RESUMO
GTP-bound forms of Ras proteins (Rasâ¢GTP) assume two interconverting conformations, "inactive" state 1 and "active" state 2. Our previous study on the crystal structure of the state 1 conformation of H-Ras in complex with guanosine 5'-(ß, γ-imido)triphosphate (GppNHp) indicated that state 1 is stabilized by intramolecular hydrogen-bonding interactions formed by Gln61. Since Ras are constitutively activated by substitution mutations of Gln61, here we determine crystal structures of the state 1 conformation of H-Rasâ¢GppNHp carrying representative mutations Q61L and Q61H to observe the effect of the mutations. The results show that these mutations alter the mode of hydrogen-bonding interactions of the residue 61 with Switch II residues and induce conformational destabilization of the neighboring regions. In particular, Q61L mutation results in acquirement of state 2-like structural features. Moreover, the mutations are likely to impair an intramolecular structural communication between Switch I and Switch II. Molecular dynamics simulations starting from these structures support the above observations. These findings may give a new insight into the molecular mechanism underlying the aberrant activation of the Gln61 mutants.
Assuntos
Guanosina Trifosfato/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Cristalografia por Raios X , Guanosina Trifosfato/genética , Humanos , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
BACKGROUND: Lymphocytes circulate between peripheral lymphoid tissues via blood and lymphatic systems, and chemokine-induced migration is important in trafficking lymphocytes to distant sites. The small GTPase Rap1 is important in mediating lymphocyte motility, and Rap1-GEFs are involved in chemokine-mediated Rap1 activation. Here, we describe the roles and mechanisms of Rap1-GEFs in lymphocyte trafficking. RESULTS: In this study, we show that RA-GEF-1 and 2 (also known as Rapgef2 and 6) are key guanine nucleotide exchange factors (GEF) for Rap1 in lymphocyte trafficking. Mice harboring T cell-specific knockouts of Rapgef2/6 demonstrate defective homing and egress of T cells. Sphingosine-1-phosphate (S1P) as well as chemokines activates Rap1 in a RA-GEF-1/2-dependent manner, and their deficiency in T cells impairs Mst1 phosphorylation, cell polarization, and chemotaxis toward S1P gradient. On the other hand, B cell-specific knockouts of Rapgef2/6 impair chemokine-dependent retention of B cells in the bone marrow and passively facilitate egress. Phospholipase D2-dependent production of phosphatidic acid by these chemotactic factors determines spatial distribution of Rap1-GTP subsequent to membrane localization of RA-GEFs and induces the development of front membrane. On the other hand, basal de-phosphorylation of RA-GEFs is necessary for chemotactic factor-dependent increase in GEF activity for Rap1. CONCLUSIONS: We demonstrate here that subcellular distribution and activation of RA-GEFs are key factors for a directional movement of lymphocytes and that phosphatidic acid is critical for membrane translocation of RA-GEFs with chemokine stimulation.
Assuntos
Movimento Celular , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Linfócitos/fisiologia , Ácidos Fosfatídicos/metabolismo , Animais , Linhagem Celular , Feminino , Humanos , Masculino , Camundongos , FosforilaçãoRESUMO
Integrin activation by Rap1-GTP is pivotal for lymphocyte trafficking. In this study, we show the phosphatidic acid (PA)-dependent membrane distribution of RA-GEF-1 and -2 (also known as Rapgef2 and 6), which are guanine nucleotide exchange factors for Rap1, plays important roles in lymphocyte migration. RA-GEF-1 associates with PA through 919-967 aa within CDC25 homology domain, and the deletion of this region of RA-GEF-1 inhibits chemokine-dependent migration. Chemokine stimulation induces temporal production of PA on the plasma membrane, which is not necessary for Rap1 activation, but the translocation of RA-GEFs. Thus, chemokine-dependent generation of PA is critical for lymphocyte migration through membrane localization of RA-GEFs.
Assuntos
Movimento Celular , Quimiocinas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ácidos Fosfatídicos/metabolismo , Animais , Linhagem Celular , Células HEK293 , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , CamundongosRESUMO
BACKGROUND: We have shown that phospholipase Cε (PLCε), an effector of Ras and Rap1 small GTPases, plays pivotal roles in inflammation and inflammation-associated carcinogenesis by augmenting proinflammatory cytokine production from epithelial cells of various organs. The purpose of this study is to analyze its role in neutrophilic alveolar inflammation accompanying acute lung injury (ALI), focusing on that in alveolar epithelial cells (AECs), which are known to make a major contribution to the pathogenesis of ALI. METHODS: We examine the effect of the PLCε genotypes on the development of ALI induced by intratracheal administration of lipopolysaccharide (LPS) to PLCε wild-type (PLCε+/+) and knockout (PLCεΔX/ΔX) mice. Pathogenesis of ALI is analyzed by histological examination of lung inflammation and measurements of the levels of various cytokines, in particular neutrophil-attracting chemokines such as Cxcl5, by quantitative reverse transcription-polymerase chain reaction and immunostaining. Primary cultures of AECs, established from PLCε+/+ and PLCεΔX/ΔX mice, are used to analyze the roles of PLCε, protein kinase D (PKD) and nuclear factor-κB (NF-κB) in augmentation of LPS-induced Cxcl5 expression. RESULTS: Compared to PLCε+/+ mice, PLCεΔX/ΔX mice exhibit marked alleviation of lung inflammation as shown by great reduction in lung wet/dry weight ratios, accumulation of inflammatory cells in the alveolar space and thickening of alveolar walls as well as the number of neutrophils and the protein concentration in bronchoalveolar lavage fluid. Also, LPS-induced expression of the CXC family of chemokines, in particular Cxcl5, is substantially diminished in the total lung and AECs of PLCεΔX/ΔX mice. Moreover, LPS-induced Cxcl5 expression in primary cultured AECs is markedly suppressed on the PLCεΔX/ΔX background (p < 0.05 versus PLCε+/+ AECs), which is accompanied by the reduction in phosphorylation of inhibitor κB (IκB), PKD and nuclear translocation of NF-κB p65. Also, it is suppressed by the treatment with inhibitors of PKD and IκB kinase, suggesting the involvement of the PLCε-PKD-IκB-NF-κB pathway. CONCLUSIONS: PLCε-mediated augmentation of the production of the CXC family of chemokines, in particular Cxcl5, in AECs plays a crucial role in neutrophilic alveolar inflammation accompanying ALI, suggesting that PLCε may be a potential molecular target for the treatment of acute respiratory distress syndrome.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Células Epiteliais Alveolares/metabolismo , Quimiocina CXCL5/biossíntese , Neutrófilos/metabolismo , Fosfoinositídeo Fosfolipase C/fisiologia , Lesão Pulmonar Aguda/induzido quimicamente , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Células Cultivadas , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Distribuição AleatóriaRESUMO
The ras oncogene products (H-Ras, K-Ras, and N-Ras) have been regarded as some of the most promising targets for anticancer drug discovery because their activating mutations are frequently found in human cancers. Nonetheless, molecular targeted therapy for them is currently unavailable. Here, we report the discovery of a small-molecule compound carrying a naphthalene ring, named KBFM123, which binds to the GTP-bound form of H-Ras. The solution structure of its complex with the guanosine 5'-(ß,γ-imide) triphosphate-bound form of H-RasT35S (H-RasT35S·GppNHp) indicates that the naphthalene ring of KBFM123 interacts directly with a hydrophobic pocket located between switch I and switch II and allosterically inhibits the effector interaction by inducing conformational changes in switch I and its flanking region in strand ß2, which are directly involved in recognition of the effector molecules, including c-Raf-1. In particular, Asp38 of H-Ras, a crucial residue for the interaction with c-Raf-1 via the formation of a salt bridge with Arg89 of the Ras-binding domain (RBD) of c-Raf-1, shows a drastic conformational change: its side chain orients toward the opposite direction. Consistent with these results, KBFM123 exhibits an activity to inhibit, albeit weakly, the association of H-RasG12V·GppNHp with the c-Raf-1 RBD. The binding of the naphthalene ring to the hydrophobic pocket of H-RasT35S·GppNHp is further supported by nuclear magnetic resonance analyses showing that two other naphthalene-containing compounds with distinct structures also exhibit similar binding properties with KBFM123. These results indicate that the naphthalene ring could become a promising scaffold for the development of Ras inhibitors.
Assuntos
Guanosina Trifosfato/metabolismo , Naftalenos/química , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Sítios de Ligação , Catálise , Domínio Catalítico , Descoberta de Drogas , Humanos , Modelos Moleculares , Conformação 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
Rapgef2 and Rapgef6 define a subfamily of guanine nucleotide exchange factors for Rap1, characterized by possession of the Ras/Rap-associating domains and implicated in the etiology of schizophrenia. We previously found that dorsal telencephalon-specific Rapgef2 conditional knockout mice exhibits severe defects in formation of apical surface adherence junctions (AJs) and localization of radial glial cells (RGCs). In this study, we analyze the underlying molecular mechanism by using primary cultures of RGCs established from the developing cerebral cortex. The results show that Rapgef2-deficient RGCs exhibit a decreased ability of neurosphere formation, morphological changes represented by regression of radial glial (RG) fibers and reduced expression of AJ-constituent proteins such as N-cadherin, zonula occludens-1, E-cadherin and ß-catenin. Moreover, siRNA-mediated knockdown of Rapgef2 or Rap1A inhibits the AJ protein expression and RG fiber formation while overexpression of Rapgef2, Rapgef6, Rap1AG12V or Rap1BG12V in Rapgef2-deficient RGCs restores them. Furthermore, Rapgef2-deficient RGCs exhibit a reduction in phosphorylation of extracellular signal-regulated kinase (ERK) leading to downregulation of the expression of c-jun, which is implicated in the AJ protein expression. These results indicate a crucial role of the Rapgef2-Rap1A-ERK-c-jun pathway in regulation of the AJ formation in RGCs.
Assuntos
Junções Aderentes/fisiologia , Junções Aderentes/ultraestrutura , Células Ependimogliais/metabolismo , Células Ependimogliais/ultraestrutura , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Células Cultivadas , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Knockout , Regulação para Cima/fisiologiaRESUMO
Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of Rasâ GTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-Rasâ GTP carrying an H-Ras-type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-Rasâ GTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H-ras(G12V)-transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras(G12V) gene by oral administration. The NMR structure of a complex of the compound with H-Rasâ GTP(T35S), exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple Rasâ GTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target Rasâ GTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Proteínas ras/antagonistas & inibidores , Proteínas ras/metabolismo , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Biologia Computacional/métodos , Glutationa Transferase/metabolismo , Guanosina Trifosfato/química , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Conformação Molecular , Mutação , Células NIH 3T3 , Transplante de Neoplasias , Ligação Proteica , Conformação Proteica , Transdução de SinaisRESUMO
Small GTPase Rap1 has been implicated in the proper differentiation of testicular germ cells. In the present study, we investigated the functional significance of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in testicular differentiation using mice lacking RA-GEF-2. RA-GEF-2 was expressed predominantly on the luminal side of the seminiferous tubules in wild-type mice. No significant differences were observed in the body weights or hormonal parameters of RA-GEF-2(-)(/)(-) and wild-type mice. However, the testes of RA-GEF-2(-)(/)(-) male mice were significantly smaller than those of wild-type mice and were markedly atrophied as well as hypospermatogenic. The concentration and motility of epididymal sperm were also markedly reduced and frequently had an abnormal shape. The pregnancy rate and number of fetuses were markedly lower in wild-type females after they mated with RA-GEF-2(-)(/)(-) males than with wild-type males, which demonstrated the male infertility phenotype of RA-GEF-2(-)(/)(-) mice. Furthermore, a significant reduction and alteration were observed in the expression level and cell junctional localization of N-cadherin, respectively, in RA-GEF-2(-)(/)(-) testes, which may, at least in part, account for the defects in testicular differentiation and spermatogenesis in these mice.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/fisiologia , Infertilidade Masculina/fisiopatologia , Espermatogênese/fisiologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Atrofia , Caderinas/genética , Caderinas/metabolismo , Epididimo/metabolismo , Epididimo/patologia , Feminino , Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Imuno-Histoquímica , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Motilidade dos Espermatozoides/genética , Motilidade dos Espermatozoides/fisiologia , Espermatogênese/genética , Espermatozoides/metabolismo , Espermatozoides/ultraestrutura , Testículo/metabolismo , Testículo/patologiaRESUMO
Ras small GTPases undergo dynamic equilibrium of two interconverting conformations, state 1 and state 2, in the GTP-bound forms, where state 2 is recognized by effectors, whereas physiological functions of state 1 have been unknown. Limited information, such as static crystal structures and (31)P NMR spectra, was available for the study of the conformational dynamics. Here we determine the solution structure and dynamics of state 1 by multidimensional heteronuclear NMR analysis of an H-RasT35S mutant in complex with guanosine 5'-(ß, γ-imido)triphosphate (GppNHp). The state 1 structure shows that the switch I loop fluctuates extensively compared with that in state 2 or H-Ras-GDP. Also, backbone (1)H,(15)N signals for state 2 are identified, and their dynamics are studied by utilizing a complex with c-Raf-1. Furthermore, the signals for almost all the residues of H-Ras·GppNHp are identified by measurement at low temperature, and the signals for multiple residues are found split into two peaks corresponding to the signals for state 1 and state 2. Intriguingly, these residues are located not only in the switch regions and their neighbors but also in the rigidly structured regions, suggesting that global structural rearrangements occur during the state interconversion. The backbone dynamics of each state show that the switch loops in state 1 are dynamically mobile on the picosecond to nanosecond time scale, and these mobilities are significantly reduced in state 2. These results suggest that multiconformations existing in state 1 are mostly deselected upon the transition toward state 2 induced by the effector binding.
Assuntos
Guanosina Trifosfato/química , Proteínas Proto-Oncogênicas p21(ras)/química , Substituição de Aminoácidos , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Humanos , Mutação de Sentido Incorreto , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Relação Estrutura-AtividadeRESUMO
GTP-bound forms of Ras family small GTPases exhibit dynamic equilibrium between two interconverting conformations, "inactive" state 1 and "active" state 2. A great variation exists in their state distribution; H-Ras mainly adopts state 2, whereas M-Ras predominantly adopts state 1. Our previous studies based on comparison of crystal structures representing state 1 and state 2 revealed the importance of the hydrogen-bonding interactions of two flexible effector-interacting regions, switch I and switch II, with the γ-phosphate of GTP in establishing state 2 conformation. However, failure to obtain both state structures from a single protein hampered further analysis of state transition mechanisms. Here, we succeed in solving two crystal structures corresponding to state 1 and state 2 from a single Ras polypeptide, M-RasD41E, carrying an H-Ras-type substitution in residue 41, immediately preceding switch I, in complex with guanosine 5'-(ß,γ-imido)triphosphate. Comparison among the two structures and other state 1 and state 2 structures of H-Ras/M-Ras reveal two new structural features playing critical roles in state dynamics; interaction of residues 31/41 (H-Ras/M-Ras) with residues 29/39 and 30/40, which induces a conformational change of switch I favoring its interaction with the γ-phosphate, and the hydrogen-bonding interaction of switch II with its neighboring α-helix, α3-helix, which induces a conformational change of switch II favoring its interaction with the γ-phosphate. The importance of the latter interaction is proved by mutational analyses of the residues involved in hydrogen bonding. These results define the two novel functional regions playing critical roles during state transition.
Assuntos
Simulação de Dinâmica Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Camundongos , Proteínas Monoméricas de Ligação ao GTP/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Homologia Estrutural de Proteína , Proteínas rasRESUMO
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 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
Ras family small GTPases assume two interconverting conformations, "inactive" state 1 and "active" state 2, in their GTP-bound forms. Here, to clarify the mechanism of state transition, we have carried out x-ray crystal structure analyses of a series of mutant H-Ras and M-Ras in complex with guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp), representing various intermediate states of the transition. Crystallization of H-RasT35S-GppNHp enables us to solve the first complete tertiary structure of H-Ras state 1 possessing two surface pockets unseen in the state 2 or H-Ras-GDP structure. Moreover, determination of the two distinct crystal structures of H-RasT35S-GppNHp, showing prominent polysterism in the switch I and switch II regions, reveals a pivotal role of the guanine nucleotide-mediated interaction between the two switch regions and its rearrangement by a nucleotide positional change in the state 2 to state 1 transition. Furthermore, the (31)P NMR spectra and crystal structures of the GppNHp-bound forms of M-Ras mutants, carrying various H-Ras-type amino acid substitutions, also reveal the existence of a surface pocket in state 1 and support a similar mechanism based on the nucleotide-mediated interaction and its rearrangement in the state 1 to state 2 transition. Intriguingly, the conformational changes accompanying the state transition mimic those that occurred upon GDP/GTP exchange, indicating a common mechanistic basis inherent in the high flexibility of the switch regions. Collectively, these results clarify the structural features distinguishing the two states and provide new insights into the molecular basis for the state transition of Ras protein.
Assuntos
Guanosina Trifosfato/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Aminoácidos/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Guanosina Difosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Humanos , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas rasRESUMO
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
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
The Rho family GTPase Rac1 has been implicated in the regulation of glucose uptake in myoblast cell lines. However, no evidence for the role of Rac1 has been provided by a mouse model. The purpose of this study is to test the involvement of Rac1 in insulin action in mouse skeletal muscle. Intravenous administration of insulin indeed elicited Rac1 activation in gastrocnemius muscle, suggesting the involvement of Rac1 in this signaling pathway. We then examined whether insulin-stimulated translocation of the facilitative glucose transporter GLUT4 from its storage sites to the skeletal muscle sarcolemma depends on Rac1. We show that ectopic expression of constitutively activated Rac1, as well as intravenous administration of insulin, caused translocation of GLUT4 to the gastrocnemius muscle sarcolemma, as revealed by immunofluorescent staining of a transiently expressed exofacial epitope-tagged GLUT4 reporter. Of particular note, insulin-dependent, but not constitutively activated Rac1-induced, GLUT4 translocation was markedly suppressed in skeletal muscle-specific rac1-knockout mice compared to control mice. Immunogold electron microscopic analysis of endogenous GLUT4 gave similar results. Collectively, we propose a critical role of Rac1 in insulin-dependent GLUT4 translocation to the skeletal muscle sarcolemma, which has heretofore been predicted solely by cell culture studies.
Assuntos
Transportador de Glucose Tipo 4/metabolismo , Insulina/farmacologia , Sarcolema/metabolismo , Proteínas rac1 de Ligação ao GTP/fisiologia , Animais , Camundongos , Microscopia Eletrônica , Proteínas Monoméricas de Ligação ao GTP , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Transporte ProteicoRESUMO
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
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
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.