Hedgehog Proteins Act Through a Receptor Complex of Patched and Smoothened, Which Oppose Each Other

Like Wnt proteins, the Hedgehog proteins are a family of secreted signal molecules that act as local mediators in many developmental processes in both invertebrates and vertebrates. Abnormalities in the Hedgehog pathway during development can be lethal and in adult cells can also lead to cancer. The Hedgehog proteins were discovered in Drosophila, where a mutation in the only gene encoding such a protein produces a larva with spiky processes (denticles) resembling a hedgehog. At least three genes encode Hedgehog proteins in vertebrates—sonic, desert, and indian hedgehog. The active form of all Hedgehog proteins is unusual in that it is covalently coupled to cholesterol, which helps to restrict its diffusion following secretion. The cholesterol is added during a remarkable processing step, in which the protein cleaves itself. The proteins are also modified by the addition of a fatty acid chain, which, for unknown reasons, can be required for their signaling activity.

Two transmembrane proteins, Patched and Smoothened, mediate the responses to all Hedgehog proteins. Patched is predicted to cross the plasma membrane 12 times, and it is the receptor that binds the Hedgehog protein. In the absence of a Hedgehog signal, Patched inhibits the activity of Smoothened, which is a 7-pass transmembrane protein with a structure similar to a Frizzled protein. This inhibition is relieved when a Hedgehog protein binds to Patched, allowing Smoothened to relay the signal into the cell. Most of what we know about the downstream signaling pathway activated by Smoothened comes from genetic studies in flies, and it is the fly pathway that we summarize here.

In some respects the Hedgehog signaling pathway in Drosophila operates similarly to the Wnt pathway. In the absence of a Hedgehog signal, a gene regulatory protein called Cubitus interruptus (Ci) is proteolytically cleaved in proteasomes. Instead of being completely degraded, however, it is processed to form a smaller protein that accumulates in the nucleus, where it acts as a transcriptional repressor, helping to keep some Hedgehog-responsive genes silent. The proteolytic processing of the Ci protein depends on a large multiprotein complex. The complex contains a serine/threonine kinase (called Fused) of unknown function, an anchoring protein (called Costal) that binds the complex to microtubules (keeping Ci out of the nucleus), and an adaptor protein (called Suppressor of Fused)



Figure 15-73. A model for Hedgehog signaling in Drosophila. (A) In the absence of Hedgehog, the Patched receptor inhibits Smoothened probably by promoting the degradation or intracellular sequestration of Smoothened. The Ci protein is located in a protein complex and is cleaved to form a transcriptional repressor, which accumulates in the nucleus to help keep Hedgehog target genes inactive. The protein complex includes the serine/threonine kinase Fused, the anchoring protein Costal (which binds the complex to microtubules), and the adaptor protein Suppressor of Fused. (B) Hedgehog binding to Patched relieves the inhibition of Smoothened, which now signals to the protein complex to stop processing Ci, to dissociate from microtubules, and to release the unprocessed Ci so it can accumulate in the nucleus and activate the transcription of Hedgehog-responsive genes. Most of the molecular events in the pathway are unknown.

When Hedgehog binds to Patched to activate the signaling pathway, Ci processing is suppressed, and the unprocessed Ci protein is released from its complex and enters the nucleus, where it activates the transcription of Hedgehog target genes (Figure 15-73B).

Among the genes activated by Ci is the gene that encodes the Wnt protein Wingless, which helps pattern tissues in the fly embryo (discussed in Chapter 21). Another target gene is patched itself; the resulting increase in Patched protein on the cell surface inhibits further Hedgehog signaling—a form of negative feedback.

Many gaps in the Hedgehog signaling pathway still remain to be filled in. It is not known, for example, how Patched inhibits Smoothened, how Smoothened activates the pathway, how the proteolysis of Ci is regulated (although it is known that Ci phosphorylation by PKA is required for the processing), or how the release of the complex from microtubules and unprocessed Ci from the complex is controlled.

Even less is known about the Hedgehog pathway in vertebrate cells. In addition to there being at least three types of vertebrate Hedgehog proteins, there are two forms of Patched and three Ci-like proteins (Gli1, Gli2, and Gli3). Unlike in flies, Hedgehog signaling stimulates the transcription of the Gli genes, and it is unclear whether all of the Gli proteins undergo proteolytic processing, although there is evidence that Gli3 does. Inactivating mutations in one of the human patched genes, which leads to excessive Hedgehog signaling, occur frequently in the most common form of skin cancer (basal cell carcinoma), suggesting that Patched normally helps to keep skin cell proliferation in check.

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Sonic Hedgehog (Shh)
SONIC HEDGEHOG (SHH)
First printed in R&D Systems' 2002 catalog.
Contents

* Introduction
* Hedgehog Ligands
* Signaling Pathway
* Long- and Short-range Signaling
* Shh in Vertebrate Development
* Holoprosencephaly
* Left-Right Asymmetry
* Hematopoiesis
* Vascularization
* References
Introduction

Cell-cell signaling is a crucial aspect of development and yet just five signal transduction pathways mediate the early development of most animals. These intercellular signaling pathways consist of: Wnt, TGF-ß, Notch, RTK (receptor tyrosine kinase) and Hedgehog pathways.1 The most common target of signaling in development is transcription. Different pathways activate or repress different genes at distinct times and places in the embryo.1 Signaling pathways have important roles in determining embryonic patterning and cell fate decisions. Analysis of Drosophila development has been vital in elucidating the components and functions of these signaling pathways. Research in vertebrates revealed that not only are the same signaling components also found, but often the developmental roles were similar to those in Drosophila. This review will focus on one of the vertebrate Hedgehogs, Sonic hedgehog, by addressing its signaling mechanism and roles in vertebrate development.
Hedgehog Ligands

The vertebrate hedgehog family is represented by at least three members: Desert hedgehog (Dhh), Indian hedgehog (Ihh) and Sonic hedgehog (Shh). The original hedgehog gene, identified in Drosophila, is named after its mutant phenotype: the embryo is covered with pointy denticles, resembling a hedgehog. Two vertebrate genes are named after species of hedgehogs (dhh, ihh) and shh after a video game character. Shh is the most extensively characterized vertebrate homolog, and is involved in a wide variety of embryonic events. It can act as both a short-range, contact-dependent factor and as a long-range, diffusible morphogen.2,3 Shh genes are highly conserved and have been identified within a variety of species, including human, mouse, frog, fish, and chicken. Mouse and human Shh proteins are 92% identical at the amino acid level.4 In the human embryo, Shh is expressed in the notochord, the floorplate of the neural tube, the gut, and in the developing limbs.5
[img]http://www.rndsystems.com/DAM_public/5201.jpg[img]
Fig. 1. Shh undergoes autocatalytic processing prior to secretion. The Shh precursor protein is cleaved to yield an ~20 kDa N-terminal domain (signaling domain) and an ~25 kDa C-terminal domain (catalytic domain). Cholesterol modification is important for secretion and activity of the Shh protein. [Note: figure is adapted from reference 7.]

Hedgehog proteins undergo autocatalytic processing and modification that is critical for signaling activity (see Figure 1). The ~45 kDa precursor protein is cleaved to yield an ~20 kDa N-terminal domain and an ~25 kDa C-terminal domain.6 Autoprocessing of Hedgehog also causes the covalent attachment of cholesterol onto the carboxy terminus of the N-terminal domain. The N-terminal domain retains all known signaling capabilities while the C-terminal domain is responsible for the intramolecular precursor processing, acting as a cholesterol transferase.6 The cholesterol moiety is thought to direct Hedgehog protein traffic in the secretory cell.7 Another form of human Shh is also produced by mammalian and insect cells. This form contains both cholesterol and palmitoyl groups. The site of the palmitic acid modification was identified at Cys-24, on the N-terminal domain.8 These lipid modifications do not affect binding affinity of Shh to the Patched-1 receptor but do significantly enhance the activity of Shh protein in the C3H10T1/2 alkaline phosphatase assay.8 Thus, cholesterol may be important in the Hedgehog pathway not only for signal generation, affecting secretion, but also for signal reception, affecting activity.9
Signaling Pathway

The canonical Hedgehog signaling pathway is a tale of two transmembrane proteins (see Figure 2). Patched (Ptc), a twelve-pass membrane protein binds Hedgehog ligand. Smoothened (Smo), a seven-pass membrane protein is a signal transducer. In the absence of ligand, Ptc interacts with and inhibits Smo, either directly or indirectly. This repression culminates in a transcription factor acting as a transcriptional repressor. The transcription factor is called Cubitus interruptus (Ci) in Drosophila and Gli in vertebrates. There are 3 Gli genes in vertebrates, each with distinct transcriptional functions. When Hedgehog binds Ptc, Ptc's interactions with Smo are altered such that Smo is no longer inhibited. This leads to Ci/Gli protein entering the nucleus and acting as a transcriptional activator for the same genes it represses when Ptc is free to interact with and inhibit Smo. Studies in vertebrates indicate that the determination of diverse cell fates by Shh signaling occurs by regulating the combination of Gli genes expressed in a cell (for reviews, see references 7 and 10-12).


Fig. 2. The Shh signaling pathway involves two transmembrane proteins, Patched (Ptc) and Smoothened (Smo). Ptc binds Shh, whereas Smo acts as a signal transducer. In the absence of ligand, Ptc interacts with and inhibits Smo. This inhibition activates a transcriptional repressor (e.g. Gli in vertebrates). In the presence of ligand, the interaction of Ptc and Smo is altered and Smo is no longer inhibited. Gli protein may then enter the nucleus and function as a transcriptional activator. [Note: figure is adapted from reference 7.]

This model of Hedgehog signal transduction was predominantly worked out in Drosophila, where mutational analysis is easier and faster than in mammals; however, the model is holding up well in vertebrate studies. Binding of Ptc to Smo has been demonstrated for vertebrate homologs, although it is unknown whether binding is direct, since it was observed in the context of other cellular proteins.12 Mammals may express at least two Patched proteins, Patched-1 (Ptc-1) and Patched-2 (Ptc-2), both of which bind vertebrate Hedgehogs with similar affinity.13 As expected from the model, mice lacking Ptc-1 are lacking the inhibition of Smo activity and have constitutively activated Shh response genes in target tissues.14 Ptc-2 is likely to play a role in Dhh signaling as the expression patterns are similar, but a mutant Ptc-2 phenotype has not been reported.13 Analysis of Smo null mutant mice reveals phenotypes identical to Shh mutants and additional phenotypes not associated with Shh. These results indicate that Smo activity during development is likely not limited to activation of the Shh pathway.15 Interestingly, in Drosophila, Hedgehog-independent Ptc function and Ptc- independent Hedgehog function have also been observed.10 Thus, the Hedgehog signaling pathway may be more complicated than the canonical model suggests, as it is likely that pathway components interact with additional ligands and receptors that have yet to be identified.
Long- and Short-range Signaling

In flies and vertebrates, Hedgehog ligands can act either in short-range or long-range signaling. Both short and long-range signaling mechanisms are mediated by the N-terminal peptide after autoprocessing.16 Due to the addition of lipid moieties during processing, Shh N-terminal peptide is localized to the cell membrane.17 Short-range signaling events, over 1 or 2 cell diameters and in a contact-dependent manner, are easier to reconcile with a membrane-associated ligand. How then does the lipid-modified Shh mediate long-range signaling, where different concentrations of Shh induce different cell fates? Three mechanisms have been proposed to explain the long-range effects of Shh: simple diffusion of Shh; a relay mechanism in which Shh activates secondary signals; and direct delivery of Shh through cytoplasmic extensions.18

Cell culture and in vivo studies support the simple diffusion model of long-range signaling. Initial studies using a soluble, N-terminal processed form of Shh demonstrate that it is sufficient to direct long-range effects in vertebrate motor neuron induction in chick embryos.19 Although this soluble form lacks lipid modifications, a recent report provides evidence that a native, cholesterol-modified form of Shh is biologically potent and mediates long range signaling.18 Soluble, lipid processed Shh (s-ShhNp) is formed by concentrating cholesterol-modified, processed Shh into lipid rafts, where it multimerizes with the lipid attachments on the inside of the multimer. This soluble form of Shh can be isolated from chick limb buds, a biologically relevant source of Shh. In such tissues, s-ShhNp is thought to be freely diffusible and able to form a gradient to facilitate long-range signaling.18 Likewise in the mouse limb bud, cholesterol modification is essential for the normal range of signaling.20 Further studies in the chick neural tube using a deleted form of Ptc-1 to block Hedgehog signals, also support a gradient mechanism where Shh acts directly and at long-range.21 Importantly, visualizing Shh at a distance from sites of expression has now been achieved. Using optimized immunohistochemistry techniques in the mouse, Shh ligand is detectable over considerable distances depending on the tissue. Visualization of Shh proteins in target tissues is achieved under conditions allowing proteoglycan/glycosaminoglycan (PG/GAG) preservation, suggesting a role for PG/GAG in controlling Shh movement during long-range signaling.22 These results all support the model of direct, diffusible, long-range action by Shh.
Shh in Vertebrate Development

The Shh signaling pathway functions throughout development. Shh is involved in the determination of cell fate and embryonic patterning during early vertebrate development. An example of this activity is the patterning of the neural tube such that motor neurons are derived from the ventral region and sensory neurons are formed from the dorsal region.10 Later in development, Shh is involved in the proper formation and function of a variety of tissues and organs. Table 1 shows a list of Shh-dependent organs and tissues in vertebrate development. It should be noted that in some cases Shh works with other signaling factors such as FGFs, Wnts, and BMPs to mediate developmental processes.

Vertebrate embryonic development utilizes both short- and long-range mechanisms of Shh signaling. Short-range signaling by Shh is apparent during floor plate induction by the notochord within the neural tube.2 Long-range signaling by Shh occurs during motor neuron formation in the neural tube, sclerotome induction and proliferation in the somites, and limb patterning along the anterior-posterior axis.23 These developmental events were some of the first to be characterized for Shh signaling. In other embryonic patterning events where Shh is known to have activity, it is still unclear whether signaling acts locally or at a distance from Shh-producing cells. For this review, only several of the multiple developmental events in which Shh plays a role will be described.
Holoprosencephaly

The importance of Shh signaling in human development became evident by the discovery that mutations in Shh cause Holoprosencephaly (HPE). HPE is a developmental disorder that affects the midline of the face and nervous system. The disorder is characterized by cleft lip and palate, single central incisor, impaired CNS septation, and in severe cases complete cyclopia.5,10 Similar characteristics along with additional phenotypes are observed in mice with a targeted disruption of Shh. The Shh signal is shown to be required for maintenance of the notochord, induction of floorplate and motor neurons, induction of axial skeleton and promotion of distal limb fates.27 Since Shh secreted from the prechordal mesoderm normally signals to inhibit eye formation in the center of eye rudiment, cyclopia results when this signal is defective or missing.1,27 Further confirmation of Shh function in this capacity comes from a plant teratogen, cyclopamine, which causes cyclopia in vertebrate embryos. Cyclopamine, a steroidal alkaloid analogous to cholesterol, acts by inhibiting the ability of target tissues to respond to Shh signaling, probably by antagonizing Smo.28
-Right Asymmetry

Note: This table was adapted from Chuang, P.-T. & T.B. Kornberg,23 and the references marked with an asterisk (*) are contained within that review. This table and associated references are not meant to be comprehensive, but are merely listed to highlight contributions made within the last several years.

The first evidence of a role for Shh signaling in vertebrate left-right asymmetry came from experiments in chick embryos. Shh is expressed asymmetrically in the chick node, on the left side during gastrulation. This expression pattern is correlated with normal heart situs (positioning of the heart) later in development.29 Ectopic expression of Shh on the right side causes a randomization of heart situs and induces ectopic expression in the right lateral plate mesoderm of a left-sided marker, nodal.29 Although conservations of left-right asymmetry and of left-sided nodal expression have been described in a variety of vertebrates, there is no evidence of asymmetric hedgehog expression in these other organisms.30 Recent experiments and observations suggest that Shh signaling may still be asymmetric in other vertebrates, even if ligand expression is not. Shh mutant mice exhibit misexpression of left-right markers as well as laterality defects in morphologies of the heart and other organs.31 Interestingly, Shh/Ihh compound mutants have the same phenotypes as Smo mutants. The embryos fail to undergo the normal rightward looping of the heart that is responsible for generating the asymmetry of heart situs.15 In addition, expressions of nodal and other asymmetric left-right markers are absent in Smo mutants.15 Since neither Hedgehog ligands (Shh, Ihh) nor receptors (Smo, Ptc) are asymmetrically expressed,15 it is not clear where the asymmetry of Shh signaling originates. Nonetheless, these results suggest that chick and mouse may utilize similar signaling pathways, such as Shh, to initiate left-right development even though the details of signaling regulation may be different.
Hematopoiesis

Hematopoiesis is initiated by a population of stem cells that arise from the mesoderm during embryogenesis. Hematopoiesis is sustained by uncommitted stem cells that give rise to progenitors capable of producing mature blood cells (differentiation) and of regenerating additional pluripotent stem cells (self-renewal). Combinations of cytokines regulate the decision to differentiate into specific lineages or to renew the pluripotent, hematopoietic cells capable of repopulating.32 Cytokines that control proliferation of stem cells usually do so by promoting differentiation, thereby losing a pluripotent stem cell capability that can repopulate blood lineages in deficient mice. Shh and its receptors, Ptc-1 and Smo, are expressed in highly purified populations of primitive human blood cells.24 Cytokine-induced proliferation of this highly purified primitive cell population can be inhibited with Hedgehog antibodies. Conversely, addition of soluble Shh results in an increase in the number of hematopoietic cells capable of repopulating function. These results indicate that Hedgehog proteins regulate the expansion of hematopoietic stem cells in a dose-dependent manner.24 Partially neutralizing the amount of Hedgehog signals with antibodies inhibits differentiation and proliferation of primitive blood cells, while enhancing the amount of Shh signaling expands the population of cells having pluripotent stem cell capabilities. It turns out that Hedgehog signaling acts upstream of BMP-4 to modulate BMP signaling and induce proliferation without differentiation in primitive blood cells.24 Since BMP-4 cannot induce this expansion of repopulating cells, Shh must have additional effects not mediated by BMP-4. Shh appears to be the first factor capable of inducing proliferation of primitive blood cells that remain in an undifferentiated state.24
Vascularization

Shh is involved in de novo vascularization of certain embryonic tissues as well as in inducing angiogenesis (formation of new blood vessels by remodeling and building on older ones) in an adult mammalian system. The role of Shh in embryonic vascularization was discovered by gain- and loss-of-function studies in mice. Transgenic mice reveal that overexpression of Shh in the dorsal neural tube results in hypervascularization of neuroectoderm.33 On the other hand, mice lacking Shh function exhibit poor vascularization of the developing lung.34 Recent work in the adult mouse indicates that Shh acts as an angiogenic factor, inducing robust neovascularization in hindlimbs damaged by ischemia.35 The mechanism responsible for this effect is indirect. Shh induces up-regulation of proteins required for the formation of blood vessels: all three VEGF-1 (vascular endothelial growth factor-1) isoforms and angiopoietins-1 and -2.<SUP>35 The ability to up-regulate these cytokines in concert so far seems unique to Shh. This concerted up-regulation of cytokines is likely to be physiologically relevant: Shh induces a rather complex vascular system and increased blood flow, resulting in complete limb salvage in half the mice, a significant increase compared to VEGF165 controls.35 How Shh upregulates angiogenic growth factors remains to be determined. Though Ptc-1 is necessarily upregulated for this effect, no Gli response elements are present in VEGF-1 or angiopoietin-1, suggesting a Gli-independent pathway.35 These results present the possibility of using Shh for pro-angiogenic therapy in ischemic disorders.
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