Three Hedgehog proteins are found in mammals; these are sonic hedgehog (gene SHH), Indian hedgehog (gene IHH) and desert hedgehog (gene DHH). They are first synthesised as a 45 kDa precursor, which undergoes an autocatalytic cleavage releasing a 19 kDa N-terminal peptide involved in signalling and a 45 kDa C-terminal peptide, which is involved in cleavage and bears a catalytic activity of cholesterol transferase.
HHG proteins are subjected to post-translational modifications that are required for their activity; on the C-terminal side, they are covalently bound to a cholesterol molecule; on the N-terminal side, a specific palmitoyltransferase, HHAT (hedgehog acyltransferase) or SKI (skinny hedgehog), binds them to a palmitic acid moiety at the level of a cysteine residue. When equipped with these two hydrophobic ligands, HHG proteins can be inserted in the peripheral phospholipid layer of a lipoprotein and thus form a multimeric complex (Fig. 9.1).
Fig. 9.1. HHG ligands and their post-translational modifications. (a) Native HHG proteins (45 kDa) are autocatalytically cleaved to generate a signalling fragment, which is covalently bound to a cholesterol molecule on its C-terminal serine. Afterwards, a palmitoyltransferase HHAT adds a palmitic acid to a cysteine residue located at the N-terminal extremity. (b) The molecule, with two lipid substituents, is inserted in phospholipid micelles, as part of lipoprotein structures
The mechanism of secretion of the HHG ligands is not precisely known. It involves a 12-transmembrane-domain protein called dispatched (DISP), which is indispensable and behaves as a membrane transporter of HHG proteins. This protein contains a sterol-binding domain, which should intervene in the processes of recognition and secretion of the HHG proteins.
Patched receptors and their activation
The HHG ligands with their two lipid substituents are recognised on a target cell by a receptor called patched (PTCH). There are two distinct PTCH proteins, encoded by the genes PTCH1 and PTCH2, which seem to have different functions, the first one being the best known and the only one to be considered here. PTCH belongs to the same transporter family as DISP and also presents 12 transmembrane domains and a cholesterol-binding domain. It also presents two large extracellular domains, which ensure HHG binding. PTCH is only an intermediary in HHG signal reception, since it transfers the information thus received to another membrane protein called smoothened (gene SMO), by releasing the constitutive inhibition exerted in this protein. The mechanism of this release is not intimately known, but it seems that no direct interaction exists between PTCH and SMO. PTCH would only be a transport protein and would control the localisation of a small inhibitory or excitatory molecule that remains to be identified. In the absence of signal, PTCH would pump an SMO inhibitory molecule from the inside to the outside of the cell; when bound to HHG, PTCH would cease pumping and SMO would then be functional; the reverse would be true if the molecule could conversely activate SMO (Fig. 9.2). Some authors hypothesise that the small molecule transported by PTCH and interacting with SMO could be vitamin D3 or an oxysterol.
Fig. 9.2. PTCH receptor information transduction to SMO proteins. This figure presents a possible model for SMO activation by the PTCH receptor following HHG ligand binding. The SMO membrane protein is inactive in its endosomal localisation and is activated after migration to the primary cilium. In a first hypothesis, a substrate of PTCH transport activity (oxysterol?) would favour SMO migration to primary cilia and stabilise the active form of SMO. In the absence of HHG ligand (schema a), PTCH would pump oxysterol outside the cell, preventing thus its positive action on SMO ciliary localisation. In the presence of the HHG ligand (schema b), PTCH pumping activity would be inhibited, and the oxysterol would be available for maintaining SMO under its active form. In a second hypothesis, the substrate of the transport activity of PTCH (vitamin D3?) would be an inhibitor of SMO and would favour its inactive, endosomal localisation. In the absence of HHG ligand (schema a), PTCH would pump this sterolic molecule outside the cell where they would inhibit SMO. In the presence of HHG ligands (schema b), PTCH pumping activity would be inhibited, and SMO would no longer be destabilised by the inhibitory sterol. The two hypotheses are presented on the same schemas, the putative SMO activator (oxysterol?) as yellow circles and the putative SMO inhibitor (vitamin D3?) by red circles
SMO is a membrane protein with seven transmembrane domains, belonging to the fifth family of GPCRs (G-protein-coupled receptors) (Chap. 6) as FZD receptors (Chap. 7). When PTCH binds an HHG ligand, SMO accumulates in primary cilia, which are subcellular organelles present in all vertebrate cells and form protrusions at the surface of the plasma membrane, devoid of motility and based upon a microtubule cytoskeleton. SMO migration to primary cilia is required for Hedgehog signalling. This migration is accompanied by SMO phosphorylation by GRK2 (GPCR kinase 2, gene ADRBK1), which is thought to be required for migration. Proteins other than PTCH are able to bind HHG ligands and modulate the signal brought to the target cell: CDO (cell adhesion molecule-related/downregulated by oncogenes) and BOC (brother of CDO), which are members of the immunoglobulin superfamily and function as accessory receptors to HHG; it is not clear whether or not they are required for signal transmission. They operate via a glycosaminoglycan (heparan sulphate)-binding site. Another protein, HHIP (hedgehog-interacting protein), would be a decoy receptor for HHG ligands and would thus inhibit this signalling pathway.
HHG signal transduction
Whereas the previous steps of Hedgehog pathway signalling are highly similar in Drosophila and mammals, the subsequent steps of signal transduction, downstream SMO activation, appear to be different, and the proteins identified in Drosophila have no exact orthologs in humans. In the absence of HHG signal, zinc-finger transcription factors called GLI (glioblastoma-associated oncogene) (genes GLI1, GLI2 and GLI3) are sequentially phosphorylated in the primary cilia by serine/ threonine kinases: PKA (protein kinase A), CK1 (casein kinase 1) and GSK3β (glycogen synthase kinase 3β ), organised with an adapter protein called SUFU (suppressor of fused) in a destruction complex; these phosphorylations lead the GLI transcription factors to the proteasome. In the presence of a HHG signal, the kinases are inhibited, and the GLI transcription factors can migrate to the nucleus and activate their target genes (Fig. 9.3).
PKA inhibition occurring upon SMO activation could be due to the recruitment by SMO of a large heterotrimeric G-protein with an ai subunit, whose activation inhibits adenylyl cyclase and thus cAMP formation and cAMP-induced PKA activation (see Chap. 6). In Drosophila, an assembly protein, cos2, is involved in the concerted activation of the kinases; its recruitment by SMO prevents this activation. In mammals, there is no cos2 ortholog and this function could be assumed by the kinesin KIF7; in fact, a protein called SUFU (suppressor of fused) is the main negative regulator of the GLI transcription factors in mammals, whereas it plays a minor role in Hedgehog signal transduction in Drosophila. In addition, a truncated variant of GLI3 could play a negative regulation of Hedgehog signalling by negative dominance.
The target genes of the GLI transcription factors are involved in morphogenesis, especially Homeobox (HOX) gene family members. PTCH1 and HHIP are also target genes, which explains positive and negative retroaction control of this pathway. Genes encoding proteins involved in cell proliferation, such as BCL2 (B-cell lymphoma 2, Chap. 18), cyclin D1 (Chap. 17) or PDGFRa (platelet-derived growth factor receptor a, Chap. 1), are also target genes of the Hedgehog signalling pathway.
Fig. 9.3. The Hedgehog signalling pathway. (a) In the absence of HHG ligands, PTCH inhibits SMO and prevents it to migrate to the primary cilia. The GLI transcription factors are phosphorylated in the primary cilia by several kinases (CK1, PKA, GSK3β ), a process that drives them to the proteasome. (b) The HHG ligands are matured after translation and leave the producing cell via the membrane protein DISP. (c) In the presence of HHG ligands on PTCH, SMO can migrate to the primary cilia, preventing thus the phosphorylation of the GLI transcription factors. These factors can then migrate to the nucleus and activate transcription programmes. One of the major negative regulators of this pathway is the SUFU protein. A positive regulator (not represented here) could be a large heterotrimeric G-protein with ai subunit, which would inhibit cAMP production and consequently PKA activity
The Hedgehog pathway was first shown to be involved in a syndrome of predisposition to skin basocellular carcinomas (BCCs), called Gorlin syndrome. A heterozygous mutation in the PTCH1 gene has been identified in normal cells of these patients, and an additional loss of the non-mutated allele has been detected in tumour cells. These invalidating mutations induce the loss of PTCH-mediated SMO inhibition, which is in turn responsible for the increased transcription of the GLI target genes. Similarly, sporadic BCCs also present PTCH1 inactivating mutations or SMO activating mutations. PTCH1 and SMO appear, therefore, as a tumour suppressor gene and a proto-oncogene, respectively. Other malignancies, such as medulloblastomas and rhabdomyosarcomas, also present such mutations, as well as less frequent SUFU inactivating mutations.
In other cancers, alterations can be found at the level of the HHG ligands. HHG overexpression, with subsequent increase in GLI activation, is observed in pancreatic and small-cell lung cancers, but the driver role of these alterations has not been proved; autocrine loops favouring cell proliferation could well be operating in such cases. Stromal cell interactions with the Hedgehog pathway in tumour cells have been evidenced. The Hedgehog pathway appears, therefore, as involved in basic mechanisms of oncogenesis and metastasis, especially because of its role in stem cell proliferation and epithelial-to-mesenchymal transition.
A natural inhibitor of the Hedgehog pathway, cyclopamine, was discovered early; this sterol-related alkaloid induces alterations in lamb foetal development when pregnant ewes were fed with contaminated grass. This SMO inhibitor likely interferes with an oxysterol-binding site. Cyclopamine itself does not seem usable in therapeutics because of its very low bioavailability, but other compounds, such as vismodegib, have been identified and recently patented, with marked antitumour effect in metastatic invasive BCCs and medulloblastomas. PTCH1 mutations, such as W844C, inducing a constitutive activation of the signalling pathway, have been identified in vismodegib responders; conversely, secondary SMO mutations in treated patients, such as D473H, induce resistance to vismodegib. Vitamin D3 could behave as an SMO inhibitor, which would be in agreement with its known role on skin trophicity.
Another potential target is represented by the GLI transcription factors, downstream the Hedgehog pathway. Several small molecules have been identified and may likely enter clinical trials. Finally, upstream the PTCH–SMO interaction, monoclonal antibodies targeting the HHG proteins have shown activity in preclinical models.