Fluid shear stress-induced TGF-β/ALK5 signaling in renal epithelial cells is modulated by MEK1/2

Abstract

Renal tubular epithelial cells are exposed to mechanical forces due to fluid flow shear stress within the lumen of the nephron. These cells respond by activation of mechano-sensors located at the plasma membrane or the primary cilium, having crucial roles in maintenance of cellular homeostasis and signaling. In this paper, we applied fluid shear stress to study TGF-β signaling in renal epithelial cells with and without expression of the Pkd1-gene, encoding a mechano-sensor mutated in polycystic kidney disease. TGF-β signaling modulates cell proliferation, differentiation, apoptosis, and fibrotic deposition, cellular programs that are altered in renal cystic epithelia. SMAD2/3-mediated signaling was activated by fluid flow, both in wild-type and Pkd1 −/− cells. This was characterized by phosphorylation and nuclear accumulation of p-SMAD2/3, as well as altered expression of downstream target genes and epithelial-to-mesenchymal transition markers. This response was still present after cilia ablation. An inhibitor of upstream type-I-receptors, ALK4/ALK5/ALK7, as well as TGF-β-neutralizing antibodies effectively blocked SMAD2/3 activity. In contrast, an activin-ligand trap was ineffective, indicating that increased autocrine TGF-β signaling is involved. To study potential involvement of MAPK/ERK signaling, cells were treated with a MEK1/2 inhibitor. Surprisingly, fluid flow-induced expression of most SMAD2/3 targets was further enhanced upon MEK inhibition. We conclude that fluid shear stress induces autocrine TGF-β/ALK5-induced target gene expression in renal epithelial cells, which is partially restrained by MEK1/2-mediated signaling.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-017-2460-x) contains supplementary material, which is available to authorized users.

Comprehensive transcriptome analysis of fluid shear stress altered gene expression in renal epithelial cells

“…We previously reported changes in TGF‐β signaling, involving genes encoding proteins relaying the signal from cell membrane toward the nucleus, i.e. the ligands Tgfb1‐3 and the receptor Alk5 ( Tgfbr1 ), as well as down‐stream targets, that is, Pai1 ( Serpine1 ), Fn1 , Col1a1 , and Snai1 (Kunnen et al, ). Our gene expression profile now also shows increased expression of genes encoding proteins involved in TGF‐β ligand activation ( Furin , Thbs1 ) or ligand inhibition ( Ltbp2 ), the transcription factor Smad3 , but also the inhibitors Smad7 , Smurf1 , Skil , and Tgif1 , all critical components of the pathway (Table ).…”

“…ALK4/5/7 inhibitor LY‐364947 (Calbiochem; #616451) from Merck Millipore (Darmstadt, Germany), MEK1/2 inhibitor Trametinib (GSK1120212; #S2673) from Selleckchem (Bio‐Connect, Huissen, The Netherlands) and ammonium sulfate (#A‐2939) from Sigma–Aldrich (Zwijndrecht, The Netherlands) were used as previously described (Kunnen et al, ).…”

“…SV40 large T‐antigen immortalized murine proximal tubular epithelial cells (PTEC), derived from a Pkd1 lox,lox mouse, were generated and cultured as described previously (Kunnen et al, ; Leonhard et al, ). Briefly, cells were maintained at 37°C and 5% CO 2 in DMEM/F‐12 with GlutaMAX (Gibco, Fisher Scientific, Landsmeer, The Netherlands; #31331‐093) supplemented with 100 U/ml Penicillin‐Streptomycin (Gibco, Life Technologies; #15140‐122), 2% Ultroser G (Pall Corporation, Pall BioSepra, Cergy St Christophe, France; #15950‐017), 1x Insulin‐Transferrin‐Selenium‐Ethanolamine (Gibco, Life Technologies; #51500‐056), 25 ng/L Prostaglandin E1 (Sigma–Aldrich; #P7527) and 30 ng/L Hydrocortisone (Sigma–Aldrich; #H0135).…”

“…Control cells were treated similarly, but without AS. Cilia formation was checked on a parallel slide by immunofluorescence using anti‐acetylated α‐tubulin antibodies (Sigma Aldrich; #T6793) as previously described (Kunnen et al, ).…”

“…Furthermore, mTOR signaling and cell‐size control, as well as STAT6/p100‐regulated transcription are thought to be negatively regulated upon flow‐induced bending of the cilium, independent from flow‐induced Ca 2+ influx (Boehlke et al, ; Low et al, ; Weimbs, ; Zhong et al, ). Cilia‐independent shear‐induced alterations in renal signaling include increased Na + and HCO 3 − reabsorption and autocrine TGF‐β/ALK5 signaling (Kotsis, Boehlke, & Kuehn, ; Kunnen et al, ).…”