Cholesterol accessibility at the ciliary membrane controls Hedgehog signaling

  1. Maia Kinnebrew
  2. Ellen Jean Iverson
  3. Bhaven B Patel
  4. Ganesh V Pusapati
  5. Jennifer H Kong
  6. Kristen A Johnson
  7. Giovanni Luchetti
  8. Kaitlyn M Eckert
  9. Jeffrey G McDonald
  10. Douglas F Covey
  11. Christian Siebold
  12. Arun Radhakrishnan  Is a corresponding author
  13. Rajat Rohatgi  Is a corresponding author
  1. Stanford University School of Medicine, United States
  2. Stanford University School of medicine, United States
  3. University of Texas Southwestern Medical Center, United States
  4. Washington University School of Medicine, United States
  5. University of Oxford, United Kingdom

Abstract

Previously we proposed that transmission of the Hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and Hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies Hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks Hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.

Data availability

All data generated or analyzed are included in Supplementary Files 1-5 in this manuscript.

Article and author information

Author details

  1. Maia Kinnebrew

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7344-8231
  2. Ellen Jean Iverson

    Department of Biochemistry, Stanford University School of medicine, Stanford, United States
    Competing interests
    No competing interests declared.
  3. Bhaven B Patel

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.
  4. Ganesh V Pusapati

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.
  5. Jennifer H Kong

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.
  6. Kristen A Johnson

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
  7. Giovanni Luchetti

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.
  8. Kaitlyn M Eckert

    Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
  9. Jeffrey G McDonald

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
  10. Douglas F Covey

    Department of Developmental Biology, Washington University School of Medicine, St Louis, United States
    Competing interests
    No competing interests declared.
  11. Christian Siebold

    Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6635-3621
  12. Arun Radhakrishnan

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    arun.radhakrishnan@utsouthwestern.edu
    Competing interests
    Arun Radhakrishnan, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7266-7336
  13. Rajat Rohatgi

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    For correspondence
    rrohatgi@stanford.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7609-8858

Funding

National Institutes of Health (GM118082)

  • Rajat Rohatgi

American Heart Association (14POST20370057)

  • Ganesh V Pusapati

American Heart Association (19POST34380734)

  • Jennifer H Kong

National Institutes of Health (GM13251801)

  • Jennifer H Kong

Ford Foundation (Pre-doctoral Fellowship)

  • Giovanni Luchetti

National Institutes of Health (GM106078)

  • Rajat Rohatgi

National Institutes of Health (HL20948)

  • Kristen A Johnson
  • Jeffrey G McDonald
  • Arun Radhakrishnan

Welch Foundation (I-1793)

  • Kristen A Johnson
  • Arun Radhakrishnan

Cancer Research UK (C20724/A14414)

  • Christian Siebold

Cancer Research UK (C20724/A26752)

  • Christian Siebold

European Research Council (647278)

  • Christian Siebold

National Science Foundation (Pre-doctoral Fellowship)

  • Maia Kinnebrew

National Science Foundation (Pre-doctoral Fellowship)

  • Ellen Jean Iverson

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2019, Kinnebrew et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 7,228
    views
  • 1,226
    downloads
  • 106
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Maia Kinnebrew
  2. Ellen Jean Iverson
  3. Bhaven B Patel
  4. Ganesh V Pusapati
  5. Jennifer H Kong
  6. Kristen A Johnson
  7. Giovanni Luchetti
  8. Kaitlyn M Eckert
  9. Jeffrey G McDonald
  10. Douglas F Covey
  11. Christian Siebold
  12. Arun Radhakrishnan
  13. Rajat Rohatgi
(2019)
Cholesterol accessibility at the ciliary membrane controls Hedgehog signaling
eLife 8:e50051.
https://doi.org/10.7554/eLife.50051

Share this article

https://doi.org/10.7554/eLife.50051

Further reading

    1. Biochemistry and Chemical Biology
    2. Developmental Biology
    Giovanni Luchetti, Ria Sircar ... Rajat Rohatgi
    Research Article Updated

    Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility.

    1. Cancer Biology
    2. Cell Biology
    Ida Marie Boisen, Nadia Krarup Knudsen ... Martin Blomberg Jensen
    Research Article

    Testicular microcalcifications consist of hydroxyapatite and have been associated with an increased risk of testicular germ cell tumors (TGCTs) but are also found in benign cases such as loss-of-function variants in the phosphate transporter SLC34A2. Here, we show that fibroblast growth factor 23 (FGF23), a regulator of phosphate homeostasis, is expressed in testicular germ cell neoplasia in situ (GCNIS), embryonal carcinoma (EC), and human embryonic stem cells. FGF23 is not glycosylated in TGCTs and therefore cleaved into a C-terminal fragment which competitively antagonizes full-length FGF23. Here, Fgf23 knockout mice presented with marked calcifications in the epididymis, spermatogenic arrest, and focally germ cells expressing the osteoblast marker Osteocalcin (gene name: Bglap, protein name). Moreover, the frequent testicular microcalcifications in mice with no functional androgen receptor and lack of circulating gonadotropins are associated with lower Slc34a2 and higher Bglap/Slc34a1 (protein name: NPT2a) expression compared with wild-type mice. In accordance, human testicular specimens with microcalcifications also have lower SLC34A2 and a subpopulation of germ cells express phosphate transporter NPT2a, Osteocalcin, and RUNX2 highlighting aberrant local phosphate handling and expression of bone-specific proteins. Mineral disturbance in vitro using calcium or phosphate treatment induced deposition of calcium phosphate in a spermatogonial cell line and this effect was fully rescued by the mineralization inhibitor pyrophosphate. In conclusion, testicular microcalcifications arise secondary to local alterations in mineral homeostasis, which in combination with impaired Sertoli cell function and reduced levels of mineralization inhibitors due to high alkaline phosphatase activity in GCNIS and TGCTs facilitate osteogenic-like differentiation of testicular cells and deposition of hydroxyapatite.