key: cord-0007516-b11sapor authors: Jany, Berthold; Gallup, Marianne; Tsuda, Tohru; Basbaum, Carol title: Mucin gene expression in rat airways following infection and irritation date: 1991-11-27 journal: Biochem Biophys Res Commun DOI: 10.1016/s0006-291x(05)81373-7 sha: 96174bec110bfcd670e31e1297aeabb341c6ce29 doc_id: 7516 cord_uid: b11sapor Airway mucus hypersecretion occurs in response to infection and irritation and poses an important and poorly understood clinical problem. In order to gain insight into its pathogenesis, we have focused on an mRNA encoding the major mucus glycoprotein, mucin. Northern blots showed that mucin mRNA was abundant in the intestine of specific pathogen free rats whereas it was undetectable in the airways of these rats until pathogen-free conditions were suspended and rats acquired Sendai (Parainfluenza I) virus infections. Airway mucin hybridization signals in rats that were both infected with Sendai virus and exposed to SO(2)were more intense than those in rats with infection alone. These results suggest that pathogen-and irritant-induced hypersecretion may be partly controlled at the level of mucin mRNA. accessible ad libitum. SOg-exoosed rats were exposed in plexiglass chambers to 400 ppm sulfur dioxide gas in air for 3 h/if, 5 ~week, for 1 to 3 wk [7] . SO2-concentrations were measured in the chamber outflow by a colorimetric assay [8] . Sham-exposed control rats were placed in an exposure chamber into which air was administered in place of SO2. Non-exposed time control rats were sacrificed immediately after arrival from the vendor or after living for three weeks in the animal colony. Rats were assigned to experimental and control groups for RNA analysis as follows: unexposed 0 time=5 rats (Fig. 3, Group D) ; unexposed 3 weeks=4 rats (Fig. 3, Group B ); SO2 exposed 1 week=2 rats (Fig. 3 , Group C); SO2 exposed 2 weeks=2 rats (Fig. 3 , Group C); SO2 exposed 3 weeks=3 rats (Fig. 3 , Group C; sham exposed 3 weeks=2 rats (Fig. 3, Group A) . In addition, rats used for electron microscope analysis were as follows: unexposed 0 time=2 rats (Fig 1, upper panel) ; unexposed 3 weeks--4 rats; SO2 exposed 1 week =3 rats ; SO2 exposed 2 weeks=4 rats; SO2 exposed 3 weeks=5 rats (Fig. 1, lower panel) . Electron microscopy: Electron microscopy was performed on tissues from rats that had been f'~ed by intracardiac perfusion with 2.5% glutaraldehyde in 0.08 M Na cacodylate buffer, pH 7.4. Osmication was in 2% OsO4 in 0.14 M Na veronal and block staining was in uranyl acetate in 0.2 M Na maleate buffer, pH 5.2. Tissue was dehydrated in graded ethanols, passed through propylene oxide, and embedded in Medcast resin. Silver sections were stained with uranyl acetate and lead citrate and photographed in a Zeiss EM 10 electron microscope. Serum antibody titer determination: Serum was taken from rats upon sacrifice and was assayed by ELISA (Enzyme Linked Immunosorbent Assay) for antibodies against M. pulmonis, Corona and Sendai (Parainfluenza 1) viruses, PVM, KRV, Toolan H-l, and CAR Bacillus by Microbiological Associates, Inc. (Bethesda, MD). For RNA analysis, the tracheobronchial tree, as well as the intestine and heart, were removed from anesthetized rats. RNA was extracted as previously described [9] . Ten micrograms of total RNA per lane were electrophoresed through denaturing agarose/formaldehyde gels, transferred to GeneScreen membranes, and hybridized to the [32p]_ labeled human intestinal mucin eDNA, SMUC 41 [10] . Washing conditions were 0.2 x SSC (2 x SSC = 0.3 M sodium chloride, 0.03 M sodium citrate), 1% SDS, 63°C, 30'. After washing, blots were exposed to X-OMAT film (Kodak). Blots were stripped and re-probed with a [32p]_ labeled beta-actin cDNA. As shown in Figure 1 , the airways of rats exposed to SO2 for three weeks contained numerous mucous (goblet) cells. These were similar to those seen in human chronic bronchitis [2] . Secretions adhering to the apical surfaces of airway epithelial cells were also present in SO2exposed, but not SPF control rats (Fig. 1) . In order to monitor mucin mRNA levels in the airways of experimental and control rats, it was necessary to identify a mucin eDNA that hybridized with rat mucin mRNA. At the time we began our studies, mucin cDNAs had been isolated from eDNA libraries constructed from the human intestine [10] , pig submaxillary [11] , and human mammary [12] , [13] , [14] glands. The similarity between the amino acid compositions of intestinal and airway mucins [15] , [16] , [17] , [18] led us to test the feasibility of using an intestinal cDNA for our studies. Our initial studies revealed a high degree of homology between mucin mRNAs in the intestine and the airway of man [19] . Using the intestinal mucin cDNA SMUC 41 as a probe, we isolated HAM-l, a close homologue of SMUC 41 containing threonine-and proline-rich tandem repeats, from a human airway eDNA library [19] . Although we initially expected the human airway mucin eDNA to be more favorable for probing rat airways than the human intestinal mucin eDNA, the larger size of SMUC-41 made it more favorable. Using SMUC-41 in Northern blots, we probed RNA from two rat mucin-secreting organs (intestine and airways), and used RNA from a non-mucin secreting organ (heart) as a negative control. In Northern blots, the intestinal (Fig. 2) and airway (Fig. 3 ) signals were polydisperse, a distinguishing feature of mucin mRNA [10] [19] [20] corresponding to polydispersity in the size of nascent [21] and deglycosylated [18] mucin peptides. The integrity of the RNA was confirmed by reprobing blots with a beta actin eDNA (Figs. 2 and 3, lower panel). RNA from the heart and airways of specific pathogen-free rats was negative ( Figure 2, and D lanes, Fig 3) . The airway negativity is consistent with studies showing the paucity of mucous cells and mucin glycoprotein in the airways of specific pathogen-free rats (Fig 1, control, [22] , [23] ) as well as in healthy dogs [24] , [25] and humans [26] . The failure of SMUC-41 to hybridize to RNA from SPF rat airways was conspicuously reversed when rats were housed in the Animal Colony under non-pathogen-free conditions and exposed for 1-3 weeks to SO2 (Fig. 3 , C lanes) or air alone (Fig.3, A lanes) . Serum from each rat was tested (see Table) for the presence of antibodies directed against PVM, Sendai virus, Corona virus, KRV, Toolan H-l, M. pulmonis, and CAR Bacillus. We found that all rats with significant antibody titers for Sendal virus [27] , [28] , [29] (Fig. 3) showed mucin hybridization signals. SPF rats (Fig. 3 , D lanes and the bronchus lane in Figure 2 ) as well as those infected by another pathogen (i.e. Corona virus, Fig. 3 , B lanes) did not. Mucin hybridization signals obtained from airway RNA of Sendai virus-infected rats that were exposed to SO2 for 1 wk showed signals more intense than those from rats with infection alone (Fig. 3, C lanes) . We have shown that a human intestinal mucin cDNA, SMUC-41, is homologous to mucin mRNA in the intestine and airways of the rat. This homology enabled us to use SMUC-41 to monitor rat mucin mRNA and to discover that a SMUC mucin gene analogue is constitutively Rats sham-exposed to air, housed in laboratory 3 wk; (B) rats housed in animal colony 3 wk; (C) rats exposed to SO2, 1, 2, or 3 wk, housed in laboratory, (D) rats sacrificed immediately upon arrival from vendor (D). Each lane represents an individual animal (6, 17) . "+" = significant antibody titer or SO2-exposure; "--" = no significant antibody titer or SO2-exposure; "O" = antibody titer not determined. For a complete list of pathogens assayed, see Table 1 . = 10 lag total RNA was loaded per lane. Blots were hybridized to [32P]-labeled SMUC 41 as described above, exposed to X-OMAT film (Kodak), stripped, and reprobed with a beta-actin cDNA. expressed in the intestine of SPF rats, but requires activation by infection or injury in order to be expressed in the airways. As in human tissues [10] [19] , the rat intestine and airway yielded large, polydisperse signals when hybridized with SMUC 41. The polydispersity could not be explained by overall RNA degradation since reprobing with a beta actin probe consistently produced crisp hybridization bands (Fig 3) . Polydispersity in the size of deglycosylated mucin polypeptides [ 18] suggests that polydisperse transcripts are functional. Such transcripts could arise through alternative splicing [30] , although partial degradation of message during RNA turnover [31] cannot be excluded. The constitutive expression of mucin mRNA in the rat intestine (Fig 2) is not surprising in light of the necessity in that organ for continuous lubrication. On the other hand, mucin and mucin mRNA may be only minimally produced in airways except in specific response to insult or injury. Mucin appears to be present in low abundance in the airways of healthy dogs [25] and humans [26] but is present in high abundance in the airways of chronic bronchitics [26] or dogs exposed to tobacco smoke [25] . Our results are consistent with these data as well as with data showing that mucous cells are rare in the airways of SPF rats [23] , [22] but increase when airways become injured [7] , [32] or infected [22] . The particularly high abundance of mucin mRNA observed after one week SO2 exposure is intriguing; the diminution at two and three weeks occurs while mucous (goblet) cell numbers remain elevated. This may indicate that during the early stages of injury, goblet cells rapidly discharge and resynthesize mucin, but in the later stages, tolerance occurs and ceils are relatively quiescent. The resuks of our study suggest that one of the molecular events reponsible for converting a low-mucin-secreting to a high-mucin-secreting epithelium is the induction of mucin mRNA. The increased mucin mRNA steady state does not seem to occur secondary to mucous cell mitosis because mucous cells (containing electron lucent secretory granules) do not incorporate [3H]thymidine during the period of mucin mRNA induction [33] . Further, mucous cells have not been observed to incorporate [3H] thymidine during the period of rapid mucous cell increase following cigarette smoke exposure [34] . We suggest that the increased numbers of mucous cells in injured airways arise through environmental stimulation of mucin gene transcription, presumably an early event in mucous cell differentiation. Our studies revealed that the airways of SPF rats contained undetectable mucin mRNA, as determined by lack of hybridization with SMUC-41. Under the housing conditions used in our studies, rats spontaneously became infected with Sendai virus, Corona virus, or CAR bacillus. Those infected by Sendai virus were the only rats to show mucin hybridization signals, and all rats with significant antibody titers against Sendai virus showed conspicuous signals ( Figure 3 and Table 1 ). Although correlation does not prove causality, these data strongly suggest that Sendai virus is responsible for the marked induction seen in our experiments. Although we cannot rule out potential roles of other pathogens, the rarity of rat pathogens other than those screened for makes it unlikely that an undetected pathogen accounted for the induction. Although Sendai virus-infected rats showed conspicuous hybridization signals, they were much less intense than those seen in two animals simultaneously infected with Sendal virus and exposed for 1 wk to the irritant SO2 (Fig 3, C lanes, 1 week exposure) . The relative intensities of the signals suggest that mucin mRNA induction by Sendal virus may be potentiated by SO2. Highly prevalent in rodent colonies [27] , Sendai virus is known to induce a variety of structural and functional alterations in the respiratory tract [27] . Based on our results, these may include mucin mRNA induction. Although the specific mechanisms by which pathogens (and possibly irritants) induce mucin mRNA are unknown, such mechanisms would likely act to increase the transcription rate or decrease the degradation rate of mucin mRNA. Viruses may activate the expression of multiple gene products through induction of interferons [35] . Further, irritation can introduce macrophage-, neutrophil-, and lymphocyte-derived mediators to the environment [2] [36] [37] . These could potentially stimulate epithelial cells to synthesize or activate transcription factors [31] or could influence the processing of specific mRNAs to affect their stability [30] . Although infection and irritation have long been known to initiate mucus hypersecretion in animals and humans [38] , the underlying mechanisms have remained obscure. In demonstrating that pathogens and irritants are capable of affecting mucus secretion at the level of mucin mRNA, our results provide one of the first clues to the pathogenesis of hypersecretion. 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Pathology and immunohistochemistry Alternative splicing in the control of gene expression Messenger RNA turnover in eukaryotic cells Goblet cells increase in rat bronchial epithelium after exposure to cigarette and cigar smoke Analysis of thymidine incorporation by airway epithelial cells in a rat model of chronic bronchitis Cell division and differentiation in bronchial epithelium Interferon-induced expression of different genes is mediated by distinct regulatory pathways Irreversible bronchial goblet cell metaplasia in hamsters with elastase-induced panacinar emphysema Plasticity in the airway epithelium We thank Drs. Donald McDonald and Charles Ordahl for helpful discussions and M. Uy for excellent technical assistance. We thank Drs. Young Kim and James Gum for generously providing the SMUC-41 cDNA used in these studies. Dr. Jany was supported by grants from Deutsche Forschungsgemeinschaft (DFG), Boehringer Ingelheim, Inc., and Cystic Fibrosis Research, Inc. The research was supported by the UCSF Academic Senate and NIH RO1 HL43762-01A1.