Decrease, SMO relocation leads to Decrease, GLI1/2 translocation

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). Multiple ciliopathies are associated with clefting in humans including Meckel-Gruber syndrome (OMIM 249000) and Ellis-van Creveld syndrome (OMIM 225500)(Brugmann, Cordero et al. 2010).

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).

          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

The data presented in support of this KER includes both in vitro and in vivo 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 in vivo work shows that loss of cilia in embryonic mice leads to a significant decrease in GLI1 in the nucleus. SUFU expression was found to overlap with PTCH1 expression in both mice and human embryos supporting that SUFU plays a role in SHH signal transduction.  The quantitative understanding of this linkage is low as studies including dose-response and time-course were not found.  

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 and that aberrant SHH signaling during embryonic development can cause birth defects including orofacial clefts (OFCs). For this reason, this KER is applicable to the embryonic stage with a high level of confidence.