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Hedgehog Signaling in Development and Disease
Hedgehog Signaling in Development and Disease D Jenkins, UCL Institute of Child Health, London, UK r 2016 Elsevier Inc. All rights reserved.
Introduction Key components of the hedgehog (HH) signaling pathway were first isolated as mutants affecting larval cuticle patterning in the fruit fly, Drosophila melanogaster (Nusslein-Volhard and Wieschaus, 1980). The HH pathway has served as a paradigm of signal transduction for over three decades, leading in our understanding of many of the fundamental mechanisms involved in intercellular communication. The generally accepted canonical model of HH signaling is largely conserved from fruit flies to humans, although differences in some of the biochemical details are apparent between species. One recent development is the finding that primary cilia are particularly important for regulation of HH signaling in higher vertebrates, and this has spawned a whole new field of investigation. The HH pathway also provides text-book examples of: (1) how a long-range morphogen is produced and secreted; (2) how transmembrane receptors are regulated by a morphogen gradient; and (3) how this can feed into transcriptional networks. Other lessons that have emerged from investigation of the HH pathway include: (4) how long-range regulatory elements determine the expression of a morphogen; and (5) the way in which a whole spectrum of anticancer and teratogenic compounds can influence various aspects of signaling mechanisms. It should also be noted that the HH pathway is a paradigm for human genetic disease. It is the only signaling pathway for which most of its constituent components are mutated in human birth defects, while somatic mutations within the pathway also give rise to specific pediatric and adult cancers. Importantly, a battery of therapeutic compounds targeting HH signaling components has been identified. Finally, very recent evidence also suggests added complexity in the HH pathway, in the form of so-called noncanonical pathways.
The Basic Mechanisms of HH Signaling Production and Secretion of a Morphogen – The HH Proteins By definition, a morphogen is a secreted molecule that acts at long-range. While a single HH ligand exists in fruit flies, in mammals, three homologs of Drosophila HH exist, which are known as Sonic HH (SHH), Indian HH (IHH), and Desert HH (DHH). Each HH morphogen is expressed as a 52 kDa precursor protein that undergoes autoproteolytic cleavage to generate an N-terminal 19 kDa protein that fulfils all patterning activities both at short- and long-range, and a C-terminal 25 kDa species (Lee et al., 1994; Porter et al., 1995). The C-terminal region of the unprocessed precursor serves as a catalytic domain, which cleaves the precursor protein at a highly conserved cysteine residue by an intramolecular mechanism, and this reaction exhibits concentration-independent kinetics (Porter et al., 1995). Processing of HH proteins to generate an active N-terminal morphogen requires
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normal cholesterol metabolism, and chemical inhibition of sterol metabolism following treatment of cells with inhibitors of cholesterol biosynthesis impedes HH precursor processing into the two smaller fragments (Guy, 2000). It is now known that HH morphogens are modified by the addition of cholesterol following autoproteolysis of the precursor protein; the newly made N-terminus of the 19 kDa morphogen becomes covalently modified by the addition of cholesterol while its C-terminus becomes palmitoylated at the same time (Lee et al., 1994; Porter et al., 1995). SHH and IHH have been elegantly shown to travel at a distance of many cell diameters from their source in mice using immunohistochemistry on tissues fixed in such a way that preserves proteoglycans/glycosaminoglycans and proteins in an insoluble form (Gritli-Linde et al., 2001). It is thought that addition of cholesterol is important in determining the range over which HH proteins signal. By harboring a cholesterol moiety, HH morphogens have the tendency to remain at the surface of cells that have synthesized them, owing to hydrophobic interaction between the cholesterol group and cell surface lipids. In some tissues, however, the HH-producing cell also expresses Dispatched (DISP), a sterol-sensing domain protein dedicated to the release of cholesterol modified HH ligand (Burke et al., 1999). Thus, HH signaling is long-range when expressed with DISP but juxtacrine/autocrine when DISP is absent. It is thought that DISP serves to mask the cholesterol motif of HH, reducing its affinity for the cell membrane and allowing its diffusion over many cell diameters, perhaps also promoting HH oligomerization. Given that three mammalian HH morphogens exist, each of which function in an overlapping yet distinct range of tissues, the choice of morphogen therefore represents another mechanism by which HH signaling is modulated. While SHH, IHH, and DHH bind to their receptor with equal affinity, in certain assays the concentration of each morphogen required to elicit a response can be very different (up to 60-fold); SHH has the greatest potency in most scenarios, with IHH and then DHH inducing progressively reduced effects. These relationships may also be tissue-dependent, however, because in some contexts, such as the inhibition of chondrocyte differentiation, DHH can be more potent than IHH (Pathi et al., 2001). Taken together, this combined evidence shows how processing to generate an active morphogen is also involved in the tight regulation of HH signaling.
Intracellular Effectors of Signal Transduction – Transmembrane Receptors Cells that are potentially responsive to HH morphogens express the 12-transmembrane domain receptor, Patched (PTCH), which normally serves to repress a second transmembrane protein, Smoothened (SMO) (Figure 1). Therefore, in the absence of HH morphogen, HH signaling is silenced. In contrast, binding of secreted HH to PTCH relieves its normal
Encyclopedia of Cell Biology, Volume 3
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Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease
IHH
SHH
DHH
No Shh:
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+ Shh: Dync2h1
PTCH1 PTCH1 SMO
GLI-R
PTCH1
SMOH
SMOH
Kinesin-II
GLI-A
Figure 1 HH signal transduction. Left – Scheme showing the logic of HH signaling. Black lines indicate activity in the absence of SHH, IHH, or DHH ligand. Red lines indicate actions following binding of ligand to the PTCH1 receptor. Middle – Scheme showing a primary cilium with central microtubular doublets anchored to the basal body and encased by membrane. In the absence of HH ligand, the PTCH1 receptor is transported into the cilium, leading to the export of SMO and transport away from the cilium in endocytic vesicles. Right – In the presence of ligand, PTCH1 no longer enters the cilium, allowing SMO to accumulate in this organelle. The box indicates two key molecules, Kinesin-II and Dync2h1 (a key component of dynein-2), which regulate anterograde and retrograde intraflagellar transport, respectively.
repression of SMO by inducing a conformational change (Ingham et al., 2000; Zhao et al., 2007). Extensive genetic and biochemical analyses have demonstrated the importance of protein kinases in driving changes in the subcellular localization of SMO (Jia et al., 2004; Zhang et al., 2005; Liu et al., 2007; Zhu et al., 2003; Denef et al., 2000). Inhibition of PTCH repressor function by HH leads to the cell surface accumulation of SMO, a process which requires phosphorylation at a number of protein kinase A (PKA) and casein kinase I (CKI) sites in the C-terminal tail of SMO (Jia et al., 2004). This intracellular domain of SMO interacts with a protein complex that includes PKA, CKI, fused (FU; another kinase), suppressor of fused (SUFU), and the kinesin-like molecule Costal-2 (COS2), which facilitates interaction of the complex with microtubules (Zhang et al., 2005). Cholesterol biosynthesis may also be important for regulation of signaling downstream of SMO. Whereas cells overexpressing SMO alone displayed low but significant levels of pathway activity, as assessed by a responsive reporter assay, treatment of these cells with cyclodextrin (an oligosaccharide that complexes with sterols) inhibited this response in a dosedependent manner. In contrast, the HH response was unaffected by cyclodextrin treatment in cells transfected with a constitutively active oncogenic form of SMO (SMOA1; see Section Mutations in HH Pathway Components in Congenital Disease and Cancer). These results suggest that proper cholesterol biosynthesis is required for normal SMO activity as well as morphogen autoprocessing (Cooper et al., 2003). These results are also relevant for human disease, because mutations in 7-dehydrocholesterol reductase (DHCR7), which encodes the ultimate enzyme in the production of cholesterol, cause Smith–Lemli–Opitz syndrome (SLOS). SLOS was the first inborn error of metabolism found to manifest itself as a malformation syndrome, with the range of malformations resembling those of patients with mutations in other HH pathway components (Kelley and Henekam, 2000; see Section Mutations in HH Pathway Components in Congenital Disease and Cancer). Several novel receptors for HH morphogens, HIP, BOC, CDO, and GAS1, have also been identified more recently. These molecules were identified through combined efforts that
screened cDNA libraries for genes whose protein products facilitate binding of SHH morphogen to the cell surface (Chuang and McMahon, 1999), and that used microarrays to identify genes that are up- or down-regulated in mice with mutations in HH pathway components, consistent with their being targets of HH signaling (Tenzen et al., 2006) (see also the following section). These receptors bind HH morphogens, inhibiting their diffusion and possibly internalizing and degrading them, thus regulating the steepness of the morphogen gradient. In the case of BOC, CDO, and GAS1, they also promote the response of target cells to HH morphogen (Tenzen et al., 2006; Martinelli and Fan, 2007; Allen et al., 2007; Zhang et al., 2006).
Gene Regulatory Networks Controlled by the HH Pathway – The Glioma-Associated Transcription Factors The ultimate output of HH signaling is the regulation of gene expression, thereby translating concentration-dependent signals into geometric patterns of transcription within a tissue. In the fruit fly, the main transcription factor that mediates the HH response is known as cubitus interruptus (CI), whereas there are three orthologous proteins in vertebrates, known as glioma-associated oncogenes, GLI1-3. In the absence of a morphogen, GLI2 and GLI3 undergo proteolytic cleavage to remove the C-terminus that contains a transcriptional activator domain. The resultant truncated 89 kDa protein still contains its zinc-finger domain, and thus retains its ability to bind DNA. In this context GLI2/GLI3 act as transcriptional repressors owing to N-terminal transcriptional repressor domains. Binding of HH to its receptor and derepression of SMO leads to activation of a series of cellular processes that prevent the cleavage of GLI2/GLI3. In their full-length 190 kDa forms, GLI2/GLI3 possess C-terminal transcriptional activator domains in combination with their zinc-finger domains and thereby actively transcribe target genes. Both the mouse and human GLI2/GLI3 proteins possess an N-terminal repressor and C-terminal activator domain (Roessler et al., 2005) and so are responsible for mediating the HH response. In contrast, the GLI1 protein does not possess an N-terminal transcriptional
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Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease
Neural tube: neural precursors
Spinal cord: differentiated neurons
p0
p0 p1 p2 pMN p3
Dbx1
p1
Dbx2
p2
Nkx6.1
Nkx6.2 V0 V1
Irx1
p2 pMN
Pax6
p3
Nkx2.2
SHH
Olig2
V2 MN V3
Figure 2 Neural tube patterning by long-range SHH morphogen. Left – Scheme of the neural tube. SHH is expressed by the floor plate (red) which is secreted, generating and ventral-to-dorsal gradient. This leads to the patterning of the neural tube into distinct neural precursor domains, p3–p0. Middle – Scheme showing that this SHH gradient establishes different domains expressing specific combinations of transcription factors. Some examples are given. These transcription factors have many mutually repressive activities, thereby converting the continuous SHH gradient into distinct boundaries of gene expression and neural identity. Right – Scheme of the spinal cord showing how these precursors give rise to specific neurons following this early patterning by SHH.
repressor domain and thus is not responsive to HH signaling. While GLI2/GLI3 mediate the response to HH in most developmental contexts, the GLI1 gene is dispensable for development although it does compensate for a loss of transcriptional activation in mice lacking GLI2/GLI3 (Bai and Joyner, 2001; Bai et al., 2002; Ding et al., 1998; Motoyama et al., 1998; Park et al., 2000). As such, GLI1-mediated transcriptional activation may simply serve to amplify the response to positive HH signaling mediated by GLI2/GLI3. The cellular localization of CI/GLI proteins is also regulated according to the concentration of morphogen through modification of nuclear localization and nuclear export signals within these proteins. At low concentrations of morphogen, a scaffold of proteins will tether any uncleaved, potentially transcriptionally active GLI protein to the cytoskeleton, preventing its entry into the nucleus. One component of this scaffold is Costal-2, and two vertebrate homologs, Kif7 and Kif27, have been identified as negative regulators of HH signaling in vertebrates. Regulation of HH signaling by these proteins in vertebrates also relates to cilia, which involves tethering of cargo to, and its transport along, the ciliary axoneme (see Section Mutations in HH Pathway Components in Congenital Disease and Cancer). Taken together, the data suggest a ‘three-response’ model for HH signaling (Hooper and Scott, 2005). In the absence of HH morphogen, GLI-repressor is formed through phosphorylation and proteolytic cleavage, thereby inhibiting the expression of HH target genes. In the presence of low concentrations of morphogen, the processing of GLI-repressor is blocked, and some target genes are derepressed, however, a fully functional GLI-activator is either not synthesized, or it is sequestered in the cell cytoplasm. Finally, if the levels of morphogen are sufficiently high, GLI-activator protein isoforms will additionally be released from the protein complex and will enter the nucleus to activate the expression of target genes. Thus a cell can initiate graded responses depending on the concentration of morphogen sensed by the cell. Work on patterning of the embryonic neural tube has provided an exquisite example of the way in which a gradient
of GLI-transcription factor activity can feed into a gene regulatory network that determines cellular identity. The neural tube is the anlagen of the vertebrate central nervous system. Differentiation of cell types along the dorsoventral (D–V) axis of the neural tube becomes evident as distinct classes of neurons appear at different D–V positions (Figure 2). Initial work implicating HH proteins as morphogens showed that the notochord and floor plate specifically expressed SHH and served as heterologous inducers of D–V neuronal identity. The finding that SHH could substitute for these structures to induce D– V cell fates, showed that SHH acts as a long-range morphogen within the developing neural tube (Yamada et al., 1993; Roelink et al., 1994). Subsequent work showed that the GLI-transcription factors elicit the response to SHH in this tissue. Gli2/ mice do not correctly specify the ventral-most cells in the neural tube (Ding et al., 1998; Park et al., 2000), while this structure is dorsalized in Gli3/ mutants (Persson et al., 2002; Bai et al., 2002; Mo et al., 1997; Park et al., 2000). These findings are consistent with the notion that GLI2 acts primarily as a transcriptional-activator, while GLI3 acts primarily as a repressor. In the neural tube, this is likely to be the result of differential spatial expression of Gli2 and Gli3 in the dorsal– ventral axis rather than intrinsically different biochemical properties. Different neuronal subtypes within the neural tube are derived from different precursor populations that express a number of homeodomain and basic helix–loop–helix transcription factors in response to SHH (Figure 2). Many of these factors act as transcriptional repressors, and a number of mutually cross-repressive interactions have been identified (Dessaud, 2008, for review). As such, following the initial expression of these factors at defined locations along the neural tube, a gene regulatory network is established. This network serves to reinforce the boundaries between different domains of transcription factor expression. As such, a continuous gradient of SHH morphogen is translated into a segmented pattern of neuronal fates via the GLI-transcription factors.
Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease
Newer Aspects of HH Signal Transduction The Role of Primary Cilia in Regulating Mammalian HH Signaling Analysis of mouse mutants affecting neural tube patterning has shown that primary cilia play roles in regulating HH signal transduction. Cilia are membrane-encased, microtubular extensions present on the apical surface of most cells. They consist of a ciliary axoneme, composed of nine outer microtubule doublets (Jenkins and Beales, 2013). Some cilia also have a central pair of microtubules, and are therefore known as ‘9 þ 2’ cilia, whereas others do not have a central pair and are referred to as ‘9 þ 0’ cilia. The ciliary axoneme connects at its base to a cytoplasmically located structure known as the basal body that consists of a mother and a daughter centriole. Recent evidence has now shown that there is regulated movement of both proteins, such as transmembrane receptors, and lipids into the cilium which means that the ciliary membrane composition is separated from the cell surface membrane (Rohatgi and Snell, 2010; Hu et al., 2010; Kim et al., 2010). Recent evidence has also identified vesicle trafficking proteins with highly specific roles in trafficking various components to cilia. Several HH pathway components also localize to cilia in a manner that correlates with HH pathway activity. Ciliary motor proteins are separated into complexes B or A, involved in anterograde and retrograde transport, respectively. GLI3 processing is abnormal in mice with mutations disrupting these motor proteins, with an imbalance in the levels of activator and repressor forms. Furthermore, disruption of different ciliary components can affect GLI3 processing differently. It is therefore clear that different classes of ciliary trafficking proteins regulate distinct aspects of GLI processing. The mechanisms that control this are only just beginning to be understood. Although HH signaling is highly conserved, both the mechanisms by which Ptch1 represses Smo, and those leading to the activation of the Gli-transcription factors have remained unclear, and recent evidence suggests that this may involve primary cilia. Rohatgi et al. (2007) have shown that Ptch1 and Smo exhibit mutually exclusive patterns of ciliary localization – in unstimulated fibroblasts Ptch1 is present within cilia but Smo is not, whereas Smo becomes localized to cilia following application of Shh protein to cells, and Ptch1 concomitantly becomes excluded from cilia. Shh was shown to bind directly to Ptch1 at the ciliary membrane, and Smo was constitutively localized to cilia in Ptch1/ cells. Further work has suggested that Smo is trafficked directly from the plasma membrane to the ciliary membrane via a process of lateral transport, independent of dynamin-mediated endocytosis (Milenkovic et al., 2009). β-Arrestins are also essential for trafficking of Smo into cilia following Shh treatment (Kovacs et al., 2008). β-Arrestins are key regulators of receptor internalization in clathrin-coated vesicles, and this work has suggested a model whereby Smo is internalized from the plasma membrane and recycled into the cilium through interaction with the type-II kinesin motor, Kif3a. Smo is consitutively localized to cilia in cells deficient for the dynein-heavy chain protein, Dyn2ch1 (orthologous to
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the gene mutated in asphyxiating thoracic dystrophy and short rib polydactyly) (Dagoneau et al., 2009; Merrill et al., 2009), although these cells fail to elicit a HH response (Ocbina and Anderson, 2008; Ocbina et al., 2011). Therefore, Smo translocation into cilia is necessary, but not sufficient, for HH signaling. Instead it seems that the transport of Smo, and probably also GLI proteins, through the cilium is essential to regulate HH signaling appropriately.
Noncanonical HH Signaling Early work into HH signaling was based largely on the analysis of gene expression in response to HH signaling in body segments and wing imaginal discs in transgenic flies. It is perhaps not surprising, therefore, that the large body of work that has followed has been strongly biased toward an analysis of the transcriptional outputs of signal transduction. In recent years, biochemical analyses of the PTCH1 protein and studies of cell migration/axon guidance are collectively revealing new, transcription-independent modes of response to HH morphogen. Based on this evidence, Jenkins (2009) defined three modes of ‘noncanonical’ HH signaling: Type 1 – Signaling that involves HH pathway components but which is independent of GLI-mediated transcription. An example where transcription-independent signaling has been formally demonstrated in a fibroblast cell line was described by Bijlsma et al. (2007). Soluble SHH protein was shown to act as a chemoattractant when applied to cultured fibroblasts, in a manner that was dependent on SMO. However, both chemoattraction and cytoskeletal changes were observed after SHH treatment for only 10 min, a matter of hours before GLImediated transcriptional responses to morphogen could first be observed in these cells. More recently, similar experiments in fibroblasts and endothelial cells have indicated that this transcription-independent regulation of cell migration is mediated by direct activation of Rac1 by SMO, in turn leading to modulation of the cytoskeleton (Polizio et al., 2011a,b). Further support for this mode of signaling initially came from studies of chick neural progenitor cells cultured on Fibronectin or Laminin-1 coated plates. These cells are initially adherent, and subsequently dissociate to become migratory. When cultured on plates onto which SHH had been adsorbed, these cells largely failed to adhere. In contrast, treatment of these explants with soluble SHH protein had no affect on adherence or migration (Jarov et al., 2003; Testaz et al., 2001). This suggests that SHH directly interacts with the extracellular matrix of neuroepithelial cells to stabilize them within an epithelium. Type 2 – Direct interaction of HH signaling components with other molecular pathways, as opposed to the usual indirect regulation of cellular processes by GLI-mediated transcription. One example of this mode of signaling comes from work showing that SHH and PTCH1 participate in a novel G2/M phase checkpoint independent of other downstream pathway components. Cyclin B1 is a critical regulator of mitotic cell division, entering the cell nucleus during late G2 phase. Through a direct protein–protein interaction involving its large intracellular loop, PTCH1 has been shown to sequester the active, phosphorylated form of Cyclin B1 at the cell
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Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease
surface, thereby inhibiting cell cycle progression (Barnes et al., 2001). Binding of SHH to PTCH1 leads to release of Cyclin B1, thereby allowing it to enter the cell nucleus. As such, these HH pathway components can regulate cell cycle progression in certain situations, independently of SMO or downstream pathway components. PTCH1 has also been shown to act as a so-called ‘dependence-receptor,’ inducing caspase-dependent cell death when transfected into cell lines in which it is not normally expressed (Thibert et al., 2003). This effect could be inhibited by treatment with SHH morphogen in a dose-dependent manner, thereby generating a state of cellular dependency on SHH ligand for survival. PTCH1 has now been shown to function as a dependence receptor through its intracellular C-terminal tail, by forming a multi-protein complex consisting of the adaptor protein, DRAL, and Caspase-9 (Mille et al., 2009). Formation of this complex in the absence of SHH morphogen leads to the ubiquitination and activation of Caspase-9, in turn triggering apoptosis. Once again, this pathway is thought to be independent of SMO or the transcriptional response to morphogen. Since the initial definition of noncanonical HH signaling, many more examples of noncanonical HH signaling have been reported and their roles in vivo have been studied further. However, an in-depth evaluation of each of these pathways is beyond the scope of this article.
Long-Range Transcriptional Regulation of the Mammalian SHH Gene – The ‘Zone of Polarizing Activity Regulatory Sequence’ As shown in Figure 3, the limb bud is patterned by a gradient of SHH morphogen which is normally expressed in a very specific location posteriorly, known as the zone of polarizing activity (ZPA). This gradient controls the number and identity of the digits. The mechanisms that regulate this expression
Distal
were illuminated by the identification of noncoding mutations that cause triphalangeal thumb in humans and polydactyly in mice and cats (Hill and Lettice, 2013, for review). These mutations lie within a highly conserved 780 bp cis-regulator, known as the ZPA regulatory sequence (ZRS), which is located within an intron of the ubiquitously expressed Lmbr1 gene. This element lies approximately 1 Mb away from the SHH promoter, making it one of the longest-range gene regulatory elements reported to date. Using mouse transgenic reporter assays, Lettice et al. (2014) showed that the ZRS was able to drive the expression of a LacZ reporter gene within the ZPA. Interestingly, by testing the ability of truncated forms of this element to drive this pattern of expression, it was found that the 5’ half was essential for driving expression, while the 3’ half was dispensable in these transgenic assays. However, by creating mutations within the endogenous locus, it was found that the 3’ element was required for expression of Shh within the ZPA. Therefore, the entire ZRS is required for proper regulation of Shh expression. Using 3D FISH, these authors and others have shown that the 3’ sequence of the ZRS is required for the ZRS to localize with the SHH locus, which loops out of its chromosomal territory specifically within ZPA cells (Lettice et al., 2014; Amano et al., 2009). Over 20 point mutations have been identified in the ZRS in patients with triphalangeal thumb. When these mutations have been tested in the same transgenic reporter assays, several have been shown to cause ectopic expression of LacZ within the anterior limb bud in addition to the ZPA, which would explain why the thumb takes on the identity of a finger. This result suggests that there are repressor elements within the ZRS that somehow inhibit expression in the anterior limb bud, perhaps through binding of transcriptional repressors. Indeed, one family has been described with a duplication of the ZRS. This has been interpreted as leading to the ‘dilution’ of bound repressive factors, i.e., these repressors bind to both the wildtype and duplicated ZRS, thereby halving binding at the ZRS
Proximal
Apical ectodermal ridge (AER) – expressing Fgf4/8
Anterior Fgf4/8
Bmp4 Posterior
Region of ectopic Shh expression in individuals with a ZRS mutation, causing triphalangeal thumb/polydactyly
Gremlin
Limb bud mesenchyme – expressing gremlin, Bmp4
Shh
Zone of polarising activity (ZPA) – expressing Shh
Figure 3 HH signaling in limb developing, and the effect of mutations in a long-range gene regulatory element. Scheme showing the early limb bud (B4 weeks in human fetuses), with proximo–distal (what will become shoulder-to-finger tip) and antero–posterior (little finger-to-thumb) axis indicated. SHH is secreted from the zone of polarizing activity (ZPA) where it signals to the limb bud mesenchyme and sets-up a posterior-toanterior gradient of positional information. Antagonism between SHH and BMP signaling (by regulating expression of the BMP antagonist Gremlin) modulates expression of Fgf ligand in the apical ectodermal ridge (AER) which in turn signals back to the ZPA and mesenchyme. Mutations in the long-range ZRS leads to ectopic expression in the anterior domain of the limb bud in addition to the ZPA, which disrupts these signaling events and altered digit number and identity.
Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease that interacts with the Shh promoter and allowing some ectopic expression. Initial support for the transcription factor binding hypothesis comes from analysis of a point mutation in a polydactylous mouse strain. This point mutant form of the ZRS is bound by HnRNP U, whereas the wild-type version is not. HnRNP U also binds the 5’ UTR of the Shh gene, and this dual interaction leads to ectopic association between the ZRS and the Shh promoter (Zhao et al., 2009). Taken together, the current evidence suggests that the ZRS acts as a likely platform for the binding of many transcriptional repressors and activators that interact with the Shh promoter through long-range chromatin looping.
HH Signaling in Human Disease Mutations in HH Pathway Components in Congenital Disease and Cancer Most of the molecular components that regulate the HH pathway, which have been discussed in previous sections, are constitutionally mutated in human birth defects, and a number of these components are also somatically mutated in human cancers. The HH pathway is unique, being the only signal transduction pathway for which all of its core components are mutated in human disease, as summarized in Tables 1 and 2. These tables list the phenotypes associated with each of these mutations and a summary of the clinical features of each disease. As discussed above, core HH signaling components have been shown to localize to primary cilia, and their transport through cilia is essential for the proper regulation of HH signaling. Diseases associated with mutations affecting cilia are known as ‘ciliopathies.’ More than 100 genes have currently been found to be mutated in ciliopathies, making this perhaps the largest category of disease in terms of genetic etiology, and this number is increasing rapidly with the advent of next-generation sequencing technologies. Consistent with the central role of cilia in HH signaling, patients with ciliopathies also exhibit defects that are characteristic of abnormal HH signaling, as discussed below, in particular limb (polydactyly and brachydactyly type-A1) and heart malformations. This category of disease will not be discussed
Table 1
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further as it has recently been reviewed in detail (Jenkins and Beales, 2013). As can be seen in Tables 1 and 2, different birth defects are readily attributable to the distinct roles of SHH, IHH, or DHH as the active morphogen in different tissues. For example, patients with heterozygous mutations in the SHH gene exhibit craniofacial defects including cleft lip/palate and a spectrum of midline defects ranging from solitary median central incisor to cyclopia, which reflects a failure of signaling from the notochord (as discussed in Section Gene Regulatory Networks Controlled by the HH Pathway – The Glioma-Associated Transcription Factors) to instruct the midline structures (including the eye field) to expand during fetal development. Patients also exhibit neurological defects, such as agenesis of the corpus callosum, which reflects the important role of SHH during development of the brain and nervous system. IHH is known to play prominent roles during endochondral ossification of the skeleton, as shown by studies in mice, and patients with IHH mutations exhibit defects that reflect this function. Whereas patients with heterozygous mutations in IHH exhibit a reduction in the middle phalanges of their hands (known as brachydactyly type-A1, BDA1), patients with homozygous mutations have BDA1 together with a specific form of dwarfism, known as acrocapitofemoral dysplasia (ACFD), which stems from abnormal development of the appendicular skeleton. Finally, DHH is known to have much more restricted functions during embryonic development, with a restricted pattern of expression that is limited to the testis. Consistent with this, patients with DHH mutations only display gonadal dysgenesis. Given that SHH, IHH, and DHH ligands all signal through the PTCH1 receptor, it comes as no surprise that patients with mutations in PTCH1, who have basal cell nevus syndrome (BCNS), exhibit a combination of defects that are attributable to the roles of both SHH and IHH ligands during development. These patients have skeletal defects, affecting the ribs, vertebrae and phalanges of the hands, which likely stem from defective IHH signaling. They also have polydactyly, which reflects abnormal patterning of the early limb bud by SHH (Figure 3), and abnormalities of the brain. Interestingly, whereas patients with SHH mutations, which render HH signaling in craniofacial structures less active, have a reduction in
Summary of human mutations within the hedgehog pathway
Syndrome
Mutated gene
Type of mutation
Mode of inheritance
OMIMa accession
HPE3/SMMCI BDA1 ACFD Gondal dysgenesis BCNS Carpenter Pallister–Hall GCPS/ACS/PAP P/HPE-L P/HPE-L
SHH IHH IHH DHH PTCH1 RAB23 GLI3 GLI3 GLI2 GLI2
LOF LOF LOF ND LOF LOF DN LOF DN LOF
AD AD AR AR AD AR AD AD AD AD
#142945 #112500 #607778 #607080 #109400 #201000 #146510 #175700, #200910 #610829 #610829
a
Online Mendelian Inheritance in Man − http://www.ncbi.nlm.nih.gov/omim Abbreviations: ACFD, acrocapitofemoral dysplasia; ACS, acrocallosal syndrome; BDA1, brachydactyly type-A1; GCPS, Greig cephalopolysyndactyly; HPE3, holoprosencephaly-3; PAP, pre-axial polydactyly; P/HPE-L, pituitary involvement/holoprosencephaly-like; SMMCI, solitary median maxillary central incisor.
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Summary of birth defects commonly associated with hedgehog pathway mutations
Syndrome
Phenotypes Anogenital
Heart
Craniofacial
Limb
Skeletal
Neurologic
Other
HPE3/SMMCI
Hypoplastic genitalia
Patent ductus arteriosus
–
Scoliosis
–
–
Short/absent middle phalanges
–
ACFD
–
–
Macrocephaly
–
–
Gonadal dysgenesis BCNS
– –
– –
– –
– –
Carpenter
Cryptorchidism
Complex
– Macrocephaly, hypertelorism Craniosynostosis; abnormal dentition
Short/absent middle phalanges, short/long bones, and coneshaped epiphyses – Polydactyly
Short stature; musculoskeletal defects Short stature; dysplastic pelvis, vertebrae and hips
Dysplastic pituitary, corpus callosum and cerebellum; holoprosencephaly –
–
BDA1
Hypertelorism, cyclopia, solitary central incisor, and cleft palate –
High birthweight
Pallister–Hall
–
–
Abnormal dentition
Hypoplastic corpus callosum, occasional cranial neural tube defect –
GCPS/ACS/PAP
–
–
P/HPE-L
–
–
Macrocephaly, hypertelorism –
Polydactyly; cutaneous syndactyly; club foot
Polydactyly; cutaneous syndactyly Polydactyly; cutaneous syndactyly Polydactyly
– Bifid ribs; scoliosis; dysplastic vertebrae –
– – –
Hypoplastic corpus callosum –
– – –
Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease
Table 2
Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease
midline structures (e.g., cyclopia), patients with mutations in PTCH1, which render HH signaling more active, have expanded midline structures (e.g., hypertelorism). Patients with mutations in GLI2 or GLI3 also have a similar combination of defects. The HH pathway has also been implicated in driving several human cancers. As well as exhibiting a variety of birth defects, as described above, patients with BCNS who are heterozygous for inactivating mutations in PTCH1 also have an increased risk for basal cell carcinomas (BCCs) and medulloblastomas (Hahn et al., 1996). Given the role of PTCH1 as a negative-regulator of the HH pathway, this finding links activated HH signaling to formation of these specific tumors which typically affect the adult skin and the brain of children, respectively. Consistent with this function of PTCH1 as a classical tumor suppressor acting early in tumor formation, loss-of-heterozygosity at the PTCH1 locus was reported in both familial and sporadic tumors biopsies (Gailani et al., 1992). Interestingly, while germ-line SMOH mutations have not been reported in human birth defects (presumably because they would produce nonviable fetuses), specific somatic activating mutations in SMOH are found in sporadic BCCs (Lam et al., 1999). Mutations in the negative-regulator SUFU have also been identified in these tumors type (Taylor et al., 2002). Interestingly, genetic ablation of cilia in a mouse model of basal carcinoma which carries an activating mutation in SMOH, prevents tumors formation, thereby demonstrating the essential role of cilia in the activation of HH signaling (Wong et al., 2009). Several drivers of cell proliferation and G1/S phase progression, including C-MYC and CYCND1, are positive transcriptional targets of HH signaling. Therefore, it seems logical that mutations that activate HH signaling have been described in cancer, perhaps leading to enhanced cell survival and proliferation.
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pathway. Indeed, a next-generation orally bioavailable SMO inhibitor, GDC-0449, was used to successfully clear extensive metastatic medulloblastoma nodules in a patient with PTCH1 loss-of-heterozygosity after only 2 months of treatment, although the metastases did relapse after 3 months, presumably owing to the acquisition of drug-resistant mutations (Rudin et al., 2009). Promising results were also found in the treatment of advanced and metastatic BCC (Von Hoff et al., 2009). Interestingly, different SMO antagonists have different effects on SMO accumulation within primary cilia. For example, cyclopamine inhibits the downstream HH response, but does not prevent SMO from entering the cilium (Rohatgi et al., 2009). This is analogous to the result described in Dync2h1 / mice, whereby SMO is constitutively localized to cilia but no HH response is elicited, showing that transport through the cilium necessary for HH signaling (Ocbina and Anderson, 2008; Ocbina et al., 2011). Furthermore, constitutive localization of SMO to cilia in Ptch1/ cells is reversed by two other SMO antagonists, known as SANT1&2, but not by cyclopamine. These pharmacological experiments have therefore led to a model whereby SMO is regulated by trafficking to different locations within primary cilia, within which it adopts different active or inactive conformations. Clearly, the identification of pharmacological modulators of HH pathway activity will be important both for the treatment of patients with mutations in the pathway, and for dissection of the basic molecular mechanisms that regulate HH signal transduction.
See also: Cell Communication: Growth Factor Mediated Cell Signaling: Tumor Necrosis Factor Receptors: A Brief Digestion
References Targeting the HH Pathway with Drugs Given the importance of the HH pathway in normal development, and its deregulation in many forms of human disease, it is therefore of great clinical importance that many small molecules targeting different aspects of the pathway are available for potential treatment of disease. This area of study began over 40 years ago with the identification of cyclopamine. Epidemiologists noticed that clusters of sheep in certain mountainous areas of the United States were producing offspring with cyclopia, similar to patients with SHH mutations. Further investigation revealed that the source of these teratogenic effects was the Californian corn lily, which pregnant females were feeding on. These plants produce several steroidal alkaloids, including jervine and cyclopamine, which are teratogenic. A body of work subsequently showed that this compound acts as a small molecule inhibitor of SMO and thus a negative regulator of the HH pathway (Chen et al., 2002). Subsequent chemical screening also identified other small molecules that target the HH pathway. Two such compounds, SAG and purmorphamine, activated HH signaling by agonizing SMO (Chen et al., 2002; Sinha and Chen, 2006), suggesting that SMO is a very ‘druggable’ molecule. This is of great importance for the potential treatment of a variety of cancers caused by activation of the HH
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Mille, F., Thibert, C., Fombonne, J., et al., 2009. The Patched dependence receptor triggers apoptosis through a DRAL-caspase-9 complex. Nature Cell Biology 11, 739–746. Mo, R., Freer, A.M., Zinyk, D.L., et al., 1997. Specific and redundant functions of Gli2 and Gli3 zinc finger genes in skeletal patterning and development. Development 124, 113–123. Motoyama, J., Liu, J., Mo, R., et al., 1998. Essential function of Gli2 and Gli3 in the formation of lung, trachea and oesophagus. Nature Genetics 20, 54–57. Nusslein-Volhard, C., Wieschaus, E., 1980. Mutations affecting segment number and polarity in Drosophila. Nature 287, 795–801. Ocbina, P.J., Anderson, K.V., 2008. Intraflagellar transport, cilia, and mammalian Hedgehog signaling: Analysis in mouse embryonic fibroblasts. Developmental Dynamics 237, 2030–2038. Ocbina, P.J., Eggenschwiler, J.T., Moskowitz, I., Anderson, K.V., 2011. Complex interactions between genes controlling trafficking in primary cilia. Nature Genetics 43, 547–553. Park, H.L., Bai, C., Platt, K.A., et al., 2000. Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation. Development 127, 1593–1605. Pathi, S., Pagan-Westphal, S., Baker, D.P., et al., 2001.Comparative biological responses to human Sonic, Indian and Desert Hedgehog. Mechanisms of Development 106, 107–117. Persson, M., Stamataki, D., te Welscher, P., et al., 2002. Dorsal−ventral patterning of the spinal cord requires Gli3 transcriptional repressor activity. Genes and Development 16 (22), 2865–2878. Polizio, A.H., Chinchilla, P., Chen, X., et al., 2011a. Heterotrimeric Gi proteins link Hedgehog signaling to activation of Rho small GTPases to promote fibroblast migration. Journal of Biological Chemistry 286 (22), 19589–19596. doi:10.1074/ jbc.M110.197111. Polizio, A.H., Chinchilla, P., Chen, X., Manning, D.R., Riobo, N.A., 2011b. Sonic Hedgehog activates the GTPases Rac1 and RhoA in a Gli-independent manner through coupling of smoothened to Gi proteins. Science Signaling 4 (200), pt7. Available at: http://www.ncbi.nlm.nih.gov/pubmed/22114142 (accessed 17.03.15). Porter, J.A., von Kessler, D.P., Ekker, S.C., et al., 1995.The product of hedgehog autoproteolytic cleavage active in local and long-range signaling. Nature 374, 363–366. Roelink, H., Augsburger, A., Heemskerk, J., et al., 1994. Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord. Cell 76 (4), 761–775. Roessler, E., Ermilov, A.N., Grange, D.K., et al., 2005. A previously unidentified amino-terminal domain regulates transcriptional activity of wild-type and diseaseassociated human GLI2. Human Molecular Genetics 14, 2181–2188. Rohatgi, R., Milenkovic, L., Corcoran, R.B., Scott, M.P., 2009. Hedgehog signal transduction by smoothened: Pharmacologic evidence for a 2-step activation process. Proceedings of the National Academy of Sciences of the United States of America 106, 3196–3201. Rohatgi, R., Milenkovic, L., Scott, M.P., 2007. Patched1 regulates hedgehog signaling at the primary cilium. Science 317, 372–376. Rohatgi, R., Snell, W.J., 2010. The ciliary membrane. Current Opinion in Cell Biology 22, 541–546. Rudin, C.M., Hann, C.L., Laterra, J., et al., 2009. Treatment of medulloblastoma with hedgehog pathway inhibitor GDC-0449. New England Journal of Medicine 361, 1173–1178. Sinha, S., Chen, J.K., 2006. Purmorphamine activates the Hedgehog pathway by targeting Smoothened. Nature Chemical Biology 2, 29–30. Taylor, M.D., Liu, L., Raffel, C., et al., 2002. Mutations in SUFU predispose to medulloblastoma. Nature Genetics 31, 306–310. Tenzen, T., Allen, B.L., Cole, F., et al., 2006. The cell-surface membrane proteins Cdo and Boc are components and targets of the Hedgehog signaling pathway and feedback network in mice. Developmental Cell 10, 647–656. Testaz, S., Jarov, A., Williams, K.P., et al., 2001. Sonic hedgehog restricts adhesion and migration of neural crest cells independently of the Patched-Smoothened-Gli signaling pathway. Proceedings of the National Academy of Sciences of the United States of America 98, 12521–12526. Thibert, C., Teillet, M.A., Lapointe, F., et al., 2003. Inhibition of neuroepithelial patched-induced apoptosis by sonic hedgehog. Science 301, 843–846. Von Hoff, D.D., LoRusso, P.M., Rudin, C.M., et al., 2009. Inhibition of the hedgehog pathway in advanced basal-cell carcinoma. New England Journal of Medicine 361, 1164–1172. Wong, S.Y., Seol, A.D., So, P.L., et al., 2009. Primary cilia can both mediate and suppress Hedgehog pathway-dependent tumorigenesis. Nature Medicine 15, 1055–1061.
Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease
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Introduction Key components of the hedgehog (HH) signaling pathway were first isolated as mutants affecting larval cuticle patterning in the fruit fly, Drosophila melanogaster (Nusslein-Volhard and Wieschaus, 1980). The HH pathway has served as a paradigm of signal transduction for over three decades, leading in our understanding of many of the fundamental mechanisms involved in intercellular communication. The generally accepted canonical model of HH signaling is largely conserved from fruit flies to humans, although differences in some of the biochemical details are apparent between species. One recent development is the finding that primary cilia are particularly important for regulation of HH signaling in higher vertebrates, and this has spawned a whole new field of investigation. The HH pathway also provides text-book examples of: (1) how a long-range morphogen is produced and secreted; (2) how transmembrane receptors are regulated by a morphogen gradient; and (3) how this can feed into transcriptional networks. Other lessons that have emerged from investigation of the HH pathway include: (4) how long-range regulatory elements determine the expression of a morphogen; and (5) the way in which a whole spectrum of anticancer and teratogenic compounds can influence various aspects of signaling mechanisms. It should also be noted that the HH pathway is a paradigm for human genetic disease. It is the only signaling pathway for which most of its constituent components are mutated in human birth defects, while somatic mutations within the pathway also give rise to specific pediatric and adult cancers. Importantly, a battery of therapeutic compounds targeting HH signaling components has been identified. Finally, very recent evidence also suggests added complexity in the HH pathway, in the form of so-called noncanonical pathways.
The Basic Mechanisms of HH Signaling Production and Secretion of a Morphogen – The HH Proteins By definition, a morphogen is a secreted molecule that acts at long-range. While a single HH ligand exists in fruit flies, in mammals, three homologs of Drosophila HH exist, which are known as Sonic HH (SHH), Indian HH (IHH), and Desert HH (DHH). Each HH morphogen is expressed as a 52 kDa precursor protein that undergoes autoproteolytic cleavage to generate an N-terminal 19 kDa protein that fulfils all patterning activities both at short- and long-range, and a C-terminal 25 kDa species (Lee et al., 1994; Porter et al., 1995). The C-terminal region of the unprocessed precursor serves as a catalytic domain, which cleaves the precursor protein at a highly conserved cysteine residue by an intramolecular mechanism, and this reaction exhibits concentration-independent kinetics (Porter et al., 1995). Processing of HH proteins to generate an active N-terminal morphogen requires
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normal cholesterol metabolism, and chemical inhibition of sterol metabolism following treatment of cells with inhibitors of cholesterol biosynthesis impedes HH precursor processing into the two smaller fragments (Guy, 2000). It is now known that HH morphogens are modified by the addition of cholesterol following autoproteolysis of the precursor protein; the newly made N-terminus of the 19 kDa morphogen becomes covalently modified by the addition of cholesterol while its C-terminus becomes palmitoylated at the same time (Lee et al., 1994; Porter et al., 1995). SHH and IHH have been elegantly shown to travel at a distance of many cell diameters from their source in mice using immunohistochemistry on tissues fixed in such a way that preserves proteoglycans/glycosaminoglycans and proteins in an insoluble form (Gritli-Linde et al., 2001). It is thought that addition of cholesterol is important in determining the range over which HH proteins signal. By harboring a cholesterol moiety, HH morphogens have the tendency to remain at the surface of cells that have synthesized them, owing to hydrophobic interaction between the cholesterol group and cell surface lipids. In some tissues, however, the HH-producing cell also expresses Dispatched (DISP), a sterol-sensing domain protein dedicated to the release of cholesterol modified HH ligand (Burke et al., 1999). Thus, HH signaling is long-range when expressed with DISP but juxtacrine/autocrine when DISP is absent. It is thought that DISP serves to mask the cholesterol motif of HH, reducing its affinity for the cell membrane and allowing its diffusion over many cell diameters, perhaps also promoting HH oligomerization. Given that three mammalian HH morphogens exist, each of which function in an overlapping yet distinct range of tissues, the choice of morphogen therefore represents another mechanism by which HH signaling is modulated. While SHH, IHH, and DHH bind to their receptor with equal affinity, in certain assays the concentration of each morphogen required to elicit a response can be very different (up to 60-fold); SHH has the greatest potency in most scenarios, with IHH and then DHH inducing progressively reduced effects. These relationships may also be tissue-dependent, however, because in some contexts, such as the inhibition of chondrocyte differentiation, DHH can be more potent than IHH (Pathi et al., 2001). Taken together, this combined evidence shows how processing to generate an active morphogen is also involved in the tight regulation of HH signaling.
Intracellular Effectors of Signal Transduction – Transmembrane Receptors Cells that are potentially responsive to HH morphogens express the 12-transmembrane domain receptor, Patched (PTCH), which normally serves to repress a second transmembrane protein, Smoothened (SMO) (Figure 1). Therefore, in the absence of HH morphogen, HH signaling is silenced. In contrast, binding of secreted HH to PTCH relieves its normal
Encyclopedia of Cell Biology, Volume 3
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Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease
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Figure 1 HH signal transduction. Left – Scheme showing the logic of HH signaling. Black lines indicate activity in the absence of SHH, IHH, or DHH ligand. Red lines indicate actions following binding of ligand to the PTCH1 receptor. Middle – Scheme showing a primary cilium with central microtubular doublets anchored to the basal body and encased by membrane. In the absence of HH ligand, the PTCH1 receptor is transported into the cilium, leading to the export of SMO and transport away from the cilium in endocytic vesicles. Right – In the presence of ligand, PTCH1 no longer enters the cilium, allowing SMO to accumulate in this organelle. The box indicates two key molecules, Kinesin-II and Dync2h1 (a key component of dynein-2), which regulate anterograde and retrograde intraflagellar transport, respectively.
repression of SMO by inducing a conformational change (Ingham et al., 2000; Zhao et al., 2007). Extensive genetic and biochemical analyses have demonstrated the importance of protein kinases in driving changes in the subcellular localization of SMO (Jia et al., 2004; Zhang et al., 2005; Liu et al., 2007; Zhu et al., 2003; Denef et al., 2000). Inhibition of PTCH repressor function by HH leads to the cell surface accumulation of SMO, a process which requires phosphorylation at a number of protein kinase A (PKA) and casein kinase I (CKI) sites in the C-terminal tail of SMO (Jia et al., 2004). This intracellular domain of SMO interacts with a protein complex that includes PKA, CKI, fused (FU; another kinase), suppressor of fused (SUFU), and the kinesin-like molecule Costal-2 (COS2), which facilitates interaction of the complex with microtubules (Zhang et al., 2005). Cholesterol biosynthesis may also be important for regulation of signaling downstream of SMO. Whereas cells overexpressing SMO alone displayed low but significant levels of pathway activity, as assessed by a responsive reporter assay, treatment of these cells with cyclodextrin (an oligosaccharide that complexes with sterols) inhibited this response in a dosedependent manner. In contrast, the HH response was unaffected by cyclodextrin treatment in cells transfected with a constitutively active oncogenic form of SMO (SMOA1; see Section Mutations in HH Pathway Components in Congenital Disease and Cancer). These results suggest that proper cholesterol biosynthesis is required for normal SMO activity as well as morphogen autoprocessing (Cooper et al., 2003). These results are also relevant for human disease, because mutations in 7-dehydrocholesterol reductase (DHCR7), which encodes the ultimate enzyme in the production of cholesterol, cause Smith–Lemli–Opitz syndrome (SLOS). SLOS was the first inborn error of metabolism found to manifest itself as a malformation syndrome, with the range of malformations resembling those of patients with mutations in other HH pathway components (Kelley and Henekam, 2000; see Section Mutations in HH Pathway Components in Congenital Disease and Cancer). Several novel receptors for HH morphogens, HIP, BOC, CDO, and GAS1, have also been identified more recently. These molecules were identified through combined efforts that
screened cDNA libraries for genes whose protein products facilitate binding of SHH morphogen to the cell surface (Chuang and McMahon, 1999), and that used microarrays to identify genes that are up- or down-regulated in mice with mutations in HH pathway components, consistent with their being targets of HH signaling (Tenzen et al., 2006) (see also the following section). These receptors bind HH morphogens, inhibiting their diffusion and possibly internalizing and degrading them, thus regulating the steepness of the morphogen gradient. In the case of BOC, CDO, and GAS1, they also promote the response of target cells to HH morphogen (Tenzen et al., 2006; Martinelli and Fan, 2007; Allen et al., 2007; Zhang et al., 2006).
Gene Regulatory Networks Controlled by the HH Pathway – The Glioma-Associated Transcription Factors The ultimate output of HH signaling is the regulation of gene expression, thereby translating concentration-dependent signals into geometric patterns of transcription within a tissue. In the fruit fly, the main transcription factor that mediates the HH response is known as cubitus interruptus (CI), whereas there are three orthologous proteins in vertebrates, known as glioma-associated oncogenes, GLI1-3. In the absence of a morphogen, GLI2 and GLI3 undergo proteolytic cleavage to remove the C-terminus that contains a transcriptional activator domain. The resultant truncated 89 kDa protein still contains its zinc-finger domain, and thus retains its ability to bind DNA. In this context GLI2/GLI3 act as transcriptional repressors owing to N-terminal transcriptional repressor domains. Binding of HH to its receptor and derepression of SMO leads to activation of a series of cellular processes that prevent the cleavage of GLI2/GLI3. In their full-length 190 kDa forms, GLI2/GLI3 possess C-terminal transcriptional activator domains in combination with their zinc-finger domains and thereby actively transcribe target genes. Both the mouse and human GLI2/GLI3 proteins possess an N-terminal repressor and C-terminal activator domain (Roessler et al., 2005) and so are responsible for mediating the HH response. In contrast, the GLI1 protein does not possess an N-terminal transcriptional
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Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease
Neural tube: neural precursors
Spinal cord: differentiated neurons
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Figure 2 Neural tube patterning by long-range SHH morphogen. Left – Scheme of the neural tube. SHH is expressed by the floor plate (red) which is secreted, generating and ventral-to-dorsal gradient. This leads to the patterning of the neural tube into distinct neural precursor domains, p3–p0. Middle – Scheme showing that this SHH gradient establishes different domains expressing specific combinations of transcription factors. Some examples are given. These transcription factors have many mutually repressive activities, thereby converting the continuous SHH gradient into distinct boundaries of gene expression and neural identity. Right – Scheme of the spinal cord showing how these precursors give rise to specific neurons following this early patterning by SHH.
repressor domain and thus is not responsive to HH signaling. While GLI2/GLI3 mediate the response to HH in most developmental contexts, the GLI1 gene is dispensable for development although it does compensate for a loss of transcriptional activation in mice lacking GLI2/GLI3 (Bai and Joyner, 2001; Bai et al., 2002; Ding et al., 1998; Motoyama et al., 1998; Park et al., 2000). As such, GLI1-mediated transcriptional activation may simply serve to amplify the response to positive HH signaling mediated by GLI2/GLI3. The cellular localization of CI/GLI proteins is also regulated according to the concentration of morphogen through modification of nuclear localization and nuclear export signals within these proteins. At low concentrations of morphogen, a scaffold of proteins will tether any uncleaved, potentially transcriptionally active GLI protein to the cytoskeleton, preventing its entry into the nucleus. One component of this scaffold is Costal-2, and two vertebrate homologs, Kif7 and Kif27, have been identified as negative regulators of HH signaling in vertebrates. Regulation of HH signaling by these proteins in vertebrates also relates to cilia, which involves tethering of cargo to, and its transport along, the ciliary axoneme (see Section Mutations in HH Pathway Components in Congenital Disease and Cancer). Taken together, the data suggest a ‘three-response’ model for HH signaling (Hooper and Scott, 2005). In the absence of HH morphogen, GLI-repressor is formed through phosphorylation and proteolytic cleavage, thereby inhibiting the expression of HH target genes. In the presence of low concentrations of morphogen, the processing of GLI-repressor is blocked, and some target genes are derepressed, however, a fully functional GLI-activator is either not synthesized, or it is sequestered in the cell cytoplasm. Finally, if the levels of morphogen are sufficiently high, GLI-activator protein isoforms will additionally be released from the protein complex and will enter the nucleus to activate the expression of target genes. Thus a cell can initiate graded responses depending on the concentration of morphogen sensed by the cell. Work on patterning of the embryonic neural tube has provided an exquisite example of the way in which a gradient
of GLI-transcription factor activity can feed into a gene regulatory network that determines cellular identity. The neural tube is the anlagen of the vertebrate central nervous system. Differentiation of cell types along the dorsoventral (D–V) axis of the neural tube becomes evident as distinct classes of neurons appear at different D–V positions (Figure 2). Initial work implicating HH proteins as morphogens showed that the notochord and floor plate specifically expressed SHH and served as heterologous inducers of D–V neuronal identity. The finding that SHH could substitute for these structures to induce D– V cell fates, showed that SHH acts as a long-range morphogen within the developing neural tube (Yamada et al., 1993; Roelink et al., 1994). Subsequent work showed that the GLI-transcription factors elicit the response to SHH in this tissue. Gli2/ mice do not correctly specify the ventral-most cells in the neural tube (Ding et al., 1998; Park et al., 2000), while this structure is dorsalized in Gli3/ mutants (Persson et al., 2002; Bai et al., 2002; Mo et al., 1997; Park et al., 2000). These findings are consistent with the notion that GLI2 acts primarily as a transcriptional-activator, while GLI3 acts primarily as a repressor. In the neural tube, this is likely to be the result of differential spatial expression of Gli2 and Gli3 in the dorsal– ventral axis rather than intrinsically different biochemical properties. Different neuronal subtypes within the neural tube are derived from different precursor populations that express a number of homeodomain and basic helix–loop–helix transcription factors in response to SHH (Figure 2). Many of these factors act as transcriptional repressors, and a number of mutually cross-repressive interactions have been identified (Dessaud, 2008, for review). As such, following the initial expression of these factors at defined locations along the neural tube, a gene regulatory network is established. This network serves to reinforce the boundaries between different domains of transcription factor expression. As such, a continuous gradient of SHH morphogen is translated into a segmented pattern of neuronal fates via the GLI-transcription factors.
Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease
Newer Aspects of HH Signal Transduction The Role of Primary Cilia in Regulating Mammalian HH Signaling Analysis of mouse mutants affecting neural tube patterning has shown that primary cilia play roles in regulating HH signal transduction. Cilia are membrane-encased, microtubular extensions present on the apical surface of most cells. They consist of a ciliary axoneme, composed of nine outer microtubule doublets (Jenkins and Beales, 2013). Some cilia also have a central pair of microtubules, and are therefore known as ‘9 þ 2’ cilia, whereas others do not have a central pair and are referred to as ‘9 þ 0’ cilia. The ciliary axoneme connects at its base to a cytoplasmically located structure known as the basal body that consists of a mother and a daughter centriole. Recent evidence has now shown that there is regulated movement of both proteins, such as transmembrane receptors, and lipids into the cilium which means that the ciliary membrane composition is separated from the cell surface membrane (Rohatgi and Snell, 2010; Hu et al., 2010; Kim et al., 2010). Recent evidence has also identified vesicle trafficking proteins with highly specific roles in trafficking various components to cilia. Several HH pathway components also localize to cilia in a manner that correlates with HH pathway activity. Ciliary motor proteins are separated into complexes B or A, involved in anterograde and retrograde transport, respectively. GLI3 processing is abnormal in mice with mutations disrupting these motor proteins, with an imbalance in the levels of activator and repressor forms. Furthermore, disruption of different ciliary components can affect GLI3 processing differently. It is therefore clear that different classes of ciliary trafficking proteins regulate distinct aspects of GLI processing. The mechanisms that control this are only just beginning to be understood. Although HH signaling is highly conserved, both the mechanisms by which Ptch1 represses Smo, and those leading to the activation of the Gli-transcription factors have remained unclear, and recent evidence suggests that this may involve primary cilia. Rohatgi et al. (2007) have shown that Ptch1 and Smo exhibit mutually exclusive patterns of ciliary localization – in unstimulated fibroblasts Ptch1 is present within cilia but Smo is not, whereas Smo becomes localized to cilia following application of Shh protein to cells, and Ptch1 concomitantly becomes excluded from cilia. Shh was shown to bind directly to Ptch1 at the ciliary membrane, and Smo was constitutively localized to cilia in Ptch1/ cells. Further work has suggested that Smo is trafficked directly from the plasma membrane to the ciliary membrane via a process of lateral transport, independent of dynamin-mediated endocytosis (Milenkovic et al., 2009). β-Arrestins are also essential for trafficking of Smo into cilia following Shh treatment (Kovacs et al., 2008). β-Arrestins are key regulators of receptor internalization in clathrin-coated vesicles, and this work has suggested a model whereby Smo is internalized from the plasma membrane and recycled into the cilium through interaction with the type-II kinesin motor, Kif3a. Smo is consitutively localized to cilia in cells deficient for the dynein-heavy chain protein, Dyn2ch1 (orthologous to
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the gene mutated in asphyxiating thoracic dystrophy and short rib polydactyly) (Dagoneau et al., 2009; Merrill et al., 2009), although these cells fail to elicit a HH response (Ocbina and Anderson, 2008; Ocbina et al., 2011). Therefore, Smo translocation into cilia is necessary, but not sufficient, for HH signaling. Instead it seems that the transport of Smo, and probably also GLI proteins, through the cilium is essential to regulate HH signaling appropriately.
Noncanonical HH Signaling Early work into HH signaling was based largely on the analysis of gene expression in response to HH signaling in body segments and wing imaginal discs in transgenic flies. It is perhaps not surprising, therefore, that the large body of work that has followed has been strongly biased toward an analysis of the transcriptional outputs of signal transduction. In recent years, biochemical analyses of the PTCH1 protein and studies of cell migration/axon guidance are collectively revealing new, transcription-independent modes of response to HH morphogen. Based on this evidence, Jenkins (2009) defined three modes of ‘noncanonical’ HH signaling: Type 1 – Signaling that involves HH pathway components but which is independent of GLI-mediated transcription. An example where transcription-independent signaling has been formally demonstrated in a fibroblast cell line was described by Bijlsma et al. (2007). Soluble SHH protein was shown to act as a chemoattractant when applied to cultured fibroblasts, in a manner that was dependent on SMO. However, both chemoattraction and cytoskeletal changes were observed after SHH treatment for only 10 min, a matter of hours before GLImediated transcriptional responses to morphogen could first be observed in these cells. More recently, similar experiments in fibroblasts and endothelial cells have indicated that this transcription-independent regulation of cell migration is mediated by direct activation of Rac1 by SMO, in turn leading to modulation of the cytoskeleton (Polizio et al., 2011a,b). Further support for this mode of signaling initially came from studies of chick neural progenitor cells cultured on Fibronectin or Laminin-1 coated plates. These cells are initially adherent, and subsequently dissociate to become migratory. When cultured on plates onto which SHH had been adsorbed, these cells largely failed to adhere. In contrast, treatment of these explants with soluble SHH protein had no affect on adherence or migration (Jarov et al., 2003; Testaz et al., 2001). This suggests that SHH directly interacts with the extracellular matrix of neuroepithelial cells to stabilize them within an epithelium. Type 2 – Direct interaction of HH signaling components with other molecular pathways, as opposed to the usual indirect regulation of cellular processes by GLI-mediated transcription. One example of this mode of signaling comes from work showing that SHH and PTCH1 participate in a novel G2/M phase checkpoint independent of other downstream pathway components. Cyclin B1 is a critical regulator of mitotic cell division, entering the cell nucleus during late G2 phase. Through a direct protein–protein interaction involving its large intracellular loop, PTCH1 has been shown to sequester the active, phosphorylated form of Cyclin B1 at the cell
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Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease
surface, thereby inhibiting cell cycle progression (Barnes et al., 2001). Binding of SHH to PTCH1 leads to release of Cyclin B1, thereby allowing it to enter the cell nucleus. As such, these HH pathway components can regulate cell cycle progression in certain situations, independently of SMO or downstream pathway components. PTCH1 has also been shown to act as a so-called ‘dependence-receptor,’ inducing caspase-dependent cell death when transfected into cell lines in which it is not normally expressed (Thibert et al., 2003). This effect could be inhibited by treatment with SHH morphogen in a dose-dependent manner, thereby generating a state of cellular dependency on SHH ligand for survival. PTCH1 has now been shown to function as a dependence receptor through its intracellular C-terminal tail, by forming a multi-protein complex consisting of the adaptor protein, DRAL, and Caspase-9 (Mille et al., 2009). Formation of this complex in the absence of SHH morphogen leads to the ubiquitination and activation of Caspase-9, in turn triggering apoptosis. Once again, this pathway is thought to be independent of SMO or the transcriptional response to morphogen. Since the initial definition of noncanonical HH signaling, many more examples of noncanonical HH signaling have been reported and their roles in vivo have been studied further. However, an in-depth evaluation of each of these pathways is beyond the scope of this article.
Long-Range Transcriptional Regulation of the Mammalian SHH Gene – The ‘Zone of Polarizing Activity Regulatory Sequence’ As shown in Figure 3, the limb bud is patterned by a gradient of SHH morphogen which is normally expressed in a very specific location posteriorly, known as the zone of polarizing activity (ZPA). This gradient controls the number and identity of the digits. The mechanisms that regulate this expression
Distal
were illuminated by the identification of noncoding mutations that cause triphalangeal thumb in humans and polydactyly in mice and cats (Hill and Lettice, 2013, for review). These mutations lie within a highly conserved 780 bp cis-regulator, known as the ZPA regulatory sequence (ZRS), which is located within an intron of the ubiquitously expressed Lmbr1 gene. This element lies approximately 1 Mb away from the SHH promoter, making it one of the longest-range gene regulatory elements reported to date. Using mouse transgenic reporter assays, Lettice et al. (2014) showed that the ZRS was able to drive the expression of a LacZ reporter gene within the ZPA. Interestingly, by testing the ability of truncated forms of this element to drive this pattern of expression, it was found that the 5’ half was essential for driving expression, while the 3’ half was dispensable in these transgenic assays. However, by creating mutations within the endogenous locus, it was found that the 3’ element was required for expression of Shh within the ZPA. Therefore, the entire ZRS is required for proper regulation of Shh expression. Using 3D FISH, these authors and others have shown that the 3’ sequence of the ZRS is required for the ZRS to localize with the SHH locus, which loops out of its chromosomal territory specifically within ZPA cells (Lettice et al., 2014; Amano et al., 2009). Over 20 point mutations have been identified in the ZRS in patients with triphalangeal thumb. When these mutations have been tested in the same transgenic reporter assays, several have been shown to cause ectopic expression of LacZ within the anterior limb bud in addition to the ZPA, which would explain why the thumb takes on the identity of a finger. This result suggests that there are repressor elements within the ZRS that somehow inhibit expression in the anterior limb bud, perhaps through binding of transcriptional repressors. Indeed, one family has been described with a duplication of the ZRS. This has been interpreted as leading to the ‘dilution’ of bound repressive factors, i.e., these repressors bind to both the wildtype and duplicated ZRS, thereby halving binding at the ZRS
Proximal
Apical ectodermal ridge (AER) – expressing Fgf4/8
Anterior Fgf4/8
Bmp4 Posterior
Region of ectopic Shh expression in individuals with a ZRS mutation, causing triphalangeal thumb/polydactyly
Gremlin
Limb bud mesenchyme – expressing gremlin, Bmp4
Shh
Zone of polarising activity (ZPA) – expressing Shh
Figure 3 HH signaling in limb developing, and the effect of mutations in a long-range gene regulatory element. Scheme showing the early limb bud (B4 weeks in human fetuses), with proximo–distal (what will become shoulder-to-finger tip) and antero–posterior (little finger-to-thumb) axis indicated. SHH is secreted from the zone of polarizing activity (ZPA) where it signals to the limb bud mesenchyme and sets-up a posterior-toanterior gradient of positional information. Antagonism between SHH and BMP signaling (by regulating expression of the BMP antagonist Gremlin) modulates expression of Fgf ligand in the apical ectodermal ridge (AER) which in turn signals back to the ZPA and mesenchyme. Mutations in the long-range ZRS leads to ectopic expression in the anterior domain of the limb bud in addition to the ZPA, which disrupts these signaling events and altered digit number and identity.
Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease that interacts with the Shh promoter and allowing some ectopic expression. Initial support for the transcription factor binding hypothesis comes from analysis of a point mutation in a polydactylous mouse strain. This point mutant form of the ZRS is bound by HnRNP U, whereas the wild-type version is not. HnRNP U also binds the 5’ UTR of the Shh gene, and this dual interaction leads to ectopic association between the ZRS and the Shh promoter (Zhao et al., 2009). Taken together, the current evidence suggests that the ZRS acts as a likely platform for the binding of many transcriptional repressors and activators that interact with the Shh promoter through long-range chromatin looping.
HH Signaling in Human Disease Mutations in HH Pathway Components in Congenital Disease and Cancer Most of the molecular components that regulate the HH pathway, which have been discussed in previous sections, are constitutionally mutated in human birth defects, and a number of these components are also somatically mutated in human cancers. The HH pathway is unique, being the only signal transduction pathway for which all of its core components are mutated in human disease, as summarized in Tables 1 and 2. These tables list the phenotypes associated with each of these mutations and a summary of the clinical features of each disease. As discussed above, core HH signaling components have been shown to localize to primary cilia, and their transport through cilia is essential for the proper regulation of HH signaling. Diseases associated with mutations affecting cilia are known as ‘ciliopathies.’ More than 100 genes have currently been found to be mutated in ciliopathies, making this perhaps the largest category of disease in terms of genetic etiology, and this number is increasing rapidly with the advent of next-generation sequencing technologies. Consistent with the central role of cilia in HH signaling, patients with ciliopathies also exhibit defects that are characteristic of abnormal HH signaling, as discussed below, in particular limb (polydactyly and brachydactyly type-A1) and heart malformations. This category of disease will not be discussed
Table 1
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further as it has recently been reviewed in detail (Jenkins and Beales, 2013). As can be seen in Tables 1 and 2, different birth defects are readily attributable to the distinct roles of SHH, IHH, or DHH as the active morphogen in different tissues. For example, patients with heterozygous mutations in the SHH gene exhibit craniofacial defects including cleft lip/palate and a spectrum of midline defects ranging from solitary median central incisor to cyclopia, which reflects a failure of signaling from the notochord (as discussed in Section Gene Regulatory Networks Controlled by the HH Pathway – The Glioma-Associated Transcription Factors) to instruct the midline structures (including the eye field) to expand during fetal development. Patients also exhibit neurological defects, such as agenesis of the corpus callosum, which reflects the important role of SHH during development of the brain and nervous system. IHH is known to play prominent roles during endochondral ossification of the skeleton, as shown by studies in mice, and patients with IHH mutations exhibit defects that reflect this function. Whereas patients with heterozygous mutations in IHH exhibit a reduction in the middle phalanges of their hands (known as brachydactyly type-A1, BDA1), patients with homozygous mutations have BDA1 together with a specific form of dwarfism, known as acrocapitofemoral dysplasia (ACFD), which stems from abnormal development of the appendicular skeleton. Finally, DHH is known to have much more restricted functions during embryonic development, with a restricted pattern of expression that is limited to the testis. Consistent with this, patients with DHH mutations only display gonadal dysgenesis. Given that SHH, IHH, and DHH ligands all signal through the PTCH1 receptor, it comes as no surprise that patients with mutations in PTCH1, who have basal cell nevus syndrome (BCNS), exhibit a combination of defects that are attributable to the roles of both SHH and IHH ligands during development. These patients have skeletal defects, affecting the ribs, vertebrae and phalanges of the hands, which likely stem from defective IHH signaling. They also have polydactyly, which reflects abnormal patterning of the early limb bud by SHH (Figure 3), and abnormalities of the brain. Interestingly, whereas patients with SHH mutations, which render HH signaling in craniofacial structures less active, have a reduction in
Summary of human mutations within the hedgehog pathway
Syndrome
Mutated gene
Type of mutation
Mode of inheritance
OMIMa accession
HPE3/SMMCI BDA1 ACFD Gondal dysgenesis BCNS Carpenter Pallister–Hall GCPS/ACS/PAP P/HPE-L P/HPE-L
SHH IHH IHH DHH PTCH1 RAB23 GLI3 GLI3 GLI2 GLI2
LOF LOF LOF ND LOF LOF DN LOF DN LOF
AD AD AR AR AD AR AD AD AD AD
#142945 #112500 #607778 #607080 #109400 #201000 #146510 #175700, #200910 #610829 #610829
a
Online Mendelian Inheritance in Man − http://www.ncbi.nlm.nih.gov/omim Abbreviations: ACFD, acrocapitofemoral dysplasia; ACS, acrocallosal syndrome; BDA1, brachydactyly type-A1; GCPS, Greig cephalopolysyndactyly; HPE3, holoprosencephaly-3; PAP, pre-axial polydactyly; P/HPE-L, pituitary involvement/holoprosencephaly-like; SMMCI, solitary median maxillary central incisor.
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Summary of birth defects commonly associated with hedgehog pathway mutations
Syndrome
Phenotypes Anogenital
Heart
Craniofacial
Limb
Skeletal
Neurologic
Other
HPE3/SMMCI
Hypoplastic genitalia
Patent ductus arteriosus
–
Scoliosis
–
–
Short/absent middle phalanges
–
ACFD
–
–
Macrocephaly
–
–
Gonadal dysgenesis BCNS
– –
– –
– –
– –
Carpenter
Cryptorchidism
Complex
– Macrocephaly, hypertelorism Craniosynostosis; abnormal dentition
Short/absent middle phalanges, short/long bones, and coneshaped epiphyses – Polydactyly
Short stature; musculoskeletal defects Short stature; dysplastic pelvis, vertebrae and hips
Dysplastic pituitary, corpus callosum and cerebellum; holoprosencephaly –
–
BDA1
Hypertelorism, cyclopia, solitary central incisor, and cleft palate –
High birthweight
Pallister–Hall
–
–
Abnormal dentition
Hypoplastic corpus callosum, occasional cranial neural tube defect –
GCPS/ACS/PAP
–
–
P/HPE-L
–
–
Macrocephaly, hypertelorism –
Polydactyly; cutaneous syndactyly; club foot
Polydactyly; cutaneous syndactyly Polydactyly; cutaneous syndactyly Polydactyly
– Bifid ribs; scoliosis; dysplastic vertebrae –
– – –
Hypoplastic corpus callosum –
– – –
Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease
Table 2
Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease
midline structures (e.g., cyclopia), patients with mutations in PTCH1, which render HH signaling more active, have expanded midline structures (e.g., hypertelorism). Patients with mutations in GLI2 or GLI3 also have a similar combination of defects. The HH pathway has also been implicated in driving several human cancers. As well as exhibiting a variety of birth defects, as described above, patients with BCNS who are heterozygous for inactivating mutations in PTCH1 also have an increased risk for basal cell carcinomas (BCCs) and medulloblastomas (Hahn et al., 1996). Given the role of PTCH1 as a negative-regulator of the HH pathway, this finding links activated HH signaling to formation of these specific tumors which typically affect the adult skin and the brain of children, respectively. Consistent with this function of PTCH1 as a classical tumor suppressor acting early in tumor formation, loss-of-heterozygosity at the PTCH1 locus was reported in both familial and sporadic tumors biopsies (Gailani et al., 1992). Interestingly, while germ-line SMOH mutations have not been reported in human birth defects (presumably because they would produce nonviable fetuses), specific somatic activating mutations in SMOH are found in sporadic BCCs (Lam et al., 1999). Mutations in the negative-regulator SUFU have also been identified in these tumors type (Taylor et al., 2002). Interestingly, genetic ablation of cilia in a mouse model of basal carcinoma which carries an activating mutation in SMOH, prevents tumors formation, thereby demonstrating the essential role of cilia in the activation of HH signaling (Wong et al., 2009). Several drivers of cell proliferation and G1/S phase progression, including C-MYC and CYCND1, are positive transcriptional targets of HH signaling. Therefore, it seems logical that mutations that activate HH signaling have been described in cancer, perhaps leading to enhanced cell survival and proliferation.
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pathway. Indeed, a next-generation orally bioavailable SMO inhibitor, GDC-0449, was used to successfully clear extensive metastatic medulloblastoma nodules in a patient with PTCH1 loss-of-heterozygosity after only 2 months of treatment, although the metastases did relapse after 3 months, presumably owing to the acquisition of drug-resistant mutations (Rudin et al., 2009). Promising results were also found in the treatment of advanced and metastatic BCC (Von Hoff et al., 2009). Interestingly, different SMO antagonists have different effects on SMO accumulation within primary cilia. For example, cyclopamine inhibits the downstream HH response, but does not prevent SMO from entering the cilium (Rohatgi et al., 2009). This is analogous to the result described in Dync2h1 / mice, whereby SMO is constitutively localized to cilia but no HH response is elicited, showing that transport through the cilium necessary for HH signaling (Ocbina and Anderson, 2008; Ocbina et al., 2011). Furthermore, constitutive localization of SMO to cilia in Ptch1/ cells is reversed by two other SMO antagonists, known as SANT1&2, but not by cyclopamine. These pharmacological experiments have therefore led to a model whereby SMO is regulated by trafficking to different locations within primary cilia, within which it adopts different active or inactive conformations. Clearly, the identification of pharmacological modulators of HH pathway activity will be important both for the treatment of patients with mutations in the pathway, and for dissection of the basic molecular mechanisms that regulate HH signal transduction.
See also: Cell Communication: Growth Factor Mediated Cell Signaling: Tumor Necrosis Factor Receptors: A Brief Digestion
References Targeting the HH Pathway with Drugs Given the importance of the HH pathway in normal development, and its deregulation in many forms of human disease, it is therefore of great clinical importance that many small molecules targeting different aspects of the pathway are available for potential treatment of disease. This area of study began over 40 years ago with the identification of cyclopamine. Epidemiologists noticed that clusters of sheep in certain mountainous areas of the United States were producing offspring with cyclopia, similar to patients with SHH mutations. Further investigation revealed that the source of these teratogenic effects was the Californian corn lily, which pregnant females were feeding on. These plants produce several steroidal alkaloids, including jervine and cyclopamine, which are teratogenic. A body of work subsequently showed that this compound acts as a small molecule inhibitor of SMO and thus a negative regulator of the HH pathway (Chen et al., 2002). Subsequent chemical screening also identified other small molecules that target the HH pathway. Two such compounds, SAG and purmorphamine, activated HH signaling by agonizing SMO (Chen et al., 2002; Sinha and Chen, 2006), suggesting that SMO is a very ‘druggable’ molecule. This is of great importance for the potential treatment of a variety of cancers caused by activation of the HH
Allen, B.L., Tenzen, T., McMahon, A.P., 2007. The Hedgehog-binding proteins Gas1 and Cdo cooperate to positively regulate Shh signaling during mouse development. Genes & Development 21, 1244–1257. Amano, T., Sagai, T., Tanabe, H., et al., 2009. Chromosomal dynamics at the Shh locus: Limb bud-specific differential regulation of competence and active transcription. Developmental Cell 16, 47–57. Bai, C.B., Auerbach, W., Lee, J.S., Stephen, D., Joyner, A.L., 2002. Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway. Development 129, 4753–4761. Bai, C.B., Joyner, A.L., 2001. Gli1 can rescue the in vivo function of Gli2. Development 128, 5161–5172. Barnes, E.A., Kong, M., Ollendorff, V., Donoghue, D.J., 2001. Patched1 interacts with cyclin B1 to regulate cell cycle progression. EMBO Journal 20, 2214–2223. Bijlsma, M.F., Borensztajn, K.S., Roelink, H., Peppelenbosch, M.P., Spek, C.A., 2007. Sonic hedgehog induces transcription-independent cytoskeletal rearrangement and migration regulated by arachidonate metabolites. Cellular Signalling 19, 2596–2604. Burke, R., Nellen, D., Bellotto, M., et al., 1999. Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells. Cell 99, 803–815. Chen, J.K., Taipale, J., Young, K.E., Maiti, T., Beachy, P.A., 2002. Small molecule modulation of smoothened activity. Proceedings of the National Academy of Sciences of the United States of America 99, 14071–14076. Chuang, P.T., McMahon, A.P., 1999. Vertebrate Hedgehog signalling modulated by induction of a Hedgehog-binding protein. Nature 397 (6720), 617–621. Cooper, M.K., Wassif, C.A., Krakowiak, P.A., et al., 2003. A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis. Nature Genetics 33, 508–513.
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Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease
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Cell Communication: Growth Factor Mediated Cell Signaling: Hedgehog Signaling in Development and Disease
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