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Although basal cell carcinoma (BCC) is often managed successfully with surgery, patients with locally advanced BCC (laBCC) or metastatic BCC (mBCC) who are not candidates for surgery or radiotherapy have limited treatment options. Most BCCs result from aberrant Hedgehog pathway activation in keratinocyte tumor cells, caused by sporadic or inherited mutations. Mutations in the patched homologue 1 gene that remove its inhibitory regulation of Smoothened homologue (SMO) or mutations in SMO that make it constitutively active, lead to Hedgehog pathway dysregulation and downstream activation of GLI1/2 transcription factors, promoting cell differentiation and proliferation. Hedgehog inhibitors (HHIs) block overactive signaling of this pathway by inhibiting SMO and are currently the only approved treatments for advanced BCC. Two small-molecule SMO inhibitors, vismodegib and sonidegib, have shown efficacy and safety in clinical trials of advanced BCC patients. Although these agents are effective and tolerable for many patients, HHI resistance occurs in some patients. Mechanisms of resistance include mutations in SMO, noncanonical cell identity switching leading to tumor cell resistance, and non-canonical pathway crosstalk causing Hedgehog pathway activation. Approaches to managing HHI resistance include switching HHIs, HHI and radiotherapy combination therapy, photodynamic therapy, and targeting Hedgehog pathway downstream effectors. Increasing understanding of the control of downstream effectors has identified new therapy targets and potential agents for evaluation in BCC. Identification of biomarkers of resistance or response is needed to optimize HHI use in patients with advanced BCC. This review examines HHI resistance, its underlying mechanisms, and methods of management for patients with advanced BCC.

Vertebrate and invertebrate Hedgehog (Hh) morphogens signal over short and long distances to direct cell fate decisions during development and to maintain tissue homeostasis after birth. One of the most important questions in Hh biology is how such Hh signaling to distant target cells is achieved, because all Hh proteins are secreted as dually lipidated proteins that firmly tether to the outer plasma membrane leaflet of their producing cells. There, Hhs multimerize into light microscopically visible storage platforms that recruit factors required for their regulated release. One such recruited release factor is the soluble glycoprotein Scube2 (Signal sequence, cubulin domain, epidermal-growth-factor-like protein 2), and maximal Scube2 function requires concomitant activity of the resistance-nodulation-division (RND) transporter Dispatched (Disp) at the plasma membrane of Hh-producing cells. Although recently published cryo-electron microscopy-derived structures suggest possible direct modes of Scube2/Disp-regulated Hh release, the mechanism of Disp-mediated Hh deployment is still not fully understood. In this review, we discuss suggested direct modes of Disp-dependent Hh deployment and relate them to the structural similarities between Disp and the related RND transporters Patched (Ptc) and Niemann-Pick type C protein 1. We then discuss open questions and perspectives that derive from these structural similarities, with particular focus on new findings that suggest shared small molecule transporter functions of Disp to deplete the plasma membrane of cholesterol and to modulate Hh release in an indirect manner.

The Patched 1 (PTCH1) gene encodes a membrane receptor involved in the Hedgehog (Hh) signaling pathway, an abnormal state of which may result in congenital defects or human tumors. In this study, we conducted whole-exome sequencing on a three-generation Chinese family characterized with variable penetrance of orofacial clefts. A rare heterozygous variant in the PTCH1 gene (c.2833C > T p.R945X) was identified as a disease-associated mutation. Structural modeling revealed a truncation starting from the middle of the second extracellular domain of PTCH1 protein. This may damage its ligand recognition and sterol transportation abilities, thereby affecting the Hh signaling pathway. Biochemical assays indicated that the R945X protein had reduced stability compared to the wild-type in vitro. In addition, we reviewed the locations and mutation types of PTCH1 variants in individuals with clefting phenotypes, and analyzed the associations between clefts and locations or types of variants within PTCH1. Our findings provide further evidence that PTCH1 variants result in orofacial clefts, and contributed to genetic counseling and clinical surveillance in this family.

Perinatal alcohol exposure induces fetal alcohol syndrome partially through Sonic Hedgehog (SHH) impairment; however, the relationship between SHH signaling cascade and alcohol drinking pattern in adulthood remains obscure. We studied the expression of SHH and components of respective signaling cascade [PTCH receptor (Patched), SMO co-receptor (Smoothened) and downstream transcriptional factor Glioma-associated oncogene (GLI)] during early abstinence in brain regions of rats demonstrating different drinking patterns in intermittent access two-bottle choice paradigm (IA2BC). Male Wistar rats were subjected to twenty 24-h sessions of free access to two-bottle choice (water or 20% ethanol) with 24-h withdrawal periods (water only). Control animals had access to water only. Quantitative PCR and western blotting were used to assess transcript and protein levels in the brain, respectively. During the course of the IA2BC, one part of animals demonstrated gradual escalation from low to high alcohol intake and preference of alcohol over water (group I), while the other one consumed alcohol at stable high level (group II) (Peregud et al., 2021). Three days after the last drinking session, PTCH mRNA elevated in the hippocampus of group I rats as compared to the control group. However, SHH, SMO and GLI mRNA levels in the hippocampus did not change. The protein content of PTCH in the hippocampus of group I rats was higher as compared to both control and group II. PTCH elevation is a known marker of SHH cascade activity. Thus, activated hippocampal SHH signaling cascade is a hallmark of rats demonstrating gradual escalation of alcohol intake in the IA2BC procedure.

Although adequate periconceptional folic acid (FA) supplementation has reduced the occurrence of pregnancies affected by neural tube defects (NTDs), the mechanisms underlying FA-resistant NTDs are poorly understood, and thus NTDs still remain a global public health concern. A high level of Kruppel-like factor 12 (KLF12) exerts deleterious effects on heath in most cases, but evidence for its roles in development has not been published. We observed KLF12-overexpressing mice showed disturbed neural tube development. KLF12-overexpressing fetuses died in utero at approximately 10.5 days post-coitus, with 100% presenting cranial NTDs. Neither FA nor formate promoted normal neural tube closure in mutant fetuses. The RNA-seq results showed that a high level of KLF12 caused NTDs in mice via overactivating the sonic hedgehog (Shh) signaling pathway, leading to the upregulation of patched 1, GLI-Kruppel family member GLI1, hedgehog-interacting protein, etc., whereas FA metabolism-related enzymes did not express differently. PF-5274857, an antagonist of the Shh signaling pathway, significantly promoted dorsolateral hinge point formation and partially rescued the NTDs. The regulatory hierarchy between a high level of KLF12 and FA-resistant NTDs might provide new insights into the diagnosis and treatment of unexplained NTDs in the future. Summary sentence A high level of KLF12 in the early stage of embryogenesis caused FA-resistant neural tube defects by overactivating the Shh signaling pathway, which could be partially rescued by PF-5274857.

Background Microcystic adnexal carcinoma (MAC) is a rare skin neoplasm that has not been characterized on a molecular basis. Objectives We aimed to assess expression profiles of hedgehog (HH) signaling molecules in MAC and control tumours. Methods Immunohistochemistry was performed for Sonic HH (SHH), Indian HH (IHH), Patched 1 (PTCH1), and Smoothened (SMO) on patients' MAC (n=26) tissue and control tumour tissue, including syringoma (SyG; n=11), trichoepithelioma (TE; n=11), and basal cell carcinoma (BCC; n=12). Results PTCH1 and SMO immunoreactivity was significantly higher in BCC than in SyG, TE, and MAC (P = 0.000002 and P = 0.029, respectively). The highest IHH expression was observed in BCC and TE when compared to SyG and MAC (P = 0.038). Notably, the highest SHH protein expression was observed in SyG as compared to MAC, TE, and even BCC (P = 0.00075). In MAC patients, SMO immunoreactivity significantly (r = 0.51; P = 0.0086) correlated with PTCH1 expression. Further correlation studies did not show significant associations between the HH expression markers assessed (P > 0.05). Conclusions Our results indicate that alterations of the HH signaling are unlikely to play a major role in the pathogenesis of MAC, which is in contrast to the morphologically similar BCC and TE. Our observation provides additional information to the limited molecular pathology knowledge on this rare tumour.

Ubiquitous overactivation of Hedgehog signaling in adult pituitaries results in increased expression of proopiomelanocortin (Pomc), growth hormone (Gh) and prolactin (Prl), elevated adrenocorticotropic hormone (Acth) production and proliferation of Sox2+ cells. Moreover, ACTH, GH and PRL-expressing human pituitary adenomas strongly express the Hedgehog target GLI1. Accordingly, Hedgehog signaling seems to play an important role in pathology and probably also in homeostasis of the adult hypophysis. However, the specific Hedgehog-responsive pituitary cell type has not yet been identified. We here investigated the Hedgehog pathway activation status and the effects of deregulated Hedgehog signaling cell-specifically in endocrine and non-endocrine pituitary cells. We demonstrate that Hedgehog signaling is unimportant for the homeostasis of corticotrophs, whereas it is active in subpopulations of somatotrophs and folliculo-stellate cells in vivo. Reinforcement of Hedgehog signaling activity in folliculo-stellate cells stimulates growth hormone production/release from somatotrophs in a paracrine manner, which most likely is mediated by the neuropeptide vasoactive intestinal peptide. Overall, our data show that Hedgehog signaling affects the homeostasis of pituitary hormone production via folliculo-stellate cell-mediated regulation of growth hormone production/secretion.

Mammalian cerebellar astrocytes critically regulate the differentiation and maturation of neuronal Purkinje cells and granule precursors. The G protein-coupled receptor 37-like 1 (Gpr37l1) is expressed by Bergmann astrocytes and interacts with patched 1 (Ptch1) at peri-ciliary membranes. Cerebellar primary astrocyte cultures from wild-type and Gpr37l1 null mutant mouse pups were established and studied. Primary cilia were produced by cultures of both genotypes, as well as Ptch1 and smoothened (Smo) components of the sonic hedgehog (Shh) mitogenic pathway. Compared to wild-type cells, Gpr37l1-/- astrocytes displayed striking increases in proliferative activity, Ptch1 protein expression and internalization, intracellular cholesterol content, ciliary localization of Smo, as well as a marked production of active Shh. Similar effects were reproduced by treating wild-type astrocytes with a putative prosaptide ligand of Gpr37l1. These findings indicate that Gpr37l1-Ptch1 interactions specifically regulate Ptch1 internalization and trafficking, with consequent stimulation of Shh production and activation of proliferative signaling.

Non-small cell lung cancer (NSCLC) has been identified as a leading cause of tumor-associated death around the world. Presently, it is necessary to find effective and safe therapy for its treatment in clinic. Jervine (Jer), a sterodial alkaloid from rhizomes of Veratrum album, exhibits anti-inflammatory and anti-cancer effects. However, its effects on lung cancer progression are still unknown. In this study, we explored if Jer showed any influences on NSCLC development, as well as the underlying molecular mechanisms. The results showed that Jer time- and dose-dependently reduced the proliferation of NSCLC cells, along with inhibited colony formation capacity. Apoptosis was highly induced by Jer in NSCLC cells through promoting the expression of cleaved Caspase-3. Furthermore, Jer treatment led to autophagy in cancer cells, as evidenced by the fluorescence microscopy results and increases of LC3II. Autophagy inhibitor bafilomycinA1 (BafA1) abrogated the inhibitory effects of Jer on cell proliferation and apoptosis induction, showing that Jer triggered autophagy-mediated apoptosis in NSCLC cells. Additionally, AKT and mammalian target of Rapamycin (mTOR) signaling pathway was highly repressed in cancer cells. Importantly, promoting AKT activation greatly rescued the cell survival, while attenuated autophagy and apoptosis in Jer-incubated NSCLC cells, revealing that Jer-modulated autophagic cell death was through the blockage of AKT signaling. Hedgehog signaling pathway was then found to be suppressed by Jer, as proved by the decreased expression of Sonic Hedgehog (Shh), Hedgehog receptor protein patched homolog 1 (PTCH1), smoothened (SMO) and glioma-associated oncogene homolog 1 (Gli1) in NSCLC cells. Of note, enhancing Shh signaling dramatically diminished the stimulative effects of Jer on autophagy-mediated apoptosis in vitro, demonstrating the importance of Hedgehog signaling in Jer-regulated cell death. Moreover, Jer treatment effectively reduced tumor growth in A549-bearing mice with few toxicity. Together, Jer may be a promising and effective therapeutic strategy for NSCLC treatment.

The relevance of Hedgehog signaling in Merkel cell carcinoma has only been addressed by a few studies with conflicting results. Thus, we aimed to establish the expression of Hedgehog signaling molecules in Merkel cell carcinoma to characterize causes of aberrant expression and to correlate these findings with the clinical course of the patients. Immunohistochemistry was performed for Sonic, Indian, Patched 1 (PTCH1) and Smoothened on patients' tumor tissue. Respective mRNA expression was analyzed in 10 Merkel cell carcinoma cell lines using quantitative real-time polymerase chain reaction. PTCH1 sequencing and DNA methylation microarray analyses were carried out on tumor tissues as well as cell lines. PTCH1 immunoreactivity in Merkel cell carcinoma was similar to that of basal cell carcinomas, which both significantly differed from PTCH1 immunoreactivity in healthy skin. Most PTCH1 mutations found were synonymous or without known functional impact. However, on average, the promoter regions of both PTCH1 were hypomethylated independently from PTCH1 gene expression or Merkel cell polyomavirus status. PTCH1 and GLI1/2/3 genes were differently expressed in different cell lines; notably, there was a significant correlation between GLI2 and PTCH1 mRNA expression. Similar to PTCH1 protein expression in patient tissues, PTCH1 gene expression in Merkel cell carcinoma cell lines is highly variable, but due to the similar methylation pattern across Merkel cell carcinoma cell lines, effects other than methylation seem to be the reason for the differential expression and PTCH1 appears to be upregulated by GLI as a classical Hedgehog target gene.

Background Hyperoxia increases Sonic hedgehog (Shh) expression in neonatal rat lungs. The effect of mesenchymal stem cells (MSCs) on the hedgehog signaling pathway in hyperoxia-induced lung injury is unknown. This study evaluated whether MSCs could inhibit hedgehog signaling and improve established hyperoxia-induced lung injury in newborn rats. Methods Newborn rats were assigned to room air (RA) or hyperoxia (85% O2) groups from postnatal day 4–15, and some received intravenous injection of human MSCs (9 × 105 cells) in 90 μL of normal saline (NS) through the tail vein on postnatal day 15. We obtained four study groups as follows: RA + NS, RA + MSCs, O2 + NS, and O2 + MSCs. Pups from each group were sacrificed on postnatal days 15 and 29, and lungs were removed for histological and Western blot analyses. Results Neonatal hyperoxia on postnatal days 4–15 reduced the body weight, increased the mean linear intercept, and decreased the vascular density of the rats, and these effects were associated with increased Shh and Smoothened (Smo) expression and decreased Patched expression. By contrast, the MSC-treated hyperoxic pups exhibited improved alveolarization, increased vascularization, and decreased Shh and Smo expression on postnatal day 29. Conclusion Human MSC treatment reversed established hyperoxia-induced lung injury in newborn rats, probably through suppression of the hedgehog pathway.

The Hedgehog (Hh) family of lipid-modified signaling proteins directs embryonic tissue patterning and postembryonic tissue homeostasis, and dysregulated Hh signaling drives familial and sporadic cancers. Hh ligands bind to and inhibit the tumor suppressor Patched and allow the oncoprotein Smoothened (SMO) to accumulate in cilia, which in turn activates the GLI family of transcription factors. Recent work has demonstrated that endogenous cholesterol and oxidized cholesterol derivatives (oxysterols) bind and modulate SMO activity. Here we discuss the myriad sterols that activate or inhibit the Hh pathway, with emphasis on endogenous 24(S),25-epoxycholesterol and 3β,5α-dihydroxycholest-7-en-6-one, and propose models of sterol regulation of SMO. Synthetic inhibitors of SMO have long been the focus of drug development efforts. Here, we discuss the possible utility of steroidal SMO ligands or inhibitors of enzymes involved in sterol metabolism as cancer therapeutics.

The evolutionarily conserved hedgehog (Hh) pathway is essential for organogenesis and plays critical roles in postnatal tissue maintenance and renewal. A unique feature of the vertebrate Hh pathway is that signal transduction requires the primary cilium (PC) where major pathway components are dynamically enriched. These factors include smoothened (SMO) and patched, which constitute the core reception system for sonic hedgehog (SHH) as well as GLI transcription factors, the key mediators of the pathway. Here, we report biallelic loss-of-function variations in SMO in seven individuals from five independent families; these variations cause a wide phenotypic spectrum of developmental anomalies affecting the brain (hypothalamic hamartoma and microcephaly), heart (atrioventricular septal defect), skeleton (postaxial polydactyly, narrow chest, and shortening of long bones), and enteric nervous system (aganglionosis). Cells derived from affected individuals showed normal ciliogenesis but severely altered Hh-signal transduction as a result of either altered PC trafficking or abnormal activation of the pathway downstream of SMO. In addition, Hh-independent GLI2 accumulation at the PC tip in cells from the affected individuals suggests a potential function of SMO in regulating basal ciliary trafficking of GLI2 when the pathway is off. Thus, loss of SMO function results in abnormal PC dynamics of key components of the Hh signaling pathway and leads to a large continuum of malformations in humans.

Background Sedum sarmentosum is traditionally used to treat various inflammatory diseases in China. It has protective effects against acute liver injury, but the exact mechanism of such effects remains unclear. This study investigated the protective effects of S. sarmentosum extract on lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced acute liver injury in mice and the mechanism of such effects. Methods Mice were randomly divided into control, treatment, model, and model treatment groups. Acute liver injury was induced in model mice via intraperitoneal injection of LPS and D-GalN with doses of 10 μg/kg of LPS and 500 mg/kg, respectively. The mRNA expression levels of miR-124, Hedgehog, Patched (Ptch), Smoothened (Smo), and glioma-associated oncogene homolog (Gli) in liver tissues were determined through RT-PCR, and the protein levels of Hedgehog, Ptch, Smo, Gli, P13k, Akt, HMGB1, TLR4, IkB-α, p-IkB-α, and NF-kB65 were evaluated via Western blot analysis. The serum levels of IL-6, TNF-α, CRP, IL-12, and ICAM-1 were determined via ELISA. TLR4 and NF-κBp65 activity and the levels of DNA-bound NF-KB65 and TLR4 in LPS/D-GalN-induced liver tissues were also determined. We recorded the time of death, plotted the survival curve, and calculated the liver index. We then observed the pathological changes in liver tissue and detected the levels of liver enzymes (alanine aminotransferase [ALT] and aspartate transaminase [AST]) in the serum and myeloperoxidase (MPO) and plasma inflammatory factors in the liver homogenate. Afterward, we evaluated the protective effects of S. sarmentosum extracts on acute liver injury in mice. Results Results showed that after S. sarmentosum extract was administered, the expression level of miR-124 increased in liver tissues. However, the protein expression levels of Hedgehog, Ptch, Smo, Gli, P13k, p-Akt, HMGB1, TLR4, p-IκB-α, and NF-κB65 and the mRNA expression levels of Hedgehog, Ptch, Smo, and Gli decreased. The MPO level in the liver, the IL-6, TNF-α, CRP, IL-12, and MMP-9 levels in the plasma, and the serum ALT and AST levels also decreased, thereby reducing LPS/D-GalN-induced liver injury and improving the survival rate of liver-damaged animals within 24 h. Conclusions S. sarmentosum extract can alleviate LPS/D-GalN-induced acute liver injury in mice and improve the survival rate of mice. The mechanism may be related to the increase in miR-124 expression, decrease in Hedgehog and HMGB1 signaling pathway activities, and reduction in inflammatory responses in the liver. Hedgehog is a regulatory target for miR-124.

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.

Hedgehog signaling pathway originally identified in the fruit fly Drosophila is an evolutionarily conserved signaling mechanism with crucial roles in embryogenesis, growth and patterning. It exerts its biological effect through a signaling mechanism that terminates at glioma-associated oncogene (GLI) transcription factors which alternate between activator and repressor forms and mediate various responses. The important components of the pathway include the hedgehog ligands (SHH), the Patched (PTCH) receptor, Smoothened (SMO), Suppressor of Fused (SuFu) and GLI transcription factors. Activating or inactivating mutations in key genes cause uncontrolled activation of the pathway in a ligand independent manner. The ligand-dependent aberrant activation of the hedgehog pathway causing overexpression of hedgehog pathway components and its target genes occurs in autocrine as well as paracrine fashion. In adults, aberrant activation of hedgehog signaling has been linked to birth defects and multiple solid cancers. In this review, we assimilate data from recent studies to understand the mechanism of functioning of the hedgehog signaling pathway, role in cancer, its association in various solid malignancies and the current strategies being used to target this pathway for cancer treatment.

Patched (Ptc) and Patched-related (Ptr) proteins containing sterol-sensing domains (SSD) and Patched domains are highly conserved in eukaryotes for lipid transport and metabolism. Four proteins containing predicted SSD and Patched domains were simultaneously found by searching thePhytophthora sojaegenome database, and one of them was identified as a Patched-like (PTL) protein. Here, we investigated the biological function ofPsPTL. The expression level ofPsPTLwas higher during mycelial and sporulation stages, compared to zoospore (ZO), cyst, and germinated-cyst stages, without significant change during infection. However, deletion ofPsPTLusing CRISPR/Cas9 had no significant effect on the growth, development, or virulence ofP. sojae. Further investigations showed thatPsPTLis not essential forP. sojaeto cope with external stresses such as temperature, pH, oxidative and osmotic pressure. In addition, this gene did not appear to play an essential role inP. sojae’s response to exogenous sterols. The transcript levels of the other three proteins containing predicted SSD and Patched domains were also not significantly upregulated inPsPTLdeletion transformants. Our studies demonstrated that PsPTL is not an essential protein forP. sojaeunder the tested conditions, and more in-depth research is required for revealing the potential functions ofPsPTLunder special conditions or in other signaling pathways.

A long-standing mystery in vertebrate Hedgehog signaling is how Patched 1 (PTCH1), the receptor for Hedgehog ligands, inhibits the activity of Smoothened, the protein that transmits the signal across the membrane. We previously proposed (Kinnebrew et al., 2019) that PTCH1 inhibits Smoothened by depleting accessible cholesterol from the ciliary membrane. Using a new imaging-based assay to directly measure the transport activity of PTCH1, we find that PTCH1 depletes accessible cholesterol from the outer leaflet of the plasma membrane. This transport activity is terminated by binding of Hedgehog ligands to PTCH1 or by dissipation of the transmembrane potassium gradient. These results point to the unexpected model that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export in the opposite direction. Our study provides a plausible solution for how PTCH1 inhibits SMO by changing the organization of cholesterol in membranes and establishes a general framework for studying how proteins change cholesterol accessibility to regulate membrane-dependent processes in cells. Ministry of Education (MOE) Published version Christian Siebold - Cancer Research UK grant reference numbers C20724 and A26752, European Research Council grant reference number 647278. Rajat Rohatgi - National Institutes of Health grant reference numbers GM118082 and GM106078. Arun Radhakrishnan - National Institutes of Health grant reference number HL20948, Welch Foundation grant reference number I-1793 and Leducq Foundation grant reference number 19CVD04. Yasunori Saheki - Ministry of Education, Singapore grant reference numbers MOE2017-T2-2-001 and MOE-T2EP30120-0002. Maia Kinnebrew - National Science Foundation Predoctoral Fellowship. Giovanni Luchetti - Ford Foundation Predoctoral Fellowship.

In this article we discuss the concept of accessible plasma membrane cholesterol and its involvement as a signalling molecule. Changes in plasma membrane accessible cholesterol, although only being minor in the context of total cholesterol plasma membrane cholesterol and total cell cholesterol, are a key regulator of overall cellular cholesterol homeostasis by the SREBP pathway. Accessible cholesterol also provides the second messenger between patched 1 and smoothened in the hedgehog signalling pathway important during development, and its depletion may provide a mechanism of resistance to microbial pathogens including SARS-CoV-2. We revise the hypothesis that oxysterols are a signalling form of cholesterol, in this instance as a rapidly acting and paracrine version of accessible cholesterol.

Malignant melanoma is the deadliest skin cancer, with a poor prognosis in advanced stages. We recently showed that the extracellular signal-regulated kinase 5 (ERK5), encoded by the MAPK7 gene, plays a pivotal role in melanoma by regulating cell functions necessary for tumour development, such as proliferation. Hedgehog-GLI signalling is constitutively active in melanoma and is required for proliferation. However, no data are available in literature about a possible interplay between Hedgehog-GLI and ERK5 pathways. Here, we show that hyperactivation of the Hedgehog-GLI pathway by genetic inhibition of the negative regulator Patched 1 increases the amount of ERK5 mRNA and protein. Chromatin immunoprecipitation showed that GLI1, the major downstream effector of Hedgehog-GLI signalling, binds to a functional non-canonical GLI consensus sequence at the MAPK7 promoter. Furthermore, we found that ERK5 is required for Hedgehog-GLI-dependent melanoma cell proliferation, and that the combination of GLI and ERK5 inhibitors is more effective than single treatments in reducing cell viability and colony formation ability in melanoma cells. Together, these findings led to the identification of a novel Hedgehog-GLI-ERK5 axis that regulates melanoma cell growth, and shed light on new functions of ERK5, paving the way for new therapeutic options in melanoma and other neoplasms with active Hedgehog-GLI and ERK5 pathways.

The Hedgehog signaling pathway coordinates early development and is important in various cancers. Classic approaches to test pathway activation rely on transcriptional readouts or ciliary accumulation of specific pathway components. Although these assays have laid the foundation for studying Hedgehog pathway activation, they integrate the complex molecular actions of the transporter Patched and the seven transmembrane protein Smoothened. Though it is clear that cellular sterols are critical for pathway activity, direct dissection of which sterols drive Smoothened activity is precluded by the complex biosynthetic pathways responsible for cellular sterols. Here we describe a direct method of measuring Smoothened activity in vitro. This assay measures the binding of Smoothened to NbSmo8, a single-domain antibody that is selective for the active-state of Smoothened. Binding of purified Smoothened, reconstituted with specific sterols, to fluorescently labelled NbSmo8 can be rapidly evaluated using a fluorescence-detection size-exclusion chromatography assay. This approach enables a reductionist approach to precisely interrogate the regulatory activities of cellular lipids and sterols during Hedgehog signaling.

The Patched-related superfamily of transmembrane proteins can transport lipids or other hydrophobic molecules across cell membranes. While the Hedgehog receptor Patched has been intensively studied, much less is known about the biological roles of other Patched-related family members. Caenorhabditis elegans has a large number of Patched-related proteins, despite lacking a canonical Hedgehog pathway. Here, we show that PTR-4 promotes the assembly of the precuticle apical extracellular matrix, a transient and molecularly distinct matrix that precedes and patterns the later collagenous cuticle or exoskeleton. ptr-4 mutants share many phenotypes with precuticle mutants, including defects in eggshell dissolution, tube shaping, alae (cuticle ridge) structure, molting, and cuticle barrier function. PTR-4 localizes to the apical side of a subset of outward-facing epithelia, in a cyclical manner that peaks when precuticle matrix is present. Finally, PTR-4 is required to limit the accumulation of the lipocalin LPR-3 and to properly localize the Zona Pellucida domain protein LET-653 within the precuticle. We propose that PTR-4 transports lipids or other hydrophobic components that help to organize the precuticle and that the cuticle and molting defects seen in ptr-4 mutants result at least in part from earlier disorganization of the precuticle.

The Sonic hedgehog (Shh) pathway controls embryonic development and tissue homeostasis after birth. Long-standing questions about this pathway include how the dual-lipidated, firmly plasma membrane-associated Shh ligand is released from producing cells to signal to distant target cells and how the resistance–nodulation–division transporter Dispatched 1 (Disp, also known as Disp1) regulates this process. Here, we show that inactivation of Disp in Shh-expressing human cells impairs proteolytic Shh release from its lipidated terminal peptides, a process called ectodomain shedding. We also show that cholesterol export from Disp-deficient cells is reduced, that these cells contain increased cholesterol amounts in the plasma membrane, and that Shh shedding from Disp-deficient cells is restored by pharmacological membrane cholesterol extraction and by overexpression of transgenic Disp or the structurally related protein Patched 1 (Ptc, also known as Ptch1; a putative cholesterol transporter). These data suggest that Disp can regulate Shh function via controlled cell surface shedding and that membrane cholesterol-related molecular mechanisms shared by Disp and Ptc exercise such sheddase control., Summary: The resistance–nodulation–division protein Dispatched 1 expels plasma membrane cholesterol to indirectly increase Sonic hedgehog morphogen release from the cell surface.

This report is a synthesis of 22 studies which concludes that coronary artery disease is caused by free radical damage to tumor suppressor genes, which mutation allows cells to proliferate out of control to create benign mini tumors between the endothelium lining and the smooth muscle walls of arteries. As these tumors grow, they cause tiny tears in the endothelium that are patched with interlacing filaments of fibrin. This rough scar tissue traps minerals (especially calcium), heavy metals, macrophages, and cellular debris. The final smooth layer of cholesterol that is applied over this arterial plaque plays no causal part in its development. Recent studies indicate that nearly 75 percent of patients hospitalized for a heart attack had cholesterol levels which indicate they were not at high risk for a cardiovascular event, according to current national cholesterol guidelines. Thus, there is no causal relation between serum cholesterol levels and coronary artery disease.

Background Cerebellar neurogenesis involves the generation of large numbers of cerebellar granule neurons (GNs) throughout development of the cerebellum, a process that involves tight regulation of proliferation and differentiation of granule neuron progenitors (GNPs). A number of transcriptional regulators, including Math1, and the signaling molecules Wnt and Shh have been shown to have important roles in GNP proliferation and differentiation, and deregulation of granule cell development has been reported to be associated with the pathogenesis of medulloblastoma. While the progenitor/differentiation states of cerebellar granule cells have been broadly investigated, a more detailed association between developmental differentiation programs and spatial gene expression patterns, and how these lead to differential generation of distinct types of medulloblastoma remains poorly understood. Here, we provide a comparative single-cell spatial transcriptomics analysis to better understand the similarities and differences between developing granule and medulloblastoma cells. Results To acquire an enhanced understanding of the precise cellular states of developing cerebellar granule cells, we performed single-cell RNA sequencing of 24,919 murine cerebellar cells from granule neuron-specific reporter mice (Math1-GFP; Dcx-DsRed mice). Our single-cell analysis revealed that there are four major states of developing cerebellar granule cells, including two subsets of granule progenitors and two subsets of differentiating/differentiated granule neurons. Further spatial transcriptomics technology enabled visualization of their spatial locations in cerebellum. In addition, we performed single-cell RNA sequencing of 18,372 cells from Patched+/− mutant mice and found that the transformed granule cells in medulloblastoma closely resembled developing granule neurons of varying differentiation states. However, transformed granule neuron progenitors in medulloblastoma exhibit noticeably less tendency to differentiate compared with cells in normal development. Conclusion In sum, our study revealed the cellular and spatial organization of the detailed states of cerebellar granule cells and provided direct evidence for the similarities and discrepancies between normal cerebellar development and tumorigenesis.

Intrinsic mechanisms such as temporal series of transcription factors orchestrate neurogenesis from a limited number of neural progenitors in the brain. Extrinsic regulations, however, remain largely unexplored. Here we describe a two-step glia-derived signal that regulates neurogenesis in the Drosophila mushroom body (MB). In a temporal manner, glial-specific ubiquitin ligase dSmurf activates non-cell-autonomous Hedgehog signaling propagation by targeting the receptor Patched to suppress and promote the exit of MB neuroblast (NB) proliferation, thereby specifying the correct α/β cell number without affecting differentiation. Independent of NB proliferation, dSmurf also stabilizes the expression of the cell-adhesion molecule Fasciclin II (FasII) via its WW domains and regulates FasII homophilic interaction between glia and MB axons to refine α/β-lobe integrity. Our findings provide insights into how extrinsic glia-to-neuron communication coordinates with NB proliferation capacity to regulate MB neurogenesis; glial proteostasis is likely a generalized mechanism in orchestrating neurogenesis.

Coordination of neurite extension with surrounding glia development is critical for neuronal function, but the underlying molecular mechanisms remain poorly understood. Through a genome-wide mutagenesis screen in C. elegans, we identified dyf-4 and daf-6 as two mutants sharing similar defects in dendrite extension. DAF-6 encodes a glia-specific patched-related membrane protein that plays vital roles in glial morphogenesis. We cloned dyf-4 and found that DYF-4 encodes a glia-secreted protein. Further investigations revealed that DYF-4 interacts with DAF-6 and functions in a same pathway as DAF-6 to regulate sensory compartment formation. Furthermore, we demonstrated that reported glial suppressors of daf-6 could also restore dendrite elongation and ciliogenesis in both dyf-4 and daf-6 mutants. Collectively, our data reveal that DYF-4 is a regulator for DAF-6 which promotes the proper formation of the glial channel and indirectly affects neurite extension and ciliogenesis., Author summary In C. elegans sensory organ, the ciliated neuronal endings are wrapped in a luminal channel formed by glial cells, forming a specialized sensory compartment critical for sensory activity. Coordination of glial channel formation, dendritic tip anchoring and ciliogenesis are critical for the formation of a functional sensory compartment. DAF-6, a patched-related glial membrane protein, was reported to play an important role in glial channel morphogenesis, but the molecular function and regulatory mechanism of DAF-6 remain poorly understood. Here, we found that DYF-4, a glia-secreted protein, interacts and colocalizes with DAF-6, and functions in a same pathway as DAF-6 to regulate sensory compartment formation. We propose that DYF-4 is a novel regulator for DAF-6 to control sensory compartment formation.

The mechanisms that determine the final topology of skeletal muscles remain largely unknown. We have been developingDrosophilabody wall musculature as a model to identify and characterize the pathways that control muscle size, shape, and orientation during embryogenesis (Johnson et al., 2013; Williams et al., 2015; Yang et al., 2020a; Yang et al., 2020b). Our working model argues muscle morphogenesis is regulated by (1) extracellular guidance cues that direct muscle cells toward muscle attachment sites, and (2) contact dependent interactions between muscles and tendons. While we have identified several pathways that regulate muscle morphogenesis, our understanding is far from complete. Here we report the results of a recent EMS-based forward genetic screen that identified a myriad of loci not previously associated with muscle morphogenesis. We recovered new alleles of known muscle morphogenesis genes, includingbsd, kon, ths, andtum, arguing our screening strategy was effective and efficient. We also identified and sequenced new alleles ofsalm,barr, andptcthat presumably disrupt independent pathways directing muscle morphogenesis. Equally as important, our screen shows that at least 11 morphogenetic loci remain to be identified. This screen has developed exciting new tools to study muscle morphogenesis, which may provide future insights into the mechanisms that determine skeletal muscle topology.

Objective The Hedgehog pathway is a fundamental signaling pathway in organogenesis. The expression patterns of the ligand Sonic Hedgehog (Shh) and key pathway components have been studied in many tissues but direct spatial comparisons across tissues with different cell compositions and structural organization are not common and could reveal tissue-specific differences in pathway dynamics. Results We directly compared the expression characteristics of Shh, and four genes with functional roles in signaling and whose expression levels serve as readouts of pathway activity in multiple tissues of the embryonic mouse head at embryonic day 15.5 by serial in situ hybridization. The four readout genes were the positive feedback regulator Gli1, and three negative feedback regulators, Patched1, Patched2, and Hedgehog Interacting Protein. While the relative abundance of Gli1 was similar across tissues, the relative expression levels and spatial distribution of Shh and the negative feedback regulators differed, suggesting that feedback regulation of hedgehog signaling is context dependent. This comparative analysis offers insight into how consistent pathway activity could be achieved in tissues with different morphologies and characteristics of ligand expression.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated mortality worldwide. Hepatocarcinogenesis involves numerous interlinked factors and processes, including the Sonic hedgehog (Shh) signaling pathway, which participates in the carcinogenesis, progression, invasiveness, recurrence and cancer stem cell maintenance of HCC. The Shh signaling pathway is activated by ligands that bind to their receptor protein, Protein patched homolog (Ptch). The process of Shh ligand binding to Ptch weakens the inhibition of smoothened homolog (SMO) and activates signal transduction via glioma-associated oncogene homolog (Gli) transcription factors. The overexpression of Shh pathway molecules, including Shh, Ptch-1, Gli and SMO has been indicated in patients with HCC. It has also been suggested that the Shh signaling pathway exhibits cross-talk between numerous other signaling pathways. The inactivation of the Shh signaling pathway reduces HCC growth, increases radio-sensitivity and increases the beneficial effect of chemotherapy in HCC treatment. Therefore, inhibition of the Shh pathway may be an effective target therapy that can be used in the treatment of HCC.

The Hedgehog (Hh) signaling pathway is crucial for the development of vertebrate and invertebrate animals alike. Hh ligand binds its receptor Patched (Ptc), allowing the activation of the obligate signal transducer Smoothened (Smo). The levels and localizations of both Ptc and Smo are regulated by ubiquitination, and Smo is under additional regulation by phosphorylation and SUMOylation. Downstream of Smo, the Ci/Gli family of transcription factors regulates the transcriptional responses to Hh. Phosphorylation, ubiquitination and SUMOylation are important for the stability and localization of Ci/Gli proteins and Hh signaling output. Finally, Suppressor of Fused directly regulates Ci/Gli proteins and itself is under proteolytic regulation that is critical for normal Hh signaling.

Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids—a palmitoyl group at the N terminus and a cholesteroyl group at the C terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with reassessment of previous cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic hedgehog (Shh) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signaling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, Shh inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core. Cholesterol can function as both a substrate and an inhibitor of the Hedgehog receptor Patched. Structural analysis and molecular dynamics simulations reveal that cholesterol inhibits Patched by inserting into its extracellular domain

Radioresistance is the primary cause for the low efficacy of radiotherapy in the treatment of esophageal cancer (EC). Increasing evidence has demonstrated that the Sonic Hedgehog (Shh) signaling pathway may be involved in the pathology of various tumors, including EC. The present study aimed to examine the association between radioresistance in EC and the Sonic Hedgehog pathway, and to determine whether a downstream transcription factor of the Shh pathway, glioma-associated oncogene family zinc finger 1 (Gli1), serves a primary role in radioresistance. The radiation-resistant cell line Eca109R was established by repeated low dose (cumulative dose 60 Gy) irradiation of the human EC cell line Eca109. The level of cell radiosensitivity was determined by colony formation assay, and the localization of Gli1 was detected using immunofluorescence. Western blotting was used to determine the protein expression levels of Gli1, Shh, patched 1 (Ptch) and smoothened frizzled class receptor (Smo) in the two cell lines. Significantly higher levels of Gli1 were identified in the Eca109R cell line compared with those inEca109 cells (P

Continuous growth of the mouse incisor teeth is due to the life-long maintenance of epithelial stem cells (SCs) in their niche called cervical loop (CL). Several signaling factors regulate SC maintenance and/or their differentiation to achieve organ homeostasis. Previous studies indicated that Hedgehog signaling is crucial for both the maintenance of the SCs in the niche, as well as for their differentiation. How Hedgehog signaling regulates these two opposing cellular behaviors within the confinement of the CL remains elusive. In this study, we used in vitro organ and cell cultures to pharmacologically attenuate Hedgehog signaling. We analyzed expression of various genes expressed in the SC niche to determine the effect of altered Hedgehog signaling on the cellular hierarchy within the niche. These genes include markers of SCs (Sox2 and Lgr5) and transit-amplifying cells (P-cadherin, Sonic Hedgehog, and Yap). Our results show that Hedgehog signaling is a critical survival factor for SCs in the niche, and that the architecture and the diversity of the SC niche are regulated by multiple Hedgehog ligands. We demonstrated the presence of an additional Hedgehog ligand, nerve-derived Desert Hedgehog, secreted in the proximity of the CL. In addition, we provide evidence that Hedgehog receptors Ptch1 and Ptch2 elicit independent responses, which enable multimodal Hedgehog signaling to simultaneously regulate SC maintenance and differentiation. Our study indicates that the cellular hierarchy in the continuously growing incisor is a result of complex interplay of two Hedgehog ligands with functionally distinct Ptch receptors. Stem Cells 2019;37:1238–1248

Context Placental dysfunction is the underlying cause of common major disorders of pregnancy, such as fetal growth restriction and preeclampsia. However, the mechanisms of placental dysfunction are not entirely elucidated. We previously reported 10 reliable preeclampsia pathways based on multiple microarray data sets, among which was the sonic hedgehog (SHH) pathway. In this study, we describe the significant role of SHH signaling involved in placental development and fetal growth. Design The placental expression levels of surrogate markers of the SHH pathway, patched homolog 1 (PTCH1) and glioma-associated oncogene homolog (GLI) 2, were evaluated using quantitative real-time PCR, western blot analysis, and immunohistochemistry. We investigated the underlying mechanisms of the SHH pathway in trophoblast syncytialization, a critical process for placental development and maturation, using primary cytotrophoblasts. Moreover, the potential roles of placental SHH signaling in the regulation of the IGF axis were explored by pathway analysis of microarray data. Finally, the influence of SHH signaling on fetal growth was examined by placental administration of cyclopamine, an SHH pathway inhibitor, to pregnant mice. Results The SHH pathway was downregulated in preeclampsia placentas, and its activation was highly correlated with birth weight. Trophoblast syncytialization was modulated by noncanonical SHH–adenylate cyclase (ADCY) signaling rather than canonical SHH–GLI signaling. The IGF1 receptor pathway was regulated by both noncanonical SHH–ADCY signaling and canonical SHH–GLI signaling. Inhibition of placental SHH signaling significantly reduced fetal weight in mice. Conclusion Placental development and fetal growth were regulated through the SHH pathway via the IGF axis.

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.

Significance Primary cilia are antenna-like sensory organelles critical for many signal transduction pathways. One such pathway, called Hedgehog signaling, is essential for normal embryonic development and linked to tissue homeostasis and tumorigenesis. While the tightly regulated spatial and temporal localization of proteins in cilia controls activity, the mechanistic details are not fully understood. To characterize the movements of the Hedgehog receptor Patched1 in the cilium, we used single-molecule fluorescence microscopy, a tool that separates the behavior of individual proteins from the ensemble average, unveiling the underlying hidden physical behaviors. Our study reveals Hedgehog-induced changes in the motion of individual Patched1 molecules, which precede the exodus of Patched1 from cilia. These changes constitute one of the earliest measurable steps of Hedgehog-signal transduction., The Hedgehog-signaling pathway is an important target in cancer research and regenerative medicine; yet, on the cellular level, many steps are still poorly understood. Extensive studies of the bulk behavior of the key proteins in the pathway established that during signal transduction they dynamically localize in primary cilia, antenna-like solitary organelles present on most cells. The secreted Hedgehog ligand Sonic Hedgehog (SHH) binds to its receptor Patched1 (PTCH1) in primary cilia, causing its inactivation and delocalization from cilia. At the same time, the transmembrane protein Smoothened (SMO) is released of its inhibition by PTCH1 and accumulates in cilia. We used advanced, single molecule-based microscopy to investigate these processes in live cells. As previously observed for SMO, PTCH1 molecules in cilia predominantly move by diffusion and less frequently by directional transport, and spend a fraction of time confined. After treatment with SHH we observed two major changes in the motional dynamics of PTCH1 in cilia. First, PTCH1 molecules spend more time as confined, and less time freely diffusing. This result could be mimicked by a depletion of cholesterol from cells. Second, after treatment with SHH, but not after cholesterol depletion, the molecules that remain in the diffusive state showed a significant increase in the diffusion coefficient. Therefore, PTCH1 inactivation by SHH changes the diffusive motion of PTCH1, possibly by modifying the membrane microenvironment in which PTCH1 resides.

Annually, 48,000 people die from pancreatic ductal adenocarcinoma (PDAC), ranking it the fourth among cancer-related deaths in the United States. Currently, anti-cancer drugs are not effective against PDAC, and only extends survival by 3 months. Aberrant DNA methylation has been shown to play an important role during carcinogenesis in PDAC, with approximately 80% of tumor overexpressing the DNA methyltransferase 1 (DNMT1) protein. In the present study, we used DNMTs as a screening platform to find a new DNMT inhibitor, n-butylidenephthalide (n-BP), which is identified from a Chinese herbal drug. n-BP could inhibit DNMT1 expression in both dose-dependent and time-dependent manner. It also displays an effect in suppressing growth of PDAC cells and inducing cell cycle arrest at G0/G1 phase leading apoptosis. Growth suppression can be restored by the overexpression of DNMT1 in PDAC cells. Furthermore, we found n-BP-mediated DNMT1 suppression influenced the protein stability rather than changing the RNA expression. Through microarray studies, we found that the patched domain contained 4 (PTCHD4) is the potential downstream gene of DNMT1. Following silencing of PTCHD4 expression by siRNA, n-BP decreased tumor growth inhibition. Finally, in vivo, two animal models were used to evaluate the efficacy and survival after n-BP treatment by interstitial control release polymer delivery. The results show that n-BP could effectively inhibit PDAC tumor volume growth and extend animal survival. In summary, n-BP may inhibit the growth of human PDAC cells though reducing DNMT1 and increasing the expression of PTCHD4 both in vitro and in vivo.

Summary Introduction Hypospadias is one of the most common congenital anomalies of the male genitalia. Sonic hedgehog homologue (SHH) signalling pathway is believed to be involved in the development of the male genital system. Objective In this clinical prospective study, the role of the SHH pathway in hypospadias aetiology was investigated. Study design In this study, 200 healthy children (boys without hypospadias, control group), 118 patients (boys with distal hypospadias) and 82 patients (boys with proximal hypospadias) of age 0–16 years were included. The expression of the genes suppressor of fused protein (SUFU), SHH, protein patched homologue (PTCH; PTCH1 and PTCH2), glioma-associated oncogene homologue (GLI; GLI1, GLI2, GLI3 and GLI4), smoothened, frizzled-class receptor (SMO) and serine/threonine-protein kinase 36 (STK36) that are involved in SHH pathway were investigated. Furthermore, polymorphism analyses of GLI2, SHH and PTCH1 genes were performed. The history of hypospadias in the first and second-degree relatives of the patients in boys with distal hypospadias and boys with proximal hypospadias was inquired. Results Ten patients in the boys with distal hypospadias and twenty patients in the boys with proximal hypospadias had a history of hypospadias in first or second-degree relatives (p 0.05). Discussion SHH expression is required for the growth and differentiation of the genital bulge. Developmental defects in the external genital organs were demonstrated in mice with SHH deletion. It has been demonstrated that SHH mainly plays a role in the formation of sinusoid morphology of the penis. In the present study, although SHH and PTCH gene expressions were found to be decreased only in the penile tissues of proximal hypospadias, GLI2 gene expression was decreased in penile tissues of boys with both distal hypospadias and boys with proximal hypospadias. Conclusion Genes involved in the SHH pathway might play a role in the aetiology of hypospadias. Furthermore, there is a correlation between molecular defects in this pathway and severity of hypospadias. Summary Table . Fold-changes in mRNA expression in genes examined in penile tissue among cases with distal hypospadias and with proximal hypospadias. No Cases with distal hypospadias Cases with proximal hypospadias Gene name mRNA fold-change p-value∗∗∗ mRNA fold-change p-value 1 SUFU 1035 0.898 1028 0.922 2 SHH 1087 0.753 0451 0.044∗ 3 PTCH1 1087 0.754 0441 0.041∗ 4 PTCH2 1035 0.899 1035 0.901 5 GLI1 1157 0.586 1133 0.639 6 GLI2 0,330 0021∗ 0,302 0018∗ 7 GLI3 1181 0.537 1117 0.676 8 GLI4 0.818 0457 0.877 0609 9 SMO 0.920 0746 0.986 0948 10 STK36 1214 0.475 1275 0.378 p calculated as One way anowa test. ∗p

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.

Bone elongation is driven by chondrocyte proliferation and hypertrophy in the growth plate. Both processes are modulated by multiple signaling pathways including the Indian Hedgehog (IHH) signaling pathway. Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders characterized by accumulation of glycosaminoglycans (GAGs) in multiple tissues and organs, leading to a range of clinical symptoms including bone shortening through mechanisms that are not fully understood. Using MPS VII mice, we previously observed a reduction in the number of proliferating and hypertrophic chondrocytes and a reduced gene expression of Ihh in the tibial growth plate. We further demonstrate here that IHH secretion by MPS VII chondrocytes was reduced both in vitro and in vivo. While normal chondrocytes showed no response to exogenous IHH, proliferation of MPS VII chondrocytes was stimulated in response to exogenous IHH in vitro. This was accompanied by an elevated gene expression of patched receptor (Ptch1). The results from this study suggested that reduced proliferation in MPS VII growth plate may be partially due to dysfunction of the IHH signaling pathway., Highlights • Production of IHH by MPS VII mouse chondrocytes is reduced. • Distribution of IHH is altered across the growth plate in MPS VII mice. • MPS VII chondrocytes are responsive to the addition of exogenous IHH.

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.

Nevoid basal cell carcinoma syndrome (NBCCS), also known as Gorlin syndrome, is inherited in an autosomal dominant manner and is characterized by a combination of developmental abnormalities and a predisposition to tumor formation. Hedgehog receptor Patched 1 (PTCH1) has been identified as the mutated gene in NBCCS. We identified the PTCH1_c.3298_3299insAAG_p.1099_1100insE mutation in the transmembrane region, which comprises a sterol transporter whose abnormal function is reportedly related to pathogenicity.

SummaryThe Sonic hedgehog (Shh) pathway controls embryonic development and tissue homeostasis after birth. Long-lasting questions about this pathway are how dual-lipidated, firmly plasma membrane-associated Shh ligand is released from producing cells to signal to distant target cells, and how the resistance-nodulation-division transporter Dispatched (Disp) regulates this process. Here we show that Disp inactivation in Shh expressing cells specifically impairs proteolytic Shh release from its lipidated terminal peptides, a process called ectodomain shedding. Shh shedding from Disp-deficient cells was restored by pharmacological membrane cholesterol extraction and by overexpressed transgenic Disp or structurally related Patched (Ptc, a putative cholesterol transporter). These data suggest that Disp regulates physiological Shh function via controlled cell surface shedding and that molecular mechanisms shared by Disp and Ptc exercise such sheddase control.

A prototypic pediatric cancer that frequently shows activation of RAS signaling is embryonal rhabdomyosarcoma (ERMS). ERMS also show aberrant Hedgehog (HH)/GLI signaling activity and can be driven by germline mutations in this pathway. We show, that in ERMS cell lines derived from sporadic tumors i.e. from tumors not caused by an inherited genetic variant, HH/GLI signaling plays a subordinate role, because oncogenic mutations in HRAS, KRAS, or NRAS (collectively named oncRAS) inhibit the main HH target GLI1 via the MEK/ERK-axis, but simultaneously increase proliferation and tumorigenicity. oncRAS also modulate expression of stem cell markers in an isoform- and context-dependent manner. In Hh-driven murine ERMS that are caused by a Patched mutation, oncHRAS and mainly oncKRAS accelerate tumor development, whereas oncNRAS induces a more differentiated phenotype. These features occur when the oncRAS mutations are induced at the ERMS precursor stage, but not when induced in already established tumors. Moreover, in contrast to what is seen in human cell lines, oncRAS mutations do not alter Hh signaling activity and marginally affect expression of stem cell markers. Together, all three oncRAS mutations seem to be advantageous for ERMS cell lines despite inhibition of HH signaling and isoform-specific modulation of stem cell markers. In contrast, oncRAS mutations do not inhibit Hh-signaling in Hh-driven ERMS. In this model, oncRAS mutations seem to be advantageous for specific ERMS populations that occur within a specific time window during ERMS development. In addition, this window may be different for individual oncRAS isoforms, at least in the mouse.

Two distinct mechanisms for primordial germ cell (PGC) specification are observed within Bilatera: early determination by maternal factors or late induction by zygotic cues. Here we investigate the molecular basis for PGC specification inNematostella, a representative pre-bilaterian animal where PGCs arise as paired endomesodermal cell clusters during early development. We first present evidence that the putative PGCs delaminate from the endomesoderm upon feeding, migrate into the gonad primordia, and mature into germ cells. We then show that the PGC clusters arise at the interface betweenhedgehog1andpatcheddomains in the developing mesenteries and use gene knockdown, knockout and inhibitor experiments to demonstrate that Hh signaling is required for both PGC specification and general endomesodermal patterning. These results provide evidence that theNematostellagermline is specified by inductive signals rather than maternal factors, and support the existence of zygotically-induced PGCs in the eumetazoan common ancestor.

The hedgehog (HH) signaling pathway regulates normal cell growth and differentiation. As a consequence of improper control, aberrant HH signaling results in tumorigenesis and supports aggressive phenotypes of human cancers, such as neoplastic transformation, tumor progression, metastasis, and drug resistance. Canonical activation of HH signaling occurs through binding of HH ligands to the transmembrane receptor Patched 1 (PTCH1), which derepresses the transmembrane G protein-coupled receptor Smoothened (SMO). Consequently, the glioma-associated oncogene homolog 1 (GLI1) zinc-finger transcription factors, the terminal effectors of the HH pathway, are released from suppressor of fused (SUFU)-mediated cytoplasmic sequestration, permitting nuclear translocation and activation of target genes. Aberrant activation of this pathway has been implicated in several cancer types, including medulloblastoma, rhabdomyosarcoma, basal cell carcinoma, glioblastoma, and cancers of lung, colon, stomach, pancreas, ovarian, and breast. Therefore, several components of the HH pathway are under investigation for targeted cancer therapy, particularly GLI1 and SMO. GLI1 transcripts are reported to undergo alternative splicing to produce truncated variants: loss-of-function GLI1ΔN and gain-of-function truncated GLI1 (tGLI1). This review covers the biochemical steps necessary for propagation of the HH activating signal and the involvement of aberrant HH signaling in human cancers, with a highlight on the tumor-specific gain-of-function tGLI1 isoform.

Acquisition of metastatic potential by cancer cells is related to cancer stemness and anchorage-independent growth. The onset and progression of cancer are known to involve Hedgehog (HH) signaling that is activated by the binding of HH to the Patched 1 (PTCH1) receptor. However, the functions and mechanisms of action of PTCH1 in the context of bone metastasis remain to be elucidated. In this study, lentivirally-delivered shRNA was used to deplete PTCH1 levels, which resulted in the inhibition of spherical colony formation by the human non-small cell lung cancer (NSCLC) cell line; this suggested that PTCH1 promotes anchorage-independent growth. Concordantly, knockdown of PTCH1 resulted in significantly reduced migration and invasion of NSCLC cells; this was accompanied by the downregulation of MMP7 and SOX2. PTCH1 knockdown resulted in decreased bone destruction and osteoclastogenesis in a mouse bone metastasis model. These results indicate that PTCH1 may be an important regulator of bone invasion, and strongly suggest that knockdown of PTCH1 may decrease the anchorage-independent growth and metastatic potential of NSCLC.

The aberrant activation of hedgehog (Hh) signaling pathway is closely related to human diseases. The upstream protein, N-terminal product of sonic hedgehog (ShhN) is overexpressed in many cancers and considered as a promising antitumor target. Inhibitors that bind to ShhN and break its interaction with the 12-transmembrane glycoprotein patched (Ptch) protein are highly wanted to tune down the abnormal Hh pathway activation. However, research of ShhN inhibitors remains lacking. In this paper, we computationally screened potential inhibitors against the ShhN-Ptch interaction interface, and tested their activities by experimental studies. Seven compounds (1-7) with diverse scaffolds, showed inhibition in cellular assays and directly bound to ShhN in vitro. The compounds were verified to modulate the Hh pathway activity. Furthermore, we studied the structure-activity relationship of the pyrimidine (7) derivatives and identified a potent compound (7_3d3) with IC50 of 0.4 ± 0.1 μM in cellular assays. These small molecule inhibitors of ShhN provide novel chemical probes for future investigations of Hh signaling.

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.