ABSTRACT
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.
Subject(s)
Antigens, CD/metabolism , Cytokines/pharmacology , Hypothalamo-Hypophyseal System/immunology , Membrane Glycoproteins/metabolism , Neuroimmunomodulation/immunology , Pituitary Gland, Anterior/immunology , Adrenocorticotropic Hormone/metabolism , Animals , Antigens, CD/drug effects , Antigens, CD/immunology , Cell Line , Cytokine Receptor gp130 , Cytokines/immunology , Cytokines/metabolism , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Gene Expression/drug effects , Gene Expression/immunology , Hypothalamo-Hypophyseal System/drug effects , Leukemia Inhibitory Factor Receptor alpha Subunit , Ligands , Membrane Glycoproteins/drug effects , Membrane Glycoproteins/immunology , Mice , Phosphorylation/drug effects , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/drug effects , Pro-Opiomelanocortin/genetics , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/immunology , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Receptor, Ciliary Neurotrophic Factor/drug effects , Receptor, Ciliary Neurotrophic Factor/immunology , Receptor, Ciliary Neurotrophic Factor/metabolism , Receptors, Cytokine/drug effects , Receptors, Cytokine/immunology , Receptors, Cytokine/metabolism , Receptors, OSM-LIF , Repressor Proteins/genetics , STAT1 Transcription Factor , STAT3 Transcription Factor , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins , Trans-Activators/drug effects , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Tyrosine/metabolismABSTRACT
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.
