Shh is one of three proteins in the mammalian signaling pathway called hedgehog. Shh plays an important instructional role in mammalian development, localizing in and controlling the activity of the organizing centers that coordinate the specification and growth of numerous structures (such as the growth of digits on limbs and the organization of the brain). Mutations in various components of the Shh signaling pathway are thought to cause developmental disorders in humans. We recently showed that arsenic modulates Shh signaling, and that this modulation occurs at doses relevant to human exposure. Arsenic has been shown to cause birth defects in various animal models, and to increase the risk of birth defects in a small cohort of As exposed patients. Project 4 aims to find out how arsenic changes Shh signaling, and how the extent of this modulation correlates with the incidence of a selection of arsenic-induced development effects.
Project 4 will use a series of pharmacological inhibitors and molecular genetic tools, which activate or attenuate Shh signaling at discrete steps along its signal transduction pathway, to identify how arsenic is able to modulate Shh signaling. These experiments will be performed on As-treated mouse NIH3T3 cells and on immortalized human bronchial epithelial cells (BEAS-2B), both of which respond to arsenic treatment by activating Shh signaling. Results obtained with these various small-molecule modulators will be validated using specific lentiviral RNA constructs and mouse embryo fibroblasts derived from mice lacking various components of the Shh signaling pathway. These latter reagents will result in either increased or decreased Shh signaling, depending on whether the signaling component targeted is a positive or negative acting component of the Shh pathway. Noncanonical Shh signaling pathways will be explored if As modulation of Shh target genes is unaffected.