Stimulatory Role of PKCα in Extracellular Regulated Kinase 1/2 Pathway in Conjunctival Goblet Cell Proliferation

Large gel-forming mucins including MUC5AC, -5B, and -2 secreted by goblet cells protect the airways, gastrointestinal tract (GI), and ocular surface from the external environment. The quantity of mucin production is tightly regulated in each of these tissues as mucin overproduction and/or underproduction can cause disease. In the airways, mucin overproduction from an increase in goblet cell number and increase in mucin synthesis occurs in chronic obstructive pulmonary disease, asthma, and bacterial infection.1 Alterations in the cell number and mucin content occur in the GI tract in Crohn’s disease, ulcerative colitis, and colonic neoplasia.2–4 On the ocular surface, both a decrease and an increase in conjunctival goblet cell mucin lead to disease. Conjunctival goblet cell mucin is decreased in dry eye disease, herpes keratitis, and anesthetic cornea and increased in allergy, atrophy, and vernal conjunctivitis.5,6 The amount of mucin secretion in the airways, GI tract, and ocular surface is regulated by controlling the rate of mucin synthesis, the rate of mucin secretion, and the number of goblet cells (cell proliferation). These three processes are differentially controlled in these tissues with allergic, fungal, or viral inflammation causing goblet cell differentiation and mucin synthesis in the airways,1 enteric nerves, enteroendocrine cells, and resident immune cells stimulating colonic mucin secretion,7 and nerves and growth factors stimulating conjunctival mucin secretion and number of goblet cells.8–11 One regulatory compound common to many of these processes is EGF. It stimulates goblet cell mucin synthesis and hyperplasia in airway goblet cells,12 regulates goblet cell differentiation as the cells move from the base to the tops of colonic crypts,13 and induces growth in a goblet cell line from human colorectal adenocarcinoma.14 It also plays a pivotal role in conjunctival mucin production by inducing goblet cell mucin secretion and goblet cell proliferation.8,10 EGF is a critical growth factor for epithelial cells. Its over-expression plays a role in many cancers as well as normal development. EGF can activate multiple signaling pathways including: (1) phospholipase Cβ, which increases the intracellular [Ca2+] and activates protein kinase C (PKC); (2) the nonreceptor tyrosine kinases Grb2, Shc, and Sos, which ultimately activate extracellular signal-regulated kinase 1/2 (ERK 1/2 also known as p44/p42 mitogen-activated protein kinase [MAPK]); (3) phosphoinositide-3 kinase and protein kinase B (AKT); (4) p38 MAPK; and (5) c-Jun NH-(2) terminal kinase (JNK).15,16 Not only can EGF activate each of these pathways individually, but these pathways can themselves interact. In particular, activated PKC isoforms can stimulate ERK1/2.17 The PKC superfamily of lipid regulated serine/threonine kinases includes 10 different isoforms. Specific isoforms play critical roles in the signal transduction pathways that regulate cell proliferation, transformation, differentiation, and secretion.18 The location, activation, and function of each isoform are tissue specific. The PKC isoforms can be divided into three classes based on structure and cofactor. Classic or conventional PKCs (cPKCα, -β1, -β2, and -γ) are activated by Ca2+ and diacylglycerol. Novel PKCs (nPKCδ, -e, -η, and -θ) are activated by diacylglycerol, but not Ca2+. Atypical PKCs (aPKCζand -ι/λ) are unresponsive to both diacylglycerol and Ca2+. Using Cos-7 cells transfected with Raf, MEK, and p42 MAPK, Schonwasser et al.17 found that all PKC isoforms activated MEK and p42 MAPK, with classic and novel, but not atypical, PKC isoforms activating Raf. However, the actions of PKC isoforms are cell-type and agonist specific. In lung fibroblasts, PKCe activates ERK, but PKCα inhibits it.19 In conjunctival goblet cells EGF induces proliferation by activating both ERK1/2 and PKCα and –e.20 In the present study we used constitutively active PKCα (myrPKCα) to determine whether PKCα, which stimulates proliferation by itself, activates ERK1/2 to induce goblet cell proliferation. In both human and rat conjunctival goblet cells myrPKCα phosphorylated ERK1/2 and translocated it to the nucleus, thereby stimulating proliferation.