Hepatocyte Growth Factor Releases Mink Epithelial Cells from Transforming Growth Factor β1-Induced Growth Arrest by Restoring Cdk6 Expression and Cyclin E-Associated Cdk2 Activity

Abstract

Transforming growth factor β (TGF-β) potently suppresses Mv1Lu mink epithelial cell growth, whereas hepatocyte growth factor (HGF) counteracts TGF-β-mediated growth inhibition and induces Mv1Lu cell proliferation (J. Taipale and J. Keski-Oja, J. Biol. Chem. 271:4342–4348, 1996). By addressing the cell cycle regulatory mechanisms involved in HGF-mediated release of Mv1Lu cells from TGF-β inhibition, we show that increased DNA replication is accompanied by phosphorylation of the retinoblastoma protein and alternative regulation of cyclin-Cdk-inhibitor complexes. While TGF-β treatment decreased the expression of Cdk6, this effect was counteracted by HGF, followed by partial restoration of cyclin D2-associated kinase activity. Notably, HGF failed to prevent TGF-β induction of p15 and its association with Cdk6. However, HGF reversed the TGF-β-mediated decrease in Cdk6-associated p27 and cyclin D2-associated Cdk6, suggesting that HGF modifies the TGF-β response at the level of G₁ cyclin complex formation. Counteraction of TGF-β regulation of Cdk6 by HGF may in turn affect the association of p27 with Cdk2-cyclin E complexes. Though HGF did not differentially regulate the total levels of p27 in TGF-β-treated cells, p27 immunodepletion experiments suggested that upon treatment with both growth factors, less p27 is associated with Cdk2-cyclin E complexes, in parallel with restoration of the active form of Cdk2 and the associated kinase activity. The results demonstrate that HGF intercepts TGF-β cell cycle regulation at multiple points, affecting both G₁and G₁-S cyclin kinase activities.

ACKNOWLEDGMENTS

We thank Joan Massagué and Jiri Bartek for generous gifts of antibodies, Tomi Mäkelä for critical review of the manuscript, and Annamari Heiskanen for fine technical assistance.

This work was supported by the Academy of Finland, the Ida Montin Foundation, Novo Nordisk Foundation, Sigrid Juselius Foundation, Finnish Cancer Foundation, and Biocentrum Helsinki.