CCN3 (NOV) interacts with connexin43 in C6 glioma cells: possible mechanism of connexin-mediated growth suppression.

Abstract	Many tumor cells exhibit aberrant gap junctional intercellular communication, which can be restored by transfection with connexin genes. We have previously discovered that overexpression of connexin43 (Cx43) in C6 glioma cells not only reduces proliferation but also leads to production of soluble growth-inhibitory factors. We identified that several members of the CCN (Cyr61/connective tissue growth factor/nephroblastoma-overexpressed) family are up-regulated following Cx43 expression, including CCN3 (NOV). We now report evidence for an association between CCN3 and Cx43. Western blot analysis demonstrated that the 48-kDa full-length CCN3 protein was present in the lysate and conditioned medium of growth-suppressed C6-Cx43 cells, as well as primary astrocytes, but not in C6 parental and human glioma cells. Immunocytochemical examination of CCN3 revealed diffuse localization in parental C6 cells, whereas transfection of C6 cells with Cx43 (C6-Cx43) or with a modified Cx43 tagged to green fluorescent protein on its C terminus (Cx43-GFP) resulted in punctate staining, suggesting that CCN3 co-localizes with Cx43 in plaques at the plasma membrane. In cells expressing a C-terminal truncation of Cx43 (Cx43Delta244-382), this co-localization was lost. Glutathione S-transferase pull-down assay and co-immunoprecipitation demonstrated that CCN3 was able to physically interact with Cx43. In contrast, CCN3 was not found to associate with Cx43Delta244-382. Similar experiments revealed that CCN3 did not co-localize or associate with other connexins, including Cx40 or Cx32. Taken together, these data support an interaction of CCN3 with the C terminus of Cx43, which could play an important role in mediating growth control induced by specific gap junction proteins.
Authors	Christine T Fu, John F Bechberger, Mark A Ozog, Bernard Perbal, Christian C Naus
Journal	The Journal of biological chemistry (J Biol Chem) Vol. 279 Issue 35 Pg. 36943-50 (Aug 27 2004) ISSN: 0021-9258 [Print] United States
PMID	15213231 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References	CCN2 protein, human CCN2 protein, mouse CCN3 protein, human Ccn3 protein, mouse Connexin 43 Connexins Culture Media, Conditioned Immediate-Early Proteins Intercellular Signaling Peptides and Proteins Luminescent Proteins Nephroblastoma Overexpressed Protein Protein Isoforms connexin 32 connexin 40 Connective Tissue Growth Factor Green Fluorescent Proteins Glutathione Transferase
Topics	Animals Blotting, Western Brain Neoplasms (pathology) Cell Division Cell Line, Tumor Cell Membrane (metabolism) Cells, Cultured Cloning, Molecular Connective Tissue Growth Factor Connexin 43 (metabolism) Connexins (metabolism) Culture Media, Conditioned (pharmacology) Glioma (pathology) Glutathione Transferase (metabolism) Green Fluorescent Proteins Humans Immediate-Early Proteins (metabolism) Immunohistochemistry Intercellular Signaling Peptides and Proteins (metabolism) Luminescent Proteins (metabolism) Mice Microscopy, Fluorescence Nephroblastoma Overexpressed Protein Precipitin Tests Protein Binding Protein Isoforms Protein Structure, Tertiary Retroviridae (genetics) Transfection Up-Regulation

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