Erk 1,2 phosphorylates p27(Kip1): Functional evidence for a role in high glucose-induced hypertrophy of mesangial cells.

Abstract

Mesangial cell hypertrophy is one of the earliest morphological abnormalities of diabetic nephropathy. We have previously shown that high glucose induces p27(Kip1) by a post-transcriptional mechanism and that mesangial cell hypertrophy depends on G(1)-phase arrest mediated by this CDK-inhibitor. However, it remains poorly understood how high glucose stimulates p27(Kip1) expression in mesangial cells. Mesangial cells were isolated from p27(Kip1) +/+ and -/- mice and characterized by light microscopy and immunohistochemistry. It was tested by Western blotting and autoradiography whether high glucose medium activates Erk 1,2 and whether this activation phosphorylates p27(Kip1). The three consensus phosphorylation sites of p27(Kip1) were mutated and these constructs were expressed in p27(Kip1) -/- mesangial cells. Hypertrophy was assessed by different methods. High glucose stimulates phosphorylation of MAP kinases Erk 1,2 in p27(Kip1 )+/+ and -/- mesangial cells. Activation of Erk 1,2 leads to phosphorylation of p27(Kip1 )in vitro and in vivo. Mutations of serine(10) or threonine(187) still supported high glucose-induced hypertrophy. In contrast, a mutation of serine(178) converted the hypertrophic response into a proliferative phenotype. Mutation of serine(178) leads to the attenuated expression of p27(Kip1) protein in the presence of high glucose. Our study shows that high glucose stimulates Erk 1,2 that phosphorylate p27(Kip1) at serine(178) increasing its expression. This is an important molecular mechanism of high glucose-induced hypertrophy of mesangial cells.