Control del ciclo celular y fisiopatología vascularPapel de los supresores de crecimiento p27Kip1 y p53.

Abstract

The mammalian cell cycle is positively regulated by holoenzymes constituted by a regulatory subunit (called cyclin) and a cyclin-dependent kinase (CDK). Cyclin/CDK complexes are sequentally activated and inhibited in different phases of the cell cycle. CDK inhibitory proteins (CKIs) constitute an important family of growth suppressors. The p27Kip1 protein is a CKI that belongs to the CIP/KIP family which acts as an universal CDK inhibitor. Its levels are elevated in quiescent cells and decreased during mitogen-dependent entry into the cell cycle. Phosphorylation of p27Kip1 at threonine 187 (T187) is an important mechanism by which p27Kip1 levels are regulated. The tumour suppressor protein p53, which can be activated in response to several cellular insults, also modulates cell number by negatively regulating the cell cycle and inducing apoptosis. Several animal models of atherosclerois, mechanical injury and transplant-atherosclerosis have demonstrated that p27Kip1 and p53 protect against vascular obstructive lesion development, which depends in part on a hiperproliferative response of arterial wall cells, mainly smooth muscle cells (SMCs) and macrophages. The work carried out in this Doctoral Thesis has been focused in the elucidaton of molecular mechanisms implicated in the control of cellular proliferation, with especial interest in cultured SMCs and murine models of atherosclerosis and mechanical injury of the vessel wall. The following specific objectives were pursued: 1) To study the molecular mechanism by which the cytostatic agents PCA-4230 and STI571 inhibit SMC and cancer cell proliferation (Paper 1 and Paper 2). 2) To investigate whether p27Kip1 phosphorylation on T187 regulates atheroma progression in apolipoptotein E-null mice (apoE-KO) fed a high-fat and cholesterol-rich diet (Paper 3) 3) To analyze in apoE-KO mice the consequences of hightening p53 function on the development of aortic atherosclerosis and neointimal lesions induced by mechanical injury of the femoral artery (Manuscript 4). Results from paper 1 and paper 2 demostrated that E2F inhibition and transcriptional repression of cyclin A contribute to PCA-4230- and STI571-dependent SMC growth arrest. STI571 also inhibited ERK1/2 activation, whereas forced activation of these MAPKs in femoral artery SMCs impaired STI571-dependent inhibition of both cyclin A promoter activity and SMC proliferation. Results from paper 3 in the experimental model of diet-induced atherosclerosis showed identical atheroma size, lesion cellularity, proliferation, and apoptotic rates when comparing apoE-KO and p27T187A-apoE-KO mice. Moreover, our findings demonstrate that phosphorylation of p27Kip1 on T187 was not implicated in the control of aortic p27 expression. These findings are in contrast with previous studies by other investigators that have implicated this phosphorylation event on cell cycle regulation and p27Kip1 protein expression in other cell types. Thus, the relevance of p27Kip1 phosphorylation on T187 level seems to depend on the cell type and tissues and on pathologic conditions both in vitro and in vivo. Results from manuscript 4 revealed that gain of p53 function can reduce neointimal hyperplasia after mechanical injury, but has no effect on atherosclerosis, thus highlighting profound differences in the role of p53 in different models of vascular injury that may have important implications for therapeutic purposes.