Depression of Intraocular Pressure Following Inactivation of Connexin43 in the Nonpigmented Epithelium of the Ciliary Body

The aqueous humor provides oxygen and metabolites to the ocular tissues, and the balance between the rates of formation and absorption of the aqueous humor determines intraocular pressure (IOP), which maintains the curvature of the cornea and the refractive properties of the eye. The aqueous humor is produced in the ciliary body1 by a double-layered epithelium in which apical surfaces of the epithelial cells are opposed. Gap junctions are abundant at these opposing surfaces between pigmented epithelia (PE) and nonpigmented epithelia (NPE; heterologous junctions) and are present between adjoining cells in each layer (homologous junctions).2 In a current model of ciliary epithelium function, the heterologous junctions join the two epithelia in an ionic syncytium, permitting them to function coordinately to produce and secrete the aqueous humor.3 Gap junctions are composed of intercellular channels that mediate the movement of ions and low-molecular–weight metabolites directly between the cytoplasm of adjacent cells.4–6 Intercellular channels are composed of connexins. Twenty mouse genes and 21 human genes for connexins have been identified.7 Connexins are differentially expressed, with some present in only a few cell types and others more widespread.8–14 Connexin43 (Cx43) is prominently expressed in diverse eye structures such as the ciliary body15–17 and in lens and corneal epithelia.18–20 Cx43 mutations are associated with a human disease called oculodentodigital dysplasia (ODDD), which is characterized by pleiotropic developmental anomalies of the eyes, face, limbs, and teeth.21–24 Although glaucoma is found in some of the families, it is not possible to link changes in intraocular pressure directly with the mutations in Cx43. A mouse model of oculodentodigital dysplasia has been generated in which a mutation of a highly conserved amino acid G60S, in Cx43, leads to some of the symptoms of this disease, including corneal opacities and malformation of the iris.25 A range of phenotypes, however, is consistent with the pleiotropic nature of the clinical syndrome. Our laboratory has previously found that the deletion of Cx43 in mice causes no morphologic alterations in the prenatal eye.26 However, these animals die at birth as a result of a cardiac malformation.27 To explore the role of Cx43 in post-natal eye development, we produced a mouse line in which Cx43 was selectively inactivated in the PE but not the NPE of the ciliary body. These animals exhibited severe eye abnormalities, including complete loss of the vitreous humor from the posterior chamber.17 This was a surprising result in that both the PE and the NPE have been reported to express Cx40.16 If so, Cx40 in the PE of our conditional knockout should have been able to form intercellular channels with either Cx4028 or the remaining Cx43 in the NPE and thus support metabolic coupling between the layers of epithelia. Together, the data suggest that the production of aqueous humor requires the presence of gap junctions between PE and NPE containing solely Cx43. To test this idea further, we produced a mouse line in which Cx43 was selectively inactivated in the NPE by crossing a floxed Cx43 mouse line to a transgenic line expressing cre recombinase under the control of the retina-specific regulatory element α of the murine Pax6 promoter.29 In this study, we compared Pax6α-cre/Cx43+/flox (referred to as control) with Pax6α-cre/Cx43flox/flox (referred to as knockout) animals. Transgenic mice display cre activity in all retinal progenitor cells in the distal neuroretina and in the iris and ciliary epithelium (which derive from the distal retina) but not in the retinal pigment epithelium, lens, or cornea.30 Knockout mice showed a substantive loss of Cx43 expression in the NPE but not the PE, with a subsequent mild reduction in intraocular pressure. We were unable to confirm the presence of Cx40 in the ciliary epithelium or its potential to rescue the loss of Cx43. Our results confirm that Cx43 plays a key role in gap junction formation between the two epithelia and the production of aqueous humor. The critical functional role of Cx43 in the regulation of IOP suggests a putative pharmacologic target in the treatment of glaucoma, a pathologic state in which high IOP is a well-known risk factor.