The Hedgehog (Hh) signaling pathway coordinates cell–cell communication in development and regeneration. Defects in this pathway underlie diseases ranging from birth defects to cancer. Hh signals are transmitted across the plasma membrane by two proteins, Patched 1 (PTCH1) and Smoothened (SMO). PTCH1, a transporter-like tumor-suppressor protein, binds to Hh ligands, but SMO, a G-protein-coupled-receptor family oncoprotein, transmits the Hh signal across the membrane. Recent structural, biochemical and cell-biological studies have converged at the surprising model that a specific pool of plasma membrane cholesterol, termed accessible cholesterol, functions as a second messenger that conveys the signal between PTCH1 and SMO. Beyond solving a central puzzle in Hh signaling, these studies are revealing new principles in membrane biology: how proteins respond to and remodel cholesterol accessibility in membranes and how the cholesterol composition of organelle membranes is used to regulate protein function.
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Kong, J. H., Siebold, C. & Rohatgi, R. Biochemical mechanisms of vertebrate hedgehog signaling. Development 146, dev166892 (2019).
Huangfu, D. et al. Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature 426, 83–87 (2003).
Goetz, S. C., Ocbina, P. J. R. & Anderson, K. V. The primary cilium as a Hedgehog signal transduction machine. Methods Cell Biol. 94, 199–222 (2009).
Wang, C. et al. Structure of the human smoothened receptor bound to an antitumour agent. Nature 497, 338–343 (2013).
Porter, J. A., Young, K. E. & Beachy, P. A. Cholesterol modification of Hedgehog signaling proteins in animal development. Science 274, 255–259 (1996).
Pepinsky, R. B. et al. Identification of a palmitic acid-modified form of human Sonic hedgehog. J. Biol. Chem. 273, 14037–14045 (1998).
Chiang, C. et al. Cyclopia and defective axial patterning in mice lacking Sonic Hedgehog gene function. Nature 383, 407–413 (1996).
Cooper, M. K., Porter, J. A., Young, K. E. & Beachy, P. A. Teratogen-mediated inhibition of target tissue response to Shh signaling. Science 280, 1603–1607 (1998).
Cooper, M. K. et al. A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis. Nat. Genet. 33, 508–513 (2003). Cholesterol depletion using MβCD or mutations in genes encoding terminal cholesterol biosynthetic enzymes impair Hh signaling in target cells.
Blassberg, R., Macrae, J. I., Briscoe, J. & Jacob, J. Reduced cholesterol levels impair Smoothened activation in Smith-Lemli-Opitz syndrome. Hum. Mol. Genet. 25, 693–705 (2016).
Sharpe, H. J., Wang, W., Hannoush, R. N. & de Sauvage, F. J. Regulation of the oncoprotein Smoothened by small molecules. Nat. Chem. Biol. 11, 246–255 (2015).
Wang, C. et al. Structural basis for Smoothened receptor modulation and chemoresistance to anticancer drugs. Nat. Commun. 5, 4355 (2014).
Corcoran, R. B. & Scott, M. P. Oxysterols stimulate Sonic hedgehog signal transduction and proliferation of medulloblastoma cells. Proc. Natl Acad. Sci. USA 103, 8408–8413 (2006).
Rohatgi, R., Milenkovic, L. & Scott, M. P. Patched1 regulates hedgehog signaling at the primary cilium. Science 317, 372–376 (2007). PTCH1 is localized in and around primary cilia and inhibits SMO activity and accumulation in the ciliary membrane.
Dwyer, J. R. et al. Oxysterols are novel activators of the hedgehog signaling pathway in pluripotent mesenchymal cells. J. Biol. Chem. 282, 8959–8968 (2007).
Nachtergaele, S. et al. Oxysterols are allosteric activators of the oncoprotein Smoothened. Nat. Chem. Biol. 8, 211–220 (2012). Side chain oxysterols induce Hh responses in cultured cells by binding and activating SMO.
Raleigh, D. R. et al. Cilia-associated oxysterols activate Smoothened. Mol. Cell 72, 316–327.e5 (2018).
Chen, W., Chen, G., Head, D. L., Mangelsdorf, D. J. & Russell, D. W. Enzymatic reduction of oxysterols impairs LXR signaling in cultured cells and the livers of mice. Cell Metab. 5, 73–79 (2007).
Kinnebrew, M. et al. Cholesterol accessibility at the ciliary membrane controls hedgehog signaling. eLife 8, e50051 (2019). A CRISPR screen and sphingomyelin (SM) depletion showed that Hh signaling is activated selectively by the accessible pool of membrane cholesterol. Fluorescent probes described in ref. 23 showed that Hh ligands trigger an increase in accessible cholesterol in the ciliary membrane by inactivating PTCH1, allowing SMO activation.
McConnell, H. M. & Radhakrishnan, A. Condensed complexes of cholesterol and phospholipids. Biochim. Biophys. Acta 1610, 159–173 (2003).
Demel, R. A., Jansen, J. W., van Dijck, P. W. & van Deenen, L. L. The preferential interaction of cholesterol with different classes of phospholipids. Biochim. Biophys. Acta 465, 1–10 (1977).
Finean, J. B. Phospholipid-cholesterol complex in the structure of myelin. Experientia 9, 17–19 (1953).
Endapally, S. et al. Molecular discrimination between two conformations of sphingomyelin in plasma membranes. Cell 176, 1040–1053.e17 (2019). Fluorescently labeled probes derived from microbial and fungal toxins that can be used to distinguish between the accessible and sequestered pools of cholesterol were developed.
Klein, U., Gimpl, G. & Fahrenholz, F. Alteration of the myometrial plasma membrane cholesterol content with beta-cyclodextrin modulates the binding affinity of the oxytocin receptor. Biochemistry 34, 13784–13793 (1995).
Byrne, E. F. X. et al. Structural basis of Smoothened regulation by its extracellular domains. Nature 535, 517–522 (2016). A 3.2 Å multi-domain crystal structure of SMO unexpectedly revealed a cholesterol molecule bound in the cysteine-rich domain (CRD). Clinically used SMO inhibitors induce a conformational change that prevents cholesterol access to the CRD.
Luchetti, G. et al. Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling. eLife 5, e20304 (2016). Refs. 26 and 27 showed that cholesterol can function as an instructive agonist for SMO and is both necessary and sufficient to activate Hh signaling.
Huang, P. et al. Cellular cholesterol directly activates Smoothened in Hedgehog signaling. Cell 166, 1176–1187.e14 (2016).
Nedelcu, D., Liu, J., Xu, Y., Jao, C. & Salic, A. Oxysterol binding to the extracellular domain of Smoothened in Hedgehog signaling. Nat. Chem. Biol. 9, 557–564 (2013).
Myers, B. R. et al. Hedgehog pathway modulation by multiple lipid binding sites on the smoothened effector of signal response. Dev. Cell 26, 346–357 (2013).
Nachtergaele, S. et al. Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling. eLife 2, e01340 (2013).
Xiao, X. et al. Cholesterol modification of Smoothened is required for Hedgehog signaling. Mol. Cell 66, 154–162.e10 (2017). Mutations in the CRD cholesterol-binding site abolished SMO function in mouse embryos, and PTCH1 can attenuate cholesterol access to the SMO CRD.
Zhang, X. et al. Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand. Nat. Commun. 8, 15383 (2017).
Deshpande, I. et al. Smoothened stimulation by membrane sterols drives Hedgehog pathway activity. Nature 571, 284–288 (2019). An active-state structure of SMO bound to two synthetic agonists revealed a second cholesterol-binding site in the middle of the TMD, in addition to the previously identified site in the CRD.
Huang, P. et al. Structural basis of Smoothened activation in Hedgehog signaling. Cell 175, 295–297 (2018).
Qi, X. et al. Cryo-EM structure of oxysterol-bound human Smoothened coupled to a heterotrimeric Gi. Nature 571, 279–283 (2019).
Yauch, R. L. et al. Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma. Science 326, 572–574 (2009).
Myers, B. R., Neahring, L., Zhang, Y., Roberts, K. J. & Beachy, P. A. Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium. Proc. Natl Acad. Sci. USA 114, E11141–E11150 (2017).
Loftus, S. K. et al. Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene. Science 277, 232–235 (1997).
Carstea, E. D. et al. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science 277, 228–231 (1997).
Davies, J. P. & Ioannou, Y. A. Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identical to those of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein cleavage-activating protein. J. Biol. Chem. 275, 24367–24374 (2000).
Tseng, T. T. et al. The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins. J. Mol. Microbiol. Biotechnol. 1, 107–125 (1999).
Gong, X. et al. Structural basis for the recognition of Sonic Hedgehog by human Patched1. Science 361, eaas8935 (2018). Refs. 42-45 report liganded and unliganded cryo-EM structures of PTCH1, suggesting a tunnel through the protein that may function in sterol transport. Ref. 45 demonstrated that overexpression of PTCH1 reduces recruitment of a cholesterol-binding probe to the inner leaflet of the plasma membrane.
Qi, X., Schmiege, P., Coutavas, E. & Li, X. Two Patched molecules engage distinct sites on Hedgehog yielding a signaling-competent complex. Science 362, aas8843 (2018).
Qi, X., Schmiege, P., Coutavas, E., Wang, J. & Li, X. Structures of human Patched and its complex with native palmitoylated sonic hedgehog. Nature 560, 128–132 (2018).
Zhang, Y. et al. Structural basis for cholesterol transport-like activity of the Hedgehog receptor patched. Cell 175, 1352–1364.e14 (2018).
Li, X. et al. Structure of human Niemann-Pick C1 protein. Proc. Natl Acad. Sci. USA 113, 8212–8217 (2016).
Gong, X. et al. Structural insights into the Niemann-Pick C1 (NPC1)-mediated cholesterol transfer and Ebola infection. Cell 165, 1467–1478 (2016).
Qian, H. et al. Structural basis of low-pH-dependent lysosomal cholesterol egress by NPC1 and NPC2. Cell 182, 98–111.e18 (2020).
Winkler, M. B. L. et al. Structural insight into eukaryotic sterol transport through Niemann-Pick type C proteins. Cell 179, 485–497.e18 (2019).
Qi, C., Di Minin, G., Vercellino, I., Wutz, A. & Korkhov, V. M. Structural basis of sterol recognition by human hedgehog receptor PTCH1. Sci. Adv. 5, eaaw6490 (2018).
Rudolf, A. F. et al. The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism. Nat. Chem. Biol. 15, 975–982 (2019).
Qian, H. et al. Inhibition of tetrameric Patched1 by Sonic Hedgehog through an asymmetric paradigm. Nat. Commun. 10, 2320 (2019).
Long, T. et al. Structural basis for itraconazole-mediated NPC1 inhibition. Nat. Commun. 11, 152 (2020).
Infante, R. E. et al. Purified NPC1 protein. I. Binding of cholesterol and oxysterols to a 1278-amino acid membrane protein. J. Biol. Chem. 283, 1052–1063 (2008).
Infante, R. E. et al. Purified NPC1 protein: II. Localization of sterol binding to a 240-amino acid soluble luminal loop. J. Biol. Chem. 283, 1064–1075 (2008).
Infante, R. E. et al. NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes. Proc. Natl Acad. Sci. USA 105, 15287–15292 (2008).
Kwon, H. J. et al. Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol. Cell 137, 1213–1224 (2009).
Bidet, M. et al. The hedgehog receptor patched is involved in cholesterol transport. PLoS One 6, e23834 (2011). PTCH1 can promote efflux of a fluorescent cholesterol analog (BODIPY-cholesterol) from cells.
Litz, J. P., Thakkar, N., Portet, T. & Keller, S. L. Depletion with cyclodextrin reveals two populations of cholesterol in model lipid membranes. Biophys. J. 110, 635–645 (2016).
Watts, A., Volotovski, I. D. & Marsh, D. Rhodopsin-lipid associations in bovine rod outer segment membranes. Identification of immobilized lipid by spin-labels. Biochemistry 18, 5006–5013 (1979).
Leathes, J. B. Condensing effect of cholesterol on monolayers. Lancet 208, 853–856 (1925).
Finegold, L.X. Cholesterol in Membrane Models. (CRC Press, 1992).
McConnell, H. & Radhakrishnan, A. Theory of the deuterium NMR of sterol-phospholipid membranes. Proc. Natl Acad. Sci. USA 103, 1184–1189 (2006).
Radhakrishnan, A. & McConnell, H. Condensed complexes in vesicles containing cholesterol and phospholipids. Proc. Natl Acad. Sci. USA 102, 12662–12666 (2005).
Lange, Y., Tabei, S. M. A., Ye, J. & Steck, T. L. Stability and stoichiometry of bilayer phospholipid-cholesterol complexes: relationship to cellular sterol distribution and homeostasis. Biochemistry 52, 6950–6959 (2013).
Keller, S. L., Radhakrishnan, A. & McConnell, H. M. Saturated phospholipids with high melting temperatures form complexes with cholesterol in monolayers. J. Phys. Chem. B 104, 7522–7527 (2000).
Lönnfors, M., Doux, J. P. F., Killian, J. A., Nyholm, T. K. M. & Slotte, J. P. Sterols have higher affinity for sphingomyelin than for phosphatidylcholine bilayers even at equal acyl-chain order. Biophys. J. 100, 2633–2641 (2011).
Radhakrishnan, A. & McConnell, H. M. Chemical activity of cholesterol in membranes. Biochemistry 39, 8119–8124 (2000).
Ahn, K.-W. & Sampson, N. S. Cholesterol oxidase senses subtle changes in lipid bilayer structure. Biochemistry 43, 827–836 (2004).
Flanagan, J. J., Tweten, R. K., Johnson, A. E. & Heuck, A. P. Cholesterol exposure at the membrane surface is necessary and sufficient to trigger perfringolysin O binding. Biochemistry 48, 3977–3987 (2009).
Gay, A., Rye, D. & Radhakrishnan, A. Switch-like responses of two cholesterol sensors do not require protein oligomerization in membranes. Biophys. J. 108, 1459–1469 (2015).
Haynes, M. P., Phillips, M. C. & Rothblat, G. H. Efflux of cholesterol from different cellular pools. Biochemistry 39, 4508–4517 (2000).
Lange, Y., Ye, J. & Steck, T. L. How cholesterol homeostasis is regulated by plasma membrane cholesterol in excess of phospholipids. Proc. Natl Acad. Sci. USA 101, 11664–11667 (2004).
Das, A., Goldstein, J. L., Anderson, D. D., Brown, M. S. & Radhakrishnan, A. Use of mutant 125I-perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells. Proc. Natl Acad. Sci. USA 110, 10580–10585 (2013).
Das, A., Brown, M. S., Anderson, D. D., Goldstein, J. L. & Radhakrishnan, A. Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis. eLife 3, e02882 (2014). Sphingomyelin regulates the partitioning of cholesterol between the accessible and sequestered pools.
Infante, R. E. & Radhakrishnan, A. Continuous transport of a small fraction of plasma membrane cholesterol to endoplasmic reticulum regulates total cellular cholesterol. eLife 6, e25466 (2017).
Skočaj, M. et al. Tracking cholesterol/sphingomyelin-rich membrane domains with the ostreolysin A-mCherry protein. PLoS One 9, e92783 (2014).
Goldstein, J. L. & Brown, M. S. A century of cholesterol and coronaries: from plaques to genes to statins. Cell 161, 161–172 (2015).
Slotte, J. P. & Bierman, E. L. Depletion of plasma-membrane sphingomyelin rapidly alters the distribution of cholesterol between plasma membranes and intracellular cholesterol pools in cultured fibroblasts. Biochem. J. 250, 653–658 (1988).
Scheek, S., Brown, M. S. & Goldstein, J. L. Sphingomyelin depletion in cultured cells blocks proteolysis of sterol regulatory element binding proteins at site 1. Proc. Natl Acad. Sci. USA 94, 11179–11183 (1997).
Corbit, K. C. et al. Vertebrate Smoothened functions at the primary cilium. Nature 437, 1018–1021 (2005). Hh ligands promote the accumulation of SMO in primary cilia, the likely subcellular location from which it signals to the cytoplasm.
Nachury, M. V. & Mick, D. U. Establishing and regulating the composition of cilia for signal transduction. Nat. Rev. Mol. Cell Biol. 20, 389–405 (2019).
Weiss, L. E., Milenkovic, L., Yoon, J., Stearns, T. & Moerner, W. E. Motional dynamics of single Patched1 molecules in cilia are controlled by Hedgehog and cholesterol. Proc. Natl Acad. Sci. USA 116, 5550–5557 (2019).
Kaiser, F., Huebecker, M. & Wachten, D. Sphingolipids controlling ciliary and microvillar function. FEBS Lett. https://doi.org/10.1002/1873-3468.13816 (2020).
Serricchio, M. et al. Flagellar membranes are rich in raft-forming phospholipids. Biol. Open 4, 1143–1153 (2015).
Tyler, K. M. et al. Flagellar membrane localization via association with lipid rafts. J. Cell Sci. 122, 859–866 (2009).
Kaneshiro, E. S., Matesic, D. F. & Jayasimhulu, K. Characterizations of six ethanolamine sphingophospholipids from Paramecium cells and cilia. J. Lipid Res. 25, 369–377 (1984).
Breslow, D. K., Koslover, E. F., Seydel, F., Spakowitz, A. J. & Nachury, M. V. An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier. J. Cell Biol. 203, 129–147 (2013).
Kowatsch, C., Woolley, R. E., Kinnebrew, M., Rohatgi, R. & Siebold, C. Structures of vertebrate Patched and Smoothened reveal intimate links between cholesterol and Hedgehog signalling. Curr. Opin. Struct. Biol. 57, 204–214 (2019).
Nager, A. R. et al. An actin network dispatches ciliary GPCRs into extracellular vesicles to modulate signaling. Cell 168, 252–263.e14 (2017).
Phua, S. C. et al. Dynamic remodeling of membrane composition drives cell cycle through primary cilia excision. Cell 168, 264–279.e15 (2017).
He, C. et al. Macrophages release plasma membrane-derived particles rich in accessible cholesterol. Proc. Natl Acad. Sci. USA 115, E8499–E8508 (2018).
Mondal, M., Mesmin, B., Mukherjee, S. & Maxfield, F. R. Sterols are mainly in the cytoplasmic leaflet of the plasma membrane and the endocytic recycling compartment in CHO cells. Mol. Biol. Cell 20, 581–588 (2009).
Courtney, K. C. et al. C24 sphingolipids govern the transbilayer asymmetry of cholesterol and lateral organization of model and live-cell plasma membranes. Cell Reports 24, 1037–1049 (2018).
Liu, S.-L. et al. Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol. Nat. Chem. Biol. 13, 268–274 (2017).
Steck, T. L. & Lange, Y. Transverse distribution of plasma membrane bilayer cholesterol: Picking sides. Traffic 19, 750–760 (2018).
Hausmann, G., von Mering, C. & Basler, K. The hedgehog signaling pathway: where did it come from? PLoS Biol. 7, e1000146 (2009). A hypothesis that Hh signaling may have evolved from an ancient pathway for sensing and transporting hopanoids.
Bazan, J. F. & de Sauvage, F. J. Structural ties between cholesterol transport and morphogen signaling. Cell 138, 1055–1056 (2009).
Chovancova, E. et al. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLOS Comput. Biol. 8, e1002708 (2012).
Sokolov, A. & Radhakrishnan, A. Accessibility of cholesterol in endoplasmic reticulum membranes and activation of SREBP-2 switch abruptly at a common cholesterol threshold. J. Biol. Chem. 285, 29480–29490 (2010).
We acknowledge G. Pusapati for help with the figures and comments on the manuscript. C.S. was supported by grants from Cancer Research UK (C20724/A26752) and the European Research Council (647278), R.R. by grants from the National Institutes of Health (GM118082 and GM106078), and A.R. by grants from the NIH (HL20948), Welch Foundation (I-1793) and Leducq Foundation (19CVD04).
The authors declare no competing interests.
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Radhakrishnan, A., Rohatgi, R. & Siebold, C. Cholesterol access in cellular membranes controls Hedgehog signaling. Nat Chem Biol 16, 1303–1313 (2020). https://doi.org/10.1038/s41589-020-00678-2
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