RESUMO
Shp2 and Shp1 make up a small family of protein tyrosine phosphatases. Finding selective inhibitors for Shp2 is useful because although its inhibition is advantageous for the treatment of some types of cancer, inhibition of Shp1 may have the opposite effect, since it acts as a suppressor of tumors. We combined molecular docking and semiempirical molecular orbital-based calculations to produce data that were effective for the identification of selective inhibitors for Shp2. After definition of the interaction modes of the inhibitors with Shp2 and Shp1 by molecular docking, the resulting interaction enthalpy values were calculated following refinement of the enzyme/inhibitor complexes' geometries with the PM7 semiempirical molecular orbital method. Despite the complexity of the thermodynamics involved in the enzyme/inhibitor interaction, the selectivity for Shp2 of a series of 76 inhibitors, divided in two groups, could be effectively correlated with the difference in their interaction enthalpy values with both enzymes. For the first group, composed by 52 Shp2-selective indoline inhibitors for which only Shp2 inhibition activity data are available, we demonstrated that the interaction enthalpy can be used as a criterion for identification of selective Shp2 inhibitors, as it is significantly more favorable for Shp2 than Shp1 at a 99% confidence level. For the second group, composed by 24 oxindole derivatives with available Shp2 and Shp1 inhibition activity data, a satisfactory correlation (R = 0.70) could be obtained between the selectivity, based on the IC50 data, and the relative percentage difference of the calculated interaction enthalpies with the two enzymes.
Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Oxindóis/química , Oxindóis/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/antagonistas & inibidores , TermodinâmicaRESUMO
Methylation pattern is presented here for first time as a potential molecular marker of changes on SSTR2A and SHP-1(I) gene promoter related to breast and prostate carcinoma. Our results have shown low concordances with SSTR2A and methylated state in prostate cancer and moderate relationship with unmethylated CpG-27 in breast cancer. We found significant concordances for both cancers and SHP-1(I) unmethylation, and increased HER2 expression and SSTR2A methylation in breast cancer. Moreover, we found a correlation between methylation patterns of two genes in normal breast tissue. These data might assist to select subgroups of patients for the administration of alternative therapies.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Metilação de DNA , Neoplasias da Próstata/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Receptores de Somatostatina/genética , Idoso , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Isoformas de Proteínas , Receptores de Somatostatina/metabolismoRESUMO
We investigated the effect of Jianpi Bushen prescription (JBP) on the expression of the SHP-1 and apoptosis-related genes in chemically damaged model mice and a compound e-jiao slurry (EJS) group (positive control). Kunming mice received an abdominal injection of 100 mg/kg cyclophosphamide once a day for 3 consecutive days to induce chemical damage. The mice underwent lavage at a suspension of 0.1 g/kg low-dose JBP (100%), high-dose JBP (200%), and 0.2 mL/10 g EJS twice a day for 9 days. mRNA and protein expression of SHP-1 in bone marrow mononuclear cells was detected using real-time polymerase chain reaction and Western blot; mRNA expression of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax) protein was detected by in situ hybridization. Expression of SHP-1 and Bax mRNA was significantly upregulated in the model group compared to the control group (P < 0.05). Expression in the low-dose JBP, high-dose JBP, and EJS groups was significantly downregulated compared with the model group (P < 0.05). The low-dose JBP group exhibited much lower SHP-1 and Bax mRNA expression levels. Compared with controls, Bcl-2 mRNA expression was significantly reduced in the model group (P < 0.05). Expression in the low-dose JBP, high-dose JBP, and EJS groups significantly increased compared with the model group (P < 0.05). The low-dose JBP group showed much higher Bcl-2 mRNA expression. Therefore, JBP regulates the expression of the SHP- 1, Bax, and Bcl-2 genes in chemically damaged mice.
Assuntos
Apoptose/genética , Medicamentos de Ervas Chinesas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Regulação para Baixo , Medicamentos de Ervas Chinesas/administração & dosagem , Feminino , Masculino , Camundongos , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Nitric oxide (NO) is involved in angiogenesis and stimulates the EGF-R signaling pathway. Stimulation of different endothelial cell lines with bradykinin (BK) activates the endothelial NO synthase (eNOS) and promotes EGF-R tyrosine phosphorylation. Increase in NO production correlated with enhanced phosphorylation of tyrosine residues and S-nitrosylation of the EGF-R. NO-mediated stimulatory effects on tyrosine phosphorylation of the EGF-R, where cGMP independent. Inhibition of soluble guanylyl cyclase followed by BK stimulation of human umbilical vein endothelial cells (HUVECs) did not change tyrosine phosphorylation levels of EGF-R. BK-stimulation of HUVEC promoted S-nitrosylation of the phosphatase SHP-1 and of p21Ras. Phosphorylation and activation of the ERK1/2 MAP kinases mediated by BK was dependent on the activation of the B2 receptor, of the EGF-R, and of p21 Ras. Inhibition of BK-stimulated S-nitrosylation prevented the activation of the ERK1/2 MAP kinases. Furthermore, activated ERK1/2 MAP kinases inhibited internalization of EGF-R by phosphorylating specific Thr residues of its cytoplasmic domain. BK-induced proliferation of endothelial cells was partially inhibited by the NOS inhibitor (L-NAME) and by the MEK inhibitor (PD98059). BK stimulated the expression of vascular endothelial growth factor (VEGF). VEGF expression was dependent on the activation of the EGF-R, the B2 receptor, p21Ras, and on NO generation. A Matrigel®-based in vitro assay for angiogenesis showed that BK induced the formation of capillary-like structures in HUVEC, but not in those cells expressing a mutant of the EGF-R lacking tyrosine kinase activity. Additionally, pre-treatment of BK-stimulated HUVEC with L-NAME, PD98059, and with SU5416, a specific inhibitor of VEGFR resulted in inhibition of in vitro angiogenesis. Our findings indicate that BK-mediated angiogenesis in endothelial cells involves the induction of the expression of VEGF associated with the activation of the NO/EGF-R/p21Ras/ERK1/2 MAP kinases signaling pathway.
Assuntos
Indutores da Angiogênese/farmacologia , Bradicinina/farmacologia , Receptores ErbB/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Óxido Nítrico/biossíntese , Fosforilação/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Coelhos , S-Nitrosotióis/metabolismo , Tirosina/metabolismoRESUMO
This study investigated the effect of the Jianpi Bushen Prescription (JBP) on the expression of 3 major proteins in chemically damaged model mice. The 3 proteins were the Wnt3a, the SHP-1, and the transcription factors (NF-E2, c-jun, and c-fos) of the AP-1 protein family. Kunming mice were randomly divided into chemically damaged group (N=48), which received an abdominal injection of (100 mg/kg) cyclophosphamide once a day for 3 consecutive days, and control group (N=12), which received the same amount of saline. Then, the chemically damaged mice were randomly divided into chemically damaged model group (N=12), which received 0.2 mL/10 g of saline twice a day for 9 days, positive control group (N=12), which received 0.2 mL/10 g of the e-jiao slurry (EJS) compound twice a day for 9 days, low dose JBP group (N=12), which received 0.1 g/kg suspension JBP (100% concentration) twice a day for 9 days and high dose JBP group (N=12), which received 0.1 g/kg suspension JBP (200% concentration) twice a day for 9 days. The bilateral femur and tibia bone marrow were collected from the mice in all groups. The protein expression of the specified proteins and transcription factors in the bone marrow mononuclear cells were detected by Western blot analysis. The results showed that the protein expression of Wnt3a was significantly downregulated in the chemically damaged model group compared to the control group (P<0.05). The low dose JBP, high dose JBP, and e-jiao slurry treatments significantly upregulated the protein expression of Wnt3a compared to the chemically damaged model group (P<0.05), with the low dose JBP producing the best results. Compared to the control group, the protein expressions of SHP-1, c-fos, c-jun, and NF-E2 were significantly higher in the chemically damaged model group (all P<0.05). The protein expressions of SHP-1, c-fos, c-jun, and NF-E2 were significantly lower in the chemically damaged model+the low dose JBP, chemically damaged model+high dose JBP, or chemically damaged model+EJS group compared to chemically damaged model (all P<0.05), with the low dose JBP producing the best results. These results indicate that JBP regulates the expressions of SHP-1, Wnt3a, and AP-1 proteins in chemically damaged mice.
Assuntos
Medicamentos de Ervas Chinesas/administração & dosagem , Proteína Tirosina Fosfatase não Receptora Tipo 6/biossíntese , Fator de Transcrição AP-1/biossíntese , Proteína Wnt3A/biossíntese , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Ciclofosfamida/toxicidade , Fêmur/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Tíbia/efeitos dos fármacosRESUMO
BACKGROUND: Many cases of autoimmune hemolytic anemia have been reported after viral infection. Phagocyte activation and accompanying erythrophagocytosis are thought to result from proinflammatory cytokines released during viral infection. SIRP-α (signal regulatory protein-α), a receptor expressed on phagocytes, inhibits phagocytosis when bound to CD47 on the erythrocyte membrane. Ligation with CD47 results in SHP-1 recruitment to SIRP-α and dephosphorylation of specific downstream substrates involved in phagocytosis. SIRP-α ligation by CD47 may be inhibited by proinflammatory cytokines. OBJECTIVES: The aim of this work was to evaluate the effect of IFN-ß, IFN-γ, and TNF-α on erythrophagocytosis and assess the effect on expression of SIRP-α and SHP-1 in human monocytes. MATERIALS AND METHODS: Monocytes were cultured ex vivo with IFN-ß or IFN-γ/TNF-α. Erythrophagocytosis was determined by flow cytometry. SIRP-α and SHP-1 gene expression was determined by real time-PCR, while SIRP-α and SHP-1 protein expression was determined by western blot. RESULTS: Erythrophagocytosis by monocytes significantly decreased after treatment with either IFN-ß or IFN-γ/TNF-α. Monocytes cultured with IFN-γ/TNF-α showed increased SIRP-α gene and protein expression and SHP-1 gene expression. Monocytes cultured with IFN-ß did not show any alteration in SIRP-α or SHP-1 expression. CONCLUSION: We conclude that IFN-ß and IFN-γ/TNF-α decrease erythrophagocytosis by human monocytes in vitro, and this effect does not apparently require an increase in SIRP-α or SHP-1 expression.
Assuntos
Anemia Hemolítica Autoimune/imunologia , Antígenos de Diferenciação/imunologia , Membrana Eritrocítica/imunologia , Interferon beta/farmacologia , Interferon gama/farmacologia , Monócitos/imunologia , Fagocitose/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Receptores Imunológicos/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Anemia Hemolítica Autoimune/metabolismo , Anemia Hemolítica Autoimune/patologia , Antígenos de Diferenciação/biossíntese , Membrana Eritrocítica/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Masculino , Monócitos/metabolismo , Monócitos/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/biossíntese , Receptores Imunológicos/biossínteseRESUMO
The functional role of AT(2) receptors is unclear and it activates unconventional signaling pathways, which in general do not involve a classical activation of a G-protein. In the present study, we aimed to investigate the transduction mechanism of AT(2) Ang II receptors in PND15 rat hindbrain membrane preparations, which represents a physiological developmental condition. To determine whether Ang II AT(2) receptors induced association to SHP-1 in rat hindbrain, co-immunoprecipitation assays were performed. Stimulation of Ang II AT(2) receptors induced both a transient tyr-phosphorylation and activation of SHP-1. The possible participation of c-Src in Ang II-mediated SHP-1 activation, we demonstrated by recruitment of c-Src in immunocomplexes obtained with anti AT(2) or anti-SHP-1 antibodies. The association of SHP-1 to c-Src was inhibited by PD123319 and the c-Src inhibitor PP2. Similarly, SHP-1 activity determined in AT(2)-immunocomplexes was inhibited by PD123319 and the c-Src inhibitor PP2. Following stimulation with Ang II, AT(2) receptors recruit c-Src, which was responsible for SHP-1 tyr-phosphorylation and activation. Since AT(2) receptors are involved in neuron migration, we tested the presence of FAK in immunocomplexes. Surprisingly, AT(2)-immunocomplexes contained mainly the 85kDa fragment of FAK. Besides, p125FAK associated to SHP-1. In summary, we demonstrated the presence of an active signal transduction mechanism in PND15 rat hindbrain, a developmental stage critical for cerebellar development. In this model, we showed a complex containing AT(2)/SHP-1/c-Src/p85FAK, suggesting a potential role of Ang II AT(2) receptors in cerebellar development and neuronal differentiation.
Assuntos
Angiotensina II/fisiologia , Quinase 1 de Adesão Focal/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor Tipo 2 de Angiotensina/fisiologia , Rombencéfalo/citologia , Rombencéfalo/metabolismo , Animais , Animais Recém-Nascidos , Proteína Tirosina Quinase CSK , Movimento Celular/fisiologia , Cerebelo/citologia , Cerebelo/enzimologia , Cerebelo/metabolismo , Quinase 1 de Adesão Focal/química , Substâncias Macromoleculares/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Fosforilação/fisiologia , Transporte Proteico/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Ratos , Ratos Wistar , Receptor Tipo 2 de Angiotensina/agonistas , Rombencéfalo/enzimologia , Transdução de Sinais/fisiologia , Quinases da Família srcRESUMO
Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS) during transmission from the sandfly vector (ambient temperature, 25-26°C) to the mammalian host (37°C). We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis) within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 proteins, the majority of which lack a signal peptide and are thus thought to be secreted via nonconventional mechanisms. Interestingly, this protein release is accompanied by alterations in parasite morphology including an augmentation in the budding of exovesicles from its surface. Here we show that the exoproteome of L. mexicana upon TS induces cleavage and activation of the host protein tyrosine phosphatases, specifically SHP-1 and PTP1-B, in a murine bone-marrow-derived macrophage cell line. Furthermore, translocation of prominent inflammatory transcription factors, namely NF-κB and AP-1 is altered. The exoproteome also caused inhibition of nitric oxide production, a crucial leishmanicidal function of the macrophage. Overall, our results provide strong evidence that within early moments of interaction with the mammalian host, L. mexicana rapidly releases proteins and exovesicles that modulate signalling and function of the macrophage. These modulations can result in attenuation of the inflammatory response and deactivation of the macrophage aiding the parasite in the establishment of infection.
Assuntos
Leishmania mexicana/metabolismo , Macrófagos/metabolismo , Macrófagos/parasitologia , Animais , Cromatografia Líquida , Meio Ambiente , Inflamação , Camundongos , Modelos Biológicos , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteoma , Proteômica , Transdução de Sinais , Espectrometria de Massas em Tandem , TemperaturaRESUMO
Leishmania alternates between two morphologically different stages, promastigotes and amastigotes. While the majority of reports focused on how the promastigote form can alter macrophage (Mphi) signaling and function, fewer reports investigated signaling alterations mediated by amastigotes, and there is a lack of comparative studies. In this study, we performed a comparison between the ability of both forms of the parasite to alter Mphi signaling and functions. Here, we show that both promastigotes and amastigotes were able to rapidly activate host protein tyrosine phosphatases (PTPs), importantly the Src homology 2 domain-containing PTP (SHP-1). However, we found that PTP-1B is specifically activated by promastigote but not amastigote infection and that lmcpb(-/-) promastigotes were no longer able to activate PTP-1B. We also show a similarity in the way promastigotes and amastigotes inactivate the transcription factors (TFs) STAT-1alpha and AP-1, but we show differences in the modulation of NF-kappaB, with promastigotes cleaving the p65 subunit, generating a smaller p35 subunit, and amastigotes fully degrading the p65 subunit with no p35 production. Importantly, we show that the cysteine proteinase LmCPb plays a key role in the alteration of NF-kappaB, STAT-1alpha, and AP-1 by promastigote and amastigote infections, ultimately leading to the inability of these TFs to translocate to the nucleus in response to gamma interferon (IFN-gamma) stimulation and thus contributing to the ability of both parasite forms to effectively block IFN-gamma-mediated nitric oxide (NO) production in Mphis.
Assuntos
Leishmania mexicana/imunologia , Leishmania mexicana/patogenicidade , Macrófagos/imunologia , Macrófagos/parasitologia , Transdução de Sinais , Animais , Linhagem Celular , Cisteína Proteases/metabolismo , Humanos , Fator Gênico 3 Estimulado por Interferon/antagonistas & inibidores , Interferon gama/imunologia , Camundongos , Óxido Nítrico/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas de Protozoários/metabolismo , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição RelA/metabolismoRESUMO
The present study examined whether chronic treatment with angiotensin (ANG)-(1-7) reduces cardiac remodeling and inhibits growth-promoting signaling pathways in the heart of fructose-fed rats (FFR), an animal model of insulin resistance. Sprague-Dawley rats were fed either normal rat chow (control) or the same diet plus 10% fructose in drinking water. For the last 2 wk of a 6-wk period of the corresponding diet, control and FFR were implanted with osmotic pumps that delivered ANG-(1-7) (100 ng.kg(-1).min(-1)). A subgroup of each group of animals (control or FFR) underwent a sham surgery. We determined heart weight, myocyte diameter, interstitial fibrosis, and perivascular collagen type III deposition as well as the phosphorylation degree of ERK1/2, JNK1/2, and p38MAPK. FFR showed a mild hypertension that was significantly reduced after ANG-(1-7) treatment. Also, FFR displayed higher ANG II circulating and local levels in the heart that remained unaltered after chronic ANG-(1-7) infusion. An increased heart-to-body weight ratio, myocyte diameter, as well as left ventricular fibrosis and perivascular collagen type III deposition were detected in the heart of FFR. Interestingly, significant improvements in these cardiac alterations were obtained after ANG-(1-7) treatment. Finally, FFR that received ANG-(1-7) chronically displayed significantly lower phosphorylation levels of ERK1/2, JNK1/2, and p38MAPK. The beneficial effects obtained by ANG-(1-7) were associated with normal values of Src-homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) activity in the heart. In conclusion, chronic ANG-(1-7) treatment ameliorated cardiac hypertrophy and fibrosis and attenuated the growth-promoting pathways in the heart. These findings show an important protective role of ANG-(1-7) in the heart of insulin-resistant rats.
Assuntos
Angiotensina I/farmacologia , Frutose/efeitos adversos , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/prevenção & controle , Resistência à Insulina , Fragmentos de Peptídeos/farmacologia , Remodelação Ventricular/efeitos dos fármacos , Angiotensina II/metabolismo , Animais , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/fisiologia , Carboidratos da Dieta/efeitos adversos , Modelos Animais de Doenças , Hipertensão/etiologia , Hipertensão/metabolismo , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Insulina/sangue , Masculino , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Aberrant methylation in promoter-associated CpG islands has been recognized as a major mechanism for tumor suppressor gene silencing in several malignancies. We determined the methylation status of nine tumor suppressor genes in 68 newly diagnosed MM patients by methylation-specific PCR. The frequency of promoter hypermethylation for individual genes was: CDH1, 50%; p16 INK4a, 42.8%; p15 INK4b, 16.2%; SHP1, 14.7%; ER and BNIP3, 13.2%; RAR beta, 11.8%; DAPK 5.9%; and MGMT 0%. Overall, 79% of patients presented at least one hypermethylated gene. By univariate analysis, hypermethylation of DAPK (P < 0.001) and RAR beta (P = 0.01) genes were identified as adverse prognostic features. Median OS of patients with hypermethylation in DAPK (4 months) and RAR beta (34 months) was significantly lower than in patients without hypermethylation (median survival not reached), with values of P < 0.001 and P = 0.01, respectively. Our data suggest that DAPK and RAR beta hypermethylation are adverse prognostic factors in MM. The relevance of these findings as poor prognosis indicators requires confirmation in a larger sample with longer follow-ups.
Assuntos
Metilação de DNA/fisiologia , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor/fisiologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/mortalidade , Adulto , Idoso , Antígenos CD , Proteínas Reguladoras de Apoptose/genética , Caderinas/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas Quinases Associadas com Morte Celular , Feminino , Inativação Gênica , Humanos , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Prognóstico , Regiões Promotoras Genéticas/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteínas Proto-Oncogênicas/genética , Receptores de Estrogênio/genética , Receptores do Ácido Retinoico/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
The aim of this work was to evaluate the regulation of SIRP alpha, an inhibitory phagocyte receptor, and the phosphatase SHP-1 in monocytes of patients with autoimmune hemolytic anemia, and the role of dexamethasone on SIRP alpha and SHP-1 gene expression and erythrophagocytosis in vitro. SIRP alpha and SHP-1 expression was higher in monocytes from AIHA patients compared with normal, returning to normal after glucocorticoid therapy. SIRP alpha and SHP-1 mRNA expression was upregulated in healthy monocytes treated with dexamethasone compared with basal; however, the erythrophagocytic ability was not altered. Our results point to a minor role of SIRP alpha and SHP-1 in determining AIHA.
Assuntos
Anemia Hemolítica Autoimune/metabolismo , Antígenos de Diferenciação/fisiologia , Regulação da Expressão Gênica/imunologia , Glucocorticoides/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologia , Receptores Imunológicos/fisiologia , Anemia Hemolítica Autoimune/genética , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Células Cultivadas , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Fagocitose/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/biossíntese , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Receptores Imunológicos/biossíntese , Receptores Imunológicos/genéticaRESUMO
Angiotensin II (Ang II) AT(2) receptors are abundantly expressed in rat fetal tissues where they probably contribute to development. In the present study we examine the effects of Ang II type 2 receptor stimulation on SHP-1 activation. Ang II (10(-7) M) elicits a rapid and transient tyrosine phosphorylation of SHP-1, maximal at 1 min, in a dose-dependent form, blocked by the AT(2) antagonist, PD123319. SHP-1 phosphorylation is followed in time by tyrosine dephosphorylation of different proteins, suggesting a sequence of events. Ang II induces association of SHP-1 to AT(2) receptors as shown by co-immunoprecipitation, Western blot and binding assays. SHP-1 activity was determined in immunocomplexes obtained with either anti-AT(2) or anti-SHP-1 antibodies, after Ang II stimulation (1 min), in correlation with the maximal level of SHP-1 phosphorylation. Interestingly, following receptor stimulation (1 min) c-Src was associated to AT(2) or SHP-1 immunocomplexes. Preincubation with the c-Src inhibitor PP2 inhibited SHP-1 activation and c-Src association, thus confirming the participation of c-Src in this pathway. We demonstrated here for the first time the involvement of c-Src in SHP-1 activation via AT(2) receptors present in an ex vivo model expressing both receptor subtypes. In this model, AT(2) receptors are not constitutively associated to SHP-1 and SHP-1 is not constitutively activated. Thus, we clearly establish that SHP-1 activation, mediated by the AT(2) subtype, involves c-Src and precedes protein tyrosine dephosphorylation, in rat fetal membranes.
Assuntos
Feto/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Animais , Feminino , Fosforilação , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
BACKGROUND AND PURPOSE: Vascular endothelial growth factor (VEGF) is the most important proangiogenic protein. We have demonstrated that ATL-1, a synthetic analogue of aspirin-triggered lipoxin A(4), inhibits VEGF-induced endothelial cell (EC) migration. In the present study, we investigated the effects of ATL-1 in several other actions stimulated by VEGF. METHODS: Human umbilical vein ECs were treated with ATL-1 for 30 min before stimulation with VEGF. Cell proliferation was measured by thymidine incorporation. Adherent cells were determined by fluorescence intensity using a Multilabel counter. Expression and activity of matrix metalloproteinases (MMP) were analysed by western blot and zymography. KEY RESULTS: ATL-1 inhibited EC adhesion to fibronectin via interaction with its specific receptor. Furthermore, VEGF-induced MMP-9 activity and expression were reduced by pretreatment with ATL-1. Because the transcription factor NF-kappaB has been implicated in VEGF-mediated MMP expression and EC proliferation, we postulated that ATL-1 might modulate the NF-kappaB pathway and, indeed, ATL-1 inhibited NF-kappaB nuclear translocation. Pretreatment of EC with ATL-1 strongly decreased VEGF-dependent phosphorylation of phosphainositide 3-kinase (PI3-K) and extracellular signal-regulated kinase-2 (ERK-2), two signalling kinases involved in EC proliferation. Inhibition of VEGF-induced EC proliferation by ATL-1 was antagonized by sodium orthovanadate, suggesting that this inhibitory activity was mediated by a protein tyrosine phosphatase. This was confirmed by showing that ATL-1 inhibition of VEGF receptor-2 (VEGFR-2) phosphorylation correlates with SHP-1 association with VEGFR-2. CONCLUSIONS AND IMPLICATIONS: The synthetic 15-epi-lipoxin analogue, ATL-1, is a highly potent molecule exerting its effects on multiple steps of the VEGF-induced angiogenesis.
Assuntos
Lipoxinas/farmacologia , Neovascularização Patológica/prevenção & controle , Proteína Tirosina Fosfatase não Receptora Tipo 6/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Transporte Ativo do Núcleo Celular , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Neovascularização Patológica/fisiopatologia , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Veias UmbilicaisRESUMO
Gallbladder disease (GBD) is one of the major digestive diseases. Its risk factors include age, sex, obesity, type 2 diabetes, and metabolic syndrome (MS). The prevalence of GBD is high in minority populations, such as Native and Mexican Americans. Ethnic differences, familial aggregation of GBD, and the identification of susceptibility loci for gallstone disease by use of animal models suggest genetic influences on GBD. However, the major susceptibility loci for GBD in human populations have not been identified. Using ultrasound-based information on GBD occurrence and a 10-cM gene map, we performed multipoint variance-components analysis to localize susceptibility loci for GBD. Phenotypic and genotypic data from 715 individuals in 39 low-income Mexican American families participating in the San Antonio Family Diabetes/Gallbladder Study were used. Two GBD phenotypes were defined for the analyses: (1) clinical or symptomatic GBD, the cases of cholecystectomies due to stones confirmed by ultrasound, and (2) total GBD, the clinical GBD cases plus the stone carriers newly diagnosed by ultrasound. With use of the National Cholesterol Education Program/Adult Treatment Panel III criteria, five MS risk factors were defined: increased waist circumference, hypertriglyceredemia, low high-density lipoprotein cholesterol, hypertension, and high fasting glucose. The MS risk-factor score (range 0-5) for a given individual was used as a single, composite covariate in the genetic analyses. After accounting for the effects of age, sex, and MS risk-factor score, we found stronger linkage signals for the symptomatic GBD phenotype. The highest LOD scores (3.7 and 3.5) occurred on chromosome 1p between markers D1S1597 and D1S407 (1p36.21) and near marker D1S255 (1p34.3), respectively. Other genetic locations (chromosomes 2p, 3q, 4p, 8p, 9p, 10p, and 16q) across the genome exhibited some evidence of linkage (LOD >or=1.2) to symptomatic GBD. Some of these chromosomal regions corresponded with the genetic locations of Lith loci, which influence gallstone formation in mouse models. In conclusion, we found significant evidence of major genetic determinants of symptomatic GBD on chromosome 1p in Mexican Americans.
Assuntos
Cromossomos Humanos Par 1/genética , Doenças da Vesícula Biliar/epidemiologia , Doenças da Vesícula Biliar/genética , Predisposição Genética para Doença , Americanos Mexicanos/genética , Adulto , Idoso , Doenças da Vesícula Biliar/etnologia , Genoma Humano , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Escore Lod , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Obesidade/genética , Fenótipo , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , Receptores Tipo II do Fator de Necrose Tumoral/genética , Fatores de RiscoRESUMO
Insulin resistance is known to play a pivotal role in type 2 diabetes. Senile individuals, besides being prone to insulin resistance and, consequently, to type 2 diabetes, manifest diseases of the central nervous system (CNS) that may be influenced by disturbances of insulin signaling in the brain, such as memory impairment, Parkinson disease, and Alzheimer disease. We investigated the expression and response to insulin of elements involved in the insulin-signaling pathway in the forebrain cortex and cerebellum of rats ages 1 d to 60 wk. The protein content of insulin receptors and SRC homology adaptor protein (SHC) did not change significantly along the time frame analyzed. However, insulin-induced tyrosine phosphorylation of the insulin receptor and SHC, and the association of SHC/growth factor receptor binding protein-2 (GRB2) decreased significantly from d 1 to wk 60 of life in both types of tissues. Moreover, the expression of SH protein tyrosine phosphatase-2 (SHP2), a tyrosine phosphatase involved in insulin signal transduction and regulation of the insulin signal, decreased significantly with age progression, in both the forebrain cortex and the cerebellum of rats. Thus, elements involved in the insulin-signaling pathway are regulated at the expression and/or functional level in the CNS, and this regulation may play a role in insulin resistance in the brain.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Envelhecimento , Encéfalo/metabolismo , Insulina/metabolismo , Transdução de Sinais , Animais , Cerebelo/metabolismo , Proteína Adaptadora GRB2 , Insulina/sangue , Insulina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Fosforilação , Fosfotirosina/metabolismo , Prosencéfalo/metabolismo , Proteína Fosfatase 2 , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/análise , Proteínas/análise , Proteínas/metabolismo , Ratos , Ratos Wistar , Receptor de Insulina/análise , Receptor de Insulina/metabolismo , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Domínios de Homologia de srcRESUMO
Insulin induces phosphorylation and activation of JAK2 tyrosine, as well as its association with STAT1 and SHP2 in insulin-sensitive tissues of intact rats, thus demonstrating a new pathway in transduction of insulin signals. We investigated this pathway in hearts of rats in three situations of insulin resistance: 72 h of fasting, chronic treatment with dexamethasone, and acute treatment with epinephrine. The acute treatment with epinephrine showed no difference in insulin-induced JAK2 tyrosine phosphorylation or JAK2/STAT1 and JAK2/SHP2 association in comparison with the control. In fasted rats the JAK2 protein concentration decreased, accompanied by a decrease in the stoichiometry of the phosphorylation to 70%, an increase in association of JAK2/STAT1 to 160%, and a decrease in JAK2/SHP2 association to 85%. In the dexamethasone-treated group, the JAK2 protein concentrations increased but the stoichiometry of its phosphorylation decreased to 20%, whereas the JAK2/STAT1 and JAK2/SHP2 associations changed by 70% and 170%, respectively. In fasting and dexamethasone-treated rats, therefore, insulin-induced JAK2 tyrosine phosphorylation decreases, and the JAK2 protein expression is differentially regulated such that the insulin-induced JAK2 association with SHP2 and STAT1 shows opposite interactions with the kinase.
Assuntos
Resistência à Insulina/fisiologia , Miocárdio/enzimologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Animais , Proteínas de Ligação a DNA/metabolismo , Dexametasona/farmacologia , Ativação Enzimática/efeitos dos fármacos , Epinefrina/farmacologia , Jejum , Peptídeos e Proteínas de Sinalização Intracelular , Janus Quinase 2 , Masculino , Miocárdio/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/química , Ratos , Ratos Wistar , Fator de Transcrição STAT1 , Transdução de Sinais , Transativadores/metabolismo , Tirosina/metabolismoRESUMO
GH stimulates the tyrosine phosphorylation of various cellular polypeptides, including the GH receptor itself, in an early part of the intracellular response. Some of these phosphorylations are catalyzed by a GH receptor-associated kinase identified as JAK2, a member of the Janus family of tyrosine kinases. In cultured cells, GH stimulates the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-2, and Shc. This study investigated whether GH could cause the tyrosine phosphorylation of IRSs and Shc proteins in fasted rat tissues in vivo. GH was administered to fasted Wistar rats via a portal vein, and extracts of different tissues were immunoprecipitated with specific antibodies. GH increased the tyrosine phosphorylation of IRS-1, IRS-2, JAK2, and Shc proteins in the liver, heart, kidney, muscle, and adipose tissue of rats. The roles of these substrates as signaling molecules for GH were further demonstrated by the finding that GH stimulated the association of IRS-1/2 with phosphatidylinositol 3-kinase, Grb2, and phosphotyrosine phosphatase and of Shc with Grb2. The correlation between JAK2 tyrosyl phosphorylation and IRS-1 tyrosyl phosphorylation in response to GH together with the results of the in vitro tyrosine kinase assay are consistent with the hypothesis that JAK2 may mediate GH-induced phosphorylation of IRS-1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Hormônio do Crescimento/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas , Receptor de Insulina/metabolismo , Tirosina/metabolismo , Animais , Proteína Adaptadora GRB2 , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Janus Quinase 2 , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , Ratos , Ratos Wistar , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Domínios de Homologia de srcRESUMO
Insulin stimulates the tyrosine kinase activity of its receptor resulting in the phosphorylation of its cytosolic substrate, insulin receptor substrate-1 (IRS-1) which, in turn, associates with proteins containing SH2 domains. It has been shown that IRS-1 associates with the tyrosine phosphatase SHPTP2 in cell cultures. While the effect of the IRS-1/SHPTP2 association on insulin signal transduction is not completely known, this association may dephosphorylate IRS-1 and may play a critical role in the mitogenic actions of insulin. However, there is no physiological demonstration of this pathway of insulin action in animal tissues. In the present study we investigated the ability of insulin to induce association between IRS-1 and SHPTP2 in liver and muscle of intact rats, by co-immunoprecipitation with anti-IRS-1 antibody and anti-SHPTP2 antibody. In both tissues there was an increase in IRS-1 association with SHPTP2 after insulin stimulation. This association occurred when IRS-1 had the highest level of tyrosine phosphorylation and the decrease in this association was more rapid than the decrease in IRS-1 phosphorylation levels. The data provide evidence against the participation of SHPTP2 in IRS-1 dephosphorylation in rat tissues, and suggest that the insulin signal transduction pathway in rat tissues is related mainly to the mitogenic effects of the hormone.