Lawrence Donald W, Comerford Katrina M, Colgan Sean P
Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115, USA.
Am J Physiol Cell Physiol. 2002 Jun;282(6):C1235-45. doi: 10.1152/ajpcell.00288.2001.
Epithelial permeability is tightly regulated by intracellular messengers. Critical to maintaining barrier integrity is the formation of tight junction complexes. A number of signaling pathways have been implicated in tight junction biogenesis; however, the precise molecular mechanisms are not fully understood. A growing body of evidence suggests a role for intracellular cAMP in tight junction assembly. Using an epithelial model, we investigated the role of cAMP signal transduction in barrier recovery after Ca2+ switch. Our data demonstrate that elevation of intracellular cAMP levels significantly enhanced barrier recovery after Ca2+ switch. Parallel experiments revealed that epithelial barrier recovery is diminished by H-89, a specific and potent inhibitor of cAMP-dependent protein kinase (protein kinase A) activity. Of the possible PKA effector proteins, the vasodilator-stimulated phosphoprotein (VASP) is an attractive candidate, since it has been implicated in actin-binding and cross-linking functions. We therefore hypothesized that VASP may play a role in the cAMP-mediated regulation of epithelial junctional reassembly after Ca2+ switch. We demonstrate here that VASP is phosphorylated via a PKA-dependent process under conditions that enhance barrier recovery. Confocal laser scanning microscopy studies revealed that VASP localizes with ZO-1 at the tight junction and at cell-cell borders and that phospho-VASP appears at the junction after Ca2+ switch. Subsequent transfection studies utilizing epithelial cells expressing truncated forms of VASP abnormal in oligomerization or actin-binding activity revealed a functional diminution of barrier recovery after Ca2+ chelation. Our present studies suggest that VASP may provide a link between cAMP signal transduction and epithelial permeability.
上皮细胞通透性受细胞内信使严格调控。紧密连接复合物的形成对于维持屏障完整性至关重要。许多信号通路与紧密连接的生物发生有关；然而，确切的分子机制尚未完全阐明。越来越多的证据表明细胞内cAMP在紧密连接组装中发挥作用。利用上皮细胞模型，我们研究了cAMP信号转导在Ca2+转换后屏障恢复中的作用。我们的数据表明，细胞内cAMP水平升高显著增强了Ca2+转换后的屏障恢复。平行实验显示，cAMP依赖性蛋白激酶（蛋白激酶A）活性的特异性强效抑制剂H-89可减弱上皮屏障的恢复。在可能的蛋白激酶A效应蛋白中，血管舒张剂刺激磷蛋白（VASP）是一个有吸引力的候选者，因为它与肌动蛋白结合和交联功能有关。因此，我们推测VASP可能在Ca2+转换后cAMP介导的上皮连接重新组装调节中发挥作用。我们在此证明，在增强屏障恢复的条件下，VASP通过蛋白激酶A依赖性过程被磷酸化。共聚焦激光扫描显微镜研究显示，VASP与紧密连接蛋白ZO-1共定位于紧密连接和细胞-细胞边界，并且磷酸化的VASP在Ca2+转换后出现在连接处。随后利用表达寡聚化或肌动蛋白结合活性异常的VASP截短形式的上皮细胞进行的转染研究表明，Ca2+螯合后屏障恢复功能减弱。我们目前的研究表明，VASP可能在cAMP信号转导与上皮通透性之间提供联系。
