Rho signaling is involved in the formation of stress fibers and focal adhesions
27 28 29 and consequentially, several actin filament-mediated processes. Recently, several downstream targets of Rho have been identified and among them is a family of isozymes of Rho-associated serine/threonine kinases (collectively referred to as ROCK), including ROCK-I (p160ROCK/ROK-beta) and ROCK-II (ROK-alpha/Rho-kinase). ROCK has been shown to induce focal adhesions and stress fibers in cultured fibroblasts and epithelial cells.
38 40 52 53 Uehata et al.
54 have reported that a new pyridine derivative, Y-27632, is a specific inhibitor of the ROCK family. When ROCK was inhibited by treating corneal epithelial cells with Y-27632, it disrupted actin stress fibers in corneal epithelial cells, as expected. Based on the immunofluorescence analysis, ROCK inhibition resulted in the elimination of E-cadherin-based adherens junctions at the cell-cell contacts. However, it did not inhibit the assembly of Cx43 gap junctions. In fact, inhibition of ROCK activity resulted in an increase in the number of gap junctions, as evident from immunofluorescence and Western blot analyses. There was also an increase in functional gap junctions as evident from the dye diffusion study. We currently do not know whether other gap junctions, such as connexin 50 gap junctions, also accounted for an increase in functional gap junctions in ROCK-inhibited cells. Although E-cadherin was diminished from the cell-cell contacts in Y-27632-treated cells, it was detectable in the cytoplasm of the cells. The total levels of E-cadherin in the inhibitor-treated cells were not significantly different from the controls. Thus, ROCK-I signaling most likely participates in the actin filament-mediated translocation of E-cadherin to the cell membrane. Contrary to the present observation, the inhibition of ROCK with Y-27632 did not prevent clustering of cadherin during induction of cell-cell adhesion in keratinocytes in culture.
33 However, blocking of ROCK function by using a dominant negative approach was reported to partially perturb the localization of cadherin receptors in MDCK cells.
55 In the absence of E-cadherin adherens junctions, Cx43 gap junctions were detected even after 18 hours of inhibitor treatment. E-cadherin is a transmembrane protein, and its adhesive function is brought about by Ca
2+-dependent homophilic interaction between E-cadherin molecules of the adjacent cells. The cytoplasmic tail of E-cadherin interacts with the actin cytoskeleton via β-catenin, and this complex formation aides in the clustering of the receptors and strengthening of cell-cell adhesion.
18 19 20 The interaction of the cytoskeleton not only is involved in the clustering of the E-cadherin receptors but also in providing a framework for the different cytoskeletal proteins and signaling molecules at cell-cell junctions.
56 The lowering of the Ca
2+ concentration in tissue culture media prevents homophilic interactions between E-cadherin molecules of the neighboring cells. When corneal epithelial cells were cultured in low Ca
2+ medium, E-cadherin interactions were inhibited, but Cx43 gap junctions were not inhibited, confirming that the gap junction assembly did not require E-cadherin interactions between the adjacent cells. When the cells were then provided with Ca
2+ in the medium, they formed the E-cadherin-based adherens junctions in the absence of the ROCK inhibitor but not in its presence. However, there was a significant increase in the Cx43 gap junctions when the ROCK activity was inhibited. Thus, downregulation of the Rho/ROCK signaling pathway was found to promote Cx43 gap junction formation. On the basis of this finding we would have expected to see increased Cx43 gap junctions in the limbal epithelium, which lacks ROCK-I activity. Quite the opposite, the limbal epithelium has been reported to be devoid of Cx43 and gap junctions. It is likely that the expression of Cx43 is suppressed in the limbal cells, independently of Rho/ROCK signaling pathways. However, Rho signaling via another downstream target is likely to be involved in promoting Cx43 gap junction assembly because inactivation of Rho was found to inhibit Cx43 gap junction assembly. In addition to ROCK-I and ROCK-II, several other Rho effectors have been identified including PKN/PRK1/PRK2,
57 citron kinase,
58 p140mDia (mDia1),
59 PIP5K,
60 61 myosin phosphatase,
62 and rhophilin, rhotekin, and kinectin.
63 Of these Rho targets, mDia1 has received much attention in recent years. It is a mammalian homolog of Drosophila diaphanous, a protein required for cytokinesis.
64 We speculate that mDia1may be involved in regulation of the assembly of Cx43 gap junction based on the findings that mDia1
65 is involved in the formation and orientation of stable microtubules which also interact with Cx43. Tubulin binds the juxtamembrane region of Cx43, which also sediments with tubulin in pull down experiments.
66 If microtubules are involved in the assembly of Cx43 gap junctions, mDia1, which is involved in the formation of microtubules, is likely to play a role in the assembly of the gap junctions. One way to test this speculation would be to find out whether overexpression of constitutively inactive mDia1 results in inhibition of Cx43 gap junction assembly.