RESUMO
Hematopoietic cell phosphatase (HCP) is a tyrosine phosphatase with two Src homology 2 (SH2) domains that is predominantly expressed in hematopoietic cells, including cells whose growth is regulated by interleukin-3 (IL-3). The potential effects of HCP on IL-3-induced protein tyrosine phosphorylation and growth regulation were examined to assess the role of HCP in hematopoiesis. Our studies demonstrate that, following ligand binding, HCP specifically associates with the beta chain of the IL-3 receptor through the amino-terminal SH2 domain of HCP, both in vivo and in vitro, and can dephosphorylate the receptor chain in vitro. The effects of increasing or decreasing HCP levels in IL-3-dependent cells were assessed with dexamethasone-inducible constructs containing an HCP cDNA in sense and antisense orientations. Increased HCP levels were found to reduce the levels of IL-3-induced tyrosine phosphorylation of the receptor and to dramatically suppress cell growth. Conversely, decreasing the levels of HCP increased IL-3-induced tyrosine phosphorylation of the receptor and marginally increased growth rate. These results support a role for HCP in the regulation of hematopoietic cell growth and begin to provide a mechanistic explanation for the dramatic effects that the genetic loss of HCP, which occurs in motheaten (me) and viable motheaten (mev) mice, has on hematopoiesis.
Assuntos
Proteínas Tirosina Fosfatases/metabolismo , Receptores de Interleucina-3/metabolismo , Tirosina/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Regulação para Baixo , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Interleucina-3/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Mutantes , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , TransfecçãoRESUMO
The functions of wild-type and mutant mouse interleukin-10 receptors (mIL-10R) expressed in murine Ba/F3 cells were studied. As observed previously, IL-10 stimulates proliferation of IL-10R-expressing Ba/F3 cells. Accumulation of viable cells in the proliferation assay is to a significant extent balanced by concomitant cell death. Moreover, growth in IL-10 also induces a previously unrecognized response, differentiation of the cells, as evidenced both by formation of large clusters of cells in cultures with IL-10 and by induction or enhancement of expression of several cell surface antigens, including CD32/16, CD2, LECAM-1 (v-selectin), and heat-stable antigen. Two distinct functional regions near the C terminus of the mIL-10R cytoplasmic domain which mediate proliferation were identified; one of these regions also mediates the differentiation response. A third region proximal to the transmembrane domain was identified; removal of this region renders the cell 10- to 100-fold more sensitive to IL-10 in the proliferation assay. In cells expressing both wild-type and mutant IL-10R, stimulation with IL-10 leads to tyrosine phosphorylation of the kinases JAK1 and TYK2 but not JAK2 or JAK3 under the conditions tested.
Assuntos
Linfócitos B/metabolismo , Interleucina-10/farmacologia , Interleucina-3/farmacologia , Receptores de Interleucina/metabolismo , Animais , Sequência de Bases , Antígenos CD2/biossíntese , Moléculas de Adesão Celular/biossíntese , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Citometria de Fluxo , Selectina L , Camundongos , Dados de Sequência Molecular , Fosforilação , Conformação Proteica , Proteínas Tirosina Quinases/metabolismo , Receptores de IgG/biossíntese , Receptores de Interleucina/genética , Receptores de Interleucina-10 , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Members of the recently discovered SOCS/CIS/SSI family have been proposed as regulators of cytokine signaling, and while targets and mechanisms have been suggested for some family members, the precise role of these proteins remains to be defined. To date no SOCS proteins have been specifically implicated in interleukin-2 (IL-2) signaling in T cells. Here we report SOCS-3 expression in response to IL-2 in both T-cell lines and human peripheral blood lymphocytes. SOCS-3 protein was detectable as early as 30 min following IL-2 stimulation, while CIS was seen only at low levels after 2 h. Unlike CIS, SOCS-3 was rapidly tyrosine phosphorylated in response to IL-2. Tyrosine phosphorylation of SOCS-3 was observed upon coexpression with Jak1 and Jak2 but only weakly with Jak3. In these experiments, SOCS-3 associated with Jak1 and inhibited Jak1 phosphorylation, and this inhibition was markedly enhanced by the presence of IL-2 receptor beta chain (IL-2Rbeta). Moreover, following IL-2 stimulation of T cells, SOCS-3 was able to interact with the IL-2 receptor complex, and in particular tyrosine phosphorylated Jak1 and IL-2Rbeta. Additionally, in lymphocytes expressing SOCS-3 but not CIS, IL-2-induced tyrosine phosphorylation of STAT5b was markedly reduced, while there was only a weak effect on IL-3-mediated STAT5b tyrosine phosphorylation. Finally, proliferation induced by both IL-2- and IL-3 was significantly inhibited in the presence of SOCS-3. The findings suggest that when SOCS-3 is rapidly induced by IL-2 in T cells, it acts to inhibit IL-2 responses in a classical negative feedback loop.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interleucina-2/metabolismo , Proteínas do Leite , Proteínas/metabolismo , Proteínas Repressoras , Linfócitos T/citologia , Transativadores/metabolismo , Fatores de Transcrição , Tirosina/metabolismo , Animais , Divisão Celular , Linhagem Celular , Linhagem Celular Transformada , Humanos , Interleucina-2/farmacologia , Interleucina-3/metabolismo , Janus Quinase 1 , Janus Quinase 3 , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas/genética , Coelhos , Receptores de Interleucina-2/metabolismo , Fator de Transcrição STAT5 , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de CitocinaRESUMO
Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a critical role in growth and differentiation of myeloid cells. We previously reconstituted high-affinity human GM-CSF receptor (hGM-CSFR) in a pro-B cell line, BA/F3, by cotransfecting alpha- and beta-chain cDNA clones and showed that the reconstituted receptor could transduce growth-promoting signals. The high-affinity hGM-CSFR was also reconstituted in mouse NIH 3T3 cells, but its ability to transduce signals in fibroblasts remained undetermined. In the present study, we further characterized signal transduction by the reconstituted hGM-CSFR in both NIH 3T3 cells and BA/F3 cells. We found that the reconstituted hGM-CSFR transduces signals in NIH 3T3 fibroblasts and BA/F3 cells in response to hGM-CSF to activate transcription of the c-fos, c-jun, and c-myc proto-oncogenes. hGM-CSF also induces protein tyrosine phosphorylation and DNA synthesis in both cell types. These results indicated that hGM-CSFR is functional in fibroblasts, that signal transduction via hGM-CSFR in fibroblasts involves tyrosine kinase(s), and that association of hGM-CSFR with a factor(s) specific to hematopoietic cell lineage is not essential to transduce growth-promoting signals.
Assuntos
Linfócitos B/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Transdução de Sinais , Células 3T3 , Animais , Transporte Biológico , Divisão Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Genes fos/genética , Genes jun/genética , Genes myc/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Substâncias Macromoleculares , Camundongos , Fosforilação , Regiões Promotoras Genéticas/genética , Proto-Oncogenes/genética , Receptor de Fator Estimulador de Colônias de Macrófagos , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
STAT (signal transducers and activators of transcription) proteins are transcription factors which are activated by phosphorylation on tyrosine residues upon stimulation by cytokines. Seven members of the STAT family are known, including the closely related STAT5A and STAT5B, which are activated by various cytokines. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5 activation in various systems are not clear. We applied PCR-driven random mutagenesis and a retrovirus-mediated expression screening system to identify constitutively active forms of STAT5. By this strategy, we have identified a constitutively active STAT5 mutant which has two amino acid substitutions; one is located upstream of the putative DNA binding domain (H299R), and the other is located in the transactivation domain (S711F). The mutant STAT5 was constitutively phosphorylated on tyrosine residues, localized in the nucleus, and was transcriptionally active. Expression of the mutant STAT5 partially dispenses with interleukin 3 (IL-3) as a growth stimulant of IL-3-dependent cell lines. Further analyses of the mutant STAT5 have demonstrated that both of the mutations are required for nuclear localization, efficient transcriptional activation, and induction of IL-3-independent growth of an IL-3-dependent cell line, Ba/F3, and have indicated that a molecular basis for the constitutive activation is the stability of the phosphorylated form of the mutant STAT5.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas do Leite , Transativadores/genética , Transativadores/metabolismo , Animais , Divisão Celular , Linhagem Celular , Camundongos , Mutagênese , Fosforilação , Proteínas Proto-Oncogênicas c-raf/metabolismo , Fator de Transcrição STAT5RESUMO
The phosphatidylinositol (PI)-3 kinase (PI3K) pathway plays a central role in regulating many biological processes via the generation of the key second messenger PI-3,4,5-trisphosphate (PI-3,4,5-P3). This membrane-associated phospholipid, which is rapidly, albeit transiently, synthesized from PI-4,5-P2 by PI3K in response to a diverse array of extracellular stimuli, attracts pleckstrin homology (PH) domain-containing proteins to membranes to mediate its many effects. To ensure that the activation of this pathway is appropriately suppressed/terminated, the ubiquitously expressed tumor suppressor PTEN hydrolyzes PI-3,4,5-P3 back to PI-4,5-P2 while the 145-kDa hemopoietic-restricted SH2-containing inositol 5'- phosphatase, SHIP (also known as SHIP1), the 104-kDa stem cell-restricted SHIP (sSHIP) and the more widely expressed 150-kDa SHIP2 hydrolyze PI-3,4,5-P3 to PI-3,4-P2. In this review we will concentrate on the properties of the three SHIPs, with special emphasis being placed on the role that SHIP plays in cytokine-induced signaling.
Assuntos
Citocinas/fisiologia , Monoéster Fosfórico Hidrolases/fisiologia , Animais , Humanos , Camundongos , Camundongos Knockout , Modelos Biológicos , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/genética , Transdução de Sinais , Domínios de Homologia de srcRESUMO
N-Acetyldopamine is a relatively nontoxic analog of dopamine that has shown significant antitumor activity in experimental L1210 and P388 leukemias. A convenient two-step chemical synthesis of this derivative provided a sufficient quantity of material to study the effects of the administration of this drug by a more frequent schedule. The antitumor activity in the L1210 and P388 systems was significantly improved when N-acetyldopamine was administered to (C57BL/6J x DBA/2)F1 (B6D2F1) mice three times daily for 4 days. Long-term survivors appeared, which indicated tumor kills in excess of 10(5) cells. The effects of dopamine and N-acetyldopamine on the incorporation of radioactively labeled thymidine (dThd) by proliferating tissues (tumor, bone marrow, and gastrointestinal mucosa) were examined in tumor-bearing B6D2F1 mice. The dThd incorporation into tumor cells was selectively inhibited in both the L1210 and P388 leukemias with less inhibition of bone marrow and gastrointestinal mucosa cells. One hour after a dose of 400 mg N-acetyldopamine/kg, dThd incorporation was completely suppressed by both P388 and L1210 tumor cells with minimal effects on bone marrow or gastrointestinal mucosa cells. The synthetic method reported here is applicable to the preparation of various derivatives for study.
Assuntos
Dopamina/análogos & derivados , Leucemia Experimental/tratamento farmacológico , Animais , Medula Óssea/efeitos dos fármacos , Dopamina/administração & dosagem , Dopamina/síntese química , Dopamina/uso terapêutico , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Mucosa Gástrica/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Leucemia L1210/tratamento farmacológico , Masculino , CamundongosRESUMO
Interleukin-9 (IL-9) is a growth factor for T cells and various hematopoietic and lymphoid tumor cells. IL-9 signaling involves activation of Janus kinase (JAK)1 and JAK3 kinases, and signal transducer and activator of transcription (STAT)1, STAT3 and STAT5. Using a dominant negative form of STAT5 (STAT5delta), we demonstrated that this factor is an important mediator of IL-9-dependent Ba/F3 cell growth. Mutation of the STAT binding site of the IL-9 receptor (tyr116phe) results in an important decrease in STAT activation and inhibition of proliferation in the presence of IL-9. A small number of cells escape this inhibition, and IL-9-dependent cell lines could be derived. The selected cells required activation of STAT5 for growth, which was blocked by STAT5delta expression and enhanced by overexpression of wild-type STAT5. In contrast to parental cells, Ba/F3-Phe116 cells growing in the presence of IL-9 further progress to cytokine-independent tumorigenic clones. These tumorigenic clones exhibited a strong cytokine-independent activation of JAK1 and STAT5, which most likely supports their proliferation. Transfection of a constitutively activated variant of STAT5 promoted the growth of wild-type Ba/F3 cells in the absence of cytokine. Finally, the expression of the proto-oncogene pim-1 was correlated with STAT5 activation and cell growth. Our data suggest that STAT5 is an important mediator of IL-9-driven proliferation and that dysregulation of STAT5 activation favors tumorigenesis of lymphoid cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interleucina-9/metabolismo , Linfócitos/metabolismo , Proteínas do Leite , Proteínas Serina-Treonina Quinases , Transativadores/metabolismo , Animais , Sítios de Ligação , Células CHO , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Cricetinae , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta a Droga , Ativação Enzimática , Feminino , Humanos , Janus Quinase 1 , Janus Quinase 2 , Linfócitos/patologia , Camundongos , Camundongos SCID , Mutação , Transplante de Neoplasias , Plasmídeos , Testes de Precipitina , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-pim-1 , Fator de Transcrição STAT5 , Tetraciclina/farmacologia , Fatores de Tempo , Transativadores/química , Transativadores/genética , Transfecção , Células Tumorais CultivadasRESUMO
Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a critical role in growth and differentiation of myeloid cells. We previously reconstituted high affinity human GM-CSF receptor (hGM-CSFR) in a proB cell line BA/F3 by cotransfecting alpha and beta chain cDNA clones and showed that the reconstituted receptor could transduce growth promoting signals. The high affinity hGM-CSFR was also reconstituted in mouse NIH3T3 cells, but its ability to transduce signals in fibroblasts remained unanswered. In the present study, we further characterized signal transduction by the reconstituted hGM-CSFR both in NIH3T3 cells and BA/F3 cells. We found that the reconstituted hGM-CSFR transduces signals in NIH3T3 fibroblasts and BA/F3 cells in response to human GM-CSF to activate transcription of c-fos, c-jun and c-myc protooncogenes. hGM-CSF also induces protein tyrosine phosphorylation and DNA synthesis in both cell types. The ability of hGM-CSFR to transduce signals was affected by inhibitors of tyrosine kinase. These results indicated that the hGM-CSFR is functional in fibroblasts, that signal transduction via the hGM-CSFR in fibroblasts involves tyrosine kinase(s) and that association of hGM-CSFR with factor(s) specific to hematopoietic cell lineage is not essential to transduce growth promoting signals.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/fisiologia , Transdução de Sinais/fisiologia , Transcrição Gênica , Células 3T3 , Animais , Benzoquinonas , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Isoflavonas/farmacologia , Lactamas Macrocíclicas , Luciferases/genética , Luciferases/metabolismo , Camundongos , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Quinonas/farmacologia , RNA Mensageiro/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/química , Rifabutina/análogos & derivados , Especificidade da Espécie , TransfecçãoRESUMO
Recently, JAK2 kinase was found to be one of the tyrosine kinases activated by interleukin-3 (IL-3) in target cells. JAK2 belongs to a family of kinases that act upstream of transcription factors called STATs. STATs exist in the cytoplasm as latent, transcriptionally inactive forms until, in response to extracellular signals, they become phosphorylated on tyrosine residues, translocate to the nucleus, and bind to specific DNA elements. Because IL-3 activates JAK2, we searched for the STAT(s) that might transduce IL-3 signals. Several lines of evidence suggest that IL-3 uses the murine homologue of STAT5, a factor originally purified from sheep. Unexpectedly, during isolation of the murine homologue, we found two highly related molecules that we have designated STAT5A and STAT5B.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Interleucina-3/fisiologia , Interleucina-5/fisiologia , Proteínas do Leite , Transativadores/fisiologia , Animais , Proteínas de Ligação a DNA/química , Camundongos , Fosforilação , Fosfotirosina , Fator de Transcrição STAT5 , Ovinos , Transdução de Sinais , Transativadores/química , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
A variety of homobifunctional crosslinking agents have been used to gain insight into the nature of the murine interleukin 3 (mIL-3) receptor. When [125I]mIL-3 was cross-linked to receptor sites on the surfaces of intact B6SUtA1 cells with disuccinimidyl suberate (DSS), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed the existence of two radiolabeled species with molecular weights of 140 (p140) and 70 (p70) kd (after subtraction of [125I]mIL-3). The relative intensities of the two bands did not change when the [125I]mIL-3 concentration was varied, confirming Scatchard results which suggested only one affinity class. However, when [125I]mIL-3 was crosslinked to intact cells and then incubated at 37 degrees C, the intensity of p140 decreased relative to p70, suggesting a conversion of p140 to p70. This conversion could be inhibited by sodium azide, methylamine, and bacitracin and could also be prevented by first boiling for 1 min in 2% SDS and 5% 2-mercaptoethanol. The putative protease that carried out this apparent conversion appeared to be associated both with plasma membranes prepared from these cells and also with solubilized receptors. Moreover, when p140, crosslinked with both dithiobis succinimidylpropionate and glutaraldehyde, was purified and reelectrophoresed under reducing conditions, p70 could be generated. N-glycanase digestion of p140 and p70 revealed a similar level of N-linked carbohydrate, which upon closer study appeared to consist of two chains, a 3-kd and an 8-kd moiety. Consistent with this data, we propose that the receptor is a 140-kd glycoprotein that is cleaved to a 70-kd surface protein upon mIL-3 binding and chemical crosslinking.
Assuntos
Interleucina-3/metabolismo , Receptores Imunológicos/análise , Animais , Membrana Celular/análise , Reagentes de Ligações Cruzadas , Glicosilação , Radioisótopos do Iodo , Camundongos , Modelos Estruturais , Receptores de Interleucina-3RESUMO
The growth hormone regulated serine protease inhibitor (SPI) 2.1 and 2.2 gene promoters have been shown to contain a response element similar to the gamma-interferon activated sequence (GAS) family of signal transducer and activator of transcription (STAT) response elements. We have investigated the STAT and cytokine specificity of the SPI 2.1 STAT responsive element using a luciferase (LUC) reporter construct and a cDNA complementation strategy in the COS 7 cell line. Growth hormone was found to stimulate SPI-LUC reporter gene expression via activation of STAT 5, but not STATs 1 or 3, which indicates that the SPI 2.1 STAT responsive element is STAT 5 specific. In addition to the growth hormone receptor, the receptors for prolactin and erythropoietin enhanced gene transcription via the SPI 2.1 STAT responsive element, which indicates that this element is, on the other hand, not cytokine specific. Activation of STAT 5 was also observed after growth hormone treatment of cells transfected with cDNA expression plasmids for several different truncated growth hormone receptor mutants, although this activation was less efficient than with the wild type receptor. Point mutation of individual tyrosines in the growth hormone receptor intracellular domain to phenylalanines had no significant effect on signal transduction via STAT 5. These data, taken together with results from experiments using the phosphatase inhibitor sodium orthovanadate, suggest that STAT 5 may not have an absolute requirement for specific phosphorylated receptor tyrosine docking sites. That receptor tyrosine residues in a variety of amino acid contexts, or phosphorylated Janus kinase (JAK) 2 alone, can facilitate STAT 5 activation could explain the observed lack of cytokine specificity in STAT 5 activation.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas do Leite , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas , Inibidores de Serina Proteinase/genética , Serpinas , Transativadores/genética , Animais , Sequência de Bases , Células COS , DNA Complementar/genética , Eritropoetina/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Janus Quinase 2 , Camundongos , Prolactina/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Receptores da Prolactina/genética , Receptores da Prolactina/metabolismo , Receptores da Somatotropina/genética , Receptores da Somatotropina/metabolismo , Fator de Transcrição STAT5 , Deleção de Sequência , Transdução de Sinais , Ativação Transcricional , Transfecção , Vanadatos/farmacologiaRESUMO
We have investigated the possible involvement of the MAPK pathway in the growth hormone(GH)-induced activation of one of the members of signal transducers and activators of transcription, STAT5, by using the MAPK kinase (MEK) inhibitor PD98059. PD98059 treatment of Chinese hamster ovarian cells, stably transfected with the GH receptor (CHOA cells), abolished the GH-induced MAPK activity. PD98059 decreased the amount of GH-induced STAT5 in nuclear extract with DNA-binding capacity. Furthermore, GH dependent transcription of a STAT5 regulated reporter gene was inhibited by PD98059. The MEK inhibitor did not reduce GH-stimulated nuclear translocation of STAT5. We also investigated if PD98059 differentially influences the activation of the two STAT5 homologs, STAT5a and STAT5b, which differ mainly at the C-terminal end, one of the differences being the presence of a possible MAPK phosphorylation site in STAT5a. Expression plasmids for these transcription factors were transfected into CHOA cells together with a reporter gene. GH-stimulated fold induction of transcription was reduced by PD98059 in STAT5a but not in STAT5b overexpressing cells. A MAPK phosphorylation site-mutated version of STAT5a was also transfected into CHOA cells. GH-stimulated fold induction of cotransfected reporter gene was not reduced by PD98059 in cells overexpressing mutant STAT5a. The above data show that the MAPK pathway is required for the full activation of one of the STAT5 isoforms (STAT5a).
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Ligação a DNA/fisiologia , Hormônio do Crescimento/farmacologia , Proteínas do Leite , Inibidores de Proteínas Quinases , Transativadores/fisiologia , Ativação Transcricional/fisiologia , Sequência de Aminoácidos , Animais , Células CHO , Extratos Celulares , Núcleo Celular/metabolismo , Cricetinae , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Dados de Sequência Molecular , Proteínas Quinases/fisiologia , Receptores da Somatotropina/genética , Proteínas Recombinantes de Fusão , Fator de Transcrição STAT5 , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
BACKGROUND: Previous work has demonstrated prolonged allograft survival after donor-specific portal vein immunization before the transplantation. The purpose of this study was to examine the potential mechanism of portal vein-induced hyporesponsiveness after portal vein immunization with the soluble protein ovalbumin. METHODS: Balb/c mice were immunized with a portal vein injection of ovalbumin. After the immunization, in vivo delayed-type hypersensitivity response and in vitro proliferative response of ovalbumin-specific T cells were assessed to determine host immune response. Type 1 (IL-2, IL-12, IFN-gamma) and type 2 (IL-4, TGF-beta) regulatory cytokines were assessed by semiquantitative reverse transcriptase polymerase chain reaction. Sera anti-ovalbumin IgG, IgG1, and IgG2a were measured by enzyme-linked immunosorbent assay, and the antigen-presenting ability of liver nonparenchymal cells (NPCs) was assessed by T-cell proliferation to ovalbumin in vitro. RESULTS: There was significant inhibition of ovalbumin-specific delayed-type hypersensitivity and T-cell proliferation in portal vein-immunized mice compared with intraperitoneal-immunized or control mice. Reverse transcriptase polymerase chain reaction analysis results showed that lymphocytes from portal vein-immunized mice exhibited decreased type 1 and increased type 2 cytokine messenger RNA expression compared with intraperitoneal-immunized or control animals. The type 2 cytokine response of lymphocytes from ovalbumin portal vein-immunized mice correlated with increased sera ovalbumin-IgG1 and decreased IgG2a. The results of an antigen-presenting assay revealed that liver NPCs were deficient antigen-presenting cells compared with adherent cells from heart or spleen. CONCLUSIONS: Processing of ovalbumin by hepatic NPCs results in hyporesponsiveness to ovalbumin by an impaired type 1 cytokine response and a preferential shift toward a type 2 cytokine response, possibly because of defective antigen presentation by hepatic NPCs. Intrahepatic processing of antigen may play an important role in the development of strategies to reduce host immunoreactivity against transplanted allografts.
Assuntos
Antígenos/administração & dosagem , Tolerância Imunológica , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Animais , Apresentação de Antígeno , Citocinas/genética , Feminino , Facilitação Imunológica de Enxerto , Hipersensibilidade Tardia , Imunização , Imunoglobulina G/sangue , Imunoglobulina G/classificação , Técnicas In Vitro , Fígado/citologia , Fígado/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Veia Porta , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linfócitos T/imunologia , Imunologia de Transplantes , Transplante HomólogoRESUMO
The emotional strain associated with caregiving as experienced by both black (n = 117) and white (n = 464) daughter caregivers was examined from a role theory perspective. Black daughters reported less role strain overall. Conflict between caregiving duties and the caregivers' personal and social life was a predictor for both groups. For black women the unique predictors were: poor perceived health, unavailability of respite support, and lower caregiving role demand. For white women poor quality of parent-daughter relationship and work conflict were the unique predictors.
Assuntos
Negro ou Afro-Americano/psicologia , Cuidadores/psicologia , Geriatria , Papel (figurativo) , Estresse Psicológico/epidemiologia , População Branca/psicologia , Mulheres/psicologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Cuidadores/estatística & dados numéricos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Relações Pais-Filho , Teoria Psicológica , Estados UnidosRESUMO
This research examines the impact of various factors on perceived emotional strain of adult son and daughter caregivers of frail elderly parents. Daughters experienced higher levels of emotional strain than did sons. Perceived interference between caregiving and the caregiver's personal and social life predicted emotional strain for both sons and daughters. For daughters the most important predictors of emotional strain were interference with work and quality of relationship with the parent. For sons the most important predictors were behavioral problems of the parent and few informal helpers.
Assuntos
Cuidadores/psicologia , Assistência Domiciliar/psicologia , Núcleo Familiar/psicologia , Estresse Psicológico/psicologia , Atividades Cotidianas , Adulto , Idoso , Idoso de 80 Anos ou mais , Cuidadores/estatística & dados numéricos , Efeitos Psicossociais da Doença , Estudos Transversais , Feminino , Idoso Fragilizado , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Análise de Regressão , Fatores Sexuais , Estados Unidos , Recursos HumanosRESUMO
Using data from the 1982-84 National Long-Term Care Channeling Demonstration, this study examines factors associated with long-term care service use by African American, Hispanic, and white frail elders living in the community. Findings indicate that in addition to predisposing, enabling, and need factors, race/ethnicity is a significant predictor of each type of service use.
Assuntos
Etnicidade , Idoso Fragilizado/psicologia , Assistência de Longa Duração/estatística & dados numéricos , Negro ou Afro-Americano , Idoso , Idoso de 80 Anos ou mais , Feminino , Hispânico ou Latino , Humanos , Masculino , Estados UnidosRESUMO
This national interview study examined the health impact of caring for frail elders in a sample of 437 spouse caregivers. The principal findings were that (a) caregiver emotional strain was the strongest common predictor of both poor perceived health and functional limitations, (b) wife caregivers' poor health was associated with care recipients' perceived unmet needs and increased depression, (c) husband caregivers' poor health status was predicted by longer caregiving duration, and (d) non-White wife caregivers reported poorer perceived health than did their White counterparts.