Susan M. Baker
Florida Hospital College of Health Sciences, Orlando, FL, USA

Matthias M Falk
Lehigh University, Bethlehem, PA, USA

Series: Cell Biology Research Progress
BISAC: SCI017000

The vascular endothelium provides a barrier that regulates the flow of proteins and other blood-borne solutes between the vasculature and surrounding tissues. During inflammation, this barrier can become compromised by circulating inflammatory mediators, such as thrombin and endothelin. In vascular endothelial cells, regulated surface expression of gap junction (GJ) channels and proper modulation of gap junctional intercellular communication (GJIC) are required for blood vessels to maintain normal function. Inflammatory mediators such as thrombin and endothelin can elicit inhibition of GJIC and increased permeability of vascular endothelial cells, causing blood vessels to become “leaky.” In severe instances, patients may develop life-threatening conditions such as pulmonary edema and acute respiratory distress syndrome (ARDS) in response to these inflammatory mediators.

The molecular mechanisms for this down-regulation of GJIC and loss of cellular adhesion are poorly understood, and, to date, there are no effective drug therapies for such conditions. Combining high-resolution fluorescence microscopy and functional assays, we investigated the mechanism of GJIC inhibition and the loss of cell-to-cell adhesion in primary vascular endothelial cells in response to thrombin and endothelin. Activation of endothelial receptors PAR-1 and ETA/B by their natural inflammatory mediator agonists, thrombin and endothelin-1, respectively, or by mastoparan, a constitutive agonist of G-protein coupled receptors (GPCRs), resulted in a rapid and acute internalization of GJs that coincided with the inhibition of GJIC followed by increased vascular endothelial cell permeability. The process is mediated by the endocytic coat protein, clathrin, and the scaffold protein, ZO-1, and specifically requires binding of connexin 43 (Cx43) to the PDZ2 domain of ZO-1 apparently only on one side of the internalizing GJ plaque.

Disruption of Cx43/ZO-1 interaction effectively abolishes GJ internalization in response to thrombin and endothelin, suggesting a novel role for ZO-1 in the internalization process. Moreover, inhibition of GJIC, internalization of GJs and increased vascular endothelial cell permeability were all prevented with the G-protein antagonist, suramin. These findings demonstrate that GJ channel internalization is an efficient mechanism for modulating GJIC and facilitating physical cell-cell uncoupling in vascular endothelium in response to thrombin and endothelin. Additionally, the results presented here identify the PDZ2 domain of ZO-1 as a potential therapeutic target to prevent GJ internalization in vascular endothelial cells during inflammatory response. Overall, we have identified a novel role for the natural inflammatory mediators, thrombin and endothelin, in the regulation of GJIC and physical cell-to-cell adhesion. (Imprint: Nova)