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1.  Identification of a Novel Mucin Gene HCG22 Associated With Steroid-Induced Ocular Hypertension 
The pathophysiology of ocular hypertension (OH) leading to primary open-angle glaucoma shares many features with a secondary form of OH caused by treatment with glucocorticoids, but also exhibits distinct differences. In this study, a pharmacogenomics approach was taken to discover candidate genes for this disorder.
A genome-wide association study was performed, followed by an independent candidate gene study, using a cohort enrolled from patients treated with off-label intravitreal triamcinolone, and handling change in IOP as a quantitative trait.
An intergenic quantitative trait locus (QTL) was identified at chromosome 6p21.33 near the 5′ end of HCG22 that attained the accepted statistical threshold for genome-level significance. The HCG22 transcript, encoding a novel mucin protein, was expressed in trabecular meshwork cells, and expression was stimulated by IL-1, and inhibited by triamcinolone acetate and TGF-β. Bioinformatic analysis defined the QTL as an approximately 4 kilobase (kb) linkage disequilibrium block containing 10 common single nucleotide polymorphisms (SNPs). Four of these SNPs were identified in the National Center for Biotechnology Information (NCBI) GTEx eQTL browser as modifiers of HCG22 expression. Most are predicted to disrupt or improve motifs for transcription factor binding, the most relevant being disruption of the glucocorticoid receptor binding motif. A second QTL was identified within the predicted signal peptide of the HCG22 encoded protein that could affect its secretion. Translation, O-glycosylation, and secretion of the predicted HCG22 protein was verified in cultured trabecular meshwork cells.
Identification of two independent QTLs that could affect expression of the HCG22 mucin gene product via two different mechanisms (transcription or secretion) is highly suggestive of a role in steroid-induced OH.
A pharmacogenomics approach was taken to discover candidate genes for steroid-induced ocular hypertension. Two independent quantitative trait loci were identified in association with a novel mucin gene HCG22, which was characterized. The loci could affect expression of the HCG22 mucin gene product via two different mechanisms (transcription or secretion).
PMCID: PMC4416661  PMID: 25813999
ocular hypertension; glucocorticoid; GWAS; gene
2.  Mining Retrospective Data for Virtual Prospective Drug Repurposing: L-DOPA and Age-related Macular Degeneration 
The American journal of medicine  2015;129(3):292-298.
Age-related macular degeneration (AMD) is a leading cause of visual loss among the elderly. A key cell type involved in AMD, the retinal pigment epithelium, expresses a G protein–coupled receptor that, in response to its ligand, L-DOPA, up-regulates pigment epithelia–derived factor, while down-regulating vascular endothelial growth factor. In this study we investigated the potential relationship between L-DOPA and AMD.
We used retrospective analysis to compare the incidence of AMD between patients taking vs not taking L-DOPA. We analyzed 2 separate cohorts of patients with extensive medical records from the Marshfield Clinic (approximately 17,000 and approximately 20,000) and the Truven MarketScan outpatient and databases (approximately 87 million) patients. We used International Classification of Diseases, 9th Revision codes to identify AMD diagnoses and L-DOPA prescriptions to determine the relative risk of developing AMD and age of onset with or without an L-DOPA prescription.
In the retrospective analysis of patients without an L-DOPA prescription, AMD age of onset was 71.2, 71.3, and 71.3 in 3 independent retrospective cohorts. Age-related macular degeneration occurred significantly later in patients with an L-DOPA prescription, 79.4 in all cohorts. The odds ratio of developing AMD was also significantly negatively correlated by L-DOPA (odds ratio 0.78; confidence interval, 0.76–0.80; P <.001). Similar results were observed for neovascular AMD (P <.001).
Exogenous L-DOPA was protective against AMD. L-DOPA is normally produced in pigmented tissues, such as the retinal pigment epithelium, as a byproduct of melanin synthesis by tyrosinase. GPR143 is the only known L-DOPA receptor; it is therefore plausible that GPR143 may be a fruitful target to combat this devastating disease.
PMCID: PMC4841631  PMID: 26524704
Age-related macular degeneration (AMD); GPR143; L-DOPA; Movement disorder; Parkinson’s disease; Retrospective study; Retinal pigment epithelium (RPE)
3.  Expression and Functional Role of Orphan Receptor GPR158 in Prostate Cancer Growth and Progression 
PLoS ONE  2015;10(2):e0117758.
Prostate cancer (PCa) is the second-leading cause of cancer-related mortality, after lung cancer, in men from developed countries. In its early stages, primary tumor growth is dependent on androgens, thus generally can be controlled by androgen deprivation therapy (ADT). Eventually however, the disease progresses to castration-resistant prostate cancer (CRPC), a lethal form in need of more effective treatments. G-protein coupled receptors (GPCRs) comprise a large clan of cell surface proteins that have been implicated as therapeutic targets in PCa growth and progression. The findings reported here provide intriguing evidence of a role for the newly characterized glutamate family member GPR158 in PCa growth and progression. We found that GPR158 promotes PCa cell proliferation independent of androgen receptor (AR) functionality and that this requires its localization in the nucleus of the cell. This suggests that GPR158 acts by mechanisms different from other GPCRs. GPR158 expression is stimulated by androgens and GPR158 stimulates AR expression, implying a potential to sensitize tumors to low androgen conditions during ADT via a positive feedback loop. Further, we found GPR158 expression correlates with a neuroendocrine (NE) differentiation phenotype and promotes anchorage-independent colony formation implying a role for GPR158 in therapeutic progression and tumor formation. GPR158 expression was increased at the invading front of prostate tumors that formed in the genetically defined conditional Pten knockout mouse model, and co-localized with elevated AR expression in the cell nucleus. Kaplan-Meier analysis on a dataset from the Memorial Sloan Kettering cancer genome portal showed that increased GPR158 expression in tumors is associated with lower disease-free survival. Our findings strongly suggest that pharmaceuticals targeting GPR158 activities could represent a novel and innovative approach to the prevention and management of CRPC.
PMCID: PMC4333349  PMID: 25693195
4.  Glaucomatous MYOC mutations activate the IL-1/NF-κB inflammatory stress response and the glaucoma marker SELE in trabecular meshwork cells 
Molecular Vision  2015;21:1071-1084.
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.
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-β 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).
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.
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.
PMCID: PMC4575906  PMID: 26396484
5.  Lacritin-mediated regeneration of the corneal epithelia by protein polymer nanoparticles 
The avascular corneal epithelium plays an important role in maintaining normal vision and protecting the corneal interior from environmental infections. Delayed recovery of ocular wounds caused by trauma or refractive surgery strengthens the need to accelerate corneal wound healing and better restore the ocular surface. To address this need, we fused elastin-like polypeptide (ELP) based nanoparticles SI with a model mitogenic protein called lacritin. Lacritin fused at the N-terminus of the SI diblock copolymer is called LSI. This LSI fusion protein undergoes thermo-responsive assembly of nanoparticles at physiologically relevant temperatures. In comparison to ELP nanoparticles without lacritin, LSI showed potent signs of lacritin specific effects on a human corneal epithelial cell line (HCE-T), which included enhancement of cellular uptake, calcium-mediated signaling, and closure of a scratch. In vivo, the corneas of non-obese diabetic mice (NOD) were found to be highly responsive to LSI. Fluorescein imaging and corneal histology suggested that topical administration of LSI onto the ocular surface significantly promoted corneal wound healing and epithelial integrity compared to mice treated with or without plain ELP. Most interestingly, it appears that ELP-mediated assembly of LSI is essential to produce this potent activity. This was confirmed by comparison to a control lacritin ELP fusion called LS96, which does not undergo thermally-mediated assembly at relevant temperatures. In summary, fusion of a mitogenic protein to ELP nanoparticles appears to be a promising new strategy to bioengineer more potent biopharmaceuticals with potential applications in corneal wound healing.
PMCID: PMC4270104  PMID: 25530855
Elastin-like polypeptide (ELPs); Lacritin; wound healing; nanoparticle; cornea
6.  Identification of Multipotent Stem/Progenitor Cells in Murine Sclera 
This study indicated that the sclera contains multipotent mesenchymal stem cells. The results may elucidate the cellular and molecular mechanism of scleral diseases.
The sclera forms the fibrous outer coat of the eyeball and acts as a supportive framework. The purpose of this study was to examine whether the sclera contains mesenchymal stem/progenitor cells.
Scleral tissue from C57BL6/J mice was separated from the retina and choroid and subsequently enzyme digested to release single cells. Proliferation capacity, self-renewal capacity, and ability for multipotent differentiation were analyzed by BrdU labeling, flow cytometry, reverse transcriptase–polymerase chain reaction, immunocytochemistry, and in vivo transplantation.
The scleral stem/progenitor cells (SSPCs) possessed clonogenic and high doubling capacities. These cells were positive for the mesenchymal markers Sca-1, CD90.2, CD44, CD105, and CD73 and negative for the hematopoietic markers CD45, CD11b, Flk1, CD34, and CD117. In addition to expressing stem cell genes ABCG2, Six2, Notch1, and Pax6, SSPCs were able to differentiate to adipogenic, chondrogenic, and neurogenic lineages.
This study indicates that the sclera contains multipotent mesenchymal stem cells. Further study of SSPCs may help elucidate the cellular and molecular mechanism of scleral diseases such as scleritis and myopia.
PMCID: PMC3262553  PMID: 21788434
7.  Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye 
PLoS ONE  2015;10(9):e0138958.
Dry eye is a common disorder caused by inadequate hydration of the ocular surface that results in disruption of barrier function. The homeostatic protein clusterin (CLU) is prominent at fluid-tissue interfaces throughout the body. CLU levels are reduced at the ocular surface in human inflammatory disorders that manifest as severe dry eye, as well as in a preclinical mouse model for desiccating stress that mimics dry eye. Using this mouse model, we show here that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration. 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 the galectin LGALS3, 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. These findings define a fundamentally new mechanism for ocular surface protection and suggest CLU as a biotherapeutic for dry eye.
PMCID: PMC4581869  PMID: 26402857
8.  GPR158, an Orphan Member of G Protein-Coupled Receptor Family C: Glucocorticoid-Stimulated Expression and Novel Nuclear Role 
PLoS ONE  2013;8(2):e57843.
Members of the large G protein-coupled receptor (GPCR) clan are implicated in many physiological and disease processes, making them important therapeutic drug targets. In the present study, we follow up on results of a pilot study suggesting a functional relationship between glucocorticoid (GC)-induced ocular hypertension and GPR158, one of three orphan members of the GPCR Family C. GC treatment increases levels of GPR158 mRNA and protein through transcriptional mechanisms, in cultured trabecular meshwork (TBM) cells derived from the eye's aqueous outflow pathway. Like treatment with GCs, transient overexpression of GPR158 stimulates cell proliferation, while siRNA knockdown of endogenous GPR158 has the opposite effect. Both endogenous and overexpressed GPR158 show an unusual subcellular localization pattern, being found almost entirely in the nucleus. However, when cells are treated with inhibitors of clathrin-mediated endocytosis, GPR158 is shifted to the plasma membrane. Mutation of a bipartite nuclear localization signal (NLS) in the 8th helix also shifts GPR158 out of the nucleus, but in this case the protein is found in vesicles localized in the cytoplasm. These results suggest that newly synthesized GPR158 first traffics to the plasma membrane, where it rapidly undergoes endocytosis and translocation to the nucleus. Significantly, mutation of the NLS abrogates GPR158-mediated enhancement of cell proliferation, indicating a functional requirement for nuclear localization. GPR158 overexpression upregulates levels of the cell cycle regulator cyclin D1, but mutation of the NLS reverses this. Overexpression of GPR158 enhances the barrier function of a TBM cell monolayer, which is associated with an increase in the levels of tight junction proteins ZO-1 and occludin, similar to reported studies on GC treatment. Regulated paracellular permeability controls aqueous outflow facility in vivo. Since GCs stimulate GPR158 expression, the result is consistent with a role for elevation of GPR158 expression in GC-induced ocular hypertension.
PMCID: PMC3581496  PMID: 23451275
9.  A Novel Mechanism of Increased Infections in Contact Lens Wearers 
The authors have identified a molecular mechanism that may be the underlying cause of the increase in corneal damage and infection observed in contact lens wearers. The data highlight the biological and financial potential for better contact lens solutions.
It is well documented that contact lens wearers have much higher incidences of corneal infections compared with those of non–contact lens wearers, although the exact cause(s) of this increased susceptibility has not been identified. A distinct subset of mucins (MUCs) is present on the ocular surface, acting to protect the integrity of the corneal epithelium. This study was performed to determine whether multipurpose contact lens solutions (MPCLSs) can cause increased infections in the cornea by destroying the protective cell-bound mucin layer.
An immortalized human corneal limbal epithelial cell line was treated in the presence of four commonly used MPCLSs or PBS and the expression and release of MUC-16 was assessed. Cells were also cultured with Pseudomonas aeruginosa after MPCLS treatment and internalization of bacteria was assessed by quantitative genomic PCR. Loss of MUC-16 was then correlated with infection rates.
Each of the four commonly used MPCLSs examined in this study differentially affected mucin release. The relative effect was correlated with an increase in infection of corneal epithelial cells by P. aeruginosa.
The results of this study are consistent with the hypothesis that MPCLSs cause increased infections in the cornea by destroying the protective cell-bound mucin layer.
PMCID: PMC3253543  PMID: 22039229
10.  Chondrogenesis in scleral stem/progenitor cells and its association with form-deprived myopia in mice 
Molecular Vision  2015;21:138-147.
Purpose: Previously, we demonstrated that scleral stem/progenitor cells (SSPCs) from mice have a chondrogenic differentiation potential, which is stimulated by transforming growth factor-β (TGF-β). 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-β2 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-β mRNA expression.
Results: The treatment of SSPCs in vitro with TGF-β2 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-β2-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-β 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-β mRNA in the RPE-choroid complex might induce the chondrogenic change in the sclera during myopia development.
PMCID: PMC4323720  PMID: 25684979
11.  Cytokines and Signaling Pathways Regulating Matrix Metalloproteinase-9 (MMP-9) Expression in Corneal Epithelial Cells 
Journal of cellular physiology  2009;221(2):402-411.
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, TGF-β and IL-1, act additively in an isoform non-specific manner to induce MMP-9 in this cell type. Our data suggest TGF-β mediated MMP-9 induction may be regulated by the NF-kB, Smad3, and JNK pathways, whereas the IL-1β mediated induction may be regulated by the NF-kB and p38 pathways. Inhibition of the p38, NF-kB, 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.
PMCID: PMC2990951  PMID: 19626678
Corneal epithelial cells; MMP-9; signal transduction; TGF-β; IL-1
12.  Mechanisms for PDGF, a Serum Cytokine, Stimulating Loss of Corneal Keratocyte Crystallins 
Cornea  2013;32(9):10.1097/ICO.0b013e318296e0b9.
As corneal stromal cells (keratocytes) become activated prior to transition to the fibroblastic repair phenotype in response to injury (in situ) or serum (in culture), the corneal crystallins, transketolase (TKT) and aldehyde dehydrogenase (ALDH1A1), are lost. We previously showed that the serum cytokine platelet-derived growth factor-BB (PDGF), but not transforming growth factor beta2 (TGF-beta2), stimulates TKT loss. Our goal in this study was to further define molecular mechanisms for PDGF-stimulated loss of crystallins, in order to elucidate the pathway for keratocyte activation.
Freshly isolated rabbit corneal keratocytes (RCK) were plated in serum-free medium with or without PDGF and/or specific inhibitors of the PDGF-relevant signal pathway components PDGF-receptor, PI3K/AKT, or ras-initiated MAPK proteins. Intracellular TKT protein levels were quantified by immunoblotting. Ubiquinated-TKT levels were assessed by immunoprecipitation and TKT mRNA levels were quantified by quantitative RT-PCR.
PDGF treatment at the same time as inhibition of PDGF-receptor, Akt, JNK and ubiquitin-proteasome pathway (UPP) prevented PDGF-induced TKT protein loss. In contrast, treatment with PDGF did not affect TKT mRNA levels.
The results suggest that PDGF-stimulated TKT loss is mediated via cross talk between PI3K-independent Akt and JNK. This signaling pathway leads to degradation of existing TKT protein, but does not compromise the accumulation of TKT mRNA. Therefore, cells retain the potential to reaccumulate TKT protein that is enabled by PDGF removal. These findings suggest that targeting PDGF signaling could improve repair outcomes following surgical procedures in the cornea.
PMCID: PMC3874843  PMID: 23846408
corneal keratocytes; platelet-derived growth factor-BB; aldehyde dehydrogenase; transketolase; signal transduction pathway
13.  Matrix metalloproteinase gelatinase B (MMP-9) is associated with leaking glaucoma filtering blebs☆ 
Experimental eye research  2005;81(4):429-436.
The goal of glaucoma filtering surgery is to create a low resistance pathway for aqueous outflow. The result is a blister or ‘bleb’ on the conjunctiva, from which fluid drains into the vasculature. Filtering surgery results may be compromised if blebs develop leaks, a problem that surfaces more frequently when antimetabolites are used to control the wound healing response. We investigated the role of tissue remodelling enzymes of the Matrix metalloproteinase (MMP) family in the development of bleb leaks. Our design was a case series. We enrolled glaucoma patients with leaking blebs, glaucoma patients with overhanging blebs and normal eyes. Leaking bleb tissues (n = 11) and bleb leak fluid were collected from patients undergoing bleb revision surgery. Overhanging bleb tissues (from non-leaking blebs, n = 3), normal conjunctiva (n = 8), and aqueous humour (n = 4) were collected for comparison. Samples were analysed for MMP content and proteinase activity by the methods of zymography, westernblotting, immunohistochemistry, and in situ zymography. Our main outcome measures were presence and activity of MMP in sample.
Zymography revealed the presence of a high molecular weight caseinase and a 92-kDa gelatinase of a size appropriate for the proenzyme form of gelatinase B (gelB; MMP-9), in extracts from leaking bleb tissue, but not in bleb leak fluid or aqueous humour samples. In contrast, a 65-kDa gelatinase of a size appropriate for gelatinase A (MMP-2) proenzyme was observed in all samples. All proteinases disappeared when 10 mM EDTA was added to the development buffer, consistent with their identity as MMPs. Western blotting and immunohistochemical analyses confirmed the identity of the 92 kDa proteinase as gelB, and further revealed its absence from extracts of overhanging bleb tissue and normal conjunctiva. In situ zymography demonstrated strong gelatinolytic activity in leaking bleb tissue, but not overhanging bleb tissue or normal conjunctiva.
MMP-g may be involved in the mechanism of formation of bleb leaks. Precise description of the cascade of events leading to bleb leakage may allow the design of therapeutic interventions to prevent, stabilize or reverse bleb leakage.
PMCID: PMC1941659  PMID: 16185954
glaucoma; trabeculectomy; matrix metalloproteinase; gelatinase B; filtering surgery; bleb; bleb leak; zymography; western blot; immunohistochemistry; tetracycline
14.  Isoform- and Dose-sensitive Feedback Interactions between Paired Box 6 gene and δ-Catenin in Cell Differentiation and Death 
Experimental cell research  2010;316(6):1070-1081.
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 δ-catenin (CTNND2), a neural specific member of the armadillo/β-catenin superfamily. Pax6(+5a) elicited stronger effects on δ-catenin than Pax6(−5a). Inducible Pax6(+5a) expression demonstrated a biphasic and dose-dependent regulation of δ-catenin expression and cell fates. A moderate upregulation of Pax6(+5a) promoted δ-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 δ-catenin by ectopic expression elicited a feedback suppression on Pax6(+5a) expression and reduced Pax6(+5a) induced apoptosis. Therefore, δ-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.
PMCID: PMC2885963  PMID: 20074565
Paired box gene; δ-catenin; cell fates; apoptosis; feedback regulation
15.  Collagen Fibril Growth During Chicken Tendon Development: Matrix Metalloproteinase-2 and Its Activation 
Cell and tissue research  2009;336(1):79-89.
The role of matrix metalloproteinases (MMPs) in collagen fibrillogenesis during development was studied in the well-characterized chicken metatarsal tendon. Collagen fibrils are initially assembled as intermediates and the mature fibrils assemble by linear and lateral growth from intermediates. We hypothesize that this involves the turnover of fibril-associated molecules mediated by expression and activation of matrix metalloproteinase-2 (MMP-2). We demonstrate changes in the ratio of full-length to truncated MMP-2 during tendon development, consistent with enzyme activation. The level of full length proMMP-2 remains relatively unchanged, however, the truncated form of MMP-2 is highest prior to and during fibril growth. Membrane type matrix metalloproteinase-3 (MT3-MMP, MMP-16) is fibroblast-associated and involved in the regulation of MMP-2 as well as in direct matrix turnover. The ratio of full-length proMT3-MMP/truncated (active) MT3-MMP has a pattern similar to that of full-length proMMP-2/truncated (active) MMP-2 during tendon development. Regulation of proMMP-2 activation involves complex formation with active MT3-MMP and TIMP-2. The constant low TIMP-2 expression seen in tendon development is consistent with this role. Isolation of collagen fibrils from pre-fibril growth tendons (14 day) in the presence of activated MMP-2 is associated with premature fibril growth seen as increased fibril diameters compared to controls. These data implicate MMP-2/MT3-MMP in the initiation of and progression of fibril growth, matrix assembly and tendon development. This may involve turnover of fibril-associated molecules involved in regulating linear and lateral growth, such as small leucine-rich proteoglycans and fibril-associated collagens. Activation of proMMP-2 dependent on MT3-MMP would allow a focal control of turnover.
PMCID: PMC2746393  PMID: 19221802
Matrix Metalloproteinases; MMP-2; Collagen Fibril Growth; Tendon; Development
16.  Identification of E2F1 as a positive transcriptional regulator for δ-catenin 
δ-Catenin is upregulated in human carcinomas. However, little is known about the potential transcriptional factors that regulate δ-catenin expression in cancer. Using a human δ-catenin reporter system, we have screened several nuclear signaling modulators to test whether they can affect δ-catenin transcription. Among β-catenin/LEF-1, Notch1, and E2F1, E2F1 dramatically increased δ-catenin-luciferase activities while β-catenin/LEF-1 induced only a marginal increase. Rb suppressed the upregulation of δ-catenin-luciferase activities induced by E2F1 but did not interact with δ-catenin. RT-PCR and Western blot analyses in 4 different prostate cancer cell lines revealed that regulation of δ-catenin expression is controlled mainly at the transcriptional level. Interestingly, the effects of E2F1 on δ-catenin expression were observed only in human cancer cells expressing abundant endogenous δ-catenin. These studies identify E2F1 as a positive transcriptional regulator for δ-catenin, but further suggest the presence of strong negative regulator(s) for δ-catenin in prostate cancer cells with minimal endogenous δ-catenin expression.
PMCID: PMC2614404  PMID: 18302937
δ-Catenin/NPRAP; E2F1; Wnt; Notch; Prostate; catenin; LEF-1; cancer; transcription
17.  Positive influence of AP-2α transcription factor on cadherin gene expression and differentiation of the ocular surface 
The family of transcription factors Activating protein-2 (AP-2) are known to play important roles in numerous developmental events, including those associated with differentiation of stratified epithelia. However, to date, the influence of the AP-2 genes on endogenous gene expression in the stratified epithelia and how this affects differentiation has not been well defined. The following study examines the detailed expression of the AP-2α and AP-2β proteins in the stratified epithelia of the ocular surface, including that in the cornea and developing eyelids. The effect of altered levels of the AP-2α gene on ocular surface differentiation was also examined using a corneal epithelial cell line and AP-2α chimeric mice. Immunolocalization studies revealed that, while AP-2β was broadly expressed throughout all cell layers of the stratified corneal epithelium, AP-2α expression was confined to cell compartments more basally located. AP-2α was also highly expressed in the less differentiated cell layers of the eyelid epidermis. Overexpression of the AP-2α gene in the corneal cell line, SIRC, resulted in a dramatic change in cell phenotype including a clumping growth behavior that was distinct from the smooth monolayer of the parent cell line. Accompanying this change was an up-regulation in levels of the cell adhesion molecule, N-cadherin. Examination of the ocular surface of AP-2α chimeric mice, derived from a mixed population of AP-2α−/− and AP-2α+/+, revealed that a down-regulation in E-cadherin expression is correlated with location of the AP-2α−/− null cells. Together, these findings demonstrate that AP-2α participates in regulating differentiation of the ocular surface through induction in cadherin expression.
PMCID: PMC2517417  PMID: 12694203
ocular surface; cell adhesion; transcription factors; AP-2; cornea; eyelids; differentiation
18.  Conditional Deletion of Activating Protein 2α (AP-2α) in the Developing Retina Demonstrates Non-Cell-Autonomous Roles for AP-2α in Optic Cup Development▿  
Molecular and Cellular Biology  2007;27(21):7497-7510.
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.
PMCID: PMC2169054  PMID: 17724084
19.  Ultrasound Activates the TM ELAM-1/IL-1/NF-κB Response: A Potential Mechanism for Intraocular Pressure Reduction after Phacoemulsification 
Elevated intraocular pressure (IOP), the major causal risk factor for glaucoma, often decreases after cataract removal by phacoemulsification ultrasound. In this study, the hypothesis that ultrasound energy propagated through a fluid medium induces a stress response with the potential to lower IOP was investigated.
Normal and glaucomatous trabecular meshwork (TM) cell culture lines were initiated from tissue isolated from human cadaveric eyes or trabeculectomy specimens. Cultured cells were treated for 60 seconds with a phacoemulsification ultrasound probe set to a power of 70%. Activation of the TM cell-specific stress response was assayed by enzyme-linked immunosorbent assay (ELISA) and immunolocalization.
Normal TM cell cultures did not release detectable levels of the stress response protein, IL-1α, into their culture medium. In contrast, IL-1α was easily detected after treatment with ultrasound energy. Consistent with earlier findings, glaucomatous TM cells produced IL-1α constitutively, and the level of expression was increased after treatment with phacoemulsification ultrasound. As was previously demonstrated, the stressregulated transcription factor NF-κB was present in the cytoplasm of normal cells, but in the nucleus of glaucomatous cells. After treatment with ultrasound energy, NF-κB translocated to the nucleus in the normal cells. Endothelial leukocyte-adhesion molecule (ELAM)-1 was not detected in normal TM cells, but was constitutively present on glaucomatous TM cells, consistent with findings in previous work. ELAM-1 expression was induced in normal cells by ultrasound treatment.
A potentially IOP-lowering stress response is induced in TM cells by ultrasound. The findings suggest that this response may be induced clinically during cataract removal by phacoemulsification, and may be one mechanism responsible for the reduction in IOP that often follows this procedure.
PMCID: PMC1950284  PMID: 12714632
20.  Activation of a tissue-specific stress response in the aqueous outflow pathway of the eye defines the glaucoma disease phenotype 
Nature medicine  2001;7(3):304-309.
The glaucomas are a group of optic neuropathies comprising the leading cause of irreversible blindness worldwide. Elevated intraocular pressure due to a reduction in normal aqueous outflow is a major causal risk factor. We found that endothelial leukocyte adhesion molecule-1 (ELAM-1), the earliest marker for the atherosclerotic plaque in the vasculature, was consistently present on trabecular meshwork (TM) cells in the outflow pathways of eyes with glaucomas of diverse etiology. We determined expression of ELAM-1 to be controlled by activation of an interleukin-1 (IL-1) autocrine feedback loop through transcription factor NF-κB, and activity of this signaling pathway was shown to protect TM cells against oxidative stress. These findings characterize a protective stress response specific to the eye’s aqueous outflow pathways and provide the first known diagnostic indicator of glaucomatous TM cells. They further indicate that common mechanisms contribute to the pathophysiology of the glaucomas and vascular diseases.
PMCID: PMC1945815  PMID: 11231628
21.  Transcription Factors Pax6 and AP-2α Interact To Coordinate Corneal Epithelial Repair by Controlling Expression of Matrix Metalloproteinase Gelatinase B 
Molecular and Cellular Biology  2004;24(1):245-257.
Pax6 is a paired box containing transcription factor that resides at the top of a genetic hierarchy controlling eye development. It continues to be expressed in tissues of the adult eye, but its role in this capacity is unclear. Pax6 is present in the adult corneal epithelium, and we showed that the amount of Pax6 is increased at the migrating front as the epithelium resurfaces the cornea after injury (J. M. Sivak, R. Mohan, W. B. Rinehart, P. X. Xu, R. L. Maas, and M. E. Fini, Dev. Biol. 222:41-54, 2000). We also showed that Pax6 controls activity of the transcriptional promoter for the matrix metalloproteinase, gelatinase B (gelB; MMP-9) in cell culture transfection studies. gelB expression is turned on at the migrating epithelial front in the cornea, and it coordinates and effects aspects of epithelial regeneration (R. Mohan, S. K. Chintala, J. C. Jung, W. V. Villar, F. McCabe, L. A. Russo, Y. Lee, B. E. McCarthy, K. R. Wollenberg, J. V. Jester, M. Wang, H. G. Welgus, J. M. Shipley, R. M. Senior, and M. E. Fini, J. Biol. Chem. 277:2065-2072). We define here two positively acting Pax6 response elements in the gelB promoter. Pax6 binds directly to one of these sites through the paired DNA-binding domain. It binds the second site indirectly by interaction with AP-2α, a transcription factor that also exerts control over eye development. Pax6 control of gelB expression was examined in vivo by using a corneal reepithelialization model in mice heterozygous for a Pax6 paired-domain mutation (Sey+/−). A reduced Pax6 dosage in these mice resulted in a loss of gelB expression at the migrating epithelial front. This effect was correlated with an increase in inflammation and the rate of reepithelialization, a finding consistent with the phenotype of gelB knockout mice. Together, these data indicate that Pax6 controls activity of the gelB promoter through cooperative interactions with AP-2α and support an active role for Pax6 in maintenance and repair of the adult corneal epithelium.
PMCID: PMC303332  PMID: 14673159

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