A

Structure

The glycocalyx is a network of polysaccharides that project from cellular surfaces. In corneal and conjunctival epithelia, the glycocalyx can be found on the apical portion of the microvilli that project from the apical plasma membrane of the superficial cell layer ( Fig. 15.2 ). Mucins are a critical component of the glycocalyx. Mucins consist of a protein core of amino acids linked by O -glycosylation to carbohydrate side chains of varying length and complexity. Mucins are classified by the nomenclature MUC1–21 and are divided into secreted and membrane-spanning categories ( Fig. 15.4 ). Membrane-spanning mucins consist of a short intracellular tail, membrane-spanning domain, and large, extended extracellular domain that forms the glycocalyx. Secreted mucins are either gel-forming or small soluble. Gel-forming mucins are large molecules (20–40 million Da) secreted by exocytosis from goblet cells. Small soluble mucins are secreted by the lacrimal gland.

Structural motifs of the secreted and membrane-spanning mucins.

MUC5AC consists of four cysteine-rich D domains for disulfide cross-linking and flank a region of variable number tandem repeats (TR). MUC7 is monomeric, has a variable number of tandem repeats. The membrane-spanning mucins have an SIG domain, a variable number of tandem repeats, a site for cleavage of the extracellular domain, a transmembrane domain (TM) for insertion of the molecule into membranes, and a carboxyterminus (CT) that is the intracellular domain. SIG: peptide signal sequence; CK: cysteine knot.

(Modified from Gipson IK and Argueso P. 2003. Role of mucins in the function of the corneal and conjunctival epithelia. Int Rev Cytol. 231, 1–49. Copyright Elsevier 2003.)

The ocular surface contains the membrane-spanning mucins, MUC1, MUC4, and MUC16. These mucins are produced by stratified squamous cells of the cornea and conjunctiva and are stored in small clear secretory vesicles in the cytoplasm ( Fig. 15.1 ). Fusion of secretory vesicles with plasma membrane inserts these molecules into the plasma membrane. Mucin molecules are localized to the tips of the squamous cell microplicae.

There is limited information on regulation of membrane-spanning mucin synthesis and secretion. Regulation of secretion would be by regulation of insertion of mucins into plasma membranes or by control of ectodomain shedding whereby matrix metalloproteinases (MMPs) cleave the mucin releasing the extracellular, active domain of the protein into the extracellular space. In immortalized stratified corneal-limbal cells, tumor necrosis factor induced the ectodomain shedding of MUC1, MUC4, and MUC16, whereas MMP-7 and neutrophil elastase induced the shedding of MUC16 only. These compounds that caused shedding are elevated in the tears of dry eye patients and thus may cause the increase in Rose-Bengal staining found in dry eye and associated with loss of MUC16 ( Box 15.1 ).

B

Function

The membrane-spanning mucins function to hydrate the ocular surface and serve as a barrier to pathogens. Membrane-spanning mucins are considered to be disadhesive, allowing the mucous layer to move over the ocular surface. The carbohydrate side chains hold water at the surface of the apical cell membranes. The function of individual membrane-spanning mucins remains unclear.

Membrane-spanning mucins appear to be altered in dry eye. MUC16 protein levels decreased in conjunctival epithelium and increased in tears of patients with Sjogren’s syndrome. MUC1 splice variants also play a role in dry eye. Human cornea and conjunctiva contain five previously identified MUC1 splice variants and a new splice variant. These splice variants have unique changes that could affect their ectodomain shedding, signaling properties of the intracellular domain, and water retention, lubrication, and barrier properties of the extracellular domains. When the type of MUC1 splice variant was determined in dry eye patients (both evaporative dry eye and Sjogren’s syndrome) compared to control patients there was a reduced frequency of MUC1/A variant and an increase in MUC1/B variant in dry eye patients.

A

Structure

The mucous layer backbone is the gel-forming mucin MUC5AC, synthesized and secreted by conjunctival goblet cells. MUC5AC is encoded by one of the largest genes known, producing a protein of about 600 kDa. The protein backbone of MUC5AC consists of four D domains (cysteine-rich domains) ( Fig. 15.4 ) that flank a tandem repeat sequence in which amino acids in the protein backbone are O -glycosylated and linked to carbohydrate side chains. The D domains provide sites for disulfide bonds cross-linking multiple MUC5AC molecules that forms the framework of the mucous layer. Also contained in the mucous layer are shed ectodomains of membrane-spanning mucins, membrane-spanning mucins secreted by a soluble pathway, other proteins synthesized and secreted by goblet cells, and electrolytes and water secreted by goblet and stratified squamous cells.

B

Conjunctival goblet cells

Goblet cells are interspersed among stratified squamous cells of the conjunctiva (see Fig. 15.1 ). Goblet cells occur in clusters in rat and mouse, but singly in rabbit and humans. In all species studied, goblet cells are unevenly distributed over the conjunctiva.

Goblet cells are identified by the large accumulation of mucin granules in the apex (see Fig. 15.1 ). Secretory product can be visualized using Alcian blue-periodic acid stain, the lectins Ulex europeaus agglutinin I (UEA-I) or Helix pomatia agglutinin (HPA), or antibodies to MUC5AC. MUC5AC is synthesized in the endoplasmic reticulum and carbohydrate side chains added in the Golgi apparatus. The mature proteins are stored in secretory granules. Upon stimulation secretory granules fuse with each other and apical membrane releasing secretory product into the tear film. Upon cell stimulation the entire complement of granules is released, known as apocrine secretion. The amount of secretion is controlled by regulating the number of cells that are activated by a given stimulus.

C

Regulation of goblet cell mucin production

Regulation of goblet cell secretion

Nerves are the primary regulators of conjunctival goblet cell secretion. The conjunctiva is innervated by afferent sensory nerves and efferent sympathetic and parasympathetic nerves. Sensory nerves end as free nerve endings between the stratified squamous cells. The parasympathetic and sympathetic nerve endings also surround the goblet cells at the level of the secretory granules ( Fig. 15.5 ). Stimulation of the sensory nerves in the cornea by a neural reflex induces goblet cell secretion via the efferent nerves. Goblet cells have receptors for neurotransmitters from both parasympathetic and sympathetic nerves. Parasympathetic nerves release both acetylcholine (Ach) and vasoactive intestinal peptide (VIP). Muscarinic receptors of the M ₃ AchR and M ₂ AchR subtypes are located similarly to the location of efferent nerves ( Fig. 15.5 ). VIP receptors of the VIPAC2 receptor subtype are located in the same area as M ₃ AchR. Sympathetic nerves release norepinephrine and NPY. Several subtypes of both α ₁ – and β-adrenergic receptors are present on goblet cells. It appears that parasympathetic nerves using Ach and VIP are the primary stimuli of goblet cell secretion. The function of the sympathetic nerves remains unknown.

Immunofluorescence micrographs of parasympathetic nerves and M ₃ Ach receptors in rat conjunctiva.

( A ) The parasympathetic neurotransmitter VIP is shown in green. The lectin Ulex europeaus is shown in red and indicates the location of goblet cells. The goblet cell body is subjacent to the secretory granules. The cell nuclei are shown in blue.

(Reprinted with permission from Diebold, Y., J. D. Rios, R. R. Hodges, I. Rawe and D. A. Dartt. 2001. Invest Ophthalmol Vis Sci. 42(10), 2270–82.)

Components of the signaling pathways used by Ach, but not VIP, have been delineated. Cholinergic agonists use M ₃ AchR and M ₂ AchR to stimulate goblet cell secretion. Cholinergic agonists presumably use the G _αq/11 subtype of G protein that activate phospholipase C to breakdown phosphatidylinositol 4,5 bisphosphate to produce inositol 1,4,5 trisphosphate (IP ₃ ) and diacylglycerol. IP ₃ would then release intracellular Ca ²⁺ by binding to its receptors on the endoplasmic reticulum. Based on a variety of experiments cholinergic agonists are known to increase the intracellular [Ca ²⁺ ] to stimulate secretion.

The diacylglycerol (DAG) released with IP ₃ activates protein kinase C (PKC). Nine PKC isoforms are present in conjunctival goblet cells and phorbol esters, activators of PKC isoforms, stimulate goblet cell secretion. Although a role for PKC isoforms in secretion could not be substantiated, it is likely that cholinergic agonists use PKC isoforms to stimulate secretion as PKC inhibitors block a distal step in the signaling pathway, activation of extracellular regulated kinase (ERK1/2) ( Fig. 15.6 ).

Signaling pathways used by cholinergic agonists to stimulate conjunctival goblet cell secretion – the pathway is described in the text. TK-tyrosine kinase, P-phosphorylation site.

(Modified from Dartt DA. Prog Ret Eye Res 2002; 21:555–76.)

Cholinergic agonists are known to activate the epidermal growth factor (EGF) signaling pathway. In goblet cells, cholinergic agonists activate the non-receptor tyrosine kinases Pyk2 and p60Src. These kinases transactivate (phosphorylate) the EGF receptor (EGFR). This transactivation is usually mediated by ectodomain shedding by MMP causing the release of the extracellular, active domain of one member of the EGF family of growth factors. This has yet to be tested in goblet cells. The released growth factor would bind to the EGF receptor inducing two receptors to associate and be autophosphorylated. This attracts the adapter proteins Shc and Grb2 that are phosphorylated, activating the guanine nucleotide exchange factor SOS to increase Ras activity. Ras activates MAPK kinase (Raf) that phosphorylates MAPK kinase (MEK) that phosphorylates ERK1/2. In both rat and human goblet cells, cholinergic agonists increase the intracellular [Ca ²⁺ ] and activate PKC to stimulate goblet cell secretion by activating Pyk2 and p60Src to transactivate the EGF receptor, inducing the signaling cascade that activates ERK1/2 ( Fig. 15.6 ).

Other factors that stimulate goblet cell secretion include P2Y ₂ purinergic receptors that are activated by ATP and the neurotrophin family of growth factors. Of this family nerve growth factor and brain-derived neurotrophic factor stimulate secretion.

Regulation of goblet cell proliferation

The amount of goblet cell mucin on the ocular surface can also be modulated by controlling goblet cell proliferation. Serum, which contains a variety of growth factors, stimulates proliferation of cultured conjunctival goblet cells, both human and rat. EGF, transforming growth factor (TGFα), and heparin binding-EGF (HB-EGF), but not heregulin stimulate proliferation. EGF, TGFα, and HB-EGF bind to the EGF receptor (EGFR or erb-B1), HB-EGF binds to erb-B4 and heregulin binds to erb-B3 and erb-B4. That EGF, TGFα, and HB-EGF are equipotent in stimulating conjunctival goblet cell suggests that the EGFR and erb-B4 are the primary receptors used.

EGF was used as the prototype of the EGF family. EGF increases the activation of the EGFR in rat conjunctival goblet cells and activates ERK1/2 (also known as p44/p42 mitogen activated protein kinase (MAPK)) ( Fig. 15.7 ) causing its biphasic translocation to the nucleus. The slower sustained second peak response is responsible for cell proliferation, but the role of the rapid transient first peak is not known.

Schematic of EGF-dependent signaling pathways.

Activation of PKC isoforms also mediates EGF-stimulated goblet cell proliferation ( Fig. 15.7 ). In rat and human goblet cells, EGF uses PKCα and PKCε to stimulate conjunctival goblet cell proliferation.

D

Regulation of conjunctival electrolyte and water secretion

The electrolyte and water composition of the external environment has an important effect on release of mucins from secretory granules. Both the stratified squamous and goblet cells of the conjunctiva express ion and water transport proteins and appear to secrete electrolytes and water. Mucin and electrolyte and water secretion may not be coordinated as stimuli of mucin secretion appear to differ from those of electrolyte and water secretion.

The conjunctival epithelium secretes Cl ⁻ and absorbs Na ⁺ with the ratio of 1.5 to 1 causing a net secretion of fluid into the tear film. Interestingly, both absorption and secretion occur within the same cell in the conjunctiva. The ion transport proteins are distributed evenly in all areas of the conjunctiva, suggesting that the entire conjunctiva participates in fluid secretion.

The driving force for conjunctival fluid secretion is the basolaterally located Na ⁺ ,K ⁺ -ATPase that extrudes 3 Na ⁺ ions for the influx of 2 K ⁺ ions and generates a negative intracellular voltage ( Fig. 15.8 ). Also on the basolateral side is the Na ⁺ -K ⁺ -2Cl ⁻ cotransporter (NKCC1). The Cl ⁻ /HCO ₃ ⁻ exchanger takes up Cl ⁻ into the cell. Cl ⁻ is secreted from the apical side of the cell through a variety of Cl ⁻ channels. K ⁺ is also secreted from the apical side with Na ⁺ moving through the paracellular pathway to the apical side. The net effect is isotonic secretion of Na ⁺ , K ⁺ , and Cl ⁻ into the tears.

Schematic of the mechanism of electrolyte and water secretion by conjunctival epithelial cells – a description of the process is found in the text.

(Modified from Dartt DA. Prog Ret Eye Res 2002; 21:555–76.)

Na ⁺ is absorbed across the apical membrane into the cell by the Na ⁺ -dependent glucose cotransporter. The Na ⁺ that enters the cell on the apical side is extruded on the basolateral side by the Na ⁺ ,K ⁺ -ATPase. The Na ⁺ influx mechanism can also be used to transport amino acids (AA) into the cell.

Fluid secretion by the conjunctiva can be stimulated and contribute a substantial amount to the tear film. Increasing the intracellular [Ca ²⁺ ] or the cellular level of cAMP causes Cl ⁻ secretion. The β-adrenergic agonist epinephrine stimulates secretion via cAMP. Increasing cellular cAMP levels activate protein kinase A (PKA) that stimulates both Cl ⁻ secretion and Na ⁺ absorption by activating K ⁺ channels in the basolateral membrane to hyperpolarize the cell. Hyperpolarization stimulates Na ⁺ influx (into the cell) and Cl ⁻ efflux (into tears) across the apical membrane. PKA also stimulates apical Cl ⁻ channels. Because there is a higher rate of Cl ⁻ secretion relative to Na ⁺ absorption, water moves into tears in the basolateral to apical direction. In addition Na ⁺ moves paracellularly to produce an isotonic NaCl secretion into tears. Water moves through water channels known as aquaporins (AQP). AQP 5 is expressed in the apical membrane and AQP 3 in the lateral membrane.

Activation of the purinergic receptor P2Y ₂ by UTP and ATP also stimulates electrolyte and water secretion from the conjunctiva. P2Y ₂ receptors increase intracellular [Ca ²⁺ ].

E

Mucous layer function

The major roles of the conjunctiva are to contribute to tear production by secreting electrolytes and water, modify composition of the tear film by secreting mucins, absorb various organic compounds found in tears (including ophthalmic therapeutics), and contribute to resistance of the eye to infection by providing protection against microorganisms. Mucins serve as wetting agents that keep the apical epithelia hydrated. Finally, conjunctival cells release paracrine signaling agents such as growth factors that affect ocular surface properties.