Porphyromonas gingivalis Activates Gap Junction Hemichannels in Oral Keratinocytes

Communication between mucosal keratinocytes and proximal keratinocytes or neighboring immune cells is a crucial response to infection. To enable communication, undocked gap junction hemichannels, which are composed of connexins, are opened to release mediators in response to depletion of extracellular calcium, membrane depolarization or mechanical stimulation. Objective: To determine whether hemichannels function in response to infection, we tested the hypothesis that Porphyromonas gingivalis (Pg) induces opening of hemichannels in oral keratinocytes. Methods: Primary human gingival keratinocytes, tonsil epithelial cells and immortalized human oral keratinocytes (TERT-2) were analyzed for connexin43 and connexin26 expression using immunofluorescence and western blotting. To determine hemichannel activity, TERT-2 cells were cultured overnight, preloaded with calcein-AM to visualize and count total cells, and then incubated with Pg (wild type strain ATCC 33277, an arginine and lysine protease-negative mutant strain KDP 136, paraformaldehyde-fixed or heat-killed wild-type cells), purified lipopolysaccharide (LPS) or a Gram-positive Streptococcus gordonii. Next, sulfo-rhodamine B was added to the cells with or without mechanical stimulation, incubated for 5 minutes, washed and photographed. The fluorescent rhodamine uptake marked the opening of hemichannels in single cells as analyzed using Image-Pro Plus. Results: All cells expressed connexin43 and connexin26. Mechanically-stimulated TERT-2 cells showed 3-fold greater uptake (p <0.001) of rhodamine in the presence of Pg than without bacteria. Consistent with hemichannel activity, dye uptake was inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA), a hemichannel blocker. Viable Pg induced significantly more dye uptake than fixed or heat-killed Pg, a protease-negative Pg mutant (KDP 136), or S. gordonii. LPS from Pg or E. coli also induced hemichannel opening. Conclusions: Pg induces hemichannel opening in TERT-2 keratinocytes. Pg LPS and proteases are putative factors that activate hemichannels, in synergy with mechanical stimulation, and may contribute to dissemination of mucosal epithelial inflammation.

Supported by NIH R01DE11831, GM46277 and a Royal Thai Government Scholarship.