Nguyen Vu Thuong, Chernyavsky Alexander I, Arredondo Juan, Bercovich Dani, Orr-Urtreger Avi, Vetter Douglas E, Wess Jürgen, Beaudet Arthur L, Kitajima Yasuo, Grando Sergei A
Department of Dermatology, University of California, Davis, Sacramento, CA 95817, USA.
Exp Cell Res. 2004 Apr 1;294(2):534-49. doi: 10.1016/j.yexcr.2003.12.010.
The biological mechanisms involved in initiating, coordinating, and ultimately terminating cell-cell adhesion in the stratified epithelium are not well understood at present. This study was designed to elucidate the roles of the muscarinic M3, the nicotinic alpha3, and the mixed muscarinic-nicotinic alpha9 acetylcholine receptors in physiologic control of keratinocyte adhesion. Both muscarinic and nicotinic antagonists caused keratinocyte detachment and reversibly increased the permeability of keratinocyte monolayers, indicative of the involvement of both muscarinic and nicotinic pathways in the cholinergic control of keratinocyte adhesion. Since phosphorylation of adhesion proteins plays an important role in rapid assembly and disassembly of intercellular junctions, we measured muscarinic and nicotinic effects on phosphorylation of keratinocyte adhesion molecules. The phosphorylation levels of E-cadherin, beta-catenin, and gamma-catenin increased following pharmacological blockage of muscarinic receptors. Long-term blocking of alpha3, alpha9, and M3 receptor signaling pathways with antisense oligonucleotides resulted in cell-cell detachment and changes in the expression levels of E-cadherin, beta-catenin, and gamma-catenin in cultured human keratinocytes. Simultaneous inhibition of several receptor subtypes with a mixture of antisense oligonucleotides produced intensified abnormalities with cell adhesion. Moreover, altered cell-cell adhesion was found in the stratified epithelium of alpha3, alpha9, and M3 receptor knockout mice. Keratinocytes from these mice exhibited abnormal expression of adhesion molecules at both the protein and the mRNA levels. Thus, our data indicate that the alpha3, alpha9, and M3 acetylcholine receptors play key roles in regulating in a synergistic mode keratinocyte adhesion, most probably by modulating cadherin and catenin levels and activities. These findings may aid in the development of novel methods useful for the treatment of skin adhesion diseases and tumor metastasis.
目前，对于在复层上皮中启动、协调并最终终止细胞间黏附的生物学机制，人们尚未完全了解。本研究旨在阐明毒蕈碱型M3、烟碱型α3以及毒蕈碱-烟碱混合型α9乙酰胆碱受体在角质形成细胞黏附生理调控中的作用。毒蕈碱型和烟碱型拮抗剂均导致角质形成细胞脱离，并使角质形成细胞单层的通透性可逆性增加，这表明毒蕈碱型和烟碱型途径均参与了胆碱能对角质形成细胞黏附的调控。由于黏附蛋白的磷酸化在细胞间连接的快速组装和拆卸中起重要作用，我们检测了毒蕈碱型和烟碱型对角质形成细胞黏附分子磷酸化的影响。毒蕈碱受体药理学阻断后，E-钙黏蛋白、β-连环蛋白和γ-连环蛋白的磷酸化水平升高。用反义寡核苷酸长期阻断α3、α9和M3受体信号通路，导致培养的人角质形成细胞发生细胞间脱离，并使E-钙黏蛋白、β-连环蛋白和γ-连环蛋白的表达水平发生变化。用反义寡核苷酸混合物同时抑制几种受体亚型，会使细胞黏附异常加剧。此外，在α3、α9和M3受体基因敲除小鼠的复层上皮中发现了细胞间黏附改变。这些小鼠的角质形成细胞在蛋白质和mRNA水平上均表现出黏附分子的异常表达。因此，我们的数据表明，α3、α9和M3乙酰胆碱受体以协同模式在调节角质形成细胞黏附中起关键作用，很可能是通过调节钙黏蛋白和连环蛋白的水平及活性来实现的。这些发现可能有助于开发用于治疗皮肤黏附疾病和肿瘤转移的新方法。
