Technical Reference #1903

1903.

Identification of select glucocorticoids as Smoothened agonists: Potential utility for regenerative medicine Jiangbo Wang; Jiuyi Lu; Michael C. Bond; Minyong Chen; Xiu-Rong Ren; H. Kim Lyerly; Larry S. Barak; and Wei Chen, Duke University Medical Center, PNAS, 107(1903), (2010)
Link To Paper

Abstract:
Regenerative medicine holds the promise of replacing damaged tissues largely by stem cell activation. Hedgehog signaling through the plasma membrane receptor Smoothened (Smo) is an important process for regulating stem cell proliferation. The development of Hedgehog-related therapies has been impeded by a lack of US Food and Drug Administration (FDA)-approved Smo agonists. Using a high-content screen with cells expressing Smo receptors and a β-arrestin2-GFP reporter we identified four FDA-approved drugs halcinonide fluticasone clobetasol and fluocinonide as Smo agonists that activate Hedgehog signaling. These drugs demonstrated an ability to bind Smo promote Smo internalization activate Gli and stimulate the proliferation of primary neuronal precursor cells alone and synergistically in the presence of Sonic Hedgehog protein. Halcinonide fluticasone clobetasol and fluocinonide provide an unprecedented opportunity to develop unique clinical strategies to treat Hedgehog-dependent illnesses

Keywords:
Steroids; Hedgehog signaling; Gli; Stem cell proliferation; Arrestin

Materials & Methods:
Bodipy-Cyclopamine Binding Analysis of Smo Agonists in Smo-Overexpressing HEK293 Cells. HEK293 cells stably expressing WTSmo were split at 166000 cells per well in the center well (glass bottom 10-mm diameter) of collagen-coated dishes (MatTek) followed by overnight incubation. Details are provided in SI Materials and Methods.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
HEK293 Cells