Role of cPKCα and nPKCε in EGF-stimulated goblet cell proliferation.

The protein kinase C (PKC) superfamily of lipid-regulated serine/threonine protein kinases includes 10 different isoforms.1 Specific isoforms play critical roles in the signal transduction pathways that regulate cell proliferation, transformation, differentiation, and secretion. The PKC isoforms can be divided into three classes based on structure and cofactor requirements. Classical or conventional PKCs (cPKCα, -βI, -βII, and -γ) are activated by Ca2+ and diacylglycerol. Novel PKCs (nPKCδ, -e, -η, and -θ) are activated by diacylglycerol but not Ca2+. Atypical PKCs (aPKCζ and -ι/λ) are unresponsive to diacylglycerol and Ca2+. It is well established that PKC isoforms play important roles in cell proliferation2–5 and are cell type specific.6 Overexpression of PKCα has been shown to increase proliferation in thymocytes, MCF-7 cells, and U87 cells.7 In contrast, in intestinal, pancreatic, and mammary cells, PKCα has been shown to have an antiproliferative effects.6 nPKCe increases the proliferation of lactotrophs through ERK1/28 and promotes survival in lung cancer cells.6 Indeed, nPKCe has been shown to be an oncogene.4 The conjunctiva is an epithelium that surrounds the cornea and lines the eyelids. We previously showed that conjunctival goblet cells in vivo contained at least seven PKC isoforms.9 Cholinergic agonists, which are known to activate PKC, stimulate mucin secretion, as do phorbol esters.10 In this tissue, the phorbol ester PMA activates the nonreceptor tyrosine kinases Pyk2 and Src to phosphorylate the EGF receptor, which then activates ERK1/2 to cause secretion.10 Large oligomeric mucins, such as MUC5AC and MUC5B (two gel-forming mucins), are produced in the airway, gastrointestinal tract, and ocular surface and protect these wet-surfaced mucosa from the external environment.11,12 Gel-forming mucins are synthesized and secreted by goblet cells located in the wet-surfaced epithelia in response to stimuli from the extracellular environment. The amount of mucin produced by the goblet cells is dependent on the number of cells present (proliferation or differentiation), the amount of mucin synthesized and stored in the secretory granules (synthesis), and the release of mucin from the secretory granules (secretion). Each tissue has its own unique response, leading to an increase or a decrease in mucin production. Goblet cells in the conjunctiva are responsible for production of the large soluble mucin MUC5AC, the major soluble mucin of the tear film.13 Ocular mucin is increased in allergy and inflammation but decreased in diseases of impaired corneal sensitivity such as herpes keratitis and anesthetic cornea.14,15 Thus, the amount of ocular surface mucin is highly regulated. In the conjunctiva, the regulation of goblet cell mucin synthesis has not been investigated. It is known that the parasympathetic neuro-transmitters acetylcholine, vasoactive intestinal peptide and the nerve growth factor (NGF) and its family member brain-derived nerve factor (BDNF) induce goblet cell secretion.16,17 It is also known that the epidermal growth factor (EGF) family of growth factors stimulates conjunctival goblet cell proliferation when measured in primary cell culture.18,19 EGF and its family members TGFα and HB-EGF are stimuli of conjunctival goblet cell proliferation.10 EGF activates the EGF receptor and stimulates the ERK1/2 pathway, translocating ERK1/2 to the nucleus and causing proliferation in rat and human goblet cells.10 In the present study, we investigated the role of PKC isoforms in EGF stimulation of goblet cell proliferation and found that EGF activation of cPKCα and nPKCe induces cell proliferation.