Deletion of transcription factor AP-2ß from the developing murine trabecular meshwork region leads to progressive glaucomatous changes.

Glaucoma is one of the leading causes of irreversible blindness and can result from abnormalities in anterior segment structures required for aqueous humor outflow, including the trabecular meshwork (TM) and Schlemm's canal (SC). Transcription factors such as AP-2ß play critical roles in anterior segment development. Here, we show that the Mgp-Cre knock-in (Mgp-Cre.KI) mouse can be used to target the embryonic periocular mesenchyme giving rise to the TM and SC. Fate mapping of male and female mice indicates that AP-2ß loss causes a decrease in iridocorneal angle cells derived from Mgp-Cre.KI-expressing populations compared to controls. Moreover, histological analyses revealed peripheral iridocorneal adhesions in AP-2ß mutants that were accompanied by a decrease in expression of TM and SC markers, as observed using immunohistochemistry. In addition, rebound tonometry showed significantly higher intraocular pressure (IOP) that was correlated with a progressive significant loss of retinal ganglion cells, reduced retinal thickness, and reduced retinal function, as measured using an electroretinogram, in AP-2ß mutants compared with controls, reflecting pathology described in late-stage glaucoma patients. Importantly, elevated IOP in AP-2ß mutants was significantly reduced by treatment with latanoprost, a prostaglandin analog that increases unconventional outflow. These findings demonstrate that AP-2ß is critical for TM and SC development, and that these mutant mice can serve as a model for understanding and treating progressive human primary angle-closure glaucoma.

Conditional Deletion of AP-2ß in the Periocular Mesenchyme of Mice Alters Corneal Epithelial Cell Fate and Stratification.

The cornea is an anterior eye structure specialized for vision. The corneal endothelium and stroma are derived from the periocular mesenchyme (POM), which originates from neural crest cells (NCCs), while the stratified corneal epithelium develops from the surface ectoderm. Activating protein-2ß (AP-2ß) is highly expressed in the POM and important for anterior segment development. Using a mouse model in which AP-2ß is conditionally deleted in the NCCs (AP-2ß NCC KO), we investigated resulting corneal epithelial abnormalities. Through PAS and IHC staining, we observed structural and phenotypic changes to the epithelium associated with AP-2ß deletion. In addition to failure of the mutant epithelium to stratify, we also observed that Keratin-12, a marker of the differentiated epithelium, was absent, and Keratin-15, a limbal and conjunctival marker, was expanded across the central epithelium. Transcription factors PAX6 and P63 were not observed to be differentially expressed between WT and mutant. However, growth factor BMP4 was suppressed in the mutant epithelium. Given the non-NCC origin of the epithelium, we hypothesize that the abnormalities in the AP-2ß NCC KO mouse result from changes to regulatory signaling from the POM-derived stroma. Our findings suggest that stromal pathways such as Wnt/ß-Catenin signaling may regulate BMP4 expression, which influences cell fate and stratification.

Progressive Loss of Retinal Ganglion Cells in Activating Protein-2ß Neural Crest Cell Knockout Mice.

Purpose: Our lab has shown that conditionally disrupting the transcription factor activating protein 2ß (Tfap2b) gene, responsible for the activating protein-2ß (AP-2ß) transcription factor, exclusively in cranial neural crest cells (AP-2ß NCC KO), leads to anterior segment dysgenesis and a closed angle phenotype. The purpose of the current study is to determine if there is a progressive loss of retinal ganglion cells (RGCs) in the mutant over time and whether this loss was associated with macroglial activity changes and elevated intraocular pressure (IOP).Methods: Using the Cre-loxP system, we generated a conditional knockout of Tfap2b exclusively in cranial NCC (AP-2ß NCC KO). Immunohistochemistry was performed using anti-Brn3a, anti-GFAP and anti-Vimentin antibodies. IOP was measured using a tonometer and the data was analyzed using GraphPad Prism software. Brn3a and DAPI positive cells were counted using Image-J and statistical analysis was performed with GraphPad Prism software.Results: Our findings revealed that while no statistical difference in Brn3a expression was observed between wild-type and mutant mice at postnatal day (P) 4 or P10, at P40 (p < .01) and P42 (p < .0001) Brn3a expression was significantly reduced in the mutant retina at the region of the ONH. There was also increased expression of glial fibrillary acidic protein (GFAP) by Müller cells in the AP-2ß NCC KO mice at P35 and P40, indicating the presence of neuroinflammation. Moreover, increased IOP was observed starting at P35 and continuing at P40 and P42 (p < .0001 for all three ages examined).Conclusions: Together, these findings suggest that the retinal damage observed in the KO mouse becomes apparent by P40 after increased IOP was observed at P35 and progressed over time. The AP-2ß NCC KO mouse may therefore be a novel experimental model for glaucoma.

AP-2ß is required for formation of the murine trabecular meshwork and Schlemm's canal.

Previously, we have shown that Tfap2b, the gene encoding transcription factor AP-2ß, is needed for normal mouse eye development. Specifically, targeted loss of Tfap2b in neural crest cells (NCCs)1 and their derivatives, particularly the periocular mesenchyme (POM), resulted in anterior segment defects affecting the cornea and angle tissue. These defects were further associated with an increase in intraocular pressure (IOP). The present study investigates the underlying changes in embryonic and postnatal POM cell development and differentiation caused by loss of AP-2ß in the NCCs, particularly in the structures that control aqueous outflow, using Wnt1Cre+/-; Tfap2b-/lox; tdTomatolox/+ mice (AP-2ß neural crest cell knockout or AP-2ß NCC KO). Toluidine blue-stained sections and ultrathin sections stained with uranyl acetate and lead citrate were used to assess morphology and ultrastructure, respectively. Immunohistochemistry of KO and control eyes was performed at embryonic day (E) 15.5, E18.5, postnatal day (P) 1, P7 and P14 using phospho-histone H3 (PH3), α-smooth muscle actin (α-SMA), myocilin and endomucin antibodies, as well as a TUNEL assay. Conditional deletion of AP-2ß in the NCC-derived POM resulted in defects that appeared during both embryogenesis and postnatal stages. Fate mapping of the knockout cells in the mutants revealed that the POM migrated appropriately into the eye during embryogenesis. However, during postnatal stages a significant reduction in POM proliferation in the angle region was observed in the mutants compared to controls. This was accompanied by a lack of expression of appropriate trabecular meshwork and Schlemm's canal markers. This is the first study to show that AP-2ß is required for development and differentiation of the trabecular meshwork and Schlemm's canal. Together, these defects likely contributed to the elevated intraocular pressure (IOP) previously reported in the AP-2ß NCC KO mice.

Conditional Deletion of AP-2α and AP-2ß in the Developing Murine Retina Leads to Altered Amacrine Cell Mosaics and Disrupted Visual Function.

Purpose: The combined action of the activating protein-2 (AP-2) transcription factors, AP-2α and AP-2ß, is important in early retinal development, specifically in the formation of horizontal cells. However, in previous studies, it was not possible to analyze postnatal development and function of additional retinal subtypes. Methods: We used a double conditional deletion of AP-2α and AP-2ß from the retina to further examine the combinatory role of these genes in retinal cell patterning and function in postnatal adult mice as measured by Voronoi domain area and nearest-neighbor distance spatial analyses and ERGs, respectively. Results: Conditional deletion of both AP-2α and AP-2ß from the retina resulted in a variety of abnormalities, including the absence of horizontal cells, defects in the photoreceptor ribbons in which synapses failed to form, along with evidence of aberrant amacrine cell arrangement. Although no significant changes in amacrine cell population numbers were observed in the double mutants, significant irregularities in the mosaic patterning of amacrine cells was observed as demonstrated by both Voronoi domain areas and nearest-neighbor distances analyses. These changes were further accompanied by an alteration in the retinal response to light as recorded by ERGs. In particular, in the double-mutant mice lacking AP-2α and AP-2ß, the b-wave amplitude, representative of interneuron signal processing, was significantly reduced compared with control littermates. Conclusions: Together these findings demonstrate the requirement for both AP-2α and AP-2ß in proper amacrine mosaic patterning and a normal functional light response in the retina.

ß-Catenin/CBP-Dependent Signaling Regulates TGF-ß-Induced Epithelial to Mesenchymal Transition of Lens Epithelial Cells.

PURPOSE: Transforming growth factor-ß-induced epithelial-mesenchymal transition (EMT) is one of the main causes of posterior capsular opacification (PCO) or secondary cataract; however, the signaling events involved in TGF-ß-induced PCO have not been fully characterized. Here, we focus on examining the role of ß-catenin/cyclic AMP response element-binding protein (CREB)-binding protein (CBP) and ß-catenin/T-cell factor (TCF)-dependent signaling in regulating cytoskeletal dynamics during TGF-ß-induced EMT in lens epithelial explants. METHODS: Rat lens epithelial explants were cultured in medium M199 in the absence of serum. Explants were treated with TGF-ß2 in the presence or absence of the ß-catenin/CBP interaction inhibitor, ICG-001, or the ß-catenin/TCF interaction inhibitor, PNU-74654. Western blot and immunofluorescence experiments were carried out and analyzed. RESULTS: An increase in the expression of fascin, an actin-bundling protein, was observed in the lens explants upon stimulation with TGF-ß, and colocalized with F-actin filaments. Inhibition of ß-catenin/CBP interactions, but not ß-catenin/TCF interactions, led to a decrease in TGF-ß-induced fascin and stress fiber formation, as well as a decrease in the expression of known markers of EMT, α-smooth muscle actin (α-SMA) and matrix metalloproteinase 9 (MMP9). In addition, inhibition of ß-catenin/CBP-dependent signaling also prevented TGF-ß-induced downregulation of epithelial cadherin (E-cadherin) in lens explants. CONCLUSIONS: We show that ß-catenin/CBP-dependent signaling regulates fascin, MMP9, and α-SMA expression during TGF-ß-induced EMT. We demonstrate that ß-catenin/CBP-dependent signaling is crucial for TGF-ß-induced EMT in the lens.

Conditional deletion of AP-2ß in mouse cranial neural crest results in anterior segment dysgenesis and early-onset glaucoma.

Anterior segment dysgenesis (ASD) encompasses a group of developmental disorders in which a closed angle phenotype in the anterior chamber of the eye can occur and 50% of patients develop glaucoma. Many ASDs are thought to involve an inappropriate patterning and migration of the periocular mesenchyme (POM), which is derived from cranial neural crest cells (NCCs) and mesoderm. Although, the mechanism of this disruption is not well understood, a number of transcriptional regulatory molecules have previously been implicated in ASDs. Here, we investigate the function of the transcription factor AP-2ß, encoded by Tfap2b, which is expressed in NCCs and their derivatives. Wnt1-Cre-mediated conditional deletion of Tfap2b in NCCs resulted in post-natal ocular defects typified by opacity. Histological data revealed that the conditional AP-2ß NCC knockout (KO) mutants exhibited dysgenesis of multiple structures in the anterior segment of the eye including defects in the corneal endothelium, corneal stroma, ciliary body and disruption in the iridocorneal angle with adherence of the iris to the cornea. We further show that this phenotype leads to a significant increase in intraocular pressure and a subsequent loss of retinal ganglion cells and optic nerve degeneration, features indicative of glaucoma. Overall, our findings demonstrate that AP-2ß is required in the POM for normal development of the anterior segment of the eye and that the AP-2ß NCC KO mice might serve as a new and exciting model of ASD and glaucoma that is fully penetrant and with early post-natal onset.

Matrix metalloproteinase-9-null mice are resistant to TGF-ß-induced anterior subcapsular cataract formation.

Epithelial-mesenchymal transition (EMT) is associated with fibrotic diseases in the lens, such as anterior subcapsular cataract (ASC) formation. Often mediated by transforming growth factor (TGF)-ß, EMT in the lens involves the transformation of lens epithelial cells into a multilayering of myofibroblasts, which manifest as plaques beneath the lens capsule. TGF-ß-induced EMT and ASC have been associated with the up-regulation of two matrix metalloproteinases (MMPs): MMP-2 and MMP-9. The current study used MMP-2 and MMP-9 knockout (KO) mice to further determine their unique roles in TGF-ß-induced ASC formation. Adenoviral injection of active TGF-ß1 into the anterior chamber of all wild-type and MMP-2 KO mice led to the formation of distinct ASC plaques that were positive for α-smooth muscle actin, a marker of EMT. In contrast, only a small proportion of the MMP-9 KO eyes injected with adenovirus-expressing TGF-ß1 exhibited ASC plaques. Isolated lens epithelial explants from wild-type and MMP-2 KO mice that were treated with TGF-ß exhibited features indicative of EMT, whereas those from MMP-9 KO mice did not acquire a mesenchymal phenotype. MMP-9 KO mice were further bred onto a TGF-ß1 transgenic mouse line that exhibits severe ASC formation, but shows a resistance to ASC formation in the absence of MMP-9. These findings suggest that MMP-9 expression is more critical than MMP-2 in mediating TGF-ß-induced ASC formation.

Overlapping expression patterns and redundant roles for AP-2 transcription factors in the developing mammalian retina.

BACKGROUND: We have previously shown that the transcription factor AP-2α (Tcfap2a) is expressed in postmitotic developing amacrine cells in the mouse retina. Although retina-specific deletion of Tcfap2a did not affect retinogenesis, two other family members, AP-2ß and AP-2γ, showed expression patterns similar to AP-2α. RESULTS: Here we show that, in addition to their highly overlapping expression patterns in amacrine cells, AP-2α and AP-2ß are also co-expressed in developing horizontal cells. AP-2γ expression is restricted to amacrine cells, in a subset that is partially distinct from the AP-2α/ß-immunopositive population. To address possible redundant roles for AP-2α and AP-2ß during retinogenesis, Tcfap2a/b-deficient retinas were examined. These double mutants showed a striking loss of horizontal cells and an altered staining pattern in amacrine cells that were not detected upon deletion of either family member alone. CONCLUSIONS: These studies have uncovered critical roles for AP-2 activity in retinogenesis, delineating the overlapping expression patterns of Tcfap2a, Tcfap2b, and Tcfap2c in the neural retina, and revealing a redundant requirement for Tcfap2a and Tcfap2b in horizontal and amacrine cell development.

Cell autonomous roles for AP-2alpha in lens vesicle separation and maintenance of the lens epithelial cell phenotype.

In this study, we have created a conditional deletion of AP-2alpha in the developing mouse lens (Le-AP-2alpha mutants) to determine the cell-autonomous requirement(s) for AP-2alpha in lens development. Embryonic and adult Le-AP-2alpha mutants exhibited defects confined to lens placode derivatives, including a persistent adhesion of the lens to the overlying corneal epithelium (or lens stalk). Expression of known regulators of lens vesicle separation, including Pax6, Pitx3, and Foxe3 was observed in the Le-AP-2alpha mutant lens demonstrating that these genes do not lie directly downstream of AP-2alpha. Unlike germ-line mutants, Le-AP-2alpha mutants did not exhibit defects in the optic cup, further defining the tissue specific role(s) for AP-2alpha in eye development. Finally, comparative microarray analysis of lenses from the Le-AP-2alpha mutants vs. wild-type littermates revealed differential expression of 415 mRNAs, including reduced expression of genes important for maintaining the lens epithelial cell phenotype, such as E-cadherin.

The role of Hsp70 and Hsp90 in TGF-beta-induced epithelial-to-mesenchymal transition in rat lens epithelial explants.

PURPOSE: This study investigates the effects of heat shock treatment and the role of Hsp70 and Hsp90 on tranforming growth factor beta 2 (TGF-beta2)-induced epithelial-to-mesenchymal transition (EMT) in rat lens epithelial explants. METHODS: Rat lens epithelial explants from 7 to 10 day-old Wistar rats were dissected and incubated for 24 h before treatment. The explants were divided into eight treatment groups: control (culture medium), fibroblast growth factor-2 (FGF-2), TGF-beta2, and TGF-beta2+FGF-2 under normal culture conditions and heat shocked conditions. The explants were heat shocked at 45 degrees C before treatment with the respective media. H&E staining was performed on whole-mount epithelial explants from each group. Immunofluorescence staining for alpha-smooth muscle actin (alpha-SMA), F-actin, and E-cadherin was also used to determine EMT and fibrotic plaque formation in the lens epithelial explants. Apoptotic cell death was determined using the TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) assay. Confocal microscopy was used to visualize immunoreactivity in the whole-mount epithelial explants. Western blot analysis of alpha-SMA, E-cadherin, Hsp70, and Hsp90 were also performed. RESULTS: TGF-beta2-induced EMT and plaque formation in the lens epithelial explants. The simultaneous treatment of epithelial explants with TGF-beta2+FGF-2-induced the most significant morphological changes and EMT. Heat shock treatment of lens epithelial explants before TGF-beta2 treatment did not inhibit plaque formation, but there was significant reduction of alpha-SMA expression and greater E-cadherin expression when compared to the non-heat shocked TGF-beta2-treated explants. Interestingly, TGF-beta-induced apoptotic cell death was significantly lower in the heat shocked explants compared to the non-heat shock lens explants. Heat-induced accumulation of Hsp70 and Hsp90 expression was reduced in the heat shocked groups at day 4 of treatment. CONCLUSIONS: TGF-beta2-induced EMT was significantly reduced in the heat shocked TGF-beta2 lens epithelial explants. After four days of culture, there is a reduction in expression of Hsp70 and Hsp90 in the heat-shocked groups, indicating that the lens epithelial cells are under a less stressful condition than the non-heat shocked groups. In conclusion, molecular chaperones can play a protective role against TGF-beta2-induced EMT and enhance cell survival.

Lens-specific expression of TGF-beta induces anterior subcapsular cataract formation in the absence of Smad3.

PURPOSE: Smad3, a mediator of TGF-beta signaling has been shown to be involved in the epithelial-to-mesenchymal transformation (EMT) of lens epithelial cells in a lens injury model. In this study, the role of Smad3 in anterior subcapsular cataract (ASC) formation was investigated in a transgenic TGF-beta/Smad3 knockout mouse model. METHODS: TGF-beta1 transgenic mice (containing a human TGF-beta1 cDNA construct expressed under the alphaA-crystallin promoter) were bred with mice on a Smad3-null background to generate mice with the following genotypes: TGF-beta1/Smad3(-/-) (null), TGF-beta1/Smad3(+/-), TGF-beta1/Smad3(+/+), and nontransgenic/Smad3(+/+). Lenses from mice of each genotype were dissected and prepared for histologic or optical analyses. RESULTS: All transgenic TGF-beta1 lenses demonstrated subcapsular plaque formation and EMT as indicated by the expression of alpha-smooth muscle actin. However, the sizes of the plaques were reduced in the TGF-beta1/Smad3(-/-) lenses, as was the level of type IV collagen deposition when compared with TGF-beta1/Smad3(+/-) and TGF-beta1/Smad3(+/+) lenses. An increased number of apoptotic figures was also observed in the plaques of the TGF-beta1/Smad3(-/-) lenses compared with TGF-beta1/Smad3(+/+) littermates. CONCLUSIONS: Lens-specific expression of TGF-beta1 induced ASC formation in the absence of the Smad3 signaling mediator, suggests that alternative TGF-beta-signaling pathways participate in this ocular fibrotic model.

Neutralization of interleukin-11 activity decreases osteoclast formation and increases cancellous bone volume in ovariectomized mice.

The issue of whether interleukin-11 (IL-11) contributes to bone loss during states of estrogen deficiency has not been previously determined. We therefore randomized ovariectomized (OVX) mice to once daily interperitoneal injections of either sheep anti-murine IL-11 Ab or normal sheep IgG (NSIgG) for 21 days, and then determined the effects on bone using bone histomorphometry. Here we report that treatment of OVX mice with anti-IL-11 Ab significantly increases both trabecular width and cancellous bone volume. Osteoblast activity, as measured by the percentage of trabecular surface covered by osteoid and rates of bone formation, were also significantly increased following treatment with anti-IL-11 Ab. In contrast, treatment of OVX mice with anti-IL-11 Ab significantly decreased both osteoclast number and activity. Ex-vivo assays of osteoclast formation and activity confirmed the histomorphometric data. Thus, bone marrow cells isolated from anti-IL-11 Ab treated OVX mice formed fewer osteoclasts and resorbed less bone in culture than did marrow cells isolated from either untreated or NSIgG-treated OVX mice. Based on these results we conclude that IL-11 contributes to the bone loss which is observed during states of estrogen deficiency.

Heparin acts synergistically with interleukin-11 to induce STAT3 activation and in vitro osteoclast formation.

We have previously demonstrated that long-term heparin treatment causes cancellous bone loss in rats due in part to an increase in the number of osteoclasts lining the trabecular bone surface. In the present study, we investigated this phenomenon by examining the ability of heparin to synergistically enhance interleukin-11 (IL-11)-induced osteoclast formation. Treatment of murine calvaria and bone marrow cells with IL-11 was found to induce the formation of tartrate-resistant acid phosphatase-positive (TRAP(+)) multinucleated cells (MNCs) in a dose-dependent fashion. No effect was seen when cocultures were treated with heparin alone. However, when cocultures were treated with both IL-11 and heparin, IL-11's ability to induce TRAP(+) MNC formation was enhanced 6-fold. In an attempt to resolve the mechanism responsible for this effect, we examined the ability of heparin to influence IL-11 signaling using murine calvaria cells. Heparin was found to enhance both IL-11-induced STAT3-DNA complex formation and transactivation without altering either STAT3 (signal transducer and activator of transcription-3) tyrosine or serine phosphorylation. Heparin was also found to enhance IL-11's ability to induce the expression of both receptor activator of nuclear factor-kappaB ligand (RANKL) and glycoprotein (gp) 130. When taken together, these findings suggest a plausible mechanism by which heparin may cause increased osteoclastogenesis and therefore bone loss when administered long-term.