Drying and cicatrizing ocular surface diseases can be debilitating and, in advanced disease states, cause blindness. Goblet cells, mucin-secreting cells that are intercalated within the stratified epithelium of the conjunctiva disappear in these diseases, as the epithelium becomes keratinized. Little is known regarding factors regulating goblet cell differentiation in the conjunctiva. With this application, we present evidence that mice null for SAM pointed domain Ets transcription factor, Spdef, lack conjunctival goblet cells. We further demonstrate by microarray comparison of fornicial conjunctiva of Spedf-null and wild type mice that, in addition to downregulation of goblet cell-specific genes, there is a significant downregulation of 4 genes in the Wnt signaling pathway. Of these, Sfrp3 (Frzb), an antagonist of canonical Wnt signaling, is downregulated 115 fold. Immunofluorescence microscopy indicates that the antagonist is expressed in the conjunctival goblet cell, raising the possibility that the goblet cell secretes the protein to effect epithelial homeostasis and/or goblet cell differentiation. Based on these findings, we propose the following specific aims to test hypotheses relevant to goblet cell function and differentiation at the ocular surface.
Aim 1. Determine the effect of lack of goblet cells on ocular surface health in the Spdef-null mice as indicated by comparison of tear volume, fluorescein staining, exposure to dry eye chamber conditions and susceptibility to infection to that of wild type mice.
Aim 2. Compare expression levels of genes down/upregulated in fornicial conjunctiva of Spdef-null mice (goblet cell mucin biosynthesis genes, Wnt signaling pathway genes and epithelial differentiation genes) to RNA from fornicial conjunctiva of mice with smoke induced goblet cell differentiation to verify genes in goblet cell differentiation/function as opposed to those involved in epithelial response to lack of goblet cells.
Aim 3. Examine the role of Wnt signaling in goblet cell/conjunctival cell differentiation by determining if the goblet cell is the source of the Wnt inhibitor Sfrp3 identified by microarray analysis as downregulated in Spdef null mice and by assaying the function of the soluble Wnt inhibitor in Sfrp3 (Frzb) null mice and in vitro.
Aim 4. Determine whether goblet cell differentiation can be induced by overexpression of SPDEF in conjunctival epithelial cell culture and whether downregulation of the Wnt pathway influences goblet cell differentiation in vitro.
Aim 5. Determine whether expression of Spdef, the Wnt pathway antagonist Sfrp3 identified as downregulated in the absence of Spdef, and the epithelial differentiation gene Sprr2h that is upregulated in Spdef-/- mice, are altered in human dry eye diseases where goblet cell numbers are reduced.

Public Health Relevance

Drying eye diseases often cause irritation and poor vision but in severe forms cause blindness. Research in this application seeks to learn the cellular and molecular changes that occur in the outer ocular surface cell layers that are associated with the diseases. With these insights future therapies may be developed.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY003306-34
Application #
8578099
Study Section
Anterior Eye Disease Study Section (AED)
Program Officer
Mckie, George Ann
Project Start
1979-08-01
Project End
2015-11-30
Budget Start
2013-12-01
Budget End
2014-11-30
Support Year
34
Fiscal Year
2014
Total Cost
$436,500
Indirect Cost
$211,500
Name
Schepens Eye Research Institute
Department
Type
DUNS #
073826000
City
Boston
State
MA
Country
United States
Zip Code
02114
Gipson, Ilene K; Spurr-Michaud, Sandra; Tisdale, Ann (2016) Human conjunctival goblet cells express the membrane associated mucin MUC16: Localization to mucin granules. Exp Eye Res 145:230-4
Robert, Marie-Claude; Arafat, Samer N; Spurr-Michaud, Sandra et al. (2016) Tear Matrix Metalloproteinases and Myeloperoxidase Levels in Patients With Boston Keratoprosthesis Type I. Cornea 35:1008-14
Gipson, Ilene K (2016) Goblet cells of the conjunctiva: A review of recent findings. Prog Retin Eye Res 54:49-63
Menon, B B; Kaiser-Marko, C; Spurr-Michaud, S et al. (2015) Suppression of Toll-like receptor-mediated innate immune responses at the ocular surface by the membrane-associated mucins MUC1 and MUC16. Mucosal Immunol 8:1000-8
Marko, Christina Kaiser; Tisdale, Ann S; Spurr-Michaud, Sandra et al. (2014) The ocular surface phenotype of Muc5ac and Muc5b null mice. Invest Ophthalmol Vis Sci 55:291-300
Gipson, Ilene K; Spurr-Michaud, Sandra; Tisdale, Ann et al. (2014) Comparison of the transmembrane mucins MUC1 and MUC16 in epithelial barrier function. PLoS One 9:e100393
Arafat, Samer N; Suelves, Ana M; Spurr-Michaud, Sandra et al. (2014) Neutrophil collagenase, gelatinase, and myeloperoxidase in tears of patients with stevens-johnson syndrome and ocular cicatricial pemphigoid. Ophthalmology 121:79-87
Marko, Christina K; Menon, Balaraj B; Chen, Gang et al. (2013) Spdef null mice lack conjunctival goblet cells and provide a model of dry eye. Am J Pathol 183:35-48
Spurr-Michaud, Sandra J; Gipson, Ilene K (2013) Methods for culture of human corneal and conjunctival epithelia. Methods Mol Biol 945:31-43
Argueso, Pablo; Gipson, Ilene K (2012) Assessing mucin expression and function in human ocular surface epithelia in vivo and in vitro. Methods Mol Biol 842:313-25

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