Rat calvarial cell lines immortalized with SV-40 large T antigen: constitutive and retinoic acid-inducible expression of osteoblastic features.
Two new bone cell lines were established by immortalizing cells derived from embryonic rat calvariae with a recombinant retrovirus containing the cDNA for SV-40 large T antigen and the neomycin resistance gene. One cell line, RCT-1, isolated from early digest cells, a population which typically does not express osteoblastic features, displayed osteoblastic characteristics only after 3 days of treatment with 1 microM retinoic acid: alkaline phosphatase activity increased from 0.003 to 0.25 mumol/min.mg protein, the steady state level of type I procollagen mRNA increased 4-fold, and the cells acquired a PTH-stimulatable adenylate cyclase (EC50, 10 nM). mRNA for osteopontin, an abundant bone matrix protein, was induced in RCT-1 cells by 1,25-dihydroxyvitamin D3 (10 nM). The second cell line, RCT-3, isolated from late digest cells, a population previously shown to be enriched with differentiated osteoblasts, expressed constitutively the properties described above. In addition, RCT-3 cells responded to interleukin-1 by increased prostaglandin production (EC50, 20 pM) and to prostaglandin E2 by enhanced cAMP accumulation, features exhibited by calvarial cells in organ culture. Thus, the SV-40 immortalized cell lines we describe retained many of the characteristics of osteoblasts in primary culture, including hormonal regulation of phenotype-related genes. In RCT-1 cells the coordinate induction of several properties by retinoic acid offers a new model for the study of differentiation-related gene expression in bone cells.
Heath
J K
JK
Department of Bone Biology and Osteoporosis Research, Merck, Sharp, and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Rodan
S B
SB
Yoon
K
K
Rodan
G A
GA
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D001842:Bone and Bones; D002116:Calcitonin / Q000494:pharmacology; D002460:Cell Line; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics; D007545:Isoproterenol / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects; D053495:Osteopontin; D010281:Parathyroid Hormone / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D012795:Sialoglycoproteins / Q000096:biosynthesis / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics*; D019379:Teriparatide; D014212:Tretinoin / Q000494:pharmacology*
D000952:Antigens, Polyomavirus Transforming; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D010750:Phosphoproteins; D012333:RNA, Messenger; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D053495:Osteopontin; D019379:Teriparatide; D014212:Tretinoin; D002116:Calcitonin; D003094:Collagen; D000262:Adenylyl Cyclases; D007545:Isoproterenol; D015056:1-Methyl-3-isobutylxanthine