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Diping Wang,
Gina M Warner,
Ping Yin,
Bruce E Knudsen, Jingfei Cheng,
Kim Butters,
Karen R Lien,
Catherine E Gray,
Vesna D Garovic,
Lilach O Lerman,
Stephen C Textor,
Karl A Nath,
Robert D Simari,
Joseph P Grande
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ABSTRACT: Renal artery stenosis (RAS) is an important cause of chronic renal dysfunction. Recent studies have underscored a critical role for CCL2 (MCP-1)-mediated inflammation in the progression of chronic renal damage in RAS and other chronic renal diseases. In vitro studies have implicated p38 mitogen activated protein kinase (MAPK) as a critical intermediate for the production of CCL2. However, a potential role of p38 signaling in the development and progression of chronic renal disease in RAS has not been previously defined. We sought to test the hypothesis that inhibition of p38 MAPK ameliorates chronic renal injury in mice with RAS. We established RAS by placing a cuff on the right renal artery and treated mice with the p38 inhibitor SB203580 or vehicle for 2 weeks. In mice treated with vehicle, the cuffed kidney developed interstitial fibrosis, tubular atrophy, and interstitial inflammation, in accordance with our previous findings. SB203580 reduced renal atrophy in cuffed kidneys (70% vs. 39%, p < 0.05). SB203580 reduced interstitial inflammation and extracellular matrix deposition but had no effect on the development of hypertension. SB203580 prevented induction of CCL2, CCL7 (MCP-1), CC chemokine receptor 2 (CCR2), and collagen 4 mRNA expression in the cuffed kidney. In vitro, blockade of p38 prevented both TNF-α and TGF-β-induced CCL2 upregulation. Based on these observations, we conclude that p38 MAPK plays a critical role in the induction of CCL2/CCL7/CCR2 system and the development of interstitial inflammation in RAS.
AJP Renal Physiology 01/2013; · 4.42 Impact Factor
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ABSTRACT: Although the two-kidney, one-clip (2K1C) model is widely used as a model of human renovascular hypertension, mechanisms leading to the development of fibrosis and atrophy in the cuffed kidney and compensatory hyperplasia in the contralateral kidney have not been defined. Based on the well-established role of the transforming growth factor (TGF)-β signaling pathway in renal fibrosis, we tested the hypothesis that abrogation of TGF-β/Smad3 signaling would prevent fibrosis in the cuffed kidney. Renal artery stenosis (RAS) was established in mice with a targeted disruption of exon 2 of the Smad3 gene (Smad3 KO) and wild-type (WT) controls by placement of a polytetrafluoroethylene cuff on the right renal artery. Serial pulse-wave Doppler ultrasound assessments verified that blood flow through the cuffed renal artery was decreased to a similar extent in Smad3 KO and WT mice. Two weeks after surgery, systolic blood pressure and plasma renin activity were significantly elevated in both the Smad3 KO and WT mice. The cuffed kidney of WT mice developed renal atrophy (50% reduction in weight after 6 wk, P < 0.0001), which was associated with the development of interstitial fibrosis, tubular atrophy, and interstitial inflammation. Remarkably, despite a similar reduction of renal blood flow, the cuffed kidney of the Smad3 KO mice showed minimal atrophy (9% reduction in weight, P = not significant), with no significant histopathological alterations (interstitial fibrosis, tubular atrophy, and interstitial inflammation). We conclude that abrogation of TGF-β/Smad3 signaling confers protection against the development of fibrosis and atrophy in RAS.
AJP Renal Physiology 02/2012; 302(11):F1455-64. · 4.42 Impact Factor
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ABSTRACT: Monocyte chemoattractant protein 1 (MCP-1) is a CC cytokine that fundamentally contributes to the pathogenesis of inflammatory renal disease. MCP-1 is highly expressed in cytokine-stimulated mesangial cells in vitro and following glomerular injury in vivo. Interventions to limit MCP-1 expression are commonly effective in assorted experimental models. Fish oil, an abundant source of n-3 fatty acids, has anti-inflammatory properties, the basis of which remains incompletely defined. We examined potential mechanisms whereby fish oil reduces MCP-1 expression and thereby suppresses inflammatory responses to tissue injury. Cultured mesangial cells were treated with TNF-α in the presence of the n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA); equimolar concentrations of the n-6 fatty acids LA and OA served as controls. MCP-1 mRNA expression was assessed by Northern blotting, and transcriptional activity of the MCP-1 promoter was assessed by transient transfection. The involvement of the ERK and NF-κB pathways was evaluated through transfection analysis and the use of the MEK inhibitor U0126. DHA and EPA decreased TNF-α-stimulated MCP-1 mRNA expression by decreasing transcription of the MCP-1 gene. DHA and EPA decreased p-ERK expression and nuclear translocation of NF-κB, both of which are necessary for TNF-α-stimulated MCP-1 expression. Both NF-κB and AP-1 sites were involved in transcriptional regulation of the MCP-1 gene by DHA and EPA. We conclude that DHA and EPA inhibit TNF-α-stimulated transcription of the MCP-1 gene through interaction of signaling pathways involving ERK and NF-κB. We speculate that such effects may contribute to the salutary effect of fish oil in renal and vascular disease.
AJP Renal Physiology 03/2011; 300(5):F1142-51. · 4.42 Impact Factor
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ABSTRACT: In addition to their critical role in embryogenesis of the kidney, members of the transforming growth factor (TGF)-β superfamily direct a number of pathways important in the maintenance of homeostasis in the differentiated kidney. TGF-β family members also play an important role in cell-cycle regulation. Through induction of cyclin-dependent kinase inhibitors, TGF-β promotes a hypertrophic response of renal tubular epithelial cells and glomerular mesangial cells. This TGF-β-driven hypertrophic response, which occurs in diabetic nephropathy, may have deleterious effects on the kidney. In contrast, many human cancers are associated with loss of the growth inhibitory effects of TGF-β. TGF-β may promote or inhibit inflammation, an outcome which appears to depend on the cell type(s) involved and on potential interactions with other signaling pathways that regulate inflammatory responses. In recent studies, TGF-β has been implicated as a key mediator of the epithelial to mesenchymal transition, a process through which epithelial cells acquire characteristics of myofibroblasts which synthesize and deposit extracellular matrix macromolecules and lead to the development of fibrosis, a characteristic feature of chronic renal disease irrespective of etiology. In this brief overview, we highlight recent advances in our understanding of TGF-β signaling that contribute to the development and progression of chronic renal disease.
09/2009; 5(3):182-192.
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Jingfei Cheng,
Wei Zhou,
Gina M Warner,
Bruce E Knudsen,
Vesna D Garovic,
Catherine E Gray,
Lilach O Lerman,
Jeffrey L Platt,
J Carlos Romero,
Stephen C Textor,
Karl A Nath,
Joseph P Grande
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ABSTRACT: Unilateral renal artery stenosis (RAS) leads to atrophy of the stenotic kidney and compensatory enlargement of the contralateral kidney. Although the two-kidney, one-clip (2K1C) model has been extensively used to model human RAS, the cellular responses in the stenotic and contralateral kidneys, particularly in the murine model, have received relatively little attention. We studied mice 2, 5, and 11 wk after unilateral RAS. These mice became hypertensive within 1 wk. The contralateral kidney increased in size within 2 wk after surgery. This enlargement was associated with a transient increase in expression of phospho-extracellular signal-regulated kinase (p-ERK), the proliferation markers proliferating cell nuclear antigen and Ki-67, the cell cycle inhibitors p21 and p27, and transforming growth factor-beta, with return to baseline levels by 11 wk. The size of the stenotic kidney was unchanged at 2 wk but progressively decreased between 5 and 11 wk. Unlike the contralateral kidney, which showed minimal histopathological alterations, the stenotic kidney developed progressive interstitial fibrosis, tubular atrophy, and interstitial inflammation. Surprisingly, the stenotic kidney showed a proliferative response, which involved largely tubular epithelial cells. The atrophic kidney had little evidence of apoptosis, despite persistent upregulation of p53; expression of cell cycle regulatory proteins in the stenotic kidney was persistently increased through 11 wk. These studies indicate that in the 2K1C model, the stenotic kidney and contralateral, enlarged kidney exhibit a distinct temporal expression of proteins involved in cell growth, cell survival, apoptosis, inflammation, and fibrosis. Notably, an unexpected proliferative response occurs in the stenotic kidney that undergoes atrophy.
AJP Renal Physiology 08/2009; 297(4):F1055-68. · 4.42 Impact Factor
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ABSTRACT: In addition to their critical role in embryogenesis of the kidney, members of the transforming growth factor (TGF)-β superfamily direct a number of pathways important in the maintenance of homeostasis in the differentiated kidney. TGF-β family members also play an important role in cell-cycle regulation. Through induction of cyclin-dependent kinase inhibitors, TGF-β promotes a hypertrophic response of renal tubular epithelial cells and glomerular mesangial cells. This TGF-β-driven hypertrophic response, which occurs in diabetic nephropathy, may have deleterious effects on the kidney. In contrast, many human cancers are associated with loss of the growth inhibitory effects of TGF-β. TGF-β may promote or inhibit inflammation, an outcome which appears to depend on the cell type(s) involved and on potential interactions with other signaling pathways that regulate inflammatory responses. In recent studies, TGF-β has been implicated as a key mediator of the epithelial to mesenchymal transition, a process through which epithelial cells acquire characteristics of myofibroblasts which synthesize and deposit extracellular matrix macromolecules and lead to the development of fibrosis, a characteristic feature of chronic renal disease irrespective of etiology. In this brief overview, we highlight recent advances in our understanding of TGF-β signaling that contribute to the development and progression of chronic renal disease.
Journal of Organ Dysfunction 01/2009; 5(3):182 - 192.
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ABSTRACT: Although many studies have indicated that fish oil (FO) improves cardiovascular risk factors and reduces histopathological manifestations of injury in experimental renal injury models, potential mechanisms underlying this protective effect have not been adequately defined. The objective of this study was to identify potential signaling pathways that confer protection in the Dahl rat model of salt-sensitive hypertension. Male Dahl salt-sensitive rats (n = 10/group) were provided with formulated diets containing 8% NaCl, 20% protein, and 25% FO or 25% corn oil (CO) for 28 days. FO reduced blood pressure (-11% at 4 wk; P < 0.05), urine protein excretion (-45% at 4 wk; P < 0.05), plasma cholesterol and triglyceride levels (-54%, P < 0.001; and -58%, P < 0.05), and histopathological manifestations of renal injury, including vascular hypertrophy, segmental and global glomerular sclerosis, interstitial fibrosis, and tubular atrophy. Interstitial inflammation was significantly reduced by FO (-32%; P < 0.001), as assessed by quantitative analysis of ED1-positive cells in sections of the renal cortex. FO reduced tubulointerstitial proliferative activity, as assessed by Western blot analysis of cortical homogenates for PCNA (-51%; P < 0.01) and quantitative analysis of Mib-1-stained sections of the renal cortex (-42%; P < 0.001). Decreased proliferative activity was associated with reduced phospho-ERK expression (-37%; P < 0.005) and NF-kappaB activation (-42%; P < 0.05). FO reduced cyclooxygenase (COX)-2 expression (-63%; P < 0.01) and membrane translocation of the NADPH oxidase subunits p47(phox) and p67(phox) (-26 and -34%; P < 0.05). We propose that FO ameliorates renal injury in Dahl salt-sensitive rats through the inhibition of ERK, decreased NF-kappaB activation, inhibition of COX-2 expression, and decreased NADPH oxidase activation.
American journal of physiology. Renal physiology 07/2008; 294(6):F1323-35. · 3.68 Impact Factor
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Narayana S Murali,
Allan W Ackerman,
Anthony J Croatt, Jingfei Cheng,
Joseph P Grande,
Shari L Sutor,
Richard J Bram,
Gary D Bren,
Andrew D Badley,
Jawed Alam,
Karl A Nath
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ABSTRACT: Proteinuria contributes to chronic kidney disease by stimulating renal tubular epithelial cells to produce cytokines such as monocyte chemoattractant protein-1 (MCP-1). The present study determined whether cellular overexpression of heme oxygenase-1 (HO-1) can influence albumin-stimulated MCP-1 production. In response to bovine serum albumin, NRK-52E cells constitutively overexpressing HO-1 (HO-1 OE cells) exhibit less induction of MCP-1 mRNA and less production of MCP-1 protein compared with similarly treated, control NRK-52E cells (CON cells). In wild-type NRK-52E cells, and under these conditions, we demonstrate that the induction of MCP-1 is critically dependent on intact NF-kappaB binding sites in the MCP-1 promoter. In response to albumin, CON cells exhibit activation of NF-kappaB, and this is reduced in HO-1 OE cells. Albumin also activates ERK1/2 and increases ERK activity, both of which are exaggerated in HO-1 OE cells. Studies with an inhibitor of MAPK/ERK kinase (U0126) demonstrate that the inhibitory effects of U0126 on MCP-1 production are attenuated in HO-1 OE cells. We conclude that HO-1 overexpression in the proximal tubule reduces MCP-1 production in response to albumin, and this occurs, at least in part, by inhibiting an ERK-dependent, NF-kappaB-dependent pathway at a site that is distal to the activation of ERK. These findings suggest that the induction of HO-1 in the proximal tubule, as occurs in chronic kidney disease, may be a countervailing response that reduces albumin-stimulated production of cytokines such as MCP-1.
American journal of physiology. Renal physiology 03/2007; 292(2):F837-44. · 3.68 Impact Factor
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ABSTRACT: Cyclic nucleotides are recognized as critical mediators of many renal functions, including solute transport, regulation of vascular tone, proliferation of parenchymal cells, and inflammation. Although most studies have linked elevated cAMP levels to activation of protein kinase A, cAMP can also directly activate cyclic nucleotide gated ion channels and can signal through activation of GTP exchange factors. Cyclic AMP signaling is highly compartmentalized through plasma membrane localization of adenylyl cyclase and expression of scaffolding proteins that anchor protein kinase A to specific intracellular locations. Cyclic nucleotide levels are largely regulated through catabolic processes directed by phosphodiesterases (PDEs). The PDE superfamily is large and complex, with over 60 distinct isoforms that preferentially hydrolyze cAMP, cGMP, or both. PDEs contribute to compartmentalized cyclic nucleotide signaling. The unique cell- and tissue-specific distribution of PDEs has prompted the development of highly specific PDE inhibitors to treat a variety of inflammatory conditions. In experimental systems, PDE inhibitors have been employed to demonstrate functional compartmentalization of cyclic nucleotide signaling in the kidney. For example, mitogenesis in glomerular mesangial cells and normal tubular epithelial cells is negatively regulated by an intracellular pool of cAMP that is metabolized by PDE3, but not by other PDEs. In Madin-Darby canine kidney cells, an in vitro model of polycystic kidney disease, an intracellular pool of cAMP directed by PDE3 stimulates mitogenesis. In mesangial cells, an intracellular pool of cAMP directed by PDE4 inhibits reactive oxygen species and expression of the potent proin-flammatory cytokine monocyte chemoattractant protein 1. An intracellular pool of cGMP directed by PDE5 regulates solute transport. PDE5 inhibitors ameliorate renal injury in a chronic renal disease model. In this overview, we highlight recent studies to define relationships between PDE expression and renal function and to provide evidence that PDE inhibitors may be effective agents in treating chronic renal disease.
Experimental Biology and Medicine 02/2007; 232(1):38-51. · 2.64 Impact Factor
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ABSTRACT: Polycystic kidney diseases (PKD) are characterized by excessive proliferation of renal tubular epithelial cells, development of fluid-filled cysts, and progressive renal insufficiency. cAMP inhibits proliferation of normal renal tubular epithelial cells but stimulates proliferation of renal tubular epithelial cells derived from patients with PKD. Madin-Darby canine kidney (MDCK) epithelial cells, which are widely used as an in vitro model of cystogenesis, also proliferate in response to cAMP. Intracellular cAMP levels are tightly regulated by phosphodiesterases (PDE). Isoform-specific PDE inhibitors have been developed as therapeutic agents to regulate signaling pathways directed by cAMP. In other renal cell types, we have previously demonstrated that cAMP is hydrolyzed by PDE3 and PDE4, but only PDE3 inhibitors suppress proliferation by inhibiting Raf-1 activity (Cheng J, Thompson MA, Walker HJ, Gray CE, Diaz Encarnacion MM, Warner GM, Grande JP. Am J Physiol Renal Physiol 287:F940-F953, 2004.) A potential role for PDE isoform(s) in cAMP-mediated proliferation of MDCK cells has not previously been established. Similar to what we have previously found in several other renal cell types, cAMP hydrolysis in MDCK cells is directed primarily by PDE4 (85% of total activity) and PDE3 (15% of total activity). PDE4 inhibitors are more effective than PDE3 inhibitors in increasing intracellular cAMP levels in MDCK cells. However, only PDE3 inhibitors, and not PDE4 inhibitors, stimulate mitogenesis of MDCK cells. PDE3 but not PDE4 inhibitors activate B-Raf but not Raf-1, as assessed by an in vitro kinase assay. PDE3 but not PDE4 inhibitors activate the ERK pathway and activate cyclins D and E, as assessed by histone H1 kinase assay. We conclude that mitogenesis of MDCK cells is regulated by a functionally compartmentalized intracellular cAMP pool directed by PDE3. Pharmacologic agents that stimulate PDE3 activity may provide the basis for new therapies directed toward reducing cystogenesis in patients with PKD.
Experimental Biology and Medicine 04/2006; 231(3):288-95. · 2.64 Impact Factor
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ABSTRACT: Monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor (TGF)-beta1 are critical mediators of renal injury by promoting excessive inflammation and extracellular matrix deposition, thereby contributing to progressive renal disease. In renal disease models, MCP-1 stimulates the production of TGF-beta1. However, a potential role for TGF-beta1 in the regulation of MCP-1 production by mesangial cells (MCs) has not previously been evaluated. The objectives of this study were to define the role of TGF-beta1 in regulation of MCP-1 expression in cultured MCs and to define mechanisms through which rolipram (Rp), a phosphodiesterase isoenzyme 4 (PDE4) inhibitor with anti-inflammatory properties, alters MCP-1 expression. TGF-beta1 induced MCP-1 in a time- and dose-dependent manner without increasing transcription of the MCP-1 gene. TGF-beta1-mediated induction of MCP-1 occurred without activation of the NF-kappaB pathway. Rp blocked TGF-beta1-stimulated MCP-1 expression via a protein kinase A-dependent process, at least in part, by decreasing MCP-1 message stability. Rp exerted no effect on activation of the Smad pathway by TGF-beta1. TGF-beta1-mediated induction of MCP-1 required activation of ERK and p38, both of which were suppressed by a PDE4 inhibitor. TGF-beta1-stimulated reactive oxygen species (ROS) generation by MCs, and Rp inhibited ROS generation in TGF-beta1-stimulated MCs; in addition, both Rp and ROS scavengers blocked TGF-beta1-stimulated MCP-1 expression. We conclude that TGF-beta1 stimulates MCP-1 expression through pathways involving activation of ERK, p38, and ROS generation. Positive cross-talk between TGF-beta1 and MCP-1 signaling in MCs may underlie the development of progressive renal disease. Rp, by preventing TGF-beta1-stimulated MCP-1 production, may offer a therapeutic approach in retarding the progression of renal disease.
AJP Cell Physiology 11/2005; 289(4):C959-70. · 3.54 Impact Factor
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ABSTRACT: Mesangial cell (MC) mitogenesis is regulated through "negative cross talk" between cAMP-PKA and ERK signaling. Although it is widely accepted that cAMP inhibits mitogenesis through PKA-mediated phosphorylation of Raf-1, recent studies have indicated that cAMP-mediated inhibition of mitogenesis may occur independently of Raf-1 phosphorylation or without inhibiting ERK activity. We previously showed that MCs possess functionally compartmentalized intracellular pools of cAMP that are differentially regulated by cAMP phosphodiesterases (PDE); an intracellular pool directed by PDE3 but not by PDE4 suppresses mitogenesis. We therefore sought to determine whether there was a differential effect of PDE3 vs. PDE4 inhibitors on the Ras-Raf-MEK-ERK pathway in cultured MC. Although PDE3 and PDE4 inhibitors activated PKA and modestly elevated cAMP levels to a similar extent, only PDE3 inhibitors suppressed MC mitogenesis (-57%) and suppressed Raf-1 kinase and ERK activity (-33 and -68%, respectively). Both PDE3 and PDE4 inhibitors suppressed B-Raf kinase activity. PDE3 inhibitors increased phosphorylation of Raf-1 on serine 43 and serine 259 and decreased phosphorylation on serine 338; PDE4 inhibitors were without effect. Overexpression of a constitutively active MEK-1 construct reversed the antiproliferative effect of PDE3 inhibitors. PDE3 inhibitors also reduced cyclin A levels (-27%), cyclin D and cyclin E kinase activity (-30 and -50%, respectively), and induced expression of the cell cycle inhibitor p21 (+90%). We conclude that the antiproliferative effects of PDE3 inhibitors are mechanistically related to inhibition of the Ras-Raf-MEK-ERK pathway. Additional cell cycle targets of PDE3 inhibitors include cyclin A, cyclin D, cyclin E, and p21.
American journal of physiology. Renal physiology 12/2004; 287(5):F940-53. · 3.68 Impact Factor
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ABSTRACT: Although dietary fish oil supplementation has been used to prevent the progression of kidney disease in patients with IgA nephropathy, relatively few studies provide a mechanistic rationale for its use. Using an antithymocyte (ATS) model of mesangial proliferative glomerulonephritis, we recently demonstrated that fish oil inhibits mesangial cell (MC) activation and proliferation, reduces proteinuria, and decreases histologic evidence of glomerular damage. We therefore sought to define potential mechanisms underlying the antiproliferative effect of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the predominant omega-3 polyunsaturated fatty acids found in fish oil, in cultured MC. DHA and EPA were administered to MC as bovine serum albumin fatty-acid complexes. Low-dose (10-50 micromol/L) DHA, but not EPA, inhibited basal and epidermal growth factor (EGF)-stimulated [(3)H]-thymidine incorporation in MCs. At higher doses (100 micromol/L), EPA and DHA were equally effective in suppressing basal and EGF-stimulated MC mitogenesis. Low-dose DHA, but not EPA, decreased ERK activation by 30% (P <.01), as assessed with Western-blot analysis using phosphospecific antibodies. JNK activity was increased by low-dose DHA but not by EPA. p38 activity was not significantly altered by DHA or EPA. Cyclin E activity, as assessed with a histone H1 kinase assay, was inhibited by low-dose DHA but not by EPA. DHA increased expression of the cell cycle inhibitor p21 but not p27; EPA had no effect on p21 or p27. We propose that the differential effect of low-dose DHA vs EPA in suppressing MC mitogenesis is related to down-regulation of ERK and cyclin E activity and to induction of p21.
Journal of Laboratory and Clinical Medicine 06/2003; 141(5):318-29. · 2.62 Impact Factor
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ABSTRACT: Transforming growth factor-beta (TGF-beta) superfamily members are multifunctional growth factors that play pivotal roles in development and tissue homeostasis. Recent studies have underscored the importance of TGF-beta in regulation of cell proliferation and extracellular matrix synthesis and deposition. TGF-beta signaling is initiated by ligand binding to a membrane-associated receptor complex that has serine/threonine kinase activity. This receptor complex phosphorylates specific Smad proteins, which then transduce the ligand-activated signal to the nucleus. Smad complexes regulate target gene transcription either by directly binding DNA sequences, or by complexing with other transcription factors or co-activators. There is extensive crosstalk between the TGF-beta signaling pathway and other signaling systems, including the mitogen-activated protein kinase pathways. The importance of TGF-beta in regulation of cell growth has been emphasized by recent observations that mutations of critical elements of the TGF-beta signaling system are associated with tumor progression in patients with many different types of epithelial neoplasms. TGF-beta has emerged as a predominant mediator of extracellular matrix production and deposition in progressive renal disease and in other forms of chronic tissue injury. In this overview, recent advances in our understanding of TGF-beta signaling, cell cycle regulation by TGF-beta, and the role of TGF-beta in progressive renal injury are highlighted.
Experimental Biology and Medicine 01/2003; 227(11):943-56. · 2.64 Impact Factor
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ABSTRACT: Recent studies in cultured cells have provided evidence that a variety of pathobiologic stimuli, including high glucose, angiotensin II, and thromboxane A(2), trigger a signaling pathway leading to autocrine induction of TGF-beta1. TGF-beta1 production through this pathway may profoundly affect cell growth, matrix synthesis, and response to injury. This study examines the role of autocrine versus exogenously added TGF-beta1 in cellular proliferation and collagen IV production, critical targets of TGF-beta1 signaling, using renal cells derived from TGF-beta1 knockout (KO) animals or wild-type (WT) controls. Growth of WT and KO cells was assessed by cell counting and [(3)H]thymidine uptake. Basal and TGF-beta1-stimulated collagen production was assessed by Northern and Western blotting; transcriptional activity of the alpha1(IV) collagen gene was assessed by transient transfection analysis. KO cells grew at a faster rate than WT cells carefully matched for plating density and passage number. This increased growth rate was paralleled by increases in [(3)H]thymidine uptake. KO cells expressed lower levels of the cell cycle inhibitors p21 and p27 than WT cells. KO cells failed to express TGF-beta1, as expected. Basal TGF-beta3 mRNA levels were higher in KO cells than in WT cells. WT cells expressed higher basal levels of TGF-beta2 mRNA than KO cells. Basal alpha1(IV) and alpha2(IV) collagen mRNA and protein expression were significantly lower in KO cells than WT cells. Administration of exogenous TGF-beta1 induced collagen IV production in both KO and WT cells. Although basal transcriptional activity of an alpha1(IV) collagen-CAT construct was lower in KO cells than WT cells, administration of exogenous TGF-beta1 was associated with significant increases in transcriptional activity of this construct in both KO and WT cells. These studies provide evidence that autocrine production of TGF-beta1 may play a critical role in regulation of growth and basal collagen IV production by renal tubular epithelial cells.
Experimental Biology and Medicine 04/2002; 227(3):171-81. · 2.64 Impact Factor
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ABSTRACT: The release of Ca(2+) from intracellular stores is a fundamental element of signaling pathways involved in regulation of vascular tone, proliferation, apoptosis, and gene expression. Studies of sea urchin eggs have led to the identification of three functionally distinct Ca(2+) signaling pathways triggered by IP3, cADPR, and NAADP. The coexistence and functional relevance of these distinct intracellular Ca(2+) release systems has only been described in a few mammalian cell types. The purpose of this study was to determine whether the IP3, cADPR, and NAADP Ca(2+) release systems coexist in smooth muscle cells (SMC) and to determine the specificity of these intracellular Ca(2+) release pathways. Microsomes were prepared from rat aortic SMC (VSMC) and were loaded with 45Ca(2+). cADPR, NAADP, and IP3 induced Ca(2+) release from VSMC microsomes in a dose-dependent fashion. Heparin blocked only IP3-mediated Ca(2+) release, whereas the ryanodine channel inhibitors 8-Br-cADPR and ruthenium red blocked only cADPR-induced Ca(2+) release. Nifedipine, an L-type Ca(2+) channel blocker, inhibited NAADP elicited Ca(2+) release, but had no effect on IP3- or cADPR-mediated Ca(2+) release. An increase in pH from 7.2 to 8.2 inhibited cADPR-mediated Ca(2+) release, but had no effect on IP3- or NAADP-induced Ca(2+) release. By RT-PCR, VSMC expressed ryanodine receptor types 1, 2, and 3. Ca(2+)-dependent binding of [3H]-ryanodine to VSMC microsomes was enhanced by the ryanodine receptor agonists 4-chloro-methyl-phenol (CMP) and caffeine, but was inhibited by ruthenium red and cADPR. We conclude that VSMC possess at least three functionally distinct pathways that promote intracellular Ca(2+) release. IP3-, cADPR-, and NAADP-induced intracellular Ca(2+) release may play a critical role in the maladaptive responses of VSMC to environmental stimuli that are characteristically associated with hypertension and/or atherogenesis.
Experimental Biology and Medicine 02/2002; 227(1):36-44. · 2.64 Impact Factor
