Airway epithelial cells are the first line of defense against viral infections and are instrumental in coordinating the inflammatory response. In this study, we demonstrate the synergistic stimulation of CXCL10 mRNA and protein, a key chemokine responsible for the early immune response to viral infection, following treatment of airway epithelial cells with IFN γ and influenza virus. The synergism also occurred when the cells were treated with IFN γ and a viral replication mimicker (dsRNA) both in vitro and in vivo. Despite the requirement of type I interferon (IFNAR) signaling in dsRNA-induced CXCL10, the synergism was independent of the IFNAR pathway since it wasn't affected by the addition of a neutralizing IFNAR antibody or the complete lack of IFNAR expression. Furthermore, the same synergistic effect was also observed when a CXCL10 promoter reporter was examined. Although the responsive promoter region contains both ISRE and NFκB sites, western blot analysis indicated that the combined treatment of IFN γ and dsRNA significantly augmented NFκB but not STAT1 activation as compared to the single treatment. Therefore, we conclude that IFN γ and dsRNA act in concert to potentiate CXCL10 expression in airway epithelial cells via an NFκB-dependent but IFNAR-STAT independent pathway and it is at least partly regulated at the transcriptional level.
PMID: 22205631;PMCID: PMC3359907;Abstract:
Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 (TICAM1, also called TRIF) is an important adaptor protein in TLR3 and TLR4 signaling pathways that mediate proinflammatory cytokine and IFN responses. Negative regulation of TICAM1 by exogenous viral protease or by endogenous caspase and proteasome have been reported to shut down TICAM1-mediated signaling. In this study, we discovered that down-regulation of TICAM1, but not other components in this signaling pathway, occurred in a natural process of TLR3 activation induced by double-stranded RNA or human rhinovirus (RV) infection in airway epithelial cells and various other cell types. TICAM1 was essential for IFN expression, and the loss of TICAM1 significantly elevated RV production. The low level of TICAM1 protein expression, caused by the prior double-stranded RNA treatment, led to a lack of IFN production upon additional treatment, suggesting receptor desensitization. In follow-up studies, TICAM1 down-regulation was found to be dependent on TLR3 but not RIG1, MDA5, or PKR and appeared to be regulated post-translationally. Neither proteasome nor caspase inhibitors could prevent TICAM1 down-regulation. Instead, a lysosome-mediated process appeared to be involved, suggesting a novel mechanism that is different from previous reports. In conclusion, TICAM1 down-regulation is an essential step in TLR3 activation, and its function is to stop TLR3-mediated IFN production. Copyright © 2012 by the American Thoracic Society.
Development of the high-density DNA microarray technique permits the analysis of thousands of genes simultaneously for their differential expression patterns in various biological processes. Through clustering analysis and pattern recognition, the significance of differentially expressed genes can be recognized and correlated with biological events that may take place inside the cell and tissue. With this notion in mind, high-density DNA microarray nylon membrane with colorimetry detection was used to profile the expression of smoke- and hydrogen peroxide-inducible genes in a human bronchial epithelial cell line, HBE1. On the basis of the time course of expression, at least three phases of change in gene expression could be recognized. The first phase is an immediate event in response to oxidant injury. This phase includes induction of the bcl-2 and mdm-2 genes, which are involved in the regulation of apoptosis, and the mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) gene, that functions as a regulator of various mitogen-activated protein kinase activities. The second phase, usually 5 h later, includes the induction of various stress proteins and ubiquitin, which are important in providing the chaperone mechanism and the turnover of damaged macromolecules. The third phase, which is 5-10 h later, includes the induction of genes that are apparently involved in reducing oxidative stress by metabolizing reactive oxygen species. In this phase, enzymes associated with tissue and cell remodeling are also elevated. These results demonstrate a complex gene expression array by bronchial epithelial cells in response to the insult of oxidants that are relevant to environmental pollutants.
PMID: 21075863;PMCID: PMC3175562;Abstract:
The most recently discovered gel-forming mucin, MUC19, is expressed in both salivary glands and tracheal submucosal glands. We previously cloned the 3′-end partial sequence (AY236870), and here report the complete sequencing of the entire MUC19 cDNA. One highly variable region (HVR) was discovered in the 5′ end of MUC19. A total of 20 different splicing variants were detected in HVR, and 18 variants are able to translate into proteins along with the rest of the MUC19 sequence. The longest variant of MUC19 consists of 182 exons, with a transcript of approximately 25 kb. A central exon of approximately 12 kb contains highly repetitive sequences and has no intron interruption. The deduced MUC19 protein has the bona fide gel-forming mucin structure, VWD-VWD-VWD-"threonine/serine-rich repeats"-VWC-CT. An unusual structural feature of MUC19, which is lacking in other gel-forming mucins, is its long amino terminus upstream of the first VWD domain. The long amino terminus is mostly translated from the sequences in HVR, and contains serine-rich repetitive sequences. To validate the integrity of the MUC19 sequence, primers from both the 3′ and 5′ end were used to demonstrate a similar tissue expression pattern of MUC19 in trachea and salivary glands. In addition, antibodies were developed against either the amino (N) or carboxy (C) terminus of MUC19, and similar antibody staining patterns were observed in both salivary and tracheal submucosal glands. In conclusion, we have cloned and elucidated the entire MUC19 gene, which will facilitate understanding of the function and regulation of this important, yet understudied, mucin gene in airway diseases.
Using cDNA microarray analysis, we identified a cDNA clone, DD74, from primary human bronchial epithelial cells, which exhibits increased expression in vitro after treatment with all-trans retinoic acid. This clone corresponded to MAGE D2 mRNA, a gene previously identified to be upregulated in several cancer tissues. Surprisingly, in situ hybridization of lung tissue demonstrated positive hybridization signals with sense, but not antisense, MAGE D2-specific cRNA probes. Examination of several cell lines by Northern blot hybridization confirmed significant expression of two RNA bands. With strand-specific riboprobes, we identified a 2.0 kb RNA transcript with the antisense probe as expected and identified a 4.1 kb transcript by the sense probe. Further sequence analysis of the 4.1 kb transcript revealed at least a 509 nucleotide sequence exactly complementary to the 2.0 kb MAGE D2 mRNA sequence. This MAGE D2i sequence contains unique structural features not shared with those of previously described antisense transcripts. Identification of this transcript potentially has important implications for future studies examining MAGE D2 expression patterns in cancer and normal tissues. © 2004 Elsevier Inc. All rights reserved.