Functional biology of intestinal goblet cells.

Functional

biology of intestinal

goblet cells

ROBERT D. SPECIAN AND MARY G. OLIVER Department of Cellular Biology and Anatomy, Louisiana State University Medical Center, Shreveport, Louisiana 71130 SPECIAN, ROBERT D., AND MARY G. OLIVER. Functional biology of intestinal goblet cells. Am. J. Physiol. 260 (Cell Physiol. 29): C183-C193, 1991.-Goblet cells reside throughout the length of the small and large intestine and are responsible for the production and maintenance of the protective mucus blanket by synthesizing and secreting high-molecularweight glycoproteins known as mucins. To elucidate the role of goblet cells in the biology of the intestinal tract, an overview of the physiological implications of the mucus gel is presented, including a concise review of the products secreted by the cell. Because of the unique nature of this highly polarized exocrine cell, the maturational reorganization of the cytoarchitecture and the cellular mechanisms by which goblet cells secrete their products are discussed. This includes elucidation of the baseline secretory pathway, which is dependent on the cytoskeleton for granule movement, and the accelerated secretory pathway, which is independent of the cytoskeleton but requires an extracellular signal to occur. Finally, the involvement of goblet cell mucins in the pathophysiology of intestinal neoplasia and ulcerative colitis are presented.

mucin; secretion;

mucus gel

THE INTESTINAL EPITHELIUM residegobletcells, highly polarized exocrine cells recognized by their apical accumulation of secretory granules (Fig. I). Goblet cells synthesize and secrete high-molecular-weight glycoproteins called mucins (17). Upon secretion, mucins hydrate and gel, generating a protective mucus blanket overlying the epithelial surface (Fig. 2). Within the mucus gel, other components, including water, electrolytes, sloughed epithelial cells, and secreted immunoglobulins, reside. This produces a physical and chemical barrier that protects the epithelium from luminal agents such as enteric bacteria, bacterial and environmental toxins, and some dietary components that pose a threat to the mucosa. WITHIN

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Production of mucins for the maintenance of the mucus blanket is the responsibility of goblet cells. Goblet cells arise by mitosis from multipotential stem cells at the base of the crypt (8) or from poorly differentiated cells in the lower crypt referred to as oligomucous cells (7) (Fig. 3). Kinetic analysis of goblet cell dynamics in mouse small intestine shows that once propagated, these cells migrate from the base of the crypt to the villus tip, where they are sloughed into the lumen (32). This pro0363-6143/91

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gression from birth to death occurs in mice in 2-3 days; thus the population of goblet cells is very short lived and is constantly undergoing replacement. Although intestinal goblet cells are distributed through the entire length of the mammalian intestinal tract, their contribution to the total epithelial volume is not constant. In the rat small intestine the volume density of goblet cells in the crypt is fairly consistent; in the villus epithelium, however, the volume density of goblet cells increases aborally, from the duodenum to distal ileum (21). This trend continues in the large intestine with the density of goblet cells in the colonic epithelium also increasing proximal to distal, from cecum to rectum. Goblet cells from both human colon (44) and rat colon (25) produce a heterogeneous collection of high-molecular-weight glycoproteins that can be separated into multiple mucin species by ion-exchange chromatography. The production of the multiple mucin species does not occur uniformly within the epithelium but results from the presence of biochemically distinct subpopulations of goblet cells. These goblet cell subpopulations produce and secrete selective combinations of mucin speciesthat vary by location along the length of the intestinal tract and by level of maturation along the crypt-villus axis. All human colonic goblet cells contain more than one

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FIG. 1. Light micrograph of rabbit surface colonic goblet aspect of the cell contains an ovoid nucleus surrounded by philic cytoplasm containing rough endoplasmic reticulum. clear region of the cell contains the Golgi apparatus and vacuoles. Apical region consists of an apical accumulation granules surrounded by the darkly stained theta. Periodic and iron hematoxylin; magnification = X1,400.

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cells. Basal very basoSupranucondensing of mucin acid-Schiff

mucin species. Three mucin species exist in mutually .exclusive cell populations in combination with other minor mucin species (43). Structural diversity also exists within stored mucin granules in rat small intestine. Immunologic distinctions can be found not only between adjacent goblet cells but also among the granules of an individual goblet cell (Oliver and Specian, unpublished observations). Goblet cell maturation. As evidenced in morphometric studies on rabbit colon (46), the goblet cell undergoes dramatic morphological changes during its life span. Once propagated from stem cells at the base of the crypt, the immature goblet cells rapidly begin to synthesize and secrete mucin granules. These immature goblet cells at the crypt base are large and pyramidal in shape with an expansive contact with the basal lamina and a confined contact with the crypt lumen. Synthetic organelles are loosely organized within the cell and are found interspersed with mucin granules in the apical portion of the cell (Fig. 4). As the cell progresses toward the colonic surface, it diminishes in volume, shedding cytoplasm and organelles trapped between mucin granules as the granules are secreted (Tables 1 and 2). During this volume reduction, cell morphology changes; contact with the basal lamina decreases, contact with the lumen increases, and organelles become segregated. At the mouth of the crypt, the cell volume has decreased by >60% and the cell has obtained its characteristic cup-like shape (Fig. 5); the apical portion of the cell is distended and packed with mucin granules, and the basal portion of the cell is narrowed into the “stem” of the goblet in which the nucleus and synthetic organelles reside (46). Migration to the villus tip or colonic surface produces not only morphological changes but also changes in the chemical composition of the mucins produced. As detected by histochemical methodology, immature goblet

FIG.

covered cellular exclude mucosa.

2. Light micrograph of mouse cecum. Epithelial surface is by a thick mucus blanket (between arrows) consisting of debris in a matrix of mucin glycoproteins. Blanket serves to the luminal bacteria (above top arrows) from access to the Toluidine blue; magnification = x200.

cells deep within the crypts in the small intestine produce neutral mucins containing little sialic acid. As they mature and migrate to the villus tip, the mucins become increasingly sialated; these sialic acid residues not only increase the acidity of the molecule but also are sites for further modification by N- and 0-acylation (14). Similarly, in the colon there are differences in mucin composition along the crypt-surface axis that can be detected by histochemistry. Goblet cells within the crypt contain predominantly sulfomucins; the mature goblet cells on the colonic surface contain neutral mucins (26). Structural variations detected by lectin cytochemistry demonstrate that mucins at the crypt base contain more Nacetylglucosamine and less terminal fucose than the more mature cells in the upper crypt (3). Furthermore, these mucins manifest nonreducing terminal galactose residues that are absent from mucins produced in mature goblet ceils (3, 4). These data suggest that altered glycosylation patterns accompany the maturational changes. Whether these changes are accompanied by production of distinct core peptides is as yet unresolved. Cytoarchitectural organization. The goblet cells that reside on the upper regions of the intestinal villi or on the colonic surface are the fully mature mucin-producing cells, displaying the typical goblet characteristics, a polarized and highly organized biosynthetic machinery which allows for unidirectional synthesis and secretion


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FIG. 4. Schematic representation based on three-dimensional reconstruction of an immature rabbit colonic goblet cell from the lower crypt. Goblet cell is pyramidal in shape, with an expansive contact with the basal lamina. Mucin granules are found throughout the cell, interspersed with elements of the rough endoplasmic reticulum and the Golgi apparatus. [From Radwan et al. (46).]

TABLE 1. Subcellular volume fractions after correction for nuclear volumes Subcellular Volume Fractions Lower crypt Middle crypt Upper crypt Mitochondria Lysosomes RER Golgi complex Mucin granules Nucleus Clear vesicles Cytoplasm

2.8kO.3 3.8rtO.7 2.OkO.2 0.2kO.04 0.3kO.06 0.4zko.05 5.7+0.5 7.OkO.6 3.8kO.4 6.1kl.O 6.2kO.7 7.5+1.0 36.7k3.8 36.6k2.3 47.8k3.2 12.9k1.8 10.7k1.2 11.4kl.l 0.920.3 0.6kO.2 0.2kO.l 34.9k1.7 34.8rt2.4 27.1k1.8 n 27 23 19 Values are means + SE; n, no. of cells. RER, rough reticulum.

Surface 2.7kO.3 0.4kO.l 4.OkO.5 6.7kO.8 31.6k2.7 22.5+2.7 0 33.6k2.3 16 endoplasmic

TABLE 2. Absolute volumes of the subcellular organelles

Lower crypt Mitochondria Lysosomes

FIG. 3. Light micrograph of a rabbit colonic crypt. At the base of the crypt reside numerous undifferentiated cells that contain few mucin granules (*). As goblet cells migrate toward the mucosal surface, they form a large compacted mass of mucin granules at the apical pole of the cell. Periodic acid-Schiff and iron hematoxylin; magnification = x400.

of mucin. The basal region of the cell is occupied by an ovoid nucleus surrounded basolaterally by mitochondria and rough endoplasmic reticulum (RER) (46, 53). Circumstantial evidence indicates that the protein, once synthesized in the RER, leaves this compartment as a “naked” protein. Although the “link” component of mutin does contain mannose (12, 31), a hexose typically added to protein in the RER (49), autoradiographic studies have not demonstrated glycosylation in the goblet

Absolute Volume, ym” Middle crypt Upper crypt Surface

34.7 2.7 RER 70.2 Golgi complex 74.8 Mucin granules 450.9 Nucleus 158.5 Clear vesicles 11.6 Cytoplasm 428.4 n 27 Values are means + SE; n, no. of

42.3 3.2 77.4 68.1 403.7 118.0 6.2 383.5 23 cells.

16.3 3.4 30.6 60.0 383.3 91.5 1.6 217.3 19

14.6 2.1 21.5 36.2 171.0 121.8 0 182.1 16

cell RER (2, 27, 35). Above the nucleus, the cytoplasm contains numerous Golgi stacks, the site of the stepwise addition of hexoses and hexosamines to the protein core. Localization of terminal N-acetylgalactosamine residues to the cis-cisternae and last two trans-cisternae (48) suggests that glycosyltransferases are compartmentalized in the Golgi apparatus of goblet cells as they are in other cells (49). In the supranuclear region, mucin-con-


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FIG. 5. Schematic representation based on three-dimensional reconstruction of a mature rabbit colonic goblet cell from the mucosal surface. Upon reaching the colonic surface, the cell has obtained its characteristic goblet shape. Mucin granules are condensed into an apical granule mass. Rough endoplasmic reticulum is distributed from the base of the cell into the supranuclear region of the cell, whereas the Golgi apparatus is sequestered solely in the supranuclear region. [From Radwan et al. (46).]

taining condensing vacuoles and mature secretory granules are also found in association with the Golgi complex

(53). The apical portion of the cell contains a tightly packed mass of mucin granules with the modest amounts of intervening cytoplasm confined to the angular spaces produced by multiple granule apposition. This close packing of mucin granules results in the formation of transient pentalaminar “prefusion” sites between adjacent granule membranes that in freeze-fracture replicas are distinguishable by their exclusion of intramembrane particles (37). This mass of mucin granules is separated from the lateral plasma membrane and the Golgi region by a layer of filament-rich cytoplasm known as the theta

(37, 60). The theta is composed of an organized array of microtubules and intermediate filaments (60; Fig. 6, A-C). The innermost layer of the theta contains -45-50 vertically oriented microtubules that appose the stored granule mass. These microtubules can occasionally be seen penetrating the intermediate filament layer at the base of the theta, suggesting that the microtubules arise in more basal regions of the cell. The medial and outermost layers of the theta are composed of intermediate filaments. The filaments of the outer layer are arranged circumferen-

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tially, while the filaments of the medial layer are arranged in a vertical spiral that also delimits the base of the theta (Fig. 7). The shape of the theta is maintained exclusively by the intermediate filaments and is not altered by the loss of microtubules or granules (60). Actin filaments are not prominent constituents of the theta but are present in other regions of the cell. As with other intestinal epithelial cells, the apical surface of intestinal goblet cells is populated with microvilli, although meagerly in comparison with absorptive cells. Actin filaments comprise the microvillus cores and are present in the interrootlet spaces (20). Unlike absorptive cells, actin filament bundles in goblet cells are present overlying the apical granule mass (53), similar in appearance to a cortical layer (Fig. 8). Although a variety of actin-associated proteins, such as a-actinin, myosin, villin, fodrin, and spectrin, have been localized in the brush border of absorptive cells (5), they have not been specifically localized in the goblet cell cortical layer due to the frailty of this network. Baseline secretion. Maintenance of the mucus blanket is accomplished by slow continual secretion of mucin by the goblet cell population (34,35). This baseline secretion is accomplished by periodic exocytosis of the contents of a single mucin granule. Although the apical granule mass is replete with granules, only a distinct portion of them is responsible for baseline secretion. As monitored by autoradiography in both rabbit colonic (60) and human rectal goblet cells (34), granules located along the periphery of the theta incorporate radiolabel to a far greater extent than those centrally stored. Six hours after a 30min pulse with [3H]glucosamine, radiolabeled peripheral granules are near or at the apical surface (Fig. 9, A-D). Although it is not known by what mechanism baseline secretion is regulated, it is readily apparent that the cytoskeleton does play a role. Treatment of mucosal explants of rabbit colon with nocodazole depolymerizes the microtubules throughout the cell, resulting in inhibition of granule movement and the aberrant migration of condensing vacuoles into the lower regions of the cell (Oliver and Specian, unpublished observations). In control tissues, radiolabeled granules are approaching the cell’s apical surface, whereas in nocodazole-treated tissues, labeled granules still reside in the theta (Fig. 10). Treatment of mucosal explants with taxol hyperpolymerizes microtubules in the supranuclear and basal regions of the cell; this inhibits the movement of nascent mucin glycoproteins into the apical granule mass. In the theta, however, taxol stabilizes existing microtubules, an action which does not alter granule migration within the granule mass (Fig. 10). Thus we concluded that microtubules direct granule migration to the cell apex but do not directly supply the dynamics for granule translocation (Oliver and Specian, unpublished observations). Even though actin appears to be a minor component in the goblet cell cytoskeleton (20, 60), autoradiographic evidence suggests that it also plays a role in regulating baseline secretion (41). Incubation of mucosal explants with cytochalasin D and dihydrocytochalasin B eliminates F-actin localization in the apical filament layer and increases granule translocation rates; radiolabeled granules reach the apical surface of the cell in 2 h instead


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FIG. 6. Schematic representation of the cytoskeleton of the goblet cell theta. A: innermost layer of the theca, adjacent to the mucin granules, is composed of 45-50 vertically oriented microtubules. B: middle layer of the theta is composed of a basketlike network of intermediate filament bundles that spiral vertically toward the apex of the cell and delimit the base of the theta. C: outermost layer of the theta is composed of circumferentially oriented intermediate filament bundles, appearing like barrel hoops around the theta. [From Specian and Neutra (60).]

FIG. 7. Indirect immunofluorescent localization of keratin filaments in an isolated rat colonic goblet cell. Keratin filaments, the primary structural support for the goblet cell, are present throughout the cell. As a result of mucin granule loss during processing, the theca of the cell appears as an empty cup. Magnification = ~1,600.

FIG. 8. Fluorescent phallotoxin localization of F-actin with NBDphallocidin in an isolated rat colonic goblet cell. Cytochemical localization of actin filaments demonstrates their restriction to the cytoplasm overlying the apical granule mass. Magnification = X1,600. [From Oliver and Specian (41).]

of 4-6 h as in goblet cells in control tissues (Fig. 11). It is thought that the apical filament layer functions as a physical barrier, hampering contact of granule membranes with the plasma membrane (Fig. 12). Disassembly of actin filaments disrupts this network and facilitates contact of the secretory granule with the plasma membrane, allowing baseline secretory rates to increase. Although intestinal goblet cells produce multiple mutin species, not all mucin species are secreted equally. In human colonic goblet cells from mucosal explants maintained in organ culture, six distinct mucin glycoproteins can be purified; the relative proportion of each within the tissue is constant over time. There is, however, an enhanced proportion of three of these mucin species in the media, suggesting that these are preferentially secreted from the cells (55). Maintenance of the protective mucus blanket during baseline secretion (unstimulated conditions) is the responsibility of a distinct subset of mucins, inferring the possibility of differing physiological

roles for the mucin species. Accelerated secretion. Far more is known about the regulatory mechanisms of accelerated goblet cell secretion. Goblet cells rapidly discharge most or all of their stored intracellular mucin in response to a variety of stimuli, including cholinergic stimulation (57~59), intestinal anaphylaxis (23), and chemical and physical irritation (36). Irrespective of the stimulus, the secretory mechanism is the same. Initially, fusion of a central apical granule results in the release of the contents of that granule and incorporation of the granule membrane into the apical plasma membrane (Fig. 13A). Unlike baseline secretion, where continuation of the secretory event depends on the transport of the next granule into position, accelerated secretion continues by the tandem fusion of subjacent granule membranes with the apical membrane. Continuation of this process results in the entire stored granule mass of the goblet cell being secreted in a matter of minutes (Fig. 13B). The excessive


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FIG. 9. Light microscopic autoradiography in goblet cells pulse-labeled with [“Hlglucosamine. A: after 20 min on cold chase, radiolabel incorporation, visualized by the presence of overlying silver grains, is discernible in the supranuclear region of the cell, the site of glycosylation and granule genesis. B: after 2 h on cold chase, radiolabel is still resident in the supranuclear region and is entering the base of the apical granule mass. C: after 4 h, radiolabeled glycoproteins have entered the apical granule mass and have been translocated toward the cell apex. D: by 6 h, a heavy accumulation of silver grains along the periphery of the apical granule mass demonstrates that radiolabeled mucin granules have migrated along the edge of the apical granule mass to the cell apex for secretion. Numerous silver grains overlying the epithelium suggest that some radiolabeled mucins have been secreted into the external milieu. -Magnification = X1,400.

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FIG. 10. Maximal movement of radiolabeled mucin granules in rat colonic goblet cells exposed to microtubule-disrupting agents in organ culture. In control goblet cells, [3H]glucosamine incorporation occurs in the Golgi region (leuel 1 ), and mucin granule movement progresses over time through the base of the theta (leuel3) and toward the cell apex (level 7). Treatment with taxol impedes movement of labeled granules out of the Golgi region but does not alter movement through the apical granule mass. Treatment with nocodazole severely inhibits granule movement throughout the cell. * P < 0.01. (From Oliver and Specian, unpublished observations.)

FIG. 11. Maximal movement of radiolabeled mucin granules in rat colonic goblet cells exposed to actin-depolymerizing agents in organ culture. In control goblet cells, [“Hlglucosamine is incorporated into nascent granules at level 2. By 2 h, radiolabeled granules enter the apical granule mass and by 6 h have reached the cell apex. In contrast, treatment with both cytochalasin D and dihydrocytochalasin B results in dramatic acceleration in granule movement; radiolabeled granules are nearing the cell apex after only 2 h. * P < 0.01. [From Oliver and Specian (41).]

membrane surface area resulting from the rapid secretion of mucin is not recycled, but rather sloughed into the intestinal lumen (57). Accelerated secretion is independent of intracellular motility and thus is not inhibited by drugs that impair either microtubule or actin filament function. This cavitation of the goblet cell is the hallmark of accelerated secretion. The characteristic shape of the goblet cell is maintained, even in the absence of mucin granules, by the complex three-dimensional basket-like

framework of intermediate (keratin) filaments (60). Goblet cells are resilient, recovering from an accelerated secretory event quite rapidly. When accelerated secretion is stimulated in the small intestinal crypt by the cholinomimetic drug pilocarpine, 90% of the stored intracellular mucin is secreted within 30 min (Fig. 14; Ref. 20). If the cholinergic antagonist atropine is added at the end of the 30-min secretory event, the goblet cells quickly synthesize new mucin granules and refill the cell


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FIG. 12. Schematic representation of the apical surface of a colonic goblet cell. Surface of the cell is sparsely populated with microvilli. Intercalated between the apical plasma membrane and the granule mass is a filamentous barrier consisting of actin filaments. This actin filament layer forms a physical barrier that impedes granule access to the plasma membrane.

FIG. 13. Mechanism of accelerated secretion in a rabbit colonic goblet cell. A: initiation of accelerated secretion is effected by secretion of an individual centrally stored granule. Magnification = ~22,100. B: 30 min into an accelerated secretory event, granule secretion progresses through central and peripheral granules, resulting in cavitation of the apical granule mass. Magnification = x10,500.

(Fig. 15, A-C). By 60 min after the initial secretory event, crypt goblet cells have completely refilled in the small intestine, as determined by quantitative stereology (61). Although colonic goblet cells do not refill as rapidly as those in small intestine, epithelial levels of stored mucin are back to presecretory event levels after 120 min (61); these data imply that goblet cells can undergo repeated accelerated secretory events during their 3- to 5-day life span. MUCIN

PHYSIOLOGY

The physicochemical characteristics of mucins bestow upon the mucus blanket its functional capabilities: lubrication, protection, and maintenance of normal intestinal flora (33). Mucin molecules contain a net negative

charge due to their acidic carbohydrate content; upon secretion, ionic interactions between these residues form a network overlying the epithelium. This layer functions similar to long-chain polymers in solution; they reduce the frictional drag produced by the fluid’s movement along a rough surface, thus easing the passage of particles and organisms along the surface of the mucus gel, corresponding to the length of the tube or gut (65). This network inhibits movement through the depth of the gel, producing a diffusion barrier for nutrients and small molecules (56). The mucus layer acts like a molecular sieve; absorption of molecules is directly proportional to the molecule’s diffusion rate through the mucus layer, and diffusion is indirectly proportional to molecular weight (39). The structure of mucin allows it to function to protect


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Second, interactions between secretory immunoglobulin A (IgA) and mucins trap IgA-coated bacteria within the mucus layer (29); subsequent degradation and renewal of the mucus layer, coupled with peristalsis, allow for expulsion of these trapped pathogens. Mucins also function to protect the epithelium from digestion by acting as a substrate for degradative enzymes produced by the intestinal flora. Partially purified enzyme fractions of fecal extracts contain cw-galactosidase, P-N-acetylgalactosaminidase, sialidase, P-glucuronidase, blood-group-degrading enzymes, and proteases (63). These enzymes, present in anaerobic fecal cultures, have demonstrated in vitro the ability to remove >90% of the total carbohydrate; proteases, though present, degrade protein to a far lesser degree. Although degradation to this extent has not been demonstrated in vivo, biochemical studies have demonstrated that secreted mutin does differ from intracellular mucin; secreted mucin has a lower molecular weight and contains less carbohydrate than stored mucin (11). This metabolism of mucins modulates growth of intestinal bacteria in vitro (28) and may serve in vivo to regulate the microecology of the intestinal lumen. Mucin biochemistry. Biochemically, mucins are large glycoproteins, with an average molecular weight of 2 X 106, determined by their exclusion on Sepharose 4B chromatography and minimal entry into polyacrylamide gel during electrophoresis (16). Mucin glycoproteins consist of linear or branched oligosaccharide chains attached to a protein core. The majority of the molecule consists of these carbohydrate moieties; in both rat (10) and human small intestinal mucins (64), carbohydrate comprises 80-85% of the molecule by weight. Colonic mucins

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FIG. 14. Morphometrir analysis of the mucin secretory response to pilocarpine in rat small intestine. Thirty minutes after administration of pilocarpine, total stored mucin per mucosal surface area dramatically decreases throughout the length of the small intestine. [From Kemper and Specian (211.1

the mucosa from bacterial overgrowth and penetration. It has been demonstrated in mouse ileum that only a small proportion of the intestinal flora contact and adhere to the epithelial surface. The mucus layer, covering most of the tissue surface, produces an environment richly populated by bacteria and protozoa (50) and can contribute to inhibiting microbial overgrowth. Mucins bind and trap pathogens by two distinct methods. First, certain carbohydrate moieties on the mucin molecule can immobilize pathogens within the mucus layer by either mimicking epithelial cell membrane glycoproteins that are recognized and bound by a pathogen’s adherence lectin (6) or by binding to other membrane components, such as type 1 pili expressed by Escherichia coli (51, 52).

15. Light micrographs of rat jejunum stained with periodic acid-Schiff and toluidine blue. A: during unstimconditions, small intestinal goblet cells in the crypts and on the villi are filled with mucin granules. B: 30 min stimulation with pilocarpine in vivo, crypt goblet cells have emptied their complement of mucin granules, and of the surface cells are cavitated, suggesting accelerated secretion (arrows). C: treatment with pilocarpine for 30 followed by administration of pilocarpine and a 60-min recovery period, goblet cells in the crypt have refilled mucin granules, and surface cells demonstrate no cavitation. Magnification = X200.

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are less glycosylated than small intestinal mucins, com- tine stomach (47), and human small intestine (30). The prising 62% of mucin by weight in rat colon (25) and 67biochemical and structural profile of the link component is consistent across tissue and species lines; indeed, an80% in human colon (44). The protein core of intestinal mucins has a very dis- tibodies to link component from rat and human small tinctive amino acid composition. Serine (Ser) and threintestine react with those from other species (47). The onine (Thr) comprise 25% of total amino acid residues link component is a glycoprotein; however, carbohydrate in rat colonic mucin (25); in small intestinal mucins, Ser comprises only 50% of the total molecule compared with and Thr are even more prevalent, making up 41% of the 70-85% in native mucin (12). Similar to the core protein, molecule in human (64) and 57% in rat (10). Both Ser this peptide has high percentages of Ser, Thr, and Pro, and Thr have side chains that contain a hydroxyl group albeit lower than those of the core. As a result, the that are involved in linking oligosaccharide chains to the peptide has fewer attachment sites for oligosaccharide protein via 0-glycosidic bonds (62). This greater percent chains. Asp and Glu occur at higher percentages in the of Ser and Thr in small intestinal mucins produces a link component than in the core protein, resulting in a different primary structure and additional potential sites molecule which is more acidic (47). In addition to the for glycosylation. The heterocyclic amino acid proline sugars found in the native mucin, the link component (Pro) is also found in sizable quantities, accounting for also contains mannose (l2), a sugar usually associated with N-linked glycosylation that occurs in the RER (49). up to 15% of the total amino acid residues; it is thought that Pro prohibits a-helix formation and prevents com- In vitro studies suggest that the mannose-containing pact folding of the polypeptide, thus maintaining an carbohydrate moieties on the link glycopeptide are able expanded conformation that facilitates a high degree of to sequester pathogens by binding to type 1 pili on E. glycosylation (1). Kim and co-workers (19) have dem- coli (52). Mucin heterogeneity. Due to the complexity of the onstrated in human small intestinal mucin that three cDNA probes against the protein backbone contain a oligosaccharides, it was originally thought that mucins consensusamino acid sequence produced by 69-base pair were homogeneous and differences between molecules tandem repeats; this consensus sequence is rich in Ser, were due to polydispersity of the carbohydrate content. Thr, and Pro and may be the site for glycosylation of the Using ion-exchange chromatography in conjunction with molecule. Core proteins also contain a high percentage molecular sieve chromatography, however, Lamont and Ventola (25) demonstrated that rat colonic mucin conof acidic amino acids; aspartic acid (Asp) and glutamic acid (Glu) combined account for -20% of the total pro- tained more than one species of mucin glycoprotein that tein in small and large intestinal mucins of both human differed in both amino acid and carbohydrate content. Immunochemical studies on human colonic mucin aland rats. The oligosaccharide side chains are formed by five luded to the presence of more than one species of mucin (18). Ion-exchange chromatography of human colonic sugars: fucose (Fu), galactose (Gal), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and mucin resulted in isolation of six different species of sialic acid (SA). They contain no glucose and trace mucin, each with a distinctive carbohydrate profile (44), amounts of mannose (1). These sugars form linear or as well as oligosaccharide structures (42). Studies on branched arrays of 2-12 sugar residues that are attached human small intestinal mucin have revealed the presence via an 0-glycosidic bond between Ser or Thr in the of two distinct mucin speciesthat not only have different protein and GalNAc. The core region of the oligosaccha- carbohydrate composition but also different core pepride chains consists of a linear arrangement of Gal- tides (64). RNA blot analysis using cDNA probes has demonstrated restriction fragment polymorphism; GlcNAc-GalNAc (42); upon this core more complex structures are built. These consist of a backbone region whether this results from length polymorphism in the gene or from sequence differences has yet to be deterof alternating Gal and GlcNAc residues and a peripheral region of Gal, GalNAc, and the terminal sugars Fu and mined (19). It does appear, however, that mucins exist SA (13). Isolation and characterization of oligosaccha- as a true heterogeneous population. Other secretory products. In addition to mucins, other rides from human colonic mucin have revealed the presence of 21 discreet oligosaccharide structures, 10 that are potentially physiologically significant products have acidic and 11 that are neutral. The oligosaccharide profile been localized within intestinal goblet cells. Ingobsin, a Ser protease capable of cleaving both epidermal growth from rat colonic mucin is less expansive, demonstrating the presence of only eight distinct structures (56). These factor and the cobalamin-binding protein haptocorrin, structures are not species specific, however; four of the has been localized within both human and rat goblet oligosaccharides isolated from rat colonic mucin have cells (38). The presence of enzyme-positive cells dealso been isolated from human colonic mucin (44, 56). creases from duodenum to colon. Additionally, more mature goblet cells (those in the upper crypt and surface Detailed description of the oligosaccharide structures cells) appear to contain little or no ingobsin (38). Almay be found in Refs. 42 and 54. Link peptide. Intestinal mucins do not exist solely as though it does not appear to be copackaged with mucins, it is present in small quantities in luminal washes; secremonomers; they exist as polymers joined by a glycopeption of ingobsin is stimulated by acetylcholine, a known tide that is both biochemically and structurally distinct from the remainder of the molecule and is liberated by secretagogue for mucin; addition of vasoactive intestinal reduction (10). This putative link component has been peptide to this system further increases the secretion of ingobsin (22). Immunolocalization of IgA in human inisolated from numerous mucins, including those from rabbit colon (31), human colon, rat small intestine, por- testinal goblet cells has demonstrated that IgA can be


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copackaged with mucin in secretory granules (40); the mechanism whereby this phenomenon occurs is unknown.

REVIEW Address for reprint requests: R. D. Specian, Dept. of Cellular Biology and Anatomy, LSU Medical Center, 1501 Kings Highway, Shreveport, LA 71130. REFERENCES

PATHOPHYSIOLOGY

In response to pathophysiological alterations in the intestinal mucosa, the mucus gel changes as a result of perturbed synthesis/secretion by the goblet cell population. During neoplasia, there are both quantitative and qualitative alterations in mucin production. Mucin secretion in malignant human small intestinal tumors is dramatically decreased or absent (15). Mucins that are produced by goblet cells in transformed mucosa, whether malignant or benign, demonstrate an altered histochemical staining pattern. In the small intestine, goblet cells in the crypts produce sulfated mucins, which are normally not present (15); in the colon, crypt goblet ceils demonstrate an increase in sialomucin production (9). Lectin cytochemistry in both human and mouse colonic mucosa corroborate that neoplasia is manifested in altered mucin oligosaccharide structure (4). Malignant transformation results in the unmasking of nonreducing Gal residues in colonic mucins as demonstrated by peanut agglutinin binding; furthermore, these residues are present in regions of the mucosa that appear histologically normal and in the mucus gel overlying the epithelium. Normally, nonreducing Gal residues are demonstrable only in immature goblet cells and are not present in the mucus gels (3, 4). Accompanying the changes in mucin composition are alterations in glycosyltransferase activity that may account for the altered oligosaccharide content (24). These data suggest that malignant transformation is manifested by dedifferentiation of the goblet cell population; this results in alterations of the normal glycosylation patterns and the production of immature mucins. A far more specific disease-associated alteration in mucin production occurs in patients with ulcerative colitis. Quantitative analysis of colonic mucin fractions produced by colonic goblet cells during baseline secretion reveals that there is increased secretion of one mucin species that is normally retained within intracellular pools (55) and that this alteration in stored mucin is present during all phases of disease activity, including quiescence (45), and appears to be diseasespecific; mucin profiles from patients with Crohn’s colitis resemble those of controls (44). Because the fundamental function of mucus layer is to protect the epithelium from assault, physical or chemical, by conjecture, the production of abnormal mucin may impair the layer’s defense capability, allowing bacterial toxins to penetrate the epithelium and initiate the sequelae of ulcerative colitis. Because maintenance of the mucus layer is achieved by baseline secretion entailing the preferential secretion of mucous granules stored in the periphery of the theta along microtubular tracks (60; Oliver and Specian, unpublished observations), the abnormal secretion of a mucin species usually stored suggests a defect in the cell’s ability to sort and retain this product.

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Acid mucins in human intestinal goblet cells.

Secretion of intestinal goblet cells: a novel excretion pathway of nanoparticles.

β-Casein(94-123)-derived peptides differently modulate production of mucins in intestinal goblet cells.

Natural anti-intestinal goblet cell autoantibody production from marginal zone B cells.

Both crypt and villus intestinal goblet cells secrete mucin in response to cholinergic stimulation.

Goblet cells secretion and mucogenesis.

Nippostrongylus brasiliensis: intestinal goblet-cell response in adoptively immunized rats.

Cytotoxicity and metabolic stress induced by acetaldehyde in human intestinal LS174T goblet-like cells.

Is the intestinal goblet cell a major immune cell?

Isolation and functional characterization of human intestinal mucosal lymphoid cells.

Effects of Yersinia enterocolitica infection on rabbit intestinal and colonic goblet cells and mucin: morphometrics, histochemistry, and biochemistry.

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Intestinal NHE8 is highly expressed in goblet cells and its expression is subject to TNF-α regulation.

Goblet Cell Derived RELM-β Recruits CD4+ T Cells during Infectious Colitis to Promote Protective Intestinal Epithelial Cell Proliferation.

Immunomodulatory cross-talk between conjunctival goblet cells and dendritic cells.

Sedimentation velocity studies on microgram quantities of rat intestinal goblet cell mucin.

Radioimmunoassay of human intestinal goblet cell mucin. Investigation of mucus from different organs and species.

Cytoarchitectural reorganization of rabbit colonic goblet cells during baseline secretion.

The functional approach in biology.

Goblet cells of the conjunctiva: A review of recent findings.

mTORC1 signaling controls Paneth and goblet cell differentiation in the intestinal epithelium.

The influence of 400 r x-irradiation on the number and the localization of mature and immature goblet cells and Paneth cells in intestinal crypt and villus.

Effect of topical rebamipide on human conjunctival goblet cells.

Intestinal goblet cell differentiation in Nippostrongylus-infected rats after transfer of fractionated thoracic duct lymphocytes.