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Relationship: 2735

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, SMO relocation leads to Decrease, GLI1/2 translocation

Upstream event

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

Decrease, SMO relocation

Downstream event

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

Decrease, GLI1/2 translocation

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	Moderate	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 Do not cite	Under Review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
mice	Mus sp.	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 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).

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). ).

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 and appear below in Table 1. Search results were initially screened through review of the title and abstract for potential for data relating SMO relocation and GLI1/2 translocation. 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.

Table 1: KER 2735 evidence collection strategy

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

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). It has been shown that SMO localization to the tip of the primary cilia is essential for the SHH signaling cascade via the GLI transcription factors(Corbit, Aanstad et al. 2005, Rohatgi, Milenkovic et al. 2007, Rohatgi, Milenkovic et al. 2009)

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
- NIH 3T3 clones with stable HA-Gli2 expression were created and a line with low HA-Gli2 expression was selected for further study. The reporter activity was induced by ShhN and fully inhibited by cyclopamine. When stimulated with ShhN, antibody staining was used to verify that Gli2 accumulates at the tip of the primary cilia. Immunostaining was also used to find that Gli2 accumulated in the nucleus of cells treated with ShhN. Using nuclear extracts of unstimulated cells HA-Gli2R was predominantly localized in the nucleus while in stimulated cells HA-Gli2 increased and HA-Gli2 decreased. Cells treated with Shh agonist SAG also had SMO accumulation in the primary cilia and increased HA-Gli2A in the nucleus (Kim, Kato et al. 2009).
- NIH 3T3 cells were used to study whether the oxysterols and/or cholesterol are required for SHH signaling. Cells were depleted of sterols via incubation with methyl-β-cyclodextrin (MCD). Fluorinated sterols were added back as soluble components and the cells were stimulated with Shh ligand. Assays were performed for recruitment of endogenous SMO to the primary cilia and for pathway activation using a transcriptional reporter assay. Sterol depletion blocked relocation of SMO to the cilia and SHH activation. Cholesterol and 25-fluorocholesterol both rescued sterol depleted cells and restored SHH pathway activation (Huang, Nedelcu et al. 2016).
- MMS1 (human myeloma) cells were used to study whether activation of Gli1 is required for its’ translocation to the nucleus. Forskolin (FSK) which acts by blocking GLI1 access to PKA was added to culture for 24h at 10µm. The nuclear localization of GLI1 was significantly decreased in the Prescence of FSK (Blotta, Jakubikova et al. 2012).
In vivo
- none identified

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

While we know that entry to the cilia is tightly controlled, the exact mechanism of SMO ciliary trafficking is not fully understood. The 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).

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 data presented in support of this KER includes in vitro studies. The in vitro work offers data that SMO relocates to the tip of the primary cilium and that this plays a role in the translocation of the GLI transcription factors to the nucleus. The quantitative understanding of this linkage is low as studies including dose-response and time-course were not found.

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

Relocation of SMO to the PC typically occurs within ~20 minutes of agonist stimulation (Arensdorf, Marada et al. 2016). No data was found with regards to GLI1/2 translocation.

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

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 relationship between a decrease in translocation of SMO and a decrease in GLI1/2 translocation to the nucleus 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. 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

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

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.

Blotta, S., J. Jakubikova, T. Calimeri, A. M. Roccaro, N. Amodio, A. K. Azab, U. Foresta, C. S. Mitsiades, M. Rossi, K. Todoerti, S. Molica, F. Morabito, A. Neri, P. Tagliaferri, P. Tassone, K. C. Anderson and N. C. Munshi (2012). "Canonical and noncanonical Hedgehog pathway in the pathogenesis of multiple myeloma." Blood 120(25): 5002-5013.

Brugmann, S. A., D. R. Cordero and J. A. Helms (2010). "Craniofacial ciliopathies: A new classification for craniofacial disorders." Am J Med Genet A 152A(12): 2995-3006.

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.

Goetz, S. C., P. J. Ocbina and K. V. Anderson (2009). "The primary cilium as a Hedgehog signal transduction machine." Methods Cell Biol 94: 199-222.

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.

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.

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

Kim, J., E. Y. Hsia, A. Brigui, A. Plessis, P. A. Beachy and X. Zheng (2015). "The role of ciliary trafficking in Hedgehog receptor signaling." Sci Signal 8(379): ra55.

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.

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

Milenkovic, L., M. P. Scott and R. Rohatgi (2009). "Lateral transport of Smoothened from the plasma membrane to the membrane of the cilium." J Cell Biol 187(3): 365-374.

Milenkovic, L., L. E. Weiss, J. Yoon, T. L. Roth, Y. S. Su, S. J. Sahl, M. P. Scott and W. E. Moerner (2015). "Single-molecule imaging of Hedgehog pathway protein Smoothened in primary cilia reveals binding events regulated by Patched1." Proc Natl Acad Sci U S A 112(27): 8320-8325.

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.

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, 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 W. J. Snell (2010). "The ciliary membrane." Curr Opin Cell Biol 22(4): 541-546.

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.

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.