ABSTRACT
PURPOSE: To obtain experimental in vivo information on the functional properties of myocilin for aqueous humor (AH) outflow and to study in vivo the processing of mutated Tyr437His myocilin. Myocilin is a secreted glycoprotein that is mutated in some forms of primary open-angle glaucoma (POAG), and patients with the Tyr437His mutation have severe phenotypes. METHODS: The chicken betaB1-crystallin promoter was used to overexpress wild-type human myocilin and mutated Tyr437His myocilin in the lenses of transgenic mice. Expression of transgenic mRNA was monitored by Northern blot analysis and in situ hybridization. The localization and secretion of transgenic myocilin was investigated by Western blot analysis and light and electron microscopy. Intraocular pressure (IOP) was measured by anterior chamber cannulation. RESULTS: Two independent lines were established with each of the constructs that showed a strong expression of transgenic mRNA in their lenses. Transgenic expression resulted in a 4.7 +/- 1.8-fold increase of secreted normal myocilin in mouse AH, compared with its concentration in human AH. Immunoreactivity for transgenic myocilin was observed along the surfaces of lens and corneal endothelium, and in the chamber angle. At 12 weeks of age, the ultrastructure of the trabecular meshwork in mice expressing normal myocilin was not different from that of control eyes, and IOP of transgenic animals did not significantly differ from that of control littermates. In contrast, mutated Tyr437His myocilin was not secreted from lens fibers, but accumulated in dilated cisterns of rough endoplasmic reticulum. Although no structural changes were observed in lenses of animals expressing normal myocilin, lenses with Tyr437His expression developed nuclear cataracts, completely lost transparency, and eventually ruptured. Structural changes in lenses of Tyr437His expressing mice were correlated with a significant increase in IOP. CONCLUSIONS: The results do not support the concept that increasing amounts of myocilin in the outflow tissues obstruct the system and directly cause an increase in outflow resistance. Mutated Tyr437His myocilin is not secreted in vivo and causes severe alterations of cellular structure and function. A comparable mechanism may cause POAG in patients with myocilin mutations.
Subject(s)
Aqueous Humor/metabolism , Eye Proteins/genetics , Gene Expression Regulation/physiology , Glycoproteins/genetics , Lens, Crystalline/metabolism , Point Mutation , Trabecular Meshwork/metabolism , Animals , Blotting, Northern , Blotting, Western , Cytoskeletal Proteins , Eye Proteins/metabolism , Gene Transfer Techniques , Glycoproteins/metabolism , Histidine , In Situ Hybridization , In Situ Nick-End Labeling , Intraocular Pressure , Mice , Mice, Transgenic , RNA, Messenger/metabolism , Trabecular Meshwork/pathology , Transgenes , TyrosineABSTRACT
PURPOSE: The degeneration of retinal pigment epithelial (RPE) cells is considered to be a crucial event in the pathophysiology of age-related macular degeneration (AMD). Cumulative oxidative damage has been implicated in the development of the changes seen in AMD. The present study was undertaken to evaluate the expression of the small heat shock protein alphaB-crystallin in the RPE in response to oxidative stress and to explore whether alphaB-crystallin expression confers an antiapoptotic cytoprotective effect on RPE cells. METHODS: Native human RPE cells from the macula and retinal periphery were analyzed by RT-PCR and Western blot analysis for expression of alphaB-crystallin. Monolayer cultures of human RPE cells were stressed by heat shock (42 degrees C for 20 minutes) or oxidant-mediated injury (50-300 micro M H(2)O(2) for 1 hour). Induction of alphaB-crystallin and the corresponding mRNA was assessed by Western and Northern blot analyses. To study the cytoprotective effect of alphaB-crystallin, human RPE cells were transfected with either a neomycin-selectable expression vector containing alphaB-crystallin cDNA or a control vector without alphaB-crystallin cDNA. Caspase-3 activity was determined by observing the cleavage of a colorimetric peptide substrate. Cell viability was quantified by combined propidium iodide and Hoechst 33342 staining. RESULTS: alphaB-crystallin is constitutively expressed in RPE under in vivo and in vitro conditions. Western blot analysis of freshly isolated RPE showed greater baseline expression levels in RPE derived from the macular area than in that from the more peripheral regions. Heat shock treatment and oxidative stress caused a significant increase in alphaB-crystallin mRNA and protein. Oxidant-mediated injury in RPE cells with baseline expression levels of alphaB-crystallin resulted in apoptotic cell death, as measured by caspase-3 activity, whereas RPE cells that had been stably transfected with alphaB-crystallin were more resistant to H(2)O(2)-induced cellular injury. CONCLUSIONS: alphaB-crystallin may function as a stress-inducible antiapoptotic protein in human RPE and is inducible by oxidative stress, a condition implicated in the pathogenesis of AMD. Overexpression of alphaB-crystallin may be an important mechanism for the RPE to prevent apoptotic cell death in response to cellular stress.
