Simvastatin suppresses self-renewal of mouse embryonic stem cells by inhibiting RhoA geranylgeranylation

Abstract

Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, were originally developed to lower cholesterol. Their pleiotropic (or cholesterol-independent) effects at the cellular and molecular levels are highly related to numerous cellular functions, such as proliferation and differentiation. However, they are hardly studied in embryonic stem cells. In this study, we evaluated the effects of statins on mouse ESCs (J1, D3, and RW.4) to enhance our understanding of the molecular basis of ESC self-renewal. Treatment of ESCs with simvastatin, mevastatin, atorvastatin, or pravastatin induced morphological change and decreased cell proliferation. We observed that the use of simvastatin was most effective in all three ESCs. Loss of ESC self-renewal by simvastatin was determined by marked downregulation of ESC markers alkaline phosphatase, Oct4, Nanog, Rex-1, and SSEA-1. Simvastatin effects were selectively reversed by either rnevalonate or its metabolite geranylgeranyl pyro-phosphate (GGPP) but not by cholesterol or farnesyl pyrophosphate. These results suggest that simvastatin effects were mainly derived from depletion of intracellular pools of GGPP, the substrate required for the geranylgeranylation. Using this approach, we found that GGPP, a derivative of the rnevalonate pathway, is critical for ESC self-renewal. Furthermore, we identified that simvastatin selectively blocked cytosol-to-membrane translocalization of RhoA small guanosine triphosphate-binding protein, known to be the major target for geranylgeranylation, and lowered the levels of Rho-kinase (ROCK)2 protein in ESCs. In addition, simvastatin downregulated the ROCK activity, and this effect was reversed by addition of GGPP. Our data suggest that sirnvastatin, independently of its cholesterol-lowering properties, impairs the ESC self-renewal by modulating RhoA/ROCK-dependent cell-signaling.