Cross‐talk between ERK MAP kinase and Smad‐signaling pathways enhances TGF‐β dependent responses in human mesangial cells

Abstract

Transforming growth factor β (TGF‐β) stimulates renal cell fibrogenesis by a poorly understood mechanism. Previously, we suggested a synergy between TGF‐β1 activated extracellular signal‐regulated kinase (ERK) and Smad signaling in collagen production by human glomerular mesangial cells. In a heterologous DNA binding transcription assay, biochemical or dominant‐negative ERK blockade reduced TGF‐β1 induced Smad3 activity. Total serine phosphorylation of Smad2/3, but not phosphorylation of the C‐terminal SS P XS P motif, was decreased by pretreatment with the MEK/ERK inhibitors, PD98059 (10 µM) or U0126 (25 µM). This effect was not seen in the mouse mammary epithelial NMuMG cell line, indicating that ERK‐dependent activation of Smad2/3 occurs only in certain cell types. TGF‐β stimulated phosphorylation of an expressed Smad3A construct, with a mutated C‐terminal SS P XS P motif, was reduced by a MEK/ERK inhibitor. In contrast, MEK/ERK inhibition did not affect phosphorylation of a Smad3 construct mutated at consensus phosphorylation sites in the linker region (Smad3EPSM). Constitutively active MEK (caMEK) induced α2(I) collagen promoter activity, an effect blocked by co‐transfected Smad3EPSM, but not Smad3A. The effects of caMEK and TGF‐β1 on collagen promoter activity were additive. These results indicate that ERK‐dependent R‐Smad linker region phosphorylation enhances collagen I synthesis and imply positive cross talk between the ERK and Smad pathways in human mesangial cells.