Abstract
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 PKCα, 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 PKCα, 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.
Footnotes
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This work was supported in part by National Institutes of Health (National Institute of Arthritis & Musculoskeletal & Skin Diseases) Grant AR45212. Presented in part at the 61st meeting of the Society for Investigative Dermatology in May 10–14, 2000 in Chicago, IL.
- Abbreviations:
- PKC
- protein kinase C
- Stsp
- staurosporine
- PKD
- protein kinase D
- D3
- 1,25-dihydroxyvitamin D3
- BisI
- bisindolylmaleimide I
- SFKM
- serum-free keratinocyte media
- PBS−
- phosphate-buffered saline lacking divalent cations
- DMSO
- dimethyl sulfoxide
- TRK A
- tyrosine kinase A
- NGF
- nerve growth factor
- Ro 31-8220
- 3-[1-[3-(amidinothio)propyl-1H-indol]-3-(1-methyl-1H-indol-3-yl)maleimide
- Gö6983
- 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide
- Gö6976
- 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole
- BisI
- bisindolylmaleimide
- Received December 20, 2001.
- Accepted March 18, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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