RESUMO
Fluoride is known to increase bone mass in vivo, probably through stimulation of osteoblast proliferation; however, the mechanisms of fluoroaluminate action in osteoblasts have not yet been elucidated. We have previously shown that in osteoblastic MC3T3-E1 cells, fluoroaluminate stimulates G protein-mediated protein tyrosine phosphorylation (Scaronuscarona, M., Standke, G. J. R., Jeschke, M., and Rohner, D. (1997) Biochem. Biophys. Res. Commun. 235, 680-684). Although the Ser/Thr kinases Erk1, Erk2, and p70(S6K) were activated in response to fluoroaluminate, the identity of fluoroaluminate-activated tyrosine kinase(s) remained elusive. In this study, we show that in MC3T3-E1 cells, fluoroaluminate induces a 110-kDa tyrosine-phosphorylated protein that we identify as Pyk2, a cytoplasmic tyrosine kinase related to Fak (focal adhesion kinase). The tyrosine phosphorylation of Pyk2 increased in a dose- and time-dependent manner. The autophosphorylation activity of Pyk2 increased 3-fold and reached its maximum within 10 min of fluoroaluminate treatment. Fluoroaluminate also induced activation of Src and the association of Pyk2 with Src. The phosphorylation of Src-associated Pyk2 increased >20-fold in in vitro kinase assays, suggesting that Pyk2 is phosphorylated by Src. Although MC3T3-E1 cells express much more Fak than Pyk2, Src preferentially associated with Pyk2. In vitro, Pyk2 bound to the Src SH2 domain, suggesting that this interaction mediates the Src-Pyk2 association in cells. These data indicate that osteoblastic cells express Pyk2, which is tyrosine-phosphorylated and activated in response to G protein activation by fluoroaluminate, and that the mechanism of Pyk2 activation most likely involves Src. Thus, Src and Pyk2 are tyrosine kinases involved in G protein-mediated tyrosine phosphorylation in osteoblastic cells and may be important for the osteogenic action of fluoroaluminate.
Assuntos
Alumínio/farmacologia , Flúor/farmacologia , Osteoblastos/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Quinases da Família src/metabolismo , Células 3T3 , Animais , Relação Dose-Resposta a Droga , Ativação Enzimática , Quinase 2 de Adesão Focal , Camundongos , Osteoblastos/enzimologia , Fosforilação , Tirosina/metabolismoRESUMO
Stat (signal transducers and activators of transcription) and Jak (Janus kinases) proteins are central components in the signal transduction events in hematopoietic and epithelial cells. They are rapidly activated by various cytokines, hormones, and growth factors. Upon ligand binding and cytokine receptor dimerization, Stat proteins are phosphorylated on tyrosine residues by Jak kinases. Activated Stat proteins form homo- or heterodimers, translocate to the nucleus, and induce transcription from responsive genes. Stat5 and Stat6 are transcription factors active in mammary epithelial cells and immune cells. Prolactin activates Stat5, and interleukin-4 (IL-4) activates Stat6. Both cytokines are able to stimulate cell proliferation, differentiation, and survival. We investigated the transactivation potential of Stat6 and found that it is not restricted to lymphocytes. IL-4-dependent activation of Stat6 was also observed in HC11 mammary epithelial cells. In these cells, Stat6 activation led to the induction of the beta-casein gene promoter. The induction of this promoter was confirmed in COS7 cells. The glucocorticoid receptor was able to further enhance IL-4-induced gene transcription through the action of Stat6. Deletion analysis of the carboxyl-terminal region of Stat6 and recombination of this region with a heterologous DNA binding domain allowed the delimitation and characterization of the transactivation domain of Stat6. The potencies of the transactivation domains of Stat5, Stat6, and viral protein VP16 were compared. Stat6 had a transactivation domain which was about 10-fold stronger than that of Stat5. In pre-B cells (Ba/F3), the transactivation domain of Stat6 was IL-4 regulated, independently from its DNA binding function.
Assuntos
Proteínas de Ligação a DNA/genética , Linfócitos/fisiologia , Glândulas Mamárias Animais/fisiologia , Proteínas do Leite , Transativadores/genética , Sequência de Aminoácidos , Animais , Caseínas/genética , Células Cultivadas , Proteínas de Ligação a DNA/fisiologia , Epitélio/fisiologia , Regulação da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/genética , Interleucina-4/fisiologia , Camundongos , Dados de Sequência Molecular , Oncostatina M , Peptídeos/genética , Prolactina/fisiologia , Receptores de Glucocorticoides/fisiologia , Fator de Transcrição STAT5 , Fator de Transcrição STAT6 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Supressoras da Sinalização de Citocina , Ativação TranscricionalRESUMO
Fluoride is an acknowledged bone-forming agent that may act through stimulation of osteoblast proliferation. Fluoride's action on osteoblasts and bone is potentiated by aluminum, which can form a complex with fluoride (fluoroaluminate) and activate heterotrimeric G proteins. Here we examined signaling pathways activated by fluoroaluminate in MC3T3-E1 osteoblastic and in NIH3T3 fibroblastic cells. In MC3T3-E1 cells, fluoroaluminate induced a decrease in cAMP levels and an increase in MAP and p70 S6 kinase phosphorylations. These responses were partially or completely prevented by pertussis toxin, an inhibitor of G alpha i proteins. In NIH3T3 cells, fluoroaluminate induced weaker tyrosine and MAP kinase phosphorylations. Fluoroaluminate, but not PDGF, induced a long-lasting tyrosine phosphorylation of a 130 kDa protein only in MC3T3-E1 cells. The expression of G alpha i2, but not of G alpha s and G alpha q/11 proteins was about 10-fold higher in MC3T3-E1 cells. Thus, different signaling in osteoblastic and fibroblastic cells may be due to differential expression of G alpha i proteins and tyrosine kinase substrates and could underlie fluoride's pharmacological action in bone.
Assuntos
Alumínio/farmacologia , AMP Cíclico/metabolismo , Flúor/farmacologia , Osteoblastos/metabolismo , Toxina Pertussis , Transdução de Sinais/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologia , Células 3T3 , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Colforsina/farmacologia , Fibroblastos/metabolismo , Cinética , Camundongos , Fosforilação , Fosfotirosina/análise , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases S6 RibossômicasRESUMO
We have studied transcription factors that are coupled to the activation of cytokine receptors in liver and in mammary epithelial cells. Interleukin-6 (IL-6) causes the rapid activation of the acute-phase response factor (APRF) in the liver of animals during acute inflammation and in cultured human hepatoma cells (HepG2) and induces the transcription of the acute-phase protein genes, e.g. alpha 2-macroglobulin (alpha 2-M). In the mammary gland and in cultured HC11 mammary epithelial cells, milk protein genes, e.g. beta-casein, are induced by the lactogenic hormones, insulin, glucocorticoids, and PRL. The induction of the beta-casein gene promoter is preceded by the activation of the mammary gland factor (MGF). We have compared the DNA binding sequences of APRF and MGF, 5'-CTTCTT/GGGAATT-3', and have found that they coincide in 11 of 12 positions. Bandshift experiments and oligonucleotide competition experiments showed that both factors, MGF and APRF, are able to bind to the IL-6 response element of the alpha 2-M gene promoter and to the lactogenic hormone response element of the beta-casein gene promoter with very similar specificities. Partial proteolytic digestion of APRF and MGF DNA complexes yielded similar clipping patterns. The UV cross-linked DNA complexes of both transcription factors were of the same apparent molecular mass. IL-6 activation of APRF in HepG2 cells can be observed within minutes. MGF induction by PRL in HC11 cells occurs with similar kinetics. The synergistic action of glucocorticoids and PRL is necessary for the induction of the beta-casein gene, but PRL is sufficient for MGF activation.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-6/farmacologia , Fígado/efeitos dos fármacos , Proteínas do Leite , Prolactina/farmacologia , Regiões Promotoras Genéticas , Receptores de Interleucina/efeitos dos fármacos , Transativadores , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Sequência de Bases , Sítios de Ligação , Carcinoma Hepatocelular/patologia , Caseínas/biossíntese , Caseínas/genética , Linhagem Celular , Sequência Consenso , Células Epiteliais , Humanos , Fígado/citologia , Fígado/metabolismo , Neoplasias Hepáticas/patologia , Glândulas Mamárias Animais/citologia , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Ratos , Receptores de Interleucina/fisiologia , Receptores de Interleucina-6 , Fator de Transcrição STAT3 , Fator de Transcrição STAT5 , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor , alfa-Macroglobulinas/biossíntese , alfa-Macroglobulinas/genéticaRESUMO
Treatment of HC11 mouse mammary epithelial cells with the lactogenic hormones dexamethasone, insulin, and prolactin (DIP) leads to cellular differentiation and production of the milk protein beta-casein. The following experimental evidence suggests the involvement of protein kinase C (PKC) in DIP induced signal transduction. Down-regulation of PKC by 12-O-tetradecanoylphorbol-13-acetate or addition of CGP 41251, a selective inhibitor of PKC, inhibited beta-casein protein expression induced by DIP in HC11 cells. This inhibition occurs at the level of transcription, since the DIP mediated activation of a beta-casein promoter-luciferase reporter construct or of mammary gland specific factor (MGF), an essential transcription factor for beta-casein promoter activity, was also inhibited by CGP 41251. Inhibition or down-regulation of PKC reduced the activation of MGF by prolactin as well. PKC-alpha, the only conventional PKC isoform expressed in HC11 cells, is most likely involved in the DIP induced beta-casein expression. (a) Only PKC-alpha and PKC-epsilon are down-regulated by 12-O-tetradecanoylphorbol-13-acetate whereas PKC-delta and PKC-zeta are not. (b) Of the PKC isoforms expressed in HC11 cells, CGP 41251 inhibits PKC-alpha more potently than PKC-delta, PKC-epsilon, and PKC-zeta. The IC50 for the inhibition of beta-casein synthesis, MGF activation, and beta-casein promoter activity by CGP 41251 correlated well with the IC50 of PKC-alpha inhibition. (c) Finally, only PKC-alpha translocated to membrane fractions after DIP or prolactin treatment. Taken together, these data indicate that PKC-alpha plays an important role in the signaling pathway activated by prolactin during beta-casein induction.