Promotion of lens epithelial-fiber differentiation by the C-terminus of connexin 45.6 - a role independent of gap junction communication

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 Cx45.6dependent 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.