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(Circulation Research. 1996;78:395-404.)
© 1996 American Heart Association, Inc.

Articles

Preferential Differentiation of P19 Mouse Embryonal Carcinoma Cells Into Smooth Muscle Cells

Use of Retinoic Acid and Antisense Against the Central Nervous System–Specific POU Transcription Factor Brn-2

Toru Suzuki, Hyo-Soo Kim, Masahiko Kurabayashi, Hiroshi Hamada, Hideta Fujii, Masanori Aikawa, Masafumi Watanabe, Noboru Watanabe, Yasunari Sakomura, Yoshio Yazaki, Ryozo Nagai

From the Third Department of Internal Medicine (T.S., H.-S.K., M.K., M.A., M.W., N.W., Y.S., Y.Y., R.N.), University of Tokyo (Japan) and the Department of Developmental Biology and Cancer Prevention (H.H., H.F.), Tokyo Metropolitan Institute for Medical Science.

Correspondence to Ryozo Nagai, MD, Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-15, Showa-machi, Maebashi, Gunma 371, Japan. E-mail nagai@sb.gunma-u.ac.jp.

Abstract Investigation of the molecular mechanisms that control smooth muscle cell (SMC) development and differentiation is a prerequisite in understanding the regulatory mechanisms of physiological and pathological SMC-associated vascular processes. The pluripotent murine embryonal carcinoma P19 cell, whose developmental potential resembles that of early embryonic cells, can develop into cell types derived from the neuroectoderm, mesoderm, and endoderm. In the present study, we have shown a unique strategy to enhance SMC differentiation in P19 cells. Under chemical induction of high concentrations of retinoic acid (1 µmol/L), P19 cells showed optimum differentiation into SMCs. Because the P19 cells thus induced also showed differentiation into neuronal cells, a strategy to block neuronal lineage differentiation was developed using a stable transformant antisense RNA construct against Brn-2, a neuronal lineage–specific POU-domain transcription factor; thus, by specifically inhibiting neuronal differentiation, enhanced SMC differentiation by P19 cells was attained. SMC expression was confirmed by immunohistochemical staining, RNA analysis (RNase protection assay), and protein analysis (Western blot) using SMC-specific markers (eg, SM1 and calponin) and -smooth muscle actin. Our results show that the pathway of SMC differentiation may provide an in vitro system useful in the investigation of SMC regulatory mechanisms (eg, transcriptional regulation) and in the further understanding of SMC development and differentiation.

Key Words: P19 cells • smooth muscle cells • retinoic acid • transcription factors

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