Cyclopamine inhibition of sonic hedgehog signal transduction is not mediated through effects on cholesterol transport

Cyclopamine is a teratogenic steroidal alkaloid that causes cyclopia by blo cking Sonic hedgehog (Shh) signal transduction. We have tested whether this activity of cyclopamine is related to disruption of cellular cholesterol t ransport and putative secondary effects on the Shh receptor, Patched (Ptc). First, we report that the potent antagonism of Shh signaling by cyclopamin e is not a general property of steroidal alkaloids with similar structure. The structural features of steroidal alkaloids previously associated with t he induction of holoprosencephaly in whole animals are also associated with inhibition of Shh signaling in vitro. Second, by comparing the effects of cyclopamine on Shh signaling with those of compounds known to block cholest erol transport, we show that the action of cyclopamine cannot be explained by inhibition of intracellular cholesterol transport. However, compounds th at block cholesterol transport by affecting the vesicular trafficking of th e Niemann-Pick C1 protein (NPC1), which is structurally similar to Ptc, are weak Shh antagonists. Rather than supporting a direct link between cholest erol homeostasis and Shh signaling, our findings suggest that the functions of both NPC1 and Ptc involve a common vesicular transport pathway. Consist ent with this model, we find that Ptc and NPC1 colocalize extensively in a vesicular compartment in cotransfected cells. (C) 2000 Academic Press.