20(S)-Hydroxycholesterol is a selective allosteric activator of the Hedgehog pathway protein Smoothened (EC50=3μM). 20(S)-Hydroxycholesterol binds to the extracellular cysteine-rich domain (CRD) of Smoothened in a stereoselective manner, activating downstream Gli transcription factors. 20(S)-Hydroxycholesterol also enhances osteogenic differentiation of bone marrow stromal cells and synergizes with simvastatin to activate the Raf/MEK/ERK pathway, promoting bone regeneration. 20(S)-Hydroxycholesterol can be used in research related to developmental biology, oncology, and bone and angiogenesis mechanisms[1-4].
In vitro, 20(S)-Hydroxycholesterol (5μM) treatment of cortical neurons cultured on reactive astrocytes for 24 hours significantly increased neurite outgrowth. Treatment of cortical neurons with 20(S)-Hydroxycholesterol (5μM) for 30 minutes significantly increased Y416-Src phosphorylation[5]. A combination of 20(S)-hydroxycholesterol and 22(S)-hydroxycholesterol (1:1) (5μM) was used to treat M2-10B4 mouse bone marrow stromal cells for 10 minutes to 8 hours, followed by culture in differentiation medium for 4 to 14 days. This treatment induced alkaline phosphatase activity, increased mineralization, elevated osteocalcin mRNA expression, and promoted nuclear protein binding to the OSE2 site of the osteocalcin promoter[6].
References:
[1] Nachtergaele S, Mydock LK, Krishnan K, et al. Oxysterols are allosteric activators of the oncoprotein Smoothened. Nat Chem Biol. 2012 Jan 8;8(2):211-20.
[2] Nedelcu D, Liu J, Xu Y, et al. Oxysterol binding to the extracellular domain of Smoothened in Hedgehog signaling. Nat Chem Biol. 2013 Sep;9(9):557-64.
[3] Cheng YS, Zhang T, Ma X, et al. A proteome-wide map of 20(S)-hydroxycholesterol interactors in cell membranes. Nat Chem Biol. 2021 Dec;17(12):1271-1280.
[4] Peyrot SM, Nachtergaele S, Luchetti G, et al. Tracking the subcellular fate of 20(s)-hydroxycholesterol with click chemistry reveals a transport pathway to the Golgi. J Biol Chem. 2014 Apr 18;289(16):11095-11110.
[5] Berretta A, Gowing EK, Jasoni CL, et al. Sonic hedgehog stimulates neurite outgrowth in a mechanical stretch model of reactive-astrogliosis. Sci Rep. 2016 Feb 23;6:21896.
[6] Aghaloo TL, Amantea CM, Cowan CM, et al. Oxysterols enhance osteoblast differentiation in vitro and bone healing in vivo. J Orthop Res. 2007 Nov;25(11):1488-97.