Inositol-requiring enzyme 1α/X-box protein 1 pathway expression is impaired in pediatric cholestatic liver disease explants.

BACKGROUND: Increased intrahepatic bile acids cause endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) is activated to maintain homeostasis. UPR dysregulation, including the inositol-requiring enzyme 1α/X-box protein 1 (IRE1α/XBP1) pathway, is associated with adult liver diseases but has not been characterized in pediatric liver diseases. We evaluated hepatic UPR expression in pediatric cholestatic liver disease (CLD) explants and hypothesize that an inability to appropriately activate the hepatic IRE1α/XBP1 pathway is associated with the pathogenesis of CLD. METHODS: We evaluated 34 human liver explants, including: pediatric CLD (Alagille, ALGS, and progressive familial intrahepatic cholestasis, PFIC), pediatric non-cholestatic liver disease controls (autoimmune hepatitis, AIH), adult CLD, and normal controls. We performed RNA-seq, quantitative PCR, and western blotting to measure expression differences of the hepatic UPR and other signaling pathways. RESULTS: Pathway analysis demonstrated that the KEGG 'protein processing in ER' pathway was downregulated in pediatric CLD compared to normal controls. Pediatric CLD had decreased hepatic IRE1α/XBP1 pathway gene expression and decreased protein expression of phosphorylated IRE1α compared to normal controls. IRE1α/XBP1 pathway gene expression was also decreased in pediatric CLD compared to AIH disease controls. CONCLUSIONS: Pediatric CLD explants have decreased expression of the protective IRE1α/XBP1 pathway and down-regulated KEGG protein processing in the ER pathways. IRE1α/XBP1 pathway expression differences occur when compared to both normal and non-cholestatic disease controls. Attenuated expression of the IRE1α/XBP1 pathway is associated with cholestatic diseases and may be a target for future therapeutics.

Hepatic Deletion of X-Box Binding Protein 1 in FXR Null Mice Leads to Enhanced Liver Injury.

FXR regulates bile acid metabolism, and FXR null (Fxr-/-) mice have elevated bile acid levels and progressive liver injury. The inositol-requiring enzyme 1α/X-box binding protein 1 (XBP1) pathway is a protective unfolded protein response pathway activated in response to endoplasmic reticulum stress. Here, we sought to determine the role of the inositol-requiring enzyme 1α/XBP1 pathway in hepatic bile acid toxicity using the Fxr-/- mouse model. Western blotting and quantitative PCR analysis demonstrated that hepatic XBP1 and other unfolded protein response pathways were activated in 24-week-old Fxr-/- compared with 10-week-old Fxr-/- mice but not in WT mice. To further determine the role of the liver XBP1 activation in older Fxr-/- mice, we generated mice with whole-body FXR and liver-specific XBP1 double KO (DKO, Fxr-/-Xbp1LKO) and Fxr-/-Xbp1fl/fl single KO (SKO) mice and characterized the role of hepatic XBP1 in cholestatic liver injury. Histologic staining demonstrated increased liver injury and fibrosis in DKO compared with SKO mice. RNA sequencing revealed increased gene expression in apoptosis, inflammation, and cell proliferation pathways in DKO mice. The proapoptotic C/EBP-homologous protein pathway and cell cycle marker cyclin D1 were also activated in DKO mice. Furthermore, we found that total hepatic bile acid levels were similar between the two genotypes. At age 60 weeks, all DKO mice and no SKO mice spontaneously developed liver tumors. In conclusion, the hepatic XBP1 pathway is activated in older Fxr-/- mice and has a protective role. The potential interaction between XBP1 and FXR signaling may be important in modulating the hepatocellular cholestatic stress responses.

Mechanisms of liver injury in high fat sugar diet fed mice that lack hepatocyte X-box binding protein 1.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of liver diseases in the United States and can progress to cirrhosis, end-stage liver disease and need for liver transplantation. There are limited therapies for NAFLD, in part, due to incomplete understanding of the disease pathogenesis, which involves different cell populations in the liver. Endoplasmic reticulum stress and its adaptative unfolded protein response (UPR) signaling pathway have been implicated in the progression from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH). We have previously shown that mice lacking the UPR protein X-box binding protein 1 (XBP1) in the liver demonstrated enhanced liver injury and fibrosis in a high fat sugar (HFS) dietary model of NAFLD. In this study, to better understand the role of liver XBP1 in the pathobiology of NAFLD, we fed hepatocyte XBP1 deficient mice a HFS diet or chow and investigated UPR and other cell signaling pathways in hepatocytes, hepatic stellate cells and immune cells. We demonstrate that loss of XBP1 in hepatocytes increased inflammatory pathway expression and altered expression of the UPR signaling in hepatocytes and was associated with enhanced hepatic stellate cell activation after HFS feeding. We believe that a better understanding of liver cell-specific signaling in the pathogenesis of NASH may allow us to identify new therapeutic targets.

Hepatic X-Box Binding Protein 1 and Unfolded Protein Response Is Impaired in Weanling Mice With Resultant Hepatic Injury.

BACKGROUND AND AIMS: The unfolded protein response (UPR) is a coordinated cellular response to endoplasmic reticulum (ER) stress that functions to maintain cellular homeostasis. When ER stress is unresolved, the UPR can trigger apoptosis. Pathways within the UPR influence bile acid metabolism in adult animal models and adult human liver diseases, however, the UPR has not been studied in young animal models or pediatric liver diseases. In this study we sought to determine whether weanling age mice had altered UPR activation compared with adult mice, which could lead to increased bile acid-induced hepatic injury. APPROACH AND RESULTS: We demonstrate that after 7 days of cholic acid (CA) feeding to wild-type animals, weanling age mice have a 2-fold greater serum alanine aminotransferase (ALT) levels compared with adult mice, with increased hepatic apoptosis. Weanling mice fed CA have increased hepatic nuclear X-box binding protein 1 spliced (XBP1s) expression, but cannot increase expression of its protective downstream target's ER DNA J domain-containing protein 4 and ER degradation enhancing α-mannoside. In response to tunicamycin induced ER stress, young mice have blunted expression of several UPR pathways compared with adult mice. CA feeding to adult liver-specific XBP1 knockout (LS-XBP1-/- ) mice, which are unable to resolve hepatic ER stress, leads to increased serum ALT and CCAAT/enhancer binding homologous protein, a proapoptotic UPR molecule, expression to levels similar to CA-fed LS-XBP1-/- weanlings. CONCLUSIONS: Weanling mice have attenuated hepatic XBP1 signaling and impaired UPR activation with resultant increased susceptibility to bile acid-induced injury.

Farnesoid X receptor signaling activates the hepatic X-box binding protein 1 pathway in vitro and in mice.

Bile acids are endogenous ligands of the nuclear receptor, farnesoid X receptor (FXR), and pharmacological FXR modulators are under development for the treatment of several liver disorders. The inositol-requiring enzyme 1α/X-box binding protein 1 (IRE1α/XBP1) pathway of the unfolded protein response (UPR) is a protective cellular signaling pathway activated in response to endoplasmic reticulum (ER) stress. We investigated the role of FXR signaling in activation of the hepatic XBP1 pathway. Mice were treated with deoxycholic acid (DCA), cholestyramine, GW4064, or underwent bile duct ligation (BDL), and hepatic UPR activation was measured. Huh7-Ntcp and HepG2 cells were treated with FXR agonists, inhibitor, small interfering RNA (siRNA), or small heterodimer partner (SHP) siRNA to determine the mechanisms of IRE1α/XBP1 pathway activation. DCA feeding and BDL increased and cholestyramine decreased expression of hepatic XBP1 spliced (XBP1s). XBP1 pathway activation increased in Huh7-Ntcp and HepG2 cells treated with bile acids, 6α-ethyl-chenodeoxycholic acid (6-ECDCA) or GW4064. This effect decreased with FXR knockdown and treatment with the FXR inhibitor guggulsterone. FXR agonists increased XBP1 splicing and phosphorylated IRE1α (p-IRE1α) expression. Overexpression of SHP similarly increased XBP1 splicing, XBP1s, and p-IRE1α protein expression. SHP knockdown attenuated FXR agonist-induced XBP1s and p-IRE1α protein expression. Co-immunoprecipitation (Co-IP) assays demonstrate a physical interaction between overexpressed green fluorescent protein (GFP)-SHP and FLAG-IRE1α in HEK293T cells. Mice treated with GW4064 had increased, and FXR and SHP null mice had decreased, basal Xbp1s gene expression. CONCLUSION: FXR signaling activates the IRE1α/XBP1 pathway in vivo and in vitro. FXR pathway activation increases XBP1 splicing and enhances p-IRE1α expression. These effects are mediated, at least in part, by SHP. IRE1α/XBP1 pathway activation by bile acids and pharmacological FXR agonists may be protective during liver injury and may have therapeutic implications for liver diseases. (Hepatology 2018;68:304-316).

Hepatic deletion of X-box binding protein 1 impairs bile acid metabolism in mice.

The unfolded protein response (UPR) is an adaptive response to endoplasmic reticulum stress and the inositol-requiring enzyme 1α/X-box binding protein 1 (IRE1α/XBP1) pathway of the UPR is important in lipid metabolism. However, its role in bile acid metabolism remains unknown. We demonstrate that liver-specific Xbp1 knockout (LS-Xbp1-/-) mice had a 45% reduction in total bile acid pool. LS-Xbp1-/- mice had lower serum 7α-hydroxy-4-cholesten-3-one (C4) levels compared with Xbp1fl/fl mice, indicating reduced cholesterol 7α-hydroxylase (CYP7A1) synthetic activity. This occurred without reductions of hepatic CYP7A1 protein expression. Feeding LS-Xbp1-/- mice cholestyramine increased hepatic CYP7A1 protein expression to levels 2-fold and 8-fold greater than cholestyramine-fed and chow-fed Xbp1fl/fl mice, respectively. However, serum C4 levels remained unchanged and were lower than both groups of Xbp1fl/fl mice. In contrast, although feeding LS-Xbp1-/- mice cholesterol did not increase CYP7A1 expression, serum C4 levels increased significantly up to levels similar to chow-fed Xbp1fl/fl mice and the total bile acid pool normalized. In conclusion, loss of hepatic XBP1 decreased the bile acid pool and CYP7A1 synthetic activity. Cholesterol feeding, but not induction of CYP7A1 with cholestyramine, increased CYP7A1 synthetic activity and corrected the genotype-specific total bile acid pools. These data demonstrate a novel role of IRE1α/XBP1 regulating bile acid metabolism.

Epidermal growth factor inhibits transforming growth factor-ß-induced fibrogenic differentiation marker expression through ERK activation.

Transforming growth factor-ß (TGF-ß) signaling plays an important and complex role in renal fibrogenesis. The seemingly simple TGF-ß/Smad cascade is intensively regulated at several levels, including crosstalk with other signaling pathways. Epidermal growth factor (EGF) is a potent mitogen for epithelial cells and is elevated in diseased kidneys. In this study, we examined its effect on TGF-ß-induced fibrotic changes in human proximal tubular epithelial cells. Simultaneous treatment with EGF specifically inhibited basal and TGF-ß-induced type-I collagen and α-smooth muscle actin (αSMA) expression at both mRNA and protein levels. These effects were prevented by inhibition of either the EGF receptor kinase or its downstream MEK kinase but not by blockade of either the JNK or PI3K pathway. Overexpression of a constitutively active MEK1 construct mimicked the inhibitory effect of EGF. Further, EGF suppressed Smad transcriptional activities, as shown by reduced activation of ARE-luc and SBE-luc. Both reductions were prevented by MEK inhibition. However, EGF did not block Smad2 or Smad3 phosphorylation by TGF-ß, or Smad2/3 nuclear import. Finally EGF induced the phosphorylation and expression of TGIF, a known TGF-ß/Smad repressor. Both the phosphorylation and the induction were blocked by a MEK inhibitor. Overexpression of TGIF abolished TGF-ß-induced αSMA promoter activity. Together these results suggest that EGF inhibits two TGF-ß-stimulated markers of EMT through EGF receptor tyrosine kinase and downstream ERK activation, but not through PI3K or JNK. The inhibition results from effector mechanisms downstream of Smads, and most likely involves the transcriptional repressor, TGIF.

Hypoxia-inducible factor-2α and TGF-ß signaling interact to promote normoxic glomerular fibrogenesis.

Hypoxia-inducible factors (HIFs) are transcription factors consisting of an oxygen-sensitive α-subunit binding to a stable ß-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)-ß induction of collagen in renal tubular cells. Here, we performed in vitro and in vivo studies of potential glomerular crosstalk between TGF-ß 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-ß1 treatment increased both HIF-1α and HIF-2α expression in normoxia. TGF-ß1 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-ß receptor (ALK5) kinase activity was required for increased, TGF-ß-stimulated HIF-α expression in response to TGF-ß, and inhibiting PI3-kinase markedly decreased HIF-α expression. Blocking HIF-1α/2α expression using siRNA decreased basal and TGF-ß1-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-ß and HIFs to promote renal fibrogenesis in normoxia and suggest that the HIF-2α isoform is more important during glomerulosclerosis.

TGF-ß/Smad3 activates mammalian target of rapamycin complex-1 to promote collagen production by increasing HIF-1α expression.

Transforming growth factor (TGF)-ß is a major mediator of kidney fibrosis. In the past decade it was recognized that, besides canonical Smad signaling, many other signaling pathways participate in the process of TGF-ß-induced fibrogenesis. One such pathway involves mammalian target of rapamycin complex (mTORC)1. We recently reported that the hypoxia-inducible factor (HIF)-1 is essential for TGF-ß-induced collagen expression regardless of ambient oxygen tension. A modulator of HIF expression other than oxygen tension is mTORC1. We therefore sought to evaluate a possible role for mTORC1 activity in TGF-ß-induced fibrogenesis. mTORC1 activity was increased in human mesangial cells treated with TGF-ß in a TGF-ß receptor-dependent manner. Short hairpin (sh)RNA to Smad3 decreased, while overexpression of Smad3 increased, the mTORC1 activity, suggesting that TGF-ß stimulation of mTORC1 also requires Smad3. Pretreatment with rapamycin or shRNA for a regulatory molecule of mTORC1, Raptor, reduced TGF-ß-induced COL1A2-luc activity and collagen I protein expression. mTORC1 inhibition also prevented the TGF-ß-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-ß-induced HRE activity. shRNA to Smad3 reduced HRE activity, while overexpression of Smad3 increased HIF-1α protein expression and activity in an mTORC1-dependent manner. Lastly, overexpression of HIF-1α bypassed the inhibitory effect of mTORC1 blockade on collagen expression. These results suggest that Smad3/mTORC1 interaction to promote HIF-1 expression is a key step in normoxic kidney fibrogenesis.

Interdependence of HIF-1α and TGF-ß/Smad3 signaling in normoxic and hypoxic renal epithelial cell collagen expression.

Increasing evidence suggests that chronic kidney disease may develop following acute kidney injury and that this may be due, in part, to hypoxia-related phenomena. Hypoxia-inducible factor (HIF) is stabilized in hypoxic conditions and regulates multiple signaling pathways that could contribute to renal fibrosis. As transforming growth factor (TGF)-ß is known to mediate renal fibrosis, we proposed a profibrotic role for cross talk between the TGF-ß1 and HIF-1α signaling pathways in kidney cells. Hypoxic incubation increased HIF-1α protein expression in cultured human renal tubular epithelial cells and mouse embryonic fibroblasts. TGF-ß1 treatment further increased HIF-1α expression in cells treated with hypoxia and also increased HIF-1α in normoxic conditions. TGF-ß1 did not increase HIF-1α mRNA levels nor decrease the rate of protein degradation, suggesting that it enhances normoxic HIF-1α translation. TGF-ß receptor (ALK5) kinase activity was required for increased HIF-1α expression in response to TGF-ß1, but not to hypoxia. A dominant negative Smad3 decreased the TGF-ß-stimulated reporter activity of a HIF-1α-sensitive hypoxia response element. Conversely, a dominant negative HIF-1α construct decreased Smad-binding element promoter activity in response to TGF-ß. Finally, blocking HIF-1α transcription with a biochemical inhibitor, a dominant negative construct, or gene-specific knockdown decreased basal and TGF-ß1-stimulated type I collagen expression, while HIF-1α overexpression increased both. Taken together, our data demonstrate cooperation in signaling between Smad3 and HIF-1α and suggest a new paradigm in which HIF-1α is necessary for normoxic, TGF-ß1-stimulated renal cell fibrogenesis.

A conceptual framework for the molecular pathogenesis of progressive kidney disease.

The data regarding the pathogenesis of progressive kidney disease implicate cytokine effects, physiological factors, and myriad examples of relatively nonspecific cellular dysfunction. The sheer volume of information being generated on this topic threatens to overwhelm our efforts to understand progression in chronic kidney disease or to derive rational strategies to treat it. Here, a conceptual framework is offered for organizing and considering these data. Disease is initiated by an injury that evokes a tissue-specific cellular response. Subsequent structural repair may be effective, or the new structure may be sufficiently changed that it requires an adaptive physiological response. If this adaptation is not successful, subsequent cycles of misdirected repair or maladaptation may lead to progressive nephron loss. To illustrate how this framework can be used to organize our approach to disease pathogenesis, the role of cytokines in proteinuria and progressive glomerular disease is discussed. Finally, this theoretical framework is reconsidered to examine its implications for the diagnosis and treatment of clinical conditions. Application of this schema could have significant relevance to both research inquiry and clinical practice.

Rac1 promotes TGF-beta-stimulated mesangial cell type I collagen expression through a PI3K/Akt-dependent mechanism.

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-beta1 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-beta1-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-beta1-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-beta1 in human mesangial cells, only Rac1 activation enhances events that contribute to mesangial cell collagen expression, through a positive feedback loop involving PI3K.

Role of SARA (SMAD anchor for receptor activation) in maintenance of epithelial cell phenotype.

By inducing epithelial-to-mesenchymal transition (EMT), transforming growth factor-beta (TGF-beta) promotes cancer progression and fibrosis. Here we show that expression of the TGF-beta receptor-associated protein, SARA (Smad anchor for receptor activation), decreases within 72 h of exposure to TGF-beta and that this decline is both required and sufficient for the induction of several markers of EMT. It has been suggested recently that expression of the TGF-beta signaling mediators, Smad2 and Smad3, may have different functional effects, with Smad2 loss being more permissive for EMT progression. We find that the loss of SARA expression leads to a concomitant decrease in Smad2 expression and a disruption of Smad2-specific transcriptional activity, with no effect on Smad3 signaling or expression. Further, the effects of inducing the loss of Smad2 mimic those of the loss of SARA, enhancing expression of the EMT marker, smooth muscle alpha-actin. Smad2 mRNA levels are not affected by the loss of SARA. However, the ubiquitination of Smad2 is increased in SARA-deficient cells. We therefore examined the E3 ubiquitin ligase Smurf2 and found that although Smurf2 expression was unaltered in SARA-deficient cells, the interaction of Smad2 and Smurf2 was enhanced. These results describe a significant role for SARA in regulating cell phenotype and suggest that its effects are mediated through modification of the balance between Smad2 and Smad3 signaling. In part, this is achieved by enhancing the association of Smad2 with Smurf2, leading to Smad2 degradation.

TGF-beta signal transduction in chronic kidney disease.

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.

Hypoxia regulates PDGF-B interactions between glomerular capillary endothelial and mesangial cells.

BACKGROUND: Platelet-derived growth factor (PDGF)-B regulates mesangial cell and vessel development during embryogenesis, and contributes to the pathogenesis of adult renal and vascular diseases. Endothelial cell PDGF-B exerts paracrine effects on mesangial cells, but its regulation is not well defined. We examined the impact of hypoxia on PDGF-B-mediated interactions between glomerular endothelial and mesangial cells, a condition of potential relevance in developing, and diseased adult, kidneys. METHODS: Glomerular endothelial or mesangial cells were subjected to hypoxia and responses compared to normoxic cells. Endothelial PDGF-B was studied by Northern and Western analysis. Mesangial proliferative responses to PDGF-B were assessed by (3)H-thymidine incorporation, and migration by a modified Boyden chamber assay. Hypoxia-induced changes in receptor specific binding capacity were studied by saturation binding assays. RESULTS: Hypoxia stimulated increases in endothelial PDGF-B mRNA and protein. In normoxic mesangial cells, PDGF-B stimulated dose-dependent proliferation, but the proliferative response of hypoxic cells was two to three times greater. Exogenous PDGF-B also caused prompter migration in hypoxic mesangial cells. Mesangial cells were treated with endothelial cell-conditioned medium. More cells migrated when hypoxic cells were stimulated with hypoxic conditioned medium, than when normoxic cells were stimulated with normoxic conditioned medium. Preincubating conditioned medium with PDGF-B neutralizing antibody greatly decreased the chemoattractant activity. Binding studies demonstrated increased specific binding capacity in hypoxic cells. CONCLUSION: Hypoxia enhances PDGF-B paracrine interactions between glomerular endothelial and mesangial cells. These hypoxia-regulated interactions may be important during glomerulogenesis in fetal life and during the pathogenesis of adult glomerular disease.

Cytoskeletal rearrangement and signal transduction in TGF-beta1-stimulated mesangial cell collagen accumulation.

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.

TGF-beta signal transduction and mesangial cell fibrogenesis.

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.