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MBC in Press, published online ahead of print September 1, 2004
Mol. Biol. Cell 10.1091/mbc.E04-03-0271

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Submitted on March 30, 2004
Revised on August 10, 2004
Accepted on August 24, 2004

The Complex of CNTF-CNTFR{alpha} Upregulates Connexin43 and Intercellular Coupling in Astrocytes via the JAK/STAT Pathway

Mark A. Ozog,* Suzanne M. Bernier,{dagger} Dave C. Bates,{dagger} Bishwanath Chatterjee,{ddagger} Cecilia W. Lo,{ddagger} and Christian C.G. Naus*{sect}

*Department of Anatomy & Cell Biology, The University of British Columbia, Vancouver, British Columbia, Canada; {dagger}CIHR Group in Skeletal Development and Remodeling, Department of Anatomy & Cell Biology, The University of Western Ontario, London, Ontario, Canada; {ddagger}Laboratory of Developmental Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892

Monitoring Editor: Daniel Goodenough

Cytokines regulate numerous cell processes, including connexin expression and gap junctional coupling. In this study, we examined the effect of CNTF on connexin43 (Cx43) expression and intercellular coupling in astrocytes. Murine cortical astrocytes matured in vitro were treated with CNTF (20 ng/ml), soluble CNTFR{alpha} (200 ng/ml), or CNTF-CNTFR{alpha}. While CNTF and CNTFR{alpha} alone had no effect on Cx43 expression, the heterodimer CNTF-CNTFR{alpha} significantly increased both Cx43 mRNA and protein levels. Cx43 immunostaining correlated with increased intercellular coupling as determined by dye transfer analysis. Using the pharmacological inhibitor AG490, the increase in Cx43 was found to be dependent on the JAK/STAT pathway. Immunocytochemical analysis revealed that CNTF-CNTFR{alpha} 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{alpha} to induce heightened expression. CNTF-CNTFR{alpha} did not alter Cx30 mRNA levels suggesting selectivity of CNTF-CNTFR{alpha} for connexin signaling. Taken together, in the presence of soluble receptor CNTF activates the JAK/STAT pathway leading to enhanced Cx43 expression and intercellular coupling.

{sect}Corresponding author. E-mail: cnaus{at}interchange.ubc.ca




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