中华皮肤科杂志 ›› 2025, Vol. 58 ›› Issue (12): 1148-1157.doi: 10.35541/cjd.20250311

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TAGLN2通过促进RhoA磷酸化及核定位介导细胞解离在寻常型天疱疮发病中的作用机制研究

胡凤侠1，2    陈文静1    肖福煬1    王倩1    张晓语1    康晓静2    梁俊琴1   

  1. 1新疆维吾尔自治区人民医院变态反应（过敏）科，乌鲁木齐  830001；2新疆维吾尔自治区人民医院皮肤性病科  新疆皮肤病临床医学研究中心  新疆皮肤病研究重点实验室（XJYS1707），乌鲁木齐  830001
  • 收稿日期:2025-05-28 修回日期:2025-10-20 发布日期:2025-12-04
  • 通讯作者: 梁俊琴 E-mail:drliangjq@163.com
  • 基金资助:
    新疆维吾尔自治区自然科学基金（2022D01C623）；国家自然科学基金（82260621）；新疆维吾尔自治区人民医院院内项目（20220136）；新疆维吾尔自治区人民医院-干细胞生物治疗基础和临床研究专项项目（GXBZX-2023004）

Mechanisms of transgelin 2-induced cell dissociation via RhoA phosphorylation and nuclear localization in pemphigus vulgaris

Hu Fengxia1,2, Chen Wenjing1, Xiao Fuyang1, Wang Qian1, Zhang Xiaoyu1, Kang Xiaojing2, Liang Junqin1   

  1. 1Department of Allergy, People′s Hospital of Xinjiang Uygur Autonomous Region, Urumqi 830001, China; 2Department of Dermatology and Venereology, People′s Hospital of Xinjiang Uygur Autonomous Region, Xinjiang Clinical Research Center for Dermatologic Diseases, Xinjiang Key Laboratory of Dermatology Research （XJYS1707）, Urumqi 830001, China
  • Received:2025-05-28 Revised:2025-10-20 Published:2025-12-04
  • Contact: Liang Junqin E-mail:drliangjq@163.com
  • Supported by:
    Natural Science Foundation of Xinjiang Uygur Autonomous Region （2022D01C623）; National Natural Science Foundation of China （82260621）; Intramural Project of People's Hospital of Xinjiang Uygur Autonomous Region （20220136）; Basic and Clinical Research Project of Stem Cell Biotherapy - People's Hospital of Xinjiang Uygur Autonomous Region （GXBZX-2023004）

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摘要： 【摘要】 目的 探讨转胶蛋白2（TAGLN2）在寻常型天疱疮（PV）发病中的调控作用及其对Ras同源基因家族成员A （RhoA）信号通路的分子调控机制。方法 采用1 mg/ml抗桥粒黏蛋白3（Dsg3）抗体和1.8 mmol/L CaCl2刺激HaCaT细胞24 h，构建PV体外模型（模型组），常规培养的HaCaT细胞作为对照组。细胞转染实验：向HaCaT细胞分别转染TAGLN2小干扰RNA（siRNA）阴性对照（si-NC）质粒、TAGLN2 siRNA1/2/3质粒48 h，并构建PV模型，分为si-NC组、siRNA1/2/3组。TAGLN2回救实验：si-NC/siRNA1与TAGLN2过表达质粒/空载质粒共转染HaCaT细胞后构建PV模型，分为si-NC + pc-NC组、siRNA1 + pc-NC组、siRNA1 + pc-TAGLN2组。RhoA抑制实验：将HaCaT细胞分为3组，即①siRNA1组；②siRNA1 + RhoA抑制剂（Rhosin）组（siRNA1组细胞在造模时加0.4 μmol/L Rhosin共处理24 h）；③Rhosin组（常规培养细胞在造模时加0.4 μmol/L Rhosin共处理24 h）。实时定量PCR检测TAGLN2 mRNA表达水平，细胞解离实验和F肌动蛋白（F-actin）免疫荧光实验检测细胞解离情况，Dsg3免疫荧光实验评估桥粒完整性，RhoA免疫荧光实验分析RhoA表达及定位，免疫共沉淀实验检测TAGLN2与磷酸化（p）-RhoA或蛋白激酶A（PKA）的相互作用，Western印迹法检测TAGLN2、RhoA及p-RhoA蛋白表达，RhoA活性检测试剂盒测定RhoA-鸟苷三磷酸（GTP）活性。两样本间比较采用独立样本t检验；多组间比较采用单因素方差分析，事后两两比较采用Tukey检验。结果 模型组TAGLN2 mRNA和蛋白表达水平均显著高于对照组（t = 9.92、36.73，均P < 0.001）。与对照组相比，模型组细胞碎片数量明显增加，F-actin荧光强度减弱并发生重组，Dsg3大量消失且荧光强度降低；与si-NC组相比，siRNA1/2组细胞碎片数量减少，F-actin和Dsg3荧光强度增强。与对照组相比，模型组p-RhoA/RhoA比值较高（2.05 ± 0.09比1.04 ± 0.05，P < 0.001），RhoA活性较低（28.33 ± 3.21比100.00 ± 3.00，P < 0.001），RhoA发生核移位；与si-NC组相比，siRNA1组p-RhoA/RhoA比值较低（0.33 ± 0.04比2.03 ± 0.08，P < 0.001），RhoA活性较高（78.00 ± 3.00比28.67 ± 2.51，P < 0.001），RhoA的磷酸化及核移位受到抑制。免疫共沉淀实验表明，TAGLN2与p-RhoA不存在相互作用，但可与RhoA上游激酶PKA相互作用。TAGLN2回救实验表明，与siRNA1 + pc-NC组相比，siRNA1 + pc-TAGLN2组TAGLN2蛋白表达水平明显较高，细胞碎片数量明显较多，F-actin荧光染色较弱，Dsg3大量消失且荧光强度较低，p-RhoA/RhoA比值较高（0.95 ± 0.07比0.31 ± 0.04，P < 0.001）。与siRNA1组相比，siRNA1 + Rhosin组细胞碎片增多，F-actin荧光染色较弱，Dsg3荧光强度较低。结论 TAGLN2沉默可能通过抑制RhoA磷酸化以及核移位，抑制抗Dsg3抗体诱导的HaCaT细胞解离及细胞间黏附丧失。

关键词: 天疱疮, 寻常型天疱疮, 转胶蛋白2, RhoA, 细胞解离, 桥粒芯糖蛋白质3

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

引用本文

胡凤侠, 陈文静 肖福煬 王倩 张晓语 康晓静 梁俊琴. TAGLN2通过促进RhoA磷酸化及核定位介导细胞解离在寻常型天疱疮发病中的作用机制研究[J]. 中华皮肤科杂志, 2025,58(12):1148-1157. doi:10.35541/cjd.20250311

Hu Fengxia, Chen Wenjing, Xiao Fuyang, Wang Qian, Zhang Xiaoyu, Kang Xiaojing, Liang Junqin. Mechanisms of transgelin 2-induced cell dissociation via RhoA phosphorylation and nuclear localization in pemphigus vulgaris[J]. Chinese Journal of Dermatology, 2025, 58(12): 1148-1157.doi:10.35541/cjd.20250311