title: 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.affiliation: Department of Anatomy and Cell Biology, University of British Columbia, Vancouver V6T 1Z3, British Columbia, Canada.
authors.forename: Christine T
authors.initials: CT
authors.lastname: Fu
authors.forename: John F
authors.initials: JF
authors.lastname: Bechberger
authors.forename: Mark A
authors.initials: MA
authors.lastname: Ozog
authors.forename: Bernard
authors.initials: B
authors.lastname: Perbal
authors.forename: Christian C
authors.initials: CC
authors.lastname: Naus
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000473:pathology; D002455:Cell Division; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D055513:Connective Tissue Growth Factor; D018031:Connexin 43 / Q000378:metabolism*; D017630:Connexins / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology; D005910:Glioma / Q000473:pathology; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D055514:Nephroblastoma Overexpressed Protein; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D015854:Up-Regulation; D000097002:Gap Junction beta-1 Protein; D000097003:Gap Junction alpha-5 Protein
chemical_list: D055513:Connective Tissue Growth Factor; D018031:Connexin 43; D017630:Connexins; D017077:Culture Media, Conditioned; D005982:Glutathione Transferase; D049452:Green Fluorescent Proteins; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D055514:Nephroblastoma Overexpressed Protein; D020033:Protein Isoforms; D000097002:Gap Junction beta-1 Protein; D000097003:Gap Junction alpha-5 Protein; C526317:CCN2 protein, human; C526318:CCN2 protein, mouse; C526319:CCN3 protein, human; C526320:Ccn3 protein, mouse