Differential Efficacy of Small Molecules Dynasore and Mdivi-1 for the Treatment of Dry Eye Epitheliopathy or as a Countermeasure for Nitrogen Mustard Exposure of the Ocular Surface.

The ocular surface comprises the wet mucosal epithelia of the cornea and conjunctiva, the associated glands, and the overlying tear film. Epitheliopathy is the common pathologic outcome when the ocular surface is subjected to oxidative stress. Whether different stresses act via the same or different mechanisms is not known. Dynasore and dyngo-4a, small molecules developed to inhibit the GTPase activity of classic dynamins DNM1, DNM2, and DNM3, but not mdivi-1, a specific inhibitor of DNM1L, protect corneal epithelial cells exposed to the oxidant tert-butyl hydroperoxide (tBHP). Here we report that, while dyngo-4a is the more potent inhibitor of endocytosis, dynasore is the better cytoprotectant. Dynasore also protects corneal epithelial cells against exposure to high salt in an in vitro model of dysfunctional tears in dry eye. We now validate this finding in vivo, demonstrating that dynasore protects against epitheliopathy in a mouse model of dry eye. Knockdown of classic dynamin DNM2 was also cytoprotective against tBHP exposure, suggesting that dynasore's effect is at least partially on target. Like tBHP and high salt, exposure of corneal epithelial cells to nitrogen mustard upregulated the unfolded protein response and inflammatory markers, but dynasore did not protect against nitrogen mustard exposure. In contrast, mdivi-1 was cytoprotective. Interestingly, mdivi-1 did not inhibit the nitrogen mustard-induced expression of inflammatory cytokines. We conclude that exposure to tBHP or nitrogen mustard, two different oxidative stress agents, cause corneal epitheliopathy via different pathologic pathways. SIGNIFICANCE STATEMENT: Results presented in this paper, for the first time, implicate the dynamin DNM2 in ocular surface epitheliopathy. The findings suggest that dynasore could serve as a new topical treatment for dry eye epitheliopathy and that mdivi-1 could serve as a medical countermeasure for epitheliopathy due to nitrogen mustard exposure, with potentially increased efficacy when combined with anti-inflammatory agents and/or UPR modulators.

Dynasore Modulates Store-Operated Calcium Entry and Mitochondrial Calcium Release in Corneal Epithelial Cells.

Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca2+) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca2+. Store-operated Ca2+ entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca2+ into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca2+ overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca2+ release from internal sources. The inefficacy of inhibitors of ER Ca2+ channels suggested that this compartment was not the source of the Ca2+ surge caused by tBHP exposure. However, using a Ca2+-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca2+ release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca2+ homeostasis, with inhibition of mitochondrial Ca2+ release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.

Dynasore Protects Corneal Epithelial Cells Subjected to Hyperosmolar Stress in an In Vitro Model of Dry Eye Epitheliopathy.

Epitheliopathy at the ocular surface is a defining sign of dry eye disease, a common disorder that affects 10% to 30% of the world's population. Hyperosmolarity of the tear film is one of the main drivers of pathology, with subsequent endoplasmic reticulum (ER) stress, the resulting unfolded protein response (UPR), and caspase-3 activation implicated in the pathway to programmed cell death. Dynasore, is a small molecule inhibitor of dynamin GTPases that has shown therapeutic effects in a variety of disease models involving oxidative stress. Recently we showed that dynasore protects corneal epithelial cells exposed to the oxidant tBHP, by selective reduction in expression of CHOP, a marker of the UPR PERK branch. Here we investigated the capacity of dynasore to protect corneal epithelial cells subjected to hyperosmotic stress (HOS). Similar to dynasore's capacity to protect against tBHP exposure, dynasore inhibits the cell death pathway triggered by HOS, protecting against ER stress and maintaining a homeostatic level of UPR activity. However, unlike with tBHP exposure, UPR activation due to HOS is independent of PERK and mostly driven by the UPR IRE1 branch. Our results demonstrate the role of the UPR in HOS-driven damage, and the potential of dynasore as a treatment to prevent dry eye epitheliopathy.

Recombinant Human Clusterin Seals Damage to the Ocular Surface Barrier in a Mouse Model of Ophthalmic Preservative-Induced Epitheliopathy.

There is a significant unmet need for therapeutics to treat ocular surface barrier damage, also called epitheliopathy, due to dry eye and related diseases. We recently reported that the natural tear glycoprotein CLU (clusterin), a molecular chaperone and matrix metalloproteinase inhibitor, seals and heals epitheliopathy in mice subjected to desiccating stress in a model of aqueous-deficient/evaporative dry eye. Here we investigated CLU sealing using a second model with features of ophthalmic preservative-induced dry eye. The ocular surface was stressed by topical application of the ophthalmic preservative benzalkonium chloride (BAC). Then eyes were treated with CLU and sealing was evaluated immediately by quantification of clinical dye uptake. A commercial recombinant form of human CLU (rhCLU), as well as an rhCLU form produced in our laboratory, designed to be compatible with U.S. Food and Drug Administration guidelines on current Good Manufacturing Practices (cGMP), were as effective as natural plasma-derived human CLU (pCLU) in sealing the damaged ocular surface barrier. In contrast, two other proteins found in tears: TIMP1 and LCN1 (tear lipocalin), exhibited no sealing activity. The efficacy and selectivity of rhCLU for sealing of the damaged ocular surface epithelial barrier suggests that it could be of therapeutic value in treating BAC-induced epitheliopathy and related diseases.

Transcriptome Analysis of Pterygium and Pinguecula Reveals Evidence of Genomic Instability Associated with Chronic Inflammation.

Solar damage due to ultraviolet radiation (UVR) is implicated in the development of two proliferative lesions of the ocular surface: pterygium and pinguecula. Pterygium and pinguecula specimens were collected, along with adjacent healthy conjunctiva specimens. RNA was extracted and sequenced. Pairwise comparisons were made of differentially expressed genes (DEGs). Computational methods were used for analysis. Transcripts from 18,630 genes were identified. Comparison of two subgroups of pterygium specimens uncovered evidence of genomic instability associated with inflammation and the immune response; these changes were also observed in pinguecula, but to a lesser extent. Among the top DEGs were four genes encoding tumor suppressors that were downregulated in pterygium: C10orf90, RARRES1, DMBT1 and SCGB3A1; C10orf90 and RARRES1 were also downregulated in pinguecula. Ingenuity Pathway Analysis overwhelmingly linked DEGs to cancer for both lesions; however, both lesions are clearly still benign, as evidenced by the expression of other genes indicating their well-differentiated and non-invasive character. Pathways for epithelial cell proliferation were identified that distinguish the two lesions, as well as genes encoding specific pathway components. Upregulated DEGs common to both lesions, including KRT9 and TRPV3, provide a further insight into pathophysiology. Our findings suggest that pterygium and pinguecula, while benign lesions, are both on the pathological pathway towards neoplastic transformation.

Therapeutic Potential of the Molecular Chaperone and Matrix Metalloproteinase Inhibitor Clusterin for Dry Eye.

Evidence is presented herein supporting the potential of the natural homeostatic glycoprotein CLU (clusterin) as a novel therapeutic for the treatment of dry eye. This idea began with the demonstration that matrix metalloproteinase MMP9 is required for damage to the ocular surface in mouse dry eye. Damage was characterized by degradation of OCLN (occludin), a known substrate of MMP9 and a key component of the paracellular barrier. Following up on this finding, a yeast two-hybrid screen was conducted using MMP9 as the bait to identify other proteins involved. CLU emerged as a strong interacting protein that inhibits the enzymatic activity of MMP9. Previously characterized as a molecular chaperone, CLU is expressed prominently by epithelia at fluid-tissue interfaces and secreted into bodily fluids, where it protects cells and tissues against damaging stress. It was demonstrated that CLU also protects the ocular surface in mouse dry eye when applied topically to replace the natural protein depleted from the dysfunctional tears. CLU is similarly depleted from tears in human dry eye. The most novel and interesting finding was that CLU binds selectively to the damaged ocular surface. In this position, CLU protects against epithelial cell death and barrier proteolysis, and dampens the autoimmune response, while the apical epithelial cell layer is renewed. When present at high enough concentration, CLU also blocks staining by vital dyes used clinically to diagnose dry eye. None of the current therapeutics have this combination of properties to "protect, seal, and heal". Future work will be directed towards human clinical trials to investigate the therapeutic promise of CLU.

Clusterin in the eye: An old dog with new tricks at the ocular surface.

The multifunctional protein clusterin (CLU) was first described in 1983 as a secreted glycoprotein present in ram rete testis fluid that enhanced aggregation ('clustering') of a variety of cells in vitro. It was also independently discovered in a number of other systems. By the early 1990s, CLU was known under many names and its expression had been demonstrated throughout the body, including in the eye. Its homeostatic activities in proteostasis, cytoprotection, and anti-inflammation have been well documented, however its roles in health and disease are still not well understood. CLU is prominent at fluid-tissue interfaces, and in 1996 it was demonstrated to be the most highly expressed transcript in the human cornea, the protein product being localized to the apical layers of the mucosal epithelia of the cornea and conjunctiva. CLU protein is also present in human tears. Using a preclinical mouse model for desiccating stress that mimics human dry eye disease, the authors recently demonstrated that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration in the tears. When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress. CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to LGALS3 (galectin-3), a key barrier component. Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure. CLU depletion from the ocular surface epithelia is seen in a variety of inflammatory conditions in humans and mice that lead to squamous metaplasia and a keratinized epithelium. This suggests that CLU might have a specific role in maintaining mucosal epithelial differentiation, an idea that can now be tested using the mouse model for desiccating stress. Most excitingly, the new findings suggest that CLU could serve as a novel biotherapeutic for dry eye disease.

A cell-based screening assay to identify pharmaceutical compounds that enhance the regenerative quality of corneal repair.

The goal of this study was to develop and validate a simple but quantitative cell-based assay to identify compounds that might be used pharmaceutically to give tissue repair a more regenerative character. The cornea was used as the model, and some specific aspects of repair in this organ were incorporated into assay design. A quantitative cell-based assay was developed based on transcriptional promoter activity of fibrotic marker genes ACT2A and TGFB2. Immortalized corneal stromal cells (HTK) or corneal epithelial cells (HCLE) were tested and compared to primary corneal stromal cells. Cells were transiently transfected with constructs containing the firefly luciferase reporter gene driven by transcriptional promoters for the selected fibrotic marker genes. A selected panel of seven chemical test compounds was used, containing three known fibrosis inhibitors: lovastatin (LOV), tyrphostin AG 1296 (6,7-dimethoxy-3-phenylquinoxaline) and SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole), and four potential fibrosis inhibitors: 5-iodotubercidin (4-amino-5-iodo-7-(ß-D-ribofuranosyl)-pyrrolo(2,3-d)pyrimidine), anisomycin, DRB (5,6-dichloro-1-ß-D-ribofuranosyl-benzimidazole) and latrunculin B. Transfected cells were treated with TGFB2 in the presence or absence of one of the test compounds. To validate the assay, compounds were tested for their direct effects on gene expression in the immortalized cell lines and primary human corneal keratocytes using RT-PCR and immunohistochemistry. Three "hits" were validated LOV, SB203580 and anisomycin. This assay, which can be applied in a high throughput format to screen large libraries of uncharacterized compounds, or known compounds that might be repurposed, offers a valuable tool for identifying new treatments to address a major unmet medical need. Anisomycin has not previously been characterized as antifibrotic, thus, this is a novel finding of the study.

Glaucomatous MYOC mutations activate the IL-1/NF-κB inflammatory stress response and the glaucoma marker SELE in trabecular meshwork cells.

PURPOSE: Activation of the IL-1/NF-κB inflammatory stress pathway and induction of SELE expression in the trabecular meshwork (TBM) is a marker for high-tension glaucomas of diverse etiology. Pathway activation stimulates aqueous outflow and protects against oxidative stress, but may be damaging in the long-term. MYOC mutations have been causally linked to high-tension forms of primary open angle glaucoma (POAG). This study investigated a possible link between MYOC mutations and activation of the IL-1/NF-κB pathway and expression of SELE. METHODS: We constructed MYOC expression vectors with mutations at sites that cause POAG. Mutations (Q368X, Y437H, A427T) were selected to represent proteins with differing POAG-causing potency (Q368X > Y437H > A427T) and intracellular retention behavior (Q368X and Y437H retained, A427T released). The constructs were made in two different kinds of vectors; one a plasmid designed for transient transfection (pCMV6), and one a doxycycline-inducible lentiviral vector (pSLIK) for stable cell transduction. The immortalized human trabecular meshwork line TM-1 was used for all expression studies. Expression of IL1A mRNA was determined by reverse transcription (RT)-PCR, as well as a set of five other genes associated with signaling pathways linked to glaucoma: IL1B and IL6 (NF-κB pathway), TGFB2 and ACTA2 (TGF-ß pathway) and FOXO1 (E2F1 apoptotic pathway). An ELISA was used to quantify IL1A protein released into culture media. To quantify intracellular NF-κB activity, we transiently transfected stably transduced cell lines with a luciferase expression vector under control of the IL8 promoter (containing an NF-κB response element). RESULTS: Transiently expressed wild-type MYOC was released into cell culture media, whereas mutant MYOCs Q368X and Y437H remained within cells. Both mutant MYOCs activated the IL-1/ NF-κB pathway, significantly stimulating expression of IL1A and IL1B. However Y437H, which causes a severe glaucoma phenotype, was less effective than Q368X, which causes a moderate glaucoma phenotype. In addition, the retained mutants stimulated expression of stress response genes ACTA2 and FOXO1. Unexpectedly, wild-type MYOC significantly decreased expression of IL6 and TGFB2, to approximately half of the control levels, and expression of IL1B and ACTA2 was also slightly decreased. Induction of MYOC mutants Q368X and Y437H in stably transduced cell lines significantly stimulated the level of IL1A protein released into culture media. Once again however, the effect of the severe MYOC mutant Y437H was less than the effect of the moderate MYOC mutant Q368X. In contrast, induced expression of the intracellularly retained mutant MYOC A427T or wild-type MYOC did not change the amount of IL1A protein in culture media. Induction of Y437H MYOC plus IL1A treatment increased NF-κB activity by 25% over IL1A alone. In contrast, induction of Q368X or A427T plus IL1A treatment did not significantly affect NF-κB activity over IL1A alone. However, wild-type MYOC expression inhibited IL1A-stimulated NF-κB activity. We also observed that endogenous MYOC expression was induced by IL1A in TM-1 cells and primary TBM cell cultures. SELE was co-expressed with MYOC in the primary cell lines. CONCLUSIONS: These results indicate that POAG-causing MYOC mutants activate the IL-1/NF-κB pathway, with activation levels correlated with intracellular retention of the protein, but not POAG-causing potency. Unexpectedly, it was also discovered that wild-type MYOC inhibits activation of the IL-1/NF-κB pathway, and that activation of the IL-1/NF-κB pathway stimulates expression of MYOC. This is the first evidence that glaucoma-causing MYOC mutants can activate the inflammatory response and that wild-type MYOC has anti-inflammatory activity.

Chondrogenesis in scleral stem/progenitor cells and its association with form-deprived myopia in mice.

PURPOSE: Previously, we demonstrated that scleral stem/progenitor cells (SSPCs) from mice have a chondrogenic differentiation potential, which is stimulated by transforming growth factor-ß (TGF-ß). In the present study, we hypothesized that chondrogenesis in the sclera could be a possible mechanism in myopia development. Therefore, we investigated the association of form-deprivation myopia (FDM) with expressions in mice sclera representing the chondrogenic phenotype: collagen type II (Col2) and α-smooth muscle actin (α-SMA). METHODS: The mRNA levels of α-SMA and Col2 in cultured murine SSPCs during chondrogenesis stimulated by TGF-ß2 were determined by real-time quantitative RT-PCR (qRT-PCR). The expression patterns of α-SMA and Col2 were assessed by immunohistochemistry in a three dimensional pellet culture. In an FDM mouse model, a western blot analysis and immunofluorescence study were used to detect the changes in the α-SMA and Col2 protein expressions in the sclera. In the RPE-choroid complex, qRT-PCR was used to detect any changes in the TGF-ß mRNA expression. RESULTS: The treatment of SSPCs in vitro with TGF-ß2 for 24 h at 1 or 10 ng/ml led to increased levels of both the α-SMA and Col2 expressions. In addition, we observed the formation of cartilage-like pellets from TGF-ß2-treated SSPCs. Both α-SMA and Col2 were expressed in the pellet. In an in-vivo study, the α-SMA and Col2 protein expressions were significantly increased in the sclera of FDM eyes in comparison to contralateral control eyes. Similarly, the levels of TGF-ß in the RPE-choroid complex of an FDM eye were also significantly elevated. CONCLUSION: Based on the concept of stem cells possessing multipotent differentiation potentials, scleral chondrogenesis induced by SSPCs may play a role in myopia development. The increased expressions of the cartilage-associated proteins Col2 and α-SMA during scleral chondrogenesis may be potential markers for myopia development. In addition, the increased levels of TGF-ß mRNA in the RPE-choroid complex might induce the chondrogenic change in the sclera during myopia development.

Interaction of clusterin and matrix metalloproteinase-9 and its implication for epithelial homeostasis and inflammation.

Uncontrolled increases of matrix metalloproteinase-9 (MMP-9) activity have been causally linked to epithelial barrier disruption and severe symptoms of inflammatory diseases such as dry eye (DE). The data presented here show that the anti-inflammatory, cytoprotective intracellular and extracellular chaperone protein clusterin (CLU) interacts with MMP-9 both inside and outside epithelial cells. CLU bound very strongly to active MMP-9, with an affinity constant K(D) of 2.63 nmol/L. Unexpectedly, CLU had a much higher affinity for pro-MMP-9 than for active MMP-9 or pro-MMP-2, requiring the N-terminal propeptide domain of pro-MMP-9. The significance of the interaction between CLU and MMP-9 was demonstrated by the observation that CLU prevents stress-induced MMP-9 aggregation and inhibits MMP-9 enzymatic activity. Furthermore, CLU inhibited MMP-9-mediated disintegration of the tight junction structure formed between human epithelial cells. Additionally, CLU inhibited enzymatic activities of MMP-2, MMP-3, and MMP-7. Treatment with proinflammatory cytokines, which are known to increase MMP-9 transcription under inflammatory conditions, reduced the expression of CLU in human epithelial cells. Similarly, in a mouse model of human DE, inflammatory stress depleted CLU in the ocular surface epithelium but allowed MMP-9 to prevail therein. The present results thus provide novel insights into previously unrecognized mechanisms by which CLU maintains fluid-epithelial interface homeostasis, thereby preventing the onset of inflammatory conditions, especially where MMP-9 is actively involved.

Roles unveiled for membrane-associated mucins at the ocular surface using a Muc4 knockout mouse model.

Membrane-associated mucins (MAMs) are proposed to play critical roles at the ocular surface; however, in vivo evidence has been lacking. Here we investigate these roles by phenotyping of a Muc4 KO mouse. Histochemical analysis for expression of the beta-galactosidase transgene replacing Muc4 revealed a spiraling ribbon pattern across the corneal epithelium, consistent with centripetal cell migration from the limbus. Depletion of Muc4 compromised transcellular barrier function, as evidenced by an increase in rose bengal staining. In addition, the corneal surface was less smooth, consistent with disruption of tear film stability. While surface cells presented with well-developed microprojections, an increase in the number of cells with fewer microprojections was observed. Moreover, an increase in skin-type keratin K10 and a decrease in transcription factor Pax6 was observed, suggesting an incipient transdifferentiation. Despite this, no evidence of inflammatory dry eye disease was apparent. In addition, Muc4 had no effect on signaling by toll-like receptor Tlr4, unlike reports for MUC1 and MUC16. Results of this study provide the first in vivo evidence for the role of MAMs in transcellular barrier function, tear film stability, apical epithelial cell architecture, and epithelial mucosal differentiation at the ocular surface.

Clusterin, other extracellular chaperones, and eye disease.

Proteostasis refers to all the processes that maintain the correct expression level, location, folding and turnover of proteins, essential to organismal survival. Both inside cells and in body fluids, molecular chaperones play key roles in maintaining proteostasis. In this article, we focus on clusterin, the first-recognized extracellular mammalian chaperone, and its role in diseases of the eye. Clusterin binds to and inhibits the aggregation of proteins that are misfolded due to mutations or stresses, clears these aggregating proteins from extracellular spaces, and facilitates their degradation. Clusterin exhibits three main homeostatic activities: proteostasis, cytoprotection, and anti-inflammation. The so-called "protein misfolding diseases" are caused by aggregation of misfolded proteins that accumulate pathologically as deposits in tissues; we discuss several such diseases that occur in the eye. Clusterin is typically found in these deposits, which is interpreted to mean that its capacity as a molecular chaperone to maintain proteostasis is overwhelmed in the disease state. Nevertheless, the role of clusterin in diseases involving such deposits needs to be better defined before therapeutic approaches can be entertained. A more straightforward case can be made for therapeutic use of clusterin based on its proteostatic role as a proteinase inhibitor, as well as its cytoprotective and anti-inflammatory properties. It is likely that clusterin works together in this way with other extracellular chaperones to protect the eye from disease, and we discuss several examples. We end this article by predicting future steps that may lead to development of clusterin as a biological drug.

Comprehensive gene expression profiling and functional analysis of matrix metalloproteinases and TIMPs, and identification of ADAM-10 gene expression, in a corneal model of epithelial resurfacing.

This study provides a comprehensive expression analysis for the entire matrix metalloproteinase (MMP) gene family during the process of epithelial resurfacing following corneal abrasion injury in the mouse. The mRNA levels for all known MMP genes expressed in mouse, the related enzyme ADAM-10, and the known tissue inhibitors of metalloproteinases (TIMPs) were determined semi-quantitatively by reverse transcriptase-polymerase chain reaction (RT-PCR) in the uninjured epithelium, and in the epithelial tissue resurfacing the abraded area or residing in its periphery at two time points: during the epithelial migration phase and immediately following wound closure. The mRNA levels for MMP-1a, -1b, -9, -10, -12, and -13 as well as TIMP-1 were significantly up-regulated in the migrating corneal epithelium. After wound resurfacing, the mRNA levels for all of these MMPs were down-regulated, although MMP-1a, -1b, and -13 remained significantly elevated in comparison to the uninjured epithelium. The only gene found to be down-regulated was TIMP-3, which occurred throughout the wound-healing process. During resurfacing, MMP-9 was localized to the front of the migrating epithelium, MMP-10 and -13 were localized throughout the migrating epithelium, and MMP-13 could also be found in the periphery. Following epithelial closure, immunoreactive MMPs-9 and -10 became undetectable, but MMP-13 continued to be found throughout the epithelium. Functional analysis of MMP-10 revealed no effects on epithelial migration or cell proliferation. In conclusion, distinct MMP temporal-spatial profiles define the uninjured corneal epithelium and the corneal epithelium at different stages of regeneration. An extensive review of the literature is also provided in the discussion.

Isoform- and dose-sensitive feedback interactions between paired box 6 gene and delta-catenin in cell differentiation and death.

Pax6, a mammalian homolog of the Drosophila paired box gene family member expressed in stem and progenitor cells, resides at the top of the genetic hierarchy in controlling cell fates and morphogenesis. While Pax6 activation can lead to mitotic arrest, premature neurogenesis, and apoptosis, the underlying molecular mechanisms have not been resolved. Here we report that either Pax6(+5a) or Pax6(-5a) was sufficient to promote, whereas their knockdown reduced the expression of delta-catenin (CTNND2), a neural specific member of the armadillo/beta-catenin superfamily. Pax6(+5a) elicited stronger effects on delta-catenin than Pax6(-5a). Inducible Pax6(+5a) expression demonstrated a biphasic and dose-dependent regulation of delta-catenin expression and cell fates. A moderate upregulation of Pax6(+5a) promoted delta-catenin expression and induced neurite-like cellular protrusions, but increasing expression of Pax6(+5a) reversed these processes. Furthermore, sustained high expression of Pax6(+5a) triggered apoptosis as determined by the reduction of phospho-Bad, Bcl-2, survivin and procaspases, as well as the increases in Bax and cleaved poly(ADP-ribose) polymerase. Importantly, re-introducing delta-catenin by ectopic expression elicited a feedback suppression on Pax6(+5a) expression and reduced Pax6(+5a) induced apoptosis. Therefore, delta-catenin expression is not only controlled by Pax6, but it also provides a feedback suppression mechanism for their functional interactions with important implications in cellular morphogenesis, apoptosis, and cancer.

Membrane-associated mucins of the human ocular surface in health and disease.

Mucins are a family of high molecular weight, heavily-glycosylated proteins produced by wet epithelial tissues, including the ocular surface epithelia. Densely-packed O-linked glycan chains added post-translationally confer the biophysical properties of hydration, lubrication, anti-adhesion and repulsion. Membrane-associated mucins (MAMs) are the distinguishing components of the mucosal glycocalyx. At the ocular surface, MAMs maintain wetness, lubricate the blink, stabilize the tear film, and create a physical barrier to the outside world. In addition, it is increasingly appreciated that MAMs function as cell surface receptors that transduce information from the outside to the inside of the cell. Recently, our team published a comprehensive review/perspectives article for molecular scientists on ocular surface MAMs, including previously unpublished data and analyses on two new genes MUC21 and MUC22, as well as new MAM functions and biological roles, comparing human and mouse (PMID: 31493487). The current article is a refocus for the audience of The Ocular Surface. First, we update the gene and protein information in a more concise form, and include a new section on glycosylation. Next, we discuss biological roles, with some new sections and further updating from our previous review. Finally, we provide a new chapter on MAM involvement in ocular surface disease. We end this with discussion of an emerging mechanism responsible for damage to the epithelia and their mucosal glycocalyces: the unfolded protein response (UPR). The UPR offers a novel target for therapeutic intervention.

Conditional deletion of activating protein 2alpha (AP-2alpha) in the developing retina demonstrates non-cell-autonomous roles for AP-2alpha in optic cup development.

Activating protein 2alpha (AP-2alpha) is known to be expressed in the retina, and AP-2alpha-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-2alpha and created a conditional deletion of AP-2alpha in the developing retina. AP-2alpha exhibited a distinct expression pattern in the developing inner nuclear layer of the retina, and colocalization studies indicated that AP-2alpha was exclusively expressed in postmitotic amacrine cell populations. Targeted deletion of AP-2alpha in the developing retina did not result in observable retinal defects. Further examination of AP-2alpha-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-2alpha 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.

Dynasore protects ocular surface mucosal epithelia subjected to oxidative stress by maintaining UPR and calcium homeostasis.

The mucosal epithelia of the ocular surface protect against external threats to the eye. Using a model of human stratified corneal epithelial cells with mucosal differentiation, we previously demonstrated that a small molecule inhibitor of dynamin GTPases, dynasore, prevents damage to cells and their transcellular barriers when subjected to oxidative stress. Investigating mechanisms, we now report the novel finding that dynasore acts by maintaining Ca+2 homeostasis, thereby inhibiting the PERK branch of the unfolded protein response (UPR) that promotes cell death. Dynasore was found to protect mitochondria by preventing mitochondrial permeability transition pore opening (mPTP), but, unlike reports using other systems, this was not mediated by dynamin family member DRP1. Necrostatin-1, an inhibitor of RIPK1 and lytic forms of programmed cell death, also inhibited mPTP opening and further protected the plasma membrane barrier. Significantly, necrostatin-1 did not protect the mucosal barrier. Oxidative stress increased mRNA for sXBP1, a marker of the IRE1 branch of the UPR, and CHOP, a marker of the PERK branch. It also stimulated phosphorylation of eIF2α, the upstream regulator of CHOP, as well as an increase in intracellular Ca2+. Dynasore selectively inhibited the increase in PERK branch markers, and also prevented the increase intracellular Ca2+ in response to oxidative stress. The increase in PERK branch markers were also inhibited when cells were treated with the cell permeable Ca2+ chelator, BAPTA-AM. To our knowledge, this is the first time that dynasore has been shown to have an effect on the UPR and suggests therapeutic applications.

Membrane-associated mucins of the ocular surface: New genes, new protein functions and new biological roles in human and mouse.

The mucosal glycocalyx of the ocular surface constitutes the point of interaction between the tear film and the apical epithelial cells. Membrane-associated mucins (MAMs) are the defining molecules of the glycocalyx in all mucosal epithelia. Long recognized for their biophysical properties of hydration, lubrication, anti-adhesion and repulsion, MAMs maintain the wet ocular surface, lubricate the blink, stabilize the tear film and create a physical barrier to the outside world. However, it is increasingly appreciated that MAMs also function as cell surface receptors that transduce information from the outside to the inside of the cell. A number of excellent review articles have provided perspective on the field as it has progressed since 1987, when molecular cloning of the first MAM was reported. The current article provides an update for the ocular surface, placing it into the broad context of findings made in other organ systems, and including new genes, new protein functions and new biological roles. We discuss the epithelial tissue-equivalent with mucosal differentiation, the key model system making these advances possible. In addition, we make the first systematic comparison of MAMs in human and mouse, establishing the basis for using knockout mice for investigations with the complexity of an in vivo system. Lastly, we discuss findings from human genetics/genomics, which are providing clues to new MAM roles previously unimagined. Taken together, this information allows us to generate hypotheses for the next stage of investigation to expand our knowledge of MAM function in intracellular signaling and roles unique to the ocular surface.

Cytokines and signaling pathways regulating matrix metalloproteinase-9 (MMP-9) expression in corneal epithelial cells.

Matrix metalloproteinase-9 (MMP-9) is a well-known regulator and effecter of many cellular processes including wound healing. In the cornea, either too much or too little MMP-9 can be detrimental to overall wound repair. We investigated the secreted factors as well as the intracellular signaling pathways and the promoter sequences that mediate this regulation. Primary culture rabbit corneal epithelial cells were treated with various cytokines alone or in different combinations and MMP-9 induction was assessed by gel zymography. Pharmacological inhibitors were used to determine the intracellular signaling pathways induced by the cytokines tested and deletion promoter constructs were created to determine the regions of the MMP-9 promoter involved in the cytokine regulation, thereby assessing the exact transcription factors binding the MMP-9 promoter. We found that two cytokine families, transforming growth factor beta (TGF-beta) and interleukin 1 (IL-1), act additively in an isoform non-specific manner to induce MMP-9 in this cell type. Our data suggest TGF-beta mediated MMP-9 induction may be regulated by the NF-kappaB, Smad3, and JNK pathways, whereas the IL-1beta mediated induction may be regulated by the NF-kappaB and p38 pathways. Inhibition of the p38, NF-kappaB, or JNK pathways significantly reduced, but did not abrogate, basal MMP-9 levels. Inhibition of the ERK pathway did not have an effect on MMP-9 mediated expression in either the treated or untreated co-transfected cells.