Affinity-based protein profiling identifies vitamin D3 as a heat shock protein 70 antagonist that regulates hedgehog...

Vitamin D3 (VD3) is a seco-steroid that inhibits the Hedgehog (Hh) signaling pathway. Initial studies suggested its anti-Hh activity results from direct inhibition of Smoothened, a seven-transmembrane cell surface receptor that is a key regulator of the Hh signaling cascade. More recently, a role for the Vitamin D Receptor in mediating inhibition of Hh-signaling by seco-steroid has been suggested. Herein, an affinity-based protein profiling study was carried out to better understand the cellular proteins that govern VD3-mediated anti-Hh activity. We synthesized a novel biotinylated VD3 analogue (8) for use as a chemical probe to explore cellular binding targets of the seco-steroidal scaffold. Through a series of pull-down experiments and follow up mass spectrum analyses, heat shock protein 70 (Hsp70) was identified as a primary binding protein of VD3. Hsp70 was validated as a binding target of VD3 through a series of biochemical and cellular assays. VD3 bound with micromolar affinity to Hsp70. In addition, both selective knockdown of Hsp70 expression and pharmacological inhibition of its activity with known Hsp70 inhibitors suppressed Hh-signaling transduction in murine basal cell carcinoma cells, suggesting that Hsp70 regulates proper Hh-signaling. Additional cellular assays suggest that VD3 and its seco-steroidal metabolites inhibit Hh-signaling through different mechanisms.