Randall K. Mann

Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine.

Basal cell carcinoma, medulloblastoma, rhabdomyosarcoma and other human tumours are associated with mutations that activate the proto-oncogene Smoothened (SMO) or that inactivate the tumour suppressor Patched (PTCH). Smoothened and Patched mediate the cellular response to the Hedgehog (Hh) secreted protein signal, and oncogenic mutations affecting these proteins cause excess activity of the Hh response pathway. Here we show that the plant-derived teratogen cyclopamine, which inhibits the Hh response, is a potential ‘mechanism-based’ therapeutic agent for treatment of these tumours. We show that cyclopamine or synthetic derivatives with improved potency block activation of the Hh response pathway and abnormal cell growth associated with both types of oncogenic mutation. Our results also indicate that cyclopamine may act by influencing the balance between active and inactive forms of Smoothened.

Hedgehog signal transduction via Smoothened association with a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2.

The seven-transmembrane protein Smoothened (Smo) transduces extracellular activation of the Hedgehog (Hh) pathway by an unknown mechanism to increase transcriptional activity of the latent cytoplasmic transcription factor Ci (Cubitus interruptus). Here, we present evidence that Smo associates directly with a Ci-containing complex that is scaffolded and stabilized by the atypical kinesin, Costal-2 (Cos2). This complex constitutively suppresses pathway activity, but Hh signaling reverses its regulatory effect to promote Ci-mediated transcription. In response to Hh activation of Smo, Cos2 mediates accumulation and phosphorylation of Smo at the membrane as well as phosphorylation of the cytoplasmic components Fu and Su(fu). Positive response of Cos2 to Hh stimulation requires a portion of the Smo cytoplasmic tail and the Cos2 cargo domain, which interacts directly with Smo.

Cholesterol modification of proteins.

The demonstration over 30 years ago that inhibitors of cholesterol biosynthesis disrupt animal development suggested an intriguing connection between fundamental cellular metabolic processes and the more global processes of embryonic tissue patterning. Adding a new dimension to this relationship is the more recent finding that the Hedgehog family of tissue patterning factors are covalently modified by cholesterol. Here we review the mechanism of the Hedgehog autoprocessing reaction that results in this modification, and compare this reaction to that undergone by other autoprocessing proteins. We also discuss the biological consequences of cholesterol modification, in particular the use of cholesterol as a molecular handle in the spatial deployment of the protein signal in developing tissues. Finally, the developmental consequences of chemical and genetic disruption of cholesterol homeostasis are summarized, along with the potential importance of cholesterol-rich lipid rafts in production of and response to the Hh signal.

Genetic and biochemical definition of the Hedgehog receptor

Although the transporter-like protein Patched (Ptc) is genetically implicated in reception of the extracellular Hedgehog (Hh) protein signal, a clear definition of the Hh receptor is complicated by the existence of additional Hh-binding proteins and, in Drosophila, by the lack of physical evidence for direct binding of Hh to Ptc. Here we show that activity of Ihog (Interference hedgehog), or of its close relative Boi (Brother of Ihog), is absolutely required for Hh biological response and for sequestration of the Hh protein to limit long-range signaling. We demonstrate that Ihog interacts directly with Ptc, is required for presentation of Ptc on the cell surface, and that Ihog and Ptc are both required for high-affinity Hh binding. On the basis of their joint roles in ligand binding, signal transduction, and receptor trafficking, we conclude that Ihog and Ptc together constitute the Drosophila Hh receptor.

Scube/You activity mediates release of dually lipid-modified Hedgehog signal in soluble form.

Owing to their covalent modification by cholesterol and palmitate, Hedgehog (Hh) signaling proteins are localized predominantly to the plasma membrane of expressing cells. Yet Hh proteins are also capable of mobilizing to and eliciting direct responses from distant cells. The zebrafish you gene, identified genetically >15 years ago, was more recently shown to encode a secreted glycoprotein that acts cell-nonautonomously in the Hh signaling pathway by an unknown mechanism. We investigated the function of the protein encoded by murine Scube2, an ortholog of you, and found that it mediates release in soluble form of the mature, cholesterol- and palmitate-modified Sonic hedgehog protein signal (ShhNp) when added to cultured cells or purified detergent-resistant membrane microdomains containing ShhNp. The efficiency of Scube2-mediated release of ShhNp is enhanced by the palmitate adduct of ShhNp and by coexpression in ShhNp-producing cells of mDispatchedA (mDispA), a transporter-like protein with a previously defined role in the release of lipid-modified Hh signals. The structural determinants of Scube2 required for its activity in cultured cell assays match those required for rescue of you mutant zebrafish embryos, and we thus conclude that the role of Scube/You proteins in Hh signaling in vivo is to facilitate the release and mobilization of Hh proteins for distant action.

Endogenous B-ring oxysterols inhibit the hedgehog component smoothened in a manner distinct from cyclopamine or side-chain oxysterols

Significance The Hedgehog protein signal (Hh), covalently modified by cholesterol, functions to coordinate embryonic tissue patterning and postembryonic tissue maintenance. Cholesterol and several of its side-chain oxidized derivatives also function in Hh response by augmenting the activity of Smoothened, an essential seven-transmembrane protein. Here, we show that a distinct class of sterols, oxidized in the B-ring, also affect Hh response, but act by a distinct mechanism to inhibit Smoothened activity. These sterols and their precursor, 7-dehydrocholesterol, accumulate in the human genetic disease Smith–Lemli–Opitz syndrome, providing a rationale for diminished Hedgehog pathway activity in Smith–Lemli–Opitz syndrome and suggesting new candidates as potential modulators of Smoothened activity in normal cells. Cellular lipids are speculated to act as key intermediates in Hedgehog signal transduction, but their precise identity and function remain enigmatic. In an effort to identify such lipids, we pursued a Hedgehog pathway inhibitory activity that is particularly abundant in flagellar lipids of Chlamydomonas reinhardtii, resulting in the purification and identification of ergosterol endoperoxide, a B-ring oxysterol. A mammalian analog of ergosterol, 7-dehydrocholesterol (7-DHC), accumulates in Smith–Lemli–Opitz syndrome, a human genetic disease that phenocopies deficient Hedgehog signaling and is caused by genetic loss of 7-DHC reductase. We found that depleting endogenous 7-DHC with methyl-β-cyclodextrin treatment enhances Hedgehog activation by a pathway agonist. Conversely, exogenous addition of 3β,5α-dihydroxycholest-7-en-6-one, a naturally occurring B-ring oxysterol derived from 7-DHC that also accumulates in Smith–Lemli–Opitz syndrome, blocked Hedgehog signaling by inhibiting activation of the essential transduction component Smoothened, through a mechanism distinct from Smoothened modulation by other lipids.

Bifurcating action of Smoothened in Hedgehog signaling is mediated by Dlg5

Binding of the Hedgehog (Hh) protein signal to its receptor, Patched, induces accumulation of the seven-pass transmembrane protein Smoothened (Smo) within the primary cilium and of the zinc finger transcription factor Gli2 at the ciliary tip, resulting ultimately in Gli-mediated changes in nuclear gene expression. However, the mechanism by which pathway activation is communicated from Smo to Gli2 is not known. In an effort to elucidate this mechanism, we identified Dlg5 (Discs large, homolog 5) in a biochemical screen for proteins that preferentially interact with activated Smo. We found that disruption of Smo-Dlg5 interactions or depletion of endogenous Dlg5 leads to diminished Hh pathway response without a significant impact on Smo ciliary accumulation. We also found that Dlg5 is localized at the basal body, where it associates with another pathway component, Kif7. We show that Dlg5 is required for Hh-induced enrichment of Kif7 and Gli2 at the tip of the cilium but is dispensable for Gpr161 exit from the cilium and the consequent suppression of Gli3 processing into its repressor form. Our findings suggest a bifurcation of Smo activity in Hh response, with a Dlg5-independent arm for suppression of Gli repressor formation and a second arm involving Smo interaction with Dlg5 for Gli activation.

Neuronal delivery of Hedgehog directs spatial patterning of taste organ regeneration

Significance The maintenance of taste sensory organs (taste buds) in the tongue has been known for 140 years to depend on sensory innervation from distant neurons by an unknown mechanism. We find that maintenance and regeneration of taste receptor cells (TRCs) within taste buds requires neuronal delivery of the Sonic hedgehog (Shh) protein signal, thus explaining loss of taste sensation associated with Hedgehog pathway antagonism in patients and illustrating the principle that spatial patterning of TRC regeneration is specified by the projection pattern of Shh-expressing sensory neurons. We also find that pharmacologic Hedgehog pathway activation accelerates TRC recovery, suggesting a means to ameliorate the loss of taste sensation and appetite and the associated delay in recovery in cancer patients undergoing chemotherapy. How organs maintain and restore functional integrity during ordinary tissue turnover or following injury represents a central biological problem. The maintenance of taste sensory organs in the tongue was shown 140 years ago to depend on innervation from distant ganglion neurons, but the underlying mechanism has remained unknown. Here, we show that Sonic hedgehog (Shh), which encodes a secreted protein signal, is expressed in these sensory neurons, and that experimental ablation of neuronal Shh expression causes loss of taste receptor cells (TRCs). TRCs are also lost upon pharmacologic blockade of Hedgehog pathway response, accounting for the loss of taste sensation experienced by cancer patients undergoing Hedgehog inhibitor treatment. We find that TRC regeneration following such pharmacologic ablation requires neuronal expression of Shh and can be substantially enhanced by pharmacologic activation of Hedgehog response. Such pharmacologic enhancement of Hedgehog response, however, results in additional TRC formation at many ectopic sites, unlike the site-restricted regeneration specified by the projection pattern of Shh-expressing neurons. Stable regeneration of TRCs thus requires neuronal Shh, illustrating the principle that neuronal delivery of cues such as the Shh signal can pattern distant cellular responses to assure functional integrity during tissue maintenance and regeneration.