Figure 8.

PGE2 downregulates N-cadherin via EP4 and Cx43 via EP1/Ca²⁺/calpain signaling. (a) HBVPs were treated with inhibitors of different EP receptors for 5 h prior to PGE2 treatment. N-cadherin and Cx43 expression was analyzed 72 h later by western blot. (b, c) EP1 or EP4 was silenced in HBVPs. Cells were treated with PGE2 24 h after siRNA transfection, and protein extracts were collected 48 h later for western blot analysis. d. The changes in intracellular calcium levels induced by PGE2 were monitored in HBVPs by Fluo4-calcium assay. One-way ANOVA test, ***p < 0.001; ns: not significant. (e) HBVPs were incubated 4 h with a μ-calpain inhibitor before PGE2 or DMSO treatment. N-cadherin and Cx43 expression was analyzed 72 h later by western blot. (f) A proposed model of Cx43 downregulation by PGE2/EP1/Ca²⁺/calpain signaling. (g) In order to verify that PGE2-mediated decrease of Cx43 is not a result of epitope loss following cleavage by calpain, Cx43 expression was analyzed in HBVPs following PGE2 treatment using an anti-Cx43 antibody targeting a N-terminal epitope. h. HBVPs were treated with various inhibitors of proteins suspected to be involved in PGE2-induced N-cadherin degradation. Pepstatin A was used as an inhibitor for γ-secretase and Gö6976 for PKC. The cells were treated with each inhibitor 4 h prior PGE2 treatment, and protein extracts were collected after a total of 72 h.