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 ␣B-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 IRE1␣ 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 EIF2␣ (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.Proteins that do not traffic properly or are misfolded can cause endoplasmic reticulum (ER) 2 stress. To compensate for the disturbance of proteostasis, cells transduce ER stress through at least three different signaling pathways: (a) the inositol-requiring enzyme 1 (IRE1␣)-dependent, (b) the activating transcription factor 6 (ATF6)-dependent, and (c) the PRKR-like ER kinase (PERK)-dependent pathways. Prolonged and uncompensated ER stress triggers the unfolded protein response (UPR), which may ultimately target the cells for apoptosis.Many disease-linked connexin mutants show impaired cellular trafficking. One such mutant has a missense mutation in the lens gap junction protein, connexin50 (Cx50), affecting amino acid residue 47 (Cx50D47N in humans and Cx50D47A in mice) and is linked to congenital cataracts (1, 2). When expressed in transfected cells, Cx50D47N and Cx50D47A localize mostly within the ER (1). Lenses from mice expressing Cx50D47A have nuclear cataracts, reduced sizes, and impaired denucleation (3). Some immunoreactive Cx50 localizes intracellularly in lens epithelial cells from these mice (3). Homozygous Cx50D47A lenses have increased levels of ␣B-crystallins (3), which are responsive to several types of stress in different cell types, including lens epithelial cells (4 -7). These observations suggest that expression of a Cx50D47 mutant may cause ER stress in the lens and contribute to pathology in this organ.The current understanding of cellular responses to ER stress is based on many studies in cultured cells...

Endoplasmic Reticulum Stress in Hearing Loss

“…In Cx50G22R or Cx50S50P mutant mice, the IRE1α-XBP1 pathway is activated, while PERK-eIF2α-ATF4 and ATF6α pathways are largely unaffected [96]. In contrast, in Cx50D47A mutant mice, the PERK-eIF2α-ATF4 pathway is activated, while the IRE1α-XBP1 and ATF6α pathways are not activated [97].…”

“…For example, skin-disease-associated Cx31 mutants were shown to induce all three UPR pathways in transfected cells [95]. ER stress is also activated in cataract-associated Cx50 mutant mouse lenses [96,97]. Interestingly, different UPR pathways are activated by different Cx50 mutations.…”

Connexin 50-R205G Mutation Perturbs Lens Epithelial Cell Proliferation and Differentiation

“…The Cx50-D47A, Cx50-G22R, and Cx50-S50P mutations have improper and reduced gap junction communication in the lens and have been implicated in inducing stress pathways or promoting calcium elevation and deposits. 17,25,26 Increased ER stress is also involved in the cell death in skin disease-associated Cx31 mutations. 32,33 However, our data indicate that the BiP expression is not significantly increased, and there is no obvious accumulation of Cx50-R205 mutant proteins in cultured Cx50(R205G/R205G) LECs (Fig.…”

“…Previous studies have reported that Cx50-S50P, Cx50-G22R, and Cx50-D47A mutations can increase ER stress. 25,26 We examined the expression of BiP, an ER protein whose increased expression can reflect the activation of unfolded protein response, to test whether ER stress is a possible underlying mechanism for the severe cataracts caused by the Cx50-R205G mutation. Immunostaining of BiP was performed in primary cultured LECs (Fig.…”

Age-related cataracts: Role of unfolded protein response, Ca 2+ mobilization, epigenetic DNA modifications, and loss of Nrf2/Keap1 dependent cytoprotection

“…There are several experimental nuclear cataract models describing the involvement of UPR in the disease pathology, and none of them provided the detailed mechanism. Thus, one can suggest that various ER stressors with different potential and exposure durations may affect the development of the different types of cataracts (Berthoud et al, 2016; Lyu et al, 2016; Mulhern et al, 2006; Palsamy et al, 2014b). Recently, it has been demonstrated the activation of the UPR through discrete pathways in the lens of age- related, high myopia-related and congenital cataracts (Yang et al, 2015).…”