The Cataract-linked Mutant Connexin50D47A Causes Endoplasmic Reticulum Stress in Mouse Lenses*

Abstract

Mice expressing connexin50D47A (Cx50D47A) exhibit nuclear cataracts and impaired differentiation. Cx50D47A does not traffic properly, and homozygous mutant lenses show increased levels of the stress-responsive αB-crystallins. Therefore, we assessed whether expression of Cx50D47A led to endoplasmic reticulum (ER) stress in the lens in vivo. Although pharmacologic induction of ER stress can be transduced by three different pathways, we found no evidence for activation of the IRE1α or ATF6 pathways in Cx50D47A-expressing lenses. In contrast, heterozygous and homozygous Cx50D47A lenses showed an increase in phosphorylated PERK immunoreactivity and in the ratio of phosphorylated to total EIF2α (2.4- and 3.3-fold, respectively) compared with wild type. Levels of ATF4 were similar in wild type and heterozygous lenses but elevated in homozygotes (391%). In both heterozygotes and homozygotes, levels of calreticulin protein were increased (184 and 262%, respectively), as was Chop mRNA (1.9- and 12.4-fold, respectively). CHOP protein was increased in homozygotes (384%). TUNEL staining was increased in Cx50D47A lenses, especially in homozygous mice. Levels of two factors that may be pro-survival, Irs2 and Trib3, were greatly increased in homozygous lenses. These results suggest that expression of Cx50D47A induces ER stress, triggering activation of the PERK-ATF4 pathway, which potentially contributes to the lens pathology and leads to increased expression of anti-apoptotic factors, allowing cell survival.