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Activation, AhR leads to Decrease, sox9 expression
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding||Point of Contact||Author Status||OECD Status|
|Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced impeded craniofacial development||non-adjacent||Moderate||Low||Prarthana Shankar (send email)||Under development: Not open for comment. Do not cite|
|Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced cardiovascular toxicity||non-adjacent||High||Low||Prarthana Shankar (send email)||Under development: Not open for comment. Do not cite|
Life Stage Applicability
Key Event Relationship Description
- The Ahr is a ligand activated transcription factor that is capable of regulating gene expression of several genes, all belonging to the Ahr signaling cascade (Larigot et al., 2018).
- Canonical Ahr signaling involves receptor translocation from the cytoplasm to the nucleus, followed by Ahr-ARNT heterodimerization. The heterodimer then recognizes Aryl hydrocarbon response elements (AHREs) in the promoter regions of different genes to regulate their expression (Swanson 2002). Indirect gene regulation is also possible, with the downstream target genes interacting with other signaling pathways (Mathew et al., 2008).
- Sox9 is one proposed indirect gene within the Ahr signaling cascade. Sox9b, one of two paralogs of the sox9 gene in zebrafish, is one of the most reduced transcripts in the jaw upon TCDD exposure in zebrafish (Xiong et al., 2008). Thus, there exists a non-adjacent relationship between Ahr activation and the repression of sox9.
Evidence Collection Strategy
Evidence Supporting this KER
KER 2688 concordance table: https://aopwiki.org/system/dragonfly/production/2022/10/20/7inngdvxht_Concordance_Table_AHR_to_sox9_clean.pdf
- Evidence for biological plausibility comes from Ahr’s ability to interact with several molecular signaling pathways, including the Wnt-beta catenin pathway (Mathew et al. 2008). Sox9 is one important member of the Wnt-beta catenin signaling pathway, specifically as it relates to chondrogenesis (Sinha et al., 2021; Topol et al., 2009).
Empirical evidence and essentiality of KEup for KEdown to occur
- Developing zebrafish exposed to 1ng/mL TCDD significantly repress sox9b (one of two paralogs of sox9 in zebrafish) by approximately 2-fold in the heart tissue at 72 hours post fertilization (hpf) (Hofsteen et al., 2013).
- sox9b is significantly repressed in 72-hpf whole animal zebrafish exposed to 2nM TCDD (Jenny et al., 2009). In the same study, knockdown of AHRRb caused a significant decrease in sox9b mRNA expression in the absence of TCDD exposure, suggesting some level of endogenous Ahr control of sox9.
- Whole animal zebrafish exposed to a concentration range of TCDD significantly repress sox9b from 0.5 ng/mL exposure concentration, with cyp1a, a biomarker of Ahr activation, significantly induced from 0.0625 ng/mL (Garcia et al., 2018b).
- A sox9b reporter zebrafish line exposed to 1ng/mL TCDD showed a trend for sox9b repression measured using qRT-PCR (Garcia et al., 2017).
- 96-hpf zebrafish exposed to 1ng/mL TCDD induces cyp1a and represses sox9b in parallel in isolated jaw tissue over multiple time points after exposure (Xiong et al., 2008).
- In regenerating fin tissue after 2 or 3 days post caudal fin amputation of 48-hpf zebrafish exposed to 1ng/mL TCDD, sox9b was one of the most repressed transcripts (Mathew et al., 2008).
- Additionally, one of the most repressed genes in caudal fins from adult zebrafish IP injected with TCDD was also sox9b (Andreasen et al., 2006).
- In whole atlantic salmon larvae exposed to 1 or 10 ng/L PCB-77, sox9 mRNA was significantly reduced (by 50% compared to controls) only till 500 dd, after which, non-significant or significant increases were detected at both concentrations (Olufsen and Arukwe 2011).
- In A549 pulmonary epithelial cells, individual exposures to TCDD, the PAHS Benzo[a]pyrene and benzo[k]fluoranthene, as well as a non-cytotoxic concentration of ambient aerosol particle fraction PM0.5, significantly repressed sox9 expression while also inducing cyp1a expression (Prochazkova et al., 2018; Simeckova et al., 2019).
- Developing zebrafish exposed to 0.5, 5, and 50 nM pyrene (a known Ahr activating chemical), had concentration-dependent significant sox9 repression in the craniofacial skeleton (seen using in situ hybridization), as well as concentration-dependent craniofacial deformations (Shi et al., 2012).
- Ahr2 knockout zebrafish with 1ng/mL TCDD exposure did not have significantly reduced sox9b expression at 48 hpf (Garcia et al., 2018a).
In one study, adult white sturgeon were exposed to equipotent concentrations of TCDD, PCB-77, and BaP. Repression of sox9 transcript was identified in the livers of fish exposed to all three chemicals (Doering et al., 2016).
Uncertainties and Inconsistencies
- Whole animal zebrafish exposed to several individual PAHs, many of whom significantly induce cyp1a by 48 hpf, do not cause significant repression of sox9b (Garcia et al., 2018b). The PAHs are retene, benzo[j]fluoranthene, benzo[k]fluoranthene, dibenzo[a,h]pyrene, benzo[b]fluoranthene, fluoranthene, phenanthrene, and 9-methylanthracene. Dibenzo[a,i]pyrene was the only PAH from the list that showed a trend for sox9b reduction. One explanation is that the possible tissue-specific sox9b repression was not enough to capture expression changes in this whole-animal study where zebrafish were exposed to Ahr activators not as strong as TCDD.
- A microarray study investigating gene expression changes in the jaw primordium of zebrafish exposed to TCDD from 1 to 24 hpf did not include either paralog of sox9 in the top downregulated gene list (Planchart and Mattingly 2010). It is possible that sox9 was not present in the microarray.
- In a human glioblastoma cell culture study, sox9 was repressed when ARNT2 was knocked down, in addition to the study identifying potential binding regions of ARNT2 in the regulatory region of sox9 (Bogeas et al., 2018). While no functional studies were conducted, it is possible that there may be cell-specific direct regulation of sox9 by Ahr/ARNT.
- In frozen human lung tumor samples, expression of sox9 was significantly higher in smokers compared to in samples from non-smokers. Additionally, in adenocarcinomas in smoking women, sox9 expression was relatively high. Of note, these results were accompanied by the lack of induction of Ahr expression (Szymanowska-Narloch et al., 2013).
Known modulating factors
Quantitative Understanding of the Linkage
- Whole animal zebrafish have sox9b significantly reduced from 0.5 ng/mL exposure concentration, with trends in repression seen from 0.125 ng/mL TCDD exposure (Garcia et al., 2018b).
- Developing zebrafish at 96 hpf were exposed to TCDD, and their jaws were isolated for measurement of gene expression by microarray. By 12 hours post exposure (hpe), sox9b was the most strongly affected gene, with an almost 15-fold reduction compared to control animals (Xiong et al., 2008).
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
- The relationship between Ahr activation and sox9 repression is best studied in developing zebrafish. Some supporting evidence comes from salmon larvae, as well as human lung cells, suggesting that this relationship is highly evolutionarily conserved among vertebrates (at least), but also likely tissue-specific.
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Bogeas A, Morvan-Dubois G, El-Habr EA, Lejeune FX, Defrance M, Narayanan A, Kuranda K, Burel-Vandenbos F, Sayd S, Delaunay V et al. 2018. Changes in chromatin state reveal arnt2 at a node of a tumorigenic transcription factor signature driving glioblastoma cell aggressiveness. Acta Neuropathol. 135(2):267-283.
Doering JA, Tang S, Peng H, Eisner BK, Sun J, Giesy JP, Wiseman S, Hecker M. 2016. High conservation in transcriptomic and proteomic response of white sturgeon to equipotent concentrations of 2, 3, 7, 8-tcdd, pcb 77, and benzo [a] pyrene. Environmental Science & Technology. 50(9):4826-4835.
Garcia GR, Goodale BC, Wiley MW, La Du JK, Hendrix DA, Tanguay RL. 2017. In vivo characterization of an ahr-dependent long noncoding rna required for proper sox9b expression. Mol Pharmacol. 91(6):609-619.
Garcia GR, Bugel SM, Truong L, Spagnoli S, Tanguay RL. 2018a. Ahr2 required for normal behavioral responses and proper development of the skeletal and reproductive systems in zebrafish. PLoS One. 13(3):e0193484.
Garcia GR, Shankar P, Dunham CL, Garcia A, La Du JK, Truong L, Tilton SC, Tanguay RL. 2018b. Signaling events downstream of ahr activation that contribute to toxic responses: The functional role of an ahr-dependent long noncoding rna (slincr) using the zebrafish model. Environ Health Perspect. 126(11):117002.
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Jenny MJ, Karchner SI, Franks DG, Woodin BR, Stegeman JJ, Hahn ME. 2009. Distinct roles of two zebrafish ahr repressors (ahrra and ahrrb) in embryonic development and regulating the response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicological Sciences. 110(2):426-441.
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Shi X, He C, Zuo Z, Li R, Chen D, Chen R, Wang C. 2012. Pyrene exposure influences the craniofacial cartilage development of sebastiscus marmoratus embryos. Mar Environ Res. 77:30-34.
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Sinha A, Fan VB, Ramakrishnan AB, Engelhardt N, Kennell J, Cadigan KM. 2021. Repression of wnt/beta-catenin signaling by sox9 and mastermind-like transcriptional coactivator 2. Sci Adv. 7(8).
Swanson HI. 2002. DNA binding and protein interactions of the ahr/arnt heterodimer that facilitate gene activation. Chem-Biol Interact. 141(1-2):63-76.
Szymanowska-Narloch A, Jassem E, Skrzypski M, Muley T, Meister M, Dienemann H, Taron M, Rosell R, Rzepko R, Jarzab M et al. 2013. Molecular profiles of non-small cell lung cancers in cigarette smoking and never-smoking patients. Adv Med Sci. 58(2):196-206.
Topol L, Chen W, Song H, Day TF, Yang Y. 2009. Sox9 inhibits wnt signaling by promoting beta-catenin phosphorylation in the nucleus. J Biol Chem. 284(5):3323-3333.
Xiong KM, Peterson RE, Heideman W. 2008. Aryl hydrocarbon receptor-mediated down-regulation of sox9b causes jaw malformation in zebrafish embryos. Mol Pharmacol. 74(6):1544-1553.