Abstract
The cAMP pathway, a major intracellular pathway mediating parathyroid hormone signal, regulates osteoblastic function. Parathyroid hormone (through activation of protein kinase A) has also been shown to stimulate ubiquitin/proteasome activity in osteoblasts. Since the osteoblast-specific transcription factor Osf2/Cbfa1 is important for differentiation of osteoblastic cells, we examined the roles of the cAMP and ubiquitin/proteasome pathways in regulation of Cbfa1. In the osteoblastic cell line, MC3T3-E1, continuous treatment with cAMP elevating agents inhibited both osteoblastic differentiation based on alkaline phosphatase assay and DNA binding ability of Cbfa1 based on a gel retardation assay. Cbfa1 inhibition was paralleled by an inhibitory 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 forskolin treatment reversed the effect of forskolin. Furthermore, addition of proteasome 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.
Highlights
The cAMP pathway, a major intracellular pathway mediating parathyroid hormone signal, regulates osteoblastic function
Since the osteoblast-specific transcription factor Osf2/Cbfa1 is important for differentiation of osteoblastic cells, we examined the roles of the cAMP and ubiquitin/proteasome pathways in regulation of Cbfa1
The results showed that alkaline phosphatase activity increased as cells progressed through differentiation in control cells, this increase was blocked by forskolin treatment (Fig. 1A)
Summary
Osteoblast-specific factor 2; Cbfa, core binding factor ␣-1; PTH, parathyroid hormone; PSI, proteasome inhibitor I; RT-PCR, reverse transcriptase-polymerase chain reaction. PTH/ cAMP have been shown to regulate expression of several osteoblastic differentiation genes including Cbfa1-regulated osteoblast-specific genes such as osteopontin [13], type I collagen [14, 15], bone sialoprotein [16], and osteocalcin [11]. The 26 S proteasome, a multicatalytic protease complex, is a site of regulatory degradation for most proteins [17, 18] It is an ATP-dependent extralysosomal protease that is necessary for viability and essential for regulation of proliferation and differentiation of eukaryotic cells [18]. It is ubiquitously distributed throughout the cell, in the nucleus, cytosol, endoplasmic reticulum, and even associated with the cytoskeleton [18]. Results showed that continuous treatment of cAMP agonists inhibited Cbfa by proteolytic degradation involving the ubiquitin/proteasome-dependent mechanism
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