Antagonism of Smoothened receptor leading to orofacial clefting

• Jacob Reynolds

• Judy Choi

Revision dates for related pages

Domain(s) 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.

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

• KER ID-Title-[Adjacency], [Evidence], [Quantitative Understanding
• KER 2734-Antagonism Smoothened leads to Decrease, SMO relocation-[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.
• KER 2735- Decrease, SMO relocation leads to Decrease, GLI1/2 translocation-[Adjacent], [Moderate], [Low]- Moderate evidence is presented to support that a loss of the primary cilia leads to a significant decrease in GLI1. GLI1 requires activation prior to nuclear translocation.
• KER 2721-Decrease,GLI1/2 translocation leads to Decrease, GLI1/2 target gene-[Adjacent], [Low], [Low]- There is high biological plausibility of this relationship but to date few studies were found to explore the relationship.
• KER 2731-Decrease, GLI1/2 target gene expression leads to Decrease, SHH second messenger production-[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.  
• KER 2732-Decrease, SHH second messenger production leads to Decrease, Cell proliferation-[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. In FGF10 deficient models SHH was found to be reduced.
• KER 2724-Decrease, Cell proliferation leads to Decrease, outgrowth-[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.
• KER 2726-Decrease, outgrowth leads to OFC-[Adjacent], [Moderate], [Low]- 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). 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.
• KER 2792-Apoptosis leads to Decrease, Outgrowth-[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.
• KER 2882-Decrase, GLI1/2 target gene expression leads to Apoptosis-[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 CNCC.
• KER 2894-Antagonism Smoothened leads to OFC-[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. 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 table 1. Many studies were found to use a single exposure.

Table 2: Concordance table

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.

1. Concentration dependent clefting with cyclopamine exposure (Omnell, Sim et al. 1990)
2. Concentration dependent neural tube malformations with cyclopamine exposure (Incardona, Gaffield et al. 1998)
3. Dose dependent binding to SMO (Chen, Taipale et al. 2002)
4. 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 MIE leading to the AO through a series of 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.

Does cyclopamine block SMO relocation-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.

Assessment of quantitative understanding of the AOP:

The quantitative understanding for this AOP except for the non-adjacent relationship between Antagonism Smoothened leads to OFC is low. The majority of the data found through the literature 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.