RESUMO
Mutational activation of the Ras family of proto-oncogenes promotes cell survival and proliferation. Studies using cells cultured in vitro have shown that ectopic expression of constitutively active Ras suppresses apoptosis induced by serum deprivation. However, in some cellular contexts, constitutively active Ras exerts the opposite effects, including apoptosis of serum-starved embryonic fibroblasts. Such observations first came over two decades ago, but the molecular mechanisms by which mutant Ras increases the susceptibility of cells to serum deprivation leading to apoptosis are still not fully understood. To revisit this issue, I investigate the effects of serum depletion and mutant Ras expression on intracellular signaling and transcriptome of cells carrying an inducible allele of constitutively active mutant Hras (HrasG12V). I identify zinc ions (Zn2+) as a serum factor that suppresses proapoptotic signaling in cells expressing HrasG12V. Mechanistically, HrasG12V expression along with Zn2+ deficiency activates c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), which are required for caspase-3 activation involved in the induction of cell death. Transcriptome analyses suggest that HrasG12V induces the unfolded protein response (UPR). Further analyses of intracellular signaling biomolecules related to the UPR indicate that HrasG12V activates inositol-requiring protein 1 (IRE1), which synergizes with Zn2+ deficiency to activate JNK and p38 MAPK signaling. These results provide insights into a role of Zn2+ that counteracts proapoptotic signaling activated by mutationally activated Ras.
Assuntos
Transdução de Sinais , Zinco , Apoptose/genética , Fibroblastos , Íons/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Zinco/metabolismo , Zinco/farmacologiaRESUMO
Ras undergoes post-translational modifications including farnesylation, proteolysis, and carboxymethylation at the C terminus, which are necessary for membrane recruitment and effector recognition. Full activation of c-Raf-1 requires cooperative interaction of the farnesylated C terminus and the activator region of Ras with its cysteine-rich domain (CRD). However, the molecular basis for this interaction remains unclear because of difficulties in preparing modified Ras in amounts sufficient for structural studies. Here, we use Sortase A-catalyzed protein ligation to prepare modified Ras in sufficient amounts for NMR and X-ray crystallographic analyses. The results show that the farnesylated C terminus establishes an intramolecular interaction with the catalytic domain and brings the farnesyl moiety to the proximity of the activator region, which may be responsible for their cooperative recognition of c-Raf-1-CRD.
Assuntos
Guanosina Trifosfato/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas ras/química , Proteínas ras/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Ligação ProteicaRESUMO
Phospholipase Cϵ (PLCϵ), an effector of Ras and Rap small GTPases, plays a crucial role in inflammation by augmenting proinflammatory cytokine expression. This proinflammatory function of PLCϵ is implicated in its facilitative role in tumor promotion and progression during skin and colorectal carcinogenesis, although their direct link remains to be established. Moreover, the molecular mechanism underlying these functions of PLCϵ remains unknown except that PKD works downstream of PLCϵ. Here we show by employing the colitis-induced colorectal carcinogenesis model, where Apc(Min) (/+) mice are administered with dextran sulfate sodium, that PLCϵ knock-out alleviates the colitis and suppresses the following tumorigenesis concomitant with marked attenuation of proinflammatory cytokine expression. In human colon epithelial Caco2 cells, TNF-α induces sustained expression of proinflammatory molecules and sustained activation of nuclear factor-κB (NF-κB) and PKD, the late phases of which are suppressed by not only siRNA-mediated PLCϵ knockdown but also treatment with a lysophosphatidic acid (LPA) receptor antagonist. Also, LPA stimulation induces these events in an early time course, suggesting that LPA mediates TNF-α signaling in an autocrine manner. Moreover, PLCϵ knockdown results in inhibition of phosphorylation of IκB by ribosomal S6 kinase (RSK) but not by IκB kinases. Subcellular fractionation suggests that enhanced phosphorylation of a scaffolding protein, PEA15 (phosphoprotein enriched in astrocytes 15), downstream of the PLCϵ-PKD axis causes sustained cytoplasmic localization of phosphorylated RSK, thereby facilitating IκB phosphorylation in the cytoplasm. These results suggest the crucial role of the TNF-α-LPA-LPA receptor-PLCϵ-PKD-PEA15-RSK-IκB-NF-κB pathway in facilitating inflammation and inflammation-associated carcinogenesis in the colon.
Assuntos
Células Epiteliais/metabolismo , NF-kappa B/metabolismo , Fosfoinositídeo Fosfolipase C/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Células CACO-2 , Colite/genética , Colite/metabolismo , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Citoplasma/enzimologia , Humanos , Proteínas I-kappa B/metabolismo , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisofosfolipídeos/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidor de NF-kappaB alfa , Fosfoinositídeo Fosfolipase C/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Interferência de RNA , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: Phospholipase Cε (PLCε) is an effector of Ras and Rap small GTPases and expressed in non-immune cells. It is well established that PLCε plays an important role in skin inflammation, such as that elicited by phorbol ester painting or ultraviolet irradiation and contact dermatitis that is mediated by T helper (Th) 1 cells, through upregulating inflammatory cytokine production by keratinocytes and dermal fibroblasts. However, little is known about whether PLCε is involved in regulation of inflammation in the respiratory system, such as Th2-cells-mediated allergic asthma. METHODS: We prepared a mouse model of allergic asthma using PLCε+/+ mice and PLCεΔX/ΔX mutant mice in which PLCε was catalytically-inactive. Mice with different PLCε genotypes were immunized with ovalbumin (OVA) followed by the challenge with an OVA-containing aerosol to induce asthmatic response, which was assessed by analyzing airway hyper-responsiveness, bronchoalveolar lavage fluids, inflammatory cytokine levels, and OVA-specific immunoglobulin (Ig) levels. Effects of PLCε genotype on cytokine production were also examined with primary-cultured bronchial epithelial cells. RESULTS: After OVA challenge, the OVA-immunized PLCεΔX/ΔX mice exhibited substantially attenuated airway hyper-responsiveness and broncial inflammation, which were accompanied by reduced Th2 cytokine content in the bronchoalveolar lavage fluids. In contrast, the serum levels of OVA-specific IgGs and IgE were not affected by the PLCε genotype, suggesting that sensitization was PLCε-independent. In the challenged mice, PLCε deficiency reduced proinflammatory cytokine production in the bronchial epithelial cells. Primary-cultured bronchial epithelial cells prepared from PLCεΔX/ΔX mice showed attenuated pro-inflammatory cytokine production when stimulated with tumor necrosis factor-α, suggesting that reduced cytokine production in PLCεΔX/ΔX mice was due to cell-autonomous effect of PLCε deficiency. CONCLUSIONS: PLCε plays an important role in the pathogenesis of bronchial asthma through upregulating inflammatory cytokine production by the bronchial epithelial cells.
Assuntos
Asma/enzimologia , Brônquios/enzimologia , Hiper-Reatividade Brônquica/enzimologia , Células Epiteliais/enzimologia , Fosfoinositídeo Fosfolipase C/imunologia , Animais , Asma/induzido quimicamente , Asma/imunologia , Asma/patologia , Brônquios/imunologia , Brônquios/patologia , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/patologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Masculino , Camundongos , Camundongos Knockout , Ovalbumina , Fosfoinositídeo Fosfolipase C/deficiência , Fosfoinositídeo Fosfolipase C/genética , Cultura Primária de Células , Mucosa Respiratória/enzimologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Transdução de Sinais , Equilíbrio Th1-Th2/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Células Th2/metabolismo , Células Th2/patologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas rap de Ligação ao GTP/genética , Proteínas rap de Ligação ao GTP/imunologia , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/imunologiaRESUMO
Phospholipase Cε (PLCε) is a unique class of PLC regulated by both Ras family small GTPases and heterotrimeric G proteins. We previously showed by using mice bearing its null or transgenic allele that PLCε plays a crucial role in various forms of skin inflammation through upregulation of proinflammatory cytokine production from keratinocytes. However, molecular mechanisms how PLCε augments cytokine production were largely unknown. We show here using cultured human keratinocyte PHK16-0b cells that induction of the expression of chemokine (C-C motif) ligand 2 (CCL2) following stimulation with tumor necrosis factor (TNF)α, which primarily depends on the activation of the NF-κB pathway, is abrogated by small interfering RNA-mediated knockdown of PLCε. Enforced expression of PLCε causes substantial CCL2 expression and cooperates with low level TNFα stimulation to induce marked overexpression of CCL2, both of which are only partially blocked by pharmacological inhibition of the NF-κB signaling. However, PLCε knockdown exhibits no effect on both the NF-κB-cis-element-mediated transcription per se and the post-translational modifications of NF-κB implicated in transcriptional regulation, suggesting that PLCε constitutes a yet unknown signaling pathway distinct from the NF-κB pathway. This pathway can cooperate with the NF-κB pathway to achieve a synergistic TNFα-stimulated CCL2 induction in keratinocytes.
Assuntos
Quimiocina CCL2/biossíntese , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Fosfoinositídeo Fosfolipase C/metabolismo , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Queratinócitos/efeitos dos fármacos , Camundongos , Fosfoinositídeo Fosfolipase C/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Phospholipase C (PLC) É is a phosphoinositide-specific PLC regulated by small GTPases including Ras and Rap. We previously demonstrated that PLCÉ has an important role in the development of phorbol ester-induced skin inflammation. In this study, we investigated the role of PLCÉ in ultraviolet (UV) B-induced acute inflammatory reactions in the skin. Wild-type (PLCÉ+/+) and PLCÉ gene knockout (PLCÉâ»/â») mice were irradiated with a single dose of UVB at 1, 2.5, and 10 kJ/m² on the dorsal area of the skin, and inflammatory reactions in the skin were histologically evaluated up to 168 h after irradiation. In PLCÉ+/+ mice, irradiation with 1 and 2.5 kJ/m² UVB resulted in dose-dependent neutrophil infiltration in the epidermis at 24 and 48 h after irradiation. When mice were irradiated with 10 kJ/m² of UVB, most mice developed skin ulcers by 48 h and these ulcers became more severe at 168 h. In PLCÉâ»/â» mice, UVB (1 or 2.5 kJ/m²)-induced neutrophil infiltration was markedly suppressed compared with PLCÉ+/+ mice. The suppression of neutrophil infiltration in PLCÉâ»/â» mice was accompanied by attenuation of UVB-induced production of CXCL1/keratinocyte-derived chemokine (KC), a potent chemokine for neutrophils, in the whole skin. Cultured epidermal keratinocytes and dermal fibroblasts produced CXCL1/KC in a PLCÉ-dependent manner after UVB irradiation, and the UVB-induced upregulation of CXCL1/KC in these cells was significantly abolished by a PLC inhibitor. Furthermore, UVB-induced epidermal thickening was noticeably reduced in the skin of PLCÉâ»/â» mice. These results indicate that PLCÉ has a crucial role in UVB-induced acute inflammatory reactions such as neutrophil infiltration and epidermal thickening by at least in part regulating the expression of CXCL1/KC in skin cells such as keratinocytes and fibroblasts.
Assuntos
Quimiocina CXCL1/genética , Dermatite/etiologia , Regulação da Expressão Gênica/fisiologia , Neutrófilos/imunologia , Fosfoinositídeo Fosfolipase C/fisiologia , Raios Ultravioleta , Animais , Sequência de Bases , Células Cultivadas , Primers do DNA , Dermatite/imunologia , Ensaio de Imunoadsorção Enzimática , Camundongos , Fosfoinositídeo Fosfolipase C/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
RBM5 (RNA-binding motif protein 5) is a nuclear RNA binding protein containing 2 RNA recognition motifs. The RBM5 gene is located at the tumor suppressor locus 3p21.3. Deletion of this locus is the most frequent genetic alteration in lung cancer, but is also found in other human cancers. RBM5 is known to induce apoptosis and cell cycle arrest but the molecular mechanisms of RBM5 function are poorly understood. Here, we show that RBM5 is important for the activity of the tumor suppressor protein p53. Overexpression of RBM5 enhanced p53-mediated inhibition of cell growth and colony formation. Expression of RBM5 augmented p53 transcriptional activity in reporter gene assays and resulted in increased mRNA and protein levels for endogenous p53 target genes. In contrast, shRNA-mediated knockdown of endogenous RBM5 led to decreased p53 transcriptional activity and reduced levels of mRNA and protein for endogenous p53 target genes. RBM5 affected protein, but not mRNA, levels of endogenous p53 after DNA damage suggest that RBM5 contributes to p53 activity through post-transcriptional mechanisms. Our results show that RBM5 contributes to p53 transcriptional activity after DNA damage and that growth suppression and apoptosis mediated by RBM5 are linked to activity of the tumor suppressor protein p53.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Ligação a RNA/fisiologia , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/fisiologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Dano ao DNA , Humanos , RNA Mensageiro/análiseRESUMO
Phospholipase Cε (PLCε) is an effector of Ras and Rap small GTPases. We showed previously using PLCε-deficient mice that PLCε plays a critical role in activation of cytokine production in non-immune skin cells in a variety of inflammatory reactions. For further investigation of its role in inflammation, we created transgenic mice overexpressing PLCε in epidermal keratinocytes. The resulting transgenic mice spontaneously developed skin inflammation as characterized by formation of adherent silvery scales, excessive growth of keratinocytes, and aberrant infiltration of immune cells such as T cells and DC. Development of the skin symptoms correlated well with increased expression of factors implicated in human inflammatory skin diseases, such as IL-23, in keratinocytes, and with the accumulation of CD4(+) T cells producing IL-22, a potent inducer of keratinocyte proliferation. Intradermal injection of a blocking antibody against IL-23 as well as treatment with the immunosuppressant FK506 reversed these skin phenotypes, which was accompanied by suppression of the IL-22-producing T-cell infiltration. These results reveal a crucial role of PLCε in the development of skin inflammation and suggest a mechanism in which PLCε induces the production of cytokines including IL-23 from keratinocytes, leading to the activation of IL-22-producing T cells.
Assuntos
Citocinas/imunologia , Dermatite/imunologia , Queratinócitos/imunologia , Fosfoinositídeo Fosfolipase C/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Bloqueadores/farmacologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Dermatite/enzimologia , Dermatite/patologia , Feminino , Humanos , Imunossupressores/farmacologia , Interleucina-23/análise , Interleucina-23/antagonistas & inibidores , Interleucina-23/imunologia , Interleucinas/análise , Interleucinas/imunologia , Queratinócitos/enzimologia , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfoinositídeo Fosfolipase C/análise , Fosfoinositídeo Fosfolipase C/metabolismo , Tacrolimo/farmacologia , Regulação para Cima , Interleucina 22RESUMO
Phospholipase C (PLC) ε is a phosphoinositide-specific PLC regulated by small guanosine triphosphatases including Ras and Rap. Our previous studies revealed that PLCε gene-knockout (PLCε(-/-)) mice exhibit marked resistance to tumor formation in two-stage skin chemical carcinogenesis using 7,12-dimethylbenz(a)anthracene as an initiator and 12-O-tetradecanoylphorbol-13-acetate as a promoter. In this model, PLCε functions in tumor promotion through augmentation of 12-O-tetradecanoylphorbol-13-acetate-induced inflammation. In this study, we have further assessed the role of PLCε in tumorigenesis using a mouse model of ultraviolet (UV) B-induced skin tumor development. We irradiated PLCε(+/+), PLCε(+/-) or PLCε(-/-) mice with doses of UVB increasing from 1 to 10 kJ/m(2) three times a week for a total of 25 weeks and observed tumor formation for up to 50 weeks. In sharp contrast to the results from the two-stage chemical carcinogenesis study, PLCε(-/-) mice developed a large number of neoplasms including malignant tumors, whereas PLCε(+/+) and PLCε(+/-) mice developed a relatively small number of benign tumors. However, UVB-induced skin inflammation was greatly suppressed in PLCε(-/-) mice, as observed with 12-O-tetradecanoylphorbol-13-acetate-induced inflammation, implying that PLCε's role in the suppression of UVB-induced tumorigenesis is not mediated by inflammation. Studies of the tumor initiation stage revealed that UVB-induced cell death in the skin was markedly suppressed in PLCε(-/-)mice. Our findings identify a novel function for PLCε as a critical molecule regulating UVB-induced cell death and suggest that resistance to UVB-induced cell death conferred by the absence of PLCε is closely related to the higher incidence of skin tumor formation.
Assuntos
Apoptose , Neoplasias Induzidas por Radiação/etiologia , Fosfoinositídeo Fosfolipase C/fisiologia , Neoplasias Cutâneas/etiologia , Raios Ultravioleta/efeitos adversos , Animais , Interleucina-10/biossíntese , Interleucina-1beta/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Induzidas por Radiação/patologia , RNA Mensageiro/análise , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol , Proteína X Associada a bcl-2/fisiologiaRESUMO
Phospholipase Cepsilon (PLCepsilon) is an effector of Ras/Rap small GTPases. We previously demonstrated that PLCepsilon plays a crucial role in development of phorbor ester-induced skin inflammation, which is intimately involved in the promotion of skin carcinogenesis. In this study, we have examined its role in local skin inflammatory reactions during development of contact hypersensitivity toward a hapten 2,4-dinitrofluorobenzene (DNFB). PLCepsilon(+/+) and PLCepsilon(-/-) mice were sensitized with DNFB, followed by a DNFB challenge on the ears. PLCepsilon(-/-) mice exhibited substantially attenuated inflammatory reactions compared with PLCepsilon(+/+) mice as shown by suppression of ear swelling, neutrophil infiltration, and proinflammatory cytokine production. In contrast, the extent and kinetics of CD4+ T cell infiltration showed no difference depending on the PLCepsilon background. Adoptive transfer of CD4+ T cells from the sensitized mice to naive mice between PLCepsilon(+/+) and PLCepsilon(-/-) backgrounds indicated that PLCepsilon exerts its function in cells other than CD4+ T cells, presumably fibroblasts or keratinocytes of the skin, to augment inflammatory reactions during the elicitation stage of contact hypersensitivity. Moreover, dermal fibroblasts and epidermal keratinocytes cultured from the skin expressed proinflammatory cytokines in a PLCepsilon-dependent manner on stimulation with T cell-derived cytokines such as IL-17, IFN-gamma, TNF-alpha, and IL-4. These results indicate that PLCepsilon plays a crucial role in induction of proinflammatory cytokine expression in fibroblasts and keratinocytes at the challenged sites, where infiltrated CD4+ T cells produce their intrinsic cytokines, thereby augmenting the local inflammatory reactions.
Assuntos
Inflamação/patologia , Fosfoinositídeo Fosfolipase C/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Quimiotaxia de Leucócito , Citocinas/biossíntese , Dermatite de Contato , Dinitrofluorbenzeno , Fibroblastos/metabolismo , Queratinócitos/metabolismo , Camundongos , Camundongos Knockout , Fosfoinositídeo Fosfolipase C/deficiênciaRESUMO
Apc(Min/+) mice, carrying an inactivated allele of the adenomatous polyposis coli gene, are widely used as an animal model for human colorectal tumorigenesis, where tumor environment, such as inflammation, is known to play a critical role in tumor progression. We previously demonstrated that phospholipase C (PLC)epsilon, an effector of Ras and Rap small GTPases, plays a crucial role in two-stage skin chemical carcinogenesis using 12-O-tetradecanoyl-phorbor-13-acetate (TPA) as a promoter through augmentation of TPA-induced inflammation. Here, we show that Apc(Min/+) mice lacking PLCepsilon (PLCepsilon(-/-)) exhibit marked resistance to spontaneous intestinal tumorigenesis compared with those with the PLCepsilon(+/+) background. Time course of the development of tumors, which are histopathologically classified into low- and high-grade adenomas with increasing dysplasia and size, and adenocarcinomas indicates that not only the low-grade adenoma formation but also the progression to high-grade adenoma are suppressed in PLCepsilon(-/-);Apc(Min/+) mice. Low-grade adenomas of PLCepsilon(-/-);Apc(Min/+) mice exhibit accelerated apoptosis and reduced cellular proliferation. They also show marked attenuation of tumor angiogenesis and reduction in expression of vascular endothelial growth factor. In contrast, high-grade adenomas of PLCepsilon(-/-);Apc(Min/+) mice exhibit marked attenuation of tumor-associated inflammation without significant differences in apoptosis and proliferation. These results suggest that PLCepsilon plays crucial roles in intestinal tumorigenesis through two distinct mechanisms, augmentation of angiogenesis and inflammation, depending on the tumor stage.
Assuntos
Gastrite/etiologia , Genes APC/fisiologia , Neoplasias Intestinais/irrigação sanguínea , Neovascularização Patológica/etiologia , Fosfoinositídeo Fosfolipase C/fisiologia , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/etiologia , Adenocarcinoma/patologia , Adenoma/irrigação sanguínea , Adenoma/etiologia , Adenoma/patologia , Animais , Apoptose , Western Blotting , Carcinógenos/toxicidade , Proliferação de Células , Feminino , Gastrite/patologia , Técnicas Imunoenzimáticas , Neoplasias Intestinais/etiologia , Neoplasias Intestinais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/toxicidade , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
In two-stage skin chemical carcinogenesis, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) acts as a promoter essential for clonal expansion of the initiated cells carrying the activated ras oncogenes. Although protein kinase C (PKC) isozymes are the main targets of TPA, their role in tumor promotion remains controversial. We previously reported that mice lacking a Ras/Rap effector phospholipase C epsilon (PLC epsilon(-/-) mice) exhibited marked resistance to tumor formation in the two-stage skin carcinogenesis. PLC epsilon(-/-) mice also failed to exhibit basal layer cell proliferation and epidermal hyperplasia induced by TPA, suggesting a role of PLC epsilon in tumor promotion. Here, we show that PLC epsilon(-/-) mice exhibit resistance to TPA-induced skin inflammation as assessed by reduction in edema, granulocyte infiltration, and expression of a proinflammatory cytokine, interleukin-1 alpha (IL-1 alpha). On the other hand, the proliferative potentials of keratinocytes or dermal fibroblasts in culture remain unaffected by the PLC epsilon background, suggesting that the PLC epsilon's role in tumor promotion may be ascribed to augmentation of inflammatory responses. In dermal fibroblast primary culture, TPA can induce activation of the PLC epsilon lipase activity, which leads to the induction of IL-1 alpha expression. Experiments using small interfering RNA-mediated knockdown indicate that this activation is mediated by Rap1, which is activated by a TPA-responsive guanine nucleotide exchange factor RasGRP3. Moreover, TPA-induced activation of Rap1 and PLC epsilon is inhibited by a PKC inhibitor GF109203X, indicating a crucial role of PKC in signaling from TPA to PLC epsilon. These results imply that two TPA targets, RasGRP3 and PKC, are involved in TPA-induced inflammation through PLC epsilon activation, leading to tumor promotion.
Assuntos
Carcinógenos/toxicidade , Dermatite de Contato/enzimologia , Fosfoinositídeo Fosfolipase C/fisiologia , Pele/efeitos dos fármacos , Acetato de Tetradecanoilforbol/toxicidade , Animais , Dermatite de Contato/genética , Dermatite de Contato/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Interleucina-1/genética , Interleucina-1/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Camundongos , Camundongos Mutantes , Fosfoinositídeo Fosfolipase C/genética , Pele/enzimologia , Pele/patologia , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/genética , Proteínas rap1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismo , Fatores ras de Troca de Nucleotídeo Guanina/metabolismoRESUMO
The Rap1 small GTPase has been implicated in regulation of integrin-mediated leukocyte adhesion downstream of various chemokines and cytokines in many aspects of inflammatory and immune responses. However, the mechanism for Rap1 regulation in the adhesion signaling remains unclear. RA-GEF-2 is a member of the multiple-member family of guanine nucleotide exchange factors (GEFs) for Rap1 and characterized by the possession of a Ras/Rap1-associating domain, interacting with M-Ras-GTP as an effector, in addition to the GEF catalytic domain. Here, we show that RA-GEF-2 is specifically responsible for the activation of Rap1 that mediates tumor necrosis factor-alpha (TNF-alpha)-triggered integrin activation. In BAF3 hematopoietic cells, activated M-Ras potently induced lymphocyte function-associated antigen 1 (LFA-1)-mediated cell aggregation. This activation was totally abrogated by knockdown of RA-GEF-2 or Rap1. TNF-alpha treatment activated LFA-1 in a manner dependent on M-Ras, RA-GEF-2, and Rap1 and induced activation of M-Ras and Rap1 in the plasma membrane, which was accompanied by recruitment of RA-GEF-2. Finally, we demonstrated that M-Ras and RA-GEF-2 were indeed involved in TNF-alpha-stimulated and Rap1-mediated LFA-1 activation in splenocytes by using mice deficient in RA-GEF-2. These findings proved a crucial role of the cross-talk between two Ras-family GTPases M-Ras and Rap1, mediated by RA-GEF-2, in adhesion signaling.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Integrinas/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Baço/citologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Marcação de Genes , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Modelos Biológicos , Baço/efeitos dos fármacosRESUMO
Phosphoinositide-specific phospholipase C (PLC) plays a pivotal role in signal transduction from various receptor molecules on the plasma membrane. PLCepsilon is characterized by possession of two Ras/Rap-associating (RA) domains and a CDC25 homology domain acting as a guanine nucleotide exchange factor for Rap1. Our recent studies using PLCepsilon-deficient mice have suggested that PLCepsilon plays crucial roles in cardiac semilunar valvulogenesis downstream of the EGF receptor, as well as in chemical carcinogen-induced skin tumor development downstream of Ha-Ras. Stimulation of cultured mammalian cells with growth factors induces translocation of PLCepsilon from the cytoplasm to the plasma membrane and to the Golgi apparatus through direct association at its RA domains with the GTP-bound forms of Ras and Rap1, respectively. These results suggest that growth factor stimulation activates PLCepsilon by means of Ras and/or Rap1. However, growth factor-induced activation of the PLCepsilon lipase activity cannot be measured accurately because of simultaneous activation of PLCgamma through receptor-dependent phosphorylation. In this article, we introduce two methods to assay Ras- or Rap1-dependent activation of PLCepsilon lipase activity, with special emphasis on the use of cells expressing a mutant platelet-derived growth factor receptor lacking the PLCgamma-binding sites.
Assuntos
Fosfoinositídeo Fosfolipase C/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Animais , Células COS , Chlorocebus aethiops , Ativação Enzimática , Proteínas Ativadoras de GTPase/genética , Genes ras/genética , Humanos , Fosfatos de Inositol/metabolismo , Proteínas Nucleares , Fosfoinositídeo Fosfolipase C/análise , Fator de Crescimento Derivado de Plaquetas/fisiologia , Ensaio Radioligante , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , TransfecçãoRESUMO
Phospholipase C (PLC) epsilon is directly regulated by Ras and Rap1 small GTPases: Ras and Rap1, in their GTP-bound form, interact with the Ras/Rap1-associationg (RA) domain of PLCepsilon, thereby translocating PLCepsilon to the plasma membrane and the Golgi apparatus, respectively. In the plasma membrane and the Golgi apparatus, PLCepsilon acts as a phosphoinositide-specific PLC, regulating various downstream signaling pathways. PLCepsilon also contains a CDC25 homology domain, which enhances guanine nucleotide exchange on Rap1. Here, we describe biochemical characterization of the CDC25 homology domain of PLCepsilon and provide insights into its physiological role in the regulation of PLCepsilon activity.
Assuntos
Fosfoinositídeo Fosfolipase C/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , ras-GRF1/metabolismo , Animais , Células COS , Chlorocebus aethiops , Guanosina Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Transporte Proteico , SpodopteraRESUMO
Phospholipase Cepsilon is a novel class of phosphoinositide-specific phospholipase C, identified as a downstream effector of Ras and Rap small GTPases. We report here the first genetic analysis of its physiological function with mice whose phospholipase Cepsilon is catalytically inactivated by gene targeting. The hearts of mice homozygous for the targeted allele develop congenital malformations of both the aortic and pulmonary valves, which cause a moderate to severe degree of regurgitation with mild stenosis and result in ventricular dilation. The malformation involves marked thickening of the valve leaflets, which seems to be caused by a defect in valve remodeling at the late stages of semilunar valvulogenesis. This phenotype has a remarkable resemblance to that of mice carrying an attenuated epidermal growth factor receptor or deficient in heparin-binding epidermal growth factor-like growth factor. Smad1/5/8, which is implicated in proliferation of the valve cells downstream of bone morphogenetic protein, shows aberrant activation at the margin of the developing semilunar valve tissues in embryos deficient in phospholipase Cepsilon. These results suggest a crucial role of phospholipase Cepsilon downstream of the epidermal growth factor receptor in controlling semilunar valvulogenesis through inhibition of bone morphogenetic protein signaling.
Assuntos
Valva Aórtica/anormalidades , Valva Aórtica/embriologia , Valva Pulmonar/anormalidades , Valva Pulmonar/embriologia , Fosfolipases Tipo C/fisiologia , Alelos , Animais , Valva Aórtica/imunologia , Defeito do Septo Aortopulmonar/genética , Proteínas Morfogenéticas Ósseas/fisiologia , Cardiomiopatia Dilatada/etiologia , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Receptores ErbB/deficiência , Receptores ErbB/genética , Receptores ErbB/fisiologia , Marcação de Genes , Doenças das Valvas Cardíacas/complicações , Doenças das Valvas Cardíacas/genética , Ventrículos do Coração/patologia , Camundongos , Camundongos Mutantes , Mutação/genética , Fosfoinositídeo Fosfolipase C , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Valva Pulmonar/imunologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas Smad , Proteína Smad1 , Proteína Smad5 , Proteína Smad8 , Transativadores/análise , Transativadores/genética , Transativadores/metabolismo , Fosfolipases Tipo C/análise , Fosfolipases Tipo C/genéticaRESUMO
Mutational activation of the ras proto-oncogenes is frequently found in skin cancers. However, the nature of downstream signaling pathways from Ras involved in skin carcinogenesis remains poorly understood. Recently, we and others identified phospholipase C (PLC) epsilon as an effector of Ras. Here we have examined the role of PLCepsilon in de novo skin chemical carcinogenesis by using mice whose PLCepsilon is genetically inactivated. PLCepsilon(-/-) mice exhibit delayed onset and markedly reduced incidence of skin squamous tumors induced by initiation with 7,12-dimethylbenz(a)anthracene followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, the papillomas formed in PLCepsilon(-/-) mice fail to undergo malignant progression into carcinomas, in contrast to a malignant conversion rate of approximately 20% observed with papillomas in PLCepsilon(+/+) mice. In all of the tumors analyzed, the Ha-ras gene is mutationally activated irrespective of the PLCepsilon background. The skin of PLCepsilon(-/-) mice fails to exhibit basal layer cell proliferation and epidermal hyperplasia in response to TPA treatment. These results indicate a crucial role of PLCepsilon in ras oncogene-induced de novo carcinogenesis and downstream signaling from TPA, introducing PLCepsilon as a candidate molecular target for the development of anticancer drugs.
Assuntos
Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/enzimologia , Fosfolipases Tipo C/fisiologia , 9,10-Dimetil-1,2-benzantraceno , Animais , Carcinógenos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fosfoinositídeo Fosfolipase C , RNA/análise , RNA/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Acetato de Tetradecanoilforbol , Fosfolipases Tipo C/biossíntese , Fosfolipases Tipo C/deficiência , Fosfolipases Tipo C/genética , Proteínas ras/genéticaRESUMO
Phospholipase C is a key enzyme of intracellular signal transduction in the central nervous system. We and others recently discovered a novel class of phospholipase C, phospholipase Cepsilon, which is regulated by Ras and Rap small GTPases. As a first step toward analysis of its function, we have examined the spatial and temporal expression patterns of phospholipase Cepsilon during mouse development by in situ hybridization and immunohistochemistry. Around embryonic day 10.5, abundant expression of phospholipase Cepsilon is observed specifically in the outermost layer of the neural tube. On embryonic day 12 and later, it is observed mainly in the marginal zone of developing brain and spinal cord as well as in other regions undergoing neuronal differentiation, such as the retina and olfactory epithelium. The phospholipase Cepsilon-expressing cells almost invariably express microtubule-associated protein 2, but hardly express nestin or glial fibrillary acidic protein, indicating that the expression of phospholipase Cepsilon is induced specifically in cells committed to the neuronal lineage. The expression of phospholipase Cepsilon persists in the terminally differentiated neurons and exhibits no regional specificity. Further, an in vitro culture system of neuroepithelial stem cells is employed to show that abundant expression of phospholipase Cepsilon occurs in parallel with the loss of nestin expression as well as with the induction of microtubule-associated protein 2 expression and neuronal morphology. Also, glial fibrillary acidic protein-positive glial lineage cells do not exhibit the high phospholipase Cepsilon expression. These results suggest that the induction of phospholipase Cepsilon expression may be a specific event associated with the commitment of the neural precursor cells to the neuronal lineage.
Assuntos
Encéfalo/embriologia , Diferenciação Celular , Neurônios/citologia , Fosfolipases Tipo C/biossíntese , Animais , Northern Blotting , Encéfalo/citologia , Encéfalo/enzimologia , Linhagem da Célula , Células Cultivadas , DNA Complementar/análise , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos ICR , Neuroglia/citologia , Neuroglia/enzimologia , Neurônios/enzimologia , Fosfoinositídeo Fosfolipase C , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência , Células-Tronco/enzimologiaRESUMO
Phospholipase C epsilon is a phosphoinositide-specific phospholipase C that selectively associates with Ras and Rap small GTPases as a target. Here we explored the molecular basis of the Rap1- as well as Ras-mediated regulation of phospholipase C epsilon upon platelet-derived growth factor stimulation by using a receptor mutant deficient in its ability to phosphorylate and activate phospholipase C gamma. Following platelet-derived growth factor treatment, this receptor induces persistent activation of ectopically expressed PLC epsilon through activation of Ras and Rap1. The rapid and initial phase of the activation is mediated by Ras, whereas Rap1 is responsible for the prolonged activation. We further demonstrate that the CDC25 homology domain, which exhibits guanine nucleotide exchange factor activity toward Rap1, but not Ras, is critical for the prolonged activation of phospholipase C epsilon. Platelet-derived growth factor prevented the hematopoietic BaF3 cells containing the mutant receptor from undergoing apoptosis, and enabled these cells to proliferate, only when phospholipase C epsilon was expressed. Therefore, the phospholipase C signal is suggested to be critical for survival and growth of BaF3 cells.
Assuntos
Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Fosfolipases Tipo C/metabolismo , Proteínas rap1 de Ligação ao GTP/fisiologia , Substituição de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Células COS , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/metabolismo , Chlorocebus aethiops , Ativação Enzimática/efeitos dos fármacos , Genes ras , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/biossíntese , Interleucina-3/farmacologia , Fosfoinositídeo Fosfolipase C , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas p21(ras)/química , Ratos , Receptores do Fator de Crescimento Derivado de Plaquetas/efeitos dos fármacos , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Recombinantes de Fusão/fisiologia , Deleção de Sequência , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Transfecção , Fosfolipases Tipo C/química , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/química , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
The interaction of activated Ras with Raf initiates signaling cascades that contribute to a significant percentage of human tumors, suggesting that agents that specifically disrupt this interaction might have desirable chemotherapeutic properties. We used a subtractive forward two-hybrid approach to identify small molecule compounds that block the interaction of Ras with Raf. These compounds (MCP1 and its derivatives, 53 and 110) reduced serum-induced transcriptional activation of serum response element as well as Ras-induced transcription by way of the AP-1 site. They also inhibited Ras-induced Raf-1 activation in human embryonic kidney 293 cells, Raf-1 and mitogen-activated protein kinase kinase 1 activities in HT1080 fibrosarcoma cells, and epidermal growth factor-induced Raf-1 activation in A549 lung carcinoma cells. The MCP compounds caused reversion of ras-transformed phenotypes including morphology, in vitro invasiveness, and anchorage-independent growth of HT1080 cells. Decreased level of matrix metalloproteinases was also observed. Further characterization showed that MCP compounds restore actin stress fibers and cause flat reversion in NIH 3T3 cells transformed with H-Ras (V12) but not in NIH 3T3 cells transformed with constitutively active Raf-1 (RafDeltaN). Finally, we show that MCP compounds inhibit anchorage-independent growth of A549 and PANC-1 cells harboring K-ras mutation. Furthermore, MCP110 caused G(1) enrichment of A549 cells with the decrease of cyclin D level. These results highlight potent and specific effects of MCP compounds on cancer cells with intrinsic Ras activation.