Mark E.C. Dockrell

The differential role of Smad2 and Smad3 in the regulation of pro-fibrotic TGFβ1 responses in human proximal-tubule epithelial cells

In chronic renal diseases, progressive loss of renal function correlates with advancing tubulo-interstitial fibrosis. TGFbeta1-Smad (transforming growth factor-beta1-Sma and Mad protein) signalling plays an important role in the development of renal tubulo-interstitial fibrosis. Secretion of CTGF (connective-tissue growth factor; CCN2) by PTECs (proximal-tubule epithelial cells) and EMT (epithelial-mesenchymal transdifferentiation) of PTECs to myofibroblasts in response to TGFbeta are critical Smad-dependent events in the development of tubulo-interstitial fibrosis. In the present study we have investigated the distinct contributions of Smad2 and Smad3 to expression of CTGF, E-cadherin, alpha-SMA (alpha-smooth-muscle actin) and MMP-2 (matrix-metalloproteinase-2) in response to TGFbeta1 treatment in an in vitro culture model of HKC-8 (transformed human PTECs). RNA interference was used to achieve selective and specific knockdown of Smad2 and Smad3. Cellular E-cadherin, alpha-SMA as well as secreted CTGF and MMP-2 were assessed by Western immunoblotting. TGFbeta1 treatment induced a fibrotic phenotype with increased expression of CTGF, MMP-2 and alpha-SMA, and decreased expression of E-cadherin. TGFbeta1-induced increases in CTGF and decreases in E-cadherin expression were Smad3-dependent, whereas increases in MMP-2 expression were Smad2-dependent. Increases in alpha-SMA expression were dependent on both Smad2 and Smad3 and were abolished by combined knockdown of both Smad2 and Smad3. In conclusion, we have demonstrated distinct roles for Smad2 and Smad3 in TGFbeta1-induced CTGF expression and markers of EMT in human PTECs. This can be of therapeutic value in designing targeted anti-fibrotic therapies for tubulo-interstitial fibrosis.

Connective tissue growth factor-(CTGF, CCN2)--a marker, mediator and therapeutic target for renal fibrosis.

Connective tissue growth factor (CTGF, CCN2) is a key mediator of tissue fibrosis. CCN2 plays an important role in the development of glomerular and tubulointerstitial fibrosis in progressive kidney diseases. In this review, we discuss the biology of CCN2 with a focus on the regulation of CCN2 gene, cellular mechanisms of profibrotic CCN2 effects and the current in vivo and in vitro evidence for the role of CCN2 in the development of renal fibrosis. We also discuss the therapeutic potential of targeting CCN2 for the treatment of renal fibrosis.

TGF-β1-Induced Connective Tissue Growth Factor (CCN2) Expression in Human Renal Proximal Tubule Epithelial Cells Requires Ras/MEK/ERK and Smad Signalling

Background: Connective tissue growth factor (CTGF, CCN2) plays a fundamental role in the development of tissue fibrosis by stimulating matrix deposition and mediating many of the pro-fibrotic effects of transforming growth factor (TGF)-β. CCN2 induction by TGF-β in renal proximal tubule epithelial cells (PTECs) is likely to play an important role in the development of tubulointerstitial fibrosis. In this study, we investigated the induction of CCN2 by TGF-β1 and the possible mechanisms of this induction in human PTECs. Methods: Experiments were performed on primary and transformed (human kidney cell (HKC)-clone 8) human PTECs. Induction of CCN2 in response to TGF-β1 was studied at the gene promoter level by reporter gene assay, mRNA by semi-quantitative RT-PCR and protein by immunoblotting. While chemical inhibitors were used to assess the role of Ras/MEK/ERK1,2 signalling, an HKC cell line over-expressing Smad7 was used to assess the role of Smad signalling in induction of CCN2 by TGF-β1. Results: TGF-β1 induced CCN2 promoter activity, mRNA and protein in human PTECs. TGF-β1-dependent CCN2 promoter activity was reduced by inhibiting Ras and MEK activation. MEK inhibition also resulted in inhibition of the TGF-β1-induced secreted CCN2 protein. There was no significant increase in CCN2 gene promoter activity or protein by TGF-β1 in Smad7 over-expressing HKCs. Conclusions: TGF-β1 induces the expression of CCN2 in human PTECs. This induction is dependent on Ras/MEK/ERK and Smad signalling. Inhibiting TGF-β induced CCN2 by targeting Smad and/or Ras/MEK/ERK1,2 signalling pathways could be of therapeutic value in renal fibrosis.

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

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.

Primary Culture of Human Renal Proximal Tubule Epithelial Cells and Interstitial Fibroblasts

Renal physiology and pathology are complex systems that are best studied in whole living organisms. This, however, is often restricted by our desire to limit the number of animal experiments undertaken and to replace them with relevant in vitro models that can be used as surrogates for the system under test. Primary culture cells are derived directly from the relevant tissue and therefore correlate more closely with the system under examination. Although the tissue of origin is not always readily available for culture and cells may quickly change their phenotype after only a few passages, they can be used in many circumstances to validate results obtained from closely related cell lines and to confirm vital protein expression patterns. This chapter outlines methods by which proximal tubular epithelial cells and renal interstitial fibroblasts can be isolated and characterized from human renal nephrectomy tissue.

TGF-β activates ERK5 in human renal epithelial cells

The role of the MAP kinase, extracellular signal-regulated kinase 5 (ERK5) remains unknown, however it is involved in cell differentiation and survival as highlighted by the embryonic lethality of the ERK5 knockout. ERK5 can be activated by growth factors and other extracellular signals. TGF-beta, a powerful controller of epithelial cell phenotype, is known to activate the MAP kinase, ERK1/2 however its effect on ERK5 remains unknown. This study demonstrates, fort the first time, ERK5 activation by TGF-beta, observed in both transformed and primary adult human PTEC; activation required ALK-5 receptor activity. In addition this work demonstrates expression of myocyte enhancer factor-2 (MEF2C) by PTEC and that TGF-beta increased the association of MEK5 with phospho-ERK5 and MEF2C. ERK5 activation by either TGF-beta or epidermal growth factor (EGF) was also inhibited by the p38 MAP kinase inhibitor, SB-202190.

The Regulation of TGFβ1 Induced Fibronectin EDA Exon Alternative Splicing in Human Renal Proximal Tubule Epithelial Cells

The EDA+ splice variant of fibronectin (Fn) is an early and important component of the extracellular matrix in renal fibrosis. In this work, we investigate cellular mechanisms of EDA+Fn production in human primary proximal tubule epithelial cells (PTECs). TGFβ1‐induced EDA+Fn production was assessed by immunocytochemistry, PCR, and Western blotting. SRp40 knockdown was achieved by siRNA. The role of the PI3 kinase‐AKT signalling and splicing regulatory protein SRp40 in the production of EDA+Fn was studied by using the chemical inhibitor LY294002 and siRNA targeted to SRp40 respectively. Interaction between PI3 kinase‐AKT signalling and SRp40 were assessed by immunofluorescence and immunoprecipitation. To assess the specificity of SRp40 in regulating the splicing of EDA+ exon, we studied the effect of SRp40 knockdown on TGFβ1 induced splicing of FGF receptor 2. Primary human PTECs expressed EDA+ and EDA‐ Fn. TGFβ1 treatment resulted in increases in the production and deposition of EDA+ Fn as well as an increase in the ratio of EDA+/EDA‐ Fn mRNA. The TGFβ1 induced EDA+ production was dependent on PI3 kinase‐AKT signalling and SRp40 expression. Immunoprecipitation experiments demonstrated direct binding between AKT and SRp40 with an increase in the amount of SRp40 bound to AKT upon TGFβ1 treatment. TGFβ1 treatment resulted in reduction in the FGF receptor2 IIIb splice variant which was unaffected by SRp40 knockdown. In this work, we have presented the first evidence for the regulation of Fn pre‐mRNA splicing by PI3 kinase‐AKT signalling and SRp40 in human PTECs. Targeting the splicing of Fn pre‐mRNA to skip the EDA exon is an attractive option to combat fibrosis. J. Cell. Physiol. 230: 286–295, 2015.