The Hedgehog (Hh) signaling pathway plays a central role in specifying the embryonic pattern of metazoan organs. Post-embryonically, the Hh pathway controls homeostatic maintenance and injury-induced regeneration of adult tissues. Drugs that block Hh pathway activity have received FDA approval or are in clinical trials for treatment of basal cell carcinoma or medulloblastoma, cancers that are associated with elevated pathway activity in primary tumor cells. In pancreatic, bladder, and other cancers of endodermal origin, Hh pathway activity in tumor-associated stroma restrains cancer growth and progression, suggesting pharmacological pathway activation as a potential therapeutic approach. In addition, pathway activation may have a therapeutic role in regenerative medicine through its ability to enhance neurogenesis (e.g., in Down Syndrome), to accelerate bone and muscle repair, and to ameliorate inflammatory bowel disease, among other emerging biological activities. Despite the importance of Hh signaling in development and physiology, and the established and growing role of pathway modulation in control of malignant and non-malignant disease, we fundamentally do not understand how the extracellular Hh protein signal is transduced across the membrane. The Hh receptor is unusual in that its signaling activity is subdivided into sensing of the extracellular Hh ligand by the transporter-like protein Patched (Ptch), and propagation of this signal into the cell by the seven- transmembrane protein Smoothened (Smo). Mechanistically, Ptch is thought to inhibit Smo by acting as a chemiosmotically-driven transmembrane transporter of a lipidic Smo modulator, but the chemiosmotic driver and lipidic substrate of Ptch remain to be elucidated, as does the mechanism by which Hh binding disrupts Ptch activity. We have developed an in vitro assay that directly monitors activity and conformation of a purified, reconstituted Smo protein allowing us to directly test Smo modulation by distinct classes of ligands, including a recently identified class that is the leading candidate for mediating Ptch suppression of Smo. We also have developed a real-time in vivo assay of Smo conformation that monitors Ptch activity, and will use this assay to further elucidate the role of a candidate chemiosmotic driver that we have recently identified. Furthermore, we have purified an active, well-behaved form of Ptch for functional reconstitution and for tests of interaction with candidate mediators; we have in addition generated a cryo-EM based structure of this Ptch protein in complex with a Hh ligand, and will refine this structure as a basis for illuminating the mechanism of Ptch-Smo regulation and understanding Hh inactivation of Ptch. Finally, we will determine how the dual lipid adducts of the mature Hh ligand are shielded within the soluble complex that allows movement of Hh from its cellular source to the Ptch receptor on target cells, and how the transition from this complex to interaction with Ptch is accomplished. Our findings will be integrated into a detailed molecular account of Hh signal transduction, and may provide a basis for improvements in cancer or regenerative therapies involving Hh pathway inhibition or activation.

Public Health Relevance

We propose a multi-disciplinary approach to address how the extracellular Hedgehog protein signal, with central roles in embryonic tissue patterning and regeneration of adult tissues, can act within tissues so that its presence is sensed and transmitted across the cell membrane. Our findings will illuminate the causes of and possible therapies for birth defects and malignant and non-malignant disease associated with derangements of Hedgehog pathway regulation and activity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM102498-06
Application #
9540030
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Dunsmore, Sarah
Project Start
2012-08-15
Project End
2021-05-31
Budget Start
2018-06-01
Budget End
2019-05-31
Support Year
6
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Stanford University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304
Zhang, Yunxiao; Bulkley, David P; Xin, Yao et al. (2018) Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched. Cell 175:1352-1364.e14
Myers, Benjamin R; Neahring, Lila; Zhang, Yunxiao et al. (2017) Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium. Proc Natl Acad Sci U S A 114:E11141-E11150
Sever, Navdar; Mann, Randall K; Xu, Libin et al. (2016) Endogenous B-ring oxysterols inhibit the Hedgehog component Smoothened in a manner distinct from cyclopamine or side-chain oxysterols. Proc Natl Acad Sci U S A 113:
Ally, Mina S; Ransohoff, Katherine; Sarin, Kavita et al. (2016) Effects of Combined Treatment With Arsenic Trioxide and Itraconazole in Patients With Refractory Metastatic Basal Cell Carcinoma. JAMA Dermatol 152:452-6
Kim, Jynho; Hsia, Elaine Y C; Brigui, Amira et al. (2015) The role of ciliary trafficking in Hedgehog receptor signaling. Sci Signal 8:ra55
Oh, Sekyung; Kato, Masaki; Zhang, Chi et al. (2015) A Comparison of Ci/Gli Activity as Regulated by Sufu in Drosophila and Mammalian Hedgehog Response. PLoS One 10:e0135804
Chong, Yong Chun; Mann, Randall K; Zhao, Chen et al. (2015) Bifurcating action of Smoothened in Hedgehog signaling is mediated by Dlg5. Genes Dev 29:262-76
Strating, Jeroen R P M; van der Linden, Lonneke; Albulescu, Lucian et al. (2015) Itraconazole inhibits enterovirus replication by targeting the oxysterol-binding protein. Cell Rep 10:600-15
Kim, Jynho; Hsia, Elaine Y C; Kim, James et al. (2014) Simultaneous measurement of smoothened entry into and exit from the primary cilium. PLoS One 9:e104070
Kim, James; Aftab, Blake T; Tang, Jean Y et al. (2013) Itraconazole and arsenic trioxide inhibit Hedgehog pathway activation and tumor growth associated with acquired resistance to smoothened antagonists. Cancer Cell 23:23-34

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