Mucins synthesized and secreted by conjunctival goblet cells provide a critical line of defense for the ocular surface by protecting it from the external environment. A decrease in conjunctival goblet cell mucin production is devastating to the ocular surface and results in a spectrum of ocular surface diseases. Mucin production depends upon two processes, the percentage of goblet cells secreting mucins in response to a given stimulus (rapid response) and the total number of goblet cells present in the conjunctiva (long-term response). Thus the long-term goal of this project is to treat diseases of ocular surface mucin deficiency by stimulating goblet cell secretion and proliferation thus increasing mucin production and replenishing the mucous layer of the tear film. The focus of the present proposal is to determine: (1) if sensory nerves in the cornea stimulate epidermal growth factor (EGF) release from the conjunctiva, perhaps from the goblet cells, to stimulate goblet cell proliferation and (2) the signaling pathways used by EGF to stimulate this proliferation. Goblet cell proliferation will be stimulated in vivo by a corneal sensory nerve stimulus and proliferating goblet cells measured by immuno-fluorescence microscopy to determine if activation of the EGF receptor is used. Pieces of conjunctiva will be used to determine if activation of nerves releases EGF and cultured goblet cells used to determine if these cells release EGF. Passaged rat and human conjunctival goblet cells will be used to determined if EGF stimulates proliferation by activating 1) p44/p42 mitogen-activated protein kinase, (2) phosphatidylinositol-3 kinase, or (3) PKC isoforms, Proliferation will be measured by a biochemical and an immunohistochemical assay. Pharmacological inhibitors and neutralizing antibodies, as well as dominant negative and constitutively active adenovirus will be used to inhibit or stimulate specific signaling pathways. Individual signaling components will be measured by immunoprecipitation, western blotting, and immunofluorescence microscopy techniques. In diseases of mucin deficiency such as anesthetic cornea, herpetic keratitis, and neurotrophic keratitis, as well as in aging and LASIK surgery, activation of neural and growth factor regulation of goblet cell proliferation is impaired. Study of the signaling pathways that stimulate conjunctival goblet cell proliferation will lead to the development of treatments to replenish the mucin layer in these diseases.
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