TGF-beta receptor-binding proteins: complex interactions.

Members of the Smad protein family are fundamental downstream mediators of TGF-beta signals. However, the basic, linear Smad signaling pathway is unlikely to be the sole contributor to the plethora of cell type-specific TGF-beta responses. Investigators have identified a number of molecules that interact with the TGF-beta receptors (TbetaRs) and may explain, at least in part, the tight regulation of TGF-beta effects. Understanding these TbetaR-interacting molecules is thus a matter of great potential significance for elucidating TGF-beta-family signal transduction. The present article reviews our current understanding of the roles and mechanisms of action of this relatively understudied group of molecules.

Cell phenotype-specific down-regulation of Smad3 involves decreased gene activation as well as protein degradation.

Signaling by transforming growth factor-beta (TGF-beta), a regulator of several biological processes, including renal fibrosis, is mediated, in part, by the Smad proteins. Tight control of Smad level and activity is critical for proper TGF-beta biological functions. Here, we have investigated the mechanisms involved in regulating Smad3 expression. In human glomerular mesangial cells, Smad3 protein levels were specifically reduced by 24 h of TGF-beta1 treatment, whereas Smad2 and Smad4 levels were not. TGF-beta1 increased endogenous Smad3 ubiquitination, and proteasome inhibitor treatment blocked TGF-beta1-mediated Smad3 down-regulation resulting in accumulation of ubiquitinated Smad3. These data support the concept that Smad3 down-regulation occurs via degradation by the ubiquitin/proteasome machinery. However, changes in Smad3 protein levels were also paralleled by changes in Smad3 mRNA expression. TGF-beta1 did not decrease Smad3 mRNA stability, but it significantly inhibited Smad3 promoter activity. In renal tubular epithelial cells, decreased Smad3 levels were observed only after exposure to TGF-beta1 for longer time periods (5-7 days) that paralleled epithelial-to-mesenchymal transition, as determined by increased expression of smooth muscle alpha-actin and decreased expression of E-cadherin. Decline in Smad3 expression also occurred in kidneys after unilateral ureteral obstruction, a model of tubulointerstitial fibrosis associated with TGF-beta up-regulation and epithelial-to-mesenchymal transition. Our data show for the first time that TGF-beta1 modulates the expression of a receptor-activated Smad at both the protein and transcriptional level. Smad3 down-regulation could represent a feedback loop controlling TGF-beta signaling in a cell phenotype-specific manner.

MAP-kinase activity necessary for TGFbeta1-stimulated mesangial cell type I collagen expression requires adhesion-dependent phosphorylation of FAK tyrosine 397.

The signals mediating transforming growth factor beta (TGFbeta)-stimulated kidney fibrogenesis are poorly understood. We previously reported TGFbeta-stimulated, Smad-mediated collagen production by human kidney mesangial cells, and that ERK MAP kinase activity optimizes collagen expression and enhances phosphorylation of the Smad3 linker region. Furthermore, we showed that disrupting cytoskeletal integrity decreases type I collagen production. Focal adhesion kinase (FAK, PTK2) activity could integrate these findings. Adhesion-dependent FAK Y397 phosphorylation was detected basally, whereas FAK Y925 phosphorylation was TGFbeta1-dependent. By immunocytochemistry, TGFbeta1 stimulated the merging of phosphorylated FAK with the ends of thickening stress fibers. Cells cultured on poly-L-lysine (pLL) to promote integrin-independent attachment spread less than those on control substrate and failed to demonstrate focal adhesion (FA) engagement with F-actin. FAK Y397 phosphorylation and ERK activity were also decreased under these conditions. In cells with decreased FAK Y397 phosphorylation from either plating on pLL or overexpressing a FAK Y397F point mutant, serine phosphorylation of the Smad linker region, but not of the C-terminus, was reduced. Y397F and Y925F FAK point mutants inhibited TGFbeta-induced Elk-Gal activity, but only the Y397F mutant inhibited TGFbeta-stimulated collagen-promoter activity. The inhibition by the Y397F mutant or by culture on pLL was prevented by co-transfection of constitutively active ERK MAP kinase kinase (MEK), suggesting that FAK Y397 phosphorylation promotes collagen expression via ERK MAP kinase activity. Finally, Y397 FAK phosphorylation, and both C-terminal and linker-region Smad3 phosphorylation were detected in murine TGFbeta-dependent kidney fibrosis. Together, these data demonstrate adhesion-dependent FAK phosphorylation promoting TGFbeta-induced responses to regulate collagen production.

The role of internalization in transforming growth factor beta1-induced Smad2 association with Smad anchor for receptor activation (SARA) and Smad2-dependent signaling in human mesangial cells.

Recent data investigating the role of the Smad anchor for receptor activation (SARA) in TGF-beta signaling have suggested that it has a crucial function in both aiding the recruitment of Smad to the TGF-beta receptor, and ensuring appropriate subcellular localization of the activated receptor-bound complex. The FYVE domain in SARA directs its localization to early endosomal compartments where it can interact with both the TGF-beta receptors and Smads. However, the necessity of endocytosis in the TGF-beta response remains controversial. We sought to examine the role of internalization in TGF-beta/Smad signaling in human kidney mesangial cells. Using co-immunoprecipitation studies, we show that endogenous Smad2 interacts with SARA after TGF-beta1 stimulation. Inhibition of clathrin-mediated internalization only slightly affects TGF-beta1-stimulated association between SARA and Smad2, Smad2 phosphorylation, or Smad2 interaction with Smad4. However, endocytosis inhibition decreases TGF-beta1-induced Smad2 nuclear translocation and thus abrogates Smad2-dependent transcriptional responses. The TGF-beta1-stimulated association between SARA and Smad2 peaks at 30 min followed by separation of the complex components. However, under conditions of inhibited endocytosis, Smad2 remains bound to SARA for at least 6 h without a significant decline in associated levels. This lack of complex dissociation correlates with a lack of Smad2 nuclear accumulation and reduction of Smad2-dependent ARE-Luc reporter activity. Our data therefore suggest that endocytosis plays a critical role in TGF-beta signaling in mesangial cells, and that internalization enhances the dissociation of Smad2 from the TGF-beta receptor-SARA complex, allowing Smad2 to accumulate in the nucleus and modulate target gene transcription.

Smad3 and PKCdelta mediate TGF-beta1-induced collagen I expression in human mesangial cells.

Transforming growth factor (TGF)-beta has been associated with fibrogenesis in clinical studies and animal models. We previously showed that Smad3 promotes COL1A2 gene activation by TGF-beta1 in human mesangial cells. In addition to the Smad pathway, it has been suggested that TGF-beta1 could also activate more classical growth factor signaling. Here, we report that protein kinase C (PKC)delta plays a role in TGF-beta1-stimulated collagen I production. In an in vitro kinase assay, TGF-beta1 treatment specifically increased mesangial cell PKCdelta activity in a time-dependent manner. Translocation to the membrane was detected by immunocytochemistry and immunoblot, suggesting activation of PKCdelta by TGF-beta1. Inhibition of PKCdelta by rottlerin decreased basal and TGF-beta1-stimulated collagen I production, mRNA expression, and COL1A2 promoter activity, whereas blockade of conventional PKCs by Gö 6976 had little or no effect. In a Gal4-LUC assay system, inhibition of PKCdelta abolished TGF-beta1-induced transcriptional activity of Gal4-Smad3 and Gal4-Smad4(266-552). Overexpression of Smad3 or Smad3D, in which the three COOH-terminal serine phosphoacceptor residues have been mutated, increased activity of the SBE-LUC construct, containing four DNA binding sites for Smad3 and Smad4. This induction was blocked by PKCdelta inhibition, suggesting that rottlerin decreased Smad3 transcriptional activity independently of COOH-terminal serine phosphorylation. Blockade of PKCdelta abolished ligand-independent and ligand-dependent stimulation of COL1A2 promoter activity by Smad3. These data indicate that PKCdelta is activated by TGF-beta1 in human mesangial cells. TGF-beta1-stimulated PKCdelta activity positively regulates Smad transcriptional activity and is required for COL1A2 gene transcription. Thus cross talk among multiple signaling pathways likely contributes to the pathogenesis of glomerular matrix accumulation.

The phosphatidylinositol 3-kinase/Akt pathway enhances Smad3-stimulated mesangial cell collagen I expression in response to transforming growth factor-beta1.

Transforming growth factor (TGF)-beta has been associated with renal glomerular matrix accumulation. We previously showed that Smad3 promotes COL1A2 gene activation by TGF-beta1 in human glomerular mesangial cells. Here, we report that the PI3K/Akt pathway also plays a role in TGF-beta1-increased collagen I expression. TGF-beta1 stimulates the activity of phosphoinositide-dependent kinase (PDK)-1, a downstream target of PI3K, starting at 1 min. Akt, a kinase downstream of PDK-1, is phosphorylated and concentrates in the membrane fraction within 5 min of TGF-beta1 treatment. The PI3K inhibitor LY294002 decreases TGF-beta1-stimulated alpha1(I) and alpha2(I) collagen mRNA expression. Similarly, LY294002 or an Akt dominant negative construct blocks TGF-beta1 induction of COL1A2 promoter activity. However, PI3K stimulation alone is not sufficient to increase collagen I expression, since neither a constitutively active p110 PI3K construct nor PDGF, which induces Akt phosphorylation, is able to stimulate COL1A2 promoter activity or mRNA expression, respectively. LY294002 inhibits stimulation of COL1A2 promoter activity by Smad3. In a Gal4-LUC assay system, blockade of the PI3K pathway significantly decreases TGF-beta1-induced transcriptional activity of Gal4-Smad3. Activity of SBE-LUC, a Smad3/4-responsive construct, is stimulated by over-expression of Smad3 or Smad3D, in which the three C-terminal serine phospho-acceptor residues are mutated. This induction is blocked by LY294002, suggesting that inhibition of the PI3K pathway decreases Smad3 transcriptional activity independently of C-terminal serine phosphorylation. However, TGF-beta1-induced total serine phosphorylation of Smad3 is decreased by LY294002, suggesting that Smad3 is phosphorylated by the PI3K pathway at serine residues other than the direct TGF-beta receptor I target site. Thus, although the PI3K-PDK1-Akt pathway alone is insufficient to stimulate COL1A2 gene transcription, its activation by TGF-beta1 enhances Smad3 transcriptional activity leading to increased collagen I expression in human mesangial cells. This cross-talk between the Smad and PI3K pathways likely contributes to TGF-beta1 induction of glomerular scarring.

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.