Anne Christine Poncelet

Sp1 and Smad Proteins Cooperate to Mediate Transforming Growth Factor-β1-induced α2(I) Collagen Expression in Human Glomerular Mesangial Cells

The mechanism(s) by which Smads mediate and modulate the transforming growth factor (TGF)-β signal transduction pathway in fibrogenesis are not well characterized. We previously showed that Smad3 promotes α2(I) collagen gene (COL1A2) activation in human glomerular mesangial cells, potentially contributing to glomerulosclerosis. Here, we report that Sp1 binding is necessary for TGF-β1-induced type I collagen mRNA expression. Deletion of three Sp1 sites (GC box) between −376 and −268 or mutation of a CAGA box at −268/−260 inhibited TGF-β1-induced α2(I) collagen promoter activity. TGF-β1 inducibility was also blocked by a Smad3 dominant negative mutant. Chemical inhibition of Sp1 binding with mithramycin A, or deletion of the GC boxes, inhibited COL1A2 activation by Smad3, suggesting cooperation between Smad3 and Sp1 in the TGF-β1 response. Electrophoretic mobility shift assay showed that Sp1 and Smads form complexes with −283/−250 promoter sequences. Coimmunoprecipitation experiments demonstrate that endogenous Sp1, Smad3, and Smad4 form complexes in mesangial cells. In a Gal4-LUC reporter assay system, Sp1 stimulated the TGF-β1-induced transcriptional activity of Gal4-Smad3, Gal4-Smad4 (266), or both. Using the transactivation domain B of Sp1 fused to the Gal4 DNA binding domain, we show that, in our system, the transcriptional activity of this Sp1 domain is not regulated by TGF-β1, but it becomes responsive to this factor when Smad3 is coexpressed. Finally, combined Sp1 and Smad3 overexpression induces marked ligand-independent and ligand-dependent promoter activity of COL1A2. Thus, Sp1 and Smad proteins form complexes and their synergy plays an important role in mediating TGF-β1-induced α2(I) collagen expression in human mesangial cells.

The Phosphatidylinositol 3-Kinase/Akt Pathway Enhances Smad3-stimulated Mesangial Cell Collagen I Expression in Response to Transforming Growth Factor-β1

Transforming growth factor (TGF)-β has been associated with renal glomerular matrix accumulation. We previously showed that Smad3 promotes COL1A2 gene activation by TGF-β1 in human glomerular mesangial cells. Here, we report that the PI3K/Akt pathway also plays a role in TGF-β1-increased collagen I expression. TGF-β1 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-β1 treatment. The PI3K inhibitor LY294002 decreases TGF-β1-stimulated α1(I) and α2(I) collagen mRNA expression. Similarly, LY294002 or an Akt dominant negative construct blocks TGF-β1 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-β1-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-β1-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-β receptor I target site. Thus, although the PI3K-PDK1-Akt pathway alone is insufficient to stimulate COL1A2 gene transcription, its activation by TGF-β1 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-β1 induction of glomerular scarring.

MAP-kinase activity necessary for TGFβ1-stimulated mesangial cell type I collagen expression requires adhesion-dependent phosphorylation of FAK tyrosine 397

The signals mediating transforming growth factor β (TGFβ)-stimulated kidney fibrogenesis are poorly understood. We previously reported TGFβ-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 TGFβ1-dependent. By immunocytochemistry, TGFβ1 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 TGFβ-induced Elk-Gal activity, but only the Y397F mutant inhibited TGFβ-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 TGFβ-dependent kidney fibrosis. Together, these data demonstrate adhesion-dependent FAK phosphorylation promoting TGFβ-induced responses to regulate collagen production.

Cell Phenotype-specific Down-regulation of Smad3 Involves Decreased Gene Activation as Well as Protein Degradation

Signaling by transforming growth factor-β (TGF-β), 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-β 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-β1 treatment, whereas Smad2 and Smad4 levels were not. TGF-β1 increased endogenous Smad3 ubiquitination, and proteasome inhibitor treatment blocked TGF-β1-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-β1 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-β1 for longer time periods (5–7 days) that paralleled epithelial-to-mesenchymal transition, as determined by increased expression of smooth muscle α-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-β up-regulation and epithelial-to-mesenchymal transition. Our data show for the first time that TGF-β1 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-β signaling in a cell phenotype-specific manner.