Vol. 302, Issue 1, 352-358, July 2002

Putative Conventional Protein Kinase C Inhibitor Gödecke 6976 [12-(2-Cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] Stimulates Transglutaminase Activity in Primary Mouse Epidermal Keratinocytes

Brian A. Shapiro, Sagarika Ray, EunMi Jung, William T. Allred and Wendy B. Bollag

Program in Cell Signaling, Department of Medicine (B.A.S., S.R., E.M.J., W.T.A., W.B.B), Institute of Molecular Medicine and Genetics, and (W.B.B.)Department of Cellular Biology and Anatomy , Medical College of Georgia, Augusta, Georgia

Much data in the literature suggest a role for protein kinase C (PKC) in regulating keratinocyte proliferation and differentiation. Nevertheless, the exact role of this family of isoenzymes is unclear, since PKC agonists (e.g., phorbol esters) are known to stimulate expression of both proliferative and differentiative markers in keratinocytes. Similarly, PKC inhibitors have been demonstrated both to inhibit [2-[1-3(aminopropyl)indol-3-yl]-3(1-methyl-1H-indol-3-yl)maleimide, acetate (Ro 31-7549) and 3-[1-[3-(amidinothio)propyl-1H-indol-3-(1-methyl-1H-indol-3yl) maleimide (Ro 31-8220)] and to induce (staurosporine) keratinocyte differentiation. In this study, we examined the role of the PKC inhibitor, Gödecke 6976 (Gö6976) [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo (3,4-c)-carbazole], on keratinocyte proliferation, as measured by DNA synthesis, and differentiation, as monitored by transglutaminase activity. This compound is reported to be selective for the conventional PKC isoforms, of which keratinocytes express only PKC, and for protein kinase D (PKD; also known as PKCµ). We report that Gö6976 stimulated transglutaminase activity. Consistent with this effect, Gö6976 also potently inhibited [³H]thymidine incorporation (a half-maximal inhibitory concentration of ~0.1 µM). In addition, Gö6976 (1 µM) was able to enhance the stimulation of transglutaminase activity by 1,25-dihydroxyvitamin D₃ but had no effect on D₃-induced expression of keratin-1. Conversely, Gö6983 [2-[1-(3-dimethylaminopropy)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide], a similar compound that also selectively inhibits conventional PKC, but not PKD, had little or no effect on DNA synthesis or transglutaminase activity (up to 1 µM). The effect of Gö6976 was not due to cytotoxicity as its effect on thymidine incorporation was largely reversible, and its stimulation of transglutaminase activity could be inhibited by another general PKC inhibitor, bisindolylmaleimide I. Therefore, our results suggest a proproliferative, antidifferentiative role for PKD in epidermal maturation.