3), which could be related to the insufficient production of fiber cell-specific membrane or other elongation-associated proteins (89). fiber cells. InCol4a1+/ex40mutant mice, the accumulation of mutant chains also caused low levels of UPR activation. However, cell death was not induced in mutant lenses, suggesting that low levels of UPR activation are not proapoptotic. Collectively, the results providein vivoevidence for a role of UPR in cataract formation in response to accumulation of terminally unfolded proteins in the endoplasmic reticulum. == Introduction == The ocular lens is a transparent, Adenosine cellular structure that refracts light onto the retina, resulting in high resolution vision. Many environmental risk factors and single gene defects are known or hypothesized to result in clouding of the lens, a condition known as cataract. Cataract is the primary cause of blindness worldwide (1,2), with autosomal dominant congenital cataract being the leading cause of treatable childhood blindness (3,4). Cataract surgery is the most commonly performed surgical operation in the United States and consumes 60% of the Medicare budget for vision (5,6). Cataract can be a multifactorial disease and is often associated with systemic or genetic disorders, such as diabetes and Lowe syndrome (79). Notably, human diseases caused by mutations in extracellular matrix (ECM)4genes are also often associated with an increased risk of cataract. Stickler and Marshall syndromes are two disorders caused by mutations in theCOL2A1gene that are associated with the early onset of distinctive cataracts (10,11). Alport syndrome, caused by mutations in either theCOL4A3, COL4A4, orCOL4A5genes, is also associated with lens capsule abnormalities and cataract formation (1214). Humans carrying mutations in theCOL4A1locus often exhibit lens abnormalities and cataracts along with porencephaly and sporadic intracerebral hemorrhage (1519). To date, approximately 13 independent mutations in the mouseCol4a1locus and three independent mutations in mouseCol4a2locus have been found to cause vacuolar cataract and lens abnormalities in mice (1921). However, the underlying mechanisms of cataract pathogenesis resulting from these collagen mutations are still unknown. In other tissues, mutations in genes encoding secretory pathway proteins have been found to cause endoplasmic reticulum (ER) stress and subsequent activation of the unfolded protein response (UPR), a set of evolutionarily conserved signaling pathways activated upon ER stress (2228). UPR has been implicated in the pathogenesis of many conformational diseases, such as Alzheimer disease, Parkinson disease, and diabetes, and is being investigated in many others (2933). UPR pathways are activated Adenosine following accumulation of unfolded Adenosine proteins in the ER lumen and attempt to relieve the stress by 1) up-regulating the ER folding capacity through increasing the levels of ER-resident molecular chaperones and expansion of the ER, 2) reducing the demand on the ER through attenuation of protein synthesis, and 3) increasing the clearance Adenosine of unfolded proteins from the ER through up-regulation of ER-associated degradation (24,34,35). However, if these mechanisms cannot relieve the stress, the UPR pathway activates apoptosis (3638). Mammalian UPR is mediated by three ER-resident transmembrane proteins, IRE1, PERK (PKR-like ER kinase), and ATF6, whose combined activation alters transcriptional and translational programs and induces profound changes in cellular processes, such as cell growth, differentiation, and survival (3840). Notably, UPR pathways are induced in the lens in response to oxidative stress; however, the relative contributions of UPR and oxidative damage to lens pathogenesis are difficult to ascertain (41,42). Here we test the hypothesis that the presence of unfolded proteins within the lens secretory pathway results in UPR activation, disrupts lens differentiation and/or lens cell survival, and contributes to cataract pathogenesis. We first investigated whether the chronic production of unassembled collagen IV chains in the lens results in ER stress, the activation of UPR pathways, and the cellular changes leading to Rabbit polyclonal to ADD1.ADD2 a cytoskeletal protein that promotes the assembly of the spectrin-actin network.Adducin is a heterodimeric protein that consists of related subunits. cataract formation. We then determined that UPR pathways were also activated in the lenses fromCol4a1mutant mice, suggesting that UPR induction may be generally important in the pathogenesis of cataracts associated with the mutation of ECM genes. == EXPERIMENTAL PROCEDURES == == == == == == Generation of Transgenic and Mutant Mice == All experiments using transgenic animals were approved by the University of Delaware Institutional Animal Care and Use Committee. The enA plasmid containing the hybrid en/A-crystallin promoter, the rabbit -globin intron, and the human growth hormone poly(A) signal was a gift from Lixing Reneker (University of Missouri, Columbia, MO) (43). The mouse cDNAs forCol4a3andCol4a4were created as described previously (44). After the cDNA for eitherCol4a3orCol4a4was placed between the rabbit -globin intron and human growth hormone poly(A) signal in enA, the fragment containing the promoter, cDNA, intron, and poly(A) signal was liberated from the backbone plasmid by restriction enzyme digestion and gel-purified. This fragment was microinjected into the pronuclei of FVB/N fertilized eggs by the University of Delaware transgenic mouse facility as described previously (45). The genotyping of the transgenic mice is described in thesupplemental material. Col4a1+/ex40mice were bred at the University of.