Myc Downregulation by Transforming Growth Factor β Required for Activation of the p15^Ink4b G₁ Arrest Pathway

Abstract

The antimitogenic action of transforming growth factor β (TGF-β) in epithelial cells involves cyclin-dependent kinase (cdk) inhibitory gene responses and downregulation of c-Myc expression. Although the cdk inhibitory responses are sufficient for G₁arrest, enforced expression of c-Myc prevents G₁ arrest by TGF-β. We investigated the basis of this antagonism by using Mv1Lu lung epithelial cell lines that conditionally express levels of human c-Myc. We show that c-Myc prevents induction of the cdk4 inhibitor p15^Ink4b and the subsequent inhibition of G₁cdks by TGF-β. We assessed the significance of this effect by analyzing the oligomeric state of cdk4 in these cells. In proliferating cells, endogenous cdk4 is distributed among three populations: an abundant high-molecular-mass (>400-kDa) pool of latent cdk4 that serves as a source of cdk4 for cyclin D, a low-abundance pool containing active cyclin D-cdk4 complexes, and an inactive population of monomeric cdk4. Cell stimulation with TGF-β converts the latent and active cdk4 pools into inactive cdk4, an effect that is specifically mimicked by overexpression of p15 but not by other forms of G₁ arrest. This process of TGF-β-induced cdk4 inactivation is completely blocked by expression of c-Myc, even though the latent and active cdk4 complexes from c-Myc-expressing cells remain sensitive to dissociation by p15 in vitro. c-Myc causes a small increase in cyclin D levels, but this effect contributes little to the loss of TGF-β responses in these cells. The evidence suggests that c-Myc interferes with TGF-β activation of the p15 G₁arrest pathway. TGF-β must therefore downregulate c-Myc in order to activate this pathway.

ACKNOWLEDGMENTS

We thank Mark Ewen for the mink cdk4 sequence, Akiko Hata and Celio Pouponnot for p15 reporter constructs and advice, Inga Reynisdóttir for tet-cyclin D1 cells, and Andy Koff for critical discussion.

S.W.B. is a Fellow of the Leukemia Society of America. J.S. is a Fellow of the Ministerio de Educación y Cultura of Spain. B.J.W. and J.M. are, respectively, a Fellow and an Investigator of the Howard Hughes Medical Institute.