Cholesterol as a Second Messenger in the Hedgehog Signaling Pathway

The Hedgehog (HH) pathway is a cell‐cell communication system essential for embryonic development; loss of proper HH signaling results in birth defects and cancer. Signaling is initiated when HH ligands are received on target cells by their receptor, Patched‐1 (PTCH1). This relieves the inhibitory effect of PTCH1 on the G protein‐coupled receptor Smoothened (SMO), allowing the HH signal to be transmitted across the membrane. The question of how PTCH1 inhibits SMO has remained a central mystery in developmental signaling for 25 years. Prior work implicated cholesterol as an endogenous lipid regulator of SMO. However, a major conundrum is presented by the abundance of cholesterol: how can a lipid that makes up 30%‐50% of the plasma membrane be used to regulate a key signaling pathway? Using a custom CRISPR library focused on genes involved in lipid regulation, we asked the more general question of which lipids regulate HH signaling in target cells. We found that Sphingomyelin (SM) levels negatively regulate HH signaling. We show that SM exerts its effect by sequestering cholesterol into complexes, altering the chemical activity (or “accessibility”) of cholesterol. This highlights a key concept in membrane biology: only the accessible pool of cholesterol is available to interact with proteins and engage in signaling reactions. Using toxin‐based sensors to measure accessible and sequestered cholesterol in cells, we find that HH ligands change cholesterol accessibility selectively in the membrane of the primary cilium. Primary cilia are antenna‐like organelles required for HH signaling in all vertebrates. By compartmentalizing HH signaling in cilia, cells can use accessible cholesterol to communicate between PTCH1 and SMO without interfering with overall cellular cholesterol homeostasis. Our work suggests that the second messenger that communicates the signal between PTCH1 and SMO is the accessibility of cholesterol in a membrane compartment.