Mechanisms underlying the reversal of acantholysis in pemphigus by a cholinergic receptor agonist

Abstract

Objective To evaluate the reversal effect of a cholinergic receptor agonist on acantholysis in pemphigus, and to investigate its mechanism. Methods Human HaCaT keratinocytes were co-cultured with pemphigus vulgaris immunoglobulin G(PV-IgG) to establish a cell model of pemphigus, then classified into two groups to be incubated with the cholinergic receptor agonist carbachol for 12 hours (test group) or remain untreated (control group). Cell dissociation assay was performed to quantitatively estimate the reversal effect of carbachol on acantholysis, and immunofluorescence assay to qualitatively assess the changes of desmosomal proteins. Radio-immunoprecipitation assay (RIPA) lysis buffer and Triton X-100 were used to lyse HaCaT cells to obtain total proteins and cytoplasmic proteins, and Western blot was conducted to determine the expression levels of adhesion-related proteins desmoglein 3(Dsg3) and plakoglobin (PG) on the surface of HaCaT cells, as well as the phosphorylation levels of p38 mitogen activated protein kinase (p38 MAPK) and epidermal growth factor receptor (EGFR) at different time points. Quantitative polymerase chain reaction (qPCR) was performed to detect the mRNA expressions of the above surface proteins, and co-immunoprecipitation assay to qualitatively evaluate the interaction between Dsg3 and PG. Results The number of cell debris was significantly lower in the test group than in the control group (18.67±2.52 vs. 46.67±2.03,t=11.22, P<0.01). Immunofluorescence assay showed that carbachol could reverse the internalization of desmosomal molecules induced by PV-IgG. In the pemphigus cell model, the levels of total Dsg3 and PG as well as non-desmosomal Dsg3 were decreased, while the level of non-desmosomal PG increased, and the interaction between Dsg3 and PG was attenuated. When the pemphigus cell model was co-cultured with carbachol, these above changes were reversed. Carbachol also increased the mRNA levels (expressed as 2-ΔΔCt) of Dsg3 and PG from 1.428±0.215 and 1.563±0.247 in the control group to 4.974±0.948 (t=3.65,P=0.01) and 13.420±1.715 (t=6.85, P<0.01) in the test group respectively. In phosphorylation assay, carbachol inhibited the phosphorylation of EGFR, but had no significant effect on that of p38 MAPK. Conclusions The cholinergic receptor agonist carbachol can reverse acantholysis in pemphigus, likely by inhibiting the internalization of Dsg3 and PG, enhancing their expressions and interaction, and suppressing the phosphorylation of the key signaling molecule for acantholysis, EGFR. Key words: Pemphigus; Cholinergic agonists; Acantholysis; Desmoglein 3; gamma Catenin; HaCaT cells