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Relationship: 987

Title

The title of the KER should clearly define the two KEs being considered and the sequential relationship between them (i.e., which is upstream and which is downstream). Consequently all KER titles take the form “upstream KE leads to downstream KE”.  More help

Activation, Sp1 leads to Increase, Mucin production

Upstream event
Upstream event in the Key Event Relationship. On the KER page, clicking on the Event name under Upstream Relationship will bring the user to that individual KE page. More help
Downstream event
Downstream event in the Key Event Relationship. On the KER page, clicking on the Event name under Upstream Relationship will bring the user to that individual KE page. More help

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes. Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

This table is automatically generated upon addition of a KER to an AOP. All of the AOPs that are linked to this KER will automatically be listed in this subsection. Clicking on the name of the AOP in the table will bring you to the individual page for that AOP. More help

Taxonomic Applicability

Select one or more structured terms that help to define the biological applicability domain of the KER. In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. Authors can indicate the relevant taxa for this KER in this subsection. The process is similar to what is described for KEs (see pages 30-31 and 37-38 of User Handbook) More help
Term Scientific Term Evidence Link
human Homo sapiens High NCBI

Sex Applicability

Authors can indicate the relevant sex for this KER in this subsection. The process is similar to what is described for KEs (see pages 31-32 of the User Handbook). More help
Sex Evidence
Mixed Low

Life Stage Applicability

Authors can indicate the relevant life stage for this KER in this subsection. The process is similar to what is described for KEs (see pages 31-32 of User Handbook). More help
Term Evidence
Adult Moderate

Key Event Relationship Description

Provide a brief, descriptive summation of the KER. While the title itself is fairly descriptive, this section can provide details that aren’t inherent in the description of the KEs themselves (see page 39 of the User Handbook). This description section can be viewed as providing the increased specificity in the nature of upstream perturbation (KEupstream) that leads to a particular downstream perturbation (KEdownstream), while allowing the KE descriptions to remain generalised so they can be linked to different AOPs. The description is also intended to provide a concise overview for readers who may want a brief summation, without needing to read through the detailed support for the relationship (covered below). Careful attention should be taken to avoid reference to other KEs that are not part of this KER, other KERs or other AOPs. This will ensure that the KER is modular and can be used by other AOPs. More help

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

Assembly and description of the scientific evidence supporting KERs in an AOP is an important step in the AOP development process that sets the stage for overall assessment of the AOP (see pages 49-56 of the User Handbook). To do this, biological plausibility, empirical support, and the current quantitative understanding of the KER are evaluated with regard to the predictive relationships/associations between defined pairs of KEs as a basis for considering WoE (page 55 of User Handbook). In addition, uncertainties and inconsistencies are considered. More help

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.

Biological Plausibility
Define, in free text, the biological rationale for a connection between KEupstream and KEdownstream. What are the structural or functional relationships between the KEs? For example, there is a functional relationship between an enzyme’s activity and the product of a reaction it catalyses. Supporting references should be included. However, it is recognised that there may be cases where the biological relationship between two KEs is very well established, to the extent that it is widely accepted and consistently supported by so much literature that it is unnecessary and impractical to cite the relevant primary literature. Citation of review articles or other secondary sources, like text books, may be reasonable in such cases. The primary intent is to provide scientifically credible support for the structural and/or functional relationship between the pair of KEs if one is known. The description of biological plausibility can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured (see page 40 of the User Handbook for further information).   More help

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).

Uncertainties and Inconsistencies
In addition to outlining the evidence supporting a particular linkage, it is also important to identify inconsistencies or uncertainties in the relationship. Additionally, while there are expected patterns of concordance that support a causal linkage between the KEs in the pair, it is also helpful to identify experimental details that may explain apparent deviations from the expected patterns of concordance. Identification of uncertainties and inconsistencies contribute to evaluation of the overall WoE supporting the AOPs that contain a given KER and to the identification of research gaps that warrant investigation (seep pages 41-42 of the User Handbook).Given that AOPs are intended to support regulatory applications, AOP developers should focus on those inconsistencies or gaps that would have a direct bearing or impact on the confidence in the KER and its use as a basis for inference or extrapolation in a regulatory setting. Uncertainties that may be of academic interest but would have little impact on regulatory application don’t need to be described. In general, this section details evidence that may raise questions regarding the overall validity and predictive utility of the KER (including consideration of both biological plausibility and empirical support). It also contributes along with several other elements to the overall evaluation of the WoE for the KER (see Section 4 of the User Handbook).  More help

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).

Response-response Relationship
This subsection should be used to define sources of data that define the response-response relationships between the KEs. In particular, information regarding the general form of the relationship (e.g., linear, exponential, sigmoidal, threshold, etc.) should be captured if possible. If there are specific mathematical functions or computational models relevant to the KER in question that have been defined, those should also be cited and/or described where possible, along with information concerning the approximate range of certainty with which the state of the KEdownstream can be predicted based on the measured state of the KEupstream (i.e., can it be predicted within a factor of two, or within three orders of magnitude?). For example, a regression equation may reasonably describe the response-response relationship between the two KERs, but that relationship may have only been validated/tested in a single species under steady state exposure conditions. Those types of details would be useful to capture.  More help

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).

Time-scale
This sub-section should be used to provide information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). This can be useful information both in terms of modelling the KER, as well as for analyzing the critical or dominant paths through an AOP network (e.g., identification of an AO that could kill an organism in a matter of hours will generally be of higher priority than other potential AOs that take weeks or months to develop). Identification of time-scale can also aid the assessment of temporal concordance. For example, for a KER that operates on a time-scale of days, measurement of both KEs after just hours of exposure in a short-term experiment could lead to incorrect conclusions regarding dose-response or temporal concordance if the time-scale of the upstream to downstream transition was not considered. More help

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
This sub-section presents information regarding modulating factors/variables known to alter the shape of the response-response function that describes the quantitative relationship between the two KEs (for example, an iodine deficient diet causes a significant increase in the slope of the relationship; a particular genotype doubles the sensitivity of KEdownstream to changes in KEupstream). Information on these known modulating factors should be listed in this subsection, along with relevant information regarding the manner in which the modulating factor can be expected to alter the relationship (if known). Note, this section should focus on those modulating factors for which solid evidence supported by relevant data and literature is available. It should NOT list all possible/plausible modulating factors. In this regard, it is useful to bear in mind that many risk assessments conducted through conventional apical guideline testing-based approaches generally consider few if any modulating factors. More help

Unknown

Known Feedforward/Feedback loops influencing this KER
This subsection should define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits? In some cases where feedback processes are measurable and causally linked to the outcome, they should be represented as KEs. However, in most cases these features are expected to predominantly influence the shape of the response-response, time-course, behaviours between selected KEs. For example, if a feedback loop acts as compensatory mechanism that aims to restore homeostasis following initial perturbation of a KE, the feedback loop will directly shape the response-response relationship between the KERs. Given interest in formally identifying these positive or negative feedback, it is recommended that a graphical annotation (page 44) indicating a positive or negative feedback loop is involved in a particular upstream to downstream KE transition (KER) be added to the graphical representation, and that details be provided in this subsection of the KER description (see pages 44-45 of the User Handbook).  More help

Unknown

Domain of Applicability

As for the KEs, there is also a free-text section of the KER description that the developer can use to explain his/her rationale for the structured terms selected with regard to taxonomic, life stage, or sex applicability, or provide a more generalizable or nuanced description of the applicability domain than may be feasible using standardized terms. More help

The available evidence is restricted to human cells.

References

List of the literature that was cited for this KER description using the appropriate format. Ideally, the list of references should conform, to the extent possible, with the OECD Style Guide (OECD, 2015). More help

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.

Van Seuningen, I., Pigny, P., Perrais, M., Porchet, N. and Aubert, J.P., 2001. Transcriptional regulation of the 11p15 mucin genes. Towards new biological tools in human therapy, in inflammatory diseases and cancer. Front Biosci 6, D1216-D1234.
 
Wan, H., Kaestner, K.H., Ang, S.L., Ikegami, M., Finkelman, F.D., Stahlman, M.T., Fulkerson, P.C., Rothenberg, M.E., and Whitsett, J.A. (2004). Foxa2 regulates alveolarization and goblet cell hyperplasia. Development 131, 953-964.
 

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.