Upstream eventActivation, Sp1
Increase, Mucin production
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|EGFR Activation Leading to Decreased Lung Function||adjacent||High||High|
Life Stage Applicability
Key Event Relationship Description
Sp1 can be phosphorylated by many kinases, resulting in its translocation to the nucleus where it then binds and activates promoters of various genes to induce their expression. Sp1 activation was shown to result in MUC5AC expression through EGFR/MAPK activation in human airway epithelial cells following stimulation with EGFR ligands (Perrais et al., 2002) or PMA (Hewson et al. 2004), and in a mouse influenza model (Barbier et al., 2012). In addition, increased Sp1 expression and nuclear translocation, resulting in enhanced Sp1-DNA binding and promoter transactivation through two in cis-elements, and ultimately leading to increased expression of MUC5AC, was shown in airway epithelial cells treated with cigarette smoke extract (Di et al., 2012).
Evidence Supporting this KER
MUC5AC expression can be induced by a variety of inflammatory mediators and growth factors, endotoxins, lipid products and hormones (van Seuningen et al., 2001). The transcription factor Sp1 has long been recognized for its role in regulating expression of mucin genes.
Confirmatory evidence for the involvement of Sp1 in MUC5AC transcription comes from electrophoretic mobility shift assay (EMSA), site-directed mutagenesis and chromosome immunoprecipitation (CHIP) experiments (Hewson et al., 2004; Di et al., 2012). These studies also show that pretreatment of cells with mithramycin A, an antibiotic that specifically blocks Sp1 binding to GC-rich recognition sequences, can suppress or abolish induced MUC5AC transcription.
Multiple studies have shown that there are Sp1 binding sites in mucin gene promoters, including those of the MUC5AC, MUC5B and MUC2 genes, and that deleting or blocking those binding sites reduces mucin gene and protein expression (Perrais et al., 2002; Hewson et al., 2004; Barbier et al., 2012) and human bronchial epithelial cells (Lee et al., 2011; Wu et al., 2007).
MUC5AC and MUC5B promoter activities were reduced by addition of Sp1 inhibitor mithramycin A in a dose- and/or time-dependent manner (Hewson et al., 2004; Lee et al., 2011; Wu et al., 2007).
Uncertainties and Inconsistencies
The MUC5AC promoter has multiple transcription factor binding sites. Therefore, it is likely that alternative pathways contribute to increased mucin production such as activation of HIF-1α or decreased FOXA2 expression (Hao et al., 2014; Kim et al., 2014; Wan et al., 2004).
Quantitative Understanding of the Linkage
Treatment of primary or immortalized bronchial epithelial cells with 10 nM TCDD increased MUC5AC expression in a time-dependent fashion and significantly so after 24 and 48 hours (three- to sevenfold, respectively). A similar behavior was noted for MUC5AC protein expression, and the effect of TCDD was subsequently linked to time-dependent activation of the MUC5AC promoter (reporter gene assay). TCDD was also found to increase Sp1 phosphorylation ca. 1.3-fold at 4-6 hours of treatment (Lee et al., 2012).
Sp1 expression in A549 cells was increased by approx. 2.5-fold following treatment with cigarette smoke extract for 2 hours, and Sp1 nuclear translocation was confirmed by immunostaining. Furthermore, Sp1-DNA complex formation was sigificantly increased in treated compared with untreated cells, with binding to the Sp1B cis element occurring slightly earlier (0.5 hours) than binding to the Sp1A cis element (2 hours); however, quantitative evidence from the EMSA and CHIP experiments was not provided in this report. Additional reporter gene assay data showed that MUC5AC gene expression increased more than 6-fold in the presence of an Sp1-expressing vector following stimulation with 3% cigarette smoke extract (Di et al., 2012).
Lee et al. (2012) reported maximal Sp1 phosphorylation within 4-6 hours and maximal MUC5AC promoter activity at 6-12 hours after stimulation of human bronchial epithelial cells with 10 nM TCDD.
Another study in A549 cells showed increased Sp1 expression and nuclear transloaction at 2 hours, significantly increased binding to the Sp1B cis element at 0.5 and to the Sp1A cis element at 2 hours, and maximally increased MUC5AC expression at 2 to 4 hours of treatment with 3% cigarette smoke extract (Di et al., 2012).
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
The available evidence is restricted to human cells.
Barbier, D., Garcia-Verdugo, I., Pothlichet, J., Khazen, R., Descamps, D., Rousseau, K., Thornton, D., Si-Tahar, M., Touqui, L., Chignard, M., et al. (2012). Influenza A Induces the Major Secreted Airway Mucin MUC5AC in a Protease–EGFR–Extracellular Regulated Kinase–Sp1–Dependent Pathway. Am J Respir Cell Mol Biol 47, 149–157.
Di, Y.P., Zhao, J., and Harper, R. (2012). Cigarette smoke induces MUC5AC protein expression through the activation of Sp1. J Biol Chem 287, 27948-27958.
Hao, Y., Kuang, Z., Jing, J., Miao, J., Mei, L.Y., Lee, R.J., Kim, S., Choe, S., Krause, D.C., and Lau, G.W. (2014). Mycoplasma pneumoniae modulates STAT3-STAT6/EGFR-FOXA2 signaling to induce overexpression of airway mucins. Infection and Immunity 82, 5246-5255.
Hewson, C., Edbrooke, M., and Johnston, S. (2004). PMA induces the MUC5AC respiratory mucin in human bronchial epithelial cells, via PKC, EGF/TGF-alpha, Ras/Raf, MEK, ERK and Sp1-dependent mechanisms. J Mol Biol 344, 683–695.
Kim, Y.-J., Cho, H.-J., Shin, W.-C., Song, H.-A., Yoon, J.-H., and Kim, C.-H. (2014). Hypoxia-mediated mechanism of MUC5AC production in human nasal epithelia and its implication in rhinosinusitis. PLoS One 9, e98136.
Lee, Y.C., Oslund, K.L., Thai, P., Velichko, S., Fujisawa, T., Duong, T., Denison, M.S., and Wu, R. (2011). 2,3,7,8-Tetrachlorodibenzo-p-dioxin–Induced MUC5AC Expression. Am J Respir Cell Mol Biol 45, 270–276.
Perrais, M., Pigny, P., Copin, M., Aubert, J., and Van Seuningen, I. (2002). Induction of MUC2 and MUC5AC mucins by factors of the epidermal growth factor (EGF) family is mediated by EGF receptor/Ras/Raf/extracellular signal-regulated kinase cascade and Sp1. J Biol Chem 277, 32258–32267.
Wu, D.Y., Wu, R., Reddy, S.P., Lee, Y.C., and Chang, M.M.-J. (2007). Distinctive epidermal growth factor receptor/extracellular regulated kinase-independent and -dependent signaling pathways in the induction of airway mucin 5B and mucin 5AC expression by phorbol 12-myristate 13-acetate. Am J Pathol 170, 20–32.