Transgenic overexpression of connexin50 induces cataracts.
To examine the effects of increased expression of Cx50 in the mouse lens, transgenic mice were generated using a DNA construct containing the human Cx50 coding region and a C-terminal FLAG epitope driven by the chicken betaB1-crystallin promoter. Expression of this protein in paired Xenopus oocytes induced gap junctional currents of similar magnitude to wild type human Cx50. Three lines of transgenic mice expressing the transgenic protein were analyzed. Lenses from transgenic mice were smaller than those from non-transgenic littermates, and had cataracts that were already visible at postnatal day 1. Expression of the transgene resulted in a 3- to 13-fold increase in Cx50 protein levels above those of non-transgenic animals. Light microscopy revealed alterations in epithelial cell differentiation, fiber cell structure, interactions between fiber cells and areas of liquefaction. Scanning electron microscopy showed fiber cells of varying widths with bulging areas along single fibers. Anti-Cx50 and anti-FLAG immunoreactivities were detected at appositional membranes and in intracellular vesicles in transgenic lenses. N-cadherin, Cx46, ZO-1 and aquaporin 0 localized mainly at the plasma membrane, although some N-cadherin and aquaporin 0 was associated with the intracellular vesicles. The abundance and solubility/integrity of alphaA-, alphaB-, beta- and gamma-crystallin were unaffected. These results demonstrate that transgenic expression of Cx50 in mice leads to cataracts associated with formation of cytoplasmic vesicles containing Cx50 and decreased or slowed epithelial differentiation without major alterations in the distribution of other integral membrane or membrane-associated proteins or the integrity/solubility of crystallins.
Chung
June
J
Department of Pediatrics, University of Chicago, IL 60637, USA. jchung@peds.bsd.uchicago.edu
Berthoud
Viviana M
VM
Novak
Layne
L
Zoltoski
Rebecca
R
Heilbrunn
Benjamin
B
Minogue
Peter J
PJ
Liu
Xiaoqin
X
Ebihara
Lisa
L
Kuszak
Jer
J
Beyer
Eric C
EC
D000818:Animals; D020346:Aquaporins / Q000032:analysis / Q000378:metabolism; D002386:Cataract / Q000209:etiology* / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D017630:Connexins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D003459:Crystallins / Q000378:metabolism; D004594:Electrophysiology; D005136:Eye Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005818:Genetic Engineering; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007908:Lens, Crystalline / Q000473:pathology / Q000648:ultrastructure; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D009865:Oocytes / Q000502:physiology; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D012995:Solubility; D014981:Xenopus; D029867:Xenopus Proteins; D062826:Zonula Occludens-1 Protein; D000099264:Connexin 50
D020346:Aquaporins; D017630:Connexins; D003459:Crystallins; D005136:Eye Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D029867:Xenopus Proteins; D062826:Zonula Occludens-1 Protein; D000099264:Connexin 50; C569045:TJP1 protein, Xenopus; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse