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Molecular and Cellular Biology, September 1998, p. 5199-5207, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Induction of Differentiation in Normal Human Keratinocytes by Adenovirus-Mediated Introduction of the eta  and delta  Isoforms of Protein Kinase C

Motoi Ohba,1 Keiko Ishino,1 Mariko Kashiwagi,2 Shoko Kawabe,3 Kazuhiro Chida,4 Nam-Ho Huh,5 and Toshio Kuroki2,*

Department of Microbiology, School of Pharmaceutical Sciences,1 and Institute of Molecular Oncology,2 Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Mitsubishi Kasei Institute of Life Science, Machida, Tokyo 194,3 Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639,4 and Department of Biochemistry, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Sugitani, Toyama-shi 930-0194,5 Japan

Received 23 February 1998/Returned for modification 13 April 1998/Accepted 29 June 1998

Protein kinase C (PKC) plays a crucial role(s) in regulation of growth and differentiation of cells. In the present study, we examined possible roles of the alpha , delta , eta , and zeta  isoforms of PKC in squamous differentiation by overexpressing these genes in normal human keratinocytes. Because of the difficulty of introducing foreign genes into keratinocytes, we used an adenovirus vector system, Ax, which allows expression of these genes at a high level in almost all the cells infected for at least 72 h. Increased kinase activity was demonstrated in the cells overexpressing the alpha , delta , and eta  isoforms. Overexpression of the eta  isoform inhibited the growth of keratinocytes of humans and mice in a dose (multiplicity of infection [MOI])-dependent manner, leading to G1 arrest. The eta -overexpressing cells became enlarged and flattened, showing squamous cell phenotypes. Expression and activity of transglutaminase 1, a key enzyme of squamous cell differentiation, were induced in the eta -overexpressing cells in dose (MOI)- and time-dependent manners. The inhibition of growth and the induction of transglutaminase 1 activity were found only in the cells that express the eta  isoform endogenously, i.e., in human and mouse keratinocytes but not in human and mouse fibroblasts or COS1 cells. A dominant-negative eta  isoform counteracted the induction of transglutaminase 1 by differentiation inducers such as a phorbol ester, 1alpha ,25-dihydroxyvitamin D3, and a high concentration of Ca2+. Among the isoforms examined, the delta  isoform also inhibited the growth of keratinocytes and induced transglutaminase 1, but the alpha  and zeta  isoforms did not. These findings indicate that the eta  and delta  isoforms of PKC are involved crucially in squamous cell differentiation.

* Corresponding author. Mailing address: Institute of Molecular Oncology, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Phone: 81-3-3784-8145. Fax: 81-3-3784-2299. E-mail: tkuroki{at}med.showa-u.ac.jp.

Molecular and Cellular Biology, September 1998, p. 5199-5207, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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