Understanding the Role of Yes-Associated Protein (YAP) Signaling in the Transformation of Lens Epithelial Cells (EMT) and Fibrosis.

Fibrotic cataracts, posterior capsular opacification (PCO), and anterior subcapsular cataracts (ASC) are mainly attributed to the transforming growth factor-ß (TGFß)-induced epithelial-to-mesenchymal transition (EMT) of lens epithelial cells (LECs). Previous investigations from our laboratory have shown the novel role of non-canonical TGFß signaling in the progression of EMT in LECs. In this study, we have identified YAP as a critical signaling molecule involved in lens fibrosis. The observed increase in nuclear YAP in capsules of human ASC patients points toward the involvement of YAP in lens fibrosis. In addition, the immunohistochemical (IHC) analyses on ocular sections from mice that overexpress TGFß in the lens (TGFßtg) showed a co-expression of YAP and α-SMA in the fibrotic plaques when compared to wild-type littermate lenses, which do not. The incubation of rat lens explants with verteporfin, a YAP inhibitor, prevented a TGFß-induced fiber-like phenotype, α-SMA, and fibronectin expression, as well as delocalization of E-cadherin and ß-catenin. Finally, LECs co-incubated with TGFß and YAP inhibitor did not exhibit an induction in matrix metalloproteinase 2 compared to those LECs treated with TGFß alone. In conclusion, these data demonstrate that YAP is required for TGFß-mediated lens EMT and fibrosis.

ß-Catenin/Smad3 Interaction Regulates Transforming Growth Factor-ß-Induced Epithelial to Mesenchymal Transition in the Lens.

Cataracts are the leading cause of blindness worldwide. Although surgery is a successful method to restore vision loss due to cataracts, post-surgical complications can occur, such as secondary cataracts, also known as posterior capsular opacification (PCO). PCO arises when lens epithelial cells (LEC) are left behind in the capsular bag following surgery and are induced to undergo epithelial to mesenchymal transition (EMT). Following EMT, LEC morphology and phenotype are altered leading to a loss of transparency and vision. Transforming growth factor (TGF)-ß-induced signaling through both canonical, TGF-ß/Smad, and non-canonical, ß-catenin/Wnt and Rho/ROCK/MRTF-A, pathways have been shown to be involved in lens EMT, and thus PCO. However, the interactions between these signaling pathways in the lens have not been thoroughly explored. In the current study we use rat LEC explants as an ex vivo model, to examine the interplay between three TGF-ß-mediated pathways using α-smooth muscle actin (α-SMA) as a molecular marker for EMT. We show that Smad3 inhibition via SIS3 prevents nuclear translocation of ß-catenin and MRTF-A, and α-SMA expression, suggesting a key role of Smad3 in regulation of MRTF-A and ß-catenin nuclear transport in LECs. Further, we demonstrate that inhibition of ß-catenin/CBP interaction by ICG-001 decreased the amount of phosphorylated Smad3 upon TGF-ß stimulation in addition to significantly decreasing the expression levels of TGF-ß receptors, TBRII and TBRI. Overall, our findings demonstrate interdependence between the canonical and non-canonical TGF-ß-mediated signaling pathways controlling EMT in the lens.

Dual function of TGFß in lens epithelial cell fate: implications for secondary cataract.

The most common vision-disrupting complication of cataract surgery is posterior capsule opacification (PCO; secondary cataract). PCO is caused by residual lens cells undergoing one of two very different cell fates: either transdifferentiating into myofibroblasts or maturing into lens fiber cells. Although TGFß has been strongly implicated in lens cell fibrosis, the factors responsible for the latter process have not been identified. We show here for the first time that TGFß can induce purified primary lens epithelial cells within the same culture to undergo differentiation into either lens fiber cells or myofibroblasts. Marker analysis confirmed that the two cell phenotypes were mutually exclusive. Blocking the p38 kinase pathway, either with direct inhibitors of the p38 MAP kinase or a small-molecule therapeutic that also inhibits the activation of p38, prevented TGFß from inducing epithelial-myofibroblast transition and cell migration but did not prevent fiber cell differentiation. Rapamycin had the converse effect, linking MTOR signaling to induction of fiber cell differentiation by TGFß. In addition to providing novel potential therapeutic strategies for PCO, our findings extend the so-called TGFß paradox, in which TGFß can induce two disparate cell fates, to a new epithelial disease state.

Matrix metalloproteinase 9 is associated with peritoneal membrane solute transport and induces angiogenesis through ß-catenin signaling.

Background: For patients using peritoneal dialysis (PD), the peritoneal membrane can develop fibrosis and angiogenesis, leading to ultrafiltration failure, chronic hypervolemia and increased risk of technique failure and mortality. Matrix metalloproteinases (MMPs), and specifically the gelatinases (MMP2 and MMP9), may be involved in peritoneal membrane injury. Methods: From stable PD patients, mesothelial cells were assayed for MMP gene expression. MMP9 was overexpressed in mouse peritoneum by adenovirus, and MMP9 -/- mice were subjected to transforming growth factor ß (TGF-ß)-induced peritoneal fibrosis. Results: MMP9 mRNA expression correlated with peritoneal membrane solute transport properties. Overexpression of MMP9 in the mouse peritoneum induced submesothelial thickening and angiogenesis. MMP9 induced mesothelial cell transition to a myofibroblast phenotype measured by increased alpha smooth muscle actin and decreased E-cadherin expression. Angiogenesis was markedly reduced in MMP9 -/- mice treated with an adenovirus expressing active TGF-ß compared with wild-type mice. TGF-ß-mediated E-cadherin cleavage was MMP9 dependent, and E-cadherin cleavage led to ß-catenin-mediated signaling. A ß-catenin inhibitor blocked the angiogenic response induced by AdMMP9. Conclusions: Our data suggest that MMP9 is involved in peritoneal membrane injury possibly through cleavage of E-cadherin and induction of ß-catenin signaling. MMP9 is a potential biomarker for peritoneal membrane injury and is a therapeutic target to protect the peritoneal membrane in PD patients.

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.

AP-2ß Is a Downstream Effector of PITX2 Required to Specify Endothelium and Establish Angiogenic Privilege During Corneal Development.

PURPOSE: The homeodomain transcription factor, PITX2, is at the apex of a genetic pathway required for corneal development, but the critical effector genes regulated by the PITX2 remain unknown. The purpose of this study was to discover and validate PITX2-dependent mechanisms required for specifying cell lineages and establishing angiogenic privilege within the developing cornea. METHODS: Microarrays were used to compare gene expression in corneas isolated from temporal Pitx2 knockout embryos and control littermates. Quantitative RT-PCR and immunohistochemistry was used to further validate Tfap2b expression differences in Pitx2 knockout versus control corneas. In situ hybridization and protein immunohistochemistry were used to assay eyes of a Tfap2b allelic series of embryos to identify differentiated cellular lineages in the cornea, blood vessel endothelium, or lymphatic vessel endothelium. RESULTS: We show that PITX2 is required for the expression of Tfap2b, encoding the AP-2ß transcription factor, in the neural crest during corneal development. Markers of differentiated corneal epithelium and stroma are expressed in the absence of AP-2ß. In contrast, markers of differentiated corneal endothelium are not expressed in the absence of AP-2ß. Endomucin+ blood vessels are present throughout the developing corneal stroma in the absence of AP-2ß, whereas LYVE1+ lymphatic vessels are not found. CONCLUSIONS: The AP-2ß transcription factor is an important effector of PITX2 function during corneal development, required for differentiation of corneal endothelium and establishment of angiogenic privilege. Unlike PITX2, AP-2ß is not required for the early expression of available lineage specific markers for the corneal epithelium and stroma during embryogenesis, nor establishment of lymphangiogenic privilege. Therefore, additional PITX2-dependent factors likely regulate these latter processes during embryonic development. These results extend our understanding of the genetic mechanisms regulating cornea development.

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.

AP-2α is required after lens vesicle formation to maintain lens integrity.

BACKGROUND: Transcription factors are critical in regulating lens development. The AP-2 family of transcription factors functions in differentiation, cell growth and apoptosis, and in lens and eye development. AP-2α, in particular, is important in early lens development, and when conditionally deleted at the placode stage defective separation of the lens vesicle from the surface ectoderm results. AP-2α's role during later stages of lens development is unknown. To address this, the MLR10-Cre transgene was used to delete AP-2α from the lens epithelium beginning at embryonic day (E) 10.5. RESULTS: The loss of AP-2α after lens vesicle separation resulted in morphological defects beginning at E18.5. By P4, a small highly vacuolated lens with a multilayered epithelium was evident in the MLR10-AP-2α mutants. Epithelial cells appeared elongated and expressed fiber cell specific ßB1 and γ-crystallins. Epithelial cell polarity and lens cell adhesion was disrupted and accompanied by the misexpression of ZO-1, N-Cadherin, and ß-catenin. Cell death was observed in the mutant lens epithelium between postnatal day (P) 14 and P30, and correlated with altered arrangements of cells within the epithelium. CONCLUSIONS: Our findings demonstrate that AP-2α continues to be required after lens vesicle separation to maintain a normal lens epithelial cell phenotype and overall lens integrity and to ensure correct fiber cell differentiation.

Nuclear translocation of myocardin-related transcription factor-A during transforming growth factor beta-induced epithelial to mesenchymal transition of lens epithelial cells.

PURPOSE: Transforming growth factor beta (TGFß) is a known inducer of epithelial to mesenchymal transition (EMT), and studies in other systems have shown that nuclear localization of the myocardin-related transcription factor (MRTF) is downstream of TGFß. In the following study, we investigated whether nuclear translocation of MRTF-A or MRTF-B is involved in TGFß-induced EMT of lens epithelial cells (LECs). We further investigated the relationship between matrix metalloproteinase-2 and -9 (MMP-2/9) and MRTF in the EMT of LECs. METHODS: Rat lens explant cultures were used as the model system. Explants were treated with TGFß, an MMP-2/9 inhibitor, or actin binding drugs and immunostained for alpha smooth muscle actin (αSMA), MRTF-A, and MRTF-B. Cytoplasmic and nuclear intensities of cells were measured using ImageJ. Production of αSMA was measured using western blot analysis and ImageJ. RESULTS: Untreated explant cells exhibited little αSMA expression, and MRTF-A and B were found to reside primarily in the cytosol. However, when stimulated with TGFß, a significantly greater number of cells exhibited nuclear expression of MRTF-A, accompanied by an increase in αSMA expression. However, MRTF-B remained in the cytoplasm following TGFß treatment. Cotreatment with an MMP-2/9 inhibitor and TGFß resulted in reduced MRTF-A nuclear localization and αSMA expression compared to cells treated with TGFß alone. CONCLUSIONS: Our results are the first to demonstrate the expression of MRTF-A in LECs and that its nuclear translocation can be stimulated by TGFß. Our data further suggest that MMP-2 and -9 are involved in the translocation of MRTF-A in LECs during TGFß-induced EMT.

Activation of the hedgehog signaling pathway in the developing lens stimulates ectopic FoxE3 expression and disruption in fiber cell differentiation.

PURPOSE: The signaling pathways and transcriptional effectors responsible for directing mammalian lens development provide key regulatory molecules that can inform our understanding of human eye defects. The hedgehog genes encode extracellular signaling proteins responsible for patterning and tissue formation during embryogenesis. Signal transduction of this pathway is mediated through activation of the transmembrane proteins smoothened and patched, stimulating downstream signaling resulting in the activation or repression of hedgehog target genes. Hedgehog signaling is implicated in eye development, and defects in hedgehog signaling components have been shown to result in defects of the retina, iris, and lens. METHODS: We assessed the consequences of constitutive hedgehog signaling in the developing mouse lens using Cre-LoxP technology to express the conditional M2 smoothened allele in the embryonic head and lens ectoderm. RESULTS: Although initial lens development appeared normal, morphological defects were apparent by E12.5 and became more significant at later stages of embryogenesis. Altered lens morphology correlated with ectopic expression of FoxE3, which encodes a critical gene required for human and mouse lens development. Later, inappropriate expression of the epithelial marker Pax6, and as well as fiber cell markers c-maf and Prox1 also occurred, indicating a failure of appropriate lens fiber cell differentiation accompanied by altered lens cell proliferation and cell death. CONCLUSIONS: Our findings demonstrate that the ectopic activation of downstream effectors of the hedgehog signaling pathway in the mouse lens disrupts normal fiber cell differentiation by a mechanism consistent with a sustained epithelial cellular developmental program driven by FoxE3.

Ocular gene transfer of active TGF-beta induces changes in anterior segment morphology and elevated IOP in rats.

Purpose. Transforming growth factor beta (TGF-beta) is known to play a crucial role in wound healing and fibrotic tissue remodeling. A large body of evidence suggests a role for this cytokine in the pathogenesis of glaucoma; however, the mechanisms by which it affects anterior segment morphology are not well understood. Therefore, the purpose of this study was to examine the effects of TGF-beta overexpression on anterior segment morphology and subsequent effects on intraocular pressure. Methods. Adenoviral gene transfer was used to deliver active TGF-beta1 to the rat eye. Measurements of intraocular pressure were taken with a tonometer on days 0, 14, 21, and 29. Histologic analysis was undertaken to examine anterior segment morphology, and markers of matrix deposition and fibrosis were used. Results. Gene transfer of TGF-beta in the anterior segment resulted in the formation of peripheral anterior synechiae (PAS), which consisted of a fibroproliferative region of corneal endothelial cells, matrix accumulation, and decrease in trabecular meshwork expression of alpha-smooth muscle actin. These features were accompanied by ocular hypertension. Conclusions. Gene transfer of TGF-beta into the anterior segment induces aberrant PAS associated with the transition of corneal endothelial cells and subsequent matrix deposition. These features are highly reminiscent of human iridocorneal endothelial (ICE) syndrome. Gene transfer of TGF-beta can, therefore, be used to induce anatomic changes in the anterior segment in a rodent model that result in ocular hypertension.

Corneal epithelial cell adhesion and growth on EGF-modified aminated PDMS.

Growth factor tethering has significant potential to mediate cellular responses in biomaterials and tissue engineering. We have previously demonstrated that epidermal growth factor (EGF) can be tethered to polydimethylsiloxane (PDMS) substrates and that these surfaces promoted interactions with human corneal epithelial cells in vitro. The goal of the current work was to better understand the specific effects of the tethered growth factor on the cells. The EGF was reacted with a homobifunctional N-hydroxysuccinimide (NHS) polyethylene glycol (PEG) derivative, and then bound to allyamine plasma-modified PDMS. Human corneal epithelial cells were seeded on the surfaces and cultured in serum-free medium for periods of up to 5 days. Cell growth was monitored and quantified by trypsinization and counting with a Coulter counter. Expression of matrix proteins and alpha(6)-integrins was assessed by immunostaining and confocal microscopy. A centrifugation assay was used to determine cell adhesion under an applied detachment force. Binding of EGF was found to significantly increase cell numbers and coverage across the surfaces at 5 days of culture in vitro. Immunofluorescence experiments indicate increased expression of fibronectin, laminin, and alpha(6)-integrins on the EGF-modified surfaces, and expression is localized at the cell-material interface as observed by confocal microscopy. In accordance with these results, the highest quantity of adherent cells is found on the EGF-modified subtrates at 5 days of culture. The results provide initial evidence that binding of EGF may be used to improve the epithelialization of and the adhesion of the cells on a polymeric artificial cornea device.

Development and use of the lens epithelial explant system to study lens differentiation and cataractogenesis.

Over the last two decades much progress has been made in identifying and characterizing many of the molecules involved in understanding normal lens biology and its pathology. Much of this has been made possible through the establishment and use of the lens epithelial explant system. This simplistic tissue culture model, comprised of a sheet of lens epithelium on its native substratum, has been used effectively to study many cellular processes, including lens epithelial cell proliferation, fiber cell differentiation, cell apoptosis as well as epithelial-to-mesenchymal transformation of cells. In doing so, a number of key growth factors and cytokines, including members of the FGF, Wnt and TGFbeta family have been shown to play essential roles in many of these cellular events. This has led to further studies exploring the signaling pathways downstream of these molecules in the lens, paving the way for the development of a number of in situ models (primarily transgenic mouse lines) to further explore in more detail the nature of these molecular and cellular interactions. To reciprocate, the lens epithelial explant system is increasingly being used to further characterize the nature of many complex phenotypes and pathologies observed in these in situ models, allowing us to selectively isolate and examine the direct impact of an individual molecule on a specific cellular response in lens cells. There is no question that the lens epithelial explant system has served as a powerful tool to further our understanding of lens biology and pathology, and there is no doubt that it will continue to serve in such a capacity, as new developments are realized and putative treatments for aberrant lens cell behavior are to be trialed.

Co-operative roles for E-cadherin and N-cadherin during lens vesicle separation and lens epithelial cell survival.

The classical cadherins are known to have both adhesive and signaling functions. It has also been proposed that localized regulation of cadherin activity may be important in cell assortment during development. In the context of eye development, it has been suggested that cadherins are important for separation of the invaginated lens vesicle from the surface ectoderm. To test this hypothesis, we conditionally deleted N-cadherin or E-cadherin from the presumptive lens ectoderm of the mouse. Conditional deletion of either cadherin alone did not produce a lens vesicle separation defect. However, these conditional mutants did exhibit common structural deficits, including microphthalmia, severe iris hyperplasia, persistent vacuolization within the fibre cell region, and eventual lens epithelial cell deterioration. To assess the co-operative roles of E-cadherin and N-cadherin within the developing lens, double conditional knockout embryos were generated. These mice displayed distinct defects in lens vesicle separation and persistent expression of another classical cadherin, P-cadherin, within the cells of the persistent lens stalk. Double mutant lenses also exhibited severe defects in lens epithelial cell adhesion and survival. Finally, the severity of the lens phenotype was shown to be sensitive to the number of wild-type E- and N-cadherin alleles. These data suggest that the co-operative expression of both E- and N-cadherin during lens development is essential for normal cell sorting and subsequent lens vesicle separation.

Temporal changes in MMP mRNA expression in the lens epithelium during anterior subcapsular cataract formation.

Transforming growth factor beta (TGFbeta) has been known to play a role in anterior subcapsular cataract (ASC) formation and posterior capsule opacification (PCO), both of which are fibrotic pathologies of the lens. Several models have been utilized to study ASC formation, including the TGFbeta1 transgenic mouse model and the ex-vivo rat lens model. A distinct characteristic of ASC development within these models includes the formation of isolated fibrotic plaques or opacities which form beneath the lens capsule. A hallmark feature of ASC formation is the epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) into myofibroblasts. Recently, the matrix metalloproteinases (MMPs) have been implicated in the formation of these cataracts through their involvement in EMT. In the present study, we sought to further investigate the role of MMPs in subcapsular cataract formation in a time course manner, through the examination of gene expression and morphological changes which occur during this process. RT-QPCR and immunohistochemical analysis was carried out on lenses treated with TGFbeta for a period of 2, 4 and 6 days. Laser capture microdissection (LCM) was utilized to specifically isolate cells within the plaque region and cells from the adjacent epithelium in lenses treated for a 6 day period. Multilayering of LECs was observed as early as day 2, which preceded the presence of alpha smooth muscle actin (alpha-SMA) immunoreactivity that was evident following 4 days of treatment with TGFbeta. A slight reduction in E-cadherin mRNA was detected at day 2, although this was not significant until the day 4 time point. Importantly, our results also indicate an early induction of MMP-9 mRNA following 2 days of TGFbeta treatment, whereas MMP-2 was found to be upregulated at the later 4 day time point. Further experiments using FHL 124 cells show an induction in MMP-2 protein levels following treatment with recombinant MMP-9. Together these findings suggest an upstream role for MMP-9 in ASC formation.

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.

Adenoviral gene transfer of bioactive TGFbeta1 to the rodent eye as a novel model for anterior subcapsular cataract.

PURPOSE: To produce a gene-transfer model of rodent anterior subcapsular cataracts (ASC) using a replication-deficient, adenoviral vector containing active TGFbeta1. Establishment of this model will be important for further investigations of TGFbeta-induced signaling cascades in ASC. METHODS: Adenovirus containing the transgene for active TGFbeta1 (AdTGFbeta1), beta-galactosidase (AdLacZ), green fluorescent protein (AdGFP) or no transgene (AdDL) was injected into the anterior chamber of C57Bl/6, Smad3 WT and Smad3 KO mice. Four and 21 days after injection, animals were enucleated and eyes were processed and examined by routine histology. Immunolocalization of markers indicative of epithelial to mesenchymal transition (EMT), fibrosis, proliferation and apoptosis was also carried out. RESULTS: By day 4, treatment with AdLacZ demonstrated transgene expression in multiple structures of the anterior chamber including the lens epithelium. In contrast to AdDL, treatment with AdTGFbeta1 produced alphaSMA-positive subcapsular plaques in all three groups of mice, which shared features reminiscent of human ASC. At day 21, plaques remained alphaSMA-positive and extensive extracellular matrix deposition was observed. The AdTGFbeta1 model was further employed in Smad3 deficient mice and this resulted in the development of small ASC. CONCLUSIONS: Gene transfer of active TGFbeta1 using an adenoviral vector produced cataractous plaques four days postinjection, which were found to develop independent of functional Smad3.

Targeted deletion of AP-2alpha leads to disruption in corneal epithelial cell integrity and defects in the corneal stroma.

PURPOSE: The present study was undertaken to create a conditional knockout of AP-2alpha in the corneal epithelium. METHODS: A line of mice expressing Cre-recombinase specifically in the early lens placode was crossed with mice in which the AP-2alpha allele is flanked by two loxP sites. The resultant Le-AP-2alpha mutants exhibited a targeted deletion of AP-2alpha in lens placode derivatives, including the differentiating corneal epithelium. RESULTS: The Le-AP-2alpha mutant mice were viable and had a normal lifespan. The adult corneal epithelium exhibited a variation in the number of stratified epithelial layers, ranging from 2 to 10 cell layers. A substantial decrease in expression of the cell-cell adhesion molecule, E-cadherin, was observed in all layers of the Le-AP-2alpha mutant corneal epithelium. The basement membrane, or Bowman's layer, was thinner in the mutant cornea and in many regions was discontinuous. These defects corresponded with altered distribution of laminin and entactin, and to a lesser degree, type IV collagen. The Le-AP-2alpha mutant cornea also exhibited stromal defects, including disrupted organization of the collagen lamellae and accumulation of fibroblasts beneath the epithelium that showed increased immunoreactivity for proliferating cell nuclear antigen (PCNA), alpha-smooth muscle actin (alpha-SMA), p-Smad2, and TGF-beta2. CONCLUSIONS: In the absence of AP-2alpha, the corneal epithelium exhibits altered cell adhesion and integrity and defects in its underlying basement membrane. These defects likely caused the alterations in the corneal stroma.

Transient overexpression of TGF-{beta}1 induces epithelial mesenchymal transition in the rodent peritoneum.

Epithelial mesenchymal transition (EMT), a process involved in many growth and repair functions, has been identified in the peritoneal tissues of patients who undergo peritoneal dialysis. The sequence of changes in gene regulation and cellular events associated with EMT after TGF-beta1-induced peritoneal fibrosis is reported. Sprague-Dawley rats received an intraperitoneal injection of an adenovirus vector that transfers active TGF-beta1 (AdTGF-beta1) or control adenovirus, AdDL. Animals were killed 0 to 21 days after infection. Peritoneal effluent and tissue were analyzed for markers of EMT. In the animals that were treated with AdTGF-beta1, an increase in expression of genes associated with EMT and fibrosis, such as type I collagen A2, alpha-smooth muscle actin, and the zinc finger regulatory protein Snail, was identified. Transition of mesothelial cells 4 to 7 d after infection, with appearance of epithelial cells in the submesothelial zone 7 to 14 d after exposure to AdTGF-beta1, was demonstrated. This phase was associated with disruption of the basement membrane and increased expression of matrix metalloproteinase 2. By 14 to 21 d after infection, there was evidence of restoration of normal submesothelial architecture. These findings suggest that EMT occurs in vivo after TGF-beta1 overexpression in the peritoneum. Cellular changes and gene regulation associated with EMT are evident throughout the fibrogenic process and are not limited to early time points. This further supports the central role of TGF-beta1 in peritoneal fibrosis and provides an important model to study the sequence of events involved in TGF-beta1-induced EMT.

Activity and expression of Xenopus laevis matrix metalloproteinases: identification of a novel role for the hormone prolactin in regulating collagenolysis in both amphibians and mammals.

Prolactin (PRL) has long been implicated in Xenopus metamorphosis as an anti-metamorphic and/or juvenilizing hormone. Numerous studies showed that PRL could prevent effects of either endogenous or exogenous thyroid hormone (TH; T(3)). It has been shown that expression of matrix metalloproteinases (MMPs) is induced by TH during Xenopus metamorphosis. Direct in vivo evidence, however, for such anti-TH effects by PRL with respect to MMPs has not been available for the early phase of Xenopus development or metamorphosis. To understand the functional role of PRL, we investigated effects of PRL on Xenopus collagenase-3 (XCL3) and collagenase-4 (XCL4) expression in a cultured Xenopus laevis cell line, XL-177. Northern blot analysis demonstrated that XCL3 and XCL4 expression were not detected in control or T(3)-treated cells, but were differentially induced by PRL in a dose- and time-dependent fashion. Moreover, treatment with IL-1alpha as well as phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, or H8, a protein kinase A (PKA) inhibitor, augmented PRL-induced collagenase expression, suggesting that multiple protein kinase pathways and cytokines may participate in PRL-induced collagenase expression. Interestingly, XCL3 expression could be induced in XL-177 cells by T(3), but only when co-cultured with prometamorphic Xenopus tadpole tails (stage 54/55), suggesting that the tails secrete a required intermediate signaling molecule(s) for T(3)-induced XCL3 expression. Taken together, these data demonstrate that XCL3 and XCL4 can be differentially induced by PRL and T(3) and further suggest that PRL is a candidate regulator of TH-independent collagenase expression during the organ/tissue remodeling which occurs in Xenopus development.