GLUCOCORTICOID INHIBITION OF FIBROBLAST PROLIFERATION AND REGULATION OF THE CYCLIN KINASE INHIBITOR P21(CIP1)

Glucocorticoids inhibit the proliferation of fibroblastic cells in viv o and in culture; however, the molecular mechanism that accounts for t his effect has remained obscure. We have undertaken to elucidate the m echanism whereby glucocorticoids decrease the rate of proliferation of mouse L929 fibroblastic cells. Addition of dexamethasone to mid-log p hase fibroblasts prolongs G1 phase. This increase in the G1 interval i s associated with, and probably due to, inhibition of phosphorylation of the product of the Rb-1 tumor suppressor gene, pRb. Inhibition of p Rb phosphorylation by cyclin D-dependent kinases can be demonstrated i n vitro. Nevertheless, there is no detectable change in the expression of cyclin D1, cyclin D2, or cyclin D3. Cyclin-dependent kinase-4 (Cdk 4) and Cdk6 are not down-regulated in L929 cells after addition of glu cocorticoids, and the abundance of cyclin D/Cdk4 complexes does not ch ange. Inhibition of pRb kinase activity is associated with an increase in the abundance of one of the Cdk inhibitors, p21(Cip1). The abundan ce of another cyclin kinase inhibitor, p27(Kip1), remains constant. Th e amount of Cdk4 that is bound to p21(Cip1) increases rapidly after ad dition of dexamethasone, and the activity of Cdk4-pRb kinase decreases in parallel. These results indicate that glucocorticoid inhibition of fibroblast proliferation is due to induction of p21(Cip1), which bind s to and inactivates cyclinD/Cdk4 complexes. The abundance of p21 mRNA increases about 5-fold within 2 h after addition of dexamethasone. Th is effect does not obtain in L929 mutants that are null for the glucoc orticoid receptor, and a variant that expresses the glucocorticoid rec eptor from a tetracycline-repressible expression vector demonstrates i nduction of p21 mRNA only in the absence of tetracycline. Cycloheximid e does not block induction of p21 mRNA, and dexamethasone has no detec table effect on the apparent rate of degradation of p21 mRNA. Nuclear run-on transcription of the Cip1 gene increases within 2 h after addit ion of dexamethasone, This effect can be blocked by tetracycline-media ted repression of the glucocorticoid receptor.