Transforming growth factor (TGF)-β-induced epithelial-mesenchymal transition (EMT) leads to the formation of ocular fibrotic pathologies, such as anterior subcapsular cataract and posterior capsule opacification. Remodeling of the actin cytoskeleton, mediated by the Rho family of GTPases, plays a key role in EMT. However, how actin dynamics affect downstream markers of EMT has not been fully determined. Our previous work suggests that myocardin-related transcription factor A (MRTF-A), an actin-binding protein, might be an important mediator of TGFβ-induced EMT in lens epithelial cells. The aim of the current study was to determine the requirement of RhoA/ROCK signaling in mediating TGFβ-induced nuclear accumulation of MRTF-A and ultimate expression of α-smooth muscle actin (αSMA), a marker of a contractile myofibroblast phenotype. Using rat lens epithelial explants, we demonstrate that ROCK inhibition using Y-27632 prevents TGFβ-induced nuclear accumulation of MRTF-A, E-cadherin/β-catenin complex disassembly, and αSMA expression. Using a novel inhibitor specifically targeting MRTF-A signaling, CCG-203971, we further demonstrate the requirement of MRTF-A nuclear localization and activity in the induction of αSMA expression. Overall, our findings suggest that TGFβ-induced cytoskeletal reorganization through RhoA/ROCK/MRTF-A signaling is critical to EMT of lens epithelial cells.

Purpose. Smad3, a mediator of TGF-β 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-β/Smad3 knockout mouse model. methods. TGF-β1 transgenic mice (containing a human TGF-β1 cDNA construct expressed under the αA-crystallin promoter) were bred with mice on a Smad3-null background to generate mice with the following genotypes: TGF-β1/Smad3^−/− (null), TGF-β1/Smad3^+/−, TGF-β1/Smad3^+/+, and nontransgenic/Smad3^+/+. Lenses from mice of each genotype were dissected and prepared for histologic or optical analyses. results. All transgenic TGF-β1 lenses demonstrated subcapsular plaque formation and EMT as indicated by the expression of α-smooth muscle actin. However, the sizes of the plaques were reduced in the TGF-β1/Smad3^−/− lenses, as was the level of type IV collagen deposition when compared with TGF-β1/Smad3^+/− and TGF-β1/Smad3^+/+ lenses. An increased number of apoptotic figures was also observed in the plaques of the TGF-β1/Smad3^−/− lenses compared with TGF-β1/Smad3^+/+ littermates. conclusions. Lens-specific expression of TGF-β1 induced ASC formation in the absence of the Smad3 signaling mediator, suggests that alternative TGF-β-signaling pathways participate in this ocular fibrotic model.

Activating protein 2α (AP-2α) is known to be expressed in the retina, and AP-2α-null mice exhibit defects in the developing optic cup, including patterning of the neural retina (NR) and a replacement of the dorsal retinal pigmented epithelium (RPE) with NR. In this study, we analyzed the temporal and spatial retinal expression patterns of AP-2α and created a conditional deletion of AP-2α in the developing retina. AP-2α exhibited a distinct expression pattern in the developing inner nuclear layer of the retina, and colocalization studies indicated that AP-2α was exclusively expressed in postmitotic amacrine cell populations. Targeted deletion of AP-2α in the developing retina did not result in observable retinal defects. Further examination of AP-2α-null mutants revealed that the severity of the RPE defect was variable and, although defects in retinal lamination occur at later embryonic stages, earlier stages showed normal lamination and expression of markers for amacrine and ganglion cells. Together, these data demonstrate that, whereas AP-2α alone does not play an intrinsic role in retinogenesis, it has non-cell-autonomous effects on optic cup development. Additional expression analyses showed that multiple AP-2 proteins are present in the developing retina, which will be important to future studies.

Appropriate development of the retina and optic nerve requires that the forebrain-derived optic neuroepithelium undergoes a precisely coordinated sequence of patterning and morphogenetic events, processes which are highly influenced by signals from adjacent tissues. Our previous work has suggested that transcription factor activating protein-2 alpha (AP-2α; Tcfap2a) has a non-cell autonomous role in optic cup (OC) development; however, it remained unclear how OC abnormalities in AP-2α knockout (KO) mice arise at the morphological and molecular level. In this study, we show that patterning and morphogenetic defects in the AP-2α KO optic neuroepithelium begin at the optic vesicle stage. During subsequent OC formation, ectopic neural retina and optic stalk-like tissue replaced regions of retinal pigment epithelium. AP-2α KO eyes also displayed coloboma in the ventral retina, and a rare phenotype in which the optic stalk completely failed to extend, causing the OCs to be drawn inward to the midline. We detected evidence of increased sonic hedgehog signaling in the AP-2α KO forebrain neuroepithelium, which likely contributed to multiple aspects of the ocular phenotype, including expansion of PAX2-positive optic stalk-like tissue into the OC. Our data suggest that loss of AP-2α in multiple tissues in the craniofacial region leads to severe OC and optic stalk abnormalities by disturbing the tissue–tissue interactions required for ocular development. In view of recent data showing that mutations in human TFAP2A result in similar eye defects, the current findings demonstrate that AP-2α KO mice provide a valuable model for human ocular disease.