Myc Downregulation by Transforming Growth Factor β Required for Activation of the p15 Ink4b G 1 Arrest Pathway

Abstract

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 G 1 arrest, enforced expression of c-Myc prevents G 1 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 G 1 cdks 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 G 1 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 G 1 arrest pathway. TGF-β must therefore downregulate c-Myc in order to activate this pathway.