Chernyavsky Alex I, Arredondo Juan, Piser Timothy, Karlsson Evert, Grando Sergei A
Department of Dermatology, University of California, Irvine, California 92697.
AstraZeneca Pharmaceuticals, Wilmington, Delaware 19850.
J Biol Chem. 2008 Feb 8;283(6):3401-3408. doi: 10.1074/jbc.M704956200. Epub 2007 Dec 11.
The mechanisms mediating and regulating assembly and disassembly of intercellular junctions is a subject of intensive research. The IgG autoantibodies produced in patients with the immunoblistering skin disease pemphigus vulgaris (PV) can induce keratinocyte (KC) dyshesion (acantholysis) via mechanisms that involve signaling kinases targeting intercellular adhesion molecules, thus providing a useful model to study the physiologic regulation of KC cohesion. Previous studies showed that activation of Src and protein kinase C are the earliest events in the PV IgG-induced intracellular phosphorylation cascades and that cholinergic agonists are effective for treating patients with pemphigus. In this study, we sought to elucidate the molecular mechanisms allowing cholinergic agonists to inhibit PV IgG-induced acantholysis and phosphorylation of KC adhesion molecules. The extent of acantholysis in KC monolayers correlated closely with the degree of PV IgG-induced phosphorylation of p120- and beta-catenins, with classic isoforms of protein kinase C mediating serine phosphorylation of beta-catenin and Src-tyrosine phosphorylation of p120-catenin. The M(1) muscarinic agonist pilocarpine blocked phosphorylation of both catenins, which could be abolised by the M(1) antagonist MT7. The alpha7 nicotinic agonist AR-R17779 inhibited phosphorylation of P120-cateinin. The alpha7 antagonist methyllycaconitine abolished the effect of AR-R17779. Okadaic acid abrogated protective effects of agonists on phosphorylation of beta-catenin, and pervanadate, on that of p120-catenin. Stimulation of KCs with pilocarpine significantly (p < 0.05) elevated both serine/threonine and tyrosine phosphatase activities in KCs. AR-R17779 both stimulated tyrosine phosphatase and decreased PV IgG-induced Src activity. Methyllycaconitine released Src activity in intact KCs and caused acantholysis. Thus, downstream signaling from M(1) abolished PV IgG-dependent catenin phosphorylation due to activation of both serine/threonine and tyrosine phosphatases, whereas alpha7 action involved both activation of tyrosine phosphatase and inhibition of Src. These findings identified novel paradigm of regulation of signaling kinases associated with cholinergic receptors and provided mechanistic explanation of therapeutic activity of cholinomimetics in PV patients.
介导和调节细胞间连接组装与拆卸的机制是深入研究的课题。免疫性疱性皮肤病寻常型天疱疮（PV）患者产生的IgG自身抗体可通过涉及靶向细胞间粘附分子的信号激酶的机制诱导角质形成细胞（KC）黏附丧失（棘层松解），从而为研究KC黏附的生理调节提供了有用模型。先前的研究表明，Src和蛋白激酶C的激活是PV IgG诱导的细胞内磷酸化级联反应中的最早事件，并且胆碱能激动剂对治疗天疱疮患者有效。在本研究中，我们试图阐明胆碱能激动剂抑制PV IgG诱导的棘层松解和KC黏附分子磷酸化的分子机制。KC单层中的棘层松解程度与PV IgG诱导的p120连环蛋白和β-连环蛋白磷酸化程度密切相关，蛋白激酶C的经典同工型介导β-连环蛋白的丝氨酸磷酸化和p120连环蛋白的Src酪氨酸磷酸化。M（1）毒蕈碱激动剂毛果芸香碱可阻断两种连环蛋白的磷酸化，这可被M（1）拮抗剂MT7消除。α7烟碱激动剂AR-R17779抑制P120连环蛋白的磷酸化。α7拮抗剂甲基lycaconitine消除了AR-R17779的作用。冈田酸消除了激动剂对β-连环蛋白磷酸化的保护作用，过钒酸盐消除了对p120连环蛋白磷酸化的保护作用。用毛果芸香碱刺激KC可显著（p<0.05）提高KC中的丝氨酸/苏氨酸和酪氨酸磷酸酶活性。AR-R17779既刺激酪氨酸磷酸酶又降低PV IgG诱导的Src活性。甲基lycaconitine释放完整KC中的Src活性并导致棘层松解。因此，M（1）的下游信号传导由于丝氨酸/苏氨酸和酪氨酸磷酸酶的激活而消除了PV IgG依赖性连环蛋白磷酸化，而α7的作用涉及酪氨酸磷酸酶的激活和Src的抑制。这些发现确定了与胆碱能受体相关的信号激酶调节的新范式，并为拟胆碱药在PV患者中的治疗活性提供了机制解释。
