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Rat Calvarial Cell Lines Immortalized with SV-40 Large T Antigen: Constitutive and Retinoic Acid- Inducible Expression of Osteoblastic Features

Department of Bone Biology and Osteoporosis Research, Merck, Sharp, and Dohme Research Laboratories West Point, Pennsylvania 19486

Abstract

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 µM retinoic acid: alkaline phosphatase activity increased from 0.003 to 0.25 µmol/min · mg protein, the steady state level of type I procollagen mRNA increased 4-fold, and the cells acquired a PTH-stimulatable adenylate cyclase (EC₆₀,10 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 (EC₆₀, 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. (Endocrinology 124: 3060-3069,1989)

Footnotes

^* This work was supported by Grant 3'576-0.87 from the Swiss National Science Foundation, by Contract 1451 from the Ares Serono group.

Received December 12, 1988.

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