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The Hedgehog (HH) pathway plays a pivotal role in regulating a diverse array of events from embryonic tissue patterning to adult organ self-renewal. Aberrant activation of the pathway is linked to carcinogenesis. Key factors in the HH pathway include the signaling ligand HH, the receptor Patched (PTCH), and the G-protein-coupled receptor-like transducer Smoothened (SMO). A long-lasting question about this pathway is how PTCH prevents SMO from being activated. Recent high-resolution structural studies provide insight into the molecular basis of HH recognition by PTCH. Moreover, cholesterol stands out as the endogenous ligand of SMO and acts by binding and/or covalently linking to SMO. In this review, we discuss current advances in HH signaling, the interplay of PTCH, SMO and cholesterol, and propose putative models of SMO activation by cholesterol binding and/or modification.

Sonic hedgehog (SHH) signaling is crucial for the maintenance of the physiological self-renewal of granule neuron progenitor cells (GNPs) during cerebellar development, and its dysregulation leads to oncogenesis. However, how SHH signaling is controlled during cerebellar development is poorly understood. Here, we show that Trim32, a cell fate determinant, is distributed asymmetrically in the cytoplasm of mitotic GNPs, and that genetic knockout of Trim32 keeps GNPs at a proliferating and undifferentiated state. In addition, Trim32 knockout enhances the incidence of medulloblastoma (MB) formation in the Ptch1 mutant mice. Mechanistically, Trim32 binds to Gli1, an effector of SHH signaling, via its NHL domain and degrades the latter through its RING domain to antagonize the SHH pathway. These findings provide a novel mechanism that Trim32 may be a vital cell fate regulator by antagonizing the SHH signaling to promote GNPs differentiation and a tumor suppressor in MB formation.

Tongxinluo capsule (TXL), a Chinese prescription, has been extensively used for treating ischaemic cerebrovascular diseases in China. Studies have demonstrated that TXL protects the blood-brain barrier (BBB) after cerebral ischaemia. However, the underlying protective mechanisms are not fully elucidated. Enlightened by the critical role of sonic hedgehog (Shh) pathway in promoting BBB integrity through up-regulating tight junction (TJ) proteins, we examined whether the Shh pathway could mediate TXL-induced up-regulation of TJ proteins and subsequent protection against BBB disruption after stroke. Ischaemic stroke was induced in adult male C57BL/6J mice by permanent middle cerebral artery occlusion (pMCAO). The mice were orally administered TXL (3.0 g/kg) at 1, 3 and 21 hours after stroke. Meanwhile, cyclopamine, a specific Shh pathway inhibitor, was intraperitoneally injected at 1 and 21 hours after stroke. The following parameters were measured at 6 and 24 hours after pMCAO: BBB permeability; TJ proteins including occludin, claudin-5 and zonula occludens-1 (ZO-1); and Shh signalling molecules such as Shh, Patched, Smoothened (Smo) and Gli-1. Our results showed that TXL protected against BBB disruption at 6 and 24 hours after pMCAO, and cyclopamine partly reversed the protective effect of TXL on BBB. Meanwhile, cyclopamine blocked the effect of TXL-up-regulated expression of occludin, claudin-5 and ZO-1. Moreover, TXL up-regulated the expression of Shh derived from astrocytes, Patched, Smo and Gli-1, and thus activated the Shh pathway. And cyclopamine inhibited TXL-induced activation of the Shh pathway. Thus, our study demonstrates that the Shh pathway mediates TXL-induced protection against BBB disruption after ischaemic stroke.

Background Aberrant activation of the Hedgehog pathway drives tumorigenesis of many cancers, including glioblastoma. However, the sensitization mechanism of the G protein-coupled-like receptor smoothened (SMO), a key component of Hedgehog signaling, remains largely unknown. Results In this study, we describe a novel protein SMO-193a.a. that is essential for Hedgehog signaling activation in glioblastoma. Encoded by circular SMO (circ-SMO), SMO-193a.a. is required for sonic hedgehog (Shh) induced SMO activation, via interacting with SMO, enhancing SMO cholesterol modification, and releasing SMO from the inhibition of patched transmembrane receptors. Deprivation of SMO-193a.a. in brain cancer stem cells attenuates Hedgehog signaling intensity and suppresses self-renewal, proliferation in vitro, and tumorigenicity in vivo. Moreover, circ-SMO/SMO-193a.a. is positively regulated by FUS, a direct transcriptional target of Gli1. Shh/Gli1/FUS/SMO-193a.a. form a positive feedback loop to sustain Hedgehog signaling activation in glioblastoma. Clinically, SMO-193a.a. is more specifically expressed in glioblastoma than SMO and is relevant to Gli1 expression. Higher expression of SMO-193a.a. predicts worse overall survival of glioblastoma patients, indicating its prognostic value. Conclusions Our study reveals that SMO-193a.a., a novel protein encoded by circular SMO, is critical for Hedgehog signaling, drives glioblastoma tumorigenesis and is a novel target for glioblastoma treatment.

Objective Astrocytes are glial cells proposed as the main Sonic hedgehog (Shh)-responsive cells in the adult brain. Their roles in mediating Shh functions are still poorly understood. In the hypothalamus, astrocytes support neuronal circuits implicated in the regulation of energy metabolism. In this study, we investigated the impact of genetic activation of Shh signaling on hypothalamic astrocytes and characterized its effects on energy metabolism. Methods We analyzed the distribution of gene transcripts of the Shh pathway (Ptc, Gli1, Gli2, and Gli3) in astrocytes using single molecule fluorescence in situ hybridization combined with immunohistofluorescence of Shh peptides by Western blotting in the adult mouse hypothalamus. Based on the metabolic phenotype, we characterized Glast-CreERT2-YFP-Ptc−/− (YFP-Ptc−/−) mice and their controls over time and under a high-fat diet (HFD) to investigate the potential effects of conditional astrocytic deletion of the Shh receptor Patched (Ptc) on metabolic efficiency, insulin sensitivity, and systemic glucose metabolism. Molecular and biochemical assays were used to analyze the alteration of key pathways modulating energy metabolism, insulin sensitivity, glucose uptake, and inflammation. Primary astrocyte cultures were used to evaluate a potential role of Shh signaling in astrocytic glucose uptake. Results Shh peptides were the highest in the hypothalamic extracts of adult mice and a large population of hypothalamic astrocytes expressed Ptc and Gli1-3 mRNAs. Characterization of Shh signaling after conditional Ptc deletion in the YFP-Ptc−/− mice revealed heterogeneity in hypothalamic astrocyte populations. Interestingly, activation of Shh signaling in Glast+ astrocytes enhanced insulin responsiveness as evidenced by glucose and insulin tolerance tests. This effect was maintained over time and associated with lower blood insulin levels and also observed under a HFD. The YFP-Ptc−/− mice exhibited a lean phenotype with the absence of body weight gain and a marked reduction of white and brown adipose tissues accompanied by increased whole-body fatty acid oxidation. In contrast, food intake, locomotor activity, and body temperature were not altered. At the cellular level, Ptc deletion did not affect glucose uptake in primary astrocyte cultures. In the hypothalamus, activation of the astrocytic Shh pathway was associated with the upregulation of transcripts coding for the insulin receptor and liver kinase B1 (LKB1) after 4 weeks and the glucose transporter GLUT-4 after 32 weeks. Conclusions Here, we define hypothalamic Shh action on astrocytes as a novel master regulator of energy metabolism. In the hypothalamus, astrocytic Shh signaling could be critically involved in preventing both aging- and obesity-related metabolic disorders., Highlights • Astrocytes exhibit differences in regulating the hedgehog signaling pathway. • Deletion of Ptc in Glast+ cells prevents body weight gain and insulin resistance. • Deletion of Ptc in Glast+ cells increases β oxidation. • Central hedgehog signaling participates in the regulation of whole-body metabolism.

Gorlin syndrome is a skeletal disorder caused by a gain of function mutation in Hedgehog (Hh) signaling. The Hh family comprises of many signaling mediators, which, through complex mechanisms, play several important roles in various stages of development. The Hh information pathway is essential for bone tissue development. It is also the major driver gene in the development of basal cell carcinoma and medulloblastoma. In this review, we first present the recent advances in Gorlin syndrome research, in particular, the signaling mediators of the Hh pathway and their functions at the genetic level. Then, we discuss the phenotypes of mutant mice and Hh signaling-related molecules in humans revealed by studies using induced pluripotent stem cells.

The Sonic Hedgehog (SHH) pathway plays a key role in cancer. Alterations of SHH canonical signaling, causally linked to tumor progression, have become rational targets for cancer therapy. However, Smoothened (SMO) inhibitors have failed to show clinical benefit in patients with cancers displaying SHH autocrine/paracrine expression. We reported earlier that the SHH receptor Patched (PTCH) is a dependence receptor that triggers apoptosis in the absence of SHH through a pathway that differs from the canonical one, thus generating a state of dependence on SHH for survival. Here, we propose a dual function for SHH: its binding to PTCH not only activates the SHH canonical pathway but also blocks PTCH-induced apoptosis. Eighty percent, 64%, and 8% of human colon, pancreatic, and lung cancer cells, respectively, overexpressed SHH at transcriptional and protein levels. In addition, SHH-overexpressing cells expressed all the effectors of the PTCH-induced apoptotic pathway. Although the canonical pathway remained unchanged, autocrine SHH interference in colon, pancreatic, and lung cell lines triggered cell death through PTCH proapoptotic signaling. In vivo, SHH interference in colon cancer cell lines decreased primary tumor growth and metastasis. Therefore, the antitumor effect associated to SHH deprivation, usually thought to be a consequence of the inactivation of the canonical SHH pathway, is, at least in part, because of the engagement of PTCH proapoptotic activity. Together, these data strongly suggest that therapeutic strategies based on the disruption of SHH/PTCH interaction in SHH-overexpressing cancers should be explored. Significance: Sonic Hedgehog–overexpressing tumors express PTCH-induced cell death effectors, suggesting that this death signaling could be activated as an antitumor strategy.

Evolutionarily conserved signaling pathways are crucial for adjusting growth, reproduction, and cell maintenance in response to altered environmental conditions or energy balance. However, we have an incomplete understanding of the signaling networks and mechanistic changes that coordinate physiological changes across tissues. We found that loss of the cAMP response element-binding protein (CREB) transcription factor significantly slows Caenorhabditis elegans reproductive decline, an early hallmark of aging in many animals. Our results indicate that CREB acts downstream of the transforming growth factor beta (TGF-{beta}) Sma/Mab pathway in the hypodermis to control reproductive aging, and that it does so by regulating a Hedgehog-related signaling factor, WRT-10. Overexpression of hypodermal wrt-10 is sufficient to delay reproductive decline and oocyte quality deterioration, potentially acting via Patched-related receptors in the germline. This TGF-{beta}/CREB/WRT-10 signaling axis allows a key metabolic tissue to communicate with the reproductive system to regulate oocyte quality and the rate of reproductive decline. HighlightsO_LIThe transcription factor CREB regulates oocyte quality and reproductive decline C_LIO_LIHypodermal CREB downstream of TGF-{beta} Sma/Mab signaling controls reproductive aging C_LIO_LICREB targets in the hypodermis include a Hedgehog-related signaling factor, wrt-10 C_LIO_LIWRT-10 regulates reproductive aging, potentially via germline Patched receptors C_LI eTOC BlurbTempleman et al. describe how CREB, a highly conserved transcription factor, is a central regulator of age-dependent reproductive decline. CREB acts downstream of the TGF-{beta} Sma/Mab pathway in the hypodermis, a key Caenorhabditis elegans metabolic tissue, to control oocyte quality maintenance and the rate of reproductive decline. Hypodermal CREB affects the expression of wrt-10, which encodes a Hedgehog-related signaling factor. The authors show that wrt-10 regulates reproductive aging, potentially via Patched-related receptors in the germline.

International audience; In the mature rodent brain, Sonic Hedgehog (Shh) signaling regulates stem and progenitor cell maintenance, neuronal and glial circuitry and brain repair. However, the sources and distribution of Shh mediating these effects are still poorly characterized. Here, we report in the adult mouse brain, a broad expression pattern of Shh recognized by the specific mono-clonal C9C5 antibody in a subset (11-12%) of CC1 + mature oligodendrocytes that do not express carbonic anhydrase II. These cells express also Olig2 and Sox10, two oligodendro-cyte lineage-specific markers, but not PDGFRα, a marker of oligodendrocyte progenitors. In agreement with oligodendroglial cells being a source of Shh in the adult mouse brain, we identify Shh transcripts by single molecule fluorescent in situ hybridization in a subset of cells expressing Olig2 and Sox10 mRNAs. These findings also reveal that Shh expression is more extensive than originally reported. The Shh-C9C5-associated signal labels the oligo-dendroglial cell body and decorates by intense puncta the processes. C9C5 + cells are distributed in a grid-like manner. They constitute small units that could deliver locally Shh to its receptor Patched expressed in GFAP + and S100β + astrocytes, and in HuC/D + neurons as shown in Ptc LacZ/+ reporter mice. Postnatally, C9C5 immunoreactivity overlaps the myelina-tion peak that occurs between P10 and P20 and is down regulated during ageing. Thus, our data suggest that C9C5 + CC1 + oligodendroglial cells are a source of Shh in the mouse post-natal brain.

Microorganisms and their hosts share the same environment, and microbial metabolic molecules (metabolites) exert crucial effects on host physiology. Environmental factors not only shape the composition of the host's resident microorganisms, but also modulate their metabolism. However, the exact molecular relationship among the environment, microbial metabolites and host metabolism remains largely unknown. Here, we discovered that environmental methionine tunes bacterial methyl metabolism to regulate host mitochondrial dynamics and lipid metabolism in Caenorhabditis elegans through an endocrine crosstalk involving NR5A nuclear receptor and Hedgehog signalling. We discovered that methionine deficiency in bacterial medium decreases the production of bacterial metabolites that are essential for phosphatidylcholine synthesis in C. elegans. Reductions of diundecanoyl and dilauroyl phosphatidylcholines attenuate NHR-25, a NR5A nuclear receptor, and release its transcriptional suppression of GRL-21, a Hedgehog-like protein. The induction of GRL-21 consequently inhibits the PTR-24 Patched receptor cell non-autonomously, resulting in mitochondrial fragmentation and lipid accumulation. Together, our work reveals an environment-microorganism-host metabolic axis regulating host mitochondrial dynamics and lipid metabolism, and discovers NR5A-Hedgehog intercellular signalling that controls these metabolic responses with critical consequences for host health and survival.

Sonic hedgehog (Shh) and its patched–smoothened receptor complex control a variety of functions in the developing central nervous system, such as neural cell proliferation and differentiation. Recently, Shh signaling components have been found to be expressed at the synaptic level in the postnatal brain, suggesting a potential role in the regulation of synaptic transmission. Using in utero electroporation of constitutively active and negative-phenotype forms of the Shh signal transducer smoothened (Smo), we studied the role of Smo signaling in the development and maturation of GABAergic transmission in the somatosensory cortex. Our results show that enhancing Smo activity during development accelerates the shift from depolarizing to hyperpolarizing GABA in a manner dependent on functional expression of potassium–chloride cotransporter type 2 (KCC2, also known as SLC12A5). On the other hand, blocking Smo activity maintains the GABA response in a depolarizing state in mature cortical neurons, resulting in altered chloride homeostasis and increased seizure susceptibility. This study reveals unexpected functions of Smo signaling in the regulation of chloride homeostasis, through control of KCC2 cell-surface stability, and the timing of the GABA excitatory-to-inhibitory shift in brain maturation., Summary: The smoothened receptor controls the time course of inhibitory transmission by regulating stability of the potassium-chloride cotransporter KCC2 at the plasma membrane.

The Hedgehog (HH) signalling pathway is a cell–cell communication system that controls the patterning of multiple tissues during embryogenesis in metazoans. In adults, HH signals regulate tissue stem cells and regenerative responses. Abnormal signalling can cause birth defects and cancer. The HH signal is received on target cells by Patched (PTCH1), the receptor for HH ligands, and then transmitted across the plasma membrane by Smoothened (SMO). Recent structural and biochemical studies have pointed to a sterol lipid, likely cholesterol itself, as the elusive second messenger that communicates the HH signal between PTCH1 and SMO, thus linking ligand reception to transmembrane signalling.

Spatiotemporal restriction of signaling plays a critical role in animal development and tissue homeostasis. All stem and progenitor cells in newly hatched C. elegans larvae are quiescent and capable of suspending their development until sufficient food is supplied. Here, we show that ptr-18, which encodes the evolutionarily conserved patched-related (PTR)/patched domain-containing (PTCHD) protein, temporally restricts the availability of extracellular hedgehog-related protein to establish the capacity of progenitor cells to maintain quiescence. We found that neural progenitor cells exit from quiescence in ptr-18 mutant larvae even when hatched under starved conditions. This unwanted reactivation depended on the activity of a specific set of hedgehog-related grl genes including grl-7. Unexpectedly, neither PTR-18 nor GRL-7 were expressed in newly hatched wild-type larvae. Instead, at the late embryonic stage, both PTR-18 and GRL-7 proteins were first localized around the apical membrane of hypodermal and neural progenitor cells and subsequently targeted for lysosomal degradation before hatching. Loss of ptr-18 caused a significant delay in GRL-7 clearance, causing this protein to be retained in the extracellular space in newly hatched ptr-18 mutant larvae. Furthermore, the putative transporter activity of PTR-18 was shown to be required for the appropriate function of the protein. These findings not only uncover a previously undescribed role of PTR/PTCHD in the clearance of extracellular hedgehog-related proteins via endocytosis-mediated degradation but also illustrate that failure to temporally restrict intercellular signaling during embryogenesis can subsequently compromise post-embryonic progenitor cell function., Author summary Inside the animal body, many “signals” are exchanged between tissues and cells to ensure healthy growth and physiological conditions. Too much or less signals have been known to cause various diseases such as cancer and diabetes. When developing animals are not fed enough food, their growth rates generally slow down. We found that when nematodes lacking the PTR-18 protein are hatched out of the egg in the absence of sufficient food, they do not appropriately halt their growth. Analyses at the molecular and cellular levels showed that immediately before hatching, PTR-18 re-uptakes a signaling molecule called GRL-7, which is known to promote growth, from the outside to the inside of cells, resulting in the degradation of GRL-7. As a result, worms lacking PTR-18 still contain excess GRL-7 after hatching, which results in unwanted growth even when ample food is not available. Thus, our findings uncover the role of PTR-18 in terminating a signal on time. Humans also possess a protein called PTCHD1, which is structurally similar to the worm’s PTR-18 and has been proposed to cause autistic spectrum disorders and learning disabilities. Our findings may provide a clue to further understand the function of human PTCHD1.

Summary Identifying the mechanisms controlling quiescence and activation of neural stem cells (NSCs) is crucial for understanding brain repair. Here, we demonstrate that Hedgehog (Hh) signaling actively regulates different pools of quiescent and proliferative NSCs in the adult ventricular-subventricular zone (V-SVZ), one of the main brain neurogenic niches. Specific deletion of the Hh receptor Patched in NSCs during adulthood upregulated Hh signaling in quiescent NSCs, progressively leading to a large accumulation of these cells in the V-SVZ. The pool of non-neurogenic astrocytes was not modified, whereas the activated NSC pool increased after a short period, before progressively becoming exhausted. We also showed that Sonic Hedgehog regulates proliferation of activated NSCs in vivo and shortens both their G1 and S-G2/M phases in culture. These data demonstrate that Hh orchestrates the balance between quiescent and activated NSCs, with important implications for understanding adult neurogenesis under normal homeostatic conditions or during injury., Highlights • Hh shortens the cell cycle of activated neural stem cells (NSCs) in culture • Hh activation depletes the pool of proliferating activated NSCs at long term • Quiescent NSCs accumulates after a long period of Hh activation in NSCs, Hedgehog (Hh) signaling is implicated in tissue regeneration. Ruat, Boussin, and colleagues show that short-term Hh pathway activation in adult neural stem cells (NSCs) results in increases in activated NSCs. Surprisingly, long-term pathway activation causes a large accumulation of quiescent NSCs and impaired neurogenesis. Thus, the Hh pathway has a novel role in balancing quiescent and activated adult NSC numbers.

The SHH signaling pathway is critical for gastrointestinal development and organic patterning, and dysregulation of SHH pathway molecules has been detected in multiple gastrointestinal neoplasms. This study investigated the role of the SHH signaling pathway in PJS. Expression of SHH, PTCH, and GLI1 was examined by real-time PCR and immunohistochemistry in 20 normal tissues and 75 colorectal lesions (25 PJPs, 25 adenomas, and 25 adenocarcinomas). Expression of SHH, PTCH, and GLI1 mRNA was higher in PJPs than in normal tissue (P < .05) and gradually increased along the PJP-adenoma-adenocarcinoma sequence (P < .05). Immunostaining indicated that SHH expression was present in 60% of PJPs, 72% of adenomas, and 84% of carcinomas, whereas 68% of PJPs, 72% of adenomas, and 88% of carcinomas exhibited cytoplasmic expression of PTCH. Moreover, high GLI1 expression was detected in 56% of PJPs, 64% of adenomas, and 80% of carcinomas; and high nuclear expression of GLI1 was observed in 8 adenomas with atypia and 15 carcinomas. Increased SHH, PTCH, and GLI1 protein correlated positively with tumor grade (P = .012, P = .003, and P = .007, respectively), tumor depth (P = .024, P = .007, and P = .01), and lymph node metastasis (P = .05, P = .015, and P = .005). This study identified aberrant expression of SHH pathway molecules in PJS, and the findings may supply a novel mechanism for the development of PJ polyps.

Klein et al. show that Ptch2 loss in either the niche or in hematopoietic cells drives myeloproliferation and accelerates JAK2V617F-driven pathogenesis, causing transformation of nonlethal chronic MPNs into aggressive lethal leukemias., JAK2V617F+ myeloproliferative neoplasms (MPNs) frequently progress into leukemias, but the factors driving this process are not understood. Here, we find excess Hedgehog (HH) ligand secretion and loss of PTCH2 in myeloproliferative disease, which drives canonical and noncanonical HH-signaling. Interestingly, Ptch2−/− mice mimic dual pathway activation and develop a MPN-phenotype with leukocytosis (neutrophils and monocytes), strong progenitor and LKS mobilization, splenomegaly, anemia, and loss of lymphoid lineages. HSCs exhibit increased cell cycling with improved stress hematopoiesis after 5-FU treatment, and this results in HSC exhaustion over time. Cytopenias, LKS loss, and mobilization are all caused by loss of Ptch2 in the niche, whereas hematopoietic loss of Ptch2 drives leukocytosis and promotes LKS maintenance and replating capacity in vitro. Ptch2−/− niche cells show hyperactive noncanonical HH signaling, resulting in reduced production of essential HSC regulators (Scf, Cxcl12, and Jag1) and depletion of osteoblasts. Interestingly, Ptch2 loss in either the niche or in hematopoietic cells dramatically accelerated human JAK2V617F-driven pathogenesis, causing transformation of nonlethal chronic MPNs into aggressive lethal leukemias with >30% blasts in the peripheral blood. Our findings suggest HH ligand inhibitors as possible drug candidates that act on hematopoiesis and the niche to prevent transformation of MPNs into leukemias.

Cardiac fibroma is an extremely rare benign tumor that remains poorly characterized genetically. Somatic copy number alterations are common in tumors and have been defined as a crucial factor leading to tumors. In this study, we present a child diagnosed with cardiac fibroma with somatic copy number losses of a total of three discontinuous segments from 9q21.33 to 9q22.33, including a mosaic deletion of PTCH1 . PTCH1 has been associated with sporadic cardiac fibroma. Sequencing analysis of the PTCH1 gene has not revealed any causative mutation. Quantitative PCR analysis of PTCH1 further confirms somatic copy number losses. Our data narrow down the critical causative deletions for sporadic cardiac fibroma to a region more precise than any other previously reported one. Our results suggest important roles of somatic copy number losses on chromosome 9q21.33q22.33 in the development of sporadic cardiac fibroma; these findings may provide a better understanding of sporadic cardiac fibroma pathogenesis and contribute to the identification of novel diagnostic biomarkers of this neoplasm.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide and the fourth most lethal cancer in China. However, although genomic studies have identified some mutations associated with ESCC, we know little of the mutational processes responsible. To identify genome-wide mutational signatures, we performed either whole-genome sequencing (WGS) or whole-exome sequencing (WES) on 104 ESCC individuals and combined our data with those of 88 previously reported samples. An APOBEC -mediated mutational signature in 47% of 192 tumors suggests that APOBEC-catalyzed deamination provides a source of DNA damage in ESCC. Moreover, PIK3CA hotspot mutations (c.1624G>A [p.Glu542Lys] and c.1633G>A [p.Glu545Lys]) were enriched in APOBEC -signature tumors, and no smoking-associated signature was observed in ESCC. In the samples analyzed by WGS, we identified focal ( CBX4 and CBX8 . In our combined cohort, we identified frequent inactivating mutations in AJUBA , ZNF750 , and PTCH1 and the chromatin-remodeling genes CREBBP and BAP1 , in addition to known mutations. Functional analyses suggest roles for several genes ( CBX4 , CBX8 , AJUBA , and ZNF750 ) in ESCC. Notably, high activity of hedgehog signaling and the PI3K pathway in approximately 60% of 104 ESCC tumors indicates that therapies targeting these pathways might be particularly promising strategies for ESCC. Collectively, our data provide comprehensive insights into the mutational signatures of ESCC and identify markers for early diagnosis and potential therapeutic targets.

Evolutionarily conserved signaling pathways are crucial for adjusting growth, reproduction, and cell maintenance in response to altered environmental conditions or energy balance. However, we have an incomplete understanding of the signaling networks and mechanistic changes that coordinate physiological changes across tissues. We found that loss of the cAMP response element-binding protein (CREB) transcription factor significantly slows Caenorhabditis elegans’ reproductive decline, an early hallmark of aging in many animals. Our results indicate that CREB acts downstream of the transforming growth factor β (TGF-β) Sma/Mab pathway in the hypodermis to control reproductive aging, and that it does so by regulating a Hedgehog-related signaling factor, WRT-10. Overexpression of hypodermal wrt-10 is sufficient to delay reproductive decline and oocyte quality deterioration, potentially acting via Patched-related receptors in the germline. This TGF-β-CREB-Hedgehog signaling axis allows a key metabolic tissue to communicate with the reproductive system to regulate oocyte quality and the rate of reproductive decline.

Hedgehog protein signals mediate tissue patterning and maintenance via binding to and inactivation of their common receptor Patched, a twelve-transmembrane protein that otherwise would suppress activity of the seven-transmembrane protein, Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. The extracellular domain mediates association of Patched monomers in an unusual dimeric architecture that implies curvature in the associated membrane. A central conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Patched expression indeed reduces cholesterol activity in the inner leaflet of the plasma membrane, in a manner antagonized by Hedgehog stimulation and with implications for regulation of Smoothened.

Skeletal myogenesis serves as a paradigm to investigate the molecular mechanisms underlying exquisitely regulated cell fate decisions in developing embryos. The evolutionarily conserved miR-133 family of microRNAs is expressed in the myogenic lineage, but how it acts remains incompletely understood. Here, we performed genome-wide differential transcriptomics of miR-133 knockdown (KD) embryonic somites, the source of vertebrate skeletal muscle. These analyses, performed in chick embryos, revealed extensive downregulation of Sonic hedgehog (Shh) pathway components: patched receptors, Hedgehog interacting protein and the transcriptional activator Gli1. By contrast, Gli3, a transcriptional repressor, was de-repressed and confirmed as a direct miR-133 target. Phenotypically, miR-133 KD impaired myotome formation and growth by disrupting proliferation, extracellular matrix deposition and epithelialization. Together, these observations suggest that miR-133-mediated Gli3 silencing is crucial for embryonic myogenesis. Consistent with this idea, we found that activation of Shh signalling by either purmorphamine, or KD of Gli3 by antisense morpholino, rescued the miR-133 KD phenotype. Thus, we identify a novel Shh/myogenic regulatory factor/miR-133/Gli3 axis that connects epithelial morphogenesis with myogenic fate specification., Summary: Here, using chick embryos, we showed that post-transcriptional silencing of the Gli3 repressor by miR-133 is required to stably establish the myogenic programme in early somites.

Summary Symmetric and asymmetric divisions are important for self-renewal and differentiation of stem cells during neurogenesis. Although cerebellar granule neurogenesis is controlled by sonic hedgehog (SHH) signaling, whether and how this process is mediated by regulation of cell division modes have not been determined. Here, using time-lapse imaging and cell culture from neuronal progenitor-specific and differentiated neuron-specific reporter mouse lines (Math1-GFP and Dcx-DsRed) and Patched+/− mice in which SHH signaling is activated, we find evidence for the existence of symmetric and asymmetric divisions that are closely associated with progenitor proliferation and differentiation. While activation of the SHH pathway enhances symmetric progenitor cell divisions, blockade of the SHH pathway reverses the cell division mode change in Math1-GFP;Dcx-DsRed;Patched+/− mice by promoting asymmetric divisions or terminal neuronal symmetric divisions. Thus, cell division mode change mediates the regulation of cerebellar granule neurogenesis controlled by SHH signaling., Highlights • Cerebellar GNPs display both symmetric and asymmetric divisions • Activation of the SHH pathway enhances symmetric progenitor divisions • A SHH pathway inhibitor reverses cell division mode change in Patched+/− mice, Gao, Yang, and colleagues demonstrates that symmetric and asymmetric divisions are crucial for the production of appropriate numbers of progenitors and differentiated neurons during cerebellar granule neurogenesis. These findings provide new insights into mechanisms of cerebellar neurogenesis and suggest that identification of molecules that regulate cell division modes might be an alternative strategy for repair of the disregulation of neurogenesis and medulloblastoma.

Sonic hedgehog (Shh), a secreted glycoprotein factor, can activate the Shh pathway, which has been implicated in neuronal polarization involving neurite outgrowth. However, little evidence is available about the effect of Shh on neurite outgrowth in primary cortical neurons and its potential mechanism. Here, we revealed that Shh increased neurite outgrowth in primary cortical neurons, while the Shh pathway inhibitor (cyclopamine, CPM) partially suppressed Shh-induced neurite outgrowth. Similar results were found for the expressions of Shh and Patched genes in Shh-induced primary cortical neurons. Moreover, Shh increased the levels of brain-derived neurotrophic factor (BDNF) not only in lysates and in culture medium but also in the longest neurites of primary cortical neurons, which was partially blocked by CPM. In addition, blocking of BDNF action suppressed Shh-mediated neurite elongation in primary cortical neurons. In conclusion, these findings suggest that Shh promotes neurite outgrowth in primary cortical neurons at least partially through modulating BDNF expression.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. In children, the 2 major RMS subtypes are alveolar and embryonal RMS. Aberrant Hedgehog/Patched1 (Hh/Ptch) signaling is a hallmark of embryonal RMS. We demonstrate that mice carrying a Ptch mutation in mesodermal Delta1-expressing cells develop embryonal-like RMS at a similar rate as mice harboring a Ptch mutation in the germline or the brachury-expressing mesoderm. The tumor incidence decreases dramatically when Ptch is mutated in Myf5- or Pax3-expressing cells. No RMS develop from Myogenin/Mef2c-expressing cells. This suggests that Hh/Ptch-associated RMS are derived from Delta1-positive, Myf5-negative, Myogenin-negative and Pax3-negative mesodermal progenitors that can undergo myogenic differentiation but lack stable lineage commitment. Additional preliminary genetic data and data on mesodermal progenitors further imply an interplay of Hh/Ptch and Delta/Notch signaling activity during RMS initiation. In contrast, Wnt signals supposedly suppress RMS formation because RMS multiplicity decreases after inactivation of the Wnt-inhibitor Wif1. Finally, our results strongly suggest that the tumor-initiating event determines the lineage of RMS origin.

Intrinsic molecular determinants of neurodevelopmental outcomes assume new, albeit related roles during adult neural regeneration. Here we studied and identified a facilitatory role for Sonic hedgehog protein (Shh), a morphogen that influences motor neuron floor plate architecture, during adult peripheral neuron regeneration. Shh and its receptors were expressed in adult dorsal root ganglia (DRG) neurons, axons and glia and trended toward higher levels following axotomy injury. Knockdown of Shh in adult sensory neurons resulted in decreased outgrowth and branching in vitro, identifying a role for Shh in facilitating outgrowth. The findings argued for an intrinsic action to support neuron regeneration. Support of advancement and turning however, were not identified in adult sensory neuron growth cones in response to local extrinsic gradients of Shh. That intrinsic Shh supported the regrowth of peripheral nerves after injury was confirmed by the analysis of axon regrowth from the proximal stumps of transected sciatic nerves. By exposing regenerating axons to local infusions of Shh siRNA in vivo within a conduit bridging the transected proximal and distal stumps, we achieved local knockdown of Shh. In response, there was attenuated axonal and Schwann cell outgrowth beyond the transection zone. Unlike its role during neurodevelopment, Shh facilitates but does not confer regenerative outgrowth properties to adult neurons alone. Exploring the differing properties of morphogens and related proteins in the adult nervous system identifies new and important roles for them.

The evolution of imaginal cells, or stem cell-like cells, contributed to the spectacular diversification of holometabolous insects, which undergo complete metamorphosis. The proliferation and differentiation of these imaginal cells is under the control of juvenile hormone (JH), but which patterning genes respond to JH is currently unknown. Here, the role of Hedgehog (Hh) signaling in the development of imaginal cells was investigated. RNA interference-mediated knockdown of the components of the Hh signaling pathway showed that Hh is required for the proliferation of polymorphic and imaginal cells in Tribolium castaneum. Hh was also necessary for the regeneration of larval appendages. In contrast, knockdown of Hh signaling antagonists, patched and costal 2 led to the overgrowth and precocious maturation of structures derived from imaginal cells and the occasional appearance of ectopic appendages from the head epidermis. In addition, JH suppressed the expression of hh both in vivo and in vitro. Our findings suggest that imaginal cells are created and maintained by modulating Hh signaling. Thus, Hh signaling may have played a critical role during the evolution of complete metamorphosis.

Accumulating evidence indicates that Hedgehog (Hh) signaling becomes activated in chronic liver injury and plays a role in the pathogenesis of hepatic fibrosis. Hepatic stellate cells (HSCs) are Hh-responsive cells and activation of the Hh pathway promotes transdifferentiation of HSCs into myofibroblasts. Targeting Hh signaling may be a novel therapeutic strategy for treatment of liver fibrosis. We previously reported that curcumin has potent antifibrotic effects in vivo and in vitro, but the underlying mechanisms are not fully elucidated. This study shows that curcumin downregulated Patched and Smoothened, two key elements in Hh signaling, but restored Hhip expression in rat liver with carbon tetrachloride-induced fibrosis and in cultured HSCs. Curcumin also halted the nuclear translocation, DNA binding, and transcription activity of Gli1. Moreover, the Hh signaling inhibitor cyclopamine, like curcumin, arrested the cell cycle, induced mitochondrial apoptosis, reduced fibrotic gene expression, restored lipid accumulation, and inhibited invasion and migration in HSCs. However, curcumin's effects on cell fate and fibrogenic properties of HSCs were abolished by the Hh pathway agonist SAG. Furthermore, curcumin and cyclopamine decreased intracellular levels of adenosine triphosphate and lactate, and inhibited the expression and/or function of several key molecules controlling glycolysis. However, SAG abrogated the curcumin effects on these parameters of glycolysis. Animal data also showed that curcumin downregulated glycolysis-regulatory proteins in rat fibrotic liver. These aggregated data therefore indicate that curcumin modulated cell fate and metabolism by disrupting the Hh pathway in HSCs, providing novel molecular insights into curcumin reduction of HSC activation.

Basal cell carcinoma (BCC), the most common type of skin cancer, is occasionally aggressive with deep invasion, destruction of adjacent structures, recurrence and, on very rare occasions, regional and distant metastases. Mutations that occur in BCC in hedgehog (Hh) pathway genes primarily involve the genes encoding patched homolog (PTCH) and smoothened homolog (SMO). Several animal models have demonstrated the functional relevance of genetic alterations in the Hh pathway during tumorigenesis. Recently, targeted therapy has become available both commercially and in the context of human clinical trials. Interestingly, Hh pathway inhibitors not only suppress BCC progression but also promote acquired immune responses. Since immune responses are crucial for long-term tumor control, new clinical trials, such as those involving a combination of Hh inhibitors with immune modifiers, are needed to supplement standard methods of tumor control.

SummarySonic hedgehog (Shh) signaling is critical in development and oncogenesis, but the mechanisms regulating this pathway remain unclear. Although protein phosphorylation clearly affects Shh signaling, little is known about phosphatases governing the pathway. Here, we conducted a small hairpin RNA (shRNA) screen of the phosphatome and identified Eya1 as a positive regulator of Shh signaling. We find that the catalytically active phosphatase Eya1 cooperates with the DNA-binding protein Six1 to promote gene induction in response to Shh and that Eya1/Six1 together regulate Gli transcriptional activators. We show that Eya1, which is mutated in a human deafness disorder, branchio-oto-renal syndrome, is critical for Shh-dependent hindbrain growth and development. Moreover, Eya1 drives the growth of medulloblastoma, a Shh-dependent hindbrain tumor. Together, these results identify Eya1 and Six1 as key components of the Shh transcriptional network in normal development and in oncogenesis.

The Hedgehog pathway is critical for animal development and has been implicated in multiple human malignancies. Despite great interest in targeting the pathway pharmacologically, many of the principles underlying the signal transduction cascade remain poorly understood. Hedgehog ligands are recognized by a unique receptor system that features the transporter-like protein Patched and the G protein-coupled receptor (GPCR)-like Smoothened (SMO). The biochemical interaction between these transmembrane proteins is the subject of intensive efforts. Recent structural and functional studies have provided great insight into the small-molecule regulation of SMO through identification of two distinct ligand-binding sites. In this Perspective, we review these recent findings and relate them to potential mechanisms for the endogenous regulation of SMO.

PTCH1 gene codes for a 12-pass transmembrane receptor with a negative regulatory role in the Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, a disorder characterized by developmental abnormalities and tumor susceptibility. The autosomal dominant inheritance, and the evidence for PTCH1 haploinsufficiency, suggests that fine-tuning systems of protein patched homolog 1 (PTC1) levels exist to properly regulate the pathway. Given the role of 5' untranslated region (5'UTR) in protein expression, our aim was to thoroughly explore cis-regulatory elements in the 5'UTR of PTCH1 transcript 1b. The (CGG)n polymorphism was the main potential regulatory element studied so far but with inconsistent results and no clear association between repeat number and disease risk. Using luciferase reporter constructs in human cell lines here we show that the number of CGG repeats has no strong impact on gene expression, both at mRNA and protein levels. We observed variability in the length of 5'UTR and changes in abundance of the associated transcripts after pathway activation. We show that upstream AUG codons (uAUGs) present only in longer 5'UTRs could negatively regulate the amount of PTC1 isoform L (PTC1-L). The existence of an internal ribosome entry site (IRES) observed using different approaches and mapped in the region comprising the CGG repeats, would counteract the effect of the uAUGs and enable synthesis of PTC1-L under stressful conditions, such as during hypoxia. Higher relative translation efficiency of PTCH1b mRNA in HEK 293T cultured hypoxia was observed by polysomal profiling and Western blot analyses. All our results point to an exceptionally complex and so far unexplored role of 5'UTR PTCH1b cis-element features in the regulation of the Hedgehog-Gli signaling pathway.

Down Syndrome (DS) is a highly complex developmental genetic disorder caused by trisomy for human chromosome 21 (Hsa21). All individuals with DS exhibit some degree of brain structural changes and cognitive impairment; mouse models such as Ts65Dn have been instrumental in understanding the underlying mechanisms. Several phenotypes of DS might arise from a reduced response of trisomic cells to the Sonic Hedgehog (SHH) growth factor. If all trisomic cells show a similar reduced response to SHH, then up-regulation of the pathway in trisomic cells might ameliorate multiple DS phenotypes. We crossed Ptch1tm1Mps/+ mice, in which the canonical SHH pathway is expected to be up-regulated in every SHH-responsive cell due to the loss of function of one allele of the pathway suppressor, Ptch1, to the Ts65Dn DS model and assessed the progeny for possible rescue of multiple DS-related phenotypes. Down-regulation of Ptch produced several previously unreported effects on development by itself, complicating interpretation of some phenotypes, and a number of structural or behavioral effects of trisomy were not compensated by SHH signaling. However, a deficit in a nest-building task was partially restored in Ts;Ptch+/- mice, as were the structural anomalies of the cerebellum seen in Ts65Dn mice. These results extend the body of evidence indicating that reduced response to SHH in trisomic cells and tissues contributes to various aspects of the trisomic phenotype.

The mammalian G-protein-coupled receptor 37 (GPR37) is expressed in brain, in adult testis, and during the early phase of gonad differentiation. Somatic Sertoli cells (SCs) are located within the seminiferous tubules where they support the germinal epithelium. An adequate number of SCs is required for the complete prepubertal differentiation of germ cells and adult fertility. This study shows that Gpr37 and its ligand prosaposin are both postnatally expressed by SCs, whose proliferation and maturation are affected in Gpr37-null mutant mice during postnatal testicular development. Mutant pups show a delayed timing in sperm cell development, with a partial arrest of spermatocytes at the meiotic pachytene (e.g., 1.5-fold increase in Gpr37(-/-) P21 pups) and their increased apoptosis (e.g., 1.8-fold and 3.5-fold increase in Gpr37(-/-) P21 and adult mice, respectively). Mutant adults have reduced testis weight (wild type, 299 ± 5 mg; knockout, 258 ± 16 mg; P0.05) and epididymal sperm count and motility (e.g., 1.5-fold and 1.45-fold decrease in Gpr37(-/-) mice, respectively). Lack of Gpr37 results in the reduction in androgen receptor levels during prepubertal testis development, alongside the altered expression of SC maturation markers. It also affects the prepubertal testis expression of desert hedgehog (Dhh) mitogenic cascade components (Dhh, 1.3-fold increase in Gpr37(-/-) P10 and P21 pups; Gli2, 1.4-fold and 1.6-fold increase in Gpr37(-/-) P10 and P21 pups, respectively) including patched homolog 1 (1.3-fold increase in Gpr37(-/-) P10 and P21 pups), which is found localized in prepubertal SCs and is associated with Gpr37 in cultured primary SC samples. These results indicate that Gpr37 is a specific modulator of murine testis Dhh mitogenic signaling and SC proliferation and maturation.

Glioblastoma Multiforme (GBM), the most common and lethal adult primary tumor of the brain, showed a link between Sonic Hedgehog (SHH) pathway in the resistance to temozolomide (TMZ). PTCH1, the SHH receptor, can tonically represses signaling by endocytosis. We asked how the decrease in PTCH1 in GBM cells could lead to TMZ-resistance. TMZ resistant GBM cells have increased PTCH1 mRNA and reduced protein. Knockdown of Dicer, a Type III RNAase, indicated that miRNAs can explain the decreased PTCH1 in TMZ resistant cells. Computational studies, real-time PCR, reporter gene studies, western blots, target protector oligos and ectopic expression identified miR-9 as the target of PTCH1 in resistant GBM cells with concomitant activation of SHH signaling. MiR-9 mediated increases in the drug efflux transporters, MDR1 and ABCG2. MiR-9 was increased in the tissues from GBM patients and in an early passage GBM cell line from a patient with recurrent GBM but not from a naïve patient. Pharmacological inhibition of SHH signaling sensitized the GBM cells to TMZ. Taken together, miR-9 targets PTCH1 in GBM cells by a SHH-independent method in GBM cells for TMZ resistance. The identified pathways could lead to new strategies to target GBM with combinations of drugs.

Background/Aims: There is interest in drugs and rehabilitation methods to enhance neurogenesis and improve neurological function after brain injury or degeneration. Resveratrol may enhance hippocampal neurogenesis and improve hippocampal atrophy in chronic fatigue mice and prenatally stressed rats. However, its effect and mechanism of neurogenesis after stroke is less well understood. Sonic hedgehog (Shh) signaling is crucial for neurogenesis in the embryonic and adult brain, but relatively little is known about the role of Shh signaling in resveratrol-enhanced neurogenesis after stroke. Methods: Neural stem cells (NSCs) before oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro were pretreated with resveratrol with or without cyclopamine. Survival and proliferation of NSCs was assessed by the CCK8 assay and BrdU immunocytochemical staining. The expressions and activity of signaling proteins and mRNAs were detected by immunocytochemistry, Western blotting, and RT-PCR analysis. Results: Resveratrol significantly increased NSCs survival and proliferation in a concentration-dependent manner after OGD/R injury in vitro. At the same time, the expression of Patched-1, Smoothened (Smo), and Gli-1 proteins and mRNAs was upregulated, and Gli-1 entered the nucleus, which was inhibited by cyclopamine, a Smo inhibitor. Conclusion: Shh signaling mediates resveratrol to increase NSCs proliferation after OGD/R injury in vitro.

Mouse models have increased our understanding of the pathogenesis of medulloblastoma (MB), the most common malignant pediatric brain tumor that often forms in the cerebellum. A major goal of ongoing research is to better understand the early stages of tumorigenesis and to establish the genetic and environmental changes that underlie MB initiation and growth. However, studies of MB progression in mouse models are difficult due to the heterogeneity of tumor onset times and growth patterns and the lack of clinical symptoms at early stages. Magnetic resonance imaging (MRI) is critical for noninvasive, longitudinal, three-dimensional (3D) brain tumor imaging in the clinic but is limited in resolution and sensitivity for imaging early MBs in mice. In this study, high-resolution (100 μm in 2 hours) and high-throughput (150 μm in 15 minutes) manganese-enhanced MRI (MEMRI) protocols were optimized for early detection and monitoring of MBs in a Patched-1 (Ptch1) conditional knockout (CKO) model. The high tissue contrast obtained with MEMRI revealed detailed cerebellar morphology and enabled detection of MBs over a wide range of stages including pretumoral lesions as early as 2 to 3 weeks postnatal with volumes close to 0.1 mm3. Furthermore, longitudinal MEMRI allowed noninvasive monitoring of tumors and demonstrated that lesions within and between individuals have different tumorigenic potentials. 3D volumetric studies allowed quantitative analysis of MB tumor morphology and growth rates in individual Ptch1-CKO mice. These results show that MEMRI provides a powerful method for early in vivo detection and longitudinal imaging of MB progression in the mouse brain.

Objectives: To examine Hedgehog signaling in cutaneous fibrosing disorders for which effective approved therapies are lacking, expand our knowledge of pathophysiology, and explore the rationale for targeted inhibition. Methods: Stain intensity and percentage of cells staining for Sonic hedgehog (Shh), Indian hedgehog (Ihh), Patched (Ptch), glycogen synthase kinase 3 β (GSK3-β), β-catenin, and Snail were evaluated in human skin biopsy specimens of keloid, hypertrophic scar (Hscar), scleroderma, nephrogenic systemic fibrosis (NSF), scar, and normal skin using a tissue microarray. Results: Ihh, but not Shh, was detected in a significantly larger proportion of cells for all case types. Ptch, GSK3-β, and β-catenin showed a gradient of expression: highest in NSF and keloid; moderate in normal skin, scar, and Hscar; and lowest in scleroderma. Snail expression was binary: low in normal skin but high in all fibrosing conditions studied. Conclusions: Differential overexpression of Hedgehog and Snail in cutaneous fibrosing disorders demonstrates a role for targeted inhibition. Ptch, GSK3-β, and β-catenin can help differentiate scleroderma from NSF in histologically subtle cases. Differences in expression between keloid and hypertrophic scar support the concept that they are pathophysiologically distinct disorders. Our findings implicate Snail as a target for the prevention of fibrogenesis or fibrosis progression and may offer a means to assess response to therapy.

Anaplastic thyroid carcinoma is the least common form of thyroid cancer; however, it accounts for the majority of deaths associated with this family of malignancies. A number of genetically engineered immunocompetent mouse models recapitulating the genetic and histological features of anaplastic thyroid cancer have been very recently generated and represent an invaluable tool to dissect the mechanisms involved in the progression from indolent, well differentiated tumors to aggressive, undifferentiated carcinomas, and to identify novel therapeutic targets. In this review, we focus on the relevant characteristics associated with these models and on what we have learned in terms of anaplastic thyroid cancer biology, genetics, and response to targeted therapy.

Nuclear factor kappa B (NF-κB) signaling plays critical roles in many physiological and pathological processes, including regulating organogenesis. Down-regulation of NF-κB signaling during development results in hypohidrotic ectodermal dysplasia. The roles of NF-κB signaling in tooth development, however, are not fully understood. We examined mice overexpressing IKKβ, an essential component of the NF-κB pathway, under keratin 5 promoter ( K5-Ikkβ). K5-Ikkβ mice showed supernumerary incisors whose formation was accompanied by up-regulation of canonical Wnt signaling. Apoptosis that is normally observed in wild-type incisor epithelium was reduced in K5-Ikkβ mice. The supernumerary incisors in K5-Ikkβ mice were found to phenocopy extra incisors in mice with mutations of Wnt inhibitor, Wise. Excess NF-κB activity thus induces an ectopic odontogenesis program that is usually suppressed under physiological conditions.

Objective Hedgehog signaling proteins were assessed in patients with cervical carcinoma receiving chemoradiation. Associations between five Hedgehog proteins and prognosis were studied. Methods In all, 131 cases of cervical carcinomas (FIGO stages I–IV) were immunohistochemically (IHC) analyzed for Patched (PTCH), Smoothened (SMO), and GLI1, GLI2 and GLI3 protein expression. Associations between Hedgehog protein expressions, clinicopathological factors, and clinical outcome data were examined. Results Positive IHC staining for the five Hedgehog proteins was recorded in 8% to 37% of the tumor cells. The highest frequency was noted for SMO and the lowest for GLI1. There was a significant association between low SMO- and GLI2-expression and KRAS-mutation. Tumors with overexpressed SMO had a higher frequency of residual tumor or local recurrences than tumors with low SMO expression. Patients with tumors expressing PTCH in more than 75% of the cells had significantly (P=0.023) better recurrence-free survival than patients with tumors with low expression. The opposite situation was true for SMO. For GLI2, there was a statistically significant difference with regard to overall (P=0.004) and distant (P=0.015) relapse rate for groups with expression of GLI2 in the range of 5–25% compared to higher rates. Conclusions A predictive and prognostic value was found for PTCH, SMO, and GLI2 with regard to residual carcinoma, local recurrences, and for GLI2 distant relapses. The Hedgehog signaling pathway also seems to play an important role in cervical carcinogenesis together with HPV16-infection and KRAS-mutation.

The Hedgehog (Hh) signaling pathway regulates normal development and cell proliferation in metazoan organisms, but its aberrant activation can promote tumorigenesis. Hh-induced tumors arise from various tissues and they may be indolent or aggressive, as is the case with skin basal cell carcinoma (BCC) or cerebellar medulloblastoma, respectively. Little is known about common cell-intrinsic factors that control the development of such diverse Hh-dependent tumors. Transcription factor Zfx is required for the self-renewal of hematopoietic and embryonic stem cells, as well as for the propagation of acute myeloid and T-lymphoblastic leukemias. We report here that Zfx facilitates the development of experimental BCC and medulloblastoma in mice initiated by deletion of the Hh inhibitory receptor Ptch1. Simultaneous deletion of Zfx along with Ptch1 prevented BCC formation and delayed medulloblastoma development. In contrast, Zfx was dispensable for tumorigenesis in a mouse model of glioblastoma. We used genome-wide expression and chromatin-binding analysis in a human medulloblastoma cell line to characterize direct, evolutionarily conserved targets of Zfx, identifying Dis3L and Ube2j1 as two targets required for the growth of the human medulloblastoma cells. Our results establish Zfx as a common cell-intrinsic regulator of diverse Hh-induced tumors, with implications for the definition of new therapeutic targets in these malignancies. Cancer Res; 74(20); 5914–24. ©2014 AACR.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide with widespread distribution. It plays pivotal role in neuronal development. PACAP-immunoreactive fibers have been found in the tooth pulp, and recently, it has been shown that PACAP may also play a role in the regeneration of the periodontium after luxation injuries. However, there is no data about the effect of endogenous PACAP on tooth development. Ectodermal organogenesis including tooth development is regulated by different members of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH), and Wnt families. There is also a growing evidence to support the hypothesis that PACAP interacts with sonic hedgehog (SHH) receptor (PTCH1) and its downstream target (Gli1) suggesting its role in tooth development. Therefore, our aim was to study molar tooth development in mice lacking endogenous PACAP. In this study morphometric, immunohistochemical and structural comparison of molar teeth in pre-eruptive developmental stage was performed on histological sections of 7-day-old wild-type and PACAP-deficient mice. Further structural analysis was carried out with Raman microscope. The morphometric comparison of the 7-day-old samples revealed that the dentin was significantly thinner in the molars of PACAP-deficient mice compared to wild-type animals. Raman spectra of the enamel in wild-type mice demonstrated higher diversity in secondary structure of enamel proteins. In the dentin of PACAP-deficient mice higher intracrystalline disordering in the hydroxyapatite molecular structure was found. We also obtained altered SHH, PTCH1 and Gli1 expression level in secretory ameloblasts of PACAP-deficient mice compared to wild-type littermates suggesting that PACAP might play an important role in molar tooth development and matrix mineralization involving influence on SHH signaling cascade.

Normally, sonic hedgehog (Shh) signaling induces high levels of Patched 1 (Ptc1) and its associated transcription factor Gli1 with genesis of specific neuronal progeny. But their roles in the neural stem cells (NSCs), including glial precursor cells (GPCs), of Alzheimer's disease (AD) are unclear. Here, we show that Ptc1 and Gli1 are significantly deficits in the hippocampus of an aged AD transgenic mouse mode, whereas these two molecules are highly elevated at young ages. Our similar findings in autopsied AD brains validate the discovery in AD mouse models. To examine whether Aβ peptides, which are a main component of the amyloid plaques in AD brains, affected Ptc1-Gli1 signaling, we treated GPCs with Aβ peptides, we found that high dose of Aβ1-42 but not Aβ1-40 significantly decreased Ptc1-Gli1, while Shh itself was elevated in hippocampal NSCs/GPCs. Furthermore, we found that deficits of Ptc1-Gli1 signaling induced NSCs/GPCs into asymmetric division, which results in an increase in the number of dividing cells including transit-amplifying cells and neuroblasts. These precursor cells commit to apoptosis-like death under the toxic conditions. By this way, adult neural precursor cell pool is exhausted and defective neurogenesis happens in AD brains. Our findings suggest that Ptc1-Gli1 signaling deregulation resulting abnormal loss of GPCs may contribute to a cognition decline in AD brains. The novel findings elucidate a new molecular mechanism of adult NSCs/GPCs on neurogenesis and demonstrate a regulatory role for Ptc1-Gli1 in adult neural circuit integrity of the brain.

Mesenchymal dysplasia (mes) mice harbour a truncation in the C-terminal region of the Hh-ligand receptor, Patched-1 (mPtch1). While the mes variant of mPtch1 binds to Hh-ligands with an affinity similar to that of wild type mPtch1 and appears to normally regulate canonical Hh-signalling via smoothened, the mes mutation causes, among other non-lethal defects, a block to mammary ductal elongation at puberty. We demonstrated previously Hh-signalling induces the activation of Erk1/2 and c-src independently of its control of smo activity. Furthermore, mammary epithelial cell-directed expression of an activated allele of c-src rescued the block to ductal elongation in mes mice, albeit with delayed kinetics. Given that this rescue was accompanied by an induction in estrogen receptor-alpha (ERα) expression and that complex regulatory interactions between ERα and c-src are required for normal mammary gland development, it was hypothesized that expression of ERα would also overcome the block to mammary ductal elongation at puberty in the mes mouse. We demonstrate here that conditional expression of ERα in luminal mammary epithelial cells on the mes background facilitates ductal morphogenesis with kinetics similar to that of the MMTV-c-srcAct mice. We demonstrate further that Erk1/2 is activated in primary mammary epithelial cells by Shh-ligand and that this activation is blocked by the inhibitor of c-src, PP2, is partially blocked by the ERα inhibitor, ICI 182780 but is not blocked by the smo-inhibitor, SANT-1. These data reveal an apparent Hh-signalling cascade operating through c-src and ERα that is required for mammary gland morphogenesis at puberty.

A reduction in the number of digits has evolved many times in tetrapods, particularly in cursorial mammals that travel over deserts and plains, yet the underlying developmental mechanisms have remained elusive. Here we show that digit loss can occur both during early limb patterning and at later post-patterning stages of chondrogenesis. In the 'odd-toed' jerboa (Dipus sagitta) and horse and the 'even-toed' camel, extensive cell death sculpts the tissue around the remaining toes. In contrast, digit loss in the pig is orchestrated by earlier limb patterning mechanisms including downregulation of Ptch1 expression but no increase in cell death. Together these data demonstrate remarkable plasticity in the mechanisms of vertebrate limb evolution and shed light on the complexity of morphological convergence, particularly within the artiodactyl lineage.

Adaptive variation in the craniofacial skeleton is a key component of resource specialization and habitat divergence in vertebrates, but the proximate genetic mechanisms that underlie complex patterns of craniofacial variation are largely unknown. Here we demonstrate that the Hedgehog (Hh) signaling pathway mediates widespread variation across a complex functional system that affects the kinematics of lower jaw depression--the opercular four-bar linkage apparatus--among Lake Malawi cichlids. By using a combined quantitative trait locus mapping and population genetics approach, we show that allelic variation in the Hh receptor, ptch1, affects the development of distinct bony elements in the head that represent two of three movable links in this functional system. The evolutionarily derived allele is found in species that feed from the water column, and is associated with shifts in anatomy that translate to a four-bar system capable of faster jaw rotation. Alternatively, the ancestral allele is found in species that feed on attached algae, and is associated with the development of a four-bar system that predicts slower jaw movement. Experimental manipulation of the Hh pathway during cichlid development recapitulates functionally salient natural variation in craniofacial geometry. In all, these results significantly extend our understanding of the mechanisms that fine-tune the craniofacial skeletal complex during adaptation to new foraging niches.

Medulloblastomas (MBs) are thought to be derived from granular cell precursors in the external granular layer (EGL) of the developing cerebellum. Heterozygous patched1 (Ptch1) knockout mice develop MBs that resemble those in humans when the sonic hedgehog (Shh) signaling pathway is activated. The present study was conducted to evaluate postnatal effects of a Shh signaling inhibitor, cyclopamine, on the development of MBs in Ptch1 mice. Ptch1 and wild-type mice were treated daily with subcutaneous cyclopamine at 40 mg/kg or vehicle from postnatal day (PND) 1 to PND14, and the subsequent development of MBs and preneoplastic lesions was examined up to week 12 (W12). Proliferative lesions in the cerebellum, MBs, and preneoplastic lesions were only detected in Ptch1 mice. Cyclopamine treatment resulted in a statistically significant reduction in the incidence and/or area of proliferative lesions at PND14 and 21. The trend of decreasing preneoplastic lesions persisted up to W12. At PND7, cyclopamine treatment reduced the width and proliferation of the EGL regardless of genotype. These results indicate that inhibition of Shh signaling during cerebellar development has prolonged inhibitory potential on MB development in Ptch1 mice. This inhibitory potential might be related to inhibition of EGL proliferation, including preneoplastic MB cells.

ATAD2 is associated with many cellular progresses such as cell growth, differentiation and apoptosis. Some studies suggest ATAD2 is highly expressed in cancer cells. In our previous studies, we found that ATAD2 is highly expressed in HCC tissues, compared with adjacent normal tissues, and patients with high expression of ATAD2 had a poorer prognosis. Moreover, we found mir-372 can regulate the expression of ATAD2 in HCC cell lines. We also detected a relationship between the mRNA expression of ATAD2 and Ptch1 by gene microarray. Here, we completed the function studies of ATAD2 in vivo and in vitro, and tested whether ATAD2 could regulate the Hh pathway. ATAD2 and Hh pathway protein expressions in 80 HCC specimens were examined by immunohistochemistry (IHC). The mRNA expression of ATAD2 and Hh pathway members in paired-HCC tissues and cell lines were, respectively, analyzed using quantitative PCR. ATAD2‑RNAi was transduced into HCCLM3 and Huh7 cells, using a lentiviral vector. The effect of ATAD2 in HCC cell lines on cell cycle and apoptosis were evaluated by flow cytometry. Tumorigenicity experiments in nude mice were performed to test the function of ATAD2 in vivo. Pharmacological regulation of Hh signaling was performed to test the relation between the ATAD2 and Hh pathways and C-myc. We found that ATAD2 and Ptch1 were both highly expressed in HCC tissues, compared with paired normal hepatic tissues. In addition, we found that ATAD2 could affect the expression of the Hh pathway by PCR and western blot anaysis in HCC cell lines, by observing the outcome before and after transfection. We speculate that ATAD2 cooperates with the MYC gene to regulate the expression of SMO and Gli, activating the Hh pathway and inducing an active feedback of the Hh pathway.

Hairless mice carrying homozygous mutations in hairless gene manifest rudimentary hair follicles (HFs), epidermal cysts, hairless phenotype, and enhanced susceptibility to squamous cell carcinomas. However, their susceptibility to basal cell carcinomas (BCCs), a neoplasm considered originated from HF-localized stem cells, is unknown. To demonstrate the role of HFs in BCC development, we bred Ptch(+/-)/C57BL6 with SKH-1 hairless mice, followed by brother-sister cross to get F2 homozygous mutant (hairless) or wild-type (haired) mice. UVB-induced inflammation was less pronounced in shaved haired than in hairless mice. In hairless mice, inflammatory infiltrate was found around the rudimentary HFs and epidermal cysts. Expression of epidermal IL1f6, S100a8, vitamin D receptor, repetin, and major histocompatibility complex II, biomarkers depicting susceptibility to cutaneous inflammation, was also higher. In these animals, HF disruption altered susceptibility to UVB-induced BCCs. Tumor onset in hairless mice was 10 weeks earlier than in haired littermates. The incidence of BCCs was significantly higher in hairless than in haired animals; however, the magnitude of sonic hedgehog signaling did not differ significantly. Overall, 100% of hairless mice developed12 tumors per mouse after 32 weeks of UVB therapy, whereas haired mice developed fewer than three tumors per mouse after 44 weeks of long-term UVB irradiation. Tumors in hairless mice were more aggressive than in haired littermates and manifested decreased E-cadherin and enhanced mesenchymal proteins. These data provide novel evidence that disruption of HFs in Ptch(+/-) mice enhances cutaneous susceptibility to inflammation and BCCs.