ERK/p44p42 mitogen-activated protein kinase mediates EGF-stimulated proliferation of conjunctival goblet cells in culture

Goblet cells are polarized secretory cells that are located in the conjunctival epithelium. They synthesize and secrete the large soluble mucin, MUC5AC, into the tear film forming the inner mucous layer.1 This tear film layer provides a physical and chemical barrier between the ocular surface (cornea and conjunctiva) and the external environment, thereby protecting it from the constantly changing external environment. The mucous layer is also critical for maintaining the health of the ocular surface. A decrease in the number of goblet cells in the conjunctiva or an inability of the goblet cells to produce mucin is associated with pathologic abnormalities of the ocular surface such as neurotrophic keratitis, dry eye syndromes, ocular cicatricial pemphigoid, vitamin A deficiency, and Stevens-Johnson syndrome.2 On the other hand, an overproduction of mucus is associated with diseases such as atopy, mucus fishing syndrome, and seasonal allergic conjunctivitis.3 Both an increase and a decrease in goblet cell mucin production occurs with ocular surface disease implying that there is an optimal amount of mucin production and suggesting that goblet cell mucin production is tightly regulated. Four different processes contribute to goblet cell mucin production: (1) the rate and amount of mucin secretion, (2) the rate of mucin synthesis, (3) the number of goblet cells present in the conjunctiva, and (4) the rate of mucin degradation. There is almost no information about the regulation of MUC5AC synthesis or the rate of mucin degradation. In contrast, the regulation of goblet cell secretion and proliferation has been addressed in several studies. Measurement of conjunctival goblet cell mucin secretion in rats and humans indicates that cholinergic agonists using M2 and M3 muscarinic receptors stimulate goblet cell secretion.4 Activation of these receptors increases intracellular [Ca2+] and activates protein kinase C isoforms. Intracellular [Ca2+] and protein kinase C isoforms activate the nonreceptor tyrosine kinases PYK2 and Src to transactivate the EGF receptor (EGFR).5 This transactivation causes phosphorylation of the homo- and heterodimerized EGF family of receptors. The phosphorylated EGFR activates the extracellular-related kinase (ERK 1/2, also known as p44/p42 mitogen-activated protein kinase [MAPK]) pathway to induce secretion. The MAPK pathway includes attraction of the adapter proteins Grb2 and Shc to the EGFR that induce SOS. SOS is a guanine nucleotide exchange factor that activates Ras. Ras then stimulates Raf (MAPK kinase kinase) and then MEK (MAPK kinase) which phosphorylates ERK 1/2. To cause secretion, ERK 1/2 remains in the cytosol and interacts with as yet unidentified target proteins. Recent studies have begun to address the complex regulation of conjunctival goblet cell proliferation. In vitro experiments using rat conjunctival goblet cells in culture demonstrated that EGF and its family members, transforming growth factor (TGF)β and heparin-binding (HB)EGF, but not heregulin, stimulate goblet cell proliferation.6 Growth factors that increase proliferation bind to the EGFR implicating its participation in proliferation as well. In vivo evidence also suggests that EGF regulates conjunctival goblet cell proliferation. In Sjogren's syndrome, the autoimmune form of dry eye, the EGF concentration in tears is decreased and correlates with a decrease in the number of conjunctival goblet cells.7 In addition to effects on the conjunctiva, it well established that EGF and its family members stimulate proliferation in a variety of cell types.8-10 Epithelial cells are the major source of EGF in tears and other body fluids. In almost all epithelia, except the submandibular gland, the EGF family members are membrane-spanning proteins. Membrane-spanning EGF is present as a 170-kDa proform that contains the 6-kDa EGF-like active fragment.11 Similarly, the lacrimal gland acinar and duct cells as well as the stratified squamous cells of the cornea contain membrane-spanning EGF.12,13 This form of EGF can be released from cells, by the action of metalloproteinases, as a 150-kDa precursor form.14 Release of EGF by this mechanism is known as ectodomain shedding. The precursor form can be further cleaved to release the 6-kDa active motif. All three forms of EGF (170, 150, and 6 kDa) are potentially active. TGFα and HB-EGF are similarly released by ectodomain shedding.15,16 The sources of EGF that could stimulate conjunctival goblet cell proliferation are the lacrimal gland, the conjunctival epithelium (stratified squamous and goblet cells), and the corneal epithelium. EGF was detected in both the basal and apical membranes of lacrimal gland acinar and duct cells.12 EGF released from the apical membrane would enter the tear film and be available to interact with goblet cells. EGF mRNA is present in corneal epithelial cells as well as in the stratified squamous and goblet cells of the conjunctiva.3,6,17,18 It is not known whether these cells can release EGF. However, HB-EGF can be released from the cornea and plays a role in corneal wound healing.19 Binding of EGF to the EGFR leads to homo- and heterodimerization and subsequent phosphorylation of multiple tyrosine residues on the C-terminal domain. Phosphotyrosines serve as docking sites for adapter proteins that are in turn phosphorylated. There are five main autophosphorylation sites: Y992, which binds phospholipase C (PLC)γ1; Y1046 which binds Cbl; Y1068 and Y1086 which bind GRB2 and GAB1, respectively; Y1148 which binds SHC; and Y1173 which binds SHP1, SHC, and PLCγ1.20-22 Attraction of each adapter protein leads to activation of specific signaling pathways. As examples, PLCγ1 uses the Ca2+ and PKC pathway, GRB2 and SHC activate the ERK signaling cascade, and the GRB2/GAB1 site stimulates the PI-3K/AKT pathway.23 Stimulation of these signaling pathways involves translocation of a signaling component into the nucleus where they differentially activate transcription factors. EGF stimulation of these signaling pathways leads to proliferation, migration, differentiation, and tumor formation.24 Using multiplex technology, we found that a 5-minute stimulation with EGF activated ERK, p38 MAPK, and JNK, but not AKT6 in conjunctival goblet cells. Thus, in the present study, we began characterizing the ERK pathway activated by EGF in cultured conjunctival goblet cells and found that EGF transactivates the EGFR to activate the ERK signaling cascade, which translocates ERK to the nucleus where it initiates goblet cell proliferation.