Abstract: 【Abstract】 Objective To investigate the role of transgelin 2 （TAGLN2） in the pathogenesis of pemphigus vulgaris （PV） and its molecular action mechanisms regulating the Ras homolog gene family member A （RhoA） signaling pathway. Methods An in vitro PV model was established by stimulating HaCaT cells with 1 mg/ml anti-desmoglein 3 （Dsg3） antibody and 1.8 mmol/L CaCl? for 24 hours （model group）, while HaCaT cells cultured under standard conditions served as a control group. In the cell transfection experiment, HaCaT cells were transfected with a TAGLN2-targeting small interfering RNA （siRNA） negative control plasmid （si-NC group） or TAGLN2-targeting siRNA1/2/3 plasmids （siRNA1/2/3 groups） for 48 hours, followed by the PV model induction. In the TAGLN2 rescue experiment, HaCaT cells were co-transfected with si-NC/siRNA1 and TAGLN2 overexpression plasmids or empty vectors, followed by the PV model induction （resulting in a si-NC + pc-NC group, a siRNA1 + pc-NC group, and a siRNA1 + pc-TAGLN2 group）. In the RhoA inhibition experiment, HaCaT cells were divided into 3 groups: a siRNA1 group, a siRNA1 + RhoA inhibitor （Rhosin） group （siRNA1-transfected cells co-treated with 0.4 μmol/L Rhosin during modeling for 24 hours）, and a Rhosin group （conventionally cultured cells co-treated with 0.4 μmol/L Rhosin during modeling for 24 hours）. TAGLN2 mRNA expression was determined by real-time quantitative PCR, cell dissociation was assessed by the cell dissociation assay and immunofluorescence assay for F-actin, desmosomal integrity was evaluated by immunofluorescence assay for Dsg3, RhoA expression and subcellular localization were analyzed by immunofluorescence assay, interactions between TAGLN2 and phosphorylated RhoA （p-RhoA） or protein kinase A （PKA） were examined using co-immunoprecipitation assay, protein expression of TAGLN2, RhoA, and p-RhoA was determined by Western blot analysis, and RhoA-guanosine triphosphate （GTP） activity was evaluated using a RhoA activity assay kit. Two-independent-sample t test was used for comparisons between two groups, and one-way analysis of variance followed by Tukey′s post hoc test was used for comparisons among multiple groups. Results TAGLN2 mRNA and protein expression levels were significantly higher in the model group than in the control group （t = 9.92, 36.73, respectively; both P < 0.001）. Compared with the control group, the model group showed markedly increased cell fragments, reduced F-actin fluorescence intensity, and F-actin reorganization, as well as substantial loss of Dsg3 with decreased fluorescence intensity; compared with the si-NC group, the siRNA1/2 groups exhibited decreased cell fragments and enhanced fluorescence intensities of F-actin and Dsg3. Compared with the control group, the model group showed a higher p-RhoA/RhoA ratio （2.05 ± 0.09 vs. 1.04 ± 0.05, P < 0.001）, lower RhoA activity （28.33 ± 3.21 vs. 100.00 ± 3.00, P < 0.001）, and nuclear translocation of RhoA; compared with the si-NC group, the siRNA1 group showed a lower p-RhoA/RhoA ratio （0.33 ± 0.04 vs. 2.03 ± 0.08, P < 0.001）, higher RhoA activity （78.00 ± 3.00 vs. 28.67 ± 2.51, P < 0.001）, and inhibition of RhoA phosphorylation and nuclear translocation. Co-immunoprecipitation assay showed no interaction between TAGLN2 and p-RhoA, but confirmed an interaction between TAGLN2 and PKA, an upstream kinase of RhoA. In the TAGLN2 rescue experiment, compared with the siRNA1 + pc-NC group, the siRNA1 + pc-TAGLN2 group showed markedly elevated TAGLN2 protein expression, increased cell fragments, weakened F-actin fluorescence intensity, substantial loss of Dsg3 with reduced fluorescence intensity, and a higher p-RhoA/RhoA ratio （0.95 ± 0.07 vs. 0.31 ± 0.04, P < 0.001）. Compared with the siRNA1 group, the siRNA1 + Rhosin group showed increased cell fragments and reduced fluorescence intensities of F-actin and Dsg3. Conclusion Knockdown of TAGLN2 may suppress anti-Dsg3 antibody-induced HaCaT cell dissociation and the loss of intercellular adhesion by inhibiting RhoA phosphorylation and nuclear translocation.

Key words: Pemphigus, Pemphigus vulgaris, Transgelin 2, RhoA, Cell dissociation, Desmoglein 3