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Downregulation of Dicer expression by serum withdrawal sensitizes human endothelial cells to apoptosis

¹Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, and ²Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto, Japan

Submitted 4 March 2008 ; accepted in final form 27 October 2008

Although the modulated expression of Dicer is documented upon neoplastic transformation, little is known of the regulation of Dicer expression by environmental stimuli and its roles in the regulation of cellular functions in primary cells. In this study, we found that Dicer expression was downregulated upon serum withdrawal in human umbilical vein endothelial cells (HUVECs). Serum withdrawal induced a time-dependent repression of Dicer expression, which was specifically rescued by vascular endothelial cell growth factor or sphingosine-1-phosphate. When Dicer expression was silenced by short-hairpin RNA against Dicer, the cells were more prone to apoptosis under serum withdrawal, whereas the rate of apoptosis was comparable with control cells in the serum-containing condition. Real-time PCR-based gene expression profiling identified several genes, the expression of which was modulated by Dicer silencing, including adhesion and matrix-related molecules, caspase-3, and nitric oxide synthase 3 (NOS3). Dicer silencing markedly impaired migratory functions without affecting cell adhesion and repressed phosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 in adherent HUVECs. Dicer knockdown upregulated caspase-3 and downregulated NOS3 expression, and serum withdrawal indeed increased caspase-3 and decreased NOS3 expression. Furthermore, the overexpression of Dicer in HUVECs resulted in a marked reduction in apoptosis upon serum withdrawal and a decreased caspase-3 and increased NOS3 expression. The inhibition of NOS activity by N-nitro-L-arginine methyl ester abrogated the effect of Dicer overexpression to rescue the cells from serum withdrawal-induced apoptosis. These results indicated that serum withdrawal decreases Dicer expression, leading to an increased susceptibility to apoptosis through the regulation of caspase-3 and NOS3 expression.

caspase 3; nitric oxide synthase 3