Biochemical and Biophysical Research Communications
ReviewMetabolites in vertebrate Hedgehog signaling
Introduction
While the Hedgehog (HH) signaling pathway plays a role in embryonic development, stem cells, cellular metabolism, axon targeting, synapse formation and nociception [1], [2], [3], [4], [5], it is also involved in human disorders and diseases, including developmental abnormalities and several forms of cancer [6], [7], [8], [9], [10], [11], [12]. The HH signaling cascade is unusual as it appears to involve metabolites while it alters the sub-cellular localization of pathway components that are closely entangled with the primary cilium [2]. Key players in this pathway, the morphogen Hedgehog, the trans-membrane proteins Patched and Smoothened and the zinc finger transcription factor Cubitus interruptus were predominantly identified through genetic screens in Drosophila in the late 1970’s and early 1980’s [13], [14], [15], [16], [17], with the subsequent discovery of the vertebrate homologs Sonic Hedgehog, Indian Hedgehog, Desert Hedgehog (SHH, IHH, DHH), Patched (PTC), Smoothened (SMO) and GLI [18], [19], [20], [21], [22], [23], [24]. As the interactions in this pathway were mapped, substantial similarities between Drosophila and vertebrates became apparent, reflecting deep evolutionary roots [2], [25], [26], [27], [28]. However, also differences exist between species; in particular many of the HH pathway components localize to the cell’s primary cilium in vertebrates, an organelle that is not present in Drosophila [28], [29], [30], [31].
In cells that produce the HH morphogen, HH undergoes cleavage, and its N-terminal peptide is dual-lipidated by cholesterol and palmitic acid (Fig. 1a). HH is then released by the resistance-nodulation division (RND) protein dispatched (DISP) either as monomeric particles, as multimeric particles or as exo-vesicles [32], [33]. The form in which HH is released appears to define its signaling range [2]. It has been proposed that lipidation of HH promotes the association of HH with sterol-rich membrane areas in receiving cells [2], [34], [35] where HH binds to a number of membrane proteins including its canonic receptor the 12 pass transmembrane protein PTC, that similar to DISP, is a member of the RND family of proteins [21], [23], [36], [37]. The binding of HH to PTC is promoted by the membrane co-receptor proteins CDO (CAM-related/downregulated by oncogens); BOC (brother of CDO) and GAS1 (growth arrest-specific 1) [2], [26], [38]. In the absence of the HH morphogen, PTC constitutively inhibits the signaling cascade [36], [39]. However, upon HH binding, PTC releases the inhibition of the pathway.
How HH signaling is progressed beyond PTC is not entirely settled and there are curious differences between Drosophila and vertebrates [28]. PTC regulates the sub-cellular localization and activity of SMO, but PTC does not appear to directly interact with SMO. When the RND domain of PTC is mutated, it is no longer capable to inhibit SMO, suggesting that metabolites may be involved in the interaction between PTC and SMO [40], [41]. What these metabolites are in vivo remains to some extent unclear and will be discussed below.
In vertebrates several sub-cellular alterations occur upon activation of the HH signaling pathway. In the absence of HH, the PTC receptor is enriched at the basis and in the primary cilium, a specialized organelle at the cellular surface that depends on the intraflagellar transport system (IFT) and that has been implied in various sensing functions [31]. When HH binds PTC, PTC leaves the primary cilium and enters the endocytotic pathway to be degraded. As PTC exits the primary cilium, also the G-protein coupled receptor GPR161, a rhodopsin family GPCR protein, is transported out of the primary cilium [42]. GPR161 negatively regulates HH signaling in the primary cilium through enhancing PKA activity by increasing cAMP levels. PKA is involved in phosphorylating the HH dependent zinc finger transcription factors GLI2 and GLI3 as described below [42]. Importantly, upon HH binding to PTC, the 7 transmembrane protein SMO, a Frizzled (FZD) class G-protein-coupled receptor with an unusually complex structure, is activated and moves in association with β-arrestin and the microtubule motor KIF3A into the primary cilium [29], [30], [35], [43], [44]. This movement originates predominantly from exocytotic SMO containing vesicles [45] but also from the plasma membrane [46]. For the translocation of SMO to the primary cilium and its activation, SMO is phosphorylated at its carboxy-terminal cytoplasmic tail by CK1 and GRK2 [47], [48] leading to a conformational switch [25].
One of the roles of vertebrate cilia is to control the proteolytic processing of members of the Ci/GLI family of zinc finger transcription factors [2]. In the inactive state of HH signaling two members of the GLI family, GLI2 and GLI3 pass through the primary cilium [31], [49] in a process that leads to their partial proteolytic processing into transcriptional repressors. GLI3 contains an N-terminal repressor domain that, in the absence of HH signaling, is sequentially phosphorylated by protein kinase A (PKA), casein kinase1 (CK1) and glycogen synthase kinase 3 (GSK3). Phosphorylated GLI3 is then recognized by βTrCP leading to ubiquitinatation of GLI3 and a partial degradation of the C-terminal trans-activation domain in the proteasome. This leads to a repressor form of GLI3 [50]. The ciliar SUFU protein assists the process by immobilizing GLI3 [51], [52]. Similar to GLI3, GLI2 is also proteolytically processed, but to a lesser extent [2], [53], [54].
When SMO enters the primary cilium it associates, phosphorylation dependent, with the EVC and EVC2 proteins (Ellis-van Creveld Syndrome protein) [55], [56], [57]. It is thought that SMO/EVC/ EVC2 interact with SUFU thereby resolving the SUFU/ GLI3 inhibitory complex [57]. In consequence, GLI3 proteolytic processing is prevented leading to a weak activator form of GLI3 that translocates to the nucleus [58]. Active GLI3 up-regulates the expression of the transcriptional activator GLI1 that, although apparently not involved in the initial Hh dependent response of the pathway, is a strong potentiator of the signal [54], [59].
Section snippets
Metabolites, PTC and SMO
The roles of metabolites in HH signaling remain only partially understood although multiple lines of evidence suggest that PTC and SMO communicate via metabolites. PTC has been proposed to be involved in sterol trafficking. PTC contains two main domains, a RND domain that is related to bacterial proteins implied in proton transport and permease activity [60], and a related sterol-sensing domain (SSD) that has been linked to sensing sterols [26], [61]. Other members of the PTC-family of RND
Analytical challenges for monitoring and discovering endogenous HH-active oxysterols and other metabolites
The uncertainty of the roles that metabolites play in HH signaling can be met to a large degree with sensitive and selective analytical methodology, which can be applied to e.g. rule out possible candidates, but also for discovering new ones. As mentioned above, one of the most potent oxysterols examined so far that activates SMO in vitro is 20S-OHC. 20S-OHC has been speculated as being the prominent activator in vivo [80], however, our group and others [68], [81], [88] have not been able to
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