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.

维生素 D3 (VD3) 是一种抑制 Hedgehog (Hh) 信号通路的seco类固醇。初步研究表明，它的抗 Hh 活性是直接抑制 Smoothened 的结果，Smoothened 是一种七跨膜细胞表面受体，是 Hh 信号级联的关键调节剂。最近，已经提出维生素 D 受体在介导seco类固醇抑制 Hh 信号传导中的作用。在此，进行了一项基于亲和力的蛋白质分析研究，以更好地了解控制 VD3 介导的抗 Hh 活性的细胞蛋白质。我们合成了一种新的生物素化 VD3 类似物 ( 8 ) 用作化学探针来探索seco的细胞结合目标。-甾体支架。通过一系列下拉实验和后续质谱分析，确定热休克蛋白 70 (Hsp70) 是 VD3 的主要结合蛋白。通过一系列生化和细胞测定，Hsp70 被证实为 VD3 的结合靶标。VD3 与 Hsp70 具有微摩尔亲和力。此外，Hsp70 表达的选择性敲低和已知 Hsp70 抑制剂对其活性的药理学抑制均抑制了小鼠基底细胞癌细胞中的 Hh 信号转导，表明 Hsp70 调节适当的 Hh 信号。额外的细胞分析表明，VD3 及其seco甾体代谢物通过不同的机制抑制 Hh 信号传导。