Nuclear orphan receptor Nurr1 directly transactivates the osteocalcin gene in osteoblasts.
Nurr1, an NGFI-B nuclear orphan receptor, which transactivates promoters through an NGFI-B response element (NBRE), is strongly induced by parathyroid hormone through the cAMP-protein kinase A signaling pathway in osteoblasts. Here, we demonstrate that multiple agents activating diverse signaling pathways in osteoblasts induce Nurr1. The strongest Nurr1 inducers were activators of cAMP-protein kinase A-coupled signaling, followed by protein kinase C- and calcium-coupled signaling activators. Receptor tyrosine kinase activators had minimal effect, whereas serine/threonine kinase activators had no effect on basal Nurr1 mRNA levels. Computer analysis of osteoblastic promoters indicated two potential NBREs in the rat osteocalcin (Ocn) promoter. Intriguingly, the proximal site maps to the cAMP-responsive cis-element. We tested whether Nurr1 induces Ocn expression through the NBRE-like site. Recombinant and endogenous Nurr1 protein from primary mouse osteoblasts bound to a consensus NBRE in EMSAs. Nurr1 induced a consensus 3 x NBRE-luciferase reporter construct in mouse osteoblasts. Recombinant and endogenous Nurr1 protein bound to the proximal NBRE-like site in the Ocn promoter in EMSAs. Endogenous Nurr1 protein bound to this site as a monomer, because neither retinoid X receptor alpha nor retinoid X receptor beta antibody supershifted the protein-DNA complex. Ocn promoter-luciferase constructs lacking or containing a mutated proximal NBRE-like site had markedly blunted responses to Nurr1 overexpression. Finally, adenovirally expressed Nurr1 protein bound to the proximal NBRE-like site in chromatin immunoprecipitation assays and induced Ocn mRNA in primary rat osteoblasts. We conclude that Ocn is a Nurr1 target gene, which positions Nurr1 in the core of transcriptional factors regulating osteoblastic gene expression.
Division of Diagnostic and Surgical Sciences and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, CA 90095, USA.
Flavia Q
FQ
Pirih
Alan
A
Tang
Ibrahim C
IC
Ozkurt
Jeanne M
JM
Nervina
Sotirios
S
Tetradis
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D007763:Lac Operon; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D010006:Osteoblasts / Q000378:metabolism*; D015675:Osteocalcin / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry; D020218:Response Elements; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D012984:Software; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; C539882:NR4A2 protein, human; C539883:Nr4a2 protein, mouse; C539884:Nr4a2 protein, rat; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D010281:Parathyroid Hormone; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014157:Transcription Factors; D015675:Osteocalcin; D000242:Cyclic AMP; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C