Transforming Growth Factor β1Selectively Inhibits the Cyclic AMP-dependent Proliferation of Primary Thyroid Epithelial Cells by Preventing the Association of Cyclin D3–cdk4 with Nuclear p27kip1

Abstract

Dog thyroid epithelial cells in primary culture constitute a physiologically relevant model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for thyrotropin (thyroid-stimulating hormone [TSH]). As previously shown in this system, the cAMP-dependent mitogenic pathway differs from growth factor cascades as it stimulates the accumulation of p27^kip1but not cyclins D. Nevertheless, TSH induces the nuclear translocations and assembly of cyclin D3 and cdk4, which are essential in cAMP-dependent mitogenesis. Here we demonstrate that transforming growth factor β₁(TGFβ₁) selectively inhibits the cAMP-dependent cell cycle in mid-G1 and various cell cycle regulatory events, but it weakly affects the stimulation of DNA synthesis by epidermal growth factor (EGF), hepatocyte growth factor, serum, and phorbol esters. EGF+serum and TSH did not interfere importantly with TGFβ receptor signaling, because they did not affect the TGFβ-induced nuclear translocation of Smad 2 and 3. TGFβ inhibited the phosphorylation of Rb, p107, and p130 induced by TSH, but it weakly affected the phosphorylation state of Rb-related proteins in EGF+serum-treated cells. TGFβ did not inhibit c-myc expression. In TSH-stimulated cells, TGFβ did not affect the expression of cyclin D3, cdk4, and p27^kip1, nor the induced formation of cyclin D3–cdk4 complexes, but it prevented the TSH-induced relocalization of p27^kip1from cdk2 to cyclin D3–cdk4. It prevented the nuclear translocations of cdk4 and cyclin D3 without altering the assembly of cyclin D3–cdk4 complexes probably formed in the cytoplasm, where they were prevented from sequestering nuclear p27^kip1away from cdk2. This study dissociates the assembly of cyclin D3–cdk4 complexes from their nuclear localization and association with p27^kip1. It provides a new mechanism of regulation of proliferation by TGFβ, which points out the subcellular location of cyclin d–cdk4 complexes as a crucial factor integrating mitogenic and antimitogenic regulations in an epithelial cell in primary culture.