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 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.
Tintut
Y
Y
Division of Cardiology, Department of Medicine, UCLA School of Medicine, Los Angeles, California 90095, USA. ytintut@ucla.edu
Parhami
F
F
Le
V
V
Karsenty
G
G
Demer
L L
LL
D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D050659:Core Binding Factor Alpha 1 Subunit; D000242:Cyclic AMP / Q000819:agonists / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism; D009363:Neoplasm Proteins; D010006:Osteoblasts / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics*; D014452:Ubiquitins / Q000378:metabolism
D050659:Core Binding Factor Alpha 1 Subunit; D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; D009097:Multienzyme Complexes; D009363:Neoplasm Proteins; D010281:Parathyroid Hormone; D012333:RNA, Messenger; D014157:Transcription Factors; D014452:Ubiquitins; D005576:Colforsin; D000242:Cyclic AMP; D000469:Alkaline Phosphatase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D002118:Calcium