Inhibition of osteoblast-specific transcription factor Cbfa1 by the cAMP pathway in osteoblastic cells - Ubiquitin/proteasome-dependent regulation

The cAMP pathway, a major intracellular pathway mediating parathyroid hormo ne signal, regulates osteoblastic function. Parathyroid hormone (through ac tivation of protein kinase A) has also been shown to stimulate ubiquitin/pr oteasome activity in osteoblasts. Since the osteoblast-specific transcripti on factor Osf2/Cbfa1 is important for differentiation of osteoblastic cells , we examined the roles of the cAMP and ubiquitin/proteasome pathways in re gulation of Cbfa1. In the osteoblastic cell line, MC3T3-E1, continuous trea tment with cAMP elevating agents inhibited both osteoblastic differentiatio n based on alkaline phosphatase assay and DNA binding ability of Cbfa1 base d on a gel retardation assay. Cbfa1 inhibition was paralleled by an inhibit ory effect of forskolin on Cbfa1-regulated genes. Northern and Western blot analyses suggested that the inhibition of Cbfa1 by forskolin was mainly at the protein level. Pretreatment with proteasome inhibitors prior to forsko lin treatment reversed the effect of forskolin. Furthermore, addition of pr oteasome inhibitors to forskolin-pretreated samples resulted in recovery of Cbfa1 protein levels and accumulation of polyubiquitinated forms of Cbfa1, indicating a role for the proteasome pathway in the degradation of Cbfa1. These results suggest that suppression of osteoblastic function by the cAMP pathway is through proteolytic degradation of Cbfa1 involving a ubiquitin/ proteasome-dependent mechanism.