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Hedgehog Signal Transduction: Key Players, Oncogenic Drivers, and Cancer Therapy

2016; Elsevier BV; Volume: 38; Issue: 4 Linguagem: Inglês

10.1016/j.devcel.2016.07.026

1878-1551

Ekaterina Pak, Rosalind A. Segal,

Epigenetics and DNA Methylation

The Hedgehog (Hh) signaling pathway governs complex developmental processes, including proliferation and patterning within diverse tissues. These activities rely on a tightly regulated transduction system that converts graded Hh input signals into specific levels of pathway activity. Uncontrolled activation of Hh signaling drives tumor initiation and maintenance. However, recent entry of pathway-specific inhibitors into the clinic reveals mixed patient responses and thus prompts further exploration of pathway activation and inhibition. In this review, we share emerging insights into regulated and oncogenic Hh signaling, supplemented with updates on the development and use of Hh pathway-targeted therapies. The Hedgehog (Hh) signaling pathway governs complex developmental processes, including proliferation and patterning within diverse tissues. These activities rely on a tightly regulated transduction system that converts graded Hh input signals into specific levels of pathway activity. Uncontrolled activation of Hh signaling drives tumor initiation and maintenance. However, recent entry of pathway-specific inhibitors into the clinic reveals mixed patient responses and thus prompts further exploration of pathway activation and inhibition. In this review, we share emerging insights into regulated and oncogenic Hh signaling, supplemented with updates on the development and use of Hh pathway-targeted therapies. The evolutionarily conserved Hedgehog (Hh) pathway serves fundamental morphogenic and mitogenic roles in tissue development, homeostasis, and repair. Disruption of Hh signaling underlies a variety of developmental disorders affecting multiple organ systems. Holoprosencephaly and cyclopia, as well as dramatic limb abnormalities, are characteristic of impaired Hh signaling during development. Moreover, ectopic activation of Hh signaling is implicated in a wide range of tumors, including medulloblastoma (MB), basal cell carcinoma (BCC), and many others. Thus, Hh signaling is an area of intense study in both developmental and cancer biology. Here, we provide updates on vertebrate Hh signal transduction and the molecular drivers of Hh pathway-dependent MB and BCC. In addition, we discuss the application of clinical and preclinical targeted therapies to treat Hh-dependent tumors. In mammals, the Hh signaling cascade is initiated by one of three spatiotemporally confined ligands: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh) (reviewed in Ingham and McMahon, 2001Ingham P.W. McMahon A.P. Hedgehog signaling in animal development: paradigms and principles.Genes Dev. 2001; 15: 3059-3087Crossref PubMed Scopus (1785) Google Scholar). Secreted Hh ligands control developmental outcomes in a concentration- and duration-dependent manner. Consequently, the reception and signal transduction system for Hh ligands must convert different levels of signal into specific levels of pathway output. Ultimately, signal transduction results in expression of a transcriptional program mediated by activator and repressor forms of the Gli transcription factors. The ability of this cascade to initiate distinct developmental outcomes in cells exposed to an Hh ligand at different concentrations or for different lengths of time is critical for Hh-dependent establishment of the dorsal-ventral axis during early neural development and formation of the anterior-posterior axis in developing limbs. Here, we summarize the vertebrate components of Hh signal transduction and focus on recent updates in this field that contribute to our current understanding of Hh signaling in development and cancer (Figure 1). For the remainder of this review, we refer to the Hh ligands for general concepts and Shh ligands for specific reports. The primary receptor for Hh ligands is the 12-pass transmembrane protein, Patched 1 (Ptch1) (Marigo et al., 1996Marigo V. Davey R.A. Zuo Y. Cunningham J.M. Tabin C.J. Biochemical evidence that patched is the hedgehog receptor.Nature. 1996; 384: 176-179Crossref PubMed Scopus (594) Google Scholar, Stone et al., 1996Stone D.M. Hynes M. Armanini M. Swanson T.A. Gu Q. Johnson R.L. Scott M.P. Pennica D. Goddard A. Phillips H. et al.The tumour-suppressor gene patched encodes a candidate receptor for sonic hedgehog.Nature. 1996; 384: 129-134Crossref PubMed Scopus (803) Google Scholar). In the absence of ligand, Ptch1 blocks pathway activity. When Hh ligand binds Ptch1, both ligand and receptor are internalized and degraded (Chen and Struhl, 1996Chen Y. Struhl G. Dual roles for patched in sequestering and transducing hedgehog.Cell. 1996; 87: 553-563Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar, Incardona et al., 2000Incardona J.P. Lee J.H. Robertson C.P. Enga K. Kapur R.P. Roelink H. Receptor-mediated endocytosis of soluble and membrane-tethered Sonic hedgehog by Patched-1.Proc. Natl. Acad. Sci. USA. 2000; 97: 12044-12049Crossref PubMed Scopus (117) Google Scholar). Thus, ligand binding not only removes pathway repression by Ptch1 but also limits the half-life of the ligand. The mechanisms by which Ptch1 is removed from the cell surface upon ligand binding are not fully understood. Recently, Shh ligands were shown to induce accumulation of Ptch1 and the E3 ubiquitin ligases Smurf1 and Smurf2 in lipid rafts, which are molecularly distinct domains of the cell membrane (Yue et al., 2014Yue S. Tang L. Tang Y. Tang Y. Shen Q. Ding J. Chen Y. Zhang Z. Yu T. Zhang Y.E. et al.Requirement of Smurf-mediated endocytosis of patched1 in sonic hedgehog signal reception.Elife. 2014; 3: e02555Crossref Scopus (7) Google Scholar). The ensuing ubiquitination of Ptch1 promoted Ptch1 endosomal trafficking to lysosomes for degradation and was important for Ptch1 clearance and graded pathway activation. Besides Ptch1, other receptors for Hh ligands modulate pathway activation (reviewed in Beachy et al., 2010Beachy P.A. Hymowitz S.G. Lazarus R.A. Leahy D.J. Siebold C. Interactions between Hedgehog proteins and their binding partners come into view.Genes Dev. 2010; 24: 2001-2012Crossref PubMed Scopus (75) Google Scholar). Positive co-receptors include Cdo, Boc, and the vertebrate-specific Gas1. Another vertebrate-specific receptor, Hhip, acts as a negative regulator of Hh signaling. Many positive co-receptors (Cdo, Boc, and Gas1) are transcriptionally repressed, while negative receptors (Ptch1 and Hhip) are activated following Hh pathway induction. In addition, proteoglycans function as co-receptors with either positive or negative effects on Hh signaling depending on their unique protein and sugar composition. The resulting network of receptors and feedback loops helps cells to properly interpret the duration and graded level of Hh signaling. In the absence of Hh ligand, Ptch1 inhibits Smoothened (Smo), a seven-pass transmembrane protein that functions as a potent pathway activator (Murone et al., 1999Murone M. Rosenthal A. de Sauvage F.J. Sonic hedgehog signaling by the patched-smoothened receptor complex.Curr. Biol. 1999; 9: 76-84Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The mechanisms by which Ptch1 inhibits Smo are unknown. The current consensus is that Ptch1 does not physically interact with Smo, but rather regulates the transport, synthesis, and/or access of a small molecule (or molecules) that affect Smo activity (Taipale et al., 2002Taipale J. Cooper M.K. Maiti T. Beachy P.A. Patched acts catalytically to suppress the activity of Smoothened.Nature. 2002; 418: 892-897Crossref PubMed Scopus (449) Google Scholar). Lipid metabolites are popular candidates for the endogenous regulators of Smo activity. The lipophilic secosteroid vitamin D3 was previously proposed to function as a Ptch1-regulated direct inhibitor of Smo (Bijlsma et al., 2006Bijlsma M.F. Spek C.A. Zivkovic D. van de Water S. Rezaee F. Peppelenbosch M.P. Repression of smoothened by patched-dependent (pro-)vitamin D3 secretion.PLoS Biol. 2006; 4: e232Crossref PubMed Scopus (49) Google Scholar). More recently, a group of cholesterol derivatives called oxysterols were shown to activate Smo by binding to its N-terminal, extracellular cysteine-rich domain (CRD) (Myers et al., 2013Myers B.R. Sever N. Chong Y.C. Kim J. Belani J.D. Rychnovsky S. Bazan J.F. Beachy P.A. Hedgehog pathway modulation by multiple lipid binding sites on the smoothened effector of signal response.Dev. Cell. 2013; 26: 346-357Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, Nachtergaele et al., 2013Nachtergaele S. Whalen D.M. Mydock L.K. Zhao Z. Malinauskas T. Krishnan K. Ingham P.W. Covey D.F. Siebold C. Rohatgi R. Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling.Elife. 2013; 2: e01340Crossref Google Scholar, Nedelcu et al., 2013Nedelcu D. Liu J. Xu Y. Jao C. Salic A. Oxysterol binding to the extracellular domain of smoothened in Hedgehog signaling.Nat. Chem. Biol. 2013; 9: 557-564Crossref PubMed Scopus (52) Google Scholar). CRD mutants fail to fully respond to Hh stimulation, but also exhibit a higher basal level of signaling compared with wild-type Smo, indicating that the CRD domain suppresses basal Smo activity. Importantly, CRD mutants are still subject to inhibition by Ptch1. Thus, oxysterol binding may be required for maximal Smo activity but not for mediating Ptch1-dependent inhibition of Smo. Additional lipid-based Smo modulators include endocannabinoids from lipoprotein particles that can bind and inhibit Smo activity (Khaliullina et al., 2015Khaliullina H. Bilgin M. Sampaio J.L. Shevchenko A. Eaton S. Endocannabinoids are conserved inhibitors of the Hedgehog pathway.Proc. Natl. Acad. Sci. USA. 2015; 112: 3415-3420Crossref PubMed Google Scholar). Despite such recent insights on lipophilic regulators of Smo activity, the mechanisms by which Ptch1 represses Smo and how Hh ligand removes this repression remain major questions in the field. Upon Hh ligand binding and Ptch1 degradation, Smo becomes phosphorylated by casein kinase 1 (CK1) and G-protein-coupled receptor kinase 2 (GRK2), moves into the primary cilium (PC), and assumes an activated conformation (Chen et al., 2011aChen Y. Sasai N. Ma G. Yue T. Jia J. Briscoe J. Jiang J. Sonic Hedgehog dependent phosphorylation by CK1alpha and GRK2 is required for ciliary accumulation and activation of smoothened.PLoS Biol. 2011; 9: e1001083Crossref PubMed Scopus (62) Google Scholar). Differential phosphorylation of Smo may help interpret Hh gradients through a Smo phosphorylation code (Chen and Jiang, 2013Chen Y. Jiang J. Decoding the phosphorylation code in Hedgehog signal transduction.Cell Res. 2013; 23: 186-200Crossref PubMed Scopus (32) Google Scholar). The relay of Hh signaling downstream of activated Smo is not yet completely understood. However, Smo activates both G-protein-dependent and -independent signals to regulate Gli transcription factors, calcium flux, and metabolic pathways (Arensdorf et al., 2016Arensdorf A.M. Marada S. Ogden S.K. Smoothened regulation: a tale of two signals.Trends Pharmacol. Sci. 2016; 37: 62-72Abstract Full Text Full Text PDF PubMed Google Scholar). Here, we focus on signals that impinge on Gli transcription factors. The negative pathway regulator, Suppressor of fused (Sufu), functions between Smo and the Gli transcription factors (Pearse et al., 1999Pearse II, R.V. Collier L.S. Scott M.P. Tabin C.J. Vertebrate homologs of Drosophila suppressor of fused Interact with the gli family of transcriptional regulators.Dev. Biol. 1999; 212: 323-336Crossref PubMed Scopus (94) Google Scholar, Stone et al., 1999Stone D.M. Murone M. Luoh S. Ye W. Armanini M.P. Gurney A. Phillips H. Brush J. Goddard A. de Sauvage F.J. et al.Characterization of the human suppressor of fused, a negative regulator of the zinc-finger transcription factor Gli.J. Cell Sci. 1999; 112: 4437-4448Crossref PubMed Google Scholar). Sufu directly interacts with and sequesters full-length Gli in the cytoplasm. Sequestration of Gli prevents its nuclear translocation and promotes phosphorylation and processing of full-length Gli into a truncated repressor (Humke et al., 2010Humke E.W. Dorn K.V. Milenkovic L. Scott M.P. Rohatgi R. The output of Hedgehog signaling is controlled by the dynamic association between Suppressor of Fused and the Gli proteins.Genes Dev. 2010; 24: 670-682Crossref PubMed Scopus (176) Google Scholar). Sequestration also stabilizes full-length Gli2 and Gli3, protecting them from proteasomal degradation and thus maintaining a pool of available Gli proteins for Shh signal transduction (Chen et al., 2009Chen M.H. Wilson C.W. Li Y.J. Law K.K. Lu C.S. Gacayan R. Zhang X. Hui C.C. Chuang P.T. Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved.Genes Dev. 2009; 23: 1910-1928Crossref PubMed Scopus (157) Google Scholar, Wang et al., 2010Wang C. Pan Y. Wang B. Suppressor of fused and Spop regulate the stability, processing and function of Gli2 and Gli3 full-length activators but not their repressors.Development. 2010; 137: 2001-2009Crossref PubMed Scopus (84) Google Scholar). Challenging the traditional cytoplasm-centric roles of Sufu, some have suggested that Sufu can regulate Gli activity in the nucleus (Lin et al., 2014Lin C. Yao E. Wang K. Nozawa Y. Shimizu H. Johnson J.R. Chen J.N. Krogan N.J. Chuang P.T. Regulation of Sufu activity by p66beta and Mycbp provides new insight into vertebrate Hedgehog signaling.Genes Dev. 2014; 28: 2547-2563Crossref PubMed Scopus (9) Google Scholar). The kinesin protein, Kif7, is also an evolutionarily conserved component of Hh signaling that modulates Gli function downstream of Smo (Cheung et al., 2009Cheung H.O.L. Zhang X. Ribeiro A. Mo R. Makino S. Puviindran V. Law K.K.L. Briscoe J. Hui C.C. The kinesin protein Kif7 is a critical regulator of gli transcription factors in mammalian hedgehog signaling.Sci. Signal. 2009; 2: ra29Crossref PubMed Scopus (105) Google Scholar, Tay et al., 2005Tay S.Y. Ingham P.W. Roy S. A homologue of the Drosophila kinesin-like protein Costal2 regulates Hedgehog signal transduction in the vertebrate embryo.Development. 2005; 132: 625-634Crossref PubMed Scopus (67) Google Scholar). Kif7 interacts with Gli proteins and exerts both positive and negative regulatory roles in Hh signaling (Endoh-Yamagami et al., 2009Endoh-Yamagami S. Evangelista M. Wilson D. Wen X. Theunissen J.W. Phamluong K. Davis M. Scales S.J. Solloway M.J. de Sauvage F.J. et al.The mammalian Cos2 homolog Kif7 plays an essential role in modulating Hh signal transduction during development.Curr. Biol. 2009; 19: 1320-1326Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, Liem et al., 2009Liem Jr., K.F. He M. Ocbina P.J. Anderson K.V. Mouse Kif7/Costal2 is a cilia-associated protein that regulates Sonic hedgehog signaling.Proc. Natl. Acad. Sci. USA. 2009; 106: 13377-13382Crossref PubMed Scopus (124) Google Scholar). Kif7 localizes at the base of the PC in the absence of Hh ligand, but moves into the PC and is important for Gli2 and Gli3 accumulation at the cilium tip when the pathway is stimulated. A recently proposed model incorporates Kif7 phosphorylation with these earlier observations of Kif7 signal transduction (Liu et al., 2014Liu Y.C. Couzens A.L. Deshwar A.R. McBroom-Cerajewski L.D.B. Zhang X. Puviindran V. Scott I.C. Gingras A.C. Hui C.C. Angers S. The PPFIA1-PP2A protein complex promotes trafficking of Kif7 to the ciliary tip and Hedgehog signaling.Sci. Signal. 2014; 7: ra117Crossref PubMed Scopus (4) Google Scholar). When the Hh pathway is inactive, Kif7 is phosphorylated and enriched at the base of the PC, while trafficking of Kif7 and Gli into the cilium is limited. When the pathway is activated, the scaffolding protein PPFIA1 and the phosphatase PP2A are recruited to and dephosphorylate Kif7, leading to increased localization of Kif7 and Gli proteins at the PC tip, Sufu dissociation from Gli proteins, and Gli activation. An intriguing addendum to this model comes from the Anderson group, who recently proposed that a major role for Kif7 in Hh signaling is to control cilium length and architecture (He et al., 2014aHe M. Subramanian R. Bangs F. Omelchenko T. Liem Jr., K.F. Kapoor T.M. Anderson K.V. The kinesin-4 protein Kif7 regulates mammalian Hedgehog signalling by organizing the cilium tip compartment.Nat. Cell Biol. 2014; 16: 663-672Crossref PubMed Scopus (43) Google Scholar). In this role, Kif7 ensures that a single cilium tip compartment is established where Gli and Sufu can localize for signal transduction. As discussed below, functional primary cilia are crucial for proper Hh signal transduction. Accordingly, understanding the roles of Kif7 in Hh signaling requires integration of the direct impact of Kif7 on pathway components with additional contributions of Kif7 activity in cilium assembly. Graded levels of Hh signaling trigger the expression of different sets of response genes, depending on the ratio of Gli activator (GliA) and Gli repressor (GliR) forms (reviewed in Hui and Angers, 2011Hui C.C. Angers S. Gli proteins in development and disease.Annu. Rev. Cell Dev. Biol. 2011; 27: 513-537Crossref PubMed Scopus (197) Google Scholar). In vertebrates, there are three Gli gene family members: Gli1, Gli2, and Gli3. Gli1 is an Hh response gene product that exists only as a transcriptional activator and functions in a positive feedback loop upon pathway activation. Gli2 functions primarily as a transcriptional activator, while Gli3 serves as the primary transcriptional repressor. Multiple mechanisms control GliA and GliR functions, including the regulation by Sufu and Kif7 described above. Post-translational modifications of Gli proteins, including phosphorylation, acetylation, ubiquitination, and sumoylation, also affect Gli output. Here, we review our current understanding of the Gli phosphorylation code. In the absence of Hh ligand, full-length Gli (GliFL) is phosphorylated by protein kinase A (PKA), glycogen synthase kinase 3 (GSK3), and CK1 (Pan et al., 2006Pan Y. Bai C.B. Joyner A.L. Wang B. Sonic hedgehog signaling regulates Gli2 transcriptional activity by suppressing its processing and degradation.Mol. Cell Biol. 2006; 26: 3365-3377Crossref PubMed Scopus (282) Google Scholar, Pan et al., 2009Pan Y. Wang C. Wang B. Phosphorylation of Gli2 by protein kinase A is required for Gli2 processing and degradation and the Sonic Hedgehog-regulated mouse development.Dev. Biol. 2009; 326: 177-189Crossref PubMed Scopus (76) Google Scholar, Tempé et al., 2006Tempé D. Casas M. Karaz S. Blanchet-Tournier M.F. Concordet J.P. Multisite protein kinase A and glycogen synthase kinase 3beta phosphorylation leads to Gli3 ubiquitination by SCFbetaTrCP.Mol. Cell Biol. 2006; 26: 4316-4326Crossref PubMed Scopus (125) Google Scholar). Hyperphosphorylated GliFL is bound by the adaptor protein β-TrCP, and the resulting complex is ubiquitinated by a Cul1-based E3 ligase and targeted for proteasomal processing to form a truncated transcriptional repressor (GliR) (Wang and Li, 2006Wang B. Li Y. Evidence for the direct involvement of {beta}TrCP in Gli3 protein processing.Proc. Natl. Acad. Sci. USA. 2006; 103: 33-38Crossref PubMed Scopus (131) Google Scholar). Alternatively, GliFL may also be completely degraded by the proteasome, facilitated by Spop-mediated Cul3-based E3 ligase ubiquitination (Wang et al., 2010Wang C. Pan Y. Wang B. Suppressor of fused and Spop regulate the stability, processing and function of Gli2 and Gli3 full-length activators but not their repressors.Development. 2010; 137: 2001-2009Crossref PubMed Scopus (84) Google Scholar). Gli2FL phosphorylation predominantly induces complete proteasomal degradation, while phosphorylated Gli3FL is more efficiently processed into Gli3R (Pan and Wang, 2007Pan Y. Wang B. A novel protein-processing domain in Gli2 and Gli3 differentially blocks complete protein degradation by the proteasome.J. Biol. Chem. 2007; 282: 10846-10852Crossref PubMed Scopus (64) Google Scholar). Smo activation blocks Gli proteolysis and simultaneously promotes Gli activator function. The unique patterns of Gli2/3 phosphorylation may be important for converting differences in Hh signal strength into discrete states of Gli activity. PKA phosphorylation of six conserved serine residues (P1 to P6) on Gli2/3 drives GliR and inhibits GliA formation (Niewiadomski et al., 2014Niewiadomski P. Kong J.H. Ahrends R. Ma Y. Humke E.W. Khan S. Teruel M.N. Novitch B.G. Rohatgi R. Gli protein activity is controlled by multisite phosphorylation in vertebrate Hedgehog signaling.Cell Rep. 2014; 6: 168-181Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Interestingly, selective phosphorylation of the first four PKA sites (P1 to P4) is sufficient to target processing of GliFL into GliR, while inhibition of GliA requires phosphorylation of all six PKA sites. Smo activation reduces phosphorylation at P1–P6, which allows PKA-independent Gli phosphorylation at a different cluster of serine/threonine sites (Pc to Pg) and results in full transcriptional activation of Gli2/3. While Gli1 is not subject to PKA-mediated proteasomal processing, several other kinases have been shown to alter Gli1 function. Phosphorylation of Gli1 by atypical protein kinase C ι/λ (aPKC-ι/λ) promotes maximal Gli1 DNA binding and transcriptional activation (Atwood et al., 2013Atwood S.X. Li M. Lee A. Tang J.Y. Oro A.E. GLI activation by atypical protein kinase C iota/lambda regulates the growth of basal cell carcinomas.Nature. 2013; 494: 484-488Crossref PubMed Scopus (69) Google Scholar). Gli1 is also phosphorylated by AMP-activated protein kinase (AMPK), which induces Gli1 degradation (Di Magno et al., 2016Di Magno L. Basile A. Coni S. Manni S. Sdruscia G. D'Amico D. Antonucci L. Infante P. De Smaele E. Cucchi D. et al.The energy sensor AMPK regulates Hedgehog signaling in human cells through a unique Gli1 metabolic checkpoint.Oncotarget. 2016; 7: 9538-9549PubMed Google Scholar, Li et al., 2015Li Y.H. Luo J. Mosley Y.Y. Hedrick V.E. Paul L.N. Chang J. Zhang G. Wang Y.K. Banko M.R. Brunet A. et al.AMP-activated protein kinase directly phosphorylates and destabilizes Hedgehog pathway transcription factor GLI1 in medulloblastoma.Cell Rep. 2015; 12: 599-609Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). Importantly, regulation of Gli1 by the energy sensor AMPK links cellular metabolic state to Hh transcriptional output, which may be crucial for the developmental roles of this pathway. Cyclic AMP (cAMP)-dependent protein kinase A (PKA) is a master negative regulator of the Hh pathway. Both Sufu and Gli2/3 transcription factors are phosphorylated by PKA. Sequential phosphorylation of Sufu by PKA and GSK3 stabilizes Sufu in a complex with Gli2/3 that moves into the PC in response to Hh ligand (Chen et al., 2011bChen Y. Yue S. Xie L. Pu X.H. Jin T. Cheng S.Y. Dual Phosphorylation of suppressor of fused (Sufu) by PKA and GSK3beta regulates its stability and localization in the primary cilium.J. Biol. Chem. 2011; 286: 13502-13511Crossref PubMed Scopus (34) Google Scholar). The roles of PKA in Gli2/3 phosphorylation and proteolysis are described above. Many inputs modulate PKA activity and thereby affect Hh pathway output. For example, production of cAMP by adenylyl cyclase and degradation of cAMP by phosphodiesterases can promote and attenuate PKA activity, respectively. Recently, degradation of cAMP was mechanistically linked to signaling by transmembrane neuropilin (Nrp) receptors (Ge et al., 2015Ge X. Milenkovic L. Suyama K. Hartl T. Purzner T. Winans A. Meyer T. Scott M.P. Phosphodiesterase 4D acts downstream of neuropilin to control hedgehog signal transduction and the growth of medulloblastoma.Elife. 2015; 4: e07068https://doi.org/10.7554/eLife.07068Crossref Scopus (2) Google Scholar). The Nrp ligand Semaphorin3 (Sema3) promotes interaction of phosphodiesterase 4D (PDE4D) with the Nrp cytoplasmic domain. Sema3-Nrp mediated translocation of PDE4D to the plasma membrane brings this phosphodiesterase close to the site of cAMP production and thus permits efficient hydrolysis of cAMP. While Sema3 alone cannot stimulate Hh signal transduction, the Sema3-Nrp-PDE4D axis enhances signaling that has been activated by Shh. Importantly, Shh pathway activation functions in a positive feedback loop to increase Nrp1 expression, although Nrp1 is not a direct target of Gli transcription factors (Hillman et al., 2011Hillman R.T. Feng B.Y. Ni J. Woo W.M. Milenkovic L. Hayden Gephart M.G. Teruel M.N. Oro A.E. Chen J.K. Scott M.P. Neuropilins are positive regulators of Hedgehog signal transduction.Genes Dev. 2011; 25: 2333-2346Crossref PubMed Scopus (33) Google Scholar). Instead, in many cellular contexts, Hh-dependent Nrp1 expression is positively regulated by the Eya1 phosphatase and the Six1 transcription factor (Eisner et al., 2015Eisner A. Pazyra-Murphy M.F. Durresi E. Zhou P. Zhao X. Chadwick E.C. Xu P.X. Hillman R.T. Scott M.P. Greenberg M.E. et al.The Eya1 phosphatase promotes Shh signaling during hindbrain development and oncogenesis.Dev. Cell. 2015; 33: 22-35Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Interestingly, while Eya1, Six1, and Nrp promote Gli2-dependent transcription, they do not alter Shh-dependent inhibition of Gli3R. In a pivotal study, production of cAMP was mechanistically coupled to Hh signaling via the orphan G-protein-coupled receptor, Gpr161 (Mukhopadhyay et al., 2013Mukhopadhyay S. Wen X. Ratti N. Loktev A. Rangell L. Scales S.J. Jackson P.K. The ciliary G-protein-coupled receptor Gpr161 negatively regulates the Sonic hedgehog pathway via cAMP signaling.Cell. 2013; 152: 210-223Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In the absence of Shh ligand, Gpr161 localizes to the PC and promotes increased levels of cAMP, probably via Gαs-mediated activation of adenylyl cyclase. In the presence of Shh ligand, Gpr161 is removed from cilia, preventing cAMP production and thus promoting pathway activation. A recent update on Gpr161 further elucidates the molecular mechanisms by which it is removed from the PC (Pal et al., 2016Pal K. Hwang S.H. Somatilaka B. Badgandi H. Jackson P.K. DeFea K. Mukhopadhyay S. Smoothened determines beta-arrestin-mediated removal of the G protein-coupled receptor Gpr161 from the primary cilium.J. Cell Biol. 2016; 212: 861-875Crossref PubMed Google Scholar). Importantly, Gpr161 unifies many components of Shh signaling: ligand stimulation, PKA regulation, and roles of the PC, which are reviewed in more detail below. The core components of vertebrate Hh signaling, including Ptch1, Smo, Sufu, Kif7, and Gli proteins, dynamically localize to the PC (reviewed in Nozawa et al., 2013Nozawa Y.I. Lin C. Chuang P.T. Hedgehog signaling from the primary cilium to the nucleus: an emerging picture of ciliary localization, trafficking and transduction.Curr. Opin. Genet. Dev. 2013; 23: 429-437Crossref PubMed Scopus (46) Google Scholar, Goetz and Anderson, 2010Goetz S.C. Anderson K.V. The primary cilium: a signalling centre during vertebrate development.Nat. Rev. Genet. 2010; 11: 331-344Crossref PubMed Scopus (644) Google Scholar). Upon Hh ligand binding, Ptch1 exits the base of the PC while Smo accumulates within the cilium. Pathway activation also promotes Sufu-Gli complex movement into the PC where they dissociate, resulting in concurrent enrichment of Gli in the distal tip of the cilium and Gli translocation to the nucleus. Smo translocation and Gli dissociation demonstrate positive regulation of Hh signaling via the PC. Cilia also participate in pathway inhibition by mediating the proteolytic processing of GliFL into GliR that turns off target genes. Thus, mice with defective or absent cilia display functional loss or altered ratios of GliA/GliR (Goetz and Anderson, 2010Goetz S.C. Anderson K.V. The primary cilium: a signalling centre during vertebrate development.Nat. Rev. Genet. 2010; 11: 331-344Crossref PubMed Scopus (644) Google Scholar). Precise localization of proteins within the PC is important for signal transduction. For example, the EvC zone, named after Ellis-van Creveld syndrome, is located at the base of the PC and defines a distinct compartment where Smo accumulates in response to Hh ligands by binding to the proteins EVC and EVC2. Anchoring of the EVC-EVC2 complex to the EvC zone is required for activation of Gli2 but not for regulating levels of Gli3R (Pusapati et al., 2014Pusapati G.V. Hughes C.E. Dorn K.V. Zhang D. Sugianto P. Aravind L. Rohatgi R. EFCAB7 and IQCE regulate hedgehog signaling by tethering the EVC-EVC2 complex to the base of primary cilia.Dev. Cell. 2014; 28: 483-496Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). This type of GliA/GliR signaling bifurcation downstream of Smo has also been demonstrated for the ciliary basal body-localized protein Dlg5 (Discs large, homolog 5) (Chong et al., 2015Chong Y.C. Mann R.K. Zhao C. Kato M. Beachy P.A. Bifurcating action of Smoothened in Hedgehog signaling is mediated by Dlg5.Genes Dev. 2015; 29: 262-276Crossref PubMed Google Scholar). Upon ligand stimulation, Dlg5 interacts with Smo to promote Kif7 and Gli2 ciliary accumulation and Gli2 activation, but Dlg5 is not required for suppression of GliR formation. Subciliary localization of Hh signaling regulators such as Dlg5 and EvC complex proteins may help coordinate the contributions of GliA/GliR functions in response to different levels of pathway activation. Recent work has highlighted how the lipid composition of the PC contributes to trafficking and signaling of Hh pathway components. The phosphoinositide phosphatidylinositol 4-phosphate (PI(4)P) is enriched in the ciliary membrane, whereas both PI(4)P and PI(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) are found in the plasma membrane (Chavez et al., 2015Chavez M. Ena S. Van Sande