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Distinct Cation Gradients Power Cholesterol Transport at Different Key Points in the Hedgehog Signaling Pathway.

ABSTRACT: Cholesterol plays two critical roles in Hedgehog signaling, a fundamental pathway in animal development and cancer: it covalently modifies the Sonic hedgehog (SHH) ligand, restricting its release from producing cells, and directly activates Smoothened in responding cells. In both contexts, a membrane protein related to bacterial RND transporters regulates cholesterol: Dispatched1 controls release of cholesterylated SHH, and Patched1 antagonizes Smoothened activation by cholesterol. The mechanism and driving force for eukaryotic RND proteins, including Dispatched1 and Patched1, are unknown. Here, we show that Dispatched1 acts enzymatically to catalyze SHH release. Dispatched1 uses the energy of the plasma membrane Na⁺ gradient, thus functioning as an SHH/Na⁺ antiporter. In contrast, Patched1 repression of Smoothened requires the opposing K⁺ gradient. Our results clarify the transporter activity of essential eukaryotic RND proteins and demonstrate that the two main cation gradients of animal cells differentially power cholesterol transport at two crucial steps in the Hedgehog pathway.

SUBMITTER: Petrov K

PROVIDER: S-EPMC7658045 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundSonic hedgehog (Shh) signaling plays a crucial role in growth and patterning during embryonic development, and also in stem cell maintenance and tissue regeneration in adults. Aberrant Shh pathway activation is involved in the development of many tumors, and one of the most affected Shh signaling steps found in these tumors is the regulation of the signaling receptor Smoothened by the Shh receptor Patched. In the present work, we investigated Patched activity and the mechanism by which Patched inhibits Smoothened.Methodology/principal findingsUsing the well-known Shh-responding cell line of mouse fibroblasts NIH 3T3, we first observed that enhancement of the intracellular cholesterol concentration induces Smoothened enrichment in the plasma membrane, which is a crucial step for the signaling activation. We found that binding of Shh protein to its receptor Patched, which involves Patched internalization, increases the intracellular concentration of cholesterol and decreases the efflux of a fluorescent cholesterol derivative (BODIPY-cholesterol) from these cells. Treatment of fibroblasts with cyclopamine, an antagonist of Shh signaling, inhibits Patched expression and reduces BODIPY-cholesterol efflux, while treatment with the Shh pathway agonist SAG enhances Patched protein expression and BODIPY-cholesterol efflux. We also show that over-expression of human Patched in the yeast S. cerevisiae results in a significant boost of BODIPY-cholesterol efflux. Furthermore, we demonstrate that purified Patched binds to cholesterol, and that the interaction of Shh with Patched inhibits the binding of Patched to cholesterol.Conclusion/significanceOur results suggest that Patched may contribute to cholesterol efflux from cells, and to modulation of the intracellular cholesterol concentration. This activity is likely responsible for the inhibition of the enrichment of Smoothened in the plasma membrane, which is an important step in Shh pathway activation.

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Distinct Cation Gradients Power Cholesterol Transport at Different Key Points in the Hedgehog Signaling Pathway.

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