Corneal epithelial development and homeostasis.

The corneal epithelium (CE), the most anterior cellular structure of the eye, is a self-renewing stratified squamous tissue that protects the rest of the eye from external elements. Each cell in this exquisite three-dimensional structure needs to have proper polarity and positional awareness for the CE to serve as a transparent, refractive, and protective tissue. Recent studies have begun to elucidate the molecular and cellular events involved in the embryonic development, post-natal maturation, and homeostasis of the CE, and how they are regulated by a well-coordinated network of transcription factors. This review summarizes the status of related knowledge and aims to provide insight into the pathophysiology of disorders caused by disruption of CE development, and/or homeostasis.

The secreted Ly-6/uPAR related protein-1 (SLURP1) stabilizes epithelial cell junctions and suppresses TNF-α-induced cytokine production.

The secreted Ly-6/uPAR related protein-1 (SLURP1) is an anti-angiogenic and anti-inflammatory peptide highly expressed by the mucosal epithelial cells. SLURP1 is abundantly expressed by the corneal epithelial cells and is significantly downregulated when these cells are transformed and adapted for culture in vitro. Here we studied the effect of overexpressing SLURP1 in Human Corneal Limbal Epithelial (HCLE) cells cultured in vitro. The expression of DSP1, DSG1, TJP1 and E-Cadherin was significantly upregulated in two different SLURP1-overexpressing HCLE cell (HCLE-SLURP1) clones. HCLE-SLURP1 cells also displayed a significant decrease in tumor necrosis factor-α (TNF-α)-induced upregulation of (i) IL-8 from 7.4- to 2.9- and 2.1-fold, (ii) IL-1ß from 4.9- to 3.9- and 2.9-fold, (iii) CXCL1 from 9- to 3.3- and 5.5-fold, and (iv) CXCL2 from 4.8- to 2.1- and 2.8-fold. ELISAs revealed a concomitant decrease in IL-8 levels in cell culture supernatants from 789 pg/ml in the control, to 503 and 352 pg/ml in HCLE-SLURP1 cells. Consistently, cytosolic IκB expression was elevated in HCLE-SLURP1 cells with a concurrent suppression of TNF-α-activated nuclear translocation of NF-κB. Collectively, these results elucidate the beneficial effects of SLURP1 in stabilizing the HCLE intercellular junctions and suppressing the TNF-α-induced upregulation of inflammatory cytokines by suppressing NF-κB nuclear translocation.

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

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-α (TNF-α)-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-α-induced (i) interleukin-8 (IL-8) and TNF-α production, (ii) adhesion to different components of the extracellular matrix, (iii) migration, and (iv) nuclear localization of NFκB. Together, these results demonstrate that SLURP1 suppresses HUVEC tube formation by blocking nuclear translocation of NFκB, and suggest a potential role for SLURP1 in promoting corneal angiogenic privilege.

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.

A CXCR3-Activating Peptide Increases Tear Break Up Time and Corrects Corneal haze in a Rabbit Model of Environmental Dry Eye.

Purpose: Environmentally-triggered dry eye disease (DED) or keratoconjunctivitis sicca (KCS), which constitutes the majority of DED cases, currently is palliatively treated with aqueous replacement solutions that do not target the dysfunction of the mucin and lipid components of tears. We tested whether a peptide that increased goblet cell numbers in a model of scleral chemical injury would also improve tear quality in environmental DED. Methods: Environmental DED was established by exposing New Zealand white rabbits (8 per group, female) to 20% humidity with rapid air replacement and b.i.d. atropine sulfate eyedrops for 3 weeks prior to test article administration; this continued for the subsequent 3 weeks of testing. Animals were dosed by (A) saline, (B) b.i.d. eyedrop of peptide in saline, (C) b.i.d. eyedrop of peptide in coacervate, or (D) weekly subconjunctival injection of peptide. In vitro, human conjunctival epithelial cells (HCjE) were exposed to TNFα in the presence or absence of peptide to determine inflammatory responsiveness. Results: The environmental DED was established with both Schirmer and TBUT being reduced at the start of test article; these levels were maintained as low through the testing period. All three treatment regimens increased TBUT approximately 3x to levels greater than prior to desiccation (P < 0.01), with little effect on Schirmer. Corneal haze was present in all eyes after induction, and completely reversed in 36 of 48 eyes across the treatments (P < 0.05). Co-treatment of HCjE with peptide reduced the production of TNFα in response to an inflammatory stimulus. Conclusions: The treatment of environmental DED/KCS with a peptide that activates CXCR3 improved tear quality and reversed corneal pathology by promoting tear stability and likely dampening the corneal inflammation, while not affecting aqueous volume of the tears.

The Secreted Ly6/uPAR-Related Protein 1 (Slurp1) Modulates Corneal Angiogenic Inflammation Via NF-κB Signaling.

Purpose: Previously we demonstrated that the secreted Ly-6/uPAR related protein 1 (SLURP1), abundantly expressed in the corneal epithelium (CE) and secreted into the tear fluid, serves as an antiangiogenic molecule. Here we describe the Slurp1-null (Slurp1X-/-) mouse corneal response to silver nitrate (AgNO3) cautery. Methods: Five days after AgNO3 cautery, we compared the wild-type (WT) and Slurp1X-/- mouse (1) corneal neovascularization (CNV) and immune cell influx by whole-mount immunofluorescent staining for CD31 and CD45, (2) macrophage and neutrophil infiltration by flow cytometry, and (3) gene expression by quantitative RT-PCR. Quantitative RT-PCR, immunofluorescent staining, and immunoblots were employed to evaluate the expression, phosphorylation status, and subcellular localization of NF-κB pathway components. Results: Unlike the WT, the Slurp1X-/- corneas displayed denser CNV in response to AgNO3 cautery, with more infiltrating macrophages and neutrophils and greater upregulation of the transcripts encoding VEGFA, MMP2, IL-1b, and vimentin. At 2, 7, and 10 days after AgNO3 cautery, Slurp1 expression was significantly downregulated in the WT corneas. Compared with the WT, naive Slurp1X-/- CE displayed increased phosphorylation of IKK(a/b), elevated phosphorylation of IκB with decreased amounts of total IκB, and higher phosphorylation of NF-κB, suggesting that NF-κB signaling is constitutively active in naive Slurp1X-/- corneas. Conclusions: Enhanced angiogenic inflammation in AgNO3 cauterized Slurp1X-/- corneas and constitutively active status of NF-κB signaling in the absence of Slurp1 suggest that Slurp1 modulates corneal angiogenic inflammation via NF-κB signaling.

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.

Molecular nature of ocular surface barrier function, diseases that affect it, and its relevance for ocular drug delivery.

The structural and functional integrity of the ocular surface, a continuous epithelial structure comprised of the cornea, the conjunctiva, and the ductal surface of the lacrimal as well as meibomian glands, is crucial for proper vision. The ocular surface barrier function (OSBF), sum of the different types of protective mechanisms that exist at the ocular surface, is essential to protect the rest of the eye from vision-threatening physical, chemical, and biological insults. OSBF helps maintain the immune privileged nature of the cornea and the aqueous humor by preventing entry of infectious agents, allergens, and noxious chemicals. Disruption of OSBF exposes the dense nerve endings of the cornea to these stimuli, resulting in discomfort and pain. This review summarizes the status of our knowledge related to the molecular nature of OSBF, describes the effect of different ocular surface disorders on OSBF, and examines the relevance of this knowledge for ocular drug delivery.

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.

Secreted Ly-6/uPAR-related protein-1 (SLURP1) is a pro-differentiation factor that stalls G1-S transition during corneal epithelial cell cycle progression.

PURPOSE: Previously we demonstrated that the secreted Ly-6/uPAR related protein-1 (SLURP1), abundantly expressed in the corneal epithelium (CE) and secreted into the tear fluid, serves as an anti-inflammatory and anti-angiogenic molecule. Here we describe the Slurp1-null (Slurp1X-/-) mouse corneal phenotype for the first time. METHODS: We compared the 10-week-old wild type (WT) and Slurp1X-/- mouse corneal (i) histology by hematoxylin-eosin and periodic acid-Schiff's reagent staining, (ii) cell proliferation by immunostaining for Ki67, (iii) cell adhesion molecules by immunostaining for desmosomal and tight junction proteins, (iv) barrier function by fluorescein staining and (v) wound-healing by epithelial debridement. Effect of SLURP1 on cell cycle was quantified in human corneal limbal epithelial (HCLE) cells engineered to express SLURP1 (HCLE-SLURP1). RESULTS: WT and Slurp1X-/- corneal histology was largely comparable, other than a few loosely attached superficial cells in Slurp1X-/- corneas. Compared with the WT, Slurp1X-/- corneas displayed (i) increase in Ki67+ cells, (ii) altered expression and/or localization of tight junction proteins Tjp1 and Pard3, and desmosomal Dsp, (iii) increased superficial fragility and (iv) slower CE wound healing. HCLE-SLURP1 cells displayed (i) decrease in Ki67+ cells, (ii) increased cell number doubling time, (iii) stalling in G1-S phase transition during cell cycle, and (iv) downregulation of cyclins CCNE and CCND1/D2, cyclin-dependent kinases CDK4 and CDK6, and upregulation of CDK inhibitor p15/CDKN2B. CONCLUSIONS: Collectively, these results elucidate that Slurp1X-/- CE cell homeostasis is altered and suggest that SLURP1 is a pro-differentiation factor that stalls G1-S transition during cell cycle progression by downregulating cyclins and upregulating p15/CDKN2B.

Krüppel-like factors: three fingers in control.

Krüppel-like factors (KLFs), members of the zinc-finger family of transcription factors capable of binding GC-rich sequences, have emerged as critical regulators of important functions all over the body. They are characterised by a highly conserved C-terminal DNA-binding motif containing three C2H2 zinc-finger domains, with variable N-terminal regulatory domains. Currently, there are 17 KLFs annotated in the human genome. In spite of their structural similarity to one another, the genes encoding different KLFs are scattered all over the genome. By virtue of their ability to activate and/or repress the expression of a large number of genes, KLFs regulate a diverse array of developmental events and cellular processes, such as erythropoiesis, cardiac remodelling, adipogenesis, maintenance of stem cells, epithelial barrier formation, control of cell proliferation and neoplasia, flow-mediated endothelial gene expression, skeletal and smooth muscle development, gluconeogenesis, monocyte activation, intestinal and conjunctival goblet cell development, retinal neuronal regeneration and neonatal lung development. Characteristic features, nomenclature, evolution and functional diversities of the human KLFs are reviewed here.

Conditional deletion of the mouse Klf4 gene results in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells.

The Krüppel-like transcription factor KLF4 is among the most highly expressed transcription factors in the mouse cornea (B. Norman, J. Davis, and J. Piatigorsky, Investig. Ophthalmol. Vis. Sci. 45:429-440, 2004). Here, we deleted the Klf4 gene selectively in the surface ectoderm-derived structures of the eye (cornea, conjunctiva, eyelids, and lens) by mating Klf4-LoxP mice (J. P. Katz, N. Perreault, B. G. Goldstein, C. S. Lee, P. A. Labosky, V. W. Yang, and K. H. Kaestner, Development 129:2619-2628, 2002) with Le-Cre mice (R. Ashery-Padan, T. Marquardt, X. Zhou, and P. Gruss, Genes Dev. 14:2701-2711, 2000). Klf4 conditional null (Klf4CN) embryos developed normally, and the adult mice were viable and fertile. Unlike the wild type, the Klf4CN cornea consisted of three to four epithelial cell layers; swollen, vacuolated basal epithelial and endothelial cells; and edematous stroma. The conjunctiva lacked goblet cells, and the anterior cortical lens was vacuolated in Klf4CN mice. Excessive cell sloughing resulted in fewer epithelial cell layers in spite of increased cell proliferation at the Klf4CN ocular surface. Expression of the keratin-12 and aquaporin-5 genes was downregulated, consistent with the Klf4CN corneal epithelial fragility and stromal edema, respectively. These observations provide new insights into the role of KLF4 in postnatal maturation and maintenance of the ocular surface and suggest that the Klf4CN mouse is a useful model for investigating ocular surface pathologies such as dry eye, Meesmann's dystrophy, and Steven's-Johnson syndrome.

Conjunctival goblet cells: Ocular surface functions, disorders that affect them, and the potential for their regeneration.

Conjunctival goblet cells (CGCs) are specialized cells that produce and secrete soluble mucins to the tear film that bathes the ocular surface. CGC numbers and functions are affected in various ocular surface diseases including dry eye disease with diverse etiologies. In this review we will (i) summarize the important functions of CGCs in ocular surface health, (ii) describe the ocular surface diseases that affect CGC numbers and function, (iii) provide an update on recent research outcomes that elucidate CGC differentiation, gene expression and functions, and (iv) present evidence in support of the prediction that restoring CGC numbers and/or functions is a viable strategy for alleviating ocular surface disorders that impact the CGCs.

KLF4 Coordinates Corneal Epithelial Apical-Basal Polarity and Plane of Cell Division and Is Downregulated in Ocular Surface Squamous Neoplasia.

Purpose: Previously, we demonstrated that Krüppel-like factor 4 (KLF4) promotes corneal epithelial (CE) homeostasis by suppressing epithelial-mesenchymal transition (EMT) and TGF-ß signaling. As TGF-ß affects epithelial apicobasal polarity (ABP) and plane of division, we investigated the role of KLF4 in these processes. Methods: Klf4 was ablated in adult ternary transgenic Klf4Δ/ΔCE (Klf4LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre) mouse CE using doxycycline chow. ABP and plane of division markers' expression in Klf4Δ/ΔCE and human ocular surface squamous neoplasia (OSSN) tissues relative to controls was evaluated by quantitative PCR, immunoblots, and/or immunofluorescent staining. Results: Klf4Δ/ΔCE CE cells displayed downregulation of apical Pals1 and Crumbs1, apicolateral Par3, and basolateral Scribble, as well as upregulation of Rho family GTPase Cdc42, suggesting disruption of ABP. Phalloidin staining revealed that the Klf4Δ/ΔCE CE actin cytoskeleton is disrupted. Klf4Δ/ΔCE cells favored vertical plane of division within 67.5° to 90° of the CE basement membrane (39% and 47% of the dividing cells relative to 23% and 26% in the control based on phospho-histone-H3 and survivin, respectively), resulting in more dividing cells within the Klf4Δ/ΔCE CE as reported previously. KLF4 was downregulated in human OSSN tissues that displayed EMT and downregulation of PAR3, PALS1, and SCRIB, consistent with a protective role for KLF4. Conclusions: By demonstrating that Klf4 ablation affects CE expression of ABP markers and Cdc42, cytoskeletal actin organization, and the plane of cell division and that KLF4 is downregulated in OSSN tissues that display EMT and lack ABP, these results elucidate the key integrative role of KLF4 in coordinating CE cell polarity and plane of division, loss of which results in OSSN.

BMP6 Regulates Corneal Epithelial Cell Stratification by Coordinating Their Proliferation and Differentiation and Is Upregulated in Pterygium.

Purpose: Proper balance between cell proliferation and differentiation is essential for corneal epithelial (CE) stratification and homeostasis. Although bone morphogenetic protein-6 (BMP6) is known to be expressed in the CE for over 25 years, its function in this tissue remains unknown. Here, we test the hypothesis that BMP6 promotes CE cell stratification and homeostasis by regulating their proliferation and differentiation. Methods: We employed postnatal day-12 (PN-12), PN-14, PN-20, and PN-90 mouse eyes; human corneal limbal epithelial (HCLE) cells; and ocular surface fibrovascular disease pterygium tissues to evaluate the role of BMP6 in CE proliferation, differentiation, and pathology by RT-qPCR, immunoblots, and/or immunofluorescent staining. Cell proliferation was quantified by immunostaining for Ki67. Results: Coincident with the mouse CE stratification between PN-12 and PN-20, BMP6 was significantly upregulated and the BMP6 antagonist Noggin downregulated. Mature CE retained high BMP6 and low Noggin expression at PN-90. BMP6 and its receptors BMPR1A and BMPR2 were upregulated during in vitro stratification of HCLE cells. Consistent with its anti-proliferative role, exogenous BMP6 suppressed HCLE cell proliferation, downregulated cyclin-D1 and cyclin-D2, and upregulated cell-cycle inhibitors Krüppel-like factor 4 (KLF4) and p21. BMP6 also upregulated the desmosomal cadherins desmoplakin and desmoglein in HCLE cells, consistent with its pro-differentiation role. Human pterygium displayed significant upregulation of BMP6 coupled with downregulation of Noggin and cell-cycle suppressors KLF4 and p21. Conclusions: BMP6 coordinates CE stratification and homeostasis by regulating their proliferation and differentiation. BMP6 is significantly upregulated in human pterygium concurrent with downregulation of Noggin, KLF4, and p21.

The secreted Ly6/uPAR-related protein-1 suppresses neutrophil binding, chemotaxis, and transmigration through human umbilical vein endothelial cells.

The secreted Ly-6/uPAR Related Protein-1 (SLURP1) is an immunomodulatory protein that promotes corneal immune- and angiogenic-privilege. Here, we have examined the influence of SLURP1 on neutrophil-vascular endothelial cell interactions using human umbilical vein endothelial cells (HUVEC) and differentiated neutrophil-like HL-60 (dHL-60) cells, or primary human neutrophils. SLURP1 blocked the tumor necrosis factor-alpha (TNF-α)-activated dHL-60 cells (i) binding to TNF-α-activated HUVEC with a concurrent reduction in endothelial cell adhesion molecule E-selectin, (ii) transmigration through TNF-α-activated confluent HUVEC monolayer by stabilizing VE-cadherin and ß-catenin on endothelial cell cytoplasmic membranes, (iii) chemotaxis towards chemoattractant formyl Met-Leu-Phe (fMLP) coupled with their decreased polarization, and (iv) TNF-α-stimulated matrix metalloproteinase-9 (MMP9) expression and activity. SLURP1 also suppressed the primary human neutrophil chemotaxis, and interaction with HUVEC. Furthermore, SLURP1 suppressed fMLP-induced phosphorylation of protein kinase-B (AKT) in dHL-60 cells. Collectively, these results provide evidence that SLURP1 suppresses neutrophil (i) docking on HUVEC cells by decreasing endothelial cell adhesion molecule E-Selectin production, (ii) transmigration through HUVEC monolayer by stabilizing endothelial cell membrane localization of VE-cadherin and ß-catenin complex and promoting their barrier function, and (iii) chemotaxis by modulating their polarization and TNF-α-stimulated MMP9 production.

KLF4 Regulates Corneal Epithelial Cell Cycle Progression by Suppressing Canonical TGF-ß Signaling and Upregulating CDK Inhibitors P16 and P27.

Purpose: Krüppel-like factor 4 (KLF4) promotes corneal epithelial (CE) cell fate while suppressing mesenchymal properties. TGF-ß plays a crucial role in cell differentiation and development, and if dysregulated, it induces epithelial-mesenchymal transition (EMT). As KLF4 and TGF-ß regulate each other in a context-dependent manner, we evaluated the role of the crosstalk between KLF4 and TGF-ß-signaling in CE homeostasis. Methods: We used spatiotemporally regulated ablation of Klf4 within the adult mouse CE in ternary transgenic Klf4Δ/ΔCE (Klf4LoxP/LoxP/ Krt12rtTA/rtTA/ Tet-O-Cre) mice and short hairpin RNA (shRNA)-mediated knockdown or lentiviral vector-mediated overexpression of KLF4 in human corneal limbal epithelial (HCLE) cells to evaluate the crosstalk between KLF4 and TGF-ß-signaling components. Expression of TGF-ß signaling components and cyclin-dependent kinase (CDK) inhibitors was quantified by quantitative PCR, immunoblots, and/or immunofluorescent staining. Results: CE-specific ablation of Klf4 resulted in (1) upregulation of TGF-ß1, -ß2, -ßR1, and -ßR2; (2) downregulation of inhibitory Smad7; (3) hyperphosphorylation of Smad2/3; (4) elevated nuclear localization of phospho-Smad2/3 and Smad4; and (5) downregulation of CDK inhibitors p16 and p27. Consistently, shRNA-mediated knockdown of KLF4 in HCLE cells resulted in upregulation of TGF-ß1 and -ß2, hyperphosphorylation and nuclear localization of SMAD2/3, downregulation of SMAD7, and elevated SMAD4 nuclear localization. Furthermore, overexpression of KLF4 in HCLE cells resulted in downregulation of TGF-ß1, -ßR1, and -ßR2 and upregulation of SMAD7, p16, and p27. Conclusions: Collectively, these results demonstrate that KLF4 regulates CE cell cycle progression by suppressing canonical TGF-ß signaling and overcomes the undesirable concomitant decrease in TGF-ß-dependent CDK inhibitors p16 and p27 expression by directly upregulating them.

Cubozoan crystallins: evidence for convergent evolution of pax regulatory sequences.

Cnidaria is the earliest-branching metazoan phylum containing a well-developed, lens-containing visual system located on specialized sensory structures called rhopalia. Each rhopalium in a cubozoan jellyfish Tripedalia cystophora has a large and a small complex, camera-type eye with a cellular lens containing distinct families of crystallins. Here, we have characterized J2-crystallin and its gene in T. cystophora. The J2-crystallin gene is composed of a single exon and encodes a 157-amino acid cytoplasmic protein with no apparent homology to known proteins from other species. The non-lens expression of J2-crystallin suggests nonoptical as well as crystallin functions consistent with the gene-sharing strategy that has been used during evolution of lens crystallins in other invertebrates and vertebrates. Although nonfunctional in transfected mammalian lens cells, the J2-crystallin promoter is activated by the jellyfish paired domain transcription factor PaxB in co-transfection tests via binding to three paired domain sites. PaxB paired domain-binding sites were also identified in the PaxB-regulated promoters of the J1A- and J1B-crystallin genes, which are not homologous to the J2-crystallin gene. Taken together with previous studies on the regulation of the diverse crystallin genes, the present report strongly supports the idea that crystallin recruitment of multifunctional proteins was driven by convergent changes involving Pax (as well as other transcription factors) in the promoters of nonhomologous genes within and between species as well as within gene families.

Regulation of the mouse alphaB-crystallin and MKBP/HspB2 promoter activities by shared and gene specific intergenic elements: the importance of context dependency.

The closely linked (863 bp), divergently arranged mouse myotonic dystrophy kinase binding protein (Mkbp)/HspB2 and small heat shock protein (shsp)/alphaB-crystallin genes have different patterns of tissue-specific expression. We showed previously that an intergenic enhancing region (-436/-257 relative to alphaB-crystallin transcription start site) selectively activates the alphaB-crystallin promoter in an orientation-dependent manner (Swamynathan, S.K. and J. Piatigorsky 2002. J. Biol. Chem. 277:49700-6). Here we show that cis-elements alphaBE1 (-420/-396) and alphaBE3 (-320/-300) functionally interact with glucocorticoid receptor (GR) and Sp1, respectively, both in vitro and in vivo. alphaBE1:GR regulates both the HspB2 and alphaB-crystallin promoters, while alphaBE3:Sp1 selectively regulates the alphaB-crystallin promoter, as judged by mutagenesis and co-transfection tests. Enhancer blocking assays indicate that the -836/-622 fragment can act as a negative regulator in transfection tests, raising the possibility that it contributes to the differential expression of the proximal HspB2 promoter and distal alphaB-crystallin promoter. Finally, experiments utilizing transiently transfected cells and transgenic mice show that two conserved E-box elements (-726/-721 and -702/-697) bind nuclear proteins and differentially regulate the HspB2 and alphaB-crystallin promoters in a tissue-specific manner. Taken together, our results indicate that the linked, differentially expressed HspB2 and alphaB-crystallin genes have evolved shared and promoter-preferred cis-control elements within the intergenic sequence. The context-dependency of cis-elements provides multiple opportunities for evolutionary novelty by small sequence changes.

Spatiotemporally Controlled Ablation of Klf5 Results in Dysregulated Epithelial Homeostasis in Adult Mouse Corneas.

Purpose: Corneal epithelial (CE) homeostasis requires coordination between proliferation and differentiation. Here we examine the role of cell proliferation regulator Krüppel-like factor 5 (Klf5) in adult mouse CE homeostasis. Methods: Klf5 was ablated in a spatiotemporally restricted manner by inducing Cre expression in 8-week-old ternary transgenic Klf5LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre (Klf5Δ/ΔCE) mouse CE by administering doxycycline via chow. Normal chow-fed ternary transgenic siblings served as controls. The control and Klf5Δ/ΔCE corneal (1) histology, (2) cell proliferation, and (3) Klf5-target gene expression were examined using (1) periodic acid Schiff reagent-stained sections, (2) Ki67 expression, and (3) quantitative PCR and immunostaining, respectively. The effect of KLF4, KLF5, and OCT1 on gastrokine-1 (GKN1) promoter activity was determined by transient transfection in human skin keratinocyte NCTC-2544 cells. Results: Klf5 expression was decreased to 23% of the controls in Klf5Δ/ΔCE corneas, which displayed increased fluorescein uptake, downregulation of tight junction proteins Tjp1 and Gkn1, desmosomal Dsg1a, and basement membrane Lama3 and Lamb1, suggesting defective permeability barrier. In transient transfection assays, KLF5 and OCT1 synergistically stimulated GKN1 promoter activity. Klf5Δ/ΔCE CE displayed significantly fewer cell layers and Ki67+ proliferative cells coupled with significantly decreased cyclin-D1, and elevated phospho(Ser-10) p27/Kip1 expression. Expression of Krt12, E-cadherin, and ß-catenin remained unaltered in Klf5Δ/ΔCE corneas. Conclusions: Klf5 contributes to adult mouse CE homeostasis by promoting (1) permeability barrier function through upregulation of Tjp1, Gkn1, Dsg1a, Lama3, and Lamb1, and (2) basal cell proliferation through upregulation of cyclin-D1 and suppression of phospho(Ser-10) p27/Kip1, without significantly affecting the expression of epithelial markers Krt12, E-cadherin, and ß-catenin.