RESUMO
OBJECTIVE: This comparative in vitro study examined the effects of all known gp130 cytokines on murine corticotroph AtT-20 cell function. METHODS: Cytokines were tested at equimolar concentrations from 0.078 to 10 nM. Tyrosine phosphorylation of the signal transducer and activator of transcription (STAT)3 and STAT1, the STAT-dependent suppressor of cytokine signaling (SOCS)-3 promoter activity, SOCS-3 gene expression, STAT-dependent POMC promoter activity and adrenocorticotropic hormone (ACTH) secretion were determined. RESULTS: Leukemia inhibitory factor (LIF), human oncostatin M (OSM) and cardiotrophin (CT)-1 (LIFR/gp130 ligands), as well as ciliary neurotrophic factor (CNTF) and novel neurotrophin-1/B-cell stimulating factor-3 (CNTFR alpha/LIFR/gp130 ligands) are potent stimuli of corticotroph cells in vitro. In comparison, interleukin (IL)-6 (IL-6R/gp130 ligand) and IL-11 (IL-11R/gp130 ligand) exhibited only modest direct effects on corticotrophs, while murine OSM (OSMR/gp130 ligand) showed no effect. CONCLUSION: (i) CNTFR complex ligands are potent stimuli of corticotroph function, comparable to LIFR complex ligands; (ii) IL-6 and IL-11 are relatively weak direct stimuli of corticotroph function; (iii) differential effects of human and murine OSM suggest that LIFR/gp130 (OSMR type I) but not OSMR/gp130 (OSMR type II) are involved in corticotroph signaling. (iv) CT-1 has the hitherto unknown ability to stimulate corticotroph function, and (v) despite redundant immuno-neuroendocrine effects of different gp130 cytokines, corticotroph cells are preferably activated through the LIFR and CNTFR complexes.
Assuntos
Antígenos CD/metabolismo , Citocinas/farmacologia , Sistema Hipotálamo-Hipofisário/imunologia , Glicoproteínas de Membrana/metabolismo , Neuroimunomodulação/imunologia , Adeno-Hipófise/imunologia , Hormônio Adrenocorticotrópico/metabolismo , Animais , Antígenos CD/efeitos dos fármacos , Antígenos CD/imunologia , Linhagem Celular , Receptor gp130 de Citocina , Citocinas/imunologia , Citocinas/metabolismo , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Subunidade alfa de Receptor de Fator Inibidor de Leucemia , Ligantes , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/imunologia , Camundongos , Fosforilação/efeitos dos fármacos , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Pró-Opiomelanocortina/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/imunologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptor do Fator Neutrófico Ciliar/efeitos dos fármacos , Receptor do Fator Neutrófico Ciliar/imunologia , Receptor do Fator Neutrófico Ciliar/metabolismo , Receptores de Citocinas/efeitos dos fármacos , Receptores de Citocinas/imunologia , Receptores de Citocinas/metabolismo , Receptores de OSM-LIF , Proteínas Repressoras/genética , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina , Transativadores/efeitos dos fármacos , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Tirosina/metabolismoRESUMO
We previously reported that ciliary neurotrophic factor (CNTF) increased the serum-free cell survival of immortalized motor neuron-like cells (NSC-34), and addition of the exogenous ganglioside GalNAc beta4(Neu5Ac alpha3)Gal beta4GlcCer (GM2) facilitated cell survival together with CNTF. Moreover beta 1,4 N-acetylgalactosaminyltransferase (GM2 synthase) activity increased in NSC-34 cells cultured with CNTF. We now have examined whether CNTF-induced cell survival is associated with the collaboration between GM2 and the CNTF receptor (CNTF-R). Despite the presence of CNTF (50 ng/ml), anti-CNTF-R antibody caused cell death and prevented the up-regulation of GM2 synthase expression. The addition of GM2 (1 to 20 microM) abrogated the anti-CNTF-R antibody effect which shortened cell survival and blocked GM2 synthase activation. Use of [125I]CNTF showed the specificity of CNTF binding in NSC-34 cells in situ. GM2 produced a 5-fold increase in the CNTF binding affinity per cell but did not change the binding site number. The study by metabolic labeling with [1-(14)C]N-acetyl-D-galactosamine ([14C]GalNAc) showed that biosynthesized GM2 was involved in the immunoprecipitation of CNTF-R. These findings indicate that up-regulated GM2 synthesis induces functional conversion of CNTF-R to the activated state, in which it has affinity for CNTF. We conclude that GM2 is a bio-regulating molecule of CNTF-R in motor neurons.
