The Complex of Ciliary Neurotrophic Factor-Ciliary Neurotrophic Factor Receptor α Up-Regulates Connexin43 and Intercellular Coupling in Astrocytes via the Janus Tyrosine Kinase/Signal Transducer and Activator of Transcription Pathway

Abstract

Cytokines regulate numerous cell processes, including connexin expression and gap junctional coupling. In this study, we examined the effect of ciliary neurotrophic factor (CNTF) on connexin43 (Cx43) expression and intercellular coupling in astrocytes. Murine cortical astrocytes matured in vitro were treated with CNTF (20 ng/ml), soluble ciliary neurotrophic factor receptor ␣ (CNTFR␣) (200 ng/ml), or CNTF-CNTFR␣. Although CNTF and CNTFR␣ alone had no effect on Cx43 expression, the heterodimer CNTF-CNTFR␣ significantly increased both Cx43 mRNA and protein levels. Cx43 immunostaining correlated with increased intercellular coupling as determined by dye transfer analysis. By using the pharmacological inhibitor ␣-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG490), the increase in Cx43 was found to be dependent on the Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Immunocytochemical analysis revealed that CNTF-CNTFR␣ treatment produced nuclear localization of phosphorylated STAT3, whereas CNTF treatment alone did not. Transient transfection of constructs containing various sequences of the Cx43 promoter tagged to a LacZ reporter into ROS 17/2.8 cells confirmed that the promoter region between ؊838 to ؊1693 was deemed necessary for CNTF-CNTFR␣ to induce heightened expression. CNTF-CNTFR␣ did not alter Cx30 mRNA levels, suggesting selectivity of CNTF-CNTFR␣ for connexin signaling. Together in the presence of soluble receptor, CNTF activates the JAK/STAT pathway leading to enhanced Cx43 expression and intercellular coupling. INTRODUCTIONGap junctions are intercellular channels between adjacent cells that permit the passage of various substances Ͻ1.2 kDa in size (Kumar and Gilula, 1996). Such junctions are formed when each cell provides a connexon, which itself is composed of six connexins (Cxs) (Kumar and Gilula, 1996). Cx43, the prominent Cx isoform expressed in the central nervous system (CNS), is highly expressed in astrocytes (Yamamoto et al., 1990;Dermietzel et al., 1991;Giaume et al., 1991), neuronal precursors (Rozental et al., 1998Bittman and LoTurco, 1999), and possibly neurons (Bruzzone and Ressot, 1997;Vaney, 1999;SiuYi et al., 2001;Rouach et al., 2002). Several studies using in vitro and in vivo models have demonstrated that impediment of gap junctional coupling and/or connexin43 expression amplifies cell death and tissue damage in the wake of injuries and diseases (Blanc et al., 1998;Naus et al., 2001;Siushansian et al., 2001;Ozog et al., 2002b;Lin et al., 2003;Nakase et al., 2003). Thus, design of an intervention to up-regulate Cx43 expression and intercellular communication may lead to novel therapies against pathological disturbances.Gap junction expression and activity can be altered on a short-term or long-term basis (reviewed by Giaume and McCarthy, 1996;Rouach and Giaume, 2001). Short-term regulation is the result of posttranslational processing such as phosphorylation or channel blocking. Long-term regulation, however, entails modification of gene transcripti...

Co-administration of Ciliary Neurotrophic Factor with Its Soluble Receptor Protects against Neuronal Death and Enhances Neurite Outgrowth

“…Culture medium was replaced every 3 days. Because reactive and newly plated (reactive-like) astrocytes express CNTFR␣, we matured our astrocytes in vitro for 6 weeks to obtain quiescent cultures (18). Neurons, obtained from the neocortices of embryonic CD-1 mice aged 16 days gestation, were then seeded on top of the astrocytes and allowed to settle for 2 h before replacing the medium with Medium I (54 ml of Neurobasal Medium, 36 ml of Dulbecco's modified Eagle's medium/F-12, 1 ml of B-27 supplements; Invitrogen).…”

“…RT-PCR-Isolated RNA was subjected to RT-PCR as described previously (18). The RT product was amplified in the presence of primers for CNTF (30), CNTFR␣ (30), gp130 (31), and LIFR␤ (32).…”

“…Cells were fixed with paraformaldehyde, processed as described previously (18), and labeled with antibodies against MAP2 and neurofilament 200 (Sigma), pSTAT3 (Tyr-705; Cell Signaling, Beverly, MA), or cleaved caspase3 (Cell Signaling), with subsequent Alexa-and fluorescein-conjugated secondary IgG antibody application (Molecular Probes Inc., Eugene, OR). Immunolabeled cells were mounted in ProLong Gold containing DAPI (Molecular Probes Inc.), viewed on a Zeiss Axioskop microscope, and analyzed using AxioVision 4.2 software.…”

“…Indeed, soluble CNTFR␣ has been identified in proximity to injured tissue and in the urine of patients with amyotrophic lateral sclerosis (4,16). Following injury-induced release, CNTF and soluble CNTFR␣ together form a heterodimer (hereafter referred to as "Complex") that can interact with the ubiquitously expressed ␤-receptor subunits, glycoprotein 130 (gp130) and leukemia inhibitory factor receptor ␤ (LIFR␤) (14,17,18). The subsequent heterodimerization of gp130 and LIFR␤ activates several signaling cascades, including the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway (19).…”

“…The subsequent heterodimerization of gp130 and LIFR␤ activates several signaling cascades, including the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway (19). In addition, we have recently found that Complex increases both the expression of the gap junctional constituent connexin43 and intercellular coupling in glia via trans-signaling (17,18), the term used to describe the ability of a ligand to bind a soluble receptor (␣ subunit), which subsequently associates with the ␤ subunits of the receptor on cells that do not normally express the ␣-receptor of the ligand (20).…”