Phorbol esters, the activators of protein kinase C (PKC), induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. The role of individual PKC isozymes as mediators of this effect has not been thoroughly examined to date. To study the involvement of the novel isozyme PKC␦, we used a replication-deficient adenovirus (PKC␦AdV), which allowed for a tightly controlled expression of PKC␦ in LNCaP cells. A significant reduction in cell number was observed after infection of LNCaP cells with PKC␦AdV. Overexpression of PKC␦ markedly enhanced the apoptotic effect of phorbol 12-myristate 13-acetate in LNCaP cells. PKC␦-mediated apoptosis was substantially reduced by the pan-caspase inhibitor z-VAD and by Bcl-2 overexpression. Importantly, and contrary to other cell types, PKC␦-mediated apoptosis does not involve its proteolytic cleavage by caspase-3, suggesting that allosteric activation of PKC␦ is sufficient to trigger apoptosis in LNCaP cells. In addition, phorbol ester-induced apoptosis was blocked by a kinase-deficient mutant of PKC␦, supporting the concept that PKC␦ plays an important role in the regulation of apoptotic cell death in LNCaP prostate cancer cells.

Inactivation of protein kinase Cdelta (PKCdelta) is associated with resistance to terminal cell death in epidermal tumor cells, suggesting that activation of PKCdelta in normal epidermis may be a component of a cell death pathway. To test this hypothesis, we constructed an adenovirus vector carrying an epitope-tagged PKCdelta under a cytomegalovirus promoter to overexpress PKCdelta in normal and neoplastic keratinocytes. While PKCdelta overexpression was detected by immunoblotting in keratinocytes, the expression level of other PKC isozymes, including PKCalpha, PKCepsilon, PKCzeta, and PKCeta, did not change. Calcium-independent PKC-specific kinase activity increased after infection of keratinocytes with the PKCdelta adenovirus. Activation of PKCdelta by 12-O-tetradecanoylphorbol-13-acetate (TPA) at a nanomolar concentration was lethal to normal and neoplastic mouse and human keratinocytes overexpressing PKCdelta. Lethality was inhibited by PKC selective inhibitors, GF109203X and Ro-32-0432. TPA-induced cell death was apoptotic as evidenced by morphological criteria, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, DNA fragmentation, and increased caspase activity. Subcellular fractionation indicated that PKCdelta translocated to a mitochondrial enriched fraction after TPA activation, and this finding was confirmed by confocal microscopy of cells expressing a transfected PKCdelta-green fluorescent protein fusion protein. Furthermore, activation of PKCdelta in keratinocytes altered mitochondrial membrane potential, as indicated by rhodamine-123 fluorescence. Mitochondrial inhibitors, rotenone and antimycin A, reduced TPA-induced cell death in PKCdelta-overexpressing keratinocytes. These results indicate that PKCdelta can initiate a death pathway in keratinocytes that involves direct interaction with mitochondria and alterations of mitochondrial function.

Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCdelta and PKCepsilon from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCdelta and PKCepsilon were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCalpha enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCdelta and PKCepsilon provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell survival pathways in response to external stimuli.

The role of intracellular Ca2+ in the regulation of Ca(2+)-induced terminal differentiation of mouse keratinocytes was investigated using the intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA). A cell permeable acetoxymethyl (AM) ester derivative BAPTA (BAPTA/AM) was loaded into primary mouse keratinocytes in 0.05 mM Ca2+ medium, and then the cells were induced to differentiate by medium containing 0.12 or 0.5 mM Ca(2+) Intracellular BAPTA loaded by BAPTA/AM (15-30 microM) inhibited the expression of epidermal differentiation-specific proteins keratin 1 (K1), keratin 10 (K10), filaggrin and loricrin as detected by immunoblotting. The differentiation-associated redistribution of E-cadherin on the cell membrane was delayed but not inhibited as determined by immunofluorescence. BAPTA also inhibited the expression of K1, K10 and loricrin mRNA. Furthermore, BAPTA prevented the decrease in DNA synthesis induced by 0.12 and 0.5 mM Ca2+, indicating the drug was inhibiting differentiation but was not toxic to keratinocytes. To evaluate the influence of BAPTA on intracellular Ca2+, the concentration of intracellular free Ca2+ (Cai) in BAPTA-loaded keratinocytes was examined by digital image analysis using the Ca(2+)-sensitive fluorescent probe fura-2, and Ca2+ influx was measured by 45Ca2+ uptake studies. Increase in extracellular Ca2+ (Cao) in the culture medium of keratinocytes caused a sustained increase in both Cai and Ca2+ localized to ionomycin-sensitive intracellular stores in keratinocytes. BAPTA lowered basal Cai concentration and prevented the Cai increase. After 12 hours of BAPTA treatment, the basal level of Cai returned to the control value, but the Ca2+ localized in intracellular stores was substantially decreased. 45Ca2+ uptake was initially (within 30 min) increased in BAPTA-loaded cells. However, the total 45Ca2+ accumulation over 24 hours in BAPTA-loaded cells remained unchanged from control values. These results indicate that keratinocytes can maintain Cai and total cellular Ca2+ content in the presence of increased amount of intracellular Ca2+ buffer (e.g., BAPTA) by depleting intracellular Ca2+ stores over a long period. The inhibition by BAPTA of keratinocyte differentiation marker expression may result from depletion of the Ca(2+)-stores since this is the major change in intracellular Ca2+ detected at the time keratinocytes express the differentiation markers. In contrast, the redistribution of E-cadherin on the cell membrane may be more directly associated with Cai change.