Connexin 50 Influences the Physiological Optics of the In Vivo Mouse Lens.

Purpose: The purpose of this study was to utilize multi-parametric magnetic resonance imaging (MRI) to investigate in vivo age-related changes in the physiology and optics of mouse lenses where Connexin 50 has been deleted (Cx50KO) or replaced by Connexin 46 (Cx50KI46). Methods: The lenses of transgenic Cx50KO and Cx50KI46 mice were imaged between 3 weeks and 6 months of age using a 7T MRI. Measurements of lens geometry, the T2 (water-bound protein ratios), the refractive index (n), and T1 (free water content) values were calculated by processing the acquired images. The lens power was calculated from an optical model that combined the geometry and the n. All transgenic mice were compared with control mice at the same age. Results: Cx50KO and Cx50KI46 mice developed smaller lenses compared with control mice. The lens thickness, volume, and surface radii of curvatures all increased with age but were limited to the size of the lenses. Cx50KO lenses exhibited higher lens power than Cx50KI46 lenses at all ages, and this was correlated with significantly lower water content in these lenses, which was probably modulated by the gap junction coupling. The refractive power tended to a steady state with age, similar to the control mice. Conclusions: The modification of Cx50 gap junctions significantly impacted lens growth and physiological optics as the mouse aged. The lenses showed delayed development growth, and altered optics governed by different lens physiology. This research provides new insights into how gap junctions regulate the development of the lens's physiological optics.

Single-Cell RNA Sequencing Analysis of the Early Postnatal Mouse Lens Epithelium.

Purpose: The lens epithelium maintains the overall health of the organ. We used single-cell RNA sequencing (scRNA-seq) technology to assess transcriptional heterogeneity between cells in the postnatal day 2 (P2) epithelium and identify distinct epithelial cell subtypes. Analysis of these data was used to better understand lens growth, differentiation, and homeostasis on P2. Methods: scRNA-seq on P2 mouse lenses was performed using the 10x Genomics Chromium Single Cell 3' Kit (v3.1) and short-read Illumina sequencing. Sequence alignment and preprocessing of data were conducted using 10x Genomics Cell Ranger software. Seurat was employed for preprocessing, quality control, dimensionality reduction, and cell clustering, and Monocle was utilized for trajectory analysis to understand the developmental progression of the lens cells. CellChat and GO analyses were used to explore cell-cell communication networks and signaling interactions. Results: Lens epithelial cells (LECs) were divided into seven subclusters, classified by specific gene markers. The expression of crystallin, cell-cycle, and metabolic genes was not uniform, indicating distinct functional roles of LECs. Trajectory analysis predicted a bifurcation of differentiating and cycling cells from an Igfbp5+ progenitor pool. We also identified heterogeneity in signaling molecules and pathways, suggesting that cycling and progenitor subclusters have prominent roles in coordinating crosstalk. Conclusions: scRNA-seq corroborated many known markers of epithelial differentiation and proliferation while providing further insight into the pathways and genes directing these processes. Interestingly, we demonstrated that the developing epithelium can be divided into distinct subpopulations. These clusters reflect the transcriptionally diverse roles of the epithelium in proliferation, signaling, and maintenance.

Age-Dependent Changes in the Water Content and Optical Power of the In Vivo Mouse Lens Revealed by Multi-Parametric MRI and Optical Modeling.

Purpose: The purpose of this study was to utilize in vivo magnetic resonance imaging (MRI) and optical modeling to investigate how changes in water transport, lens curvature, and gradient refractive index (GRIN) alter the power of the mouse lens as a function of age. Methods: Lenses of male C57BL/6 wild-type mice aged between 3 weeks and 12 months (N = 4 mice per age group) were imaged using a 7T MRI scanner. Measurements of lens shape and the distribution of T2 (water-bound protein ratios) and T1 (free water content) values were extracted from MRI images. T2 values were converted into the refractive index (n) using an age-corrected calibration equation to calculate the GRIN at different ages. GRIN maps and shape parameters were inputted into an optical model to determine ageing effects on lens power and spherical aberration. Results: The mouse lens showed two growth phases. From 3 weeks to 3 months, T2 decreased, GRIN increased, and T1 decreased. This was accompanied by increased lens thickness, volume, and surface radii of curvatures. The refractive power of the lens also increased significantly, and a negative spherical aberration was developed and maintained. Between 6 and 12 months of age, all physiological, geometrical, and optical parameters remained constant, although the lens continued to grow. Conclusions: In the first 3 months, the mouse lens power increased as a result of changes in shape and in the GRIN, the latter driven by the decreased water content of the lens nucleus. Further research into the mechanisms regulating this decrease in mouse lens water could improve our understanding of how lens power changes during emmetropization in the developing human lens.

Antibody gene transfer treatment drastically improves epidermal pathology in a keratitis ichthyosis deafness syndrome model using male mice.

BACKGROUND: Keratitis ichthyosis deafness (KID) syndrome is a rare disorder caused by hemichannel (HC) activating gain-of-function mutations in the GJB2 gene encoding connexin (Cx) 26, for which there is no cure, or current treatments based upon the mechanism of disease causation. METHODS: We applied Adeno Associated Virus (AAV) mediated mAb gene transfer (AAVmAb) to treat the epidermal features of KID syndrome with a well-characterized HC blocking antibody using male mice of a murine model that replicates the skin pathology of the human disease. FINDINGS: We demonstrate that in vivo AAVmAb treatment significantly reduced the size and thickness of KID lesions, in addition to blocking activity of mutant HCs in the epidermis in vivo. We also show that AAVmAb treatment eliminated abnormal keratinocyte proliferation and enlarged cell size, decreased apoptosis, and restored the normal distribution of keratin expression. INTERPRETATION: Our findings reinforce the critical role played by increased HC activity in the skin pathology associated with KID syndrome. They also underscore the clinical potential of anti-HC mAbs coupled with genetic based delivery systems for treating the underlying mechanistic basis of this disorder. Inhibition of HC activity is an ideal therapeutic target in KID syndrome, and the genetic delivery of mAbs targeted against mutant HCs could form the basis of new therapeutic interventions to treat this incurable disease. FUNDING: Fondazione Telethon grant GGP19148 and University of Padova grant Prot. BIRD187130 to FM; Foundation for Ichthyosis and Related Skin Types (FIRST) and National Institutes of Health grant EY 026911 to TWW.

Double Deletion of PI3K and PTEN Modifies Lens Postnatal Growth and Homeostasis.

We have previously shown that the conditional deletion of either the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), or its opposing phosphatase, phosphatase and tensin homolog (PTEN), had distinct effects on lens growth and homeostasis. The deletion of p110α reduced the levels of phosphorylated Akt and equatorial epithelial cell proliferation, and resulted in smaller transparent lenses in adult mice. The deletion of PTEN increased levels of phosphorylated Akt, altered lens sodium transport, and caused lens rupture and cataract. Here, we have generated conditional p110α/PTEN double-knockout mice, and evaluated epithelial cell proliferation and lens homeostasis. The double deletion of p110α and PTEN rescued the defect in lens size seen after the single knockout of p110α, but accelerated the lens rupture phenotype seen in PTEN single-knockout mice. Levels of phosphorylated Akt in double-knockout lenses were significantly higher than in wild-type lenses, but not as elevated as those reported for PTEN single-knockout lenses. These results showed that the double deletion of the p110α catalytic subunit of PI3K and its opposing phosphatase, PTEN, exacerbated the rupture defect seen in the single PTEN knockout and alleviated the growth defect observed in the single p110α knockout. Thus, the integrity of the PI3K signaling pathway was absolutely essential for proper lens homeostasis, but not for lens growth.

Connexin hemichannel inhibition ameliorates epidermal pathology in a mouse model of keratitis ichthyosis deafness syndrome.

Mutations in five different genes encoding connexin channels cause eleven clinically defined human skin diseases. Keratitis ichthyosis deafness (KID) syndrome is caused by point mutations in the GJB2 gene encoding Connexin 26 (Cx26) which result in aberrant activation of connexin hemichannels. KID syndrome has no cure and is associated with bilateral hearing loss, blinding keratitis, palmoplantar keratoderma, ichthyosiform erythroderma and a high incidence of childhood mortality. Here, we have tested whether a topically applied hemichhanel inhibitor (flufenamic acid, FFA) could ameliorate the skin pathology associated with KID syndrome in a transgenic mouse model expressing the lethal Cx26-G45E mutation. We found that FFA blocked the hemichannel activity of Cx26-G45E in vitro, and substantially reduced epidermal pathology in vivo, compared to untreated, or vehicle treated control animals. FFA did not reduce the expression of mutant connexin hemichannel protein, and cessation of FFA treatment allowed disease progression to continue. These results suggested that aberrant hemichannel activity is a major driver of skin disease in KID syndrome, and that the inhibition of mutant hemichannel activity could provide an attractive target to develop novel therapeutic interventions to treat this incurable disease.

Physiological Mechanisms Regulating Lens Transport.

The transparency and refractive properties of the lens are maintained by the cellular physiology provided by an internal microcirculation system that utilizes spatial differences in ion channels, transporters and gap junctions to establish standing electrochemical and hydrostatic pressure gradients that drive the transport of ions, water and nutrients through this avascular tissue. Aging has negative effects on lens transport, degrading ion and water homeostasis, and producing changes in lens water content. This alters the properties of the lens, causing changes in optical quality and accommodative amplitude that initially result in presbyopia in middle age and ultimately manifest as cataract in the elderly. Recent advances have highlighted that the lens hydrostatic pressure gradient responds to tension transmitted to the lens through the Zonules of Zinn through a mechanism utilizing mechanosensitive channels, multiple sodium transporters respond to changes in hydrostatic pressure to restore equilibrium, and that connexin hemichannels and diverse intracellular signaling cascades play a critical role in these responses. The mechanistic insight gained from these studies has advanced our understanding of lens transport and how it responds and adapts to different inputs both from within the lens, and from surrounding ocular structures.

Signaling Between TRPV1/TRPV4 and Intracellular Hydrostatic Pressure in the Mouse Lens.

Purpose: The lens uses feedback to maintain zero pressure in its surface cells. Positive pressures are detected by transient receptor potential vanilloid (TRPV4), which initiates a cascade that reduces surface cell osmolarity. The first step is opening of gap junction hemichannels. One purpose of the current study was to identify the connexin(s) in the hemichannels. Negative pressures are detected by TRPV1, which initiates a cascade that increases surface osmolarity. The increase in osmolarity was initially reported to be through inhibition of Na/K ATPase activity, but a recent study reported it was through stimulation of Na/K/2Cl (NKCC) cotransport. A second purpose of this study was to reconcile these two reports. Methods: Intracellular hydrostatic pressures were measured using a microelectrode/manometer system. Lenses from TRPV1 or Cx50 null mice were studied. Specific inhibitors of Cx50 gap junction channels, NKCC, and Akt were used. Results: Either knockout of Cx50 or blockade of Cx50 channels completely eliminated the response to positive surface pressures. Knockout of Cx50 also caused a positive drift in surface pressure. The short-term (∼20-minute) response to negative surface pressures was eliminated by blockade of NKCC, but a long-term (∼4-hour) response restored pressure to zero. Both short- and long-term responses were eliminated by knockout of TRPV1 or inhibition of Akt. Conclusions: Hemichannels made from Cx50 are required for the response to positive surface pressures. Negative surface pressures first activate NKCC, but a backup system is inhibition of Na/K ATPase activity. Both responses are initiated by TRPV1 and go through PI3K/Akt before branching.

Multi-parametric MRI of the physiology and optics of the in-vivo mouse lens.

The optics of the ocular lens are determined by its geometry (shape and volume) and its inherent gradient of refractive index (water to protein ratio), which are in turn maintained by unique cellular physiology known as the lens internal microcirculation system. Previously, magnetic resonance imaging (MRI) has been used on ex vivo organ cultured bovine lenses to show that pharmacological perturbations to this microcirculation system disrupt ionic and fluid homeostasis and overall lens optics. In this study, we have optimised in vivo MRI protocols for use on wild-type and transgenic mouse models so that the effects of genetically perturbing the lens microcirculation system on lens properties can be studied. In vivo MRI protocols and post-analysis methods for studying the mouse lens were optimised and used to measure the lens geometry, diffusion, T1 and T2, as well as the refractive index (n) calculated from T2, in wild-type mice and the genetically modified Cx50KI46 mouse. In this animal line, gap junctional coupling in the lens is increased by knocking in the gap junction protein Cx46 into the Cx50 locus. Relative to wild-type mice, Cx50KI46 mice showed significantly reduced lens size and radius of curvature, increased T1 and T2 values, and decreased n in the lens nucleus, which was consistent with the developmental and functional changes characterised previously in this lens model. These proof of principle experiments show that in vivo MRI can be applied to transgenic mouse models to gain mechanistic insights into the relationship between lens physiology and optics, and in the future suggest that longitudinal studies can be performed to determine how this relationship is altered by age in mouse models of cataract.

The Ciliary Muscle and Zonules of Zinn Modulate Lens Intracellular Hydrostatic Pressure Through Transient Receptor Potential Vanilloid Channels.

Purpose: Lenses have an intracellular hydrostatic pressure gradient to drive fluid from central fiber cells to surface epithelial cells. Pressure is regulated by a feedback control system that relies on transient receptor potential vanilloid (TRPV)1 and TRPV4 channels. The ciliary muscle transmits tension to the lens through the zonules of Zinn. Here, we have examined if ciliary muscle tension influenced the lens intracellular hydrostatic pressure gradient. Methods: We measured the ciliary body position and intracellular hydrostatic pressures in mouse lenses while pharmacologically causing relaxation or contraction of the ciliary muscle. We also used inhibitors of TRPV1 and TRPV4, in addition to phosphoinositide 3-kinase (PI3K) p110α knockout mice and immunostaining of phosphorylated protein kinase B (Akt), to determine how changes in ciliary muscle tension resulted in altered hydrostatic pressure. Results: Ciliary muscle relaxation increased the distance between the ciliary body and the lens and caused a decrease in intracellular hydrostatic pressure that was dependent on intact zonules and could be blocked by inhibition of TRPV4. Ciliary contraction moved the ciliary body toward the lens and caused an increase in intracellular hydrostatic pressure and Akt phosphorylation that required intact zonules and was blocked by either inhibition of TRPV1 or genetic deletion of the p110α catalytic subunit of PI3K. Conclusions: These results show that the hydrostatic pressure gradient within the lens was influenced by the tension exerted on the lens by the ciliary muscle through the zonules of Zinn. Modulation of the gradient of intracellular hydrostatic pressure in the lens could alter the water content, and the gradient of refractive index.

Connexin43 mutations linked to skin disease have augmented hemichannel activity.

Mutations in the gene (GJA1) encoding connexin43 (Cx43) are responsible for several rare genetic disorders, including non-syndromic skin-limited diseases. Here we used two different functional expression systems to characterize three Cx43 mutations linked to palmoplantar keratoderma and congenital alopecia-1, erythrokeratodermia variabilis et progressiva, or inflammatory linear verrucous epidermal nevus. In HeLa cells and Xenopus oocytes, we show that Cx43-G8V, Cx43-A44V and Cx43-E227D all formed functional gap junction channels with the same efficiency as wild-type Cx43, with normal voltage gating and a unitary conductance of ~110 pS. In HeLa cells, all three mutations also localized to regions of cell-cell contact and displayed a punctate staining pattern. In addition, we show that Cx43-G8V, Cx43-A44V and Cx43-E227D significantly increase membrane current flow through formation of active hemichannels, a novel activity that was not displayed by wild-type Cx43. The increased membrane current was inhibited by either 2 mM calcium, or 5 µM gadolinium, mediated by hemichannels with a unitary conductance of ~250 pS, and was not due to elevated mutant protein expression. The three Cx43 mutations all showed the same gain of function activity, suggesting that augmented hemichannel activity could play a role in skin-limited diseases caused by human Cx43 mutations.

The Phosphoinosotide 3-Kinase Catalytic Subunit p110α is Required for Normal Lens Growth.

PURPOSE: Signal transduction pathways influence lens growth, but little is known about the role(s) of the class 1A phosphoinositide 3-kinases (PI3Ks). To further investigate how signaling regulates lens growth, we generated and characterized mice in which the p110α and p110ß catalytic subunits of PI3K were conditionally deleted in the mouse lens. METHODS: Floxed alleles of the catalytic subunits of PI3K were conditionally deleted in the lens by using MLR10-cre transgenic mice. Lenses of age-matched animals were dissected and photographed. Postnatal lenses were fixed, paraffin embedded, sectioned, and stained with hematoxylin-eosin. Cell proliferation was quantified by labeling S-phase cells in intact lenses with 5-ethynyl-2'-deoxyuridine. Protein kinase B (AKT) activation was examined by Western blotting. RESULTS: Lens-specific deletion of p110α resulted in a significant reduction of eye and lens size, without compromising lens clarity. Conditional knockout of p110ß had no effect on lens size or clarity, and deletion of both the p110α and p110ß subunits resulted in a phenotype that resembled the p110α single-knockout phenotype. Levels of activated AKT were decreased more in p110α- than in p110ß-deficient lenses. A significant reduction in proliferating cells in the germinative zone was observed on postnatal day 0 in p110α knockout mice, which was temporally correlated with decreased lens volume. CONCLUSIONS: These data suggest that the class 1A PI3K signaling pathway plays an important role in the regulation of lens size by influencing the extent and spatial location of cell proliferation in the perinatal period.

Connexin channels in congenital skin disorders.

Gap junctions and hemichannels comprised of connexins influence epidermal proliferation and differentiation. Significant advances in our understanding of the functional role of connexins in the skin have been made by studying the diseases caused by connexin mutations. Eleven clinically defined cutaneous disorders with an overlapping spectrum of phenotypes are caused by mutations in five different connexin genes, highlighting that disease presentation must be deciphered with an understanding of how connexin functions are affected. Increasing evidence suggests that the skin diseases produced by connexin mutations result from dominant gains of function. In palmoplantar keratoderma with deafness, the connexin 26 mutations transdominantly alter the function of wild-type connexin 43 and create leaky heteromeric hemichannels. In keratitis-ichthyosis-deafness syndrome, different connexin 26 mutations can either form dominant hemichannels with altered calcium regulation or increased calcium permeability, leading to clinical subtypes of this syndrome. It is only with detailed understanding of these subtle functional differences that we can hope to create successful pathophysiology driven therapies for the connexin skin disorders.

Aberrant connexin26 hemichannels underlying keratitis-ichthyosis-deafness syndrome are potently inhibited by mefloquine.

Keratitis-ichthyosis-deafness (KID) syndrome is an ectodermal dysplasia caused by dominant mutations of connexin26 (Cx26). Loss of Cx26 function causes nonsyndromic sensorineural deafness, without consequence in the epidermis. Functional analyses have revealed that a majority of KID-causing mutations confer a novel expansion of hemichannel activity, mediated by connexin channels in a nonjunctional configuration. Inappropriate Cx26 hemichannel opening is hypothesized to compromise keratinocyte integrity and epidermal homeostasis. Pharmacological modulators of Cx26 are needed to assess the pathomechanistic involvement of hemichannels in the development of hyperkeratosis in KID syndrome. We have used electrophysiological assays to evaluate small-molecule analogs of quinine for suppressive effects on aberrant hemichannel currents elicited by KID mutations. Here, we show that mefloquine (MFQ) inhibits several mutant hemichannel forms implicated in KID syndrome when expressed in Xenopus laevis oocytes (IC50∼16 µM), using an extracellular divalent cation, zinc (Zn(++)), as a nonspecific positive control for comparison (IC50∼3 µM). Furthermore, we used freshly isolated transgenic keratinocytes to show that micromolar concentrations of MFQ attenuated increased macroscopic membrane currents in primary mouse keratinocytes expressing human Cx26-G45E, a mutation that causes a lethal form of KID syndrome.

Connexin 46 (cx46) gap junctions provide a pathway for the delivery of glutathione to the lens nucleus.

Maintenance of adequate levels of glutathione (GSH) in the lens nucleus is critical for protection of lens proteins from the effects of oxidative stress and for lens transparency. How GSH is transported to the nucleus is unknown. We show that GSH diffuses to the nucleus from the outer cortex, where a high concentration of the anti-oxidant is established by synthesis or uptake, via the network of gap junctions. Using electrophysiological measurements, we found that channels formed by Cx46 and Cx50, the two connexin isoforms expressed in the lens, were moderately cation-selective (P(Na)/P(Cl) ∼5 for Cx46 and ∼3 for Cx50). Single channel permeation of the larger GSH anion was low but detectable (P(Na)/P(GSH) ∼12 for Cx46 and ∼8 for Cx50), whereas permeation of divalent anion glutathione disulfide (GSSG) was undetectable. Measurement of GSH levels in the lenses from connexin knock-out (KO) mice indicated Cx46, and not Cx50, is necessary for transport of GSH to the core. Levels of GSH in the nucleus were markedly reduced in Cx46 KO, whereas they were unaffected by Cx50 KO. We also show that GSH delivery to the nucleus is not dependent on the lens microcirculation, which is believed to be responsible for extracellular transport of other nutrients to membrane transporters in the core. These results indicate that glutathione diffuses from cortical fiber cells to the nucleus via gap junction channels formed by Cx46. We present a model of GSH diffusion from outer cells to inner fiber cells through gap junctions.

AKT activation promotes PTEN hamartoma tumor syndrome-associated cataract development.

Mutations in the human phosphatase and tensin homolog (PTEN) gene cause PTEN hamartoma tumor syndrome (PHTS), which includes cataract development among its diverse clinical pathologies. Currently, it is not known whether cataract formation in PHTS patients is secondary to other systemic problems, or the result of the loss of a critical function of PTEN within the lens. We generated a mouse line with a lens-specific deletion of Pten (PTEN KO) and identified a regulatory function for PTEN in lens ion transport. Specific loss of PTEN in the lens resulted in cataract. PTEN KO lenses exhibited a progressive age-related increase in intracellular hydrostatic pressure, along with, increased intracellular sodium concentrations, and reduced Na+/K+-ATPase activity. Collectively, these defects lead to lens swelling, opacities and ultimately organ rupture. Activation of AKT was highly elevated in PTEN KO lenses compared to WT mice. Additionally, pharmacological inhibition of AKT restored normal Na+/K+-ATPase activity in primary cultured lens cells and reduced lens pressure in intact lenses from PTEN KO animals. These findings identify a direct role for PTEN in the regulation of lens ion transport through an AKT-dependent modulation of Na+/K+-ATPase activity, and provide a new animal model to investigate cataract development in PHTS patients.

The Cx26-G45E mutation displays increased hemichannel activity in a mouse model of the lethal form of keratitis-ichthyosis-deafness syndrome.

Mutations in the GJB2 gene (Cx26) cause deafness in humans. Most are loss-of-function mutations and cause nonsyndromic deafness. Some mutations produce a gain of function and cause syndromic deafness associated with skin disorders, such as keratitis-ichthyosis-deafness syndrome (KIDS). Cx26-G45E is a lethal mutation linked to KIDS that forms constitutively active connexin hemichannels. The pathomechanism(s) by which mutant Cx26 hemichannels perturb normal epidermal cornification are poorly understood. We created an animal model for KIDS by generating an inducible transgenic mouse expressing Cx26-G45E in keratinocytes. Cx26-G45E mice displayed reduced viability, hyperkeratosis, scaling, skin folds, and hair loss. Histopathology included hyperplasia, acanthosis, papillomatosis, increased cell size, and osteal plugging. These abnormalities correlated with human KIDS pathology and were associated with increased hemichannel currents in transgenic keratinocytes. These results confirm the pathogenic nature of the G45E mutation and provide a new model for studying the role of aberrant connexin hemichannels in epidermal differentiation and inherited connexin disorders.

The cataract causing Cx50-S50P mutant inhibits Cx43 and intercellular communication in the lens epithelium.

Mutations in Connexin50 (Cx50) cause cataracts in both humans and mice. The mechanism(s) behind how mutated connexins lead to a variety of cataracts have yet to be fully elucidated. Here, we tested whether the cataract inducing Cx50-S50P mutant interacts with wild-type Connexin43 (Cx43) to form mixed channels with attenuated function. Using dual whole-cell voltage clamp, immunofluorescent microscopy and in situ dye transfer analysis we identified a unique interaction between the mutant subunit and wild-type Cx43. In paired Xenopus oocytes, co-expression of Cx50-S50P with Cx43 reduced electrical coupling >/=90%, without a reduction in protein expression. In transfected cells, Cx50-S50P did not target to cell-cell interfaces by itself, but co-expression of Cx50-S50P with Cx43 resulted in its localization at areas of cell-cell contact. We used Cx43 conditional knockout, Cx50 knockout and Cx50-S50P mutant mice to examine this interaction in vivo. Mice expressing both Cx43 and Cx50-S50P in the lens epithelium revealed a unique expression pattern for Cx43 and a reduction in Cx43 protein. In situ dye transfer experiments showed that the Cx50-S50P mutant, but not the Cx50, or Cx43 conditional knockout, greatly inhibited epithelial cell gap junctional communication in a manner similar to a double knockout of Cx43 and Cx50. The inhibitory affects of Cx50-S50P lead to diminished electrical coupling in vitro, as well as a discernable reduction in epithelial cell dye permeation. These data suggest that dominant inhibition of Cx43 mediated epithelial cell coupling may play a role in the lens pathophysiology caused by the Cx50-S50P mutation.

Interaction between Connexin50 and mitogen-activated protein kinase signaling in lens homeostasis.

Both connexins and signal transduction pathways have been independently shown to play critical roles in lens homeostasis, but little is known about potential cooperation between these two intercellular communication systems. To investigate whether growth factor signaling and gap junctional communication interact during the development of lens homeostasis, we examined the effect of mitogen-activated protein kinase (MAPK) signaling on coupling mediated by specific lens connexins by using a combination of in vitro and in vivo assays. Activation of MAPK signaling pathways significantly increased coupling provided by Cx50, but not Cx46, in paired Xenopus laevis oocytes in vitro, as well as between freshly isolated lens cells in vivo. Constitutively active MAPK signaling caused macrophthalmia, cataract, glucose accumulation, vacuole formation in differentiating fibers, and lens rupture in vivo. The specific removal or replacement of Cx50, but not Cx46, ameliorated all five pathological conditions in transgenic mice. These results indicate that MAPK signaling specifically modulates coupling mediated by Cx50 and that gap junctional communication and signal transduction pathways may interact in osmotic regulation during postnatal fiber development.

Optimal lens epithelial cell proliferation is dependent on the connexin isoform providing gap junctional coupling.

PURPOSE: Gap junctions between epithelial cells are essential for normal lens growth. In mice, knockout of Cx50 or targeted replacement of Cx50 with Cx46 (knockin) caused smaller lenses because of decreased epithelial cell proliferation. However, it remains unclear whether Cx50 functionally contributes to lens epithelial coupling during maximal proliferation on postnatal day 2 (P2) and P3. To determine which connexins functionally contribute to epithelial cell coupling and proliferation, junctional coupling from epithelial cells of wild-type and knockin mice was examined. METHODS: Epithelial cells were isolated from wild-type or knockin mice at different developmental ages. Junctional currents were measured by dual whole cell voltage clamp. Cell proliferation was assayed by BrdU incorporation. Connexins were immunolocalized using specific antibodies. RESULTS: Junctional currents between lens epithelial cells exhibited a developmentally regulated sensitivity to quinine, a drug that blocks Cx50 gap junctions, but not Cx43 or Cx46. Single-channel currents had a unitary conductance of 210 pS, typical of Cx50. Immunocytochemical staining showed Cx43 and Cx50 were abundantly expressed in wild-type cells, and Cx46 replaced Cx50 in knockin cells. A correlation between functional activity of Cx50 and maximal proliferation was also found. In epithelial cells from P3 wild-type mice, there was a high density of BrdU-labeled nuclei in both the central epithelium and the equatorial epithelium, and 60% or more of total coupling was provided by Cx50. In older cells, proliferation was greatly reduced, and the contribution of Cx50 to total coupling was progressively reduced (45% or less on P12; 25% or less on P28). Coupling between epithelial cells of Cx46 knockin mice was similar in magnitude to that of wild-type mice but had pharmacologic and biophysical characteristics of Cx46. This functional replacement of Cx50 with Cx46 was correlated with 71% and 13% reductions in BrdU-labeled cells in the P3 central epithelium and equatorial epithelium, respectively. CONCLUSIONS: These results reconcile previous genetic studies showing that Cx50 influences epithelial cell proliferation, with numerous studies suggesting that Cx43 was the principal epithelial cell connexin. They further show that the contribution of Cx50 is highest during peak postnatal proliferation but progressively declines with age thereafter.