulation of the PDGF-A chain gene in mesangial cells and other vascular pericytes.
[Show abstract][Hide abstract] ABSTRACT: We have investigated the effect of phorbol 12-myristate 13-acetate (PMA) on platelet-derived growth factor (PDGF) B-chain gene transcription as well as on mRNA stability in cultured human mesangial cells. Addition of actinomycin to cells stimulated with PMA decreases steady state levels of PDGF-B chain mRNA analysed by solution hybridization assay. PDGF-B chain gene transcription was also assayed directly by measuring elongation of transcripts in isolated nuclei followed by hybridization of labeled RNA transcripts to a cDNA encoding for PDGF-B chain. Our data show that PMA induces PDGF-B chain gene transcription by approximately 2-fold. alpha-Amanitin, an RNA polymerase II inhibitor, blocked transcription by more than 70%. In addition, we determined the effect of PMA on the halflife of PDGF-B chain mRNA directly by pulse chase method. In human mesangial cells, the PDGF-B chain mRNA exhibited halflife of approximately 105 min. In the presence of PMA, the halflife of PDGF-B chain mRNA was reduced to approximately 72 min. These studies indicate that regulation of PDGF-B chain gene by PMA in human mesangial cells involves a coordinate effort at the level of transcription and mRNA stability.
[Show abstract][Hide abstract] ABSTRACT: Mesangial cells express platelet-derived growth factor (PDGF) A- and B-chain mRNA and release PDGF. Several polypeptide growth factors, including PDGF itself, induce PDGF A- and B- chain mRNA abundance. To understand the molecular mechanisms associated with the changes in mRNA abundance, we measured the effects of PDGF BB homodimer on PDGF A- and B-chain gene transcription in cultured mesangial cells. The data demonstrate 2- and 4-fold increases in PDGF A-chain gene transcription in response to PDGF BB homodimer at 5 and 24 h time points respectively. PDGF B-chain gene transcription was also induced approximately 3-fold at 2, 5 and 24 h time points in response to treatment with PDGF BB homodimer. The effect of PDGF BB on the half-life of PDGF A- as well as PDGF B-chain mRNA was measured directly by the pulse-chase method. There was no effect on PDGF A-chain mRNA half-life whereas PDGF B-chain mRNA half-life was increased 1.5-fold. These studies indicate that, in human mesangial cells, the increase in the levels of PDGF A- and B-chain mRNA in response to PDGF- receptor(s) activation is mediated at the level of gene transcription. In addition, the regulation of PDGF B- but not PDGF A-chain gene involves increased mRNA stability. Mesangial cells are a useful model for studying molecular mechanisms of PDGF- gene regulation in non-transformed human cells.