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Relationship: 2894

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Antagonism Smoothened leads to orofacial cleft

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Antagonism Smoothened

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

orofacial cleft

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

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Antagonism of Smoothened receptor leading to orofacial clefting	non-adjacent	High	Moderate	Jacob Reynolds (send email)	Under development: Not open for comment. Do not cite	Under Review

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) 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. More help

Term	Scientific Term	Evidence	Link
mouse	Mus musculus	High	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Unspecific

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Embryo	High

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

The Smoothened (SMO) receptor is Class F G protein coupled receptor involved in signal transduction of the Sonic Hedgehog (SHH) pathway. It includes distinct functional groups including ligand binding pockets, cysteine rich domain (CRD), transmembrane helix (TM), extracellular loop (ECL), intracellular loop (ICL), and a carboxyl-terminal tail (C-term tail) (Arensdorf, Marada et al. 2016). SMO signaling is dependent upon its relocation to a subcellular location. This relocation occurs in the primary cilium (PC) in vertebrates (Huangfu and Anderson 2005). Relocation of SMO to the PC typically occurs within ~20 minutes of agonist stimulation (Arensdorf, Marada et al. 2016).

In the absence of SHH ligand, the Patched (PTCH) receptor suppresses the activation of SMO. When HH ligand binds to PTCH, suppression on SMO is released and SMO can relocate, accumulate, and signal to intracellular effectors (Denef, Neubüser et al. 2000, Rohatgi and Scott 2007). It has been shown that SMO localization to the tip of the primary cilia is essential for the SHH signaling cascade in vertebrates (Corbit, Aanstad et al. 2005, Rohatgi, Milenkovic et al. 2007, Rohatgi, Milenkovic et al. 2009). This relocation then leads to signaling to effectors resulting in the activation of the GLI transcription factors and the subsequent induction of HH target gene expression (Alexandre, Jacinto et al. 1996, Von Ohlen and Hooper 1997). Antagonism of SMO disrupts the downstream signaling cascade of SHH and if disrupted during critical periods of development can lead birth defects including OFCs.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Pubmed was used as the primary database for evidence collection. Searches are organized by the date and search terms used in the supplementary table. Search results were initially screened through review of the title and abstract for potential for data relating antagonism of SMO and OFCs. Each selected publication and its’ data were then examined to determine if support or lack thereof existed for this KER. Papers that did not show any data relating to this KER were discarded. The search is detailed below in Table 1.

Table 1: KER 2894 literature search

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field 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. More help

There is high biological plausibility of this relationship.The SHH pathway is well understood to be fundamental to proper embryonic development and that aberrant SHH signaling during embryonic development can cause birth defects including orofacial clefts (OFCs).

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

in vitro- It should be noted that OFC cannot be evaluated in vitro. The evidence presented below is intended to further support the in vivo evidence and offers support of which stressors might cause an OFC and their possible mechanism.
- A small molecule screen of 10,000 compounds identified six inhibitors of SHH signaling, four of which bind directly to SMO (SANT1-4). Screening was conducted using NIH 3T3 SHH LightII cells cultured in media conditioned from HEK 293 transfected to stably express Shh-N. Cells were dosed with the compound library at 0.714ug/ml and SHH activity was quantified at 30h using Renilla luciferase activity. A fluorescent binding assay using BODIPY-cyclopamine was used to verify binding to SMO for the SANT compounds. Dose response reported as IC50 for the inhibition of SHH signaling was conducting in NIH 3T3 SHH light2, NIH 3T3 SmoA1-Light2, P2 Ptch1-/- (mouse embryonic fibroblasts) (Chen, Taipale et al. 2002).

Compound/Cell	SHH-Light2 (nM)	SmoA1-Light2 (nM)	Ptch1-/- (nM)
SANT-1	20	30	20
SANT-2	30	70	50
SANT-3	100	80	80
SANT-4	200	300	300

- Direct binding of cyclopamine to SMO was verified using a photoaffinity form of cyclopamine (PA-cyclopamine). PA-cyclopamine had previously been shown to inhibit SHH signaling in NIH 3T3 Shh-LightII cells with similar IC50 values to cyclopamine (300nm and 150nm respectively) (Taipale, Chen et al. 2000). Binding to SMO was verified using a COS-1 (fibroblast, monkey) line transfected to over express SMO. The location of cyclopamine binding was further investigated using BODIPY- cyclopamine and COS-1 cells modified to lack either a N-terminal, extracellular cysteine-rich domain, or the cytoplasmic C terminal of SMO. The findings support that cyclopamine does not require these domains and instead binds directly to the heptahelical domain (Chen, Taipale et al. 2002).
In vivo
- The presence of critical periods for disruption of SHH was investigated using C57BL/6J mice. Vismodegib was suspended at 3mg/ml in 0.5% methyl cellulose and 0.2% tween. Pregnant dams were administered 40mg/kg vismodegib at GD7.0, 7.25, 7.5, 7.75, 8.0, 8.25,8.5, 8.625, 8.75, 8.875, 9.0, 9.25, 9.5, 9.75, and 10.0. Cyclopamine was dosed at 120mg/kg/d via subcutaneous infusion between GD8.25-9.375. Pregnant dams were euthanized at GD17 and fetal specimens were collected and fixed for imaging. The control group consisted of fetuses exposed to 0.5% methyl cellulose and 0.2% tween at GD7.75, 8.875, or 9.5. Acute exposure to vismodegib resulted in a peak incidence of lateral cleft lip and palate at GD8.875 (13%). Exposure at GD9.0 and 10.0 resulted in clefts of the secondary palate only (34%). A higher penetrance (81%) was found for cyclopamine exposure (Heyne, Melberg et al. 2015).
- Timed pregnant C57B1/6J mice were treated with cyclopamine from GD 8.25-9.5 by subcutaneous infusion (160mg/kg/d) or at GD 8.5 with AZ75 (potent cyclopamine analog) via oral gavage (40 or 80mg/kg). Exposure to cyclopamine resulted in lateral cleft lip and cleft palate defects attributed to a deficiency of midline and lower medial nasal prominence tissue. Both drugs infrequently resulted in an intermediate phenotype of median CLP. Cyclopamine caused gross facial malformations in 5/14 litters with an intra-litter penetrance of clefting of 50%. AZ75 dosed at 80mg/kg caused all embryos to resorb. At 40mg/kg AZ75 caused gross facial malformations in 6/7 litters (Lipinski, Song et al. 2010).
- Timed pregnant C57B1/6J mice were administered cyclopamine via micro osmotic pumps (120mg/kg/d) surgically implanted at GD 8.25. Dams were euthanized on GD 17. 25/45 of the cyclopamine exposed fetuses presented with a cleft compared to 0/39 for the control group (Lipinski, Holloway et al. 2014).
- Pregnant Sprague Dawley rats were dosed with 240mg/kg of cyclopamine (oral gavage once daily) from GD 6.0-9.0. Craniofacial malformations were noted including cebocephaly, microphthalmia, hydrocephaly, exencephaly, and anencephaly. Parallel experimentation in golden hamsters found that 170mg/kg of cyclopamine was sufficient to cause malformations including cleft lip and palate (Keeler 1975).
- C57BL/6J and A/J mice were dosed with single doses of jervine (70, 150,300mg/kg gavage) on either GD 8, 9, 10. A dose response pattern of CLP was seen for both strains with dosing on GD 8. A dose response pattern for CP was found for C57BL/6J for treatment on GD 9 or 10 but not at GD 8(Omnell, Sim et al. 1990).

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Modulating Factor (MF)	MF Specification	Effect(s) on the KER	Reference(s)
Further work is needed to increase the understanding of this relationship and its’ modulating factors.

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Further work is needed to address these questions and create a better understanding of this relationship.

Time-scale

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?). More help

Relocation of SMO to the PC typically occurs within ~20 minutes of agonist stimulation (Arensdorf, Marada et al. 2016). No data was found on how fast antagonism of SMO will stop its’ relocation to the primary cilia. Further work is needed to increase the understanding of this relationship and its’ time scale

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Further work is needed to increase the understanding of this relationship and shed light on what other feedback/forward loops are at play.

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

The nonadjacent relationship between antagonism of SMO and orofacial clefting (OFCs) has been shown repeatedly in mice models as detailed in the empirical evidence section. The relationship is biologically plausible in human, but to date no specific experiments have addressed this question. The SHH pathway is well understood to be fundamental to proper embryonic development and that aberrant SHH signaling during embryonic development can cause birth defects including orofacial clefts (OFCs). For this reason, this KER is applicable to the embryonic stage with a high level of confidence.

References

List of the literature that was cited for this KER description. More help

Alexandre, C., A. Jacinto and P. W. Ingham (1996). "Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the cubitus interruptus protein, a member of the GLI family of zinc finger DNA-binding proteins." Genes Dev 10(16): 2003-2013.

Arensdorf, A. M., S. Marada and S. K. Ogden (2016). "Smoothened Regulation: A Tale of Two Signals." Trends Pharmacol Sci 37(1): 62-72.

Byrne, E. F. X., G. Luchetti, R. Rohatgi and C. Siebold (2018). "Multiple ligand binding sites regulate the Hedgehog signal transducer Smoothened in vertebrates." Current Opinion in Cell Biology 51: 81-88.

Byrne, E. F. X., R. Sircar, P. S. Miller, G. Hedger, G. Luchetti, S. Nachtergaele, M. D. Tully, L. Mydock-McGrane, D. F. Covey, R. P. Rambo, M. S. P. Sansom, S. Newstead, R. Rohatgi and C. Siebold (2016). "Structural basis of Smoothened regulation by its extracellular domains." Nature 535(7613): 517-522.

Chen, J. K., J. Taipale, M. K. Cooper and P. A. Beachy (2002). "Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened." Genes Dev 16(21): 2743-2748.

Chen, J. K., J. Taipale, K. E. Young, T. Maiti and P. A. Beachy (2002). "Small molecule modulation of Smoothened activity." Proc Natl Acad Sci U S A 99(22): 14071-14076.

Corbit, K. C., P. Aanstad, V. Singla, A. R. Norman, D. Y. R. Stainier and J. F. Reiter (2005). "Vertebrate Smoothened functions at the primary cilium." Nature 437(7061): 1018-1021.

Denef, N., D. Neubüser, L. Perez and S. M. Cohen (2000). "Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened." Cell 102(4): 521-531.

Dwyer, J. R., N. Sever, M. Carlson, S. F. Nelson, P. A. Beachy and F. Parhami (2007). "Oxysterols are novel activators of the hedgehog signaling pathway in pluripotent mesenchymal cells." J Biol Chem 282(12): 8959-8968.

Heyne, G. W., C. G. Melberg, P. Doroodchi, K. F. Parins, H. W. Kietzman, J. L. Everson, L. J. Ansen-Wilson and R. J. Lipinski (2015). "Definition of critical periods for Hedgehog pathway antagonist-induced holoprosencephaly, cleft lip, and cleft palate." PLoS One 10(3): e0120517.

Huang, P., D. Nedelcu, M. Watanabe, C. Jao, Y. Kim, J. Liu and A. Salic (2016). "Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling." Cell 166(5): 1176-1187.e1114.

Huang, P., S. Zheng, B. M. Wierbowski, Y. Kim, D. Nedelcu, L. Aravena, J. Liu, A. C. Kruse and A. Salic (2018). "Structural Basis of Smoothened Activation in Hedgehog Signaling." Cell 174(2): 312-324.e316.

Huangfu, D. and K. V. Anderson (2005). "Cilia and Hedgehog responsiveness in the mouse." Proc Natl Acad Sci U S A 102(32): 11325-11330.

Keeler, R. F. (1975). "Teratogenic effects of cyclopamine and jervine in rats, mice and hamsters." Proc Soc Exp Biol Med 149(1): 302-306.

Kobilka, B. K. (2007). "G protein coupled receptor structure and activation." Biochimica et Biophysica Acta (BBA) - Biomembranes 1768(4): 794-807.

Lipinski, R. J., H. T. Holloway, S. K. O'Leary-Moore, J. J. Ament, S. J. Pecevich, G. P. Cofer, F. Budin, J. L. Everson, G. A. Johnson and K. K. Sulik (2014). "Characterization of subtle brain abnormalities in a mouse model of Hedgehog pathway antagonist-induced cleft lip and palate." PLoS One 9(7): e102603.

Lipinski, R. J., C. Song, K. K. Sulik, J. L. Everson, J. J. Gipp, D. Yan, W. Bushman and I. J. Rowland (2010). "Cleft lip and palate results from Hedgehog signaling antagonism in the mouse: Phenotypic characterization and clinical implications." Birth Defects Res A Clin Mol Teratol 88(4): 232-240.

Millington, G., K. H. Elliott, Y. T. Chang, C. F. Chang, A. Dlugosz and S. A. Brugmann (2017). "Cilia-dependent GLI processing in neural crest cells is required for tongue development." Dev Biol 424(2): 124-137.

Myers, Benjamin R., N. Sever, Yong C. Chong, J. Kim, Jitendra D. Belani, S. Rychnovsky, J. F. Bazan and Philip A. Beachy (2013). "Hedgehog Pathway Modulation by Multiple Lipid Binding Sites on the Smoothened Effector of Signal Response." Developmental Cell 26(4): 346-357.

Nachtergaele, S., L. K. Mydock, K. Krishnan, J. Rammohan, P. H. Schlesinger, D. F. Covey and R. Rohatgi (2012). "Oxysterols are allosteric activators of the oncoprotein Smoothened." Nat Chem Biol 8(2): 211-220.

Omnell, M. L., F. R. Sim, R. F. Keeler, L. C. Harne and K. S. Brown (1990). "Expression of Veratrum alkaloid teratogenicity in the mouse." Teratology 42(2): 105-119.

Qi, X., H. Liu, B. Thompson, J. McDonald, C. Zhang and X. Li (2019). "Cryo-EM structure of oxysterol-bound human Smoothened coupled to a heterotrimeric Gi." Nature 571(7764): 279-283.

Rana, R., C. E. Carroll, H.-J. Lee, J. Bao, S. Marada, C. R. R. Grace, C. D. Guibao, S. K. Ogden and J. J. Zheng (2013). "Structural insights into the role of the Smoothened cysteine-rich domain in Hedgehog signalling." Nature Communications 4(1): 2965.

Rohatgi, R., L. Milenkovic, R. B. Corcoran and M. P. Scott (2009). "Hedgehog signal transduction by Smoothened: pharmacologic evidence for a 2-step activation process." Proc Natl Acad Sci U S A 106(9): 3196-3201.

Rohatgi, R., L. Milenkovic and M. P. Scott (2007). "Patched1 regulates hedgehog signaling at the primary cilium." Science 317(5836): 372-376.

Rohatgi, R. and M. P. Scott (2007). "Patching the gaps in Hedgehog signalling." Nat Cell Biol 9(9): 1005-1009.

Taipale, J., J. K. Chen, M. K. Cooper, B. Wang, R. K. Mann, L. Milenkovic, M. P. Scott and P. A. Beachy (2000). "Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine." Nature 406(6799): 1005-1009.

Von Ohlen, T. and J. E. Hooper (1997). "Hedgehog signaling regulates transcription through Gli/Ci binding sites in the wingless enhancer." Mech Dev 68(1-2): 149-156.

Wang, C., H. Wu, V. Katritch, G. W. Han, X. P. Huang, W. Liu, F. Y. Siu, B. L. Roth, V. Cherezov and R. C. Stevens (2013). "Structure of the human smoothened receptor bound to an antitumour agent." Nature 497(7449): 338-343.