Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for activation of p38α and p38δ MAPK isoforms by TGF-β1 in murine mesangial cells

Abstract

Transforming growth factor-β1 (TGF-β1) is a potent inducer of extracellular matrix (ECM) synthesis that leads to renal fibrosis. Intracellular signaling mechanisms involved in this process remain incompletely understood. Mitogen-activated protein kinase (MAPK) is a major stress signal-transducing pathway, and we have previously reported activation of p38 MAPK by TGF-β1 in rat mesangial cells and its role in the stimulation of pro-α1(I) collagen. In this study, we further investigated the mechanism of p38 MAPK activation by TGF-β1 and the role of MKK3, an upstream MAPK kinase of p38 MAPK, by examining the effect of targeted disruption of the Mkk3 gene. We first isolated glomerular mesangial cells from MKK3 AWK3-null (Mkk3-/-) and wild-type (Mkk3+/+) control mice. Treatment with TGF-β1 induced rapid phosphorylation of MKK3 as well as p38 MAPK within 15 min in cultured wild-type (Mkk3+/+) mouse mesangial cells. In contrast, TGF-β1 failed to induce phosphorylation of either MKK3 or p38 MAPK in MKK3-deficient (Mkk3-/-) mouse mesangial cells, indicating that MKK3 is required for TGF-β1-induced p38 MAPK activation. TGF-β1 selectively activated the p38 MAPK isoforms p38α and p38δ in wild-type (Mkk3+/+) mesangial cells, but not in MKK3-deficient (Mkk3-/1-) mesangial cells. Thus, activation of p38α and p38δ is dependent on the activation of upstream MKK3 by TGF-β1. Furthermore, MKK3 deficiency resulted in a selective disruption of TGF-β1-stimulated up-regulation of pro-α1(I) collagen expression but not TGF-β1 induction of fibronectin and PAI-1. These data demonstrate that the MKK3 is a critical component of the TGF-β1 signaling pathway, and its activation is required for subsequent p38α and p38δ MAPK activation and collagen stimulation by TGF-β1.