In glomerular disease, transforming growth factor-beta1 (TGF-beta1) has been demonstrated
to exert anti-mitogenic and anti-inflammatory as well as fibrogenic effects. To better
understand the TGF-beta1 action on glomerular cells at the molecular level, we investigated
mechanisms of TGF-beta1-induced growth suppression in primary cultures of rat mesangial
cells (MCs). TGF-beta1 (5 ng/ml) markedly inhibited proliferation of MCs incubated
with PDGF, endothelin-1, bFGF, serotonin, or EGF, indicating that TGF-beta1 interferes
with post-receptor signals of mitogenesis. TGF-beta1 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-beta1 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 G1 phase
of cell cycle progression at this timepoint. PDGF stimulation of MCs induced protein
expression of the G1 phase cyclin D1 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-beta1. However, TGF-beta1 prevented PDGF-elicited phosphorylation of
the retinoblastoma tumor suppressor (pRb), a negative cell cycle regulator. Moreover,
TGF-beta1 significantly reduced cyclin E-associated histone H1 kinase activity in
the presence of PDGF. These results indicate that TGF-beta1 inhibits mitogen-stimulated
MC growth by causing cell cycle arrest in late G1 phase. While TGF-beta1 does not
alter the mitogen-induced expression and abundance of G1 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 G1 phase and transition to S phase.