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 cultured renal proximal tubular cells. In previous studies, it was shown that these effects depend on oxygen radical-mediated induction of p27(Kip1), an inhibitor of cyclin-dependent kinases. The present study was undertaken to investigate whether mitogen-activated protein (MAP) kinases serve as signaling intermediates between AngII-induced oxidative stress and induction of p27(Kip1). 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 phosphorylation peak after prolong incubation (12 h) in cultured proximal tubular cells from two different species (MCT and LLC-PK(1) cells). Total protein 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 immuno-precipitation of MAP kinase. Exogenous H(2)O(2) also stimulates a biphasic phosphorylation of p44/42 MAP kinase. The flavoprotein inhibitor diphenylene iodinium, as well as the antioxidant N-acetylcysteine, prevented AngII-induced p44/42 MAP kinase phosphorylation, indicating involvement of reactive oxygen species generated by membrane-bound NAD(P)H oxidase. The MAP kinase kinase inhibitor PD98059 completely inhibits AngII-induced p27(Kip1) expression and (3)[H]leucine incorporation into proteins as a previously established marker of cell hypertrophy. PD98059 did not attenuate AngII-stimulated intracellular synthesis of oxygen radicals. Transient transfection with p44/42 MAP kinase antisense, but not sense, phosphorothioate-modified oligonucleotides also prevented AngII-induced MAP kinase phosphorylation, p27(Kip1) expression, and cell hypertrophy. Furthermore, induction of p27(Kip1) by H(2)O(2) was also abolished in the presence of PD98059. Although AngII induces phosphorylation of the stress-activated p38 MAP kinase, inhibition of this enzyme with SB203580 failed to attenuate induced p27(Kip1) expression and hypertrophy. These data provide evidence that AngII- mediated oxygen stress leads to the phosphorylation of p44/42 MAP kinase in proximal tubular cells. Activation of this enzyme is 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 tubular atrophy and tubulointerstitial fibrosis.
Hannken
Tete
T
Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, Hamburg, Germany.
Schroeder
Regine
R
Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, Hamburg, Germany.
Zahner
Gunther
G
Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, Hamburg, Germany.
Stahl
Rolf A K
RAK
Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, Hamburg, Germany.
Wolf
Gunter
G
Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, Hamburg, Germany.
D000804:Angiotensin II / Q000494:pharmacology / Q000502:physiology*; D000818:Animals; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006984:Hypertrophy; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000473:pathology*; D018374:LLC-PK1 Cells; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism* / Q000502:physiology; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism* / Q000502:physiology; D016877:Oxidants / Q000494:pharmacology; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000502:physiology*; D013552:Swine; D025521:Tumor Suppressor Proteins
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D008869:Microtubule-Associated Proteins; D016877:Oxidants; D017382:Reactive Oxygen Species; D025521:Tumor Suppressor Proteins; D000804:Angiotensin II; D050760:Cyclin-Dependent Kinase Inhibitor p27; D006861:Hydrogen Peroxide; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one