Rac and

The function and localization of both Racl and Rho in the cell have recently attracted the attention of those inves tigators studying the barrier function of the endothelium and epithelium. Kaibuchi and coworkers [8] have put forth an intriguing hypothesis that activation of Rac1 and/or Cdc42 strengthens the adherens junction of epithelial cells. Their findings support the notion that active Rac1 and Cdc42 can sequester IQGAP1, a proposed negative regulator of the junction, away from the junction allowing for strong cell-cell adhesion. The opposite occurs when Rac1 activity is decreased in Madin-Darby canine kidney II cells treated with a phorbol ester or hepatocyte growth factor, the latter inducing cell scattering. Now IQGAP1 is associated with b-catenin and not Rac1 at the cell periphery, and there is a loss of a-catenin from cell-cell borders. Expression of a consti-tutively active Rac1 blocks this disappearance of a-catenin. The binding site on b-catenin for IQGAP1 (amino-terminal 1-183 amino acids) contains the a-catenin binding domain (120-151 amino acids). a-Catenin is the key intermediary that links VE-cadherin and b-catenin to the actin cytoskele-ton to form a strong cell-cell adhesion. The presence of IQGAP1 disrupts this linear linkage and results in weak adhesion between cells. Thus, IQGAP1 may contribute to the dynamic nature of the adherens junction.

S1P induces the translocation of Rac1 and its guanine nucleotide exchange factor Tiam1 to the cell periphery in human umbilical vein endothelial cells but does not alter the spacial movement of Rho. However, inhibition of Rac1 or Rho reduces the immunolocalization of VE-cadherin and b-catenin at sites of cell-cell adhesion. Experiments with antisense oligonucleotides, have suggested that Rac1 and Rho function via Edg 1 and Edg 3, respectively. Recruitment of actin to epithelial junctions is dependent on Rac1 activation. The involvement of Rac1 and Rho in the integrity of the adherens junction of endothelial cells, however, is controversial. Inhibition of Rho kinase with Y-27632 has been reported to attenuate the increase in endothelial electrical resistance induced by S1P.

S1P increases the activity of Rac1, and this activation is sensitive to pertussis toxin. Rac1 activity is determined with an affinity precipitation assay using glutathione-^-transferase fused to the Rac1/Cdc42 binding domain of p21activated kinase (PAK). Treatment of endothelial cell monolayers with S1P results in an increase in active Rac1 by 10 minutes. Thus, S1P-induced increases in Rac1 activity and endothelial electrical resistance occur within the same time frame and both responses are sensitive to pertussis toxin, indicating the involvement of the Gi protein. As stated earlier, S1P remodels actin within minutes into stress fibers and an increased peripheral actin. Rac1 induces the formation of cortical actin at the leading edge of lamellipodia, and Rho forms stress fibers. Formation of actin stress fibers by S1P is abolished with C3 exoenzyme, an inhibitor of Rho, and with Y-27632, the Rho kinase inhibitor. A dominantnegative Rac1 inhibits both the changes in stress fibers and cortical actin.

Wojciak-Stothard and coworkers in the laboratory of Ridley [9] reported that inhibition of Rho with C3 exoenzyme or adenoviral expression of a dominant-negative Rho and inhibition of Rho kinase with Y-27632 prevent the increase in endothelial permeability and the decrease in transendothelial electrical resistance induced by thrombin and histamine. These inhibitors also prevent the disassembly of the adherens junctions induced by these inflammatory agents. Surprisingly, adenoviral expression of either a dominant-active or a dominant-negative Racl increases the permeability of unstimulated monolayers of human umbilical vein endothelial cells. Faced with the same data induced by either activation or inhibition of Racl, the authors concluded that the activity of Racl must be tightly regulated to maintain the integrity of the adherens junction between endothelial cells. According to the hypothesis of Kaibuchi, both Racl and Cdc42 can enhance the barrier function of epithelial cells. Therefore, Racl and Cdc42 may be redundant proteins with regard to the regulation of the endothelial barrier.

Beyond Racl, one study by Garcia and coworkers [5] has looked at the downstream kinase PAK, and cofilin, an actin-severing protein inactivated by LIM kinase. A Racl-bound PAK phosphorylates LIM kinase causing the subsequent inactivation of cofilin. Both PAK and cofilin translocate to the cell periphery after treatment with SlP. Expression of a dominant-negative PAK-l or wild-type cofilin reduces the increase in cortical actin. The latter also blunts the increase in endothelial electrical resistance induced by S1P. These authors concluded that a thickened, cortical actin plays a prominent role in the enhanced endothelial barrier activity of S1P. Therefore, inactivation of cofilin via Racl to PAK signaling is key to this activity of SlP.

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