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Differing responses to TNF in HeLa cells expressing vascular connexins
Author(s) -
Ouellette Yves,
Ermilov Leonid G.,
Sieck Gary C.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.594.12
Subject(s) - hela , chemistry , lysis , blot , tumor necrosis factor alpha , western blot , microbiology and biotechnology , cell , connexin , cell culture , gap junction , biology , biochemistry , endocrinology , intracellular , gene , genetics
We studied the effects of TNF on cell‐to‐cell communication in HeLa cells expressing vascular connexin 37, 40 or 43. HeLa cells were treated with TNF (20 ng/ml) for up to 2 h. In dye‐transfer experiments, carboxyfluroscein (HeLaCx40 and HeLaCx43) or Alexa Fluor‐480 (HeLaCx37) was injected into one cell for 10 s and cell transfer allowed to proceed for 10 m and the number of labeled cells counted. Cell lysates were prepared in Triton X‐100 lysate buffer and detergent‐soluble fractions collected. Cx37, 40 and 43 were detected by Western blot. After 1 hour, TNF treatment resulted in near total loss of dye‐coupling in HeLaCx37 and HeLaCx43 (p<0.02, n=14‐16) and remained constant up to 2 hours. Dye coupling in HeLaCx40 cells remained unchanged after 1 hour and decreased after 2 hours (p<0.05, n=10). Western blots indicated that TNF treatment did not affect detergent solubility of Cx40 and 43. However, TNF caused a significant increase in detergent solubility of Cx37. These results suggest that inflammatory mediators affect connexins differently, and that the loss of Cx37 function may be due to the loss of detergent resistance.