Gene Expression of Clusterin, Tissue Inhibitor of Metalloproteinase-1, and Their Receptors in Retinal Pigment Epithelial Cells and Müller Glial Cells Is Modulated by Inflammatory Stresses.

Balanced activities of matrix metalloproteinases (MMPs) and their inhibitors are essential for photoreceptor (PR) cell survival. PR rod cell survival in rodent models of inherited retinitis pigmentosa (RP) is prolonged by recombinant tissue inhibitor of metalloproteinase (TIMP)-1 or clusterin (CLU) proteins. Retinal pigment epithelial cells (RPE) and Müller glia (MG) cells support PR cells. In human RPE and MG cell lines, we measured their mRNA levels of the two genes with quantitative real-time PCR (qRT-PCR) with interleukin (IL)-1ß treatment, a key pathological component in retinal degeneration. Endogenous CLU gene expression was significantly downregulated by IL-1ß in both cell types, whereas TIMP-1 expression was upregulated in MG cells, suggesting the transcriptional control of CLU is potentially more sensitive to inflammatory conditions. The expression levels of CLU endocytic receptors revealed that the low-density lipoprotein receptor-related protein 2 (LRP2) was upregulated only in MG cells by the treatment with no detectable change in RPE cells. Like LRP2, IL-1ß upregulated TIMP-1 receptor LRP1 expression in MG cells; however, it was decreased in the expression of RPE cells. These data suggest that the gene expression of CLU and TIMP-1 and their receptors may be dynamically modulated in inflammatory conditions.

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.

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.

Matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1) are localized in the nucleus of retinal Müller glial cells and modulated by cytokines and oxidative stress.

Matrix metalloproteinases (MMPs) are involved in the pathology of numerous inflammatory retinal degenerations, including retinitis pigmentosa (RP). Our previous work revealed that intravitreal injections with tissue inhibitor of metalloproteinases 1 (TIMP-1) reduce the progression of rod cell death and inhibit cone cell remodeling that involves reactive gliosis in retinal Müller glial cells (MGCs) in rodent models. The underlying cellular and molecular mechanisms of how TIMP-1 functions in the retina remain to be resolved; however, MGCs are involved in structural homeostasis, neuronal cell survival and death. In the present study, MMP-9 and TIMP-1 expression patterns were investigated in a human MGC line (MIO-M1) under inflammatory cytokine (IL-1ß and TNF-α) and oxidative stress (H2O2) conditions. First, both IL-1ß and TNF-α, but not H2O2, have a mild in vitro pro-survival effect on MIO-M1 cells. Treatment with either cytokine results in the imbalanced secretion of MMP-9 and TIMP-1. H2O2 treatment has little effect on their secretion. The investigation of their intracellular expression led to interesting observations. MMP-9 and TIMP-1 are both expressed, not only in the cytoplasm, but also inside the nucleus. None of the treatments alters the MMP-9 intracellular distribution pattern. In contrast to MMP-9, TIMP-1 is detected as speckles. Intracellular TIMP-1 aggregation forms in the cytoplasmic area with IL-1ß treatment. With H2O2 treatments, the cell morphology changes from cobbles to spindle shapes and the nuclei become larger with increases in TIMP-1 speckles in an H2O2 dose-dependent manner. Two TIMP-1 cell surface receptors, low density lipoprotein receptor-related protein-1 (LRP-1) and cluster of differentiation 82 (CD82), are expressed within the nucleus of MIO-M1 cells. Overall, these observations suggest that intracellular TIMP-1 is a target of proinflammatory and oxidative insults in the MGCs. Given the importance of the roles for MGCs in the retina, the functional implication of nuclear TIMP-1 and MMP-9 in MGCs is discussed.

Assessment of inner retinal oxygen metrics and thickness in a mouse model of inherited retinal degeneration.

The retinal degeneration 1 (rd1) mouse is a well-established model of inherited retinal degeneration, displaying photoreceptor degeneration and retinal vasculature damage. The purpose of the current study was to determine alterations in the rate of oxygen delivery from retinal circulation (DO2), the rate of oxygen extraction from the retinal circulation for metabolism (MO2), and oxygen extraction fraction (OEF) in rd1 mice. The study was performed in a total of 18 wild type (WT) and 10 rd1 mice at both 3-weeks and 12-weeks of age. Retinal arterial and venous oxygen contents (O2A and O2V) were measured using phosphorescence lifetime imaging. Total retinal blood flow (TRBF) was determined by fluorescence and red-free imaging. DO2 and MO2 were determined as TRBF × O2A and TRBF × (O2A-O2V), respectively. OEF was calculated as MO2/DO2. The thickness of individual retinal layers was measured from histology sections and inner retina (IR) and total retina (TR) thickness were calculated. TRBF, DO2 and MO2 were lower in rd1 mice compared to WT mice (P ≤ 0.001), whereas OEF was not significantly different between rd1 and WT mice (P = 0.4). TRBF and DO2 were lower at 3-weeks of age compared to 12-weeks of age (P ≤ 0.01), while MO2 was not significantly different between age groups (P = 0.4) and OEF was higher at 3-weeks of age compared to 12-weeks of age (P = 0.003). Additionally, the outer and inner retinal cell layer thicknesses were decreased in rd1 mice at 12-weeks of age compared to both age-matched WT mice and rd1 mice at 3-weeks of age (P ≤ 0.02). MO2 was directly correlated with both IR and TR thickness (R ≥ 0.50; P ≤ 0.03, N = 20). The findings indicate that the rate oxygen is supplied by the retinal circulation is decreased and the reduction in oxygen extracted for metabolism is related to retinal cell layer thinning in rd1 mice.

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.

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.

GPR158 in the Visual System: Homeostatic Role in Regulation of Intraocular Pressure.

Purpose: GPR158 is a newly characterized family C G-protein-coupled receptor, previously identified in functional screens linked with biological stress, including one for susceptibility to ocular hypertension/glaucoma induced by glucocorticoid stress hormones. In this study, we investigated GPR158 function in the visual system. Methods: Gene expression and protein immunolocalization analyses were performed in mouse and human brain and eye to identify tissues where GPR158 might function. Gene expression was perturbed in mice, and in cultures of human trabecular meshwork cells of the aqueous outflow pathway, to investigate function and mechanism. Results:GPR158 is highly expressed in the brain, and in this study, we show prominent expression specifically in the visual center of the cerebral cortex. Expression was also observed in the eye, including photoreceptors, ganglion cells, and trabecular meshwork. Protein was also localized to the outer plexiform layer of the neural retina. Gpr158 deficiency in knockout (KO) mice conferred short-term protection against the intraocular pressure increase that occurred with aging, but this was reversed over time. Most strikingly, the pressure lowering effect of the acute stress hormone, epinephrine, was negated in KO mice. In contrast, no disruption of the electroretinogram was observed. Gene overexpression in cell cultures enhanced cAMP production in response to epinephrine, suggesting a mechanism for intraocular pressure regulation. Overexpression also increased survival of cells subjected to oxidative stress linked to ocular hypertension, associated with TP53 pathway activation. Conclusions: These findings implicate GPR158 as a homeostatic regulator of intraocular pressure and suggest GPR158 could be a pharmacological target for managing ocular hypertension.

Dynasore protects the ocular surface against damaging oxidative stress.

Water soluble "vital" dyes are commonly used clinically to evaluate health of the ocular surface; however, staining mechanisms remain poorly understood. Recent evidence suggests that sublethal damage stimulates vital dye uptake by individual living cells. Since cell damage can also stimulate reparative plasma membrane remodeling, we hypothesized that dye uptake occurs via endocytic vesicles. In support of this idea, we show here that application of oxidative stress to relatively undifferentiated monolayer cultures of human corneal epithelial cells stimulates both dye uptake and endocytosis, and that dye uptake is blocked by co-treatment with three different endocytosis inhibitors. Stress application to stratified and differentiated corneal epithelial cell cultures, which are a better model of the ocular surface, also stimulated dye uptake; however, endocytosis was not stimulated, and two of the endocytosis inhibitors did not block dye uptake. The exception was Dynasore and its more potent analogue Dyngo-4a, both small molecules developed to target dynamin family GTPases, but also having off-target effects on the plasma membrane. Significantly, while Dynasore blocked stress-stimulated dye uptake at the ocular surface of ex vivo mouse eyes when treatment was performed at the same time as eyes were stressed, it had no effect when used after stress was applied and the ocular surface was already damaged. Thus, Dynasore could not be working by inhibiting endocytosis. Employing cytotoxicity and western blotting assays, we went on to demonstrate an alternative mechanism. We show that Dynasore is remarkably protective of cells and their surface glycocalyx, preventing damage due to stress, and thus precluding dye entry. These unexpected and novel findings provide greater insight into the mechanisms of vital dye uptake and point the direction for future study. Significantly, they also suggest that Dynasore and its analogues might be used therapeutically to protect the ocular surface and to treat ocular surface disease.

Clusterin from human clinical tear samples: Positive correlation between tear concentration and Schirmer strip test results.

PURPOSE: To investigate the relationship between tear concentration of the homeostatic protein clusterin (CLU) and dry eye signs and symptoms, and to characterize tear CLU protein. METHODS: Two independent studies were conducted, one in Tucson (44 subjects), the other in Los Angeles (52 subjects). A cohort study design was employed to enroll patients without regard to dry eye diagnosis. Dry eye signs and symptoms were assessed using clinical tests. Tear samples were collected by Schirmer strip, and also by micropipette at slit lamp when possible. CLU from both sample types was quantified by immunoassay. The relationship between CLU concentration and clinical test scores was determined by Pearson's correlation coefficient (for individual eyes) and multiple linear regression analysis (including both eyes). CLU was also evaluated biochemically by western blotting. RESULTS: In the Tucson cohort, a positive correlation was observed between tear CLU concentration and results of the Schirmer strip test, a measure of tear flow (p = 0.021 includes both eyes). This result was corroborated in the Los Angeles cohort (p = 0.013). The mean tear CLU concentration was 31 ±â€¯14 µg/mL (n = 18 subjects, 33 eyes; range = 7-48 µg/mL). CLU from clinical tear samples appeared biochemically similar to CLU from a non-clinical tear sample and from blood plasma. CONCLUSIONS: Results support the hypothesis that an optimal concentration of tear CLU is important for ocular surface health, and that this drops below the effective threshold in dry eye. Tear CLU measurement might identify patients that could benefit from supplementation. Information about concentration will aid development of therapeutic dosage parameters.

Steroid-induced ocular hypertension/glaucoma: Focus on pharmacogenomics and implications for precision medicine.

Elevation of intraocular pressure (IOP) due to therapeutic use of glucocorticoids is called steroid-induced ocular hypertension (SIOH); this can lead to steroid-induced glaucoma (SIG). Glucocorticoids initiate signaling cascades ultimately affecting expression of hundreds of genes; this provides the potential for a highly personalized pharmacological response. Studies attempting to define genetic risk factors were undertaken early in the history of glucocorticoid use, however scientific tools available at that time were limited and progress stalled. In contrast, significant advances were made over the ensuing years in defining disease pathophysiology. As the genomics age emerged, it appeared the time was right to renew investigation into genetics. Pharmacogenomics is an unbiased discovery approach, not requiring an underlying hypothesis, and provides a way to pinpoint clinically significant genes and pathways that could not have been discovered any other way. Results of the first genome-wide association study to identify polymorphisms associated with SIOH, and follow-up on two novel genes linked to the disorder, GPR158 and HCG22, is discussed in the second half of the article. However, knowledge of genetic variants determining response to steroids in the eye also has value in its own right as a predictive and diagnostic tool. This article concludes with a discussion of how the Precision Medicine Initiative®, announced by U.S. President Obama in his 2015 State of the Union address, is beginning to touch the practice of ophthalmology. It is argued that SIOH/SIG may provide one of the next opportunities for effective application of precision medicine.

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.

Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye.

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.

Identification of a Novel Mucin Gene HCG22 Associated With Steroid-Induced Ocular Hypertension.

PURPOSE: 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. METHODS: 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. RESULTS: 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-ß. 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. CONCLUSIONS: 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.

Expression and functional role of orphan receptor GPR158 in prostate cancer growth and progression.

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.

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.

Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells.

BACKGROUND: DNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear. RESULTS: Here we show that G9a is not essential for maintenance of DNA methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to nucleosomes containing methylated DNA even in the absence of G9a, ensuring faithful propagation of methylated states in cooperation with DNMT1 through somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B and DNA methylation-independent manner. However, G9a knockdown synergizes with pharmacologic inhibition of DNMTs resulting in increased hypomethylation and inhibition of cell proliferation. CONCLUSIONS: Taken together, these data suggest that G9a is not involved in maintenance of DNA methylation in somatic cells but might play a role in re-initiation of de novo methylation after treatment with hypomethylating drugs, thus serving as a potential target for combinatorial treatments strategies involving DNMTs inhibitors.

A novel mechanism of increased infections in contact lens wearers.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.