CNPY4 inhibits the Hedgehog pathway by modulating membrane sterol lipids

Abstract

Introductory paragraphThe Hedgehog (HH) pathway is critical for development and adult tissue homeostasis1. Aberrant HH signaling can cause congenital malformations, such as digit anomalies and holoprosencephaly2, and other diseases, including cancer3. Signal transduction is initiated by HH ligand binding to the Patched 1 (PTCH1) receptor on primary cilia, thereby releasing inhibition of Smoothened (SMO), a HH pathway activator4. Although cholesterol and several oxysterol lipids, which are enriched in the ciliary membrane, play a crucial role in HH activation4,5, the molecular mechanisms governing the regulation of these lipid molecules remain unresolved. Here, we identify Canopy 4 (CNPY4), a Saposin-like protein, as a regulator of the HH pathway that controls membrane sterol lipid levels. Cnpy4-/- embryos exhibit multiple defects consistent with HH signaling perturbations, most notably changes in digit number. Knockdown of Cnpy4 hyperactivates the HH pathway at the level of SMO in vitro, and elevates membrane levels of accessible sterol lipids such as cholesterol, an endogenous ligand involved in SMO activation6. Thus, our data demonstrate that CNPY4 is a negative regulator that fine-tunes the initial steps of HH signal transduction, revealing a previously undescribed facet of HH pathway regulation that operates through control of membrane composition.