TGF-beta1-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-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.
Schoecklmann
H O
HO
Medizinische Klinik IV, Universität Erlangen-Nürnberg, Germany.
Rupprecht
H D
HD
Zauner
I
I
Sterzel
R B
RB
D000818:Animals; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016193:G1 Phase / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D017781:Genes, Immediate-Early / Q000187:drug effects; D005920:Glomerular Mesangium / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D008934:Mitogens / Q000494:pharmacology; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D016160:Retinoblastoma Protein / Q000378:metabolism*; D016196:S Phase / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D016213:Cyclins; D004791:Enzyme Inhibitors; D008934:Mitogens; D010982:Platelet-Derived Growth Factor; D047428:Protein Kinase Inhibitors; D016160:Retinoblastoma Protein; D016212:Transforming Growth Factor beta; D011494:Protein Kinases; C102333:histone H1 kinase; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases