TNF-α-mediated proliferation of vascular smooth muscle cells involves Raf-1-mediated inactivation of Rb and transcription of E2F1-regulated genes

Abstract

Atherosclerosis is characterized by hyperplastic neointima and an inflammatory response with cytokines such as TNFα. TNFα is a pleiotropic cytokine that mediates inflammatory, proliferative, cytostatic and cytotoxic effects in a variety of cell types, including endothelial cells and vascular smooth muscle cells (VSMCs). Interestingly, TNFα has been shown to play two very opposing roles in these cell types; it inhibits proliferation and induces apoptosis in endothelial cells, while it enhances the proliferation and migration of VSMCs. Here we show that TNFα is capable of stimulating proliferation of rat VSMCs as well as human VSMCs in a Raf-1/MAP K-dependent manner. TNFα could increase the expression of E2F-regulated proliferative cdc6, Thymidylate synthase (TS) and cdc25A genes in Aortic smooth muscle cells (AoSMC), as seen by real time PCR assays. There is an activation of the stress-induced kinase, JNK1, in VSMCs upon TNFα stimulation. TNFα was capable of inducing binding of the Raf-1 kinase to Rb, and treatment with the Rb-Raf-1 inhibitor, RRD-251, could prevent TNFα-induced S-phase entry in AoSMCs. In addition, inhibition of Raf-1 or Src kinases using pharmacologic inhibitors could also prevent S-phase entry, while inhibition of JNK was not as effective. These results suggest that inhibiting the Rb-Raf-1 interaction is a potential avenue to prevent VSMC proliferation associated with atherosclerosis.