Synergistic role of c-Myc and ERK1/2 in the mitogenic response to TGFβ-1 in cultured rat nucleus pulposus cells

INTRODUCTION Although transforming growth factor beta1 (TGFbeta1) is known to be a potent inhibitor of proliferation in most cell types, it accelerates proliferation in certain mesenchymal cells, such as articular chondrocytes and nucleus pulposus cells. The low ability for self-renewal of nucleus pulposus cells is one obstacle in developing new therapeutic options for intervertebral disc diseases, and utilizing cytokines is one of the strategies to regulate nucleus pulposus cell proliferation. However, the precise cell cycle progression and molecular mechanisms by which TGFbeta1 stimulates cell growth remain unclear. The aim of this study was to elucidate a mechanism that enables cell proliferation with TGFbeta1 stimulation. METHODS We tested cultured rat nucleus pulposus cells for proliferation and cell cycle distribution under exogenous TGFbeta1 stimulation with and without putative pharmaceutical inhibitors. To understand the molecular mechanism, we evaluated the expression levels of key regulatory G1 phase proteins, c-Myc and the cyclin-dependent kinase inhibitors. RESULTS We found that TGFbeta1 promoted proliferation and cell cycle progression while reducing expression of the cyclin-dependent kinase inhibitors p21 and p27, which are downregulators of the cell cycle. Robust c-Myc expression for 2 h and immediate phosphorylation of extra cellular signal regulated kinase (ERK1/2) were detected in cultures when TGFbeta1 was added. However, pretreatment with 10058-F4 (an inhibitor of c-Myc transcriptional activity) or PD98059 (an inhibitor of ERK1/2) suppressed c-Myc expression and ERK1/2 phosphorylation, and inhibited cell cycle promotion by TGFbeta1. CONCLUSIONS Our experimental results indicate that TGFbeta1 promotes cell proliferation and cell cycle progression in rat nucleus pulposus cells and that c-Myc and phosphorylated ERK1/2 play important roles in this mechanism. While the difference between rat and human disc tissues requires future studies using different species, investigation of distinct response in the rat model provides fundamental information to elucidate a specific regulatory pathway of TGFbeta1.