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

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, Gö decke 6976 (Gö 6976) [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 PKC). We report that Gö 6976 stimulated transglutaminase activity. Consistent with this effect, Gö 6976 also potently inhibited [3 H]thymidine incorporation (a half-maximal inhibitory concentration of ϳ0.1 M). In addition, Gö 6976 (1 M) was able to enhance the stimulation of transglutaminase activity by 1,25-dihydroxyvitamin D 3 but had no effect on D 3 -induced expression of keratin-1. Conversely, Gö 6983 [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 M). The effect of Gö 6976 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.The epidermis is composed primarily of epidermal keratinocytes, which continuously proliferate and differentiate to maintain this important tissue. Keratinocyte differentiation is characterized by a spatially and temporally regulated program of gene and protein expression, which ultimately results in terminal differentiation and cell death. This program of differentiation is essential for the function of the epidermis as a barrier to water loss, microbial invasion, and mechanical stress. Despite the importance of keratinocyte differentiation to epidermal structure, the signaling pathways that regulate this process are not well understood. Numerous data in the literature indicate a role for PKC in keratinocyte differentiation; however, the exact role of this enzyme is at present unclear (reviewed in Bollag and Bollag, 2001). Thus, PKC-activating phorbol esters elicit events associated paradoxically both with differentiati...

Phospholipase D Signaling and Extracellular Signal-Regulated Kinase-1 and -2 Phosphorylation (Activation) Are Required for Maximal Phorbol Ester-Induced Transglutaminase Activity, a Marker of Keratinocyte Differentiation

“…Similarly, we (Shapiro et al, 2002) and others (Rennecke et al, 1999) have shown that Gö 6976, but not Gö 6983, inhibits DNA synthesis in keratinocytes as well. Furthermore, we have proposed that PKD serves as a proproliferative and/or antidifferentiative signaling enzyme in these cells (Shapiro et al, 2002; M. E. Dodd, V. L. Ristich, S. Ray, R. M. Lober, and W. B. Bollag, manuscript submitted for publication; for review, see Bollag et al, 2004). Therefore, it is possible that Fig.…”

“…Nevertheless, the lack of specificity of these inhibitors complicates the interpretation of our results. Thus, for example, Gö 6976, but not Gö 6983, BisI, or Ro 31-8220, is known to inhibit the phorbol ester-responsive serine/threonine protein kinase protein kinase D (discussed in Shapiro et al, 2002). Indeed, we have previously shown that Gö 6976, but not the other PKC inhibitors mentioned above, stimulates transglutaminase activity and enhances the effect of 1,25-dihydroxyvitamin D 3 in keratinocytes (Shapiro et al, 2002).…”

“…Thus, for example, Gö 6976, but not Gö 6983, BisI, or Ro 31-8220, is known to inhibit the phorbol ester-responsive serine/threonine protein kinase protein kinase D (discussed in Shapiro et al, 2002). Indeed, we have previously shown that Gö 6976, but not the other PKC inhibitors mentioned above, stimulates transglutaminase activity and enhances the effect of 1,25-dihydroxyvitamin D 3 in keratinocytes (Shapiro et al, 2002). Similarly, we (Shapiro et al, 2002) and others (Rennecke et al, 1999) have shown that Gö 6976, but not Gö 6983, inhibits DNA synthesis in keratinocytes as well.…”

“…Keratinocytes prelabeled with 2.5 Ci/ml [ 3 H]oleate in SFKM for 20 to 24 h were incubated with SFKM or SFKM containing TPA with and without inhibitors in the presence of 0.5% ethanol for 30 min. Inhibitor concentrations were selected based on previous data from our laboratory Shapiro et al, 2002), and all samples contained the same concentration of vehicle (dimethyl sulfoxide; DMSO). Phospholipids were extracted with chloroform/methanol and separated by thin layer chromatography as described in Jung et al (1999).…”

“…We have also recently proposed that TPA activates another phorbol ester/ diacylglycerol-responsive protein kinase distinct from PKC with proproliferative (or antidifferentiative) activity (for review, see Bollag et al, 2004). The enzyme suggested to occupy this role in keratinocytes is protein kinase D (also known as PKC-), based on recent pharmacological (Shapiro et al, 2002) and transient cotransfection data (M. E. Dodd, V. L. Ristich, S. Ray, R. M. Lober, and W. B. Bollag, manuscript submitted for publication) in keratinocytes, as well as information in the literature indicating a mitogenic role for this enzyme in fibroblasts and other cell types (for review, see Bollag et al, 2004). On the other hand, TPA may activate additional enzymes other than protein kinases, such as the Ras GTP-exchange protein RasGRP (for review, see Brose and Rosenmund, 2002), involved in keratinocyte growth and differentiation (Rambaratsingh et al, 2003).…”