Reactive oxygen species stimulate p44/42 mitogen-activated protein kinase and induce p27(Kip1): Role in angiotensin II-mediated hypertrophy of proximal tubular cells

Angiotensin II (AngII) induces G(1) phase arrest and hypertrophy of culture d renal proximal tubular cells. In previous studies, it was shown that thes e effects depend on oxygen radical-mediated induction of p27(Kip1), an inhi bitor of cyclin-dependent kinases. The present study was undertaken to inve stigate whether mitogen-activated protein (MAP) kinases serve as signaling intermediates between AngII-induced oxidative stress and induction of p27(K ip1). AngII (10(-7) M) induces a biphasic phosphorylation pattern of p44/42 MAP kinase with an early phosphorylation after 2 min and a later, second p hosphorylation peak after prolong incubation (12 h) in cultured proximal tu bular cells from two different species (MCT and LLC-PK1 cells). Total prote in expression of MAP kinase was not changed by AngII. These phosphorylation patterns of p44/42 MAP kinase caused activation of the enzyme, as detected by phosphorylated MAP substrate Elk-1 after immunoprecipitation of MAP kin ase. Exogenous H2O2 also stimulates a biphasic phosphorylation of p44/42 MA P kinase. The flavoprotein inhibitor diphenylene iodinium, as well as the a ntioxidant N-acetylcysteine, prevented AngII-induced p44/42 MAP kinase phos phorylation, indicating involvement of reactive oxygen species generated by membrane-bound NAD(P)H oxidase. The MAP kinase kinase inhibitor PD98059 co mpletely inhibits AngII-induced p27(Kip1) expression and (3)[H]leucine inco rporation into proteins as a previously established marker of cell hypertro phy. PD98059 did not attenuate AngII-stimulated intracellular synthesis of oxygen radicals. Transient transfection with p44/42 MAP kinase antisense, b ut not sense, phosphorothioate-modified oligonucleotides also prevented Ang II-induced MAP kinase phosphorylation, p27(Kip1) expression, and cell hyper trophy. Furthermore, induction of p27(Kip1) by H2O2 was also abolished in t he presence of PD98059. Although AngII induces phosphorylation of the stres s-activated p38 MAP kinase, inhibition of this enzyme with SB203580 failed to attenuate induced p27(Kip1) expression and hypertrophy. These data provi de evidence that AngII-mediated oxygen stress leads to the phosphorylation of p44/42 MAP kinase in proximal tubular cells. Activation of this enzyme i s essential for p27(Kip1) expression, G(1) phase arrest, and hypertrophy of proximal tubular cells. These findings may lead to new concepts concerning interference of the development of proximal tubular hypertrophy, which may eventually turn into a maladaptive process in vivo leading ultimately to t ubular atrophy and tubulointerstitial fibrosis.