ABSTRACT
Mucous cell hyperplasia is a prevalent lesion in chronic inflammatory airway diseases including chronic bronchitis (1), asthma (2), cystic fibrosis (3), and bronchiectasis (4). Described as a hypertrophy of mucus-secreting elements in the conducting airways, mucous cell hyperplasia is characterized by an increase in goblet cell number, cell size, and mucin content. Increased epithelial height and enlargement of submucosal glands are also observed throughout the airways (re253
viewed in Refs. 5 and 6). Clinical implications of the large amount of mucus secreted into the airways include increased bacterial infections and airway obstruction that, in some cases, can lead to death (7). Even with its toll of morbidity and mortality, molecular mechanisms within epithelial cells that govern mucous cell differentiation and proliferation during development of mucous cell hyperplasia have not been elucidated.While detailed mechanisms are not currently understood, results of studies using animal models suggest that inflammation (8-13) and injury to epithelial cells (14-16) are important elements in the development of mucous cell hyperplasia. A diverse array of injurious insults including cigarette smoke (8), ozone (9), elastase (10), and sulfur dioxide (8) have been found to cause the lesion in experimental animal systems. An inflammatory component responding to this injury also appears to be important to the development of mucous cell hyperplasia, as development of the lesion can be enhanced by endotoxin (11) or previous viral infection (12). Conversely, the increase in goblet cell number induced by elastase (13), ozone, or cigarette smoke (14) can be attenuated with steroid treatment or nonsteroidal anti-inflammatory drugs (6,10). IL-13
Recent studies indicate that within the inflammatory milieu, interleukin 13 (IL-13) is a central molecule in the development of mucous cell hyperplasia. IL-13 was found to be sufficient to increase goblet cell number in the airway epithelium when administered intranasally to wild-type mice (17). Similarly, a blockade of IL-13 inhibited the increase in mucus-containing cells observed in an ovalbumin-sensitized murine model of allergic asthma (18). Selective expression of IL-13 in the lungs of transgenic mice resulted in a phenotype characterized by airway epithelial cell hypertrophy, mucous cell hyperplasia, and increased neutral and acidic mucus (19). IL-13 was also found to induce expression of Muc5/5ac mucins in the murine allergic-asthma model, with or without ovalbumin challenge (20).Increases in mucous cell number in the airways of IL-4 transgenic mice (21) have also been observed, along with induced mucin gene expression following stimulation with IL-4 in vivo and in vitro (22). Overexpression of IL-4, however, is predominantly found in the airways of allergic asthmatics, while elevated levels of IL-13 have been confirmed in both allergic and nonallergic asthma (23,24), as well as in pulmonary fibrosis (25). Thus, IL-13 may be a generalized effector molecule of the mucous cell hyperplasia observed in a wide variety of inflammatory airway diseases.Little is currently known about intracellular mechanisms governing IL-13-induced mucous cell proliferation in airway epithelium. However, IL-13-induced proliferation has been extensively examined in activated B cells (26) and hematopoietic cell lines (27), providing information about downstream events in IL-13-
mediated signal transduction pathways. Upon binding IL-13, the IL-13 receptor is known to activate one or more members of the Janus kinase (JAK) family of tyrosine kinases (28). While IL-13 can induce phosphorylation of JAK1, JAK2, or tyk2, depending on the cell type, phosphorylation of JAK3 has not been observed. IL-13 induces rapid phosphorylation of JAK2 in a human colon carcinoma cell line, followed by JAK1 and tyk2 phosphorylation (29). Tyk2 phosphorylation is also augmented in response to IL-13 stimulation in an ovarian carcinoma cell line (30). IL-13 causes phosphorylation of JAK2, but not JAK1 or tyk2 in endothelial cells (31), but in many fibroblast cell lines JAK2 and tyk2, and less frequently JAK1, become phosphorylated (29). Taken together, these findings suggest that IL-13 is unlikely to stimulate JAK3 phosphorylation in airway epithelium, but may phosphorylate JAK2, tyk2, and (possibly) JAK1.IL-13, and the related cytokine IL-4, require activation of an intracellular signaling molecule, the insulin receptor substrate (IRS-1 or IRS-2), in addition to JAKs, to provoke a proliferative response (27,28,32-34). IL-13 activates IRS-2 via phosphorylation in numerous cell lines and primary cells of lymphohemo-poietic origin (27,35-38). This suggests that during development of mucous cell hyperplasia in the airway epithelium, the IL-13 receptor with its associated JAKs may use IRS-2 as an adapter molecule for intracellular signaling. As such, IRS-2 may interact with additional signaling molecules such as the p85 regulatory subunit of phosphatidylinositol 3' kinase (PI 3' kinase) (28,34), which has been shown to activate a number of cell cycle-regulating enzymes essential for cellular proliferation (39-42). GROWTH FACTORS AND EPITHELIAL CELL INJURY While a direct proliferative effect of IL-13 on epithelial cells has not been described previously, numerous studies have noted proliferation of airway epithelial cells in response to injury that triggers inflammation (43-45). Increased amounts of transforming growth factor alpha (TGF-a) and/or epidermal growth factor receptor (EGF-R) correlate with this proliferation in damaged respiratory tissue (43,46). Interestingly, recent studies using pathogen-free rats and an airway epithelial cell line (NCI-H292) also have implicated EGF-R in the development of airway mucous cell hyperplasia (47).Proliferation of tumor cells (48,49), as well as epithelial cells in benign proliferative diseases ranging from benign prostatic hyperplasia (50) to gastroesophageal conditions (51,52) appears to involve the autocrine action of TGF-a on the EGF-R. One study even suggests that such a mechanism functions in the transformation of rat tracheal epithelial cells in vitro (53), giving precedence for TGF-a/EGF-R-activated proliferation of airway epithelium. Further support for such an autocrine/paracrine proliferative mechanism comes from observations that EGF-R and TGF-a are coincidentally localized in epithelial cells fol
lowing naphthalene-induced bronchiolar injury (43). TGF-a participating in an autocrine/paracrine proliferative mechanism could function to activate EGF-R as an uncleaved TGF-a precursor (54), or as a soluble product cleaved by an elastase-like protease (55-57). Thus, there appears to be ample precedence, supported by data regarding proliferation of respiratory tissue, to suggest that airway epithelial cell proliferation may be controlled by a TGF-a/EGF-R autocrine/ paracrine mechanism during the injury /repair process or during development of mucous cell hyperplasia. PI 3' KINASE As discussed above, recent studies implicate IL-13 and/or EGF-R as important molecules in the development of mucous cell hyperplasia. While each of these molecules may contribute to development of the lesion under different circumstances, it is also possible that another mediator coordinates their actions during the pathogenic process. Both IL-13 (60,61) and EGF-R (62-64) can activate PI 3' kinase, an enzyme shown to be activated in response to cytokines important in many proliferative responses (58,59). Thus, PI 3' kinase may serve to integrate multiple intracellular signaling pathways to affect epithelial cell proliferation during the hyperplastic response.PI y kinase, which consists of a p85 regulatory subunit and a pi 10 catalytic subunit, phosphorylates phosphoinositides at the D-3 position of the inositol, creating multiple second messengers including PI-3-P, PI-3,4-P2, and PI-3,4,5-P3 (reviewed in Ref. 65). The precise mechanistic events leading to PI 3' kinase activation currently are not well understood. Activation of PI 3' kinase appears to involve phosphorylation of the p85 regulatory subunit (63), a process mediated, in part, by IRS-2 following IL-13 stimulation (21,27,33,36,43,58). In addition, direct physical interaction with cytoplasmic proteins, such as IRS-1/IRS-2 (28), also appears to play a role in PI 3' kinase activation, although the proteins involved differ with cell type and stimulus. Activation of PI 3' kinase in response to EGF-R stimulation, for example, may involve cytoplasmic proteins as diverse as c-Cbl, src, Gabl, pi 15, and pl05 (66-69). Once the p85 subunit is phosphory-lated and associated with additional cytoplasmic proteins, it may serve to target the pi 10 catalytic subunit to the plasma membrane, where it can access its substrate, “ inositol,” or phosphatidylinositol. This translocation step appears to be required for downstream signaling via PI 3' kinase (70,71). Thus, based on current knowledge regarding PI 3' kinase activation, IL-13 stimulation is likely to result in phosphorylation of the p85 subunit and its physical association with IRS-2 and/ or other cytoplasmic proteins, followed by translocation to the plasma membrane.As a pivotal molecule coordinating IL-13-induced mucous cell hyperplasia, PI 3' kinase would likely cause an increase in mucous cell proliferation by modulating cell cycle regulatory enzymes. PI 3' kinase is known to upregulate
two such enzymes, p70S6k (72,73) and cyclin-dependent kinase-2 (cdk2) (42), both of which are essential to the transition from G1 to S phase during cellular proliferation (74,75). Increased activity of p70S6k and cdk2 induced by PI 3' kinase provokes proliferation in a variety of cell types including adipocytes, pancreatic cells, and aortic smooth muscle (39-42). Interestingly, cellular proliferation involving PI 3' kinase activation of p70S6k appears to be mediated by IRS-1 or IRS-2, signaling molecules important to IL-13-induced activation of PI 3' kinase (70,71). FUTURE DIRECTION AND SIGNIFICANCE Studies in our laboratory have begun to examine potential mechanisms by which IL-13 may induce mucous cell hyperplasia in human airway epithelium. To address this phenomenon, normal human bronchial epithelial (NHBE) cells grown in an air/liquid interface culture (76) are utilized. These NHBE cells, which begin growth in culture in an undifferentiated state, develop over time fully differentiated characteristics, including secretion of membrane-bound mucin-containing vesicles and fully developed cilia. Thus, experimentation can be carried out throughout the entire course of mucociliary differentiation, yielding a model of epithelial injury and repair. Such an in vitro model is of particular importance for studying effects of inflammatory mediators on airway epithelium, since focal points of injury are known to be interspersed with regions of normal epithelium in injured, inflamed airways (77). Thus, areas of undifferentiated and differentiated epithelium will respond differently to inflammatory mediators, a finding we have previously shown to be true when responses to such mediators (elastase, IL-13, TNF-a 4-IFN-y + IL-1 (3) are examined using the NHBE cells in vitro early and late in their course of differentiation (78). In addition, such an in vitro model of mucous cell hyperplasia can be readily manipulated to allow elucidation of signaling pathways within the epithelial cells that regulate development of the lesion.Preliminary studies from our laboratory suggest that continuous exposure of NHBE cells to IL-13 during the course of mucociliary differentiation in vitro results in a mucous phenotype with characteristics typical of mucous cell hyperplasia. In addition, IL-13 induces proliferation of differentiated NHBE cells in vitro. Thus, NHBE cells continuously exposed to IL-13 provide a good model of mucous cell hyperplasia, allowing mechanistic examination of two important phenomena leading to development of this lesion: differentiation and proliferation.Any suggested in vitro mechanism must complement well what is known from in vivo models of airway mucous cell hyperplasia (8-11). For example, elastase and ozone, two agents known to provoke mucous cell hyperplasia in vivo, can directly interact with a proposed epithelial cell TGF-a/EGF-R autocrine/ paracrine loop mechanism, as elastase can cleave TGF-a, while ozone may activate the EGF-R pathway via generation of oxidative species. In addition, any
epithelial damage inflicted in vivo results in increased airway inflammation with inflammatory mediators such as IL-13 activating additional intracellular pathways such as those mediated by IRS-2. Of more general interest may be the role PI 3' kinase plays in coordinating the mechanisms required for development of mucous cell hyperplasia. This enzyme, with its ability to be activated by several signaling pathways and to respond by producing multiple second messengers, is ideally suited to coordinate the seemingly opposing events of cellular differentiation and proliferation involved in this pathogenic process. Understanding the mechanistic details of this coordination is expected to provide a greater understanding of the ways epithelial cells regulate sequential processes to provoke permanent phenotypic changes. ACKNOWLEDGMENTS The authors would like to thank Dr. Frederic Toumier from the Laboratoire Cy-tophysiologic et Toxicologie Cellulaire, Universite Paris 7, for stimulating discussions, and Ms. Anne Crews for help in preparation of this manuscript. Dr. Macchione was funded by a fellowship from the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (Brazil). REFERENCES
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