TGF-β1-induced cell cycle arrest in renal mesangial cells involves inhibition of cyclin E-cdk 2 activation and retinoblastoma protein phosphorylation

TGF-β1-induced cell cycle arrest in renal mesangial cells involves inhibition of cyclin E-cdk 2 activation and retinoblastoma protein phosphorylation. In glomerular disease, transforming growth factor- β 1 (TGF- β 1) has been demonstrated to exert anti-mitogenic and anti-inflammatory as well as fibrogenic effects. To better understand the TGF- β 1 action on glomerular cells at the molecular level, we investigated mechanisms of TGF- β 1-induced growth suppression in primary cultures of rat mesangial cells (MCs). TGF- β 1 (5 ng/ml) markedly inhibited proliferation of MCs incubated with PDGF, endothelin-1, bFGF, serotonin, or EGF, indicating that TGF- β 1 interferes with post-receptor signals of mitogenesis. TGF- β 1 did not affect mitogen-stimulated induction of the immediate early genes, c-fos , c-jun , and Egr-1 in MCs that occurred transiently at 30 to 120 minutes. Time-course studies revealed that TGF- β 1 inhibited DNA synthesis and MC replication when added up to six to eight hours after MC stimulation with PDGF. FACS analysis demonstrated that MCs had reached middle to late G 1 phase of cell cycle progression at this timepoint. PDGF stimulation of MCs induced protein expression of the G 1 phase cyclin Dl as well as the cyclin-dependent kinases cdk 4 and cdk 2. This was not significantly altered when MCs were coincubated with both, PDGF and TGF- β 1. However, TGF- β 1 prevented PDGF-elicited phosphorylation of the retinoblastoma tumor suppressor (pRb), a negative cell cycle regulator. Moreover, TGF- β 1 significantly reduced cyclin E-associated histone H1 kinase activity in the presence of PDGF. These results indicate that TGF- β 1 inhibits mitogen-stimulated MC growth by causing cell cycle arrest in late G 1 phase. While TGF- β 1 does not alter the mitogen-induced expression and abundance of G 1 phase cyclin D1 and cdks 4 and 2 in MCs, it inhibits cyclin E-cdk 2 activity, thus preventing mitogen-elicited phosphorylation and inactivation of pRb in G 1 phase and transition to S phase.