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...

Our previous studies demonstrated that protein kinase D (PKD), a serine/threonine kinase implicated in various cell processes, is up-regulated in basal cell carcinoma (BCC), supporting a possible tumorigenic role for PKD in skin. Since the greatest risk factor for BCC is sun exposure, the ability of ultraviolet B (UVB) irradiation to activate PKD in primary mouse keratinocytes was investigated. Using western analysis with two autophosphorylation-specific antibodies, we show for the first time that UVB activated PKD in a time- and dose-dependent manner. UVB-induced PKD activation was verified using an in vitro kinase assay. Furthermore, activation was reduced by antioxidant pretreatment, suggesting a link with oxidative stress. UVB-induced PKD activation was mediated primarily by Src family tyrosine kinases rather than protein kinase C (PKC), and in fact, UVB did not alter PKC-mediated transphosphorylation. UVB induced apoptosis dose-dependently, and this death could be prevented by overexpression of wild-type PKD, but not mutant PKD or the empty adenovirus. Indeed, a mutant that cannot be phosphorylated by Src kinases exacerbated UVB-elicited apoptosis. Thus, our data indicate that UVB irradiation of keratinocytes induces Src-mediated activation of PKD, which protects cells from UVB-stimulated apoptosis, providing a possible explanation for the observed up-regulation of PKD in BCC.

SummaryDysregulation of the renin-angiotensin II (AngII)-aldosterone system can contribute to cardiovascular disease, such that an understanding of this system is critical. Diacylglycerol-sensitive serine/threonine protein kinase D (PKD) is activated by AngII in several systems, including the human adrenocortical carcinoma cell line NCI H295R, where this enzyme enhances chronic (24 hours) AngII-evoked aldosterone secretion. However, the role of PKD in acute AngII-elicited aldosterone secretion has not been previously examined. In primary cultures of bovine adrenal glomerulosa cells, which secrete detectable quantities of aldosterone in response to secretagogues within minutes, PKD was activated in response to AngII, but not an elevated potassium concentration or adrenocorticotrophic hormone. This activation was time-and dose-dependent and occurred through the AT1, but not the AT2, receptor. Adenovirus-mediated overexpression of constitutivelyactive PKD resulted in enhanced AngII-induced aldosterone secretion; whereas overexpression of a dominant-negative PKD construct decreased AngII-stimulated aldosterone secretion. Thus, we demonstrate for the first time that PKD mediates acute AngII-induced aldosterone secretion.

Two splice variants derived from the BCL-x gene via alternative 5′ splice site selection (5′SS) are pro-apoptotic Bcl-x(s) and anti-apoptotic Bcl-x(L). Previously, our laboratory demonstrated that apoptotic signaling pathways regulated the alternative 5′SS selection via protein phosphatase-1 and de novo ceramide. In this study, we examined the elusive pro-survival signaling pathways that regulate the 5′SS selection of Bcl-x pre-mRNA in cancer cells. Taking a broad-based approach by utilizing a number of small molecule inhibitors of various mitogenic/survival pathways, we found that only treatment of non-small cell lung cancer (NSCLC) cell lines with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (50 μM) or the pan-PKC inhibitor GÖ6983 (25 μM) decreased the Bcl-x(L)/Bcl-x(s) mRNA ratio. Pan-PKC inhibitors that did not target the atypical PKCs, PKCι and PKCζ, had no effect on the Bcl-x(L)/Bcl-x(s) mRNA ratio. Additional studies demonstrated that downregulation of the proto-oncogene, PKCι, in contrast to PKCζ, also resulted in a decrease in the Bcl-x(L)/Bcl-x(s) mRNA ratio. Furthermore, downregulation of PKCι correlated with a dramatic decrease in the expression of SAP155, an RNA trans-acting factor that regulates the 5′SS selection of Bcl-x pre-mRNA. Inhibition of the PI3K or atypical PKC pathway induced a dramatic loss of SAP155 complex formation at ceramide-responsive RNA cis-element 1. Lastly, forced expression of Bcl-x(L) “rescued” the loss of cell survival induced by PKCι siRNA. In summary, the PI3K/PKCι regulates the alternative splicing of Bcl-x pre-mRNA with implications in the cell survival of NSCLC cells.