Antagonism of Smoothened receptor leading to orofacial clefting

• Jacob Reynolds

• Judy Choi

Revision dates for related pages

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) , the widely used pesticide syergist piperonyl butoxide (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 empirical 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 organism. Mice are a good analog of human craniofacial development and undergo similar signaling by SHH. The plausible domain of applicability for this AOP is mammals due to the largely conserved mechanisms of orofacial development and embryonic pathway signaling.

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]

•    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. Identified SMO antagonists include 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

Agreed, Wiki updated- 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 of the studies identified while researching this AOP were performed using a single dose of antagonist making the study not suited for dose response concordance. This AOP would benefit greatly from increased studies designed to explore the dose-response concordance of the proposed relationships.  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 is summarized below. •    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 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.

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 studies that employed a single dose and were 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.