Sonic Hedgehog as a mediator of long-range signaling

The ability of Hedgehog (Hh) proteins to exert their biological effects is regulated by a series of post-translational processes. These processes include an intramolecular cleavage, covalent addition of cholesterol and/or palmitate, and conversion into a multimeric freely diffusible form. The processing of Hh proteins affects their trafficking, potency, and ability to signal over many cell diameters. Accordingly, the loss of gene products required for these processes abrogates the Hh proteins' abilities to exert their effects, which can be long range, short range, or both. We review here recent evidence demonstrating that Hh proteins are directly responsible for their long-range biological effects. Additionally, we integrate both genetic and biochemical data to delineate a model illustrating how the unusual biochemistry of Hh family members may allow them to act as morphogens, signaling over both short and long distances.     

Shifting paradigms in Hedgehog signaling

Hedgehog (Hh) signaling proteins regulate multiple developmental and adult homeostatic processes. A defining feature of Hh signaling is that relatively small changes in the concentration of Hh ligand elicit dramatically different cellular responses. As a result, the processing, release and trafficking of Hh ligands must be tightly regulated to ensure proper signaling. In addition, sensitive and specific intracellular signaling cascades are needed to interpret subtle differences in the level of Hh signal to execute an appropriate response. A detailed understanding of the mechanisms that regulate these responses is critical to shaping our view of this key regulatory system.     

Hedgehog signaling: mechanisms and evolution

The Hedgehog (Hh) family of secreted proteins plays essential roles in the development of a wide variety of animal species and underlies multiple human birth defects and cancers.To.ensure the proper ra...     

Boc and Gas1 each form distinct Shh receptor complexes with Ptch1 and are required for Shh-mediated cell proliferation

Hedgehog (Hh) proteins regulate important developmental processes, including cell proliferation and differentiation. Although Patched acts as the main Hh receptor in Drosophila, Hh signaling absolutely requires the additional Hh-binding proteins Ihog and Boi. Here we show that, unexpectedly, cerebellar granule neuron progenitors (CGNPs) lacking Boc and Cdon, the vertebrate orthologs of Ihog and Boi, still proliferate in response to Hh. This is because in their absence, Gas1, an Hh-binding protein not present in Drosophila, mediates Hh signaling. Consistently, only CGNPs lacking all three molecules-Boc, Cdon, and Gas1-have a complete loss of Hh-dependent proliferation. In a complementary manner, we find that a mutated Hh ligand that binds Patched1 but not Boc, Cdon, or Gas1 cannot activate Hh signaling. Together, this demonstrates an absolute requirement for Boc, Cdon, and Gas1 in Hh signaling and reveals a distinct requirement for ligand-binding components that distinguishes the vertebrate and invertebrate Hh receptor systems.     

Mutations in the sterol-sensing domain of Patched suggest a role for vesicular trafficking in Smoothened regulation

The tumor suppressor gene patched (ptc) encodes an approximately 140 kDa polytopic transmembrane protein [1-3] [corrected] that binds members of the Hedgehog (Hh) family of signaling proteins [4-6] [corrected] and regulates the activity of Smoothened (Smo), a G protein-coupled receptor-like protein essential for Hh signal transduction [7-9] [corrected]. Ptc contains a sterol-sensing domain (SSD) [10, 11] [corrected], a motif found in proteins implicated in the intracellular trafficking of cholesterol [12] [corrected], and/or other cargoes [13-15] [corrected]. Cholesterol plays a critical role in Hedgehog (Hh) signaling by facilitating the regulated secretion and sequestration of the Hh protein [16] [corrected], to which it is covalently coupled. In addition, cholesterol synthesis inhibitors block the ability of cells to respond to Hh [18, 19] [corrected], and this finding points to an additional requirement for the lipid in regulating downstream components of the Hh signaling pathway. Although the SSD of Ptc has been linked to both the sequestration of, and the cellular response to Hh [16, 20, 21] [corrected], definitive evidence for its function has so far been lacking. Here we describe the identification and characterization of two missense mutations in the SSD of Drosophila Ptc; strikingly, while both mutations abolish Smo repression, neither affects the ability of Ptc to interact with Hh. We speculate that Ptc may control Smo activity by regulating an intracellular trafficking process dependent upon the integrity of the SSD.     

The contrasting roles of primary cilia and cytonemes in Hh signaling

Hedgehog (Hh) is a paracrine signaling protein with major roles in development and disease. In vertebrates and invertebrates, Hh signal transduction is carried out almost entirely by evolutionarily conserved components, and in both, intercellular movement of Hh is mediated by cytonemes – specialized filopodia that serve as bridges that bring distant cells into contact. A significant difference is the role of the primary cilium, a slender, tubulin-based protuberance of many vertebrate cells. Although the primary cilium is essential for Hh signaling in cells that have one, most Drosophila cells lack a primary cilium. This perspective addresses the roles of primary cilia and cytonemes, and proposes that for Hh signaling, the role of primary cilia is to provide a specialized hydrophobic environment that hosts lipid-modified Hh and other components of Hh signal transduction after Hh has traveled from elsewhere in the cell. Implicit in this model is the idea that initial binding and uptake of Hh is independent of and segregated from the processes of signal transduction and activation.     

The zebrafish iguana locus encodes Dzip1, a novel zinc-finger protein required for proper regulation of Hedgehog signaling

Members of the Hedgehog (Hh) family of intercellular signaling molecules play crucial roles in animal development. Aberrant regulation of Hh signaling in humans causes developmental defects, and leads to various genetic disorders and cancers. We have characterized a novel regulator of Hh signaling through the analysis of the zebrafish midline mutant iguana (igu). Mutations in igu lead to reduced expression of Hh target genes in the ventral neural tube, similar to the phenotype seen in zebrafish mutants known to affect Hh signaling. Contradictory at first sight, igu mutations lead to expanded Hh target gene expression in somites. Genetic and pharmacological analyses revealed that the expression of Hh target genes in igu mutants requires Gli activator function but does not depend on Smoothened function. Our results show that the ability of Gli proteins to activate Hh target gene expression in response to Hh signals is generally reduced in igu mutants both in the neural tube and in somites. Although this reduced Hh signaling activity leads to a loss of Hh target gene expression in the neural tube, the same low levels of Hh signaling appear to be sufficient to activate Hh target genes throughout somites because of different threshold responses to Hh signals. We also show that Hh target gene expression in igu mutants is resistant to increased protein kinase A activity that normally represses Hh signaling. Together, our data indicate that igu mutations impair both the full activation of Gli proteins in response to Hh signals, and the negative regulation of Hh signaling in tissues more distant from the source of Hh. Positional cloning revealed that the igu locus encodes Dzip1, a novel intracellular protein that contains a single zinc-finger protein-protein interaction domain. Overexpression of Igu/Dzip1 proteins suggested that Igu/Dzip1 functions in a permissive way in the Hh signaling pathway. Taken together, our studies show that Igu/Dzip1 functions as a permissive factor that is required for the proper regulation of Hh target genes in response to Hh signals.     

Emerging roles for hedgehog-patched-Gli signal transduction in reproduction

Hedgehog (Hh) proteins are expressed during vertebrate development in some tissues with inductive properties and at epithelial-mesenchymal boundaries in several developing organs, including the lung, gut, hair follicle, and tooth. The Hh signaling pathway is highly conserved, and important clues to understanding the mechanism of Hh signal transduction in vertebrates have come from studies in Drosophila. In recent years, Hh signaling has been recognized during embryonic development and in some cases during postnatal life in several mammalian tissues whose functions are essential for reproduction, including the gonads, uterus, and hormonally responsive accessory sex glands such as the prostate and mammary gland. The role of the pathway in these tissues is highly reminiscent of its role at epithelial-mesenchymal-stromal boundaries in other organ systems, which has provided a framework within which to explore Hh signaling in tissues that function in reproduction. Some features unique to these tissues are emerging, including a role in proliferation and differentiation of male germline cells in mammals and apparent influences of sex steroids on Hh signaling. However, many questions remain about the function of Hh signaling in the gonads, uterus, prostate, and mammary gland, including factors regulating the signal transduction pathway, identification of downstream target genes, and roles for Hh signaling in diseases involving these tissues.     

Hedgehog secretion and signal transduction in vertebrates

Signaling by the Hedgehog (Hh) family of secreted proteins is essential for proper embryonic patterning and development. Dysregulation of Hh signaling is associated with a variety of human diseases ranging from developmental disorders such as holoprosencephaly to certain forms of cancer, including medulloblastoma and basal cell carcinoma. Genetic studies in flies and mice have shaped our understanding of Hh signaling and revealed that nearly all core components of the pathway are highly conserved. Although many aspects of the Drosophila Hh pathway are conserved in vertebrates, mechanistic differences between the two species have begun to emerge. Perhaps the most striking divergence in vertebrate Hh signaling is its dependence on the primary cilium, a vestigial organelle that is largely absent in flies. This minireview will provide an overview of Hh signaling and present recent insights into vertebrate Hh secretion, receptor binding, and signal transduction.     

The ihog cell-surface proteins bind Hedgehog and mediate pathway activation

The ihog gene (interference hedgehog), identified by RNA interference in Drosophila cultured cells, encodes a type 1 membrane protein shown here to bind and to mediate response to the active Hedgehog (Hh) protein signal. ihog mutations produce defects characteristic of Hh signaling loss in embryos and imaginal discs, and epistasis analysis places ihog action at or upstream of the negatively acting receptor component, Patched (Ptc). The first of two extracellular fibronectin type III (FNIII) domains of the Ihog protein mediates a specific interaction with Hh protein in vitro, but the second FNIII domain is additionally required for in vivo signaling activity and for Ihog-enhanced binding of Hh protein to cells coexpressing Ptc. Other members of the Ihog family, including Drosophila Boi and mammalian CDO and BOC, also interact with Hh ligands via a specific FNIII domain, thus identifying an evolutionarily conserved family of membrane proteins that function in Hh signal response.     

Cross talk between hedgehog and bone morphogenetic proteins occurs during cardiomyogenesis in P19 cells

Hedgehog (Hh) signaling plays a role in heart morphogenesis and can initiate cardiomyogenesis in P19 cells. To determine if Hh signaling is essential for P19 cell cardiomyogenesis, we determined which Hh factors are expressed and the effect of Hh signal transduction inhibitors. Here, we find that the Hh gene family and their downstream mediators are expressed during cardiomyogenesis but an active Hh signaling pathway is not essential. However, loss of Hh signaling resulted in a delay of BMP-4, GATA-4, Gli2, and Meox1 expression during cardiomyogenesis. By using Noggin-overexpressing P19 cells, we determined that Hh signaling was not active during Noggin-mediated inhibition of cardiomyogenesis. Thus, there is cross talk between the Hh and BMP signaling pathways and the Hh pathway appears important for timely cardiomyogenesis.     

Kinetic and structural studies on interactions between heparin or heparan sulfate and proteins of the hedgehog signaling pathway

Heparan sulfate (HS) proteoglycans (PGs) interact with a number of extracellular signaling proteins, thereby playing an essential role in the regulation of many physiological processes. These interactions are important for both normal signal transduction and regulation of the tissue distribution of signaling molecules. In this study, we use surface plasmon resonance (SPR) to study interactions of HS and structurally related heparin with proteins in the Hedgehog signaling pathway. SPR analysis shows that heparin binds with different affinities to active fragments of the proteins Hedgehog (Hh), Interference Hedgehog (Ihog), Cam-related/Down-regulated by Oncogenes (CDO), and Sonic Hedgehog (Shh). Solution competition studies show that the minimum size of a heparin oligosaccharide capable of interacting with Ihog is larger than a tetrasaccharide and for interacting with Shh is larger than an octasaccharide. In comparison with heparin, Ihog and Shh exhibited a lower affinity for HS than for heparin, and CDO and Hh exhibit negligible binding to HS. This study clearly demonstrates Shh and Ihog are heparin and HS binding proteins and that both molecules preferentially bind heparin or HS having a high level of sulfation.     

Regulator of G-Protein Signaling – 5 (RGS5) Is a Novel Repressor of Hedgehog Signaling

Hedgehog (Hh) signaling plays fundamental roles in morphogenesis, tissue repair, and human disease. Initiation of Hh signaling is controlled by the interaction of two multipass membrane proteins, patched (Ptc) and smoothened (Smo). Recent studies identify Smo as a G-protein coupled receptor (GPCR)-like protein that signals through large G-protein complexes which contain the Gα_i subunit. We hypothesize Regulator of G-Protein Signaling (RGS) proteins, and specifically RGS5, are endogenous repressors of Hh signaling via their ability to act as GTPase activating proteins (GAPs) for GTP-bound Gα_i, downstream of Smo. In support of this hypothesis, we demonstrate that RGS5 over-expression inhibits sonic hedgehog (Shh)-mediated signaling and osteogenesis in C3H10T1/2 cells. Conversely, signaling is potentiated by siRNA-mediated knock-down of RGS5 expression, but not RGS4 expression. Furthermore, using immuohistochemical analysis and co-immunoprecipitation (Co-IP), we demonstrate that RGS5 is present with Smo in primary cilia. This organelle is required for canonical Hh signaling in mammalian cells, and RGS5 is found in a physical complex with Smo in these cells. We therefore conclude that RGS5 is an endogenous regulator of Hh-mediated signaling and that RGS proteins are potential targets for novel therapeutics in Hh-mediated diseases.