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AOP: 460
Title
Antagonism of Smoothened receptor leading to orofacial clefting
Short name
Graphical Representation
Point of Contact
Contributors
- Jacob Reynolds
Coaches
- Judy Choi
OECD Information Table
OECD Project # | OECD Status | Reviewer's Reports | Journal-format Article | OECD iLibrary Published Version |
---|---|---|---|---|
1.101 | Under Development |
This AOP was last modified on November 14, 2023 13:47
Revision dates for related pages
Page | Revision Date/Time |
---|---|
Antagonism, Smoothened receptor | July 28, 2022 12:17 |
Decrease, GLI1/2 translocation to nucleus | October 28, 2022 15:22 |
Decrease, GLI1/2 target gene expression | March 22, 2023 10:38 |
Decrease, Cell proliferation | December 07, 2020 06:55 |
Decrease, palatal shelf outgrowth | May 16, 2023 14:37 |
Orofacial clefting | May 16, 2023 14:39 |
Decrease, Sonic Hedgehog second messenger production | March 22, 2023 12:09 |
Decrease, Smoothend relocation and activation | October 27, 2022 09:14 |
Apoptosis | February 28, 2024 09:40 |
Antagonism Smoothened leads to OFC | May 12, 2023 11:05 |
Antagonism Smoothened leads to Decrease, SMO relocation | May 17, 2023 09:12 |
Decrease, SMO relocation leads to Decrease, GLI1/2 translocation | May 22, 2023 09:22 |
Decrease, GLI1/2 translocation leads to Decrease, GLI1/2 target gene expression | June 13, 2023 14:04 |
Decrease, GLI1/2 target gene expression leads to Decrease, SHH second messenger production | May 22, 2023 09:56 |
Decrease, SHH second messenger production leads to Decrease, Cell proliferation | November 14, 2023 12:29 |
Decrease, Cell proliferation leads to Decrease, outgrowth | May 22, 2023 10:59 |
Decrease, outgrowth leads to OFC | May 22, 2023 12:36 |
Apoptosis leads to Decrease, outgrowth | April 11, 2023 10:25 |
Decrease, GLI1/2 target gene expression leads to Apoptosis | May 01, 2023 14:43 |
Vismodegib | July 14, 2022 13:04 |
Cyclopamine | August 03, 2022 09:21 |
SANT-1 | October 04, 2022 13:17 |
SANT-2 | October 04, 2022 13:17 |
SANT-3 | October 04, 2022 13:17 |
SANT-4 | October 04, 2022 13:18 |
Piperonyl butoxide | March 23, 2023 10:19 |
Abstract
The Sonic Hedgehog (SHH) is a major signaling pathway of intercellular signaling during embryonic development. Disruption of SHH during critical periods of development can lead to orofacial clefts (OFCs). In canonical SHH signaling, the SHH ligand binds to the Patched1 (PTCH1) receptor and relieves its’ suppression of Smoothened (SMO) receptor. Antagonism of SMO results in disruption of the downstream SHH signaling cascade. Disruption to the signaling cascade causes a decrease in the translocation of the GLI1/2 transcription factors to the nucleus resulting in a decrease in expression of the GLI1/2 target genes. This decrease in gene expression which causes a reduction in production of SHH secondary messengers, namely Fgf10 and members of the BMP family. This reduction in secondary messengers leads to a decrease in cellular proliferation in the palatal shelves. This reduction in cellular proliferation leads to a decrease in palatal shelf outgrowth which ultimately results in a cleft. This AOP is intended to serve as a tool for risk assessment for drug and chemical exposures during embryonic development when disruption to SHH through antagonism of SMO occurs.
AOP Development Strategy
Context
Orofacial clefts (OFCs), encompassing cleft lip with or without palate (CL/P), and cleft palate only (CPO) represent the second most common birth defect in humans with a prevalence of 1-2/1,000 births (Lidral, Moreno et al. 2008). The etiology of OFCs is complex with approximately 50% of CPO and 70% of CL/P considered non-syndromic (2011). SHH signaling is required for normal facial development and plays a critical role in the growth of the facial processes that form the upper palate and lip (Bush and Jiang 2012, Kurosaka 2015). The epithelial derived SHH drives orofacial development through an induced gradient in the underlying mesenchyme (Lan and Jiang 2009, Kurosaka 2015). This gradient of SHH induces cellular proliferation and outgrowth of the mesenchyme (Lan and Jiang 2009). The SHH pathway is sensitive to chemical disruption and can be disrupted at multiple places along the signaling cascade during critical windows for exposure and has been shown to cause OFCs (Lipinski and Bushman 2010, Heyne, Melberg et al. 2015). The targets of this disruption include ligand modification, ligand secretion, downstream sensing, and signal transduction (Jeong and McMahon 2002, Lauth, Bergström et al. 2007, Petrova, Rios-Esteves et al. 2013). Chemical modulators of the SHH pathway through antagonism of SMO have been identified including the natural alkaloid cyclopamine, both natural and synthetic pharmaceuticals, and a pesticide synergist (PBO) (Lipinski, Dengler et al. 2007, Lipinski, Song et al. 2010, Wang, Lu et al. 2012, Everson, Sun et al. 2019, Rivera-González, Beames et al. 2021).
Strategy
This AOP was developed as part of a larger network of AOPs linking disruption of SHH signaling with OFCs (EAGMST workplan project 1.101.). Orofacial clefts (OFCs) are one of the most common human birth defects and occur in approximately 1-2/1,000 live births (Lidral, Moreno et al. 2008). Early orofacial development involves epithelial ectoderm derived SHH ligand driving tissue outgrowth through an induced gradient of SHH dependent transcription in the underlying mesenchyme, which is thought to drive mesenchymal proliferation (Lan and Jiang 2009, Kurosaka 2015). The SHH pathway is sensitive to chemical disruption at multiple molecular targets along the signaling cascade, with exposure during critical windows in development leading to OFCs (Lipinski and Bushman 2010, Heyne, Melberg et al. 2015). The molecular targets of this disruption include SHH ligand modification with cholesterol and palmitoylate, ligand secretion, mesenchymal reception, and signal transduction (Jeong and McMahon 2002, Lauth, Bergström et al. 2007, Petrova, Rios-Esteves et al. 2013). This AOP focuses on the disruption to SHH signaling resulting in antagonism of the SMO receptor. To select the key events for the AOP, we used existing knowledge of the pathway along with reviews of the SHH pathway to assemble a path that was physiologically plausible. Care was taken to select events that would be of direct regulatory relevance (i.e. a method to quantify exists). To identify sources and data for each Key Event Relationship (KER), Pubmed was used. Initially results were screened for relevance off title/abstract and any of suspected relevance were reviewed in full to determine their applicability for the KER. Each KER includes a table of relevant search information (date, search terms, citations, etc). It is the hope of the authors that this AOP is used as a tool for risk assessment for drug and chemical exposures during embryonic development when disruption to SHH through antagonism of SMO occurs.
Summary of the AOP
Events:
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
Type | Event ID | Title | Short name |
---|
MIE | 2027 | Antagonism, Smoothened receptor | Antagonism Smoothened |
KE | 2044 | Decrease, Smoothend relocation and activation | Decrease, SMO relocation |
KE | 2028 | Decrease, GLI1/2 translocation to nucleus | Decrease, GLI1/2 translocation |
KE | 2040 | Decrease, GLI1/2 target gene expression | Decrease, GLI1/2 target gene expression |
KE | 1262 | Apoptosis | Apoptosis |
KE | 2043 | Decrease, Sonic Hedgehog second messenger production | Decrease, SHH second messenger production |
KE | 1821 | Decrease, Cell proliferation | Decrease, Cell proliferation |
KE | 2041 | Decrease, palatal shelf outgrowth | Decrease, outgrowth |
AO | 2042 | Orofacial clefting | OFC |
Relationships Between Two Key Events (Including MIEs and AOs)
Title | Adjacency | Evidence | Quantitative Understanding |
---|
Antagonism Smoothened leads to Decrease, SMO relocation | adjacent | Moderate | Low |
Decrease, SMO relocation leads to Decrease, GLI1/2 translocation | adjacent | Moderate | Low |
Decrease, GLI1/2 translocation leads to Decrease, GLI1/2 target gene expression | adjacent | Low | Low |
Decrease, GLI1/2 target gene expression leads to Decrease, SHH second messenger production | adjacent | Low | Low |
Decrease, SHH second messenger production leads to Decrease, Cell proliferation | adjacent | Low | Low |
Decrease, Cell proliferation leads to Decrease, outgrowth | adjacent | Low | Low |
Decrease, outgrowth leads to OFC | adjacent | Moderate | Low |
Apoptosis leads to Decrease, outgrowth | adjacent | Low | Low |
Decrease, GLI1/2 target gene expression leads to Apoptosis | adjacent | Low | Low |
Antagonism Smoothened leads to OFC | non-adjacent | High | Moderate |
Network View
Prototypical Stressors
Life Stage Applicability
Life stage | Evidence |
---|---|
Embryo | High |
Taxonomic Applicability
Term | Scientific Term | Evidence | Link |
---|---|---|---|
mouse | Mus musculus | NCBI |
Sex Applicability
Sex | Evidence |
---|---|
Unspecific | High |
Overall Assessment of the AOP
Annex 1 Table, Assessment of the relative level of confidence in the overall AOP based on rank ordered weight of evidence elements is attached in PDF format.
Domain of Applicability
Chemical: This AOP applies to antagonists of the SMO receptor. Chemical modulators of the SHH pathway have been identified including the natural alkaloid cyclopamine, both natural and synthetic pharmaceuticals (e.g. Vismodegib) , and a widely used pesticide synergist (PBO) with established human exposures (Lipinski, Dengler et al. 2007, Lipinski, Song et al. 2010, Wang, Lu et al. 2012, Everson, Sun et al. 2019, Rivera-González, Beames et al. 2021).
Sex: This AOP is unspecific to sex.
Life Stages: The relevant life stage for this AOP is embryonic development. More specifically, the development of the craniofacial region which occurs between GD 10.0 and GD 14.0 in the mouse and week 4-12 in human.
Taxonomic: At present, the assumed taxonomic applicability domain of this AOP is mouse (mus musculus). Most of the toxicological data that this AOP is based on has used mice as their model. Mice are a good analog of human craniofacial development and undergo similar signaling by SHH.
Essentiality of the Key Events
To date, few studies have addressed the essentiality of the proposed sequence of key events. Evidence linking SHH disruption through a decrease in proliferation exists. The hypothesized sequence of events has a high temporal concordance for canonical SHH signaling pathway and orofacial development. • Studies have shown that SHH signaling is required for normal facial development and plays a critical role in the growth of the facial processes that form the upper palate and lip (Bush and Jiang 2012, Kurosaka 2015). •The epithelial derived SHH drives orofacial development through an induced gradient in the underlying mesenchyme (Lan and Jiang 2009, Kurosaka 2015). This gradient of SHH induces cellular proliferation and outgrowth of the mesenchyme (Lan and Jiang 2009). • OFCs caused by disruption to SHH are believed to be due to a reduction in epithelial induced proliferation and the subsequent decrease in tissue outgrowth and the failure of the facial processes to meet and fuse (Lipinski, Song et al. 2010, Heyne, Melberg et al. 2015).
Evidence Assessment
Evidence Assessment • KER ID-Title-[Adjacency], [Evidence], [Quantitative Understanding]
• Relationship 2734: Antagonism Smoothened (Event 2027) leads to Decrease, SMO relocation (Event 2044)-[Adjacent], [Moderate], [Low]-There is a high biological plausibility of this relationship and SMO localization to the primary cilia is essential for proper SHH signaling in vertebrates (Corbit, Aanstad et al. 2005, Rohatgi, Milenkovic et al. 2007, Rohatgi, Milenkovic et al. 2009). There is good evidence that the SANT compounds block the localization of SMO to the tip of the primary cilia. Contradictory in vivo data was found regarding whether cyclopamine blocks SMO relocation to the primary cilia. Further work is required to determine if SMO antagonism via cyclopamine results in decrease in SMO relocation.
• Relationship 2735: Decrease, SMO relocation (Event 2044) leads to Decrease, GLI1/2 translocation (Event 2028)-[Adjacent], [Moderate], [Low]- Moderate evidence is presented to support that a loss of SMO relocation to the primary cilia leads to a significant decrease in GLI1. GLI1 requires activation prior to nuclear translocation.
• Relationship 2721: Decrease, GLI1/2 translocation (Event 2028) leads to Decrease, GLI1/2 target gene expression (Event 2040)-[Adjacent], [Low], [Low]- There is high biological plausibility of this relationship but to date few studies were found to explore the relationship.
• Relationship 2731: Decrease GLI1/2 target gene expression (Event 2040) leads to Decrease, SHH second messenger production (Event 2043)-[Adjacent], [Low], [Low]-Coordinated signaling is paramount for proper embryonic development and the GLI signaling cascade drives feedback/forward loops with FGF and BMP signaling pathways. Support was found for SHH having a feedforward loop with FGF10 and BMP4 however further investigation into the interaction of these pathways and their crosstalk is required.
• Relationship 2732: Decrease SHH second messenger production (Event 2043) leads to Decrease, cell proliferation (Event 1821)-[Adjacent], [Low], [Low]- SHH is a known mitogen and drives proliferation through its’ secondary messengers. SHH was found to induce proliferation and FGF10 in vivo.
• Relationship 2724: Decrease, Cell proliferation (Event 1821) leads to Decrease, outgrowth (Event 2041)-[Adjacent], [Low], [Low]-SHH is a known mitogen that helps to drive the proper development of the face which includes the outgrowth of the facial prominences. To date, few studies have measured by outgrowth of the facial prominences and proliferation. Hypoplasia of pharyngeal arch 1 was found in SHH-/- embryos supporting that outgrowth is driven by proliferation and is reduced when proliferation is decreased.
• Relationship 2726: Decrease, outgrowth (Event 2041) leads to OFC (Event 2042)-[Adjacent], [Moderate], [Low]- OFCs caused by disruption to SHH are believed to be due to a reduction in epithelial induced mesenchymal? proliferation and the subsequent decrease in tissue outgrowth and the failure of the facial processes to meet and fuse (Lipinski, Song et al. 2010, Heyne, Melberg et al. 2015). Mice with disrupted SHH signaling are found to have palatal shelves that are spaced apart supporting that the cleft results from an EMi dependent, but epithelial-mesenchyme transition (Emt) independent manner.
• Relationship 2792: Apoptosis (Event 1262) leads to Decrease, outgrowth (Event 2041)-[Adjacent], [Low], [Low]- SHH signaling is known to be associated with cell survival and there is a high biological plausibility that increasing apoptosis would cause a decrease in outgrowth. Supporting evidence is offered with increases in apoptosis in the mandibular arch seen in SHH signaling disrupted mice that exhibit decreased outgrowth.
• Relationship 2882: Decrease, GLI1/2 target gene expression (Event 2040) leads to Apoptosis (Event 1262) -[Adjacent], [Low], [Low]- To date few studies have examined the relationship of GLI1/2 target gene expression. There is a high biological plausibility that SHH plays a role in cell survival and death through GLI1/2 target gene expression. Decreased GLI1/2 target gene expression is seen in RA exposed dams alongside increased apoptosis on the cranial neural crest cells (CNCC).
• Relationship 2894: Antagonism Smoothened (Event 2027) leads to OFC (Event 2042)-[Non-adjacent], [High], [Moderate]- multiple studies have demonstrated in vivo that administration of SMO antagonists during critical windows of exposure leads to birth defects including OFC in a dose-dependent fashion.
Biological Plausibility
Biological plausibility refers to the structural and/or functional relationship that exists between the key events based on our understanding of normal biology. SHH signaling is largely conserved in mammals and is required for normal facial development and plays a critical role in the growth of the facial processes that form the upper palate and lip (Bush and Jiang 2012, Kurosaka 2015). Multiple antagonists of the SMO receptor have been identified through binding studies for including cyclopamine, vismodegib, PBO, and the SANT compounds (Lipinski, Dengler et al. 2007, Lipinski, Song et al. 2010, Wang, Lu et al. 2012, Everson, Sun et al. 2019, Rivera-González, Beames et al. 2021). While the level of support for most of the KERs is low, there is high support for the non-adjacent relationship linking antagonism of SMO and OFC. . Concordance of dose-response relationships There are a limited number of studies in which multiple key events were assessed in the same study following exposure to known SMO antagonists. These studies form the basis of the dose-response concordance of this AOP. A summary of the dose-concordance can be found in Supplementary Table 2. Many studies were found to use a single exposure. The concentration-dependence of the key event responses regarding concentration of known in vitro and/or in vivo for some of the KEs in this AOP. • Concentration dependent clefting with cyclopamine exposure (Omnell, Sim et al. 1990) • Dose dependent binding to SMO (Chen, Taipale et al. 2002) • Concentration dependent decrease in SMO-ciliary accumulation in vitro for vismodegib exposure (Wang, Arvanites et al. 2012)
Temporal concordance Temporal concordance refers to the degree to which the data supports the hypothesized sequence of Molecular Initiating Event (MIE) leading to the Adverse Outcome (AO) through a series of Key Events (KEs). The SHH pathway is a well-known developmental pathway that plays a role in embryogenesis including the development of the face. The SHH pathway is sensitive to chemical disruption at multiple molecular targets along the signaling cascade including antagonism of the SMO receptor, with exposure during critical windows in development leading to OFCs (Lipinski and Bushman 2010, Heyne, Melberg et al. 2015). Chemical modulators of the SHH pathway have been identified including the natural alkaloid cyclopamine, both natural and synthetic pharmaceuticals, and a widely used pesticide synergist (PBO) with established human exposures (Lipinski, Dengler et al. 2007, Lipinski, Song et al. 2010, Wang, Lu et al. 2012, Everson, Sun et al. 2019, Rivera-González, Beames et al. 2021). Canonical SHH signaling through PTCH-SMO-GLI is well understood and our AOP remains consistent with the pathway. SHH signaling is required for normal facial development and plays a critical role in the growth of the facial processes that form the upper palate and lip (Bush and Jiang 2012, Kurosaka 2015). The epithelial derived SHH drives orofacial development through an induced gradient in the underlying mesenchyme (Lan and Jiang 2009, Kurosaka 2015). This gradient of SHH induces cellular proliferation and outgrowth of the mesenchyme (Lan and Jiang 2009). The hypothesized sequence of events is supported by the existing data and follow the field’s current understanding of the canonical SHH signaling pathway.
Consistency The AO is not specific to this AOP. Many of the events is this AOP will overlap with AOPs linking disruption of SHH to OFC and some are expected to overlap with AOPs linking other developmental signaling pathways to OFCs.
Uncertainties, inconsistencies, and data gaps This AOP would be strengthened by studies examining the dose-response and time-course relationships for these KERs. The main data gaps for this AOP exist in the lack of studies that have examined the relationship in the context of dose response or time course. Additional studies using the mice would help to strengthen this AOP.
Data gaps: • Dose response and time course studies relating a Decrease, SMO relocation leads to Decrease, GLI1/2 translocation • Dose response and time course studies relating a decrease GLI translocation leads to decrease GLI target gene expression • Dose response and time course studies relating a Decrease, GLI1/2 target gene expression leads to Decrease, SHH second messenger production • Dose response and time course studies relating a Decrease, SHH second messenger production leads to Decrease, Cell proliferation • Dose response and time course studies relating a Decrease, Cell proliferation leads to Decrease, outgrowth • Dose response and time course studies relating a Decrease, outgrowth leads to OFC • Dose response and time course studies relating a Apoptosis leads to Decrease, Outgrowth • Dose response and time course studies relating a Decrease, GLI1/2 target gene expression leads to Apoptosis
Inconsistencies: • While it is well understood that cyclopamine is an antagonist of SMO, contradictory in vivo data was found regarding whether cyclopamine blocks SMO relocation to the primary cilia. Rohatgi et al used NIH 3T3s cell and found that cyclopamine did not inhibit the accumulation of SMO in the cilia even when dosed at 5-10um (>10 fold above kd). All three antagonists inhibited SHH pathway transduction and target gene expression (Rohatgi, Milenkovic et al. 2009). Corbit et al used a renal epithelial MDCK (Madin-Darby canine kidney) line was engineered to express Myc-tagged SMO. Following culture for 1hr in SHH conditioned media SMO presence in the primary cilium is upregulated while cells cultured in the presence of cyclopamine see a downregulation of SMO in the primary cilia (Corbit, Aanstad et al. 2005). Further work is required to determine if SMO antagonism via cyclopamine results in decrease in SMO relocation.
Uncertainties: • While we know that entry to the cilia is tightly controlled, the exact mechanism of SMO ciliary trafficking is not fully understood. The primary cilia (PC) is separated from the plasma membrane by the ciliary pockets and the transition zone which function together to regulate the movement of lipids and proteins in and out of the organelle (Goetz, Ocbina et al. 2009, Rohatgi and Snell 2010). The SHH receptor PTCH contains a ciliary localization sequence in its’ carboxy tail. Localization of PTCH to the PC is essential for inhibition of SMO as deletion of the CLS in PTCH prevents PTCH localization as well as inhibition of SMO (Kim, Hsia et al. 2015) (53). SMO also contains a CLS, but only accumulates in the PC upon ligand binding (Corbit, Aanstad et al. 2005). The entry of SMO into the PC is thought to occur either laterally through the ciliary pockets or internally via recycling endosomes (Milenkovic, Scott et al. 2009). Once inside the PC, SMO can diffuse freely, however it will usually accumulate in specific locations depending upon its’ activation state. Inactive SMO will accumulate more at the base of the PC while active SMO will accumulate in the tip of the PC (Milenkovic, Weiss et al. 2015). • The relationships and feedback/feedforward loops that exist between SHH and its’ secondary messengers primarily FGF10 and BMP4 are not well understood. More investigation into these relationships is warranted. • The exact mechanism through which SHH promotes cell survival is not well understood. Further studies are needed to illuminate the mechanism that links SHH signaling with cell survival. • The relationship between GLI1/2 target gene expression and increased apoptosis has a high biological plausibility although there is currently lack of studies that address this relationship.
Known Modulating Factors
Modulating Factor (MF) | Influence or Outcome | KER(s) involved |
---|---|---|
Quantitative Understanding
Assessment of quantitative understanding of the AOP:
The quantitative understanding for this AOP with the exception of the non-adjacent relationship between Antagonism Smoothened leads to OFC is low. Most of the data found through the literature search was obtained from doses at a single dose and was not conducted with dose-response or time-course in mind. For Antagonism Smoothend leads to OFC several studies with dose response data showing a dose-dependent incidence of clefting were found. This AOP would benefit from the generation of additional data that addresses these relationships in a dose response and time course methodology to allow for an increased quantitative understanding of the linkage.
Considerations for Potential Applications of the AOP (optional)
Considerations for potential applications of the AOP
The intended use of this AOP from a regulatory standpoint is to improve predictive potential of developmental hazards as they relate to the SHH pathway and OFCs. It is hoped that this AOP can be applied to data from in silico and in vitro high-throughput screening assays (HTS) to guide selection of agents for further investigation in more representative models of orofacial development. Disruption of the Sonic Hedgehog pathway has broader outcomes than just OFCs and SHH is known to play a role in many aspects of embryonic development including patterning of many systems and limb and digit development. This AOP can be used as part of an integrated assessment of toxicity and can help to guide risk assessment for potential exposures during development.
There is a need for development of New Approach Methodologies (NAMs) to increase understanding of the relationships that exist within this AOP to provide facilitate screenings abilities. Humans are exposed to upwards of 80,000 industrial chemicals and natural products, the majority of which have not undergone any type of toxicity testing either alone or in mixtures. Even highly regulated drugs are typically not tested for safety in pregnant women for obvious reasons despite the medical need in this population (Wise 2022). To help address this, we have engineered an in vitro microphysiological model (MPM) model of orofacial development to facilitate the study of both normal and abnormal orofacial development including disruption of SHH (Johnson, Vitek et al. 2021, Reynolds, Vitek et al. 2022). Traditional high throughput screening (HTS) assays are optimized for one pathway: one readout. This oversimplifies toxicant metabolism, intercellular pathway interactions, and ultimately makes the assay not representative of real-life exposures. Problems with HTS in drug discovery have been identified including missing intercellular interactions, co-exposures, and off target safety (Macarron, Banks et al. 2011). We can learn from these identified problems and engineer in vitro systems to more accurately recapitulate the biology to give a more thorough assessment of chemical and drug exposure.
References
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