Mammalian Patched homologs

A mouse homolog of patched has been cloned. Mouse patched is expressed close to multiple Sonic hedgehogs in mesodermal somites, brain, and spinal cord. Remarkable here is the conservation of elements of the patched-hedgehog pathway, including expression of protein kinase A and the vertebrate homolog of cubitus interruptus (Goodrich, 1996).

The human homolog of patched acts as a tumor suppressor gene. The nevoid basal cell carcinoma syndrome is an autosomal dominant disorder characterized by multiple basal cell carcinomas, pits of the palms and soles, jaw keratocysts, and developmental abnormalities. The human disease is associated with loss of heterozygosity in a region of chromosome 9. Nonneoplastic features include jaw keratocysts, dyskeratotic pitting of the hands and feet and progressive intracranial calcification. There is a broad range of skeletal defects including rib, vertebral and shoulder anomalities. Human Patched is 60% homologous to the fly protein and has two alternative first exons and a predicted 8 to 12 transmembrane domains (Hahn, 1996)

The multitransmembrane protein Patched (PTCH) is the receptor for Sonic Hedgehog (Shh), a secreted molecule implicated in the formation of embryonic structures and in tumorigenesis. Current models suggest that binding of Shh to PTCH prevents the normal inhibition of the seven-transmembrane-protein Smoothened (SMO) by PTCH. According to this model, the inhibition of SMO signaling is relieved after mutational inactivation of PTCH in the basal cell nevus syndrome. Recently, PTCH2, a molecule with sequence homology to PTCH, has been identified. To characterize both PTCH molecules with respect to the various Hedgehog proteins, the human PTCH2 gene was isolated. Biochemical analysis of PTCH and PTCH2 shows that they both bind to all hedgehog family members with similar affinity and that they can form a complex with SMO. However, the expression patterns of PTCH and PTCH2 do not fully overlap. While PTCH is expressed throughout the mouse embryo, PTCH2 is found at high levels in the skin and in spermatocytes. Because Desert Hedgehog (Dhh) is expressed specifically in the testis and is required for germ cell development, it is likely that PTCH2 mediates Dhh activity in vivo. Chromosomal localization of PTCH2 places it on chromosome 1p33-34, a region deleted in some germ cell tumors, raising the possibility that PTCH2 may be a tumor suppressor in Dhh target cells (Carpenter, 1998).

Veratrum alkaloids and distal inhibitors of cholesterol biosynthesis have been studied for more than 30 years as potent teratogens capable of inducing cyclopia and other birth defects. These compounds specifically block the Sonic hedgehog (Shh) signaling pathway. These teratogens do not prevent the sterol modification of Shh during autoprocessing but rather inhibit the response of target tissues to Shh, possibly acting through the sterol sensing domain within the Patched protein regulator of Shh response (Cooper, 1998).

Mutation of mammalian Patched homologs

Mutations of the human Patched gene (PTCH) have been identified in individuals with the nevoid basal cell carcinoma syndrome (NBCCS) as well as in sporadic basal cell carcinomas and medulloblastomas. A homolog of this tumor suppressor gene has been localized to the short arm of chromosome 1 (1p32.1-32.3). Patched 2 ( PTCH2 ) comprises 22 coding exons and spans approximately 15 kb of genomic DNA. The gene encodes a 1203 amino acid putative transmembrane protein which is highly homologous to the PTCH product. The genomic structure of PTCH2 has been characterized and single-stranded conformational polymorphism analysis was used to search for PTCH2 mutations in basal cell carcinomas and medulloblastomas in NBCCS patients. To date, one truncating mutation was identified in a medulloblastoma and a change in a splice donor site in a basal cell carcinoma, suggesting that the gene plays a role in the development of some tumors (Smyth, 1999).

Pancreas organogenesis is regulated by the interaction of distinct signaling pathways that promote or restrict morphogenesis and cell differentiation. Previous work has shown that activin, a TGFbeta signaling molecule, permits pancreas development by repressing expression of Sonic hedgehog (Shh), a member of the hedgehog family of signaling molecules that antagonize pancreas development. Indian hedgehog (Ihh), another hedgehog family member, and Patched 1 (Ptc1), a receptor and negative regulator of hedgehog activity, are expressed in pancreatic tissue. Targeted inactivation of Ihh in mice allows ectopic branching of ventral pancreatic tissue resulting in an annulus that encircles the duodenum, a phenotype frequently observed in humans suffering from a rare disorder known as annular pancreas. Shh-/- and Shh-/-;Ihh+/- mutants have a threefold increase in pancreas mass, and a fourfold increase in pancreatic endocrine cell numbers. In contrast, mutations in Ptc1 reduce pancreas gene expression and impair glucose homeostasis. Thus, islet cell, pancreatic mass and pancreatic morphogenesis are regulated by hedgehog signaling molecules expressed within and adjacent to the embryonic pancreas. Defects in hedgehog signaling may lead to congenital pancreatic malformations and glucose intolerance (Hebrok, 2000).

A recessive mouse mutation, mesenchymal dysplasia (mes), which arose spontaneously on Chromosome 13, causes excess skin, increased body weight, and mild preaxial polydactyly. Fine gene mapping in this study has indicated that mes is tightly linked to patched (ptc), which encodes a transmembrane receptor protein for Shh. Molecular characterization of the ptc gene of the mes mutant and an allelism test using a ptc knockout allele (ptc-) has demonstrated that mes is caused by a deletion of the most C-terminal cytoplasmic domain of the ptc gene. Since mes homozygous embryos exhibit normal spinal cord development as compared with ptc- homozygotes, which die around 10 dpc with severe neural tube defects, the C-terminal cytoplasmic domain lost in mes mutation is dispensable for inhibition of Shh signaling in early embryogenesis. However, compound heterozygotes of ptc- and mes alleles, that survive up to birth and die neonatally, have increased body weight and exhibit abnormal anteroposterior axis formation of the limb buds. These findings indicate that Ptc is a negative regulator of body weight and an ectopic activator of Shh signaling in the anterior mesenchyme of the limb buds, and that the C-terminal cytoplasmic domain of Ptc is involved in Ptc repressive action (Makino, 2001).

The Shh signaling pathway is required in many mammalian tissues for embryonic patterning, cell proliferation and differentiation. In addition, inappropriate activation of the pathway has been implicated in many human tumors. Based on transfection assays and gain-of-function studies in frog and mouse, the transcription factor Gli1 has been proposed to be a major mediator of Shh signaling. To address whether this is the case in mouse, a Gli1 null allele expressing lacZ was generated. Strikingly, Gli1 is not required for mouse development or viability. Of relevance, it has been shown that all transcription of Gli1 in the nervous system and limbs is dependent on Shh and, consequently, Gli1 protein is normally not present to transduce initial Shh signaling. To determine whether Gli1 contributes to the defects seen when the Shh pathway is inappropriately activated and Gli1 transcription is induced, Gli1;Ptc double mutants were generated. It has been shown that Gli1 is not required for the ectopic activation of the Shh signaling pathway or to the early embryonic lethal phenotype in Ptc null mutants. Instead, it has been found that Gli2 is required for mediating some of the inappropriate Shh signaling in Ptc mutants. These studies demonstrate that, in mammals, Gli1 is not required for Shh signaling and that Gli2 mediates inappropriate activation of the pathway due to loss of the negative regulator Ptc (Bai, 2002).

Sonic hedgehog (Shh) directs the development of ventral cell fates, including floor plate and V3 interneurons, in the mouse neural tube. The transcription factors Gli2 and Gli3, mediators of Shh signaling, are required for the development of the ventral cell fates but make distinct contributions to controlling cell fates at different locations along the rostral-caudal axis. Mutants lacking Patched1 (Ptc1), the putative receptor of Shh, were used to analyze Gli functions. Ptc1-/- mutants develop floor plate, motor neuron, and V3 interneuron progenitors in lateral and dorsal regions, suggesting that the normal role of Ptc1 is to suppress ventral cell development in dorsal neural tube. The Ptc1-/- phenotype is rescued, with restoration of dorsal cell types, by the lack of Gli2, but only in the caudal neural tube. In triple mutants of Gli2, Gli3, and Ptc1, dorsal and lateral cell fates are restored in the entire neural tube. These observations suggest that Gli2 is essential for ventral specification in the caudal neural tube, and that in more rostral regions, only Gli3 can promote development of ventral cells if Gli2 is absent. Thus, Shh signaling is mediated by overlapping but distinct functions of Gli2 and Gli3, and their relative contributions vary along the rostral-caudal axis (Moyoyama, 2003).

Hedgehog signaling is known to regulate tissue morphogenesis and cell differentiation in a dose-dependent manner. Loss of Indian hedgehog (Ihh) results in reduction in pancreas size, indicating a requirement for hedgehog signaling during pancreas development. By contrast, ectopic expression of sonic hedgehog (Shh) inhibits pancreatic marker expression and results in transformation of pancreatic mesenchyme into duodenal mesoderm. These observations suggest that hedgehog signaling activity has to be regulated tightly to ensure proper pancreas development. The function of two hedgehog inhibitors, Hhip (a type I TM protein that attenuates hedgehog signalling by binding all three mammalian hedgehog proteins) and patched 1 (Ptch), during pancreas formation has been analyzed. Loss of Hhip results in increased hedgehog signaling within the pancreas anlage. Pancreas morphogenesis, islet formation and endocrine cell proliferation is impaired in Hhip mutant embryos. Additional loss of one Ptch allele in Hhip–/–Ptch+/– embryos further impairs pancreatic growth and endodermal cell differentiation. These results demonstrate combined requirements for Hhip and Ptch during pancreas development and point to a dose-dependent response to hedgehog signaling within pancreatic tissue. Reduction of Fgf10 expression in Hhip homozygous mutants suggests that at least some of the observed phenotypes result from hedgehog-mediated inhibition of Fgf signaling at early stages (Kawahira, 2003).

Suppressor of fused is a negative regulator of Hh signaling. Targeted disruption of the murine suppressor of fused gene (Sufu) leads to a phenotype that includes neural tube defects and lethality at mid-gestation (9.0-10.5 dpc). This phenotype resembles that caused by loss of patched (Ptch1), another negative regulator of the Hh pathway. Consistent with this finding, Ptch1 and Sufu mutants display excess Hh signaling and resultant altered dorsoventral patterning of the neural tube. Sufu mutants also had abnormal cardiac looping, indicating a defect in the determination of left-right asymmetry. Marked expansion of nodal expression in 7.5 dpc embryos and variable degrees of node dysmorphology in 7.75 dpc embryos suggest that the pathogenesis of the cardiac developmental abnormalities is related to node development. Other mutants of the Hh pathway, such as Shh, Smo and Shh/Ihh compound mutants, also have laterality defects. In contrast to Ptch1 heterozygous mice, Sufu heterozygotes have no developmental defects and no apparent tumor predisposition. The resemblance of Sufu homozygotes to Ptch1 homozygotes is consistent with mouse Sufu being a conserved negative modulator of Hh signaling (Cooper, 2005).

Hedgehog (Hh) signaling plays pivotal roles in tissue patterning and development in Drosophila and vertebrates. The Patched1 (Ptc1) gene, encoding the Hh receptor, is mutated in nevoid basal cell carcinoma syndrome, a human genetic disorder associated with developmental abnormalities and increased incidences of basal cell carcinoma (BCC) and medulloblastoma (MB). Ptc1 mutations also occur in sporadic forms of BCC and MB. Mutational studies with mice have verified that Ptc1 is a tumor suppressor. A second mammalian Patched gene, Ptc2, is expressed in a distinct pattern during embryogenesis, suggesting a unique role in development. Most notably, Ptc2 is expressed in an overlapping pattern with Shh in the epidermal compartment of developing hair follicles and is highly expressed in the developing limb bud, cerebellum, and testis. This study describes the generation and phenotypic analysis of Ptc2tm1/tm1 mice. Molecular analysis suggests that Ptc2tm1 likely represents a hypomorphic allele. Despite the dynamic expression of Ptc2 during embryogenesis, Ptc2tm1/tm1 mice are viable, fertile, and apparently normal. Interestingly, adult Ptc2tm1/tm1 male animals develop skin lesions consisting of alopecia, ulceration, and epidermal hyperplasia. While functional compensation by Ptc1 might account for the lack of a strong mutant phenotype in Ptc2-deficient mice, these results suggest that normal Ptc2 function is required for adult skin homeostasis (Nieuwenhuis, 2006; full text of article).

Transcriptional regulation of mammalian Patched homologs

The tumor suppressor patched1 (PTC1), a product of the mammalian homolog of the Drosophila segment polarity gene patched, is a receptor for Hedgehog and is crucial for embryonic development. Although little is known about the signal transduction pathways leading to the activation of ptc1, increased ptc1 transcription has always been associated with elevated HH activity and decreased activity of cAMP-dependent protein kinase A. In the mammalian pineal gland, ptc1 expression exhibits a dramatic diurnal rhythm with peak expression at midnight. ptc1 mRNA expression in the pineal is regulated by a clock mechanism mediated by the superior cervical ganglion. Most important, ptc1 transcription can be induced by agents activating the cAMP signal transduction pathway both in vivo and in vitro and appears to be independent of HH signaling (Borjigin, 1999).

The cAMP-dependent and Hh-independent regulation of ptc1 in the pineal suggests PTC1 may play a role in the neuroendocrine and perhaps other systems in an activity-dependent manner. Unlike the relatively slow time course of ontogenic and oncogenic processes in which ptc1 has been previously studied, the pineal regulation of ptc occurs rapidly, with 10-20-fold changes of expression in a few hours. This swift change suggests a novel type of short term regulatory function for PTC1 in the pineal and other HH-independent PTC systems. Developmental studies indicate that ptc1 cycling is not necessary for the rhythmic transcription of serotonin N-acetyltransferase, the rate-limiting enzyme in melatonin synthesis, early in life. Perhaps PTC1 in the pineal functions in the post-transcriptional regulation of diurnal processes (i.e. mRNA stability or post-translational modifications). In addition to some diurnally determined role, PTC1 might play a part in the ontogenesis of pineal systems, including the development of circadian rhythms and a role in the oncogenesis of various pineal region tumors, consistent with the known functions of PTC1 during development and in tumor formation (Borjigin, 1999).

The Cubitus interruptus (Ci) and Gli proteins are transcription factors that mediate responses to Hedgehog proteins (Hh) in flies and vertebrates, respectively. During development of the Drosophila wing, Ci transduces the Hh signal and regulates transcription of different target genes at different locations. In vertebrates, the three Gli proteins are expressed in overlapping domains and are partially redundant. To assess how the vertebrate Glis correlate with Drosophila Ci, each was expressed in Drosophila and their behaviors and activities were monitored. Each Gli has distinct activities that are equivalent to portions of the regulatory arsenal of Ci. Gli2 and Gli1 have activator functions that depend on Hh. Gli2 and Gli3 are proteolyzed to produce a repressor form able to inhibit hh expression. However, while Gli3 repressor activity is regulated by Hh, Gli2 repressor activity is not. These observations suggest that the separate activator and repressor functions of Ci are unevenly partitioned among the three Glis, yielding proteins with related yet distinct properties (Aza-Blanc, 2000).

Although in aggregate the Gli proteins appear to embody the many different attributes of Ci, only some of the Ci activities are in each. Most intriguing, perhaps, is the differential activation of ptc and dpp expression by Gli1 and Gli2, respectively. The basis for the selectivity of Gli1 for ptc and Gli2 for dpp is not understood, but it has many conceivable causes. One is that Gli2 interacts with proteins known to associate with Ci, such as CBP, but that Gli1 does not. Alternatively, the ability of Gli2 to activate dpp more strongly could be related to the conversion of Gli2 to a repressor form. It is formally possible that the activator and repressor forms can cooperate in some manner to enhance dpp transcription, or that the repressor form competes with the activator for binding sites at the ptc promoter. Consistent with this latter proposal, the level of ptc induction in wing discs is inversely related to the level of Gli2 expression: higher levels of expression produce lower levels of ptc. Since Ci75 is abundant in A cells that express high levels of dpp, but it is not in cells closer to the compartment border where ptc is expressed, this model may be relevant to Ci. Perhaps the most interesting possibility to consider is that the reason for the differential activation of dpp and ptc may be that Gli1 and Gli2 represent different forms of Ci Act, one with a preference for ptc and the other for dpp (Aza-Blanc, 2000).

The importance of the CREB family of transcriptional activators for endochondral bone formation was evaluated by expressing a potent dominant negative CREB inhibitor (A-CREB) in growth plate chondrocytes of transgenic mice. A-CREB transgenic mice exhibit short-limbed dwarfism and die minutes after birth, apparently due to respiratory failure from a diminished rib cage circumference. Consistent with the robust Ser133 phosphorylation and, hence, activation of CREB in chondrocytes within the proliferative zone of wild-type cartilage during development, chondrocytes in A-CREB mutant cartilage exhibit a profound decrease in proliferative index and a delay in hypertrophy. Correspondingly, the expression of certain signaling molecules in cartilage, most notably the Indian hedgehog (Ihh) receptor patched (Ptch), was lower in A-CREB expressing versus wild-type chondrocytes. CREB appears to promote Ptch expression in proliferating chondrocytes via an Ihh-independent pathway; phospho-CREB levels were comparable in cartilage from Ihh -/- and wild-type mice. These results demonstrate the presence of a distinct signaling pathway in developing bone that potentiates Ihh signaling and regulates chondrocyte proliferation, at least in part, via the CREB family of activators (Long, 2001).

Cellular location Patched homologs

The Hedgehog signaling pathway is involved in early embryonic patterning as well as in cancer; however, little is known about the subcellular localization of the Hedgehog receptor complex of Patched and Smoothened. Since Hh has been found in lipid rafts in Drosophila, it was hypothesized that Patched and Smoothened might also be found in these cholesterol-rich microdomains. In this study, both Smoothened and Patched are demonstrated to be in caveolin-1-enriched/raft microdomains. Immunoprecipitation studies show that Patched specifically interacts with caveolin-1, whereas Smoothened does not. Fractionation studies show that Patched and caveolin-1 can be co-isolated from buoyant density fractions that represent caveolae/raft microdomains and that Patched and caveolin-1 co-localize by confocal microscopy. Glutathione S-transferase fusion protein experiments show that the interaction between Patched and caveolin-1 involves the caveolin-1 scaffolding domain and a Patched consensus binding site. Immunocytochemistry data and fractionation studies also show that Patched seems to be required for transport of Smoothened to the membrane. Depletion of plasmalemmal cholesterol influences the distribution of the Hh receptor complex in the caveolin-enriched/raft microdomains. These data suggest that caveolin-1 may be integral for sequestering the Hh receptor complex in these caveolin-enriched microdomains, which act as a scaffold for the interactions with the Hh protein (Karpen 2001).

Evidence for a role of vertebrate Disp1 in long-range Shh signaling

Dispatched 1 (Disp1) encodes a twelve transmembrane domain protein that is required for long-range sonic hedgehog (Shh) signaling. Inhibition of Disp1 function, both by RNAi or dominant-negative constructs, prevents secretion and results in the accumulation of Shh in source cells. Measuring the Shh response in neuralized embryoid bodies (EBs) derived from embryonic stem (ES) cells, with or without Disp1 function, demonstrates an additional role for Disp1 in cells transporting Shh. Co-cultures with Shh-expressing cells revealed a significant reduction in the range of the contact-dependent Shh response in Disp1-/- neuralized EBs. These observations support a dual role for Disp1, not only in the secretion of Shh from the source cells, but also in the subsequent transport of Shh through tissue (Etheridge, 2010).

These results suggest that Ptch1 and Disp1 act in concert to mediate the transport of Shh through tissues. The similarities between Ptch1 and Disp1, such as their ability to trimerize and their putative proton channel, indicates that their function might be conserved with that of the resistance-nodulation-cell division (RND) family of proton-driven transporters in bacteria. In general, the role of Disp1 is in the secretion of Shh, whereas Ptch1 is involved in the uptake of Shh, and the function of both is necessary for long-range Shh signaling. These observations are consistent with a model in which reiterated secretion (by Disp1) and uptake (by Ptch1) are involved in the long-range transport of Shh. The non-directionality of this process, combined with the incomplete secretion of all internalized Shh, would sufficiently distribute Shh in a gradient away from the source (Etheridge, 2010).

Based on these results the following model is proposed. Disp1 is active in MVBs and mediates the loading of Shh onto exosome/lipoprotein-like particles, which are then secreted. These particles are specifically recognized by Ptch1 at the surface of adjacent cells, which traffics them into early/late endosomes, where the particles are disassembled. Shh can either be degraded, trafficked to the apical surface or trafficked into MVBs, where it would be loaded onto exosomes again for re-secretion. This model accounts for the putative function of Disp1 as a proton-driven transporter and explains the high molecular weight complex that Shh is found in outside of cells, the pH-dependent action of Disp1 and Ptch1 in the intracellular trafficking of Shh and the role of Disp1 in the re-secretion of Shh (Etheridge, 2010).

Growth and pattern of the mammalian neural tube are governed by partially overlapping feedback activities of the hedgehog antagonists patched 1 and Hhip1

Upregulation of Patched (Ptc), the Drosophila Hedgehog (Hh) receptor in response to Hh signaling, limits the range of signaling within a target field by sequestering Hh. In vertebrates, Ptch1 also exhibits ligand-dependent transcriptional activation, but mutants lacking this response show surprisingly normal early development. The identification of Hh-interacting protein 1 (Hhip1), a vertebrate-specific feedback antagonist of Hh signaling, raises the possibility of overlapping feedback controls. The significance of feedback systems in sonic hedgehog (Shh)-dependent spinal cord patterning was addressed. Mouse embryos lacking both Ptch1 and Hhip1 feedback activities exhibit severe patterning defects consistent with an increased magnitude and range of Hh signaling, and disrupted growth control. Thus, Ptc/Ptch1-dependent feedback control of Hh morphogens is conserved between flies and mice, but this role is shared in vertebrates with Hhip1. Furthermore, this feedback mechanism is crucial in generating a neural tube that contains appropriate numbers of all ventral and intermediate neuronal cell types (Jeong, 2005).

Unlike Drosophila, vertebrates have several Hh-binding proteins that are transcriptionally regulated by Hh signaling; patched 2 (Ptch2) and Hh-interacting protein 1 (Hhip1) are positively regulated, whereas growth arrest specific gene 1 (Gas1) is negatively regulated. The role of Ptch2 or Gas1 in Hh-mediated patterning processes during normal development has yet to be established. Overexpression and loss-of-function studies in the mouse indicate that Hhip1, a cell-surface glycoprotein, is an antagonist of Hh signaling; Hhip1-/- embryos die soon after birth, owing to lung defects indicative of overactive Hh signaling. However, other parts of the body where Hh signaling plays important roles, e.g. the limb, face and spinal cord, develop normally in Hhip1 mutants. Taken together, the mild phenotypes of both MtPtch1;Ptch1-/- and Hhip1-/- embryos suggest that Ptch1 and Hhip1 may be functionally redundant in providing feedback ligand dependent antagonism (LDA) to Hh ligands. Consistent with this view, removing one copy of Ptch1 allele in Hhip1-/- embryos (Hhip1-/-;Ptch1+/-) causes earlier lethality (around E12.5) and more severe lung and pancreas defects than those observed in Hhip1-/- embryos (Jeong, 2005).

Although the previous studies point to a role for Ptch1 and Hhip1 in attenuation of paracrine Hh signaling, they did not address the issue of how LDA might contribute to controlling the magnitude (pathway activity at a given position in the tissue) or range (total distance over which the pathway is activated) of a morphogen signaling gradient to generate a specific pattern. The best evidence for Shh acting as a morphogen comes from studies in the vertebrate spinal cord. Here, Shh is first produced from the notochord that underlies the neural tube, and directs the formation of floor plate which in turn expresses Shh. Shh from these two ventral midline sources forms a concentration gradient along the dorsoventral (DV) axis of the neural tube, and represses (Class I proteins) or induces (Class II proteins) expression of several homeodomain and basic helix-loop-helix transcription factors at different thresholds. Cross-repression between the transcription factors sharing a border further sharpens the boundaries of their territories to define five neural progenitor domains in the ventral half of the spinal cord (from ventral to dorsal, p3, pMN, p2, p1, p0). Finally, cells in each domain differentiate into specific types of neurons (from ventral to dorsal, V3, motoneuron [MN], V2, V1, V0) based on the combinations of transcription factors they express. Since Hh signaling controls the specification of individual progenitor domains by a direct and dose-dependent mechanism, changes in the expression of progenitor domain-associated transcription factors provide sensitive readouts for any perturbations in the Shh morphogen gradient (Jeong, 2005).

The role of negative feedback regulation on Hh signaling was investigated in vertebrates by analyzing mouse embryos that lack both Ptch1 and Hhip1 feedback mechanisms (MtPtch1;Ptch1-/-;Hhip1-/-). The findings indicate that the LDA mediated by these components plays a crucial role in controlling the magnitude and most likely the range of Shh morphogen signaling (Jeong, 2005).

Patched1 and Patched2 inhibit Smoothened non-cell autonomously

Smoothened (Smo) inhibition by Patched (Ptch) is central to Hedgehog (Hh) signaling. Ptch, a proton driven antiporter, is required for Smo inhibition via an unknown mechanism. Hh ligand binding to Ptch reverses this inhibition and activated Smo initiates the Hh response. To determine whether Ptch inhibits Smo strictly in the same cell or also mediates non cell-autonomous Smo inhibition, genetically mosaic neuralized embryoid bodies (nEBs) were generated from mouse embryonic stem cells (mESCs). These experiments utilized novel mESC lines in which Ptch1, Ptch2, Smo, Shh and 7dhcr were inactivated via gene editing in multiple combinations, allowing measurement of non-cell autonomous interactions between cells with differing Ptch1/2 status. In several independent assays the Hh response was repressed by Ptch1/2 in nearby cells. When 7dhcr was targeted, cells displayed elevated non-cell autonomous inhibition. These findings support a model in which Ptch1/2 mediate secretion of a Smo-inhibitory cholesterol precursor (Roberts, 2016).

Mammalian Patched homologs and development

The three mouse Gli genes are putative transcription factors that are the homologs of cubitus interruptus in Drosophila. Along with the gene patched, ci has been implicated in the Hedgehog (Hh) signal transduction pathway. To assess the role of Gli in embryogenesis, its expression was compared with that of Ptc and Hh family members in mouse. Gli and Ptc are expressed in similar domains in diverse regions of the developing mouse embryo and these regions are adjacent to Hh signals. Gli and different Hh isoforms show reciprocal relationships in the limb, digits, brain, gut, and whisker follicles. Gli is expressed ectopically along with Ptc and Shh in Strong's luxoid mutant mice. It is likely that Shh is expressed ectopically in the dominant Strong's Luxoid mutation. These results are consistent with conservation of the Hh signal transduction pathway in mice with Gli potentially mediating Hh signaling in multiple regions of the developing embryo (Platt, 1997).

In Drosophila, patched encodes a negative regulator of Hedgehog signaling. Biochemical experiments have demonstrated that vertebrate patched homologs might function as a Sonic hedgehog (Shh) receptor. In mice, two Patched homologues, Ptch and Ptch2, have been identified. Sequence comparisons have suggested that they might possess distinct properties in Shh signaling. In the developing tooth, hair and whisker, Shh and Ptch2 are co-expressed in the epithelium while Ptch is strongly expressed in the mesenchymal cells. Throughout mouse development, the level of Ptch2 expression is significantly lower than that of Ptch. In early mouse embryos, Ptch and Ptch2 were found to be co-expressed in regions adjacent to Shh-expressing cells in the developing CNS. Similar to other epidermal structures, Shh and Ptch2 also show overlapping expression in the developing nasal gland and eyelids. Thus, during mouse development, Ptch2 is expressed in both Shh-producing and -nonproducing cells (Motoyama, 1998).

Branching morphogenesis of the embryonic murine lung requires interactions between the epithelium and the mesenchyme. Sonic hedgehog transcripts are present in the epithelium of the developing lung, with highest levels in the terminal buds. Transcripts of mouse patched, the putatitive Sonic hedgehog receptor, are expressed at high levels in the mesenchyme adjacent to the end buds. To investigate the function of SHH in lung development, Shh was overexpressed throughout the distal epithelium, using the surfactant protein-C (SP-C)-enhancer/promoter. Beginning around 16.5 dpc, when Shh and Ptc mRNA levels are normally both declining, this treatment causes an increase in the ratio of interstitial mesenchyme to epithelial tubules in transgenic compared to normal lungs. Transgenic newborn mice die soon after birth. Histological analysis of the lungs shows an abundance of mesenchyme and the absence of typical alveoli. Shh overexpression results in increased mesenchymal and epithelial cell proliferation at 16.5 and 17.5 dpc. However, there is no significant inhibition in the differentiation of proximal and distal epithelial cells. The expression of genes potentially regulated by SHH was also examined. No difference could be observed between transgenic and control lungs in either the level or distribution of Bmp4, Wnt2 and Fgf7 RNA. By contrast, Ptc is clearly upregulated in the transgenic lung. These results thus establish a role for SHH in lung morphogenesis, and suggest that SHH normally regulates lung mesenchymal cell proliferation in vivo (Bellusci, 1996).

Sonic hedgehog and Patched proteins are expressed in adjacent domains in the developing mouse retina. Treatment of cultures of perinatal mouse retinal cells with the amino-terminal fragment of Sonic hedgehog protein results in an increase in the proportion of cells that incorporate bromodeoxuridine, in total cell numbers, and in rod photoreceptors, amacrine cells and Muller glial cells, suggesting that Sonic hedgehog promotes the proliferation of retinal precursor cells. These finding suggest that Hedgehog and Patched are part of a conserved signaling pathway in the retinal development of both mammals and insects (Jensen, 1997).

While prostate gland development is dependent on androgens, other hormones including retinoids and estrogens can influence this process. Brief exposure to high-dose estrogen during the neonatal period in rats leads to permanent, lobe-specific aberrations in the prostate gland, a phenomenon referred to as developmental estrogenization. This response is mediated through alterations in steroid receptor expression; however, further downstream mechanisms remain unclear. Sonic hedgehog (Shh)-patched (ptc)-gli was investigated in the developing rat prostate gland, its role in branching morphogenesis, and the effects of neonatal estrogens on its expression and localization to determine whether a disturbance in this signaling pathway is involved in mediating the estrogenized phenotype. Shh is expressed in epithelial cells at the distal tips of elongating ducts in discreet, heterogeneous foci, while ptc and gli1–3 are expressed in the adjacent mesenchymal cells in the developing gland. The addition of Shh protein to cultured neonatal prostates reduces ductal growth and branching, decreases Fgf10 transcript, and increases Bmp4 expression in the adjacent mesenchyme. Shh-induced growth suppression is reversed by exogenous Fgf10, but not noggin, indicating that Fgf10 suppression is the proximate cause of the growth inhibition. A model is proposed to show how highly localized Shh expression along with regulation of downstream morphogens participates in dichotomous branching during prostate morphogenesis. Neonatal exposure to high-dose estradiol suppresses Shh, ptc, gli1, and gli3 expressions and concomitantly blocks ductal branching in the dorsal and lateral prostate lobes specifically. In contrast, ventral lobe branching and Shh-ptc-gli expression are minimally affected by estrogen exposure. Organ culture studies with lateral prostates confirms that estradiol suppresses Shh-ptc-gli expression directly at the prostatic level. Taken together, the present findings indicate that lobe-specific decreases in Shh-ptc-gli expression are involved in mediating estradiol-induced suppression of dorsal and lateral lobe ductal growth and branching during prostate morphogenesis (Pu, 2004).

Hedgehog (Hh)-Patched1 (Ptch1) signaling plays essential roles in various developmental processes, but little is known about its role in postnatal homeostasis. This study demonstrate regulation of postnatal bone homeostasis by Hh-Ptch1 signaling. Ptch1-deficient (Ptch1+/-) mice and patients with nevoid basal cell carcinoma syndrome show high bone mass in adults. In culture, Ptch1+/- cells showed accelerated osteoblast differentiation, enhanced responsiveness to the runt-related transcription factor 2 (Runx2), and reduced generation of the repressor form of Gli3 (Gli3rep). Gli3rep inhibited DNA binding by Runx2 in vitro, suggesting a mechanism that could contribute to the bone phenotypes seen in the Ptch1 heterozygotes. Moreover, systemic administration of the Hh signaling inhibitor cyclopamine decreased bone mass in adult mice. These data provide evidence that Hh-Ptch1 signaling plays a crucial role in postnatal bone homeostasis and point to Hh-Ptch1 signaling as a potential molecular target for the treatment of osteoporosis (Ohba, 2008).

Systemic hormones and local growth factor-mediated tissue interactions are essential for mammary gland development. Using phenotypic and transplantation analyses of mice carrying the mesenchymal dysplasia (mes) allele of patched 1 (Ptch1mes), it was found that Ptch1mes homozygosity led to either complete failure of gland development, failure of post-pubertal ductal elongation, or delayed growth with ductal dysplasia. All ductal phenotypes could be present in the same animal. Whole gland and epithelial fragment transplantation each yielded unique morphological defects indicating both epithelial and stromal functions for Ptch1. However, ductal elongation was rescued in all cases, suggesting an additional systemic function. Epithelial function was confirmed using a conditional null Ptch1 allele via MMTV-Cre-mediated disruption. In Ptch1mes) homozygotes, failure of ductal elongation correlated with diminished estrogen and progesterone receptor expression, but could not be rescued by exogenous ovarian hormone treatment. By contrast, pituitary isografts were able to rescue the ductal elongation phenotype. Thus, Ptch1 functions in the mammary epithelium and stroma to regulate ductal morphogenesis, and in the pituitary to regulate ductal elongation and ovarian hormone responsiveness (Moraes, 2009).

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

Patched 1 (Ptch1; see Drosophila Patched) 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; see Drosophila Smoothened). 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. 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 (Monkkonen, 2017).

Patched and limb development

The vertebrate hedgehog receptor patched 1 (Ptc1) is crucial for negative regulation of the sonic hedgehog (Shh) pathway during anterior-posterior patterning of the limb. Ptc1 was conditionally inactivated in the mesenchyme of the mouse limb using Prx1-Cre. This results in constitutive activation of hedgehog (Hh) signalling during the early stages of limb budding. The data suggest that variations in the timing and efficiency of Cre-mediated excision result in differential forelimb and hindlimb phenotypes. Hindlimbs display polydactyly (gain of digits) and a molecular profile similar to the Gli3 mutant extra-toes. Strikingly, forelimbs are predominantly oligodactylous (displaying a loss of digits), with a symmetrical, mirror-image molecular profile that is consistent with re-specification of the anterior forelimb to a posterior identity. These data suggest that this is related to very early inactivation of Ptc1 in the forelimb perturbing the gene regulatory networks responsible for both the pre-patterning and the subsequent patterning stages of limb development. These results establish the importance of the downstream consequences of Hh pathway repression, and identify Ptc1 as a key player in limb patterning even prior to the onset of Shh expression (Butterfield, 2009).

Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs

The large spectrum of limb morphologies reflects the wide evolutionary diversification of the basic pentadactyl pattern in tetrapods. In even-toed ungulates (artiodactyls, including cattle), limbs are adapted for running as a consequence of progressive reduction of their distal skeleton to symmetrical and elongated middle digits with hoofed phalanges. This study analysed bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene, which encodes a Sonic hedgehog (SHH) receptor, is disrupted specifically in the bovine limb bud mesenchyme. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded SHH signalling during bovine handplate development. This study provides a molecular explanation for the loss of digit asymmetry in bovine limb buds and suggests that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs (Lopez-Rios, 2014).

Mammalian Patched and hair development

Proper patterning of self-renewing organs, like the hair follicle, requires exquisite regulation of growth signals. Sonic hedgehog (Shh) signaling in skin controls the growth and morphogenesis of hair follicle epithelium in part through regulating the Gli transcription factors. While ectopic induction of Shh target genes leads to hair follicle tumors, such as basal cell carcinomas, how Shh signaling normally functions during the cyclic process of hair development is unknown. During the hair cycle, Shh expression and the ability of skin cells to respond to Shh signaling is spatially and temporally regulated. Induction of Shh target genes normally occurs only in the anagen (the growth phase of follicular epithelium) hair follicle in response to expression of Shh. However, in patched1 heterozygous mice, putative tumor precursors form with concomitant induction of Shh target gene transcription only during anagen in follicular and interfollicular keratinocytes. Ectopic production of Gli1 accumulates Gli protein and induces Shh target genes and epithelial tumors at anagen but not other stages, pointing to a restricted competence occurring at the level of Gli protein accumulation. Delivery and reception of growth signals among multipotent cells are restricted in time and space to facilitate cyclic pattern formation (Oro, 2003).

Mammalian Patched homologs and tooth development

The signalling peptide encoded by the sonic hedgehog gene is restricted to localised thickenings of oral epithelium, which mark the first morphological evidence of tooth development, and is known to play a crucial role during the initiation of odontogenesis. At these stages in the murine mandibular arch in the absence of epithelium, the Shh targets Ptc1 and Gli1 are upregulated in diastema mesenchyme, an edentulous region between the sites of molar and incisor tooth formation. This ectopic expression is not associated with Shh transcription but with the presence of ectopic Shh protein, undetectable in the presence of epithelium. These findings suggest that, in diastema mesenchyme, restriction of Shh activity is dependent upon the overlying epithelium. This inhibitory activity was demonstrated by the ability of transplanted diastema epithelium to downregulate Ptc1 in tooth explants, and for isolated diastema mesenchyme to express Ptc1. A candidate inhibitor in diastema mesenchyme is the glycosylphosphatidylinositol-linked membrane glycoprotein Gas1. Gas1 is normally expressed throughout mandibular arch mesenchyme; however, in the absence of epithelium this expression was downregulated specifically in the diastema where ectopic Shh protein was identified. Although Shh signalling has no effect upon Gas1 expression in mandibular arch mesenchyme, overexpression of Gas1 results in downregulation of ectopic Ptc1. Therefore, control of the position of tooth initiation in the mandibular arch involves a combination of Shh signalling at sites where teeth are required and antagonism in regions destined to remain edentulous (Cobourne, 2004).

Mammalian Patched homologs, cell cycle, cell growth and cancer

Hedgehog (Hh) proteins control many developmental events by inducing specific cell fates or regulating cell proliferation. The Patched1 (Ptc1) protein, a binding protein for Hh molecules, appears to oppose Hh signals by repressing transcription of genes that can be activated by Hh. Sonic hedgehog (Shh), one of the vertebrate homologs of Hh, controls patterning and growth of the limb but the early embryonic lethality of ptc1 mutant mice obscures the roles of ptc1 in later stages of development. ptc1 homozygous mutant embryos were partially rescued using a metallothionein promoter driving ptc1. In a wild-type background, the transgene causes a marked decrease in animal size starting during embryogenesis, and loss of anterior digits. In ptc1 homozygotes, a potent transgenic insert allows survival to E14 and largely normal morphology except for midbrain overgrowth. A less potent transgene gives rise to partially rescued embryos with massive exencephaly, and polydactyly and branched digits in the limbs. The polydactyly is preceded by unexpected anterior limb bud transcription of Shh, so one function of ptc1 is to repress Shh expression in the anterior limb bud (Milenkovic, 1999).

Transgenic mice that overexpress ptc1 are consistently smaller than control mice and the opposite is true as well -- larger body size is associated with heterozygosity at the ptc1 locus, just as it can be in humans. Another tumor suppressor gene has similar effects: overexpression of the retinoblastoma gene in mice induces dose-dependent growth retardation, as early as E15. The size of an animal generally reflects cell numbers and therefore a balance between cell proliferation and cell death. It is believed that substantially fewer cells are formed during the development of MT-ptc1 mice. Shh has mitogenic effects in several tissues including presomitic mesoderm, retina and cerebellum. Another Hh protein, Ihh controls growth in the cartilage, and Ihh-deficient mice exhibit short-limbed dwarfism. Overexpression of Hip1, another negative regulator of Hh signaling, in the cartilage, leads to a shortened skeleton that resembles that seen in Ihh loss-of-function mutants. ptc1 may counteract mitogenic activities of one or all of the Hh proteins to restrict body size. The embryos are remarkably well proportioned, so either Ptc exerts growth control in many locations or all tissues are somehow coordinated in size with those directly affected by ptc1. The generality of the effect of Ptc1 on body size raises the possibility of growth control unrelated to the Hedgehog genes. For example, there may be Ptc1 actions that cannot be blocked, or are never blocked, by a Hh signal. Since the MT-ptc1 transgenic embryos are different in size as early as the eleventh day of embryogenesis, the effects of Ptc1 are likely to precede, and be independent of, growth hormone. Major determinants of mouse embryonic growth are insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), and their cognate type 1 receptor (IGF1R). Mutations in all three genes have dwarf phenotypes. For the IGF-II and IGF-1R mutations, significant reduction in embryo size first starts at about E10.5. The IGF-I phenotype does not become evident until E13.5. Since growth-modifying effects of IGFs are apparent as early as E10.5 of embryogenesis, the IGF signaling circuitry may be involved in growth retardation effects of ptc1 (Milenkovic, 1999).

The initiation of mitosis requires the activation of M-phase promoting factor (MPF). MPF activation and its subcellular localization are dependent on the phosphorylation state of its components, cdc2 and cyclin B1. In a two-hybrid screen using a bait protein to mimic phosphorylated cyclin B1, a novel interaction was detected between cyclin B1 and patched1 (ptc1), a tumor suppressor associated with basal cell carcinoma (BCC). Ptc1 interacts specifically with constitutively phosphorylated cyclin B1 derivatives and is able to alter their normal subcellular localization. Furthermore, addition of the ptc1 ligand, sonic hedgehog (shh), disrupts this interaction and allows cyclin B1 to localize to the nucleus. Expression of ptc1 in 293T cells is inhibitory to cell proliferation; this inhibition could be relieved by coexpression of a cyclin B1 derivative that constitutively localizes to the nucleus and that could not interact with ptc1 due to phosphorylation-site mutations to Ala. In addition, endogenous ptc1 and endogenous cyclin B1 interact in vivo. The findings reported here demonstrate that ptc1 participates in determining the subcellular localization of cyclin B1 and suggest a link between the tumor suppressor activity of ptc1 and the regulation of cell division. Thus, it is proposed that ptc1 participates in a G2/M checkpoint by regulating the localization of MPF (Barnes, 2001).

Basal-cell carcinomas (BCCs) are the commonest human cancer. Insight into their genesis came from identification of mutations in the patched gene (PTCH) in patients with the basal-cell nevus syndrome, a hereditary disease characterized by multiple BCCs and by developmental abnormalities. The binding of Sonic hedgehog (SHH) to its receptor, PTCH, is thought to prevent normal inhibition by PTCH of Smoothened (SMO), a seven-span transmembrane protein. According to this model, the inhibition of SMO signaling is relieved following mutational inactivation of PTCH in basal-cell nevus syndrome. Activating somatic missense mutations have been identified in the SMO gene itself in sporadic BCCs from three patients. Mutant SMO, unlike wild type, can cooperate with adenovirus E1A to transform rat embryonic fibroblast cells in culture. Skin abnormalities similar to BCCs develop in transgenic murine skin overexpressing mutant SMO. These findings support the role of SMO as a signaling component of the SHH-receptor complex and provide direct evidence that mutated SMO can function as an oncogene in BCCs. The increased PTCH mRNA observed in BCC can be ascribed to missense mutations in SMO (Xie, 1998).

The basal cell nevus syndrome (BCNS) is characterized by developmental abnormalities and by the postnatal occurrence of cancers, especially basal cell carcinomas (BCCs), the most common human cancer. Heritable mutations in BCNS patients and a somatic mutation in a sporadic BCC were identified in a human homolog of the Drosophila patched (ptc) gene. The human PTC gene appears to be crucial for proper embryonic development and for tumor suppression (Johnson, 1996).

The Patched (Ptc) gene encodes a Sonic hedgehog (Shh) receptor and a tumor suppressor protein that is defective in basal cell nevus syndrome (BCNS). Functions of PTC were investigated by inactivating the mouse gene. Mice homozygous for the ptc mutation die during embryogenesis and have open and overgrown neural tubes. Two Shh target genes, ptc itself and Gli, are derepressed in the ectoderm and mesoderm but not in the endoderm. Shh targets that are, under normal conditions, transcribed ventrally are aberrantly expressed in dorsal and lateral neural tube cells. Thus Ptc appears to be essential for repression of genes that are locally activated by Shh. Mice heterozygous for the ptc mutation are larger than normal, and a subset of them developed hindlimb defects or cerebellar medulloblastomas, abnormalities also seen in BCNS patients (Goodrich, 1997).

About one-third of sporadic basal cell carcinomas (BCCs) of the skin and 10%-15% of primitive neuroectodermal tumors (PNETs) of the central nervous system show mutations in the PTCH tumor suppressor gene. The PTCH gene product (Ptch) functions as a transmembrane receptor for the Sonic hedgehog protein (Shh) and interacts with another transmembrane protein called Smoh. To further elucidate the significance of alterations in the Shh signaling pathway, thirty one sporadic BCCs and fifteen PNETs were investigated for the mutation and/or expression of SMOH, PTCH, SHH, and GL11. In addition, the SMOH gene locus was fine-mapped by fluorescence in situ hybridization to chromosomal band 7q32. Mutational analysis has identified four BCCs with somatic missense mutations in SMOH affecting codon 535 (TGG to TTG: Trp to Leu) in three tumors and codon 199 (CGG to TGG: Arg to Trp) in one tumor. A missense mutation at codon 533 (AGC to AAC: Ser to Asn) was found in one PNET. PTCH mutations were detected in eight BCCs and one PNET. Two BCCs demonstrate mutations in both SMOH and PTCH. The majority of tumors show an increased expression of SMOH, PTCH, and GL11 transcripts as compared with that of normal skin and nonneoplastic brain tissue, respectively. In contrast, only one BCC and one PNET express SHH mRNA at levels detectable by reverse transcription-PCR, and no SHH gene mutations were found. In summary, these results indicate that both PTCH and SMOH represent important targets for genetic alterations in sporadic BCCs and PNETs (Reifenberger, 1998).

Medulloblastoma is the most common malignant brain tumor in children. It is thought to result from the transformation of granule cell precursors (GCPs) in the developing cerebellum, but little is known about the early stages of the disease. A pre-neoplastic stage of medulloblastoma has been identified in patched heterozygous mice, a model of the human disease. Pre-neoplastic cells are present in the majority of patched mutants, although only 16% of these mice develop tumors. Pre-neoplastic cells, like tumor cells, exhibit activation of the Sonic hedgehog pathway and constitutive proliferation. Importantly, they also lack expression of the wild-type patched allele, suggesting that loss of patched is an early event in tumorigenesis. Although pre-neoplastic cells resemble GCPs and tumor cells in many respects, they have a distinct molecular signature. Genes that mark the pre-neoplastic stage include regulators of migration, apoptosis and differentiation, processes crucial for normal development but previously unrecognized for their role in medulloblastoma. The identification and molecular characterization of pre-neoplastic cells provides insight into the early steps in medulloblastoma formation, and may yield important markers for early detection and therapy of this disease (Oliver, 2005).

Studying the early stages of cancer can provide important insight into the molecular basis of the disease. A preneoplastic stage was identified in the patched (ptc) mutant mouse, a model for the brain tumor medulloblastoma. Preneoplastic cells (PNCs) are found in most ptc mutants during early adulthood, but only 15% of these animals develop tumors. Although PNCs are found in mice that develop tumors, the ability of PNCs to give rise to tumors has never been demonstrated directly, and the fate of cells that do not form tumors remains unknown. Using genetic fate mapping and orthotopic transplantation, definitive evidence was provide that PNCs give rise to tumors, and the predominant fate of PNCs that do not form tumors is differentiation. Moreover, N-myc, a gene commonly amplified in medulloblastoma, can dramatically alter the fate of PNCs, preventing differentiation and driving progression to tumors. Importantly, N-myc allows PNCs to grow independently of hedgehog signaling, making the resulting tumors resistant to hedgehog antagonists. These studies provide the first direct evidence that PNCs can give rise to tumors, and demonstrate that identification of genetic changes that promote tumor progression is critical for designing effective therapies for cancer (Kessler, 2009).

Patched and Cyclopamine inhibition of Sonic hedgehog signal

Cyclopamine is a teratogenic steroidal alkaloid that causes cyclopia by blocking Sonic hedgehog (Shh) signal transduction. Whether this activity of cyclopamine is related to disruption of cellular cholesterol transport and putative secondary effects on the Shh receptor, Patched, has been tested. The potent antagonism of Shh signaling by cyclopamine is not a general property of steroidal alkaloids with similar structure. The structural features of steroidal alkaloids previously associated with the induction of holoprosencephaly in whole animals are also associated with inhibition of Shh signaling in vitro. By comparing the effects of cyclopamine on Shh signaling with those of compounds known to block cholesterol transport, it has been shown that the action of cyclopamine cannot be explained by inhibition of intracellular cholesterol transport. However, compounds that block cholesterol transport by affecting the vesicular trafficking of the Niemann-Pick C1 protein (NPC1), which is structurally similar to Ptc, are weak Shh antagonists. Rather than supporting a direct link between cholesterol homeostasis and Shh signaling, these findings suggest that the functions of both NPC1 and Ptc involve a common vesicular transport pathway. Consistent with this model, it is found that Ptc and NPC1 colocalize extensively in a vesicular compartment in cotransfected cells (Incardona, 2000b).

Basal cell carcinoma, medulloblastoma, rhabdomyosarcoma and other human tumors are associated with mutations that activate the proto-oncogene Smoothened (SMO) or that inactivate the tumor suppressor Patched (PTCH). Smoothened and Patched mediate the cellular response to the Hedgehog (Hh) secreted protein signal, and oncogenic mutations affecting these proteins cause excess activity of the Hh response pathway. The plant-derived teratogen cyclopamine, which inhibits the Hh response, is a potential 'mechanism-based' therapeutic agent for treatment of these tumors. Cyclopamine or synthetic derivatives with improved potency block activation of the Hh response pathway and abnormal cell growth associated with both types of oncogenic mutation. These results also indicate that cyclopamine may act by influencing the balance between active and inactive forms of Smoothened (Taipale, 2000).

Whereas embryonic loss of Sonic hedgehog (Shh) signaling can result in cyclopia and other developmental defects, inappropriate activation of the Shh response pathway is associated with several types of human tumor. Current approaches to treatment of such neoplastic disorders are limited by the cytotoxic effects of therapeutic agents on proliferating tissues. Alternative 'mechanism-based' approaches specifically targeting abnormally active signaling pathways in defined types of cancer might avoid such toxicity, particularly if the pathways in question functioned primarily in embryonic development and were not required for survival in adults. Cyclopamine, a plant steroidal alkaloid, induces cyclopia in vertebrate embryos and has been shown to act by inhibiting the cellular response to the Shh signal. To evaluate the therapeutic potential of cyclopamine for the treatment of Hh-pathway-associated disorders, the mechanism by which cyclopamine acts was investigated (Taipale, 2000).

Cellular responses to the Hh signal are controlled by two transmembrane proteins, Smo and Ptch, which are predicted to have seven and twelve transmembrane spans, respectively. Genetic and biochemical evidence indicates that Ptch suppresses the activity of Smo, and that binding of Hh to Ptch relieves this suppression, allowing activation of downstream targets through the Ci/Gli family of transcriptional effectors. Because a Hh signaling assay using with Drosophila cultured cells is not sensitive to cyclopamine, several vertebrate cell lines were screened for a sensitive transcriptional response to palmitoyl- and cholesteryl-modified ShhN polypeptide (ShhNp) using a Gli-dependent luciferase reporter. Among several responsive fibroblast cell lines, NIH-3T3 mouse embryonic fibroblasts, which respond with a 20- to 150-fold induction of luciferase activity, were selected for all further studies except those requiring particular genetic backgrounds. Treatment of the cells with cyclopamine completely abolishes the response to ShhNp. To confirm the validity of this assay, the effects of overexpression of known pathway components, treatment with known pathway inhibitors, or both were analysed. The main findings of Drosophila and mouse genetic analyses were confirmed, indicating that NIH-3T3 cells provide a faithful and physiologically meaningful model for analysis of the Shh signaling pathway. Interestingly, for a full response to ShhNp cells had to be assayed after reaching saturation density (Taipale, 2000).

The steroidal nature of cyclopamine and its ability to disrupt cholesterol synthesis or transport indicates that it might affect the action of Ptch, which contains an apparent sterol-sensing domain. Having established the general characteristics of Shh response and cyclopamine inhibition in mouse embryonic fibroblasts, fibroblasts derived from Ptch-/- mouse embryos were assayed for cyclopamine sensitivity. Mice lacking functional Ptch show widespread transcriptional activation of targets of Shh signaling, including Ptch itself. As beta-galactosidase is expressed under the control of the Ptch promoter in these cells, its expression can be used to assay the state of Shh pathway activity. Addition of cyclopamine to Ptch-/- cells significantly suppresses beta-galactosidase expression and the activity of the Gli-luc reporter, indicating that cyclopamine can inhibit Shh pathway activity in the absence of Ptch function. In contrast, cyclopamine fails to prevent pathway activation induced by Gli2 overexpression (Taipale, 2000).

These results suggest that Hh-pathway-related tumors associated with loss of Ptch function might respond to treatment with cyclopamine, and that the target of cyclopamine action is likely to be a pathway component that functions between Ptch and the Gli proteins. It is unlikely that Ptch2 is the target of cyclopamine action in Ptch-/- cells, since Ptch2 is the main regulator of Shh pathway activation in embryos and Ptch2 activity appears not to be expressed in Ptch-/- cells (Taipale, 2000).

To investigate further the mechanism of cyclopamine action, NIH-3T3 cells were transiently transfected with both luciferase reporter and Smo complementary DNA, and it was found that overexpression of Smo in the absence of Shh induces reporter expression about tenfold. This Shh-independent activation of the response pathway can be suppressed by 5 µM cyclopamine, consistent with a target of cyclopamine action downstream of Ptch and with a mechanism that does not involve direct interference with Shh binding. Cyclopamine at this concentration has little effect on reporter expression induced by the oncogenic Smo mutants W539L (SmoA1) and S537N (SmoA2); cells from Ptch mutant embryos give similar results (Taipale, 2000).

These results indicate that cyclopamine may act upon Smo, and that activating mutations render Smo proteins resistant. An alternative interpretation would be that activated Smo proteins produce a high abundance of a downstream component and that a high cyclopamine level is required to suppress the increased concentration of this component. This alternative model, however, would predict that intermediate or low levels of pathway activation by oncogenic Smo proteins expressed at low levels should be subject to cyclopamine inhibition; on the contrary, it is observed that cyclopamine resistance is sustained under these conditions. In addition, cyclopamine resistance is not observed in cells expressing high levels of wild-type Smo activated by maximal Shh stimulation, again suggesting that cyclopamine does not act upon a component downstream of Smo (Taipale, 2000).

The oncogenic SmoA1 protein has been reported to resist suppression by Ptch, indicating that oncogenic Smo proteins may not be subject to normal regulation. It was found, however, that this resistance is partial. The activating effects of SmoA1 or SmoA2 can be completely inhibited by transfection of a 9-to-1 ratio of a Ptch construct or of Ptch-CTD, which encodes a carboxy-terminally deleted protein expressed at a higher level. It was also found that SmoA1, thus inhibited by Ptch-CTD, responds well to stimulation by ShhNp. Under these circumstances, induction of the Gli-responsive reporter is resistant to 5 µM cyclopamine, which would normally abolish Shh signaling. These results indicate that activated Smo molecules in the presence of sufficient Ptch can contribute to an essentially normal, albeit cyclopamine-resistant, response to the Shh signal (Taipale, 2000).

The finding that oncogenic Smo is regulated by high levels of Ptch indicates that it might also be subject to regulation by high levels of cyclopamine. To circumvent the cytotoxic effects of cyclopamine concentrations greater than 10 µM, several chemically synthesized cyclopamine derivatives were tested. The cyclopamine derivative 3-keto, N-aminoethyl aminocaproyl dihydrocinnamoyl cyclopamine (KAAD-cyclopamine), has 10-20-fold higher potency than cyclopamine in inhibition of beta-galactosidase expression in p2Ptch-/-cells, with similar or lower toxicity. This compound also has greater potency in suppression of ShhNp-induced pathway activity. Importantly, it completely suppresses SmoA1-induced reporter activity at a concentration around tenfold higher than that required for suppression of pathway activation induced by ShhNp (Taipale, 2000).

Since activation of the Hh response pathway is associated with neoplastic transformation in various types of tumor, the growth properties of response-activated cells were investigated in low serum or soft agar, conditions generally considered to reveal neoplastic transformation. p2Ptch-/- cell growth in low serum is markedly inhibited by addition of KAAD-cyclopamine, with 50% of maximal inhibition at ~50 nM. SmoA1-LIGHT cells, a cell line expressing SmoA1 clonally derived from NIH-3T3 cells, can form colonies in soft agar medium. Addition of KAAD-cyclopamine markedly inhibits colony growth, although ~500 nM is required for 50% inhibition. This concentration is higher than that required for 50% maximal inhibition of p2Ptch-/- cell growth, consistent with the higher amount of KAAD-cyclopamine required to block activation of the Hh response pathway by oncogenic Smo (Taipale, 2000).

The transition of G-protein-coupled receptors with seven transmembrane (TM) domains from the inactive to the active state is thought to involve a conformational shift in which the cytoplasmic ends of the TM6 and TM7 helices tilt outwards, exposing a binding pocket for a downstream signaling molecule, the Galpha subunit. Mutational analysis of Smo is consistent with such a conformational shift, since eight of the Smo-activating mutations introduce bulkier side chains at G533 and S537. Assuming an alpha-helical conformation for TM7, these substitutions all protrude from the same face of the helix. Furthermore, because multiple distinct substitutions at each of these two residues result in activation, Smo would appear to be activated by alteration of helix-packing interactions rather than by creation or disruption of a single critical interaction. A conformational shift of the TM7 helix with respect to other TM helices is suggestive of the type of conformational transition postulated for activation of G-protein-coupled receptors and raises the possibility that conformation-based transduction has been conserved in the evolution of seven-transmembrane-domain receptors (Taipale, 2000).

Activation of the Hh response pathway has been linked to several types of human tumor. For example, patients with basal cell nevus syndrome (also termed Gorlin syndrome), an autosomal dominant disorder associated with heterozygous loss-of-function mutations in PTCH, display increased incidence of many tumors, most notably basal cell carcinoma (BCC), medulloblastoma, rhabdomyosarcoma and fibrosarcoma. In addition, loss-of-function mutations in PTCH or activating mutations in SMO are found in around 40% of sporadic BCC and 25% of primitive neuroectodermal tumors. The results indicate that cyclopamine inhibits the Shh pathway by antagonizing Smo and that the activation of the Hh response pathway by either type of oncogenic mutation is blocked by cyclopamine or its derivatives. Levels of cyclopamine or the related compound jervine required to phenocopy the embryonic malformations in Shh-/- embryos are tolerated by pregnant females of various species, indicating that it might be possible to use these compounds or their derivatives without severe toxicity in non-pregnant adults to reverse activation of the Shh response pathway for therapeutic purposes (Taipale, 2000).

The full spectrum of developmental potential includes normal as well as abnormal and disease states. Therefore the idea seems appealing that tumors derive from the operation of paradevelopmental programs that yield consistent and recognizable morphologies. Work in frogs and mice shows that Hedgehog (Hh)-Gli signaling controls stem cell lineages and that its deregulation leads to tumor formation. Moreover, human tumor cells require sustained Hh-Gli signaling for proliferation as cyclopamine, an alkaloid of the lily Veratrum californicum that blocks the Hh pathway, inhibits the growth of different tumor cells in vitro as well as in subcutaneous xenografts. However, the evidence that systemic treatment is an effective anti-cancer therapy is missing. This study uses Ptc1+/-; p53-/- mice, in which medulloblastoma develops, to test the ability of cyclopamine to inhibit endogenous tumor growth in vivo after tumor initiation through intraperitoneal delivery: this method avoids the brain damage associated with direct injection. Systemic cyclopamine administration improves the health of Ptc1+/-;p53-/- animals. Analyses of the cerebella of cyclopamine-treated animals show a severe reduction in tumor size and a large decrease in the number of Ptc1-expressing cells, as a readout of cells with an active Hh-Gli pathway, as well as an impairment of their proliferative capacity. These data demonstrate that systemic treatment with cyclopamine inhibits tumor growth in the brain supporting its therapeutical value for human HH-dependent tumors. They also demonstrate that even the complete loss of the well-known tumor suppressor p53 does not render the tumor independent of Hh pathway function (Sanchez, 2005).

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patched: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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