Sudha Swamynathan

Conditional disruption of mouse Klf5 results in defective eyelids with malformed meibomian glands, abnormal cornea and loss of conjunctival goblet cells.

Members of the Krüppel-like family of transcription factors regulate diverse developmental processes in various organs. Previously, we have demonstrated the role of Klf4 in the mouse ocular surface. Herein, we determined the role of the structurally related Klf5, using Klf5-conditional null (Klf5CN) mice derived by mating Klf5-LoxP and Le-Cre mice. Klf5 mRNA was detected as early as embryonic day 12 (E12) in the cornea, conjunctiva and eyelids, wherein its expression increased during development. Though the embryonic eye morphogenesis was unaltered in the Klf5CN mice, postnatal maturation was defective, resulting in smaller eyes with swollen eyelids that failed to separate properly. Klf5CN palpebral epidermis was hyperplastic with 7-9 layers of keratinocytes, compared with 2-3 in the wild type (WT). Klf5CN eyelid hair follicles and sebaceous glands were significantly enlarged, and the meibomian glands malformed. Klf5CN lacrimal glands displayed increased vasculature and large number of infiltrating cells. Klf5CN corneas were translucent, thicker with defective epithelial basement membrane and hypercellular stroma. Klf5CN conjunctiva lacked goblet cells, demonstrating that Klf5 is required for conjunctival goblet cell development. The number of Ki67-positive mitotic cells was more than doubled, consistent with the increased number of Klf5CN ocular surface epithelial cells. Co-ablation of Klf4 and Klf5 resulted in a more severe ocular surface phenotype compared with Klf4CN or Klf5CN, demonstrating that Klf4 and Klf5 share few if any, redundant functions. Thus, Klf5CN mice provide a useful model for investigating ocular surface pathologies involving meibomian gland dysfunction, blepharitis, corneal or conjunctival defects.

Organization, evolution and functions of the human and mouse Ly6/uPAR family genes

Members of the lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) superfamily of proteins are cysteine-rich proteins characterized by a distinct disulfide bridge pattern that creates the three-finger Ly6/uPAR (LU) domain. Although the Ly6/uPAR family proteins share a common structure, their expression patterns and functions vary. To date, 35 human and 61 mouse Ly6/uPAR family members have been identified. Based on their subcellular localization, these proteins are further classified as GPI-anchored on the cell membrane, or secreted. The genes encoding Ly6/uPAR family proteins are conserved across different species and are clustered in syntenic regions on human chromosomes 8, 19, 6 and 11, and mouse Chromosomes 15, 7, 17, and 9, respectively. Here, we review the human and mouse Ly6/uPAR family gene and protein structure and genomic organization, expression, functions, and evolution, and introduce new names for novel family members.

Klf4 Regulates the Expression of Slurp1, Which Functions as an Immunomodulatory Peptide in the Mouse Cornea

PURPOSE The secreted Ly6/uPAR-related protein-1 (Slurp1), associated with the hyperkeratotic disorder mal de Meleda, is abundantly expressed in corneas. Here, we examine its corneal expression and functions. METHODS Gene expression was quantified by quantitative PCR (qPCR), immunoblots, and immunofluorescent staining. Effect of Kruppel-like factor 4 (Klf4) on Slurp1 promoter was evaluated by chromatin immunoprecipitation (ChIP) and transient transfections. Adenoviral vectors were used to express Slurp1 in corneas. Leukocytic infiltration in bacterial lipopolysaccharide (LPS)-, herpes simplex virus type 1 (HSV-1)-, or adenovirus (serotype 5)-treated mouse corneas was characterized by flow cytometry. RESULTS Corneal expression of Slurp1 increased sharply upon mouse eyelid opening, concurrent with the elevated expression of Klf4. Slurp1 was significantly decreased in Klf4 conditional null (Klf4CN) corneas that displayed elevated expression of cytokines and cytokine receptors, as well as neutrophil influx consistent with a proinflammatory environment. In additional models of corneal inflammation, Slurp1 expression was abrogated within 24 hours of LPS injection or HSV-1 or adenoviral infection, accompanied by a predominantly neutrophilic infiltrate. Neutrophilic infiltration was enhanced in HSV-1-infected Klf4CN corneas lacking Slurp1. SLURP1 promoter activity was stimulated by KLF4, suppressed by IL-4, IL-13, and TNFα, and unperturbed by IFN-γ. Slurp1 downregulation and neutrophil influx were comparable in HSV-1-infected wild-type (WT) and Ifng-/- mouse corneas. Mouse corneas infected with Slurp1-expressing adenoviral vectors displayed reduced signs of inflammation and restricted neutrophilic infiltration compared with those infected with control vectors. CONCLUSIONS Klf4 regulates the expression of Slurp1, a key immunomodulatory peptide that is abundantly expressed in healthy corneas and is downregulated in proinflammatory conditions.

Regulation of Corneal Epithelial Barrier Function by Krüppel-like Transcription Factor 4

PURPOSE Previously, the authors showed that Klf4-conditional null (Klf4CN) corneas display epithelial fragility. Here, they investigated the mechanism by which Klf4 regulates corneal epithelial barrier function. METHODS Klf4CN mice were generated by breeding Le-Cre with Klf4-LoxP mice. Fluorescein staining was used to test the corneal barrier function. RT-PCR, immunoblots, and immunofluorescence were used to detect the expression of cell junctional proteins. The effect of Klf4 on promoter activities was measured by transient cotransfection assays. Trans-epithelial electrical resistance (TEER) was used to measure the barrier-forming ability of control or anti-KLF4 siRNA-treated cells. RESULTS Increased fluorescein staining and decreased tight junction protein Tjp1 expression demonstrated that the Klf4CN corneal epithelial barrier function is defective. Expression of desmosomal components Dsp, Dsg-1a, and Dsg-1b was downregulated in the Klf4CN corneas, and their corresponding promoter activities were upregulated by Klf4 in transient cotransfection assays. Hemidesmosomal α3- and β4-integrin levels were not affected even though there were fewer hemidesmosomes in the Klf4CN corneas. The basement membrane components laminin-α5, -α3, -β3, and -β1-1 were downregulated, suggesting that the disrupted basement membrane is responsible for fewer hemidesmosomes in the Klf4CN cornea. Tight junction proteins OCLN1 and TJP1were downregulated in anti-KLF4 siRNA-treated cells, which failed to develop epithelial barrier function as measured by TEER. CONCLUSIONS Klf4 contributes to corneal epithelial barrier function by upregulating the expression of functionally related subsets of cell junctional proteins and basement membrane components.

Critical Role of Klf5 in Regulating Gene Expression during Post-Eyelid Opening Maturation of Mouse Corneas

Background Klf5 plays an important role in maturation and maintenance of the mouse ocular surface. Here, we quantify WT and Klf5-conditional null (Klf5CN) corneal gene expression, identify Klf5-target genes and compare them with the previously identified Klf4-target genes to understand the molecular basis for non-redundant functions of Klf4 and Klf5 in the cornea. Methodology/Principal Findings Postnatal day-11 (PN11) and PN56 WT and Klf5CN corneal transcriptomes were quantified by microarrays to compare gene expression in maturing WT corneas, identify Klf5-target genes, and compare corneal Klf4- and Klf5-target genes. Whole-mount corneal immunofluorescent staining was employed to examine CD45+ cell influx and neovascularization. Effect of Klf5 on expression of desmosomal components was studied by immunofluorescent staining and transient co-transfection assays. Expression of 714 and 753 genes was increased, and 299 and 210 genes decreased in PN11 and PN56 Klf5CN corneas, respectively, with 366 concordant increases and 72 concordant decreases. PN56 Klf5CN corneas shared 241 increases and 98 decreases with those previously described in Klf4CN corneas. Xenobiotic metabolism related pathways were enriched among genes decreased in Klf5CN corneas. Expression of angiogenesis and immune response-related genes was elevated, consistent with neovascularization and CD45+ cell influx in Klf5CN corneas. Expression of 1574 genes was increased and 1915 genes decreased in WT PN56 compared with PN11 corneas. Expression of ECM-associated genes decreased, while that of solute carrier family members increased in WT PN56 compared with PN11 corneas. Dsg1a, Dsg1b and Dsp were down-regulated in Klf5CN corneas and their corresponding promoter activities were stimulated by Klf5 in transient co-transfection assays. Conclusions/Significance Differences between PN11 and PN56 corneal Klf5-target genes reveal dynamic changes in functions of Klf5 during corneal maturation. Klf5 contributes to corneal epithelial homeostasis by regulating the expression of desmosomal components. Klf4- and Klf5-target genes are largely distinct, consistent with their non-redundant roles in the mouse cornea.

SLURP-1 Modulates Corneal Homeostasis by Serving as a Soluble Scavenger of Urokinase-Type Plasminogen Activator

PURPOSE Our previous study revealed the immunomodulatory property of the secreted lymphocyte antigen (Ly6)/urokinase-type plasminogen activator receptor (uPAR)-related protein-1 (SLURP1), abundantly expressed in the cornea and associated with the hyperkeratotic disorder Mal de Meleda. Here, we test the hypothesis that SLURP1 modulates the functions of membrane-tethered uPAR by acting as a soluble scavenger of its ligand urokinase-type plasminogen activator (uPA). METHODS Human corneal limbal epithelial (HCLE) and mouse corneal stromal fibroblast MK/T-1 cells were employed to examine the effect of SLURP1 on cell proliferation and migration. Human corneal limbal epithelial cell clones stably expressing SLURP1 under the control of cytomegalovirus (CMV) promoter were generated using lentiviral vectors. Recombinant 6× His-mouse Slurp1 and maltose-binding protein (MBP)-mouse uPA were expressed in Escherichia coli and partially purified using nickel-ion and amylose columns, respectively. Slurp1 interaction with uPA was detected using ligand blots, ELISA, pull-down assays, and immunofluorescent staining. RESULTS Stable expression of SLURP1 in HCLE cells was confirmed by immunoblots and immunofluorescent staining. Human corneal limbal epithelial and MK/T-1 cell proliferation and migration rates were suppressed by exogenous SLURP1. Ligand blots, ELISA, and pull-down assays indicated that Slurp1 efficiently interacts with uPA. Immunofluorescent staining demonstrated that exogenous SLURP1 decreased the amount of cell surface-bound uPA in the leading edges of migrating cells. In gap-filling assays, wild-type HCLE cells responded to uPA by increasing their velocity and closing larger area, while the SLURP1-expressing HCLE cells failed to do so. CONCLUSIONS SLURP1 modulates corneal homeostasis by serving as a soluble scavenger of uPA and regulating the uPA-dependent functions of uPAR.

Regulation of mouse lens maturation and gene expression by Krüppel-like factor 4.

Conditional disruption of Klf4 in the surface ectoderm-derived tissues of the eye results in defective cornea, conjunctiva and the lens. This report describes the effects of disruption of Klf4 in the lens in greater detail. Expression of Klf4, first detected in the embryonic day-12 (E12) mouse lens, peaked at E16 and was decreased in later stages. Early embryonic disruption of Klf4 resulted in a smaller lens with cortical vacuolation and nuclear opacity. Microarray comparison of Klf4CN and WT lens transcriptomes revealed fewer changes in the E16.5 (59 increases, 20 decreases of >1.5-fold) than the PN56 Klf4CN lens (239 increases, 182 decreases of >2-fold). Klf4-target genes in the lens were distinct from those previously identified in the cornea, suggesting disparate functions for Klf4 in these functionally related tissues. Transcripts encoding different crystallins were down-regulated in the Klf4CN lens. Shsp/αB-crystallin promoter activity was stimulated upon co-transfection with pCI-Klf4. Mitochondrial density was significantly higher in the Klf4CN lens epithelial cells, consistent with mitochondrial dysfunction being the most significantly affected pathway within the PN56 Klf4CN lens. The Klf4CN lens contained elevated levels of Alox12 and Alox15 transcripts, less reduced glutathione (GSH) and more oxidized glutathione (GSSG) than the WT, suggesting that it is oxidatively stressed. Although the expression of 2087 genes was modulated during WT lens maturation, transcripts encoding crystallins were abundant at E16.5 and remained stable at PN56. Among the 1065 genes whose expression increased during WT lens maturation, there were 104 Klf4-target genes (9.8%) with decreased expression in the PN56 Klf4CN lens. Taken together, these results demonstrate that Klf4 expression is developmentally regulated in the mouse lens, where it controls the expression of genes associated with lens maturation and redox homeostasis.

Corneal Expression of SLURP-1 by Age, Sex, Genetic Strain, and Ocular Surface Health

PURPOSE Although secreted Ly6/urokinase-type plasminogen activator receptor-related protein-1 (Slurp1) transcript is highly abundant in the mouse cornea, corresponding protein expression remains uncharacterized. Also, SLURP1 was undetected in previous tear proteomics studies, resulting in ambiguity about its baseline levels. Here, we examine mouse corneal Slurp1 expression in different sexes, age groups, strains, and health conditions, and quantify SLURP1 in human tears from healthy or inflamed ocular surfaces. METHODS Expression of Slurp1 in embryonic day-13 (E13), E16, postnatal day-1 (PN1), PN10, PN20, and PN70 Balb/C, FVBN, C57Bl/6, and DBA/2J mouse corneas, Klf4Δ/ΔCE corneas with corneal epithelial-specific ablation of Klf4, migrating cells in wild-type corneal epithelial wound edge, and in corneas exposed to pathogen-associated molecular patterns (PAMPs) poly(I:C), zymosan-A, or Pam3Csk4 was examined by QPCR, immunoblots, and immunofluorescent staining. Human SLURP1 levels were quantified by ELISA in tears from 34 men and women aged 18 to 80 years. RESULTS Expression of Slurp1, comparable in different strains and sexes, was low in E13, E16, PN1, and PN10 mouse corneas, and increased rapidly after eyelid opening in a Klf4-dependent manner. We found Slurp1 was downregulated in corneas exposed to PAMPs, and in migrating cells at the wound edge. Human SLURP1 expression, comparable in different sexes and age groups, was significantly decreased in tears from inflamed ocular surfaces (0.34%) than those from healthy individuals (0.77%). CONCLUSIONS These data describe the influence of age, sex, genetic background, and ocular surface health on mouse corneal expression of Slurp1, establish the baseline for human tear SLURP1 expression, and identify SLURP1 as a useful diagnostic and/or therapeutic target for inflammatory ocular surface disorders.

KLF4 Plays an Essential Role in Corneal Epithelial Homeostasis by Promoting Epithelial Cell Fate and Suppressing Epithelial–Mesenchymal Transition

Purpose The purpose of this study was to test the hypothesis that KLF4 promotes corneal epithelial (CE) cell fate by suppressing the epithelial–mesenchymal transition (EMT), using spatiotemporally regulated CE-specific ablation of Klf4 in Klf4Δ/ΔCE (Klf4LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre) mice. Methods CE-specific ablation of Klf4 was achieved by feeding Klf4Δ/ΔCE mice with doxycycline chow. The wild-type (WT; normal chow-fed littermates) and the Klf4Δ/ΔCE histology was compared by hematoxylin and eosin–stained sections; EMT marker expression was quantified by quantitative PCR, immunoblots, and immunofluorescent staining; and wound healing rate was measured by CE debridement using Algerbrush. KLF4 and EMT markers were quantified in human corneal limbal epithelial (HCLE) cells undergoing TGF-β1–induced EMT by quantitative PCR, immunoblots, and immunofluorescent staining. Results The epithelial markers E-cadherin, Krt12, claudin-3, and claudin-4 were down-regulated, whereas the mesenchymal markers vimentin, β-catenin, survivin, and cyclin-D1 and the EMT transcription factors Snail, Slug, Twist1, Twist2, Zeb1, and Zeb2 were up-regulated in the Klf4Δ/ΔCE corneas. The Klf4Δ/ΔCE cells migrated faster, filling 93% of the debrided area within 16 hours compared with 61% in the WT. After 7 days of wounding, the Klf4Δ/ΔCE cells that filled the gap failed to regain epithelial characteristics, as they displayed abnormal stratification; down-regulation of E-cadherin and Krt12; up-regulation of β-catenin, survivin, and cyclin-D1; and a 2.5-fold increase in the number of proliferative Ki67+ cells. WT CE cells at the migrating edge and the HCLE cells undergoing TGF-β1–induced EMT displayed significant down-regulation of KLF4. Conclusions Collectively, these results reveal that KLF4 plays an essential role in CE homeostasis by promoting epithelial cell fate and suppressing EMT.

Inhibition of HUVEC tube formation via suppression of NFκB suggests an anti-angiogenic role for SLURP1 in the transparent cornea

ABSTRACT Previously, we have reported that the Secreted Ly6/uPAR related protein‐1 (SLURP1) serves an important immunomodulatory function in the ocular surface. Here, we examine the involvement of SLURP1 in regulating corneal angiogenic privilege. Slurp1 expression detected by QPCR, immunoblots and immunofluorescent stain, was significantly decreased in mouse corneas subjected to alkali burn‐induced corneal neovascularization (CNV). Addition of exogenous SLURP1 (6XHis‐tagged, E. coli expressed and partially purified using Ni‐ion columns) significantly suppressed the tumor necrosis factor‐&agr; (TNF‐&agr;)‐stimulated human umbilical cord vascular endothelial cell (HUVEC) tube formation. SLURP1 suppressed the HUVEC tube length, tube area and number of branch points, without affecting their viability and/or proliferation. Exogenous SLURP1 in HUVEC also suppressed the TNF‐&agr;‐induced (i) interleukin‐8 (IL‐8) and TNF‐&agr; production, (ii) adhesion to different components of the extracellular matrix, (iii) migration, and (iv) nuclear localization of NF&kgr;B. Together, these results demonstrate that SLURP1 suppresses HUVEC tube formation by blocking nuclear translocation of NF&kgr;B, and suggest a potential role for SLURP1 in promoting corneal angiogenic privilege. HighlightsSlurp1 is downregulated during alkali burn‐induced corneal neovascularization.SLURP1 blocks HUVEC tube formation without affecting viability or proliferation.SLURP1 suppresses TNF‐&agr;‐induced expression of IL‐8 and TNF‐&agr; in HUVEC.SLURP1 modulates HUVEC interaction with the ECM, and their migration.SLURP1 suppresses TNF‐&agr;‐stimulated nuclear translocation of NF&kgr;B.