The TGFβ1-Induced Fibronectin in Human Renal Proximal Tubular Epithelial Cells Is p38 MAP Kinase Dependent and Smad Independent

Abstract

Background/Aim: Transforming growth factor beta 1 (TGFβ1) is a fibrokine implicated in the progression of renal fibrosis. Following TGFβ1 receptor activation, a number of signalling pathways are stimulated. This study investigates the role of p38 mitogen-activated protein (MAP) kinase and Smad pathways in the TGFβ1-induced fibronectin (FN) production. Methods: Transformed human proximal tubular epithelial cells of the line HKC were used. Secreted FN was analyzed by enzyme-linked immunosorbent assay and Smad proteins by Western blotting. Chemical inhibitors were used to study the role of p38 MAP kinase and the TGFβ receptor ALK5. The Smad pathway was studied using a cell line overexpressing Smad7 and small interfering RNAs (siRNA). The FN mRNA expression was assessed by reverse transcription-polymerase chain reaction. Results: TGFβ1 produced a significant increase in FN secretion in both HKC and Smad7-HKC cells, and the p38 MAP kinase inhibitor SB202190 markedly reduced this (n = 3, p < 0.05 and p < 0.01). ALK5 inhibition also reduced the TGFβ1-induced FN (n = 3, p < 0.05). Smad knockdown using the siRNA did not reduce the TGFβ1-induced FN secretion. TGFβ1 induced FN mRNA expression in HKC cells, and SB202190 decreased this induction (n = 5, p < 0.05). Conclusions: These results suggest that TGFβ1-induced FN production in HKC cells is p38 MAP kinase dependent and Smad independent. Targeting p38 MAP kinase may be of therapeutic value in renal fibrosis.

Transforming Growth Factor-β Stimulates Intestinal Epithelial Focal Adhesion Kinase Synthesis via Smad- and p38-Dependent Mechanisms

“…81 TGF-␤ enhanced fibronectin mRNA in transformed human proximal tubular epithelial HKC cells, an effect that was blocked by both the p38 inhibitor, SB202190, and the ALK5 inhibitor, SB431542 but not by Smad knockdown. 48 On the other hand, in human pancreatic adenocarcinoma PANC-1 cells, TGF-␤ regulation of biglycan gene expression via p38 activation also required Smad activation. 49 Results of the luciferase assays indicate that inhibition of either p38 or Smad signaling can abolish the TGF-␤-induced stimulation of FAK expression, suggesting that each pathway plays an individual role at the level of FAK transcription.…”

“…We then sought to determine whether activation of p38, a MAP kinase known to mediate the transcriptional effects of TGF-␤ in a number of cell types, [47][48][49] may play a role in FAK induction in our cells. p38 phosphorylation was significantly enhanced (223 Ϯ 51%, P ϭ 0.05, n ϭ 4) after 1 hour of exposure to TGF-␤, an effect that was totally abolished by TGF-␤ receptor inhibition ( Figure 9A).…”

The characteristics of Ishikawa endometrial cancer cells are modified by substrate topography with cell-like features and the polymer surface

“…In this study, the effect of blocking on phosphorylation of integrin was not evaluated. Secreted ECM components from nonstromal (Ishikawa) cells [38][39][40][41][42] or soluble factors 3,43 may be partly responsible for the interface effect. Physical signaling is complex, and a number of relevant signaling molecules and transcription factors have been identified, 43,44 but their roles are not fully defined.…”

IHG-1 Amplifies TGF-β1 Signaling and Is Increased in Renal Fibrosis

“…IHG-1 expression also increased TGF-␤1-induced fibronectin expression, which has been reported to be induced by both Smad-dependent and Smad-independent pathways. [31][32][33][34] Our data suggest that IHG-1 amplifies TGF-␤1-induced fibronectin expression by a Smad-dependent mechanism; however, there is also the possibility that this induction is Smad dependent and indirect, mediated, for instance, by CTGF.…”

“…It is proposed that induction of the profibrotic mediator connective tissue growth factor (CTGF) by TGF-␤1 is Smad3 dependent, 30 whereas TGF-␤1-induced fibronectin expression may be both Smad dependent and Smad independent. [31][32][33][34] Overexpression of IHG-1 after transduction of HK-2 cells resulted in increased levels of both CTGF (2.8-fold; Figure 5B) and fi- bronectin (2.6-fold; Figure 5C) protein after stimulation with TGF-␤1 as compared with mock-transduced cells (CTGF 1.56-fold, fibronectin 1.48-fold). These data suggest that IHG-1 facilitated increases and prolonged phosphorylation of Smad3, enhancing transcriptional responses to TGF-␤1 along a fibrotic pathway.…”