Cholesterol Modification of Smoothened Is Required for Hedgehog Signaling.

Hedgehog (Hh) has been known as the only cholesterol-modified morphogen playing pivotal roles in development and tumorigenesis. A major unsolved question is how Hh signaling regulates the activity of Smoothened (SMO). Here, we performed an unbiased biochemical screen and identified that SMO was covalently modified by cholesterol on the Asp95 (D95) residue through an ester bond. This modification was inhibited by Patched-1 (Ptch1) but enhanced by Hh. The SMO(D95N) mutation, which could not be cholesterol modified, was refractory to Hh-stimulated ciliary localization and failed to activate downstream signaling. Furthermore, homozygous SmoD99N/D99N (the equivalent residue in mouse) knockin mice were embryonic lethal with severe cardiac defects, phenocopying the Smo-/- mice. Together, the results of our study suggest that Hh signaling transduces to SMO through modulating its cholesterylation and provides a therapeutic opportunity to treat Hh-pathway-related cancers by targeting SMO cholesterylation.

Nile Tilapia (Oreochromis niloticus) Patched1 Mutations Disrupt Cardiovascular Development and Vascular Integrity through Smoothened Signaling.

Hedgehog (Hh) signaling is crucial in cardiovascular development and maintenance. However, the biological role of Patched1 (Ptch1), an inhibitory receptor of the Hh signaling pathway, remains elusive. In this study, a Ptch1 ortholog was characterized in Nile tilapia (Oreochromis niloticus), and its function was investigated through CRISPR/Cas9 gene knockout. When one-cell embryos were injected with CRISPR/Cas9 targeting ptch1, the mutation efficiency exceeded 70%. During 0-3 days post fertilization (dpf), no significant differences were observed between the ptch1 mutant group and the control group; at 4 dpf (0 day after hatching), about 10% of the larvae showed an angiogenesis defect and absence of blood flow; from 5 dpf, most larvae exhibited an elongated heart, large pericardial cavity, and blood leakage and coagulation, ultimately dying during the 6-8 dpf period due to the lack of blood circulation. Consistently, multiple differentially expressed genes related to angiogenesis, blood coagulation, and heart development were enriched in the ptch1 mutants. Furthermore, Smoothened (Smo) antagonist (cyclopamine) treatment of the ptch1 mutants greatly rescued the cardiovascular disorders. Collectively, our study suggests that Ptch1 is required for cardiovascular development and vascular integrity via Smo signaling, and excessive Hh signaling is detrimental to cardiovascular development.

Patched 1 and C-C Motif Chemokine Receptor 6 Distinguish Heterogeneous T Helper 17 Subsets in Colitic Lamina Propria.

T helper 17 (Th17) cells contribute to the pathogenesis of inflammatory bowel diseases (IBD). However, their heterogeneity and regulatory mechanisms in IBD are not completely disclosed. A mouse colitis model was established. Th17 cells were enriched from the mesenteric lymph nodes (mLN) and lamina propria (LP). The phenotypes and functions of Th17 subsets were analyzed by flow cytometry, Immunoblotting, and real-time RT-PCR. The contributions of the Th17 subsets to colitis pathogenesis were evaluated by histology, ELISA, and flow cytometry after adoptive transfer. Smoothened (SMO), GLI family zinc finger 1 (Gli1), and GLI family zinc finger 3 (Gli3) were markedly up-regulated while Patched 1 (PTCH1) was down-regulated in LP Th17 cells in colitic lamina propria. Based on the expression of PTCH1 and C-C motif chemokine receptor 6 (CCR6), LP Th17 cells were divided into a PTCH1lowCCR6low Th17 subset and a PTCH1highCCR6high Th17 subset. The former expressed higher T-bet, IFN-γ, TNF-α, IL-1ß, and GM-CSF but lower IL-17A, IL-22, IL-17F, and Gli3 than the latter. The PTCH1highCCR6high Th17 subset was more resistant to polarization towards T helper 1 (Th1) than the PTCH1lowCCR6low Th17 subset. Moreover, the PTCH1highCCR6high Th17 subset was more competent to maintain Th17 identity. The PTCH1highCCR6high Th17 subset induced less severe colitis than the PTCH1lowCCR6low Th17 subset. PTCH1highCCR6high Th17 cells are Th17 cells whereas PTCH1lowCCR6low Th17 cells are Th1-like Th17 cells. Our study deepens the understanding of Th17 heterogeneity and plasticity in colitis.

Hedgehog lipids: Promotors of alternative morphogen release and signaling?: Conflicting findings on lipidated Hedgehog transport and signaling can be explained by alternative regulated mechanisms to release the morphogen.

Two posttranslational lipid modifications present on all Hedgehog (Hh) morphogens-an N-terminal palmitate and a C-terminal cholesterol-are established and essential regulators of Hh biofunction. Yet, for several decades, the question of exactly how both lipids contribute to Hh signaling remained obscure. Recently, cryogenic electron microscopy revealed different modes by which one or both lipids may contribute directly to Hh binding and signaling to its receptor Patched1 (Ptc). Some of these modes demand that the established release factor Dispatched1 (Disp) extracts dual-lipidated Hh from the cell surface, and that another known upstream signaling modulator called Scube2 chaperones the dual-lipidated morphogen to Ptc. By mechanistically and biochemically aligning this concept with established in vivo and recent in vitro findings, this reflection identifies remaining questions in lipidated Hh transport and evaluates additional mechanisms of Disp- and Scube2-regulated release of a second bioactive Hh fraction that has one or both lipids removed.

Novel PTCH1 and concurrent TP53 mutations in four patients with numerous non-syndromic basal cell carcinomas: The paradigm of oncogenic synergy.

There has been a significant increase in basal cell carcinoma (BCC) incidence, the most common cancer in humans and the age of presentation with the first diagnosis of BCC has decreased in past decades. In this study, we investigated the possibility of genetic markers that can lead to earlier and closer observation of patients at high risk for development of multiple BCCs. The overall goal is to decrease the morbidity and the economic burden of diagnosis and treatment of recurring and/or advanced BCCs. Four patients with numerous BCCs, some of them exceptionally large, were included in this study. A sample of representative BCCs, normal non-sun-exposed skin and blood samples were obtained from each patient. Whole-exome sequencing of DNA was conducted on all samples, and a series of bioinformatics filtering was performed to identify potentially pathogenic sequence variants. The analysis of the data resulted in detection of oncogenic mutations in PTCH1, two of which being novel, and concurrent mutations in TP53 in BCC tumours of all four patients. Such mutations may explain the numerous and postexcision recurring nature of the BCCs of exceptionally large size observed in all these patients, and they can be suggested to serve as a genetic marker for high-risk patients for early detection, prognostication and close follow-up.

Patched1-ArhGAP36-PKA-Inversin axis determines the ciliary translocation of Smoothened for Sonic Hedgehog pathway activation.

The Sonic Hedgehog (Shh) pathway conducts primarily in the primary cilium and plays important roles in cell proliferation, individual development, and tumorigenesis. Shh ligand binding with its ciliary membrane-localized transmembrane receptor Patched1 results in the removal of Patched1 from and the translocation of the transmembrane oncoprotein Smoothened into the cilium, leading to Shh signaling activation. However, how these processes are coupled remains unknown. Here, we show that the Patched1-ArhGAP36-PKA-Inversin axis determines the ciliary translocation of Smoothened. We find that Patched1 interacts with and stabilizes the PKA negative regulator ArhGAP36 to the centrosome. Activating the Shh pathway results in the removal of ArhGAP36 from the mother centriole and the centrosomal PKA accumulation. This PKA then phosphorylates Inversin and promotes its interaction with and the ciliary translocation of Smoothened. Knockdown of Inversin disrupts the ciliary translocation of Smoothened and Shh pathway activation. These findings reveal a regulatory molecular mechanism for the initial step of Shh pathway activation.

Hedgehog Signalling Modulates Immune Response and Protects against Experimental Autoimmune Encephalomyelitis.

The Hedgehog (Hh) pathway is essential for the embryonic development and homeostatic maintenance of many adult tissues and organs. It has also been associated with some functions of the innate and adaptive immune system. However, its involvement in the immune response has not been well determined. Here we study the role of Hh signalling in the modulation of the immune response by using the Ptch-1-LacZ+/- mouse model (hereinafter referred to as ptch+/-), in which the hemizygous inactivation of Patched-1, the Hh receptor gene, causes the constitutive activation of Hh response genes. The in vitro TCR stimulation of spleen and lymph node (LN) T cells showed increased levels of Th2 cytokines (IL-4 and IL-10) in ptch+/-cells compared to control cells from wild-type (wt) littermates, suggesting that the Th2 phenotype is favoured by Hh pathway activation. In addition, CD4+ cells secreted less IL-17, and the establishment of the Th1 phenotype was impaired in ptch+/- mice. Consistently, in response to an inflammatory challenge by the induction of experimental autoimmune encephalomyelitis (EAE), ptch+/- mice showed milder clinical scores and more minor spinal cord damage than wt mice. These results demonstrate a role for the Hh/ptch pathway in immune response modulation and highlight the usefulness of the ptch+/- mouse model for the study of T-cell-mediated diseases and for the search for new therapeutic strategies in inflammatory diseases.

miR-511-3p promotes hepatic sinusoidal obstruction syndrome by activating hedgehog pathway via targeting Ptch1.

As a major complication of hematopoietic stem cell transplantation, the incidence of hepatic sinusoidal obstruction syndrome (HSOS) is as high as 70%. Previous evidence has demonstrated that miR-511-3p was involved in HSOS, but the mechanism remains unclear. This study aims to examine the mechanism underlying miR-511-3p regulating HSOS. Monocrotaline (MCT) was used to create an HSOS rat model and to treat liver sinusoidal endothelial cells (LSECs). Hematoxylin & eosin (H&E) and Masson staining were used to detect pathological changes in liver tissue. The expression of miR-511-3p and Hedgehog pathway-related proteins was assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The effect of miR-511-3p in regulating HSOS was investigated by 3-(4,5)-dimethylthiahiazo-2)-3,5-diphenytetrazoliumromide (MTT), enzyme-linked immunosorbent assay (ELISA) assay, and flow cytometry. Finally, the interaction between miR-511-3p and patched1 (Ptch1) was determined by luciferase reporter assay. The rats showed a typical HSOS phenotype, including LSEC damage, liver injury, and fibrosis after MCT administration. miR-511-3p was upregulated in hepatic tissue of rat HSOS model and MCT-induced LSECs. miR-511-3p directly targeted Ptch1 and suppressed Ptch1 expression to activate the Hedgehog signaling pathway. Depletion of miR-511-3p showed a protective effect against MCT-induced HSOS, as evidenced by decreased HSOS pathogenesis factors, matrix metalloproteinases-2 (MMP-2), matrix metalloproteinases-9 (MMP-9), tumor necrosis factor-α (TNF-α), and interleukin 1 ß (IL-1ß), and decreased LSEC apoptosis rates. Nevertheless, knockdown of Ptch1 reversed the protective effect of miR-511-3p depletion against MCT-induced LSEC injury and apoptosis. miR-511-3p aggravates HSOS by activating the Hedgehog signaling pathway through targeting Ptch1, and miR-511-3p may develop as the potential therapy for the treatment of HSOS.NEW & NOTEWORTHY miR-511-3p is upregulated in HSOS in vivo and in vitro models. miR-511-3p activates the Hedgehog pathway by directly targeting Ptch1. Knockdown of miR-511-3p shows a protective effect against LSEC injury and apoptosis via Hedgehog signaling pathway. Inhibition of Ptch1 reserves the effect of miR-511-3p knockdown on LSEC damage and apoptosis.

Epithelial and non-epithelial Ptch1 play opposing roles to regulate proliferation and morphogenesis of the mouse mammary gland.

Patched 1 (Ptch1) has epithelial, stromal and systemic roles in murine mammary gland organogenesis, yet specific functions remain undefined. Cre-recombinase-mediated Ptch1 ablation in mammary epithelium increased proliferation and branching, but did not phenocopy transgenic expression of activated smoothened (SmoM2). The epithelium showed no evidence of canonical hedgehog signaling, and hyperproliferation was not blocked by smoothened (SMO) inhibition, suggesting a non-canonical function of PTCH1. Consistent with this possibility, nuclear localization of cyclin B1 was increased. In non-epithelial cells, heterozygous Fsp-Cre-mediated Ptch1 ablation increased proliferation and branching, with dysplastic terminal end buds (TEB) and ducts. By contrast, homozygous Ptch1 ablation decreased proliferation and branching, producing stunted ducts filled with luminal cells showing altered ovarian hormone receptor expression. Whole-gland transplantation into wild-type hosts or estrogen/progesterone treatment rescued outgrowth and hormone receptor expression, but not the histological changes. Bone marrow transplantation failed to rescue outgrowth. Ducts of Fsp-Cre;Ptch1fl/fl mice were similar to Fsp-Cre;SmoM2 ducts, but Fsp-Cre;SmoM2 outgrowths were not stunted, suggesting that the histology might be mediated by Smo in the local stroma, with systemic Ptch1 required for ductal outgrowth and proper hormone receptor expression in the mammary epithelium.

The Vitamin D Receptor as Tumor Suppressor in Skin.

Cutaneous malignancies including melanomas and keratinocyte carcinomas (KC) are the most common types of cancer, occurring at a rate of over one million per year in the United States. KC, which include both basal cell carcinomas and squamous cell carcinomas, are substantially more common than melanomas and form the subject of this chapter. Ultraviolet radiation (UVR), both UVB and UVA, as occurs with sunlight exposure is generally regarded as causal for these malignancies, but UVB is also required for vitamin D synthesis in the skin. Keratinocytes are the major cell in the epidermis. These cells not only produce vitamin D but contain the enzymatic machinery to metabolize vitamin D to its active metabolite, 1,25(OH)2D, and express the receptor for this metabolite, the vitamin D receptor (VDR). This allows the cell to respond to the 1,25(OH)2D that it produces. Based on our own data and that reported in the literature, we conclude that vitamin D signaling in the skin suppresses UVR-induced epidermal tumor formation. In this chapter we focus on four mechanisms by which vitamin D signaling suppresses tumor formation. They are inhibition of proliferation/stimulation of differentiation with discussion of the roles of hedgehog, Wnt/ß-catenin, and hyaluronan/CD44 pathways in mediating vitamin D regulation of proliferation/differentiation, regulation of the balance between oncogenic and tumor suppressor long noncoding RNAs, immune regulation, and promotion of DNA damage repair (DDR).

The protein-specific activities of the transmembrane modules of Ptch1 and Ptch2 are determined by their adjacent protein domains.

Signaling through the Hedgehog (Hh) pathway is mediated by the Patched (Ptch) family of proteins. Although the vertebrate Ptch proteins Ptch1 and Ptch2 harbor two closely related transmembrane modules related to sterol-sensing domains (SSDs), the role of these closely related receptors in the Hh pathway are not equivalent. Ptch1 is essential for development and appears to be the principal receptor mediating responses to Hh ligands, whereas Ptch2 is nonessential, and its role in Hh-signaling remains ambiguous. We hypothesized that the SSDs of the Ptch proteins function as generic modules whose protein-specific activities are determined by the adjacent cytoplasmic and luminal domains. We first showed that individual N-terminal and C-terminal halves of Ptch1 associated noncovalently to mediate ligand-dependent regulation of Hh signaling. The analogous regions of Ptch2 also interacted noncovalently but did not repress the Hh pathway. However, the SSD of Ptch2 were capable of repressing Hh signaling, as determined using chimeric proteins where the SSDs of Ptch1 were replaced by those from Ptch2. Replacement of the SSDs of Ptch1 with the analogous regions from the cholesterol transporter NPC1 failed to produce a chimeric protein capable of Hh repression. Further refinement of the specific regions in Ptch1 and Ptch2 revealed that specific cytoplasmic domains of Ptch1 were necessary but not sufficient for repression of Hh signaling and that the two principal luminal domains of Ptch1 and Ptch2 were interchangeable. These data support a model where the SSDs of the Ptch family proteins exhibit generic activities and that the adjacent cytoplasmic and luminal domains determine their protein-specific activities.

The cell adhesion molecule CHL1 interacts with patched-1 to regulate apoptosis during postnatal cerebellar development.

The immunoglobulin superfamily adhesion molecule close homolog of L1 (CHL1) plays important roles during nervous system development. Here, we identified the hedgehog receptor patched-1 (PTCH1) as a novel CHL1-binding protein and showed that CHL1 interacts with the first extracellular loop of PTCH1 via its extracellular domain. Colocalization and co-immunoprecipitation of CHL1 with PTCH1 suggest an association of CHL1 with this major component of the hedgehog signaling pathway. The trans-interaction of CHL1 with PTCH1 promotes neuronal survival in cultures of dissociated cerebellar granule cells and of organotypic cerebellar slices. An inhibitor of the PTCH1-regulated hedgehog signal transducer, smoothened (SMO), and inhibitors of RhoA and Rho-associated kinase (ROCK) 1 and 2 prevent CHL1-dependent survival of cultured cerebellar granule cells and survival of cerebellar granule and Purkinje cells in organotypic cultures. In histological sections from 10- and 14-day-old CHL1-deficient mice, enhanced apoptosis of granule, but not Purkinje, cells was observed. The results of the present study indicate that CHL1 triggers PTCH1-, SMO-, RhoA- and ROCK-dependent signal transduction pathways to promote neuronal survival after cessation of the major morphogenetic events during mouse cerebellar development.

Loss of the PTCH1 tumor suppressor defines a new subset of plexiform fibromyxoma.

BACKGROUND: Plexiform fibromyxoma (PF) is a rare gastric tumor often confused with gastrointestinal stromal tumor. These so-called "benign" tumors often present with upper GI bleeding and gastric outlet obstruction. It was recently demonstrated that approximately one-third of PF have activation of the GLI1 oncogene, a transcription factor in the hedgehog (Hh) pathway, via a MALAT1-GLI1 fusion protein or GLI1 up-regulation. Despite this discovery, the biology of most PFs remains unknown. METHODS: Next generation sequencing (NGS) was performed on formalin-fixed paraffin-embedded (FFPE) samples of PF specimens collected from three institutions (UCSD, NCI and OHSU). Fresh frozen tissue from one tumor was utilized for in vitro assays, including quantitative RT-PCR and cell viability assays following drug treatment. RESULTS: Eight patients with PF were identified and 5 patients' tumors were analyzed by NGS. An index case had a mono-allelic PTCH1 deletion of exons 15-24 and a second case, identified in a validation cohort, also had a PTCH1 gene loss associated with a suspected long-range chromosome 9 deletion. Building on the role of Hh signaling in PF, PTCH1, a tumor suppressor protein, functions upstream of GLI1. Loss of PTCH1 induces GLI1 activation and downstream gene transcription. Utilizing fresh tissue from the index PF case, RT-qPCR analysis demonstrated expression of Hh pathway components, SMO and GLI1, as well as GLI1 transcriptional targets, CCND1 and HHIP. In turn, short-term in vitro treatment with a Hh pathway inhibitor, sonidegib, resulted in dose-dependent cell killing. CONCLUSIONS: For the first time, we report a novel association between PTCH1 inactivation and the development of plexiform fibromyxoma. Hh pathway inhibition with SMO antagonists may represent a target to study for treating a subset of plexiform fibromyxomas.

Inhibition of MicroRNA-195 Alleviates Neuropathic Pain by Targeting Patched1 and Inhibiting SHH Signaling Pathway Activation.

Trigeminal neuralgia (TN) is a type of chronic neuropathic pain that is caused by peripheral nerve lesions that result from various conditions, including the compression of vessels, tumors and viral infections. MicroRNAs (miRs) are increasingly recognized as potential regulators of neuropathic pain. Previous evidence has demonstrated that miR-195 is involved in neuropathic pain, but the mechanism remains unclear. To investigate the pathophysiological role of miR-195 and Shh signaling in TN, persistent facial pain was induced by infraorbital nerve chronic constriction injury (CCI-IoN), and facial pain responses were evaluated by Von Frey hairs. qPCR and Western blotting were used to determine the relative expression of miR-195 and Patched1, the major receptor of the Sonic Hedgehog (Shh) signaling pathway, in the caudal brain stem at distinct time points after CCI-IoN. Here, we found that the expression of miR-195 was increased in a rat model of CCI-IoN. In contrast, the expression of Patched1 decreased significantly. Luciferase assays confirmed the binding of miR-195 to Patched1. In addition, the overexpression of miR-195 by an intracerebroventricular (i.c.v) administration of LV-miR-195 aggravated facial pain development, and this was reversed by upregulating the expression of Patched1. These results suggest that miR-195 is involved in the development of TN by targeting Patched1 in the Shh signaling pathway, thus regulating extracellular glutamate.

Ventral neural patterning in the absence of a Shh activity gradient from the floorplate.

BACKGROUND: Vertebrate spinal cord development requires Sonic Hedgehog (Shh) signaling from the floorplate and notochord, where it is thought to act in concentration dependent manner to pattern distinct cell identities along the ventral-to-dorsal axis. While in vitro experiments demonstrate naïve neural tissues are sensitive to small changes in Shh levels, genetic studies illustrate that some degree of ventral patterning can occur despite significant perturbations in Shh signaling. Consequently, the mechanistic relationship between Shh morphogen levels and acquisition of distinct cell identities remains unclear. RESULTS: We addressed this using Hedgehog acetyltransferase (HhatCreface ) and Wiggable mouse mutants. Hhat encodes a palmitoylase required for the secretion of Hedgehog proteins and formation of the Shh gradient. In its absence, the spinal cord develops without floorplate cells and V3 interneurons. Wiggable is an allele of the Shh receptor Patched1 (Ptch1Wig ) that is unable to inhibit Shh signal transduction, resulting in expanded ventral progenitor domains. Surprisingly, HhatCreface/Creface ; Ptch1Wig/Wig double mutants displayed fully restored ventral patterning despite an absence of Shh secretion from the floorplate. CONCLUSIONS: The full range of neuronal progenitor types can be generated in the absence of a Shh gradient provided pathway repression is dampened, illustrating the complexity of morphogen dynamics in vertebrate patterning. Developmental Dynamics 247:170-184, 2018. © 2017 Wiley Periodicals, Inc.

Primary cilium alterations and expression changes of Patched1 proteins in niemann-pick type C disease.

Niemann-Pick type C disease (NPC) is a disorder characterized by abnormal intracellular accumulation of unesterified cholesterol and glycolipids. Two distinct disease-causing genes have been isolated, NPC1 and NPC2. The NPC1 protein is involved in the sorting and recycling of cholesterol and glycosphingolipids in the late endosomal/lysosomal system. It has extensive homology with the Patched1 (Ptc1) receptor, a transmembrane protein localized in the primary cilium, and involved in the Hedgehog signaling (Shh) pathway. We assessed the presence of NPC1 and Ptc1 proteins and evaluated the relative distribution and morphology of primary cilia in fibroblasts from five NPC1 patients and controls, and in normal fibroblasts treated with 3-ß-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A), a cholesterol transport-inhibiting drug that is widely used to mimic NPC. Immunofluorescence and western blot analyses showed a significant decrease in expression of NPC1 and Ptc1 in NPC1 fibroblasts, while they were normally expressed in U18666A-treated fibroblasts. Moreover, fibroblasts from NPC1 patients and U18666A-treated cells showed a lower percentage distribution of primary cilia and a significant reduction in median cilia length with respect to controls. These are the first results demonstrating altered cytoplasmic expression of Ptc1 and reduced number and length of primary cilia, where Ptc1 is located, in fibroblasts from NPC1 patients. We suggest that the alterations in Ptc1 expression in cells from NPC1 patients are closely related to NPC1 expression deficit, while the primary cilia alterations observed in NPC1 and U18666A-treated fibroblasts may represent a secondary event derived from a defective metabolic pathway.

Hedgehog receptor function during craniofacial development.

The Hedgehog signalling pathway plays a fundamental role in orchestrating normal craniofacial development in vertebrates. In particular, Sonic hedgehog (Shh) is produced in three key domains during the early formation of the head; neuroectoderm of the ventral forebrain, facial ectoderm and the pharyngeal endoderm; with signal transduction evident in both ectodermal and mesenchymal tissue compartments. Shh signalling from the prechordal plate and ventral midline of the diencephalon is required for appropriate division of the eyefield and forebrain, with mutation in a number of pathway components associated with Holoprosencephaly, a clinically heterogeneous developmental defect characterized by a failure of the early forebrain vesicle to divide into distinct halves. In addition, signalling from the pharyngeal endoderm and facial ectoderm plays an essential role during development of the face, influencing cranial neural crest cells that migrate into the early facial processes. In recent years, the complexity of Shh signalling has been highlighted by the identification of multiple novel proteins that are involved in regulating both the release and reception of this protein. Here, we review the contributions of Shh signalling during early craniofacial development, focusing on Hedgehog receptor function and describing the consequences of disruption for inherited anomalies of this region in both mouse models and human populations.

Activities of the Cytoplasmic Domains of Patched-1 Modulate but Are Not Essential for the Regulation of Canonical Hedgehog Signaling.

The Hedgehog (Hh) pathway is a highly conserved signaling cascade crucial for cell fate determination during embryogenesis. Response to the Hh ligands is mediated by the receptor Patched-1 (Ptch1), a 12-pass transmembrane glycoprotein. Despite its essential role in Hh signaling and its activity as a tumor suppressor, Ptch1 remains largely uncharacterized. We demonstrate here that Ptch1 binds to itself to form oligomeric structures. Oligomerization is mediated by two distinct, structurally disordered, intracellular domains spanning amino acids 584-734 ("middle loop") and 1162-1432 (C terminus). However, oligomerization is not required for Ptch1-dependent regulation of the canonical Hh pathway operating through Smo. Expression of a mutant protein that deletes both regions represses the Hh pathway and responds to the addition of Hh ligand independent of its inability to bind other factors such as Smurf2. Additionally, deletion of the cytoplasmic middle loop domain generates a Ptch1 mutant that, despite binding to Hh ligand, constitutively suppresses Hh signaling and increases the length of primary cilia. Constitutive activity because of deletion of this region is reversed by further deletion of specific sequences in the cytoplasmic C-terminal domain. These data reveal an interaction between the cytoplasmic domains of Ptch1 and that these domains modulate Ptch1 activity but are not essential for regulation of the Hh pathway.

Identification and characterization of two novel PTCH1 splice variants.

Patched-1 (PTCH1), one of the key molecules involved in the Hedgehog (HH) signaling pathway, acts as the receptor of the HH ligand. PTCH1 also inhibits the positive signal transducer Smoothened (SMO). Several PTCH1 splice variants have been identified and confirmed to play critical roles in HH pathway regulation. In the present study, two novel alternatively spliced variants of PTCH1 transcripts, designated PTCH1-Δ10 and PTCH1-Δ15, were found in humans, mice and zebrafish using RT-PCR, direct sequencing and ribonuclease protection assays. PTCH1-Δ10 lacks exon 10, which encodes part of the sterol-sensing domain (SSD), while PTCH1-Δ15 lacks 166 bp of exon 15, which causes a frame shift that generates a premature stop codon leading to a truncated PTCH1 protein. Different truncated PTCH1 proteins localized in the cytoplasm were capable of internalizing the N-terminal fragment of Sonic hedgehog (SHH-N), which was visualized using immunofluorescence microscopy. Exon skipping dramatically influenced the steady states of the proteins, with the levels of PTCH1-1B and PTCH1-Δ10 being significantly higher than those of PTCH1-Δ15, as detected using western blot. These results imply that the pronounced inhibitory signaling properties of PTCH1-1B and PTCH1-Δ10 may be partially due to high protein expression in addition to intrinsic functional differences. All isoforms examined worked as functional receptors of SHH. However, the isoforms PTCH1-1B and PTCH1-Δ10 inhibited SMO and the pathway transcription factor glioma 1 (GLI1) to a greater extent than did PTCH1-Δ15. In addition, PTCH1-1B and PTCH1-Δ10 (but not PTCH1-Δ15) can be negative regulators of the HH pathway. These results indicate that the SSD domain and the C-terminal region are essential for maximal repressor function of PTCH1. Additionally, SMO inhibition by PTCH1 occurred through a nonstoichiometric, catalytic mechanism, indicating that this inhibition was less dependent on the dose of the PTCH1 protein. Finally, all these isoforms have been revealed to inhibit GLI1 activation by either the classical HH signaling pathway or a new pathway not reliant on both SMO and apoptosis. Thus, our study clearly demonstrated the unique involvement of the two novel PTCH1 splice variants in HH signal transduction.

Ptch2 mediates the Shh response in Ptch1-/- cells.

The Hedgehog (Hh) signaling response is regulated by the interaction of three key components that include the sonic hedgehog (Shh) ligand, its receptor patched 1 (Ptch1) and the pathway activator smoothened (Smo). Under the prevailing model of Shh pathway activation, the binding of Shh to Ptch1 (the key Shh receptor) results in the release of Ptch1-mediated inhibition of Smo, leading to Smo activation and subsequent cell-autonomous activation of the Shh response. Consistent with this model, Ptch1(-/-) cells show a strong upregulation of the Shh response. Our finding that this response can be inhibited by the Shh-blocking antibody 5E1 indicates that the Shh response in Ptch1(-/-) cells remains ligand dependent. Furthermore, we find that Shh induces a strong response in Ptch1(-/-);Shh(-/-) cells, and that Ptch1(-/-) fibroblasts retain their ability to migrate towards Shh, demonstrating that Ptch1(-/-) cells remain sensitive to Shh. Expression of a dominant-negative Ptch1 mutant in the developing chick neural tube had no effect on Shh-mediated patterning, but expression of a dominant-negative form of patched 2 (Ptch2) caused an activation of the Shh response. This indicates that, at early developmental stages, Ptch2 functions to suppress Shh signaling. We found that Ptch1(-/-);Ptch2(-/-) cells cannot further activate the Shh response, demonstrating that Ptch2 mediates the response to Shh in the absence of Ptch1.