Neuropilins are membrane-spanning proteins predominantly known for acting as co-receptors for class 3 semaphorins and vascular endothelial growth factor (VEGF). Hillman et al. identified Neuropilin 1 (Nrp1) in a small interfering RNA (siRNA)–based screen for regulators of Hedgehog (Hh) signaling in NIH3T3 fibroblasts. Knocking down Nrp1 by siRNA inhibited Sonic hedgehog (Shh)–induced expression of a luciferase reporter that was driven by a promoter that responds to the activity of Gli transcription factors, which are activated by Hh signaling. Knocking down either Nrp1 or Nrp2 inhibited Shh-induced expression of the reporter as well as of endogenous markers of Hh signaling Patched 1 (Ptc1) and Gli1, and knocking down Nrp1 and Nrp2 simultaneously inhibited expression of the reporter and endogenous targets comparably to knocking down the Hh signal transducer Smoothened (Smo). Overexpression of Nrp1 in NIH3T3 cells increased Shh-stimulated activation of the reporter, and this Nrp-mediated increase in Hh signaling was not affected by the presence of the Nrp ligands Sema3A or VEGF164. Morpholino-mediated knockdown of nrp1a in zebrafish embryos caused vascular defects consistent with the role of neuropilins in VEGF signaling but also caused an axial patterning phenotype similar to those of Hh signaling mutants and reduced expression of ptc1. In mouse embryos, Nrp1 protein was detected in several cell types undergoing active Hh signaling, including the dermal papillae and overlying ectoderm of hair follicles. Shh increased the transcription of Nrp1 in NIH3T3 cells and in skin cells isolated from neonatal mice but not in Smo–/– mouse embryo fibroblasts (MEFs) or NIH3T3 cells treated with an siRNA targeting Smo, indicating the presence of positive feedback between Nrp and the Hh pathway. MEFs lacking Ptc1, a transmembrane protein that represses Smo activity until bound by a Hh ligand, exhibit enhanced expression of Gli1. Knocking down both Nrp1 and Nrp2 reduced the abundance of Gli1 at both the transcript and protein levels in Ptc1–/– MEFs but not in MEFs lacking Suppressor of Fused (SuFu), which acts downstream of Smo to inhibit the activity of Gli proteins. The authors therefore conclude that neuropilins act downstream of Smo and upstream of SuFu to affect Hh signaling.
R. T. Hillman, B. Y. Feng, J. Ni, W.-M. Woo, L. Milenkovic, M. G. H. Gephart, M. N. Teruel, A. E. Oro, J. K. Chen, M. P. Scott, Neuropilins are positive regulators of Hedgehog signal transduction. Genes Dev. 25, 2333–2346 (2011). [Abstract] [Full Text]