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Relationship: 2721

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

Decrease, GLI1/2 translocation leads to Decrease, GLI1/2 target gene expression

Upstream event

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

Decrease, GLI1/2 translocation

Downstream event

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

Decrease, GLI1/2 target gene expression

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	adjacent	Low	Low	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 Do not cite	Under Review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
human	Homo sapiens	Low	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 Glioma-associated onocogene (Gli) family of zinc finger transcription factors (Gli1, Gli2, Gli3) are the primarily downstream effectors of the Hedgehog (HH) signaling cascade. When HH ligand binds to Patched (PTCH), its’ inhibition on SMO is relieved. SMO this then able to accumulate to the tip of primary cilium in its’ active form (Corbit, Aanstad et al. 2005, Rohatgi, Milenkovic et al. 2007, Kim, Kato et al. 2009). SMO causes the GLI family to become dislodged from their complex with the negative regulator of HH signaling, Suppressor of Fused (Sufu) (Kogerman, Grimm et al. 1999, Pearse, Collier et al. 1999, Stone, Murone et al. 1999, Tukachinsky, Lopez et al. 2010). The GLI-Sufu complex maintains retention of Gli in the cytosol allowing for exposure to phosphorylation via protein kinase A (PKA) which inhibits downstream signal transduction (Tuson, He et al. 2011). When SMO is activated, the GLI2/3-Sufu complex is dismantled allowing for retrograde transport of GLI back into the nucleus (Kim, Kato 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 SHH target gene expression (Alexandre, Jacinto et al. 1996, Von Ohlen and Hooper 1997).

The GLI family is found in both a long activator form (GliA) or a proteolytically cleaved repressor form (GliR). Current understanding is that Gli3 functions primarily as a repressor while Gli1 and Gli2 function mainly as activators of the pathway and that recruitment of SMO to the cilium leads to an increase in the ratio of GliA:GliR (Hui and Angers 2011, Liu 2016). Downstream transcription is primarily activated by Gli2 and repressed by Gli3 (Wang, Fallon et al. 2000, Bai, Auerbach et al. 2002, Persson, Stamataki et al. 2002). Gli1 serves primarily as an activator of transcription and works through amplification of the activated state (Park, Bai et al. 2000).

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 GLI translocation and GLI target gene expression. 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 terms used are organized below in Table 1.

Table 1: KER 2721 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

The evidence presented for this KER is low.The relationship between GLI1/2 translocation and a decrease in GLI1/2 target gene expression relocation has been shown indirectly in multiple mouse models through disruption of SHH signaling at the level of SMO. From our understanding of the SHH pathway, we can infer that disruption of the SHH signaling pathway at the level of SMO is causing a decrease in GLI1/2 translocation and it is this that is causing the altered gene expression While clear evidence that disruption of SHH signaling leads to altered gene expression especially those of the Fox family, insufficient evidence exists for the direct relationship between GLI1/2 translocation and SHH target gene expression. The evidence also lacks direct human applicability as all presented work was performed in vitro on murine models or in vitro on murine cell lines.

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

SHH signaling is well established to be essential for proper embryonic development in vertebrates including mice and humans. Activation of the pathway results in a downstream signaling cascade resulting in the relocation of GLI to the nucleus and subsequent gene transcription (Carballo, Honorato et al. 2018).

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
- A mouse cNCC line (09-1) with the expression signature (AP-2alpha (Tfap2a, Twist1, Sox9, Cd44) was used to study whether foxf2 is a target of SHH signalling. Addition of SHH ligand (0.4µg/ml) was found to upregulate both GLI1 and Foxf2. This upregulation was completely blocked by the addition of vismodegib (120nm)(Everson, Fink et al. 2017).
- To determine if SHH pathway inhibition was downstream for GANT 61 and GANT 58, a Sufu-null MEF cell line was used. Treatment of cells with either GANT at 10µm led to a significant reduction of SHH target genes GLI1 and Hip1 as determined by qPCR. As expected, cyclopamine was unable to inhibit signalling in this system as activation occurs downstream of SMO. GANT 61 is believed to act through addition of the modification to GLI1 that compromises its’ ability to properly bind DNA (Lauth, Bergström et al. 2007).
- GLI activators bind to the GACCACCCA motif to promote transcription of GLI1, PTCH1, PTCH2, HHIP1, MYCN, CCND1, CCND2, BCL2, CFLAR, FOXF1, FOXL1, PRDM1 (BLIMP1), JAG2, GREM1, and Follistatin (Katoh and Katoh 2009)
- Using a 3D microphysiological model loaded with 3T3 SHH lightII and GMSM-K GFP SHH cells a gradient of PTCH1 correlating with the distance from the epithelium secreting SHH ligand (Johnson, Vitek et al. 2021).
In vivo
- In situ hybridization was used to determine expression of GLI1 in C57BL/6J mice to better understanding temporal SHH signalling. At GD 9.0 no difference was found between control and embryos exposed to cyclopamine (120mg/kg/day). GLI1 was downregulated in the ventral frontonasal prominence (FNP) of clomipramine exposed embryos by GD 9.25. FNP tissue was micro dissected and cDNA microarray analysis was performed. 210 genes were found to be dysregulated including a significant enrichment to the forkhead box (Fox) family. RT-PCR confirmed significant down regulation of the SHH target genes GLI1 and PTCH1 as well as nine Fox members: Foxa2, Foxb2, Foxc1, Foxc2, Foxd1, Foxe1, Foxf1, Foxf2, Foxl1. Two members of the fox family, Foxm1 and Foxo1 were not found to differentially expressed in either the cDNA microarray or RT-PCR (Everson, Fink et al. 2017).
- Using mutant Osr2-IresCre;Smo^c/cmice Foxf2 and Foxf1were found to be positively regulated by SHH-SMO signalling. Expression of Osr2 was found to be reduced by E13.5 in the mutants. Expression of Osr1, Pax9, Tbx22 were not found to be altered (Lan and Jiang 2009).
- o To study whether SHH signaling regulates the developmental fate of the ecto-mesenchyme via regulation of gene activity in the facial primordia, Wnt1-Cre;Smon/c, (removal of SHH signaling) and Wnt1-Cre;R26SmoM2 (activation of SHH signaling). Positive regulation from SHH activity was found for Foxc2, Foxd1, Foxd2, Foxf1, and Foxf2. The Fox genes were found to be dissimilar in expression pattern with spatial activation even with uniform activation of the SHH pathway. Foxc2 and Foxd1 were found to be expressed ubiquitously in the MNA except at the midline, while Foxf1 is expressed at the lateral ends. Foxd2 and Foxf2 are both expressed along the mediolateral axis with Foxd2 having an increasing gradient from medial to lateral and Foxf2 having an opposing gradient (Jeong, Mao et al. 2004). These data support that disrupting GLI1/2 translocation via disruption of the SHH signaling pathway disrupts transcription of Foxc2, Foxd1, Foxd2, Foxf1, and Foxf2.

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

None identified

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

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

The quantitative understanding for this KER is low. Studies to investigate response-response relationship as well as time scale have not been conducted or were not found in the literature review. The empirical evidence presented establishes that disruption of SHH signaling results in the altered gene expression of SHH target genes. There is a need for more studies to address the dose-response and time course relationship of this linkage.

Response-response Relationship

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

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

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

Positive feedback loop of gene expression from GLI1 and negative feedback loop for PTCH1, PTCH2, HHIP1 (Katoh and Katoh 2009)

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

All presented evidence for the relationship is performed in mice. The relationship is biologically plausible in human, but to date no specific experiments have addressed this question.

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.

Bai, C. B., W. Auerbach, J. S. Lee, D. Stephen and A. L. Joyner (2002). "Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway." Development 129(20): 4753-4761.

Carballo, G. B., J. R. Honorato, G. P. F. de Lopes and T. C. L. d. S. e. Spohr (2018). "A highlight on Sonic hedgehog pathway." Cell Communication and Signaling 16(1): 11.

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.

Everson, J. L., D. M. Fink, J. W. Yoon, E. J. Leslie, H. W. Kietzman, L. J. Ansen-Wilson, H. M. Chung, D. O. Walterhouse, M. L. Marazita and R. J. Lipinski (2017). "Sonic hedgehog regulation of Foxf2 promotes cranial neural crest mesenchyme proliferation and is disrupted in cleft lip morphogenesis." Development 144(11): 2082-2091.

Hui, C. C. and S. Angers (2011). "Gli proteins in development and disease." Annu Rev Cell Dev Biol 27: 513-537.

Jeong, J., J. Mao, T. Tenzen, A. H. Kottmann and A. P. McMahon (2004). "Hedgehog signaling in the neural crest cells regulates the patterning and growth of facial primordia." Genes Dev 18(8): 937-951.

Johnson, B. P., R. A. Vitek, M. M. Morgan, D. M. Fink, T. G. Beames, P. G. Geiger, D. J. Beebe and R. J. Lipinski (2021). "A Microphysiological Approach to Evaluate Effectors of Intercellular Hedgehog Signaling in Development." Front Cell Dev Biol 9: 621442.

Katoh, Y. and M. Katoh (2009). "Hedgehog target genes: mechanisms of carcinogenesis induced by aberrant hedgehog signaling activation." Curr Mol Med 9(7): 873-886.

Kim, J., M. Kato and P. A. Beachy (2009). "Gli2 trafficking links Hedgehog-dependent activation of Smoothened in the primary cilium to transcriptional activation in the nucleus." Proc Natl Acad Sci U S A 106(51): 21666-21671.

Kogerman, P., T. Grimm, L. Kogerman, D. Krause, A. B. Undén, B. Sandstedt, R. Toftgård and P. G. Zaphiropoulos (1999). "Mammalian suppressor-of-fused modulates nuclear-cytoplasmic shuttling of Gli-1." Nat Cell Biol 1(5): 312-319.

Lan, Y. and R. Jiang (2009). "Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth." Development 136(8): 1387-1396.

Lauth, M., A. Bergström, T. Shimokawa and R. Toftgård (2007). "Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists." Proc Natl Acad Sci U S A 104(20): 8455-8460.

Liu, K. J. (2016). "Craniofacial Ciliopathies and the Interpretation of Hedgehog Signal Transduction." PLoS Genet 12(12): e1006460.

Park, H. L., C. Bai, K. A. Platt, M. P. Matise, A. Beeghly, C. C. Hui, M. Nakashima and A. L. Joyner (2000). "Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation." Development 127(8): 1593-1605.

Pearse, R. V., 2nd, L. S. Collier, M. P. Scott and C. J. Tabin (1999). "Vertebrate homologs of Drosophila suppressor of fused interact with the gli family of transcriptional regulators." Dev Biol 212(2): 323-336.

Persson, M., D. Stamataki, P. te Welscher, E. Andersson, J. Böse, U. Rüther, J. Ericson and J. Briscoe (2002). "Dorsal-ventral patterning of the spinal cord requires Gli3 transcriptional repressor activity." Genes Dev 16(22): 2865-2878.

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

Stone, D. M., M. Murone, S. Luoh, W. Ye, M. P. Armanini, A. Gurney, H. Phillips, J. Brush, A. Goddard, F. J. de Sauvage and A. Rosenthal (1999). "Characterization of the human suppressor of fused, a negative regulator of the zinc-finger transcription factor Gli." J Cell Sci 112 ( Pt 23): 4437-4448.

Tukachinsky, H., L. V. Lopez and A. Salic (2010). "A mechanism for vertebrate Hedgehog signaling: recruitment to cilia and dissociation of SuFu-Gli protein complexes." J Cell Biol 191(2): 415-428.

Tuson, M., M. He and K. V. Anderson (2011). "Protein kinase A acts at the basal body of the primary cilium to prevent Gli2 activation and ventralization of the mouse neural tube." Development 138(22): 4921-4930.

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, B., J. F. Fallon and P. A. Beachy (2000). "Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb." Cell 100(4): 423-434.