We observed that chick lens-fiber gap-junction-forming proteins, connexin (Cx) 45.6 and Cx56, were associated with an unknown protein, which was then identified as major intrinsic protein (MIP), also known as aquaporin-0 (AQP0), the most abundant membrane protein in lens fibers. A 1063 bp cDNA of chick MIP(AQP0) was identified that encodes a 262 amino acid protein with a predicted molecular weight of 28.1 kDa. Dual immunofluorescence and confocal microscopy of sagittal and coronal sections of the lens tissues showed that MIP(AQP0) consistently localized with gap junction plaques formed by Cx45.6 and Cx56 during the early stages of embryonic chick lens development. Immunoprecipitation combined with immunoblotting analyses revealed that MIP(AQP0) was associated with Cx45.6 and Cx56 at these developmental stages. The specificity of this interaction was further confirmed with the silver staining of the protein components of immunoprecipitates. The pull-down analysis of lens lysates revealed that C-terminus of MIP(AQP0) probably interacted with these two fiber connexins. In late embryonic and adult lenses, however, uniform co-distribution of MIP(AQP0) and fiber connexins was largely disrupted, except for the area surrounding the actively differentiating bow regions, as was revealed by immunofluorescence and immunoprecipitation experiments. The interaction of MIP(AQP0) with lens fiber connexins in differentiating lens cells but not in mature lens fibers suggests a potential role for MIP(AQP0) in the facilitation of fiber connexins for the formation of gap junctions during lens development.

These results suggest that Cx45.6 plays an important role in stimulating lens cell differentiation and fiber formation, which is different from the other lens connexins, Cx43 and Cx56. This stimulatory effect is independent of gap junction-mediated intercellular communication and lens cell proliferation.

The eye lens is dependent upon a network of gap junction-mediated intercellular communication to facilitate its homeostasis and development. Three gap junctionforming proteins are expressed in the lens of which two are in lens fibers, namely connexin (Cx) 45.6 and 56. Major intrinsic protein (MIP), also known as aquaporin-0 (AQP0), is the most abundant membrane protein in lens fibers. However, its role in the lens is not clear. Our previous studies show that MIP(AQP0) associates with gap junction plaques formed by Cx45.6 and Cx56 during the early stages of embryonic chick lens development but not in late embryonic and adult lenses. We report here that MIP(AQP0) directly interacts with Cx45.6 but not with Cx56. We further identified the intracellular loop of Cx45.6 as the interacting domain for the MIP(AQP0) C terminus. Surface plasmon resonance experiments indicated that the C-terminal domain of MIP(AQP0) interacts with two binding sites within the intracellular loop region of Cx45.6 with a K D(app) of 7.5 and 10.3 M, respectively. The K D(app) for the full-length loop region is 7.7 M. The cleavage at the intracellular loop of Cx45.6 was observed during lens development, and the C terminus of MIP(AQP0) did not interact with the loop-cleaved form of Cx45.6. Thus, the dissociation between these two proteins that occurs in the mature fibers of late lens development is likely caused by this cleavage. Finally this interaction had no impact on Cx45.6-mediated intercellular communication, suggesting that the Cx45.6-MIP(AQP0) interaction plays a novel unidentified role in lens fibers.

We previously reported that, among the three connexins expressed in chick lens, overexpression of connexin (Cx) 45.6, not Cx43 or Cx56, stimulates lens cell differentiation; however, the underlying mechanism responsible for this effect is unclear. Here, we took advantage of naturally occurring loss-of-gap-junction function mutations of Cx50 (ortholog of chick Cx45.6) and generated the corresponding site mutants in Cx45.6: Cx45.6(D47A) and Cx45.6(P88S). In contrast to wild-type Cx45.6, the mutants failed to form functional gap junctions, and Cx45.6(P88S) and, to a lesser degree, Cx45.6(D47A) functioned in a dominant-negative manner. Interestingly, overexpression of both mutants incapable of forming gap junctions significantly increased epithelial-fiber differentiation to a level comparable to that of wild-type Cx45.6. To map the functional domain of Cx45.6, we generated a C-terminus chimera as well as deletion mutants. Overexpression of Cx56*45.6C, the mutant in which the C-terminus of Cx56 was replaced with that of Cx45.6, had a stimulatory effect on lens cell differentiation similar to that of Cx45.6. However, cells overexpressing Cx45.6*56C, the mutant in which C-terminus of Cx45.6 was replaced with that of Cx56, and Cx45.6(-C), in which the C-terminus was deleted, failed to promote differentiation. Taken together, we conclude that the expression of Cx45.6, but not Cx45.6-dependent gap junction channels, is involved in lens epithelial-fiber cell differentiation, and the C-terminal domain of Cx45.6 plays a predominant role in mediating this process.Supplementary material available online at

SUMMARY Connexins and connexin channels play important roles in cell growth/differentiation and tumorigenesis. Here, we identified a relationship between a connexin molecule and a critical cell-cycle regulator. Our data show that connexin (Cx) 50 regulated lens cell-cycle progression and differentiation by modulating expression of cyclin-dependent kinase inhibitor p27/p57 and E3 ubiquitin ligase Skp2. Cx50 directly interacted with and retained Skp2 in the cytosol by masking the nuclear targeting domain of Skp2, and this effect was supported by an increased nuclear localization of Skp2, disruption of Skp2 interaction with importin-7, and decreased levels of p27/p57 in mouse lenses lacking Cx50. As a result, Cx50 increased auto-ubiquitination and subsequent degradation of Skp2. A mutation (V362E) on the C terminus of Cx50 disrupted the interaction between Cx50 and Skp2 and completely abolished such effects. Therefore, this study identifies a role for connexins in regulating cell-cycle modulators and, consequently, cell growth and differentiation.