The Mechanism Of Cholinergic Receptor Pathway Played In The Adhesion Function Of HaCaT Cell And The Pathogenesis Of Pemphigus Vulgaris

Pemphigus is an autoimmune skin disease characterized by antibodies against epidermis proteins. Desmogleins play an important role in the pathogenesis of the disease. The recent studies have shown that other factors also play some role in the pathogenesis of pemphigus. Among these factors, cholinergic receptors have close relationship with pemphigus. In this study, we conducted a further research on cholinergic receptors using the cell model of pemphigus constructed by HaCaT cell to explore the role of cholinergic receptor pathway in cell adhesion to enrich the theory on pathogenesis of pemphigus.Part 1 Effects and mechanism of the drugs of cholinergic pathway on the adhesion function of HaCaT cellsObjective To study the effects and mechanism of cholinergic drugs on the adhesive function of HaCaT cells. Methods Following HaCaT cells were cultured to fusion state, the appropriate concentrations of cholinergic drugs were added into the culture. The cell dissociation assay was conducted to detect the adhesion function of HaCaT cells, and the results were expressed as the number of cellular debris. Triton RIPA and Triton X-100 were used for lysis of HaCaT cells to obtain the total and cytoplasm protein. Western blot was conducted to detect the protein levels of adhesion molecules such as desmoglein 1, desmoglein 3, plakoglobin and E-cadherin. Quantitative PCR (qPCR) was conducted to detect the mRNA levels of the corresponding molecules. Results The amount of cellular debris in the group of control, atropine, tubocurarine and carbachol was (6.33±1.15), (35.00±2.65), (6.33±1.53) and (6.00±1.00), respectively. Atropine reduced the expression of desmoglein 3 at the levels of both protein and mRNA, and made the desmolgein 3 molecule detach from the cellular membrane. Carbachol increased the expression of desmoglein 1, desmoglein 3 and plakoglobin and promote the adhesion of desmoglein 1 and plakoglobin to the membrane. Tubocurarine had no effect on these adhesion molecules. Conclusion Acetylcholine receptor on the membrane of HaCaT cell can mediate the adhesion of HaCaT cells by regulating the expression of intercellular adhesion molecules, especially the desmosomes molecules, and affect the stability of these molecules on the membrane.Part 2 Establishment of cell model of pemphigus using HaCaT cellsObjective To establish a cell model of pemphigus using co-cultured HaCaT cells with pemphigus vulgaris (PV)-IgG. Methods Typical patients with pemphigus vulgaris were enrolled. IgG in the serum of the patients was purified. Then HaCaT cells were co-cultured with PV-IgG of concentration of lmg/ml. The cell dissociation assay was conducted to detect the adhesion function of HaCaT cells and immunofluorescent assay was conducted to observe the staining patterns of desmosome proteins. Western blot was conducted to analyze the protein level of adhesion molecules. qPCR was conducted to detect desmosome mRNA level. The interaction of desmoglein3 with plakoglobin was qualitatively analyzed by co-immunoprecipitation. The phosphorylation levels of p38 MAPK and EGFR were examined. Results After co-culture of HaCaT cells with PV-IgG, the fragments of HaCaT cells increased. The molecules of desmosome protein were internalized and degraded and its stability declined. The interaction between Dsg3 and PG decreased. The expression of desmosome mRNA level was not affected. The phosphorylation of p38 MAPK and EGFR were increased and the phosphorylation of p38 MAPK occurred earlier than that of EGFR. Conclusion The cell model of pemphigus can be successfully established by co-culture of HaCaT cells with PV-IgG and desmosome molecules exhibited consistent changes such as the acantholysis process happened in vivo.Part 3 The mechanism of alleviating acantholysis by cholinergic agonist carbachol in pemphigusObjective To investigate the effect and mechanism of cholinergic receptor agonist on the alleviation of acantholysis in pemphigus. Methods HaCaT cells were co-cultured with PV-IgG and carbachol. The cell dissociation assay was used to analyze quantitatively the alleviation function of carbachol for acantholysis. The changes of desmosomal molecules were observed qualitatively by immunofluorescence assay. The cell model of pemphigus vulgaris induced by PV-IgG was used as control. HaCaT cells were lysed by RIPA and Triton X-100 respectively to obtain the total and cytoplasm protein. The variations of protein level of desmoglein 3 and plakoglobin as well as the phosphorylation level of p38 MAPK and EGFR at different time points was qualitatively detected by gray scale value analysis based on Western blot. The mRNA level of the cell surface molecules were determined by qPCR. The interaction between desmoglein3 and plakoglobin was analyzed qualitatively by co-immunoprecipitation. Results After the co-culture of HaCaT cells with PV-IgG and carbachol, the amount of cellular debris was significantly reduced compared with control group (18.67±1.45 vs 46.67±2.03, t=11.22, P<0.05). Immunofluorescence assay showed that carbachol can reverse the internalization of desmosome molecules. In the pemphigus cell model the level of desmoglein 3 and plakoglobin protein declined. In the non-desmosome part the level of desmoglein 3 declined and plakoglobin increased, and reduced interaction between desmoglein 3 and plakoglobin was observed in the cell model of pemphigus vulgaris. The addition of carbachol can alleviate all of the above changes. Carbachol can also increase the mRNA level of desmoglein 3 from (1.428±0.215) to (4.974±0.948), (t=3.65, P=0.01) and plakoglobin from (1.563±0.247) to (13.420±1.715), (t=6.85, P<0.01). Carbachol can inhibited the phosphorylation of EGFR, whereas has no obvious effect on the phosphorylation of p38 MAPK. Conclusion Cholinergic receptor agonist carbachol can alleviate acantholysis in pemphigus. The mechanism of the alleviation function may include cholinergic agonist inhibit the internalization of desmoglein 3 and plakoglobin and increase their expressions, enhance the interaction between desmoglein 3 and plakoglobin, and suppress phosphorylation of EGFR, the key signal of acantholysis process.Part 4 Effect of m3 or a9 AChR RNA interference on adhesion function of HaCaT cellsObjective To investigate the effects of m3 or a9 acetylcholine receptor (AChR) RNA interference on adhesion function of HaCaT cells and desmosome molecules. Methods RNA interfering technology was used to interfere the expression of m3 or α9 AChR, and the interference efficiency and the optimal interference time point were determined. Cell dissociation assay was conducted to detect the adhesion function of HaCaT cells after inhibition of AChR expression. Western blot was conducted to analyze the protein levels of cytoskeleton-associated or non-cytoskeleton-associated desmosome molecules. qPCR was conducted to detect the mRNA level of desmosome molecules. Results After 48h of co-culture of HaCaT cells with siRNA sequence for m3 or a9 AChR, the expressions of both m3 and a9 AChR genes were decreased. The celluar adhesion of HaCaT was reduced following the inhibition of m3 AChR expression, but the low expression of a9 AChR had no effects on the cell adhesion function. The inhibition of m3 AChR expression induced declined stability of desmosome molecules, while the low expression of α9 AChR increased the stability of desmosome. The inhibition of m3 AChR expression led to decreased level of Dsg1 mRNA and increased PG mRNA. On the status of a9 AChR interference, the mRNA expression of Dsgl and PG increased and the expression of Dsg3 mRNA decreased. Conclusion The low expression of m3 AChR induced by RNA interference reduced the adhesion ability of HaCaT cells and desmosome stability, while the low expression of a9 AChR did not affect the adhesion function of HaCaT cells and may increase the stability of desmosome.Part 5 The effects of m3 or a9 AChR interference on acantholysis in pemphigus vulgarisObjective To investigate the effects of m3 or α9 AChR interference on acantholysis in pemphigus vulgaris. Methods The expression of m3 or a9 AChR of HaCaT was inhibited by RNA interference, then the cells were co-cultured with PV-IgG. The changes of adhesion function of HaCaT cells were observed by cell dissociation assay. Immunofluorescent assay was conducted to detect the staining patterns of desmosome proteins. Western blot was conducted to detect cytoskeleton-associated or non-cytoskeleton-associated desmosome protein levels. The interactions of desmoglein3 and plakoglobin were detected qualitatively by co-immunoprecipitation. The phosphorylation levels of p38 MAPK and EGFR were examined. Results We found that under the conditions of m3AChR silencing the co-culture of HaCaT cells with PV-IgG induced more serious acantholysis. The cytoskeleton-associated desmoglein and plakoglobin significantly reduced and the phosphorylation of EGFR enhanced. However, a9 AChR silencing may exert protective effects during acantholysis process. The co-culture of a9 AChR silencing cells with PV-IgG suppressed the internalization of desmoglein protein. The levels of cytoskeleton-associated desmoglein and plakoglobin were unreduced, and more close interaction between desmoglein 3 and plakoglobin was shown. Both the p38 MAPK and EGFR signaling pathway activations were not found. Conclusion The results in this study provided evidences that m3 and a9 AChR may play certain roles in the pathogenesis of pemphigus vulgaris and the two receptors exhibit opposite effects in some special aspects during the acantholysis process.