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Transforming growth factor (TGF)-[beta] is a central mediator in the progression of glomerulosclerosis, leading to accumulation of aberrant extracellular matrix proteins and inappropriate expression of smooth muscle [alpha]-actin in the kidney. Previously, we reported that disrupting the cytoskeleton diminished TGF-[beta]-stimulated type I collagen accumulation in human mesangial cells. As cytoskeletal signaling molecules, including the Rho-family GTPases, have been implicated in fibrogenesis, we sought to determine the respective roles of RhoA and Rac1 in HMC collagen I expression. TGF-[beta]1 activated both RhoA and Rac1 within 5 min of treatment, and this activation was dependent on the kinase activity of the type I TGF-[beta] receptor. TGF-[beta]1-stimulated induction of type I collagen mRNA expression and promoter activity was diminished by inhibiting Rac1 activity and was increased by a constitutively active Rac1 mutant, whereas inhibiting RhoA activity had no such effect. Rac1 activation required phosphatidylinositol-3-kinase (PI3K) activity. Furthermore, the PI3K antagonist, LY294002, reduced TGF-[beta]1-stimulated COL1A2 promoter activity and Rac1 activation. It also partially blocked active Rac1-stimulated collagen promoter activity, suggesting that PI3K activity contributes to both TGF-[beta] activation of Rac1 and signal propagation downstream of Rac1. Thus, while both Rac1 and RhoA are rapidly activated in response to TGF-[beta]1 in human mesangial cells, only Rac1 activation enhances events that contribute to mesangial cell collagen expression, through a positive feedback loop involving PI3K. kidney; fibrosis; Rho-GTPase doi: 10.1152/ajprenal.00345.2009.

Transforming growth factor-[beta] (TGF-[beta]) is closely associated with progressive renal fibrosis. Significant progress has been accomplished in determining the cellular signaling pathways that are activated by TGF-[beta]. This knowledge is being applied to glomerular mesangial cell models of extracellular matrix (ECM) accumulation. A central component of TGF-[beta]-stimulated mesangial cell fibrogenesis is the TGF-[beta] family-specific Smad signal transduction pathway. However, while Smads play an important role in collagen accumulation, recent findings indicate that cross talk among a variety of pathways is necessary for maximal stimulation of collagen expression. Further investigation of these multiple interactions will provide insight into possible ways to interrupt cellular mechanisms of glomerular fibrogenesis. transforming growth factor-[beta]; glomerulosclerosis; Smads

Hypoxia- inducible factors (HIFs) are transcription factors consisting of an oxygen-sensitive α-subunit binding to a stable β-subunit. HIFs regulate multiple signaling pathways that could contribute to fibrogenesis, supporting their potential role in hypoxia-mediated renal fibrosis. We previously reported that HIF-1 is upregulated and required for transforming growth factor (TGF)-β induction of collagen in renal tubular cells. Here, we performed in vitro and in vivo studies of potential glomerular crosstalk between TGF-β and normoxic HIF signaling. HIF-α has two major isoforms, HIF-1α and HIF-2α with different target gene sets. In cultured human mesangial cells, TGF-β1 treatment increased both HIF-1α and HIF-2α expression in normoxia. TGF-β1 did not increase HIF-1α/2α mRNA levels nor decrease the rate of protein degradation, suggesting that it enhances HIF-1α/2α expression through translation. TGF-β receptor (ALK5) kinase activity was required for increased, TGF-β-stimulated HIF-α expression in response to TGF-β, and inhibiting PI3-kinase markedly decreased HIF-α expression. Blocking HIF-1α/2α expression using siRNA decreased basal and TGF-β1-stimulated type I collagen expression, while overexpressing nondegradable HIF-α increased the collagen response, with HIF-2α being significantly more effective than HIF-1α. In adriamycin-induced mouse glomerulosclerosis, HIF-2α target genes were upregulated in sclerosing glomeruli. Taken together, our data demonstrate potential signaling interaction between TGF-β and HIFs to promote renal fibrogenesis in normoxia and suggest that the HIF-2α isoform is more important during glomerulosclerosis. [ABSTRACT FROM AUTHOR]

Transforming growth factor (TGF)-β is a major mediator of kidney fibrosis. In the pastdecade it was recognized that, besides canonical Smad signaling, many other signaling pathways participate in the process of TGF-β-induced fibrogenesis. One such pathway involves mammalian targetof rapamycin complex (mTORC)1. We recently reported that thehypoxia-inducible factor (HIF)-1 is essential for TGF-β-induced collagenexpression regardless of ambient oxygen tension. A modulatorof HIF expression other than oxygen tension is mTORC1. We thereforesought to evaluate a possible role for mTORC1 activity inTGF-β-induced fibrogenesis, mTORC1 activity was increased inhuman mesangial cells treated with TGF-β in a TGF-β receptordependentmanner. Short hairpin (sh)RNA to Smad3 decreased, whileoverexpression of Smad3 increased, the mTORC1 activity, suggestingthat TGF-β stimulation of mTORC1 also requires Smad3. Pretreatmentwith rapamycin or shRNA for a regulatory molecule of mTORC1, Raptor, reduced TGF-β-induced COL1A2-luc activity andcollagen I protein expression, mTORC1 inhibition also prevented theTGF-β-stimulated increase in both hypoxia-responsive element(HRE) activity and HIF-1α protein expression, while activation of mTORC1 by active Rheb increased basal but not TGF-β-inducedHRE activity, shRNA to Smad3 reduced HRE activity, while overexpressionof Smad3 increased HIF-1α protein expression and activityin an mTORC1-dependent manner. Lastly, overexpression ofHIF-1α bypassed the inhibitory effect of mTORC1 blockade oncollagen expression. These results suggest that Smad3/mTORC1interaction to promote HIF-1 expression is a key step in normoxickidney fibrogenesis. [ABSTRACT FROM AUTHOR]

Transforming growth factor (TGF)-β is a central mediator in the progression of glomerulosclerosis, leading to accumulation of aberrant extracellular matrix proteins and inappropriate expression of smooth muscle α-actin in the kidney. Previously, we reported that disrupting the cytoskeleton diminished TGF-β-stimulated type I collagen accumulation in human mesangial cells. As cytoskeletal signaling molecules, including the Rho-family GTPases, have been implicated in fibrogenesis, we sought to determine the respective roles of RhoA and Rac1 in HMC collagen I expression. TGF-β1 activated both RhoA and Rac1 within 5 min of treatment, and this activation was dependent on the kinase activity of the type I TGF-β receptor. TGF-β1-stimulated induction of type I collagen mRNA expression and promoter activity was diminished by inhibiting Rac1 activity and was increased by a constitutively active Rac1 mutant, whereas inhibiting RhoA activity had no such effect. Racl activation required phosphatidylinositol-3-kinase (P13K) activity. Furthermore, the P13K antagonist, LY294002, reduced TGF-β1-stimulated COL1A2 promoter activity and Rac1 activation. It also partially blocked active Rac1-stimulated collagen promoter activity, suggesting that P13K activity contributes to both TGF-β activation of Rac1 and signal propagation downstream of Rac1. Thus, while both Rac1 and RhoA are rapidly activated in response to TGF-β1 in human mesangial cells, only Rac1 activation enhances events that contribute to mesangial cell collagen expression, through a positive feedback loop involving P13K. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β (TGF-β) is closely associated with progressive renal fibrosis. Significant progress has been accomplished in determining the cellular signaling pathways that are activated by TGF-β. This knowledge is being applied to glomerular mesangial cell models of extracellular matrix (ECM) accumulation. A central component of TGF-β-stimulated mesangial cell fibrogenesis is the TGF-β family-specific Smad signal transduction pathway. However, while Smads play an important role in collagen accumulation, recent findings indicate that cross talk among a variety of pathways is necessary for maximal stimulation of collagen expression. Further investigation of these multiple interactions will provide insight into possible ways to interrupt cellular mechanisms of glomerular fibrogenesis. [ABSTRACT FROM AUTHOR]

Transforming growth factor (TGF)-beta is a central stimulus of the events leading to chronic progressive kidney disease, having been implicated in the regulation of cell proliferation, hypertrophy, apoptosis and fibrogenesis. The fact that it mediates these varied events suggests that multiple mechanisms play a role in determining the outcome of TGF-beta signaling. Regulation begins with the availability and activation of TGF-beta and continues through receptor expression and localization, control of the TGF-beta family-specific Smad signaling proteins, and interaction of the Smads with multiple signaling pathways extending into the nucleus. Studies of these mechanisms in kidney cells and in whole-animal experimental models, reviewed here, are beginning to provide insight into the role of TGF-beta in the pathogenesis of renal dysfunction and its potential treatment.

TGF-beta1 has been implicated in glomerular extracellular matrix accumulation, although the precise cellular mechanism(s) by which this occurs is not fully understood. The authors have previously shown that the Smad signaling pathway is present and functional in human glomerular mesangial cells and plays a role in activating type I collagen gene expression. It also was determined that TGF-beta1 activates ERK mitogen-activated protein kinase in mesangial cells to enhance Smad activation and collagen expression. Here, it was shown that TGF-beta1 rapidly induces cytoskeletal rearrangement in human mesangial cells, stimulating smooth muscle alpha-actin detection in stress fibers and promoting focal adhesion complex assembly and redistribution. Disrupting the actin cytoskeleton with cytochalasin D (Cyto D) selectively decreased basal and TGF-beta1-induced cell-layer collagen I and IV accumulation. The balance of matrix metalloproteinases (MMP) and inhibitors was altered by Cyto D or TGF-beta1 alone, increasing MMP activity, increasing MMP-1 expression, and decreasing tissue inhibitor of matrix metalloproteinase-2 expression. Cyto D also decreased basal and TGF-beta1-stimulated alpha1(I) collagen mRNA but did not inhibit TGF-beta-stimulated alpha1(IV) mRNA expression. A similar decrease in alpha1(I) mRNA expression caused by the actin polymerization inhibitor latrunculin B was partially blocked by the addition of jasplakinolide, which promotes actin assembly. The Rho-family GTPase inhibitor C. difficile toxin B or the Rho-associated kinase inhibitor Y-27632 also blocked TGF-beta1-stimulated alpha1(I) mRNA expression. Cytoskeletal disruption reduced Smad2 phosphorylation but had little effect on mRNA stability, TGF-beta receptor number, or receptor affinity. Thus, TGF-beta1-mediated collagen I accumulation is associated with cytoskeletal rearrangement and Rho-GTPase signaling.