RESUMO
Melanoma differentiation-associated gene 7 (MDA-7/IL-24) exhibits cytotoxic effects on tumor cells while sparing untransformed cells, and Bcl-x(L) is reported to efficiently block the induction of cell death by MDA-7/IL-24. The expression of Bcl-x(L) is regulated at the level of RNA splicing via alternative 5' splice site selection within exon 2 to produce either the pro-apoptotic Bcl-x(s) or the anti-apoptotic Bcl-x(L). Our laboratory previously reported that Bcl-x RNA splicing is dysregulated in a large percentage of human non-small cell lung cancer (NSCLC) tumors. Therefore, we investigated whether the alternative RNA splicing of Bcl-x pre-mRNA was modulated by MDA-7/IL-24, which would suggest that specific NSCLC tumors are valid targets for this cytokine therapy. Adenovirus-delivered MDA-7/IL-24 (Ad.mda-7) reduced the viability of NSCLC cells of varying oncogenotypes, which was preceded by a decrease in the ratio of Bcl-x(L)/Bcl-x(s) mRNA and Bcl-x(L) protein expression. Importantly, both the expression of Bcl-x(L) and the loss of cell viability were "rescued" in Ad.mda-7-treated cells incubated with Bcl-x(s) siRNA. In addition, NSCLC cells ectopically expressing Bcl-x(s) exhibited significantly reduced Bcl-x(L) expression, which was again restored by Bcl-x(s) siRNA, suggesting the existence of a novel mechanism by which Bcl-x(s) mRNA restrains the expression of Bcl-x(L). In additional mechanistic studies, inhibition of SRC and PKCδ completely ablated the ability of MDA-7/IL-24 to reduce the Bcl-x(L)/(s) mRNA ratio and cell viability. These findings show that Bcl-x(s) expression is an important mediator of MDA-7/IL-24-induced cytotoxicity requiring the SRC/PKCδ signaling axis in NSCLC cells.
Assuntos
Processamento Alternativo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Interleucinas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteína Quinase C-delta/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Transdução de Sinais , Proteína bcl-X/metabolismo , Células A549 , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Interleucinas/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteína Quinase C-delta/genética , Proteínas Proto-Oncogênicas pp60(c-src)/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Proteína bcl-X/genéticaRESUMO
Keratinocytes undergo a distinct pattern of proliferation and differentiation that is essential for the function of the skin as a protective barrier. Defects in the equilibrium between proliferation and differentiation compromise the skin's barrier function and give rise to human diseases such as psoriasis and nonmelanoma skin cancer. The identification of protein kinase C (PKC) as a major cellular target for tumor-promoting phorbol esters suggested the involvement of this enzyme in the regulation of keratinocyte proliferation and tumorigenesis; however, results have demonstrated the existence in keratinocytes and other cell types of another diacylglycerol/phorbol ester-responsive protein kinase: protein kinase D (PKD) in mouse, also known as PKC micro in humans. Although numerous data suggest the importance of PKD/PKC micro in processes related to proliferation in many cell types, including keratinocytes, there are no specific inhibitors of PKD currently available. Current treatment strategies for hyperproliferative skin disorders are often suboptimal, either because of lack of efficacy or because of contraindications due to deleterious side effects or aesthetic considerations. Thus, PKD/PKC micro may represent a novel target for the development of new and more efficacious drug treatments for hyperproliferative skin disorders.
Assuntos
Queratinócitos/patologia , Proteína Quinase C/fisiologia , Animais , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/farmacologia , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/enzimologia , Camundongos , Proteína Quinase C/antagonistas & inibidores , Dermatopatias/tratamento farmacológico , Dermatopatias/patologiaRESUMO
Recent evidence has shown a role for the serine/threonine protein kinase D (PKD) in the regulation of acute aldosterone secretion upon angiotensin II (AngII) stimulation. However, the mechanism by which AngII activates PKD remains unclear. In this study, using both pharmacological and molecular approaches, we demonstrate that AngII-induced PKD activation is mediated by protein kinase C (PKC) and Src family kinases in primary bovine adrenal glomerulosa cells and leads to increased aldosterone production. The pan PKC inhibitor Ro 31-8220 and the Src family kinase inhibitors PP2 and Src-1 inhibited both PKD activation and acute aldosterone production. Additionally, like the dominant-negative serine-738/742-to-alanine PKD mutant that cannot be phosphorylated by PKC, the dominant-negative tyrosine-463-to-phenylalanine PKD mutant, which is not phosphorylatable by the Src/Abl pathway, inhibited acute AngII-induced aldosterone production. Taken together, our results demonstrate that AngII activates PKD via a mechanism involving Src family kinases and PKC, to underlie increased aldosterone production.
Assuntos
Aldosterona/biossíntese , Angiotensina II/farmacologia , Proteína Quinase C/metabolismo , Quinases da Família src/metabolismo , Adulto , Animais , Bovinos , Ativação Enzimática/efeitos dos fármacos , Humanos , Indóis , Proteínas Mutantes/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Proteína Quinase C/antagonistas & inibidores , Pirimidinas/farmacologia , Zona Fasciculada/enzimologia , Zona Glomerulosa/enzimologia , Quinases da Família src/antagonistas & inibidoresRESUMO
Two splice variants derived from the Bcl-x gene via alternative 5' splice site selection (5'SS) are proapoptotic Bcl-x(s) and antiapoptotic Bcl-x(L). Previously, our laboratory showed 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 prosurvival signaling pathways that regulate the 5'SS selection of Bcl-x pre-mRNA in cancer cells. Taking a broad-based approach by using 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 phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (50 µmol/L) or the pan-protein kinase C (PKC) inhibitor Gö6983 (25 µmol/L) decreased the Bcl-x(L)/(s) mRNA ratio. Pan-PKC inhibitors that did not target the atypical PKCs, PKCι and PKCζ, had no effect on the Bcl-x(L)/(s) mRNA ratio. Additional studies showed that downregulation of the proto-oncogene, PKCι, in contrast to PKCζ, also resulted in a decrease in the Bcl-x(L)/(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. Finally, 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.
Assuntos
Processamento Alternativo , Carcinoma Pulmonar de Células não Pequenas , Isoenzimas , Fosfatidilinositol 3-Quinases , Proteína Quinase C , Proteína bcl-X , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Ceramidas/metabolismo , Cromonas/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Indóis/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Maleimidas/farmacologia , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proto-Oncogene Mas , Sítios de Splice de RNA/genética , Fatores de Processamento de RNA , RNA Interferente Pequeno , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Transdução de Sinais , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Dysregulation 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 (24h) 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 constitutively active 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.
Assuntos
Aldosterona/metabolismo , Angiotensina II/farmacologia , Proteína Quinase C/metabolismo , Adenoviridae/genética , Hormônio Adrenocorticotrópico/farmacologia , Animais , Benzimidazóis/farmacologia , Compostos de Bifenilo , Bovinos , Ativação Enzimática/efeitos dos fármacos , Imidazóis/farmacologia , Proteínas Mutantes/metabolismo , Potássio/farmacologia , Piridinas/farmacologia , Receptor Tipo 1 de Angiotensina/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Tetrazóis/farmacologia , Fatores de Tempo , Zona Glomerulosa/citologia , Zona Glomerulosa/efeitos dos fármacos , Zona Glomerulosa/enzimologia , Zona Glomerulosa/metabolismoRESUMO
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 PKCalpha, and for protein kinase D (PKD; also known as PKCmu). We report that Gö6976 stimulated transglutaminase activity. Consistent with this effect, Gö6976 also potently inhibited [(3)H]thymidine incorporation (a half-maximal inhibitory concentration of approximately 0.1 microM). In addition, Gö6976 (1 microM) 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, Gö6983 [2-[1-(3-dimethylaminopropy)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide], a similar compound that also selectively inhibits conventional PKCalpha, but not PKD, had little or no effect on DNA synthesis or transglutaminase activity (up to 1 microM). 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.