Mouse Corneal Epithelium Research Articles

Hypoxia affects in vitro proliferation and differentiation of mouse corneal epithelial progenitor cell

This study was to investigate the proliferation and differentiation of mouse corneal epithelial progenitor cell in hypoxic airlift culture. Mouse corneal epithelial progenitor cell line progenitor cells were cultured under airlift with normoxic and hypoxic conditions for various durations up to 2wk. Under normoxic conditions when exposed to air, the hyperproliferation and abnormal epidermal-like differentiation of mouse corneal epithelium was induced, whereas when exposed to air under hypoxic conditions, although we observed augmented proliferation, the abnormal differentiation was inhibited. The mechanism by which hypoxia prevents abnormal differentiation may involve downregulation of Wnt signaling pathways, which were inhibited in cells cultured with hypoxic airlift technique. In conclusion, hypoxia can prevent abnormal differentiation while enhancing the proliferation of corneal epithelial cells by blocking Wnt/β-catenin signaling pathway.

Evaluation of dry eye syndrom in C57BL/6.NOD-Aec1Aec2 mice

Background C57BL/6. NOD-AeclAec2 mouse is considered to be an idea model for the study of the pathogenesis of Sjtigren syndrome,but the cause of dry eye in these mice is unclear. Objective This study was to investigate the histopathological change of the ocular surfaces of C57BL/6. NOD-AeclAec2 mice, and to determine whether dry eye is developed spontaneously in these mice. Methods Forty-five clean C57BL/6. NOD- AeclAee2 mice were used as the experiment group and forty-five C57BL/6J mice（ both male and female）were used as the control group in this study. Detection of fasting blood-glucose, Schirmer＇ s test I （S I t） , lissamine green staining and scoring of the corneal and conjunctival epithelium in the mice were performed at the age of 4,8,12,16 and 20 weeks. Five mice from each group were sacrificed and their corneas were obtained to measure the central corneal epithelium thickness and to count the number of conjunctival goblet cells. In addition,lymphocyte infiltration in the lacrimal gland of the mice was examined with hematoxylin-eosin staining. The ultrastructure of the corneal epithelial cells and microvilli were assessed by scanning electron microscopy. The use and care of the mice were approved by the Experimental Animal Care Committee of the Third Military Medical University. Results No sign of dry eye wasseen in both the 4-week-old C57BL/6. NOD-AeclAec2 mice and 4-week-old C57BL/6J mice. The S I t values in 8- week-old,12-week-old,16-week-old and 20 week-old mice from the experiment group were（ 2.7±0. 9 ）mm, （2.5 ± 0.8 ）mm, （1.8±0.6）mm and（ 1.9±0. 1 ）mm, respectively, showing a significant reduction in comparison with those of the control mice of the same age（ all P〈0.01 ）. The amount of lissamine green staining in the C57BL/6. NOD- AeclAec2 mice gradually increased with age, showing elevated scores in 12-week-old, 16-week-old and 20-week-old mice in the experiment group （all P〈0.01 ）. The central corneal epithelium thicknesses were （20. 18 ± 3.75 ） μm, （ 17.01±5.25 ）μm, （ 14. 19±5.72 ）μm and （ 12.00±3.25 ）μm in the 8-week-old, 12-week-old, 16-week-old and 20- week-old C57BL/6. NOD-AeclAee2 mice, respectively, which were significantly lower than those of the C57BL/6J mice of the same age（all P〈0. 01 ）. The numbers of conjunctival goblet cells were（8.2±2.4） , （6.2±2. 1 ） , （6.1± 2.2） and（4.1±2.0） in the 8-week-old, 12-week-old, 16-week-old and 20-week-old C57BL/6. NOD-AeclAec2 mice, respectively,showing a gradual decrease with age and a significant decline in comparison with those of the C57BL/6J mice of the same age（ all P〈0. 01 ）. Lymphocyte infiltration in the lacrimal gland and destruction of gland ducts were seen by hematoxylin-eosin staining,and acinar abnormality aggravated with aging. Reduction of corneal epithelial cells and the number of mierovilli were distinguished with aging under the scanning electron microscope. The fasting blood- glucose levels of the two groups were both less than 6.0 mmol/L, and no significant difference was found between them at any age（P=0. 637,0. 610,0. 163,0. 086,0. 938）. Conclusions C57BL/6. NOD-AeelAec2 mice develop dry eye spontaneously with aging. The course of disease and characteristics of dry eye in C57BL/6. NOD-AeelAec2 mice is similar to human dry eye. The C57BL/6NOD-Aecl Aec2 mouse is the perfect model to study the pathogenesis of dry eye. Key words: Sjogren syndrome; Dry eye; C57BL/6. NOD-AeclAec2 mouse; Histopathology; Inflammation

Transcriptomic Analysis of PNN- and ESRP1-Regulated Alternative Pre-mRNA Splicing in Human Corneal Epithelial Cells

We investigated the impact of PININ (PNN) and epithelial splicing regulatory protein 1 (ESRP1) on alternative pre-mRNA splicing in the corneal epithelial context. Isoform-specific RT-PCR assays were performed on wild-type and Pnn knockout mouse cornea. Protein interactions were examined by deconvolution microscopy and co-immunoprecipitation. For genome-wide alternative splicing study, immortalized human corneal epithelial cells (HCET) harboring doxycycline-inducible shRNA against PNN or ESRP1 were created. Total RNA was isolated from four biological replicates of control and knockdown HCET cells, and subjected to hGlue3_0 transcriptome array analysis. Pnn depletion in developing mouse corneal epithelium led to disrupted alternative splicing of multiple ESRP-regulated epithelial-type exons. In HCET cells, ESRP1 and PNN displayed close localization in and around nuclear speckles, and their physical association in protein complexes was identified. Whole transcriptome array analysis on ESRP1 or PNN knockdown HCET cells revealed clear alterations in transcript profiles and splicing patterns of specific subsets of genes. Separate RT-PCR validation assays confirmed successfully specific changes in exon usage of several representative splice variants, including PAX6(5a), FOXJ3, ARHGEF11, and SLC37A2. Gene ontologic analyses on ESRP1- or PNN-regulated alternative exons suggested their roles in epithelial phenotypes, such as cell morphology and movement. Our data suggested that ESRP1 and PNN modulate alternative splicing of a specific subset of target genes, but not general splicing events, in HCET cells to maintain or enhance epithelial characteristics.

Sensory and sympathetic innervation of the mouse and guinea pig corneal epithelium

This study used immunohistochemistry, retrograde tracing, and high-resolution confocal microscopy to explore the structure and neurochemistry of nerve terminals in the corneal epithelium of mice and guinea pigs. In both species, sub-basal nerves formed a plexus in the basal epithelium. Some axons had bulbar endings within the basal epithelium, but most projected perpendicularly from sub-basal nerves to within a few micrometers of the epithelial surface. Three morphologies for these nerve terminals were identified. Simple terminals did not branch after leaving the sub-basal nerves and ended with a single, bulbar swelling. Ramifying terminals branched in the squamous cell layer, forming horizontal fibers that ran parallel to the surface and terminated with single bulbar swellings. Complex terminals branched as they approached the epithelial surface, forming a cluster of highly branched fibers with multiple bulbar endings. Calcitonin gene-related peptide immunolabeled (peptidergic) axons ended mostly in simple terminals, whereas transient receptor potential cation channel subfamily M member 8 immunolabeled (cold receptor) axons ended almost exclusively in complex terminals. Retrograde labeling identified discrete subpopulations of corneal afferent neurons in the trigeminal ganglion. Tyrosine hydroxylase-immunolabeled (sympathetic) nerve terminals originating from the superior cervical ganglion occurred throughout the corneal epithelium of mice, but only in the basal epithelium of guinea pigs. These findings demonstrate that nerve terminals in the corneal epithelium of mice and guinea pigs can be distinguished on the basis of their morphology and neurochemistry, and suggest that nerve terminals with different sensory modalities can be defined on the basis of their morphology.

Cannabinoid receptor 1 suppresses transient receptor potential vanilloid 1-induced inflammatory responses to corneal injury

Cannabinoid receptor type 1 (CB1)-induced suppression of transient receptor potential vanilloid type 1 (TRPV1) activation provides a therapeutic option to reduce inflammation and pain in different animal disease models through mechanisms involving dampening of TRPV1 activation and signaling events. As we found in both mouse corneal epithelium and human corneal epithelial cells (HCEC) that there is CB1 and TRPV1 expression colocalization based on overlap of coimmunostaining, we determined in mouse corneal wound healing models and in human corneal epithelial cells (HCEC) if they interact with one another to reduce TRPV1-induced inflammatory and scarring responses. Corneal epithelial debridement elicited in vivo a more rapid wound healing response in wildtype (WT) than in CB1−/− mice suggesting functional interaction between CB1 and TRPV1. CB1 activation by injury is tenable based on the identification in mouse corneas of 2-arachidonylglycerol (2-AG) with tandem LC–MS/MS, a selective endocannabinoid CB1 ligand. Suppression of corneal TRPV1 activation by CB1 is indicated since following alkali burning, CB1 activation with WIN55,212-2 (WIN) reduced immune cell stromal infiltration and scarring. Western blot analysis of coimmunoprecipitates identified protein–protein interaction between CB1 and TRPV1. Other immunocomplexes were also identified containing transforming growth factor kinase 1 (TAK1), TRPV1 and CB1. CB1 siRNA gene silencing prevented suppression by WIN of TRPV1-induced TAK1–JNK1 signaling. WIN reduced TRPV1-induced Ca2+ transients in fura2-loaded HCEC whereas pertussis toxin (PTX) preincubation obviated suppression by WIN of such rises caused by capsaicin (CAP). Whole cell patch clamp analysis of HCEC showed that WIN blocked subsequent CAP-induced increases in nonselective outward currents. Taken together, CB1 activation by injury-induced release of endocannabinoids such as 2-AG downregulates TRPV1 mediated inflammation and corneal opacification. Such suppression occurs through protein–protein interaction between TRPV1 and CB1 leading to declines in TRPV1 phosphorylation status. CB1 activation of the GTP binding protein, Gi/o contributes to CB1 mediated TRPV1 dephosphorylation leading to TRPV1 desensitization, declines in TRPV1-induced increases in currents and pro-inflammatory signaling events.

Low humidity environmental challenge causes barrier disruption and cornification of the mouse corneal epithelium via a c-jun N-terminal kinase 2 (JNK2) pathway

Patients with tear dysfunction often experience increased irritation symptoms when subjected to drafty and/or low humidity environmental conditions. The purpose of this study was to investigate the effects of low humidity stress (LHS) on corneal barrier function and expression of cornified envelope (CE) precursor proteins in the epithelium of C57BL/6 and c-jun N-terminal kinase 2 (JNK2) knockout (KO) mice. LHS was induced in both strains by exposure to an air draft for 15 (LHS15D) or 30 days (LHS30D) at a relative humidity <30%RH. Nonstressed (NS) mice were used as controls. Oregon-green-dextran uptake was used to measure corneal barrier function. Levels of small proline-rich protein (SPRR)-2, involucrin, occludin, and MMP-9 were evaluated by immunofluorescent staining in cornea sections. Wholemount corneas immunostained for occludin were used to measure mean apical cell area. Gelatinase activity was evaluated by in situ zymography. Expression of MMP, CE and inflammatory cytokine genes was evaluated by qPCR. C57BL/6 mice exposed to LHS15D showed corneal barrier dysfunction, decreased apical corneal epithelial cell area, higher MMP-9 expression and gelatinase activity and increased involucrin and SPRR-2 immunoreactivity in the corneal epithelium compared to NS mice. JNK2KO mice were resistant to LHS-induced corneal barrier disruption. MMP-3,-9,-13, IL-1α, IL-1β, involucrin and SPRR-2a RNA transcripts were significantly increased in C57BL/6 mice at LHS15D, while no change was noted in JNK2KO mice. LHS is capable of altering corneal barrier function, promoting pathologic alteration of the TJ complex and stimulating production of CE proteins by the corneal epithelium. Activation of the JNK2 signaling pathway contributes to corneal epithelial barrier disruption in LHS.

Corneal Penetration of Topical and Subconjunctival Bevacizumab

To investigate the ability of bevacizumab to penetrate the cornea after topical application or subconjunctival injection. Bevacizumab 1% was topically applied three times a day to the corneas of mice (BALB/c) with intact corneas (n = 14), and with corneal neovascularization (n = 14). Animals were euthanized at 1, 6, 12, and 24 hours, and 2, 4, and 7 days for immunohistochemical analyses. Donkey anti-human IgG labeled with Cy3 was used for bevacizumab immunoreactivity detection. Additionally, one-time topical bevacizumab 1% was tested in corneas with denuded epithelium (n = 16). In another group (n = 16), a single dose of 0.5 mg bevacizumab was injected subconjunctivally. Animals were euthanized at 1, 6, and 24 hours, and 2, 4, 7, 14, and 21 days for immunohistochemical studies. Bevacizumab was barely detected beyond the very superficial layer of the corneal epithelium in mice with intact corneas even after 7 days of topical administration. Application of bevacizumab in mice with corneal neovascularization; however, showed variable penetration into the corneal stroma. Experimentation with single application of topical bevacizumab in corneas with denuded epithelium or subconjunctivally injected bevacizumab showed intense staining for bevacizumab. Topically applied bevacizumab has limited capacity to penetrate the corneas with intact epithelium. However, bevacizumab can penetrate the neovascularized cornea after topical application. This study demonstrates that subconjunctivally injected bevacizumab in eyes with an intact cornea penetrates well into the corneal stroma.

Crosstalk between TGF-β and MAPK Signaling during Corneal Wound Healing

The aim of this study was to elucidate the mechanisms governing epithelial cell migration and proliferation during wound healing. The authors used wound healing of mouse corneal epithelium to examine the role TGF-β signaling plays during the healing process. To achieve this goal, they used transgenic mice in which the TGF-β receptor type II (Tbr2) was conditionally ablated from the corneal epithelium. Epithelium debridement wounds were made, followed by the assessment of cell migration, proliferation, and immunostaining of various signaling pathway components. The authors showed that in the absence of TGF-β signaling corneal epithelial wound healing is delayed by 48 hours; this corresponds to a delay in p38MAPK activation. Despite the delayed p38MAPK activation, ATF2, a substrate of p38MAPK, is still phosphorylated, leading to the suppression of cell proliferation at the leading edge of the wound. These data provide evidence that in the absence of TGF-β signaling, the suppression of cell proliferation during the early stages of wound healing is maintained through the JNK activation of ATF2. CONCLUSIONS; Together the data presented here demonstrate the importance of the TGF-β and MAPK signaling pathways in corneal epithelial wound healing.

Rare corneal clones in mice suggest an age-related decrease of stem cell activity and support the limbal epithelial stem cell hypothesis

The anterior ocular surface comprises the cornea, conjunctiva and a narrow intermediate region called the limbus. It is widely accepted that the corneal epithelium is maintained by stem cells but different hypotheses propose that the stem cells that maintain the mouse corneal epithelium during normal homeostasis are located either in the basal limbal epithelium or throughout the basal corneal epithelium. There are no specific markers to help test these alternatives and new methods are required to distinguish between them. We observed that KRT5 LacZ/− transgenic mice produced rare β-galactosidase (β-gal)-positive radial stripes in the corneal epithelium. These stripes are likely to be clonal lineages of cells derived from stem cells, so they provide a lineage marker for actively proliferating stem cells. The distributions of the β-gal-positive radial stripes suggested they extended centripetally from the limbus, supporting the limbal epithelial stem cell (LESC) hypothesis. Stripe frequency declined between 15 and 30 weeks, which predicts a reduction in stem cell function with age. Pax6 +/− , KRT5 LacZ/− corneas had small patches rather than stripes, which confirms that corneal maintenance is abnormal in Pax6 +/− mice.

Transglutaminases (TGs) in Ocular and Periocular Tissues: Effect of Muscarinic Agents on TGs in Scleral Fibroblasts

ObjectiveTo investigate the expression of transglutaminases (TGs) in the ocular surface, the eyelid margin and associated glands and to determine effect of muscarinic agents on TGs in scleral fibroblasts (SF).Materials and MethodsPrimary SFs cultured from mouse and human sclera were treated with atropine and carbachol for 5 days. Lysed cell RNA was used for real-time PCR, protein was used for Western blot analysis and TG-2 transamidase activity was measured by ELISA. Immunohistochemistry was done to determine the expression of TGases.ResultsImmunohistochemistry and western blot confirmed the expression of TGs-1, 2, 3 and 5 proteins in cultured SFs and eye tissues. Real time PCR showed TG-1, 2, 5 transcript levels to be down regulated 3 fold (p<0.05) in cultured human and mouse SFs after incubation with atropine and this was reversed by carbachol. However, TG-3 expression was increased with atropine and decreased with carbachol at all concentrations. Atropine abrogated the carbachol-induced activation of SF in a dose-dependent manner. TGs-1, 3, 5 were localized in the entire mouse corneal epithelium, stroma and endothelium but TG-2 was present only in the corneal subepithelium and stroma. All TGs were localized in mouse Meibomian glands however TG-2 had a weak expression.ConclusionsOur results confirm that TGs-1, 2, 3 and 5 are expressed in human SF and murine ocular tissues, eyelid and associated Meibomian glands. Real-time PCR and Western blot results showed that muscarinic antagonist down-regulates TGs-1, 2 and 5 in both cultured human and mouse SFs and upregulates TG-3. Atropine abrogated the carbachol-induced activation of SF in a dose-dependent manner. These results suggest that manipulation of TGs by way of muscarinic receptor acting drugs may be a plausible method of intervention in wound healing and scleral remodeling.

A novel, protective Toll-like receptor independent innate immune response to HSV-1 in the cornea (105.51)

Abstract Toll-like receptors (TLRs) are innate sentinels required in clearance of bacterial and fungal infections of the cornea, but their role in viral immunity is currently ambiguous. To evaluate the role of TLRs in ocular immunity to viral infection, TLR adaptor protein deficient mice (MyD88-/- and Trif-/-) were infected with HSV-1. MyD88-/- and Trif-/- mice had similar viral loads in the cornea as those of wild type (WT) mice but substantially lower levels than type 1 interferon receptor A1 deficient (CD118-/-) mice 48-120 hours post infection (pi). A chimeric mouse model utilizing WT and CD118-/- mice was then employed and identified both a resident and bone marrow (BM)-derived immune component in viral containment 48 hours pi. The BM contribution was linked to infiltrating F4/80+ GR1+ monocytes that were severely diminished in CD118-/- mice compared to all other groups, corresponding to a loss of CCL2 production. CCL2 deficient mice were found to possess significantly more virus, consistent with a loss of F8/80+ GR1+ infiltrating monocytes in the cornea compared to WT controls. To identify the key sentinel in viral recognition of the cornea, the DNA sensor, p204, was first identified in mouse corneal epithelium by confocal microscopy and Western blot. In vivo siRNA knockdown of the sensor resulted in significant elevation of virus. Taken together, these results describe a novel TLR-independent innate immune sensor critical in the initial control of acute HSV-1 infection.

Quantitative analysis of patch patterns in mosaic tissues with ClonalTools software.

Quantitative analysis of mosaic tissues is a powerful method for following developmental lineages; however, analytical techniques are often subjective and repetitious. Here a flexible, semi-automated image analysis method for mosaic patterns is described. ClonalTools is a free customizable tool-set designed for the open-source image analysis package ImageJ. Circular, polygonal or linear one-dimensional mosaic arrays can be interrogated to provide measurements of the total number and width of positive and negative patches in a region of interest. These results are adjusted for the effects of random clumping using a previously described method to correct for differences in the contribution of the positive and negative cell type. The applicability of ClonalTools to different systems is discussed with reference to the analysis of mosaic patterns in the mouse corneal epithelium and adrenal cortex and in the outgrowth of neurites from explant cultures of mouse retina as example systems. To validate ClonalTools quantitatively, a recently published manual clonal analysis of the corneal epithelium of X-inactivation beta-Gal-mosaic mice was re-analysed. The semi-automated results did not differ significantly from the published data. Rapid quantification of such patterns to produce biologically relevant results represents a welcome improvement in terms of ease and speed of use over previous methods.

Targeted deletion of Dicer disrupts lens morphogenesis, corneal epithelium stratification, and whole eye development

Dicer, a ribonuclease essential for miRNA processing, is expressed abundantly in developing mouse cornea and lens. We studied the roles of Dicer and miRNAs in eye development by conditionally deleting the Dicer gene in the mouse lens and corneal epithelium. Adult Dicer conditional null (DicerCN) mice had severe microphthalmia with no discernible lens and a poorly stratified corneal epithelium. Targeted deletion of Dicer effectively inhibited miRNA processing in the developing lens at 12.5 day of embryogenesis (E12.5). Lens development initiated normally but underwent progressive dystrophy between E14.5 and E18.5. Microarray analysis revealed activation of P53 signaling in DicerCN lenses at E13.5, consistent with increased apoptosis and reduced cell proliferation between E12.5 and E14.5. Expression of Pax6 and other lens developmental transcription factors were not greatly affected between E12.5 and E14.5 but decreased as the lens degenerated. Our data indicated an indispensible role for Dicer and miRNAs in lens and corneal development.

A Novel Antimicrobial Peptidoglycan Recognition Protein in the Cornea

In an earlier gene expression study, the authors identified a novel antimicrobial gene, Peptidoglycan recognition protein 1 (Pglyrp1), in the mouse cornea. Here the expression of the Pglyrp1 transcript and the encoded protein, PGLYRP1, in the cornea was investigated. The role of PGLYRP1 in the cornea was further investigated using wild-type and Pglyrp1-deficient mice. This is the first report of this antimicrobial protein in the cornea. PGLYRP1 was detected in the cornea and was further localized to the epithelium by immunohistology, confocal microscopy, immunoblotting, and real-time PCR. The role of PGLYRP1 in the cornea was investigated by comparing the response of wild-type and Pglyrp1(-/-) mice to corneal epithelial wounds and Pseudomonas aeruginosa-mediated corneal infections. The antibacterial effects of corneal PGLYRP1 were assayed by measuring bacterial growth in vitro, in the presence of wild-type corneal epithelial extracts, before and after antibody-mediated blocking of PGLYRP1. PGLYRP1 is expressed at high levels in the mouse corneal epithelium. PGLYRP1 was localized to the mouse corneal epithelium and the human corneal epithelium. The Pglyrp1(-/-) mouse shows delayed healing and poor clearing of bacterial keratitis; in vitro its epithelial protein extract shows reduced bacteriostatic activity compared with wild-type mice. PGLYRP1 is a novel antimicrobial protein of the corneal epithelium and protects the ocular surface from bacterial infections.

Mosaic analysis of stem cell function and wound healing in the mouse corneal epithelium

BackgroundThe mouse corneal epithelium is a continuously renewing 5–6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age.ResultsThe initial pattern of corneal epithelial patches in XLacZ+/- X-inactivation mosaics was replaced after birth by radial stripes, indicating activation of LSCs. Stripe patterns (clockwise, anticlockwise or midline) were independent between paired eyes. Wound healing in organ culture was analysed by mosaic analysis of XLacZ+/- eyes or time-lapse imaging of GFP mosaics. Both central and peripheral wounds healed clonally, with cells moving in from all around the wound circumference without significant cell mixing, to reconstitute striping patterns. Mosaic analysis revealed that wounds can heal asymmetrically. Healing of peripheral wounds produced stripe patterns that mimicked some aberrant striping patterns observed in unwounded corneas. Quantitative analysis provided no evidence for an uneven distribution of LSC clones but showed that corrected corneal epithelial stripe numbers declined with age (implying declining LSC function) but stabilised after 39 weeks.ConclusionStriping patterns, produced by centripetal movement, are defined independently and stochastically in individual eyes. Little cell mixing occurs during the initial phase of wound healing and the direction of cell movement is determined by the position of the wound and not by population pressure from the limbus. LSC function declines with age and this may reflect reduced LSCs numbers, more quiescent LSCs or a reduced ability of older stem cells to maintain tissue homeostasis. The later plateau of LSC function might indicate the minimum LSC function that is sufficient for corneal epithelial maintenance. Quantitative and temporal mosaic analyses provide new possibilities for studying stem cell function, tissue maintenance and repair.

Basonuclin-null mutation impairs homeostasis and wound repair in mouse corneal epithelium.

At least two cellular processes are required for corneal epithelium homeostasis and wound repair: cell proliferation and cell-cell adhesion. These processes are delicately balanced to ensure the maintenance of normal epithelial function. During wound healing, these processes must be reprogrammed in coordination to achieve a rapid re-epithelialization. Basonuclin (Bnc1) is a cell-type-specific transcription factor expressed mainly in the proliferative keratinocytes of stratified epithelium (e.g., corneal epithelium, epidermis and esophageal epithelium) and the gametogenic cells in testis and ovary. Our previous work suggested that basonuclin could regulate transcription of ribosomal RNA genes (rDNA) and genes involved in chromatin structure, transcription regulation, cell-cell junction/communication, ion-channels and intracelllular transportation. However, basonuclin's role in keratinocytes has not been demonstrated in vivo. Here we show that basonuclin-null mutation disrupts corneal epithelium homeostasis and delays wound healing by impairing cell proliferation. In basonuclin-null cornea epithelium, RNA polymerase I (Pol I) transcription is perturbed. This perturbation is unique because it affects transcripts from a subset of rDNA. Basonuclin-null mutation also perturbs RNA polymerase II (Pol II) transcripts from genes encoding chromatin structure proteins histone 3 and HMG2, transcription factor Gli2, gap-junction protein connexin 43 and adheren E-cadherin. In most cases, a concerted change in mRNA and protein level is observed. However, for E-cadherin, despite a notable increase in its mRNA level, its protein level was reduced. In conclusion, our study establishes basonuclin as a regulator of corneal epithelium homeostasis and maintenance. Basonuclin likely coordinates functions of a subset of ribosomal RNA genes (rDNA) and a group of protein coding genes in cellular processes critical for the regulation of cell proliferation.

Transient receptor potential vanilloid 1 activation induces inflammatory cytokine release in corneal epithelium through MAPK signaling

In certain epithelial tissues, activation of transient receptor potential (TRP) vanilloid subtype 1 (TRPV1) by noxious stimuli induces pro-inflammatory cytokine release, which helps to mitigate the challenge. While the corneal epithelium elicits such responses to a variety of challenges, it remains unknown whether TRPV1 mediates pro-inflammatory cytokine secretion. Accordingly, we probed for TRPV1 expression and function in human (HCEC) and rabbit corneal epithelial cell (RCEC) lines, in their primary counterparts, and in human and mouse corneal epithelium in situ. Cell membrane and perinuclear TRPV1 expression was detected in all preparations and its identity verified by Western blot analysis. Capsaicin (CAP) (1-10 microM) increased nonselective cation channel whole cell currents (2.5-fold +/- 0.5-fold between -60 and 130 mV), resulting in calcium transients that were fully blocked by the TRPV1 antagonists capsazepine (CPZ) and ruthenium red, or removal of extracellular calcium. Another signaling event involved transient activation of global mitogen-activated protein kinase (MAPK) superfamily, which was followed by up to 3.3- and 9-fold increases in interleukins (IL)-6 and -8 release, respectively. Such increases in inflammatory mediators' release were suppressed by exposure to CPZ or MAPK inhibitors, or removal of Ca2+. Taken together, TRPV1 receptors may play a role in mediating corneal epithelial inflammatory mediator secretion and subsequent hyperalgesia.

Distribution of CESP-1 Protein in the Corneal Endothelium and Other Tissues

The gene expression profile of human corneal endothelium (CE) was established with the gene signature system. A novel gene, GS3582, was abundantly transcribed in the CE compared with other tissues according to a human gene expression database. This protein was designated corneal endothelium-specific protein (CESP)-1. The tissue distribution and subcellular localization of CESP-1 was assessed in humans and mice, to investigate its physiological function. Rabbit and mouse CESP-1 cDNAs were cloned, and a polyclonal anti-human CESP-1 antibody (Ab) and anti-mouse N- or C-terminal ovary-specific acidic protein (OSAP)-1 Ab were produced. CESP-1 expression was investigated in human and mouse corneas by Western blot and/or immunohistochemical analysis. The distribution of CESP-1 in human tissues was also examined by Western blot analysis. To identify the subcellular localization of CESP-1, cultured human CE was colabeled with anti-human CESP-1 Ab and anti-cytochrome c monoclonal Ab or anti-GRP78 monoclonal Ab for confocal microscopy. The rabbit and mouse CESP-1 cDNA sequences contained an open reading frame coding 242 and 283 amino acids, respectively. Mouse CESP-1 was entirely consistent with mouse OSAP. Western blot analysis showed that CESP-1 was expressed in the human corneal epithelium, CE, cultured CE, brain, testis, and ovary. Mouse CESP-1 was also expressed in mouse corneal epithelium and CE with anti-mouse C- but not N-terminal OSAP Ab according to immunohistochemical analysis. Subcellular localization of CESP-1 to the mitochondria was demonstrated in cultured human CE. The N-terminal of CESP-1, possessing a mitochondrial targeting sequence, may be processed after the protein is imported into the mitochondria. CESP-1 was distributed in the corneal epithelium, the CE and cultured human CE, as well as the brain, testis, and ovary. CESP-1 was localized in the mitochondria of cultured human CE. These findings may provide some clues about the physiological function of CESP-1.

A role for chromosomal protein HMGN1 in corneal maturation

Corneal differentiation and maturation are associated with major changes in the expression levels of numerous genes, including those coding for the chromatin-binding high-mobility group (HMG) proteins. Here we report that HMGN1, a nucleosome-binding protein that alters the structure and activity of chromatin, affects the development of the corneal epithelium in mice. The corneal epithelium of Hmgn1 −/− mice is thin, has a reduced number of cells, is poorly stratified, is depleted of suprabasal wing cells, and its most superficial cell layer blisters. In mature Hmgn1 −/− mice, the basal cells retain the ovoid shape of immature cells, and rest directly on the basal membrane which is disorganized. Gene expression was modified in Hmgn1 −/− corneas: glutathione-S-transferase (GST)α 4and GST ω 1, epithelial layer-specific markers, were selectively reduced while E-cadherin and α-, β-, and γ-catenin, components of adherens junctions, were increased. Immunofluorescence analysis reveals a complete co-localization of HMGN1 and p63 in small clusters of basal corneal epithelial cells of wild-type mice, and an absence of p63 expressing cells in the central region of the Hmgn1 −/− cornea. We suggest that interaction of HMGN1 with chromatin modulates the fidelity of gene expression and affects corneal development and maturation.

Bone Marrow-Derived Cells in Mouse and Human Cornea

Recently published experimental data on the distribution of bone marrow (BM)-derived cells in human and mouse corneas in comparison with in human skin/oral mucosa are reviewed. In mouse corneal epithelium, major histocompatibility complex (MHC) class II-negative dendritic cells (DC) are present. Immature MHC class II-negative and mature MHC class II-positive DC are present in the center and periphery of the anterior corneal stroma, respectively. Monocyte (Mo)/macrophage (MPhi) lineage cells including the MPhi marker F4/80-expressing cells reside in the posterior stroma. In human cornea, MHC class II (HLA-DR)-positive immature myeloid DC (CD11cCD16, CD11cCD16, and CD11cCD1c) and Mo/MPhi lineage cells are detectable in the corneal epithelium and stroma, respectively. Distribution of Mo/MPhi lineage cells (HLA.DRCD11bCD11cCD14) is predominant in the anterior stroma of the central cornea and all layers of the peripheral cornea. Both the phenotypes and distribution pattern of these cells in human cornea are different from those of human skin and nasal mucosa. These findings suggest that BM-derived cells in normal human cornea are present in situ in preparation for foreign antigen and pathogens and have critical roles in innate and acquired immunity of the ocular surface.