Mechanism of Lamellar Body Formation by Lung Surfactant Protein B.

Breathing depends on pulmonary surfactant, a mixture of phospholipids and proteins, secreted by alveolar type II cells. Surfactant requires lamellar bodies (LBs), organelles containing densely packed concentric membrane layers, for storage and secretion. LB biogenesis remains mysterious but requires surfactant protein B (SP-B), which is synthesized as a precursor (pre-proSP-B) that is cleaved during trafficking into three related proteins. Here, we elucidate the functions and cooperation of these proteins in LB formation. We show that the N-terminal domain of proSP-B is a phospholipid-binding and -transfer protein whose activities are required for proSP-B export from the endoplasmic reticulum (ER) and sorting to LBs, the conversion of proSP-B into lipoprotein particles, and neonatal viability in mice. The C-terminal domain facilitates ER export of proSP-B. The mature middle domain, generated after proteolytic cleavage of proSP-B, generates the striking membrane layers characteristic of LBs. Together, our results lead to a mechanistic model of LB biogenesis.

Cilia-Associated Oxysterols Activate Smoothened.

Primary cilia are required for Smoothened to transduce vertebrate Hedgehog signals, but how Smoothened accumulates in cilia and is activated is incompletely understood. Here, we identify cilia-associated oxysterols that promote Smoothened accumulation in cilia and activate the Hedgehog pathway. Our data reveal that cilia-associated oxysterols bind to two distinct Smoothened domains to modulate Smoothened accumulation in cilia and tune the intensity of Hedgehog pathway activation. We find that the oxysterol synthase HSD11ß2 participates in the production of Smoothened-activating oxysterols and promotes Hedgehog pathway activity. Inhibiting oxysterol biosynthesis impedes oncogenic Hedgehog pathway activation and attenuates the growth of Hedgehog pathway-associated medulloblastoma, suggesting that targeted inhibition of Smoothened-activating oxysterol production may be therapeutically useful for patients with Hedgehog-associated cancers.

Endogenous B-ring oxysterols inhibit the Hedgehog component Smoothened in a manner distinct from cyclopamine or side-chain oxysterols.

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-ß-cyclodextrin treatment enhances Hedgehog activation by a pathway agonist. Conversely, exogenous addition of 3ß,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.

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.

Sterol regulation of developmental and oncogenic Hedgehog signaling.

The Hedgehog (Hh) family of lipid-modified signaling proteins directs embryonic tissue patterning and postembryonic tissue homeostasis, and dysregulated Hh signaling drives familial and sporadic cancers. Hh ligands bind to and inhibit the tumor suppressor Patched and allow the oncoprotein Smoothened (SMO) to accumulate in cilia, which in turn activates the GLI family of transcription factors. Recent work has demonstrated that endogenous cholesterol and oxidized cholesterol derivatives (oxysterols) bind and modulate SMO activity. Here we discuss the myriad sterols that activate or inhibit the Hh pathway, with emphasis on endogenous 24(S),25-epoxycholesterol and 3ß,5α-dihydroxycholest-7-en-6-one, and propose models of sterol regulation of SMO. Synthetic inhibitors of SMO have long been the focus of drug development efforts. Here, we discuss the possible utility of steroidal SMO ligands or inhibitors of enzymes involved in sterol metabolism as cancer therapeutics.

Itraconazole inhibits enterovirus replication by targeting the oxysterol-binding protein.

Itraconazole (ITZ) is a well-known antifungal agent that also has anticancer activity. In this study, we identify ITZ as a broad-spectrum inhibitor of enteroviruses (e.g., poliovirus, coxsackievirus, enterovirus-71, rhinovirus). We demonstrate that ITZ inhibits viral RNA replication by targeting oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4). Consistently, OSW-1, a specific OSBP/ORP4 antagonist, also inhibits enterovirus replication. Knockdown of OSBP inhibits virus replication, whereas overexpression of OSBP or ORP4 counteracts the antiviral effects of ITZ and OSW-1. ITZ binds OSBP and inhibits its function, i.e., shuttling of cholesterol and phosphatidylinositol-4-phosphate between membranes, thereby likely perturbing the virus-induced membrane alterations essential for viral replication organelle formation. ITZ also inhibits hepatitis C virus replication, which also relies on OSBP. Together, these data implicate OSBP/ORP4 as molecular targets of ITZ and point to an essential role of OSBP/ORP4-mediated lipid exchange in virus replication that can be targeted by antiviral drugs.

Simultaneous measurement of smoothened entry into and exit from the primary cilium.

Ciliary accumulation of signaling proteins must result from a rate of ciliary entry that exceeds ciliary exit, but approaches for distinguishing ciliary entry vs. exit are lacking. Using a photoconvertible fluorescent protein tag, we establish an assay that allows a separate but simultaneous examination of ciliary entry and exit of the Hedgehog signaling protein Smoothened in individual cells. We show that KAAD-cyclopamine selectively blocks entry, whereas ciliobrevin interferes initially with exit and eventually with both entry and exit of ciliary Smoothened. Our study provides an approach to understanding regulation of ciliary entry vs. exit of Hedgehog signaling components as well as other ciliary proteins.

Hedgehog pathway modulation by multiple lipid binding sites on the smoothened effector of signal response.

Hedgehog (Hh) signaling during development and in postembryonic tissues requires activation of the 7TM oncoprotein Smoothened (Smo) by mechanisms that may involve endogenous lipidic modulators. Exogenous Smo ligands previously identified include the plant sterol cyclopamine (and its therapeutically useful synthetic mimics) and hydroxylated cholesterol derivatives (oxysterols); Smo is also highly sensitive to cellular sterol levels. The relationships between these effects are unclear because the relevant Smo structural determinants are unknown. We identify the conserved extracellular cysteine-rich domain (CRD) as the site of action for oxysterols on Smo, involving residues structurally analogous to those contacting the Wnt lipid adduct in the homologous Frizzled CRD; this modulatory effect is distinct from that of cyclopamine mimics, from Hh-mediated regulation, and from the permissive action of cellular sterol pools. These results imply that Hh pathway activity is sensitive to lipid binding at several Smo sites, suggesting mechanisms for tuning by multiple physiological inputs.

Oxysterols are novel activators of the hedgehog signaling pathway in pluripotent mesenchymal cells.

Pluripotent mesenchymal cells form a population of precursors to a variety of cell types, including osteoblasts and adipocytes. Aging tilts the balance in favor of adipocyte differentiation at the expense of osteoblast differentiation, resulting in reduced bone formation and osteopenic disorders, including osteoporosis, in humans and animals. Understanding the mechanisms involved in causing this apparent shift in differentiation and identifying factors that stimulate osteoblast formation while inhibiting adipogenesis are of great therapeutic interest. In this study we report that specific, naturally occurring oxysterols, previously shown to direct pluripotent mesenchymal cells toward an osteoblast lineage, exert their osteoinductive effects through activation of Hedgehog signaling pathway. This was demonstrated by 1) oxysterol-induced expression of the Hh target genes Gli-1 and Patched, 2) oxysterol-induced activation of a luciferase reporter driven by a multimerized Gli-responsive element, 3) inhibition of oxysterol effects by the hedgehog pathway inhibitor, cyclopamine, and 4) unresponsiveness of Smoothened-/- mouse embryonic fibroblasts to oxysterols. Using Patched-/- cells that possess high baseline Gli activity, we found that oxysterols did not dramatically shift the IC50 concentration of cyclopamine needed to inhibit Gli activity in these cells. Furthermore, binding studies showed that oxysterols did not compete with fluorescently labeled cyclopamine, BODIPY-cyclopamine, for direct binding to Smoothened. These findings demonstrate that oxysterols stimulate hedgehog pathway activity by indirectly activating the seven-transmembrane pathway component Smoothened. Osteoinductive oxysterols are, therefore, novel activators of the hedgehog pathway in pluripotent mesenchymal cells, and they may be important modulators of this critical signaling pathway that regulates numerous developmental and post-developmental processes.

Gp78, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of HMG CoA reductase.

Sterol-regulated ubiquitination is an obligatory step in ER-associated degradation (ERAD) of HMG CoA reductase, a rate-limiting enzyme in cholesterol synthesis. Accelerated degradation of reductase, one of several strategies animal cells use to limit production of cholesterol, requires sterol-induced binding of the enzyme to ER membrane proteins called Insigs. Once formed, the reductase-Insig complex is recognized by a putative membrane-associated ubiquitin ligase (E3) that mediates the reductase ubiquitination reaction. Here, we show that gp78, a membrane bound E3, binds to Insig-1 and is required for sterol-regulated ubiquitination of reductase. In addition, gp78 couples regulated ubiquitination to degradation of reductase by binding to VCP, an ATPase that plays a key role in recognition and degradation of ERAD substrates. The current results identify gp78 as the E3 that initiates sterol-accelerated degradation of reductase, and Insig-1 as a bridge between gp78/VCP and the reductase substrate.