title: BMP-2 vs. BMP-4 expression and activity in glucocorticoid-arrested MC3T3-E1 osteoblasts: Smad signaling, not alkaline phosphatase activity, predicts rescue of mineralization.
abstract: Pharmacological glucocorticoids (GCs) inhibit bone formation, leading to osteoporosis. GCs inhibit bone morphogenetic protein-2 (Bmp2) expression, and rhBMP-2 restores mineralization in GC-arrested osteoblast cultures. To better understand how GCs regulate BMPs, we investigated Bmp transcription, as well as rhBMP-induced Smad and alkaline phosphatase (ALP) activity. Bmp2 cis-regulatory regions were analyzed by reporter plasmids and LacZ-containing bacterial artificial chromosomes. We found that GCs inhibited Bmp2 via a domain > 50 kb downstream of the coding sequence. Bmp expression was evaluated by RT-PCR; whereas GCs strongly inhibited Bmp2, Bmp4 was abundantly expressed and resistant to GCs. Both rhBMP-2 and rhBMP-4 restored mineralization in GC-arrested cultures; rhBMP-2 was 5-fold more effective when dosing was based on ALP activation, however, the rhBMPs were equipotent when dosing was based on Smad transactivation. In conclusion, GCs regulate Bmp2 via a far-downstream domain, and activation of Smad, not ALP, best predicts the pro-mineralization potential of rhBMPs.
authors.affiliation: Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, Institute for Genetic Medicine, Los Angeles, CA 90033, USA.
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Luppen
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Chandler
authors.forename: Tommy
authors.initials: T
authors.lastname: Noh
authors.forename: Douglas P
authors.initials: DP
authors.lastname: Mortlock
authors.forename: Baruch
authors.initials: B
authors.lastname: Frenkel
mesh_terms: D016475:3T3 Cells; D000469:Alkaline Phosphatase / Q000032:analysis / Q000378:metabolism; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000378:metabolism*; D055415:Bone Morphogenetic Protein 4 / Q000235:genetics / Q000378:metabolism*; D002113:Calcification, Physiologic / Q000187:drug effects* / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects*; D017930:Genes, Reporter; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis / Q000378:metabolism
chemical_list: D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D005938:Glucocorticoids; D011994:Recombinant Proteins; D008156:Luciferases; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase