Mechanisms underlying the reversal of acantholysis in pemphigus by a cholinergic receptor agonist / 中华皮肤科杂志

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 coimmunoprecipitation 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-△△Ct) 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.

The expression and tyrosine phosphorylation of E-cadherin, beta-, gamma-catenin and EGFR after treatment of EGF and TGF-alpha in Cervical Cancer Cell Lines / 대한부인종양콜포스코피학회잡지

OBJECTIVES: Cadherin/catenin adhesion complex is fundamentally involved in epithelial cancer invasion and metastasis. E-cadherin and EGFR colocalize on the basolateral membrane of epithelial cell and EGF down-regulate E-cadherin expression. In the invasion and metastasis of cancer, E-cadherin expression is decreased and growth factors receptor is overexpressed. The present study was aimed to find the role of E-cadherin, beta-and gamma-catenin, growth factors and its receptors in cervical cancer cell lines. METHODS: The cervical cancer cell cultures were treated with different time duration of EGF 30 ng/ml and TGF-a 10 ng/ml(0, 10 min, 20 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr, 24 hr). The change in cancer cell morphology and the changes in E-cadherin, beta- and gamma-catenin, EGFR and activated EGFR expression were studied with a western blot analysis and an immunoprecipitation. RESULTS: Through a western blot analysis, E-cadherin 120 kDa band and EGFR 170 kDa band were expressed in CaSki, HT-3 and ME-180 cell line, which showed epithelial contact growth. 1n these 3 cell lines, expression of E-cadherin did not decrease with time dependent manner. after the treatment of EGF and TGF- alpha. The expression of EGFR decreased and activated EGFR expression increased in 30 minutes to 1 hour but decreased subsequently. When the cells treated with EGF, there were no change in beta-and gamma-catenin expression with there dependent manner. The tyrosine phosphorylation of beta-and gamma-catenin increased in 30 minutes to 1 hour but decreased subsequently with activated EGFR. CONCLUSION: This study showed that an activated EGFR which has involved with tyrosine phosphorylation of beta- and gamma-catenin influenced by growth factors rather than expression of E-cadherin, has a role in the invasion and metastasis of the cervical cancer.

Expression of E-cadherin and alpha - , beta - , gamma - catenin proteins in endometrial carcinoma / 대한산부인과학회잡지

OBJECTIVES: E-cadherin is a transmembrane protein that is one of the key players involved in cell to cell adhesion. Loss of E-cadherin expression is suggested to promote tumor invasion and distant metastasis in tumor development. Recently, it has been proposed E-cadherin function requires its linkage to the cytoskeleton through catenins. So defects in catenins may cause defective E-cadherin function and promote tumor invasion. We intend to evaluate the expression of E-cadherin and alpha-, beta-, gamma- catenin in tissues of human endometrial carcinoma to analyze the patterns of cell adhesion molecules' expression in endometrial carcinoma and to investigate the relationship between status of cell adhesion molecules and various clinicopathological factors. MATERIALS AND METHODS: The present study investigated the immunohistochemical expression of E-cadherin and alpha-, beta-, gamma- catenin in 33 paraffin embedded formalin fixed tissues of endometrial carcinomas. RESULTS: Aberrant E-cadherin, alpha-, beta-, gamma- catenin expression was observed in 33.3(11 of 33), 27.3(9 of 33), 18.2 (6 of 33), and 51.5(17 of 33) % of the specimens, respectively. Statistically significant correlation was found between aberrant expression of E-cadherin and lymph node metastasis and cell types other than endometrioid adenocarcinoma. Aberrant pattern of gamma- catenin expression also correlated with deep myometrial invasion. But alpha-, beta- catenin expression were not correlated with any clinicopathological parameters. Using Kaplan-Meier curves, abnormal expression of E-cadherin correlated closely with poor survival (p<0.05). CONCLUSION: We revealed aberrant expression of these cell adhesion molecules in part of patients with endometrial carcinoma. Aberrant expression of E-cadherin was correlated with lymph node metastasis and cell types other than endometrioid adenocarcinoma and aberrant expression of gamma-catenin was related with deep myometrial invasion.