Angiotensin II-stimulated hypertrophy of LLC-PK1 cells depends on the induction of the cyclin-dependent kinase inhibitor p27(Kip1)

Angiotensin II (Ang II) induces hypertrophy of cultured proximal tubular epithelial cells including the LLC-PK1 cell line. We have previously shown that this hypertrophy appears in the G(1)-phase of the cell cycle. Since progression through the cell cycle is controlled by a series of cyclin and cyclin-dependent kinase (CdK) complexes that may be inactivated by CdK inhibitors. we studied the expression of the CdK-inhibitor p27(Kip1) in LLC-PK1 cells challenged with Ang II. Compared to cells grown in serum-free medium. Ang II treatment enhanced p27(Kip1) protein, but not mRNA expression. This p27(Kip1) induction was mediated through AT(1)-receptors. Exogenous TGF-beta also stimulated p27(Kip1) protein expression. Immunoprecipitation experiments revealed that p27(Kip1) preferentially associated with CdK4 in Ang II-treated LLC-PK1 cells and that the activity of this kinase was inhibited after Ang II-treatment, an effect that may be generated by increased p27(Kip1) binding to cyclin D1-CdK4 complexes. In contrast. p27(Kip1) was not associated with cyclin E-CdK2 complexes in Ang II-stimulated cells. Treatment of LLC-PK1 cells with p27(Kip1) antisense. but nor missense, oligonucleotides abolished the Ang II-mediated cell hypertrophy as measured by de novo protein synthesis and total protein content. and facilitated entry into the S-phase of the cell cycle. Our findings suggest that Ang II stimulates p27(Kip1) expression in renal cells. Furthermore, this induction of the CdK-inhibitor appears pivotal in the hypertrophy induced by Ang II and elucidates the molecular mechanisms associated with this growth response in proximal tubular cells.