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Sonic hedgehog (Shh) signaling regulates embryonic morphogenesis utilizing the primary cilium, the cell's antenna, which acts as a signaling hub. Fuz, an effector of planar cell polarity signaling, regulates Shh signaling by facilitating cilia formation, and the G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling. The range of phenotypic malformations observed in mice bearing mutations in either of the genes encoding these proteins is similar; however, their functional relationship has not been previously explored. This study identified the genetic and biochemical linkage between Fuz and Gpr161 in mouse neural tube development. Fuz was found to be genetically epistatic to Gpr161 with respect to regulation of Shh signaling in mouse neural tube development. The Fuz protein biochemically interacts with Gpr161, and Fuz regulates Gpr161-mediated ciliary localization, a process that might utilize β-arrestin 2. Our study characterizes a previously unappreciated Gpr161-Fuz axis that regulates Shh signaling during mouse neural tube development. [ABSTRACT FROM AUTHOR]

AIM: To investigate the effects of Sonic hedgehog (Shh) gene-modified bone marrow mesenchymal stem cells (MSCs) on graft-induced retinal gliosis and retinal ganglion cells (RGCs) survival in diabetic mice. METHODS: Bone marrow-derived MSCs were genetically modified with the Shh gene to generate a stably transfected cell line of Shh-modified MSCs (MSC-Shh). Intravitreal injections of MSC-Shh and green fluorescent protein-modified MSCs (MSC-Gfp; control) were administered in diabetic mice. After 4wk, the effects of MSC-Shh on retinal gliosis were evaluated using fundus photography, and markers of gliosis were examined by immunofluorescence and Western blotting. The neurotrophic factors expression and RGCs survival in the host retina were evaluated using Western blotting and immunofluorescence. The mechanisms underlying the effects of MSC-Shh was investigated. RESULTS: A significant reduction of proliferative vitreoretinopathy (PVR) was observed after intravitreal injection of MSC-Shh compared to MSC-Gfp. Significant downregulation of glial fibrillary acidic protein (GFAP) was demonstrated in the host retina after MSC-Shh administration compared to MSC-Gfp. The extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase B (AKT) and phosphatidylin-ositol-3-kinase (PI3K) pathways were significantly downregulated after MSC-Shh administration compared to MSC-Gfp. Brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) levels were significantly increased in the host retina, and RGCs loss was significantly prevented after MSC-Shh administration. CONCLUSION: MSC-Shh administration reduces graft-induced reactive gliosis following intravitreal injection in diabetic mice. The ERK1/2, AKT and PI3K pathways are involved in this process. MSC-Shh also increases the levels of neurotrophic factors in the host retina and promoted RGCs survival in diabetic mice.

• Pancreatic cancer survival is limited, and 'tumor priming' strategies that increase tumor permeability and perfusion hold promise to increase delivery of therapeutic antibodies (mAb) and potentially enhance efficacy. • Tumor penetration of mAb is key to therapeutic response; this research developed methods to visualize and quantify mAb intratumor distribution in the presence and absence of tumor priming pretreatments in patient-derived xenograft models that recapitulate the desmoplastic tumor drug delivery barrier. • An integrated PK/PD model was developed to link PK and temporal PD biomarkers of the tumor priming drug (SMO-inhibitor Sonidegib) as it increased tumor permeability, and to capture plasma- and intra-tumor distribution of cetuximab, as well as its receptor occupancy, yielding insights into factors that determine mAb tumor penetration, distribution, and efficacy in primed- and un-primed tumors. • Simulations with the integrated PK/PD were implemented to test effects of tumor physiological parameters, mAb properties, dose, and treatment scheduling, and yielded testable hypotheses for increasing mAb distribution within tumors. Therapeutic antibodies have shown little efficacy in the treatment of pancreatic ductal adenocarcinomas (PDAC). Tumor desmoplasia, hypovascularity, and poor perfusion result in insufficient tumor cell exposure, contributing to treatment failure. Smoothened inhibitors of hedgehog signaling (sHHi) increase PDAC tumor permeability, perfusion, and drug delivery, and provide a tool to develop a quantitative, mechanistic understanding as to how the temporal dynamics of tumor priming can impact intratumor distribution of monoclonal antibodies (mAb). A linked pharmacokinetic (PK)/pharmacodynamic (PD) model was developed to integrate the plasma and tumor PK of a sHHi priming agent with its effects upon downstream stromal biomarkers Gli1 , hyaluronic acid, and interstitial fluid pressure in PDAC patient-derived xenograft (PDX) tumors. In parallel, in situ tumor concentrations of cetuximab (CTX: anti-epidermal growth factor receptor; EGFR) were quantified as a marker for tumor delivery of mAb or antibody-drug conjugates. A minimal, physiologically-based pharmacokinetic (mPBPK) model was constructed to link sHHi effects upon mechanistic effectors of tumor barrier compromise with the intratumor distribution of CTX, and CTX occupancy of EGFR in tumors. Integration of the mPBPK model of mAb deposition and intratumor distribution with the PK/PD model of tumor responses to priming not only identified physiological parameters that are critical for tumor antibody distribution, but also provides insight into dosing regimens that could achieve maximal tumor disposition of therapeutic antibodies under conditions of transient PDAC tumor permeability barrier compromise that mechanistically-diverse tumor priming strategies may achieve. [ABSTRACT FROM AUTHOR]

The intraflagellar transport (IFT) machinery is essential for cilia assembly, maintenance, and trans-localization of signaling proteins. The IFT machinery consists of two large multiprotein complexes, one of which is the IFT-B. TTC30A and TTC30B are integral components of this complex and were previously shown to have redundant functions in the context of IFT, preventing the disruption of IFT-B and, thus, having a severe ciliogenesis defect upon loss of one paralog. In this study, we re-analyzed the paralog-specific protein complexes and discovered a potential involvement of TTC30A or TTC30B in ciliary signaling. Specifically, we investigated a TTC30A-specific interaction with protein kinase A catalytic subunit α, a negative regulator of Sonic hedgehog (Shh) signaling. Defects in this ciliary signaling pathway are often correlated to synpolydactyly, which, intriguingly, is also linked to a rare TTC30 variant. For an in-depth analysis of this unique interaction and the influence on Shh, TTC30A or B single- and double-knockout hTERT-RPE1 were employed, as well as rescue cells harboring wildtype TTC30 or the corresponding mutation. We could show that mutant TTC30A inhibits the ciliary localization of Smoothened. This observed effect is independent of Patched1 but associated with a distinct phosphorylated PKA substrate accumulation upon treatment with forskolin. This rather prominent phenotype was attenuated in mutant TTC30B. Mass spectrometry analysis of wildtype versus mutated TTC30A or TTC30B uncovered differences in protein complex patterns and identified an impaired TTC30A–IFT57 interaction as the possible link leading to synpolydactyly. We could observe no impact on cilia assembly, leading to the hypothesis that a slight decrease in IFT-B binding can be compensated, but mild phenotypes, like synpolydactyly, can be induced by subtle signaling changes. Our systematic approach revealed the paralog-specific influence of TTC30A KO and mutated TTC30A on the activity of PRKACA and the uptake of Smoothened into the cilium, resulting in a downregulation of Shh. This downregulation, combined with interactome alterations, suggests a potential mechanism of how mutant TTC30A is linked to synpolydactyly.

Numerous intrinsic factors regulate mesenchymal progenitor commitment to a specific cell fate, such as osteogenic or adipogenic lineages. Identification and modulation of novel intrinsic regulatory factors represent an opportunity to harness the regenerative potential of mesenchymal progenitors. In the present study, the transcription factor (TF) ZIC1 was identified to be differentially expressed among adipose compared with skeletal-derived mesenchymal progenitor cells. We observed that ZIC1 overexpression in human mesenchymal progenitors promotes osteogenesis and prevents adipogenesis. ZIC1 knockdown demonstrated the converse effects on cell differentiation. ZIC1 misexpression was associated with altered Hedgehog signaling, and the Hedgehog antagonist cyclopamine reversed the osteo/adipogenic differentiation alterations associated with ZIC1 overexpression. Finally, human mesenchymal progenitor cells with or without ZIC1 overexpression were implanted in an ossicle assay in NOD-SCID gamma mice. ZIC1 overexpression led to significantly increased ossicle formation in comparison to the control, as assessed by radiographic and histologic measures. Together, these data suggest that ZIC1 represents a TF at the center of osteo/adipogenic cell fate determinations—findings that have relevance in the fields of stem cell biology and therapeutic regenerative medicine. [ABSTRACT FROM AUTHOR]

Anxiety is a prevalent and disabling psychiatric disorder. Mitochondrial dysfunction due to the high-fat diet (HFD) was regarded as a risk factor in the pathogenesis of anxiety. The Sonic hedgehog (SHH) pathway was known to improve mitochondrial dysfunction through antioxidant and anti-apoptotic effects on some neurological diseases. Nonetheless, its effect on anxiety has not been well studied. In this study, we aimed to explore whether SHH signaling pathway plays a protective role in anxiety by regulating mitochondrial homeostasis. SAG, a typical SHH signaling agonist, was administered intraperitoneally in HFD-fed mice. HFD-induced anxiety-like behavior in mice was confirmed using the open field and elevated plus maze tests. Immunofluorescence staining and Western blotting assays showed that the SHH signaling was downregulated in the prefrontal cortex neurons from HFD-fed mice. Electron microscopy results showed the mitochondria in the prefrontal cortex of HFD-fed mice were fragmented, which appeared small and spherical, and the area, perimeter and circularity of mitochondria were decreased. Mitofusin2 (Mfn2) and dynamin-related protein 1 (Drp1) were the key proteins involved in mitochondrial division and fusion. SAG treatment could rectify the imbalanced expression of Mfn2 and Drp1 in the prefrontal cortex of the HFD-fed mice, and alleviate the mitochondrial fragmentation. Furthermore, SAG decreased anxiety-like behavior in the HFD-fed mice. These findings suggested that SHH signal was neuroprotective in obesity and SAG relieved anxiety-like behavior through reducing mitochondrial fragmentation. • SHH signaling pathway promotes the mitochondrial dysfunction in HFD mice. • SAG relieved anxiety-like behavior by the imbalance between Drp1 and Mfn2. • Our findings emphasize the role of SAG in neuroprotection. [ABSTRACT FROM AUTHOR]

Abstract Sonic Hedgehog (Shh) signaling cascade is one of the complex signaling pathways that control the accurately organized developmental processes in multicellular organisms. This pathway has fundamental roles in the tumor formation and induction of resistance to conventional therapies. Numerous non-coding RNAs (ncRNAs) have been found to interact with Shh pathway to induce several pathogenic processes, including malignant and non-malignant disorders. Many of the Shh-interacting ncRNAs are oncogenes whose expressions have been increased in diverse malignancies. A number of Shh-targeting miRNAs such as miR-26a, miR-1471, miR-129-5p, miR-361-3p, miR-26b-5p and miR-361-3p have been found to be down-regulated in tumor tissues. In addition to malignant conditions, Shh-interacting ncRNAs can affect tissue regeneration and development of neurodegenerative disorders. XIST, LOC101930370, lncRNA-Hh, circBCBM1, SNHG6, LINC‐PINT, TUG1 and LINC01426 are among long non-coding RNAs/circular RNAs that interact with Shh pathway. Moreover, miR-424, miR-26a, miR-1471, miR-125a, miR-210, miR-130a-5p, miR-199b, miR-155, let-7, miR-30c, miR-326, miR-26b-5p, miR-9, miR-132, miR-146a and miR-425-5p are among Shh-interacting miRNAs. The current review summarizes the interactions between ncRNAs and Shh in these contexts.

Simple Summary: Oocyte quality is acquired during ovarian folliculogenesis including oocyte growth and maturation, and plays a key role in subsequent embryo development. However, oocytes matured in vitro do not fully support the ability to develop into the blastocyst stage due to inadequate in vitro maturation (IVM) systems which do not sufficiently mimic the in vivo microenvironment. The sonic hedgehog (SHH) signaling pathway is important for ovarian folliculogenesis and oocyte maturation. Furthermore, our previous study demonstrated that low-quality porcine cumulus–oocyte complexes (COCs) exhibit low developmental competence with a weak SHH signaling pathway. Therefore, the aim of the present study was to clarify the restorative effects of SHH protein supplementation during IVM on the developmental competence of low-quality porcine COCs, as assessed by brilliant cresyl blue staining. The results showed that the low developmental competence of low-quality porcine COCs can be improved by supplementation with the SHH protein. This indicates that an active SHH signaling pathway is required for the acquisition of developmental competence in porcine COCs. The sonic hedgehog (SHH) pathway is an important signaling pathway for mammalian ovarian folliculogenesis and oocyte maturation. A previous study demonstrated that low-quality porcine cumulus–oocyte complexes (COCs) have low developmental competence, with lower SHH signaling protein expression before and after in vitro maturation (IVM) than high-quality COCs. However, there is no reported evidence on the restorative effects of SHH protein supplementation during the IVM of low-quality porcine COCs. Therefore, this study investigated the effects of SHH protein supplementation on the IVM of low-quality porcine COCs, as assessed by brilliant cresyl blue (BCB) staining. To examine this, we designed four groups: (i) BCB− (low-quality), (ii) BCB− + SHH, (iii) BCB+ (high-quality), and (iv) BCB+ + SHH. While the supplementation of SHH protein with high-quality COCs had no effect, supplementation with low-quality COCs significantly improved cumulus cell expansion, metaphase II rate, and subsequent embryo development following parthenogenetic activation. Our results provide the first evidence that the low developmental competence of low-quality porcine COCs can be improved by supplementation with the SHH protein. These results indicate that an active SHH signaling pathway is required for the acquisition of developmental competence in porcine COCs. [ABSTRACT FROM AUTHOR]

Activated microglia act as a double-edged sword for stroke. In the acute phase of stroke, activated microglia might deteriorate neurological function. Therefore, it is of great clinical transforming potential to explore drugs or methods that can inhibit abnormal activation of microglia in the acute phase of stroke to improve neurological function after stroke. Resveratrol has a potential effect of regulating microglial activation and anti-inflammation. However, the molecular mechanism of resveratrol-inhibiting microglial activation has not been fully clarified. Smoothened (Smo) belongs to the Hedgehog (Hh) signaling pathway. Smo activation is the critical step that transmits the Hh signal across the primary cilia to the cytoplasm. Moreover, activated Smo can improve neurological function via regulating oxidative stress, inflammation, apoptosis, neurogenesis, oligodendrogenesis, axonal remodeling, and so on. More studies have indicated that resveratrol can activate Smo. However, it is currently unknown whether resveratrol inhibits microglial activation via Smo. Therefore, in this study, N9 microglia in vitro and mice in vivo were used to investigate whether resveratrol inhibited microglial activation after oxygen-glucose deprivation/reoxygenation (OGD/R) or middle cerebral artery occlusion/reperfusion (MCAO/R) injury and improved functional outcome via triggering translocation of Smo in primary cilia. We definitively found that microglia had primary cilia; resveratrol partially inhibited activation and inflammation of microglia, improved functional outcome after OGD/R and MCAO/R injury, and triggered translocation of Smo to primary cilia. On the contrary, Smo antagonist cyclopamine canceled the above effects of resveratrol. The study suggested that Smo receptor might be a therapeutic target of resveratrol for contributing to inhibit microglial activation in the acute phase of stroke. [ABSTRACT FROM AUTHOR]

Patients carrying pathogenic gene variants encoding factors linked to the sonic hedgehog (SHH) pathway suffer from severe congenital brain malformations including holoprosencephaly (HPE). A poorly understood feature of these common anomalies is the highly variable penetrance, even amongst family members, carrying the same mutation. Modifier genes–genetic variants that can affect the phenotypic outcome of the primary disease-causing gene–contribute to this variability within pedigrees. Modifier genes can confer resilience or susceptibility to a disease, but are difficult to identify in humans. Studying the complex genetic interactions in mouse models of human congenital disorders can be instrumental in the identification of genes, that powerfully modulate SHH signaling pathway capacity and ultimately the penetrance of genetic disturbances. Understanding the underlying complex molecular mechanisms of disease aetiology and can support directing future genetic linkage studies in humans. [ABSTRACT FROM AUTHOR]

Nonsyndromic cleft lip with palate (nsCLP) is a common congenital malformation; however, early prenatal diagnosis is challenging and pathogenesis remains unclear. The purpose of this study was to determine the diagnostic potential of miRNAs in plasma‐derived exosomes and whole plasma of pregnant women to identify nsCLP and an underlying mechanism. Combined RNA sequencing analysis was performed on samples from plasma exosomes and whole plasma of pregnant women carrying normal fetuses or fetuses with nsCLP in an ongoing birth cohort, in addition to lip samples from nsCLP fetuses and healthy controls. Eight let‐7 cluster miRNAs (hsa‐let‐7a‐3p, hsa‐let‐7a‐5p, hsa‐let‐7c‐5p, hsa‐let‐7d‐3p, hsa‐let‐7d‐5p, hsa‐let‐7e‐5p, hsa‐let‐7f‐5p, and hsa‐miR‐98‐5p) in plasma exosomes from pregnant women provided higher sensitivity/specificity for diagnosing fetal nsCLP than those in plasma. Area under the receiver operating characteristic curve value of the eight miRNAs from plasma exosomes was 0.992. Among them, hsa‐let‐7a‐3p showed better diagnostic capability and was downregulated in nsCLP fetal lip tissues. Upstream and downstream target genes of hsa‐let‐7a‐3p were screened and confirmed. Our work highlights the potential clinical application value of let‐7 clusters in predicting nsCLP and associates as a new regulatory axis (EN2–LIN28A–hsa‐let‐7a‐3p–HHIP–GLI2) with human nsCLP pathogenesis. [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSCs) represent a small subset of slowly dividing cells with tumor-initiating ability. They can self-renew and differentiate into all the distinct cell populations within a tumor. CSCs are naturally resistant to chemotherapy or radiotherapy. CSCs, thus, can repopulate a tumor after therapy and are responsible for recurrence of disease. Stemness manifests itself through, among other things, the expression of stem cell markers, the ability to induce sphere formation and tumor growth in vivo, and resistance to chemotherapeutics and irradiation. Stemness is maintained by keeping levels of reactive oxygen species (ROS) low, which is achieved by enhanced activity of antioxidant pathways. Here, cellular sources of ROS, antioxidant pathways employed by CSCs, and underlying mechanisms to overcome resistance are discussed. [ABSTRACT FROM AUTHOR]

In vitro maturation (IVM) of oocytes is the procedure where the immature oocytes are cultivated in a laboratory until they are mature. Since IVM oocytes generally have low developmental competence as compared to those matured in vivo, development of an optimal IVM culture system by fine-tuning culture conditions is crucial to maintain high quality. In-depth knowledge and a deep understanding of the in vivo physiology of oocyte maturation are pre-requisites to accomplish this. Within ovarian follicles, various signaling pathways that drive oocyte development and maturation regulate interaction between oocytes and surrounding somatic cells. This review discusses the sonic hedgehog (SHH) signaling pathway, which has been demonstrated to be intimately involved in folliculogenesis and oocyte maturation. Advances in elucidating the role of the SHH signaling pathway in oocyte maturation will aid attempts to improve the current inferior in vitro oocyte maturation system.

Sonic Hedgehog (Shh) signaling cascade is one of the complex signaling pathways that control the accurately organized developmental processes in multicellular organisms. This pathway has fundamental roles in the tumor formation and induction of resistance to conventional therapies. Numerous non-coding RNAs (ncRNAs) have been found to interact with Shh pathway to induce several pathogenic processes, including malignant and non-malignant disorders. Many of the Shh-interacting ncRNAs are oncogenes whose expressions have been increased in diverse malignancies. A number of Shh-targeting miRNAs such as miR-26a, miR-1471, miR-129-5p, miR-361-3p, miR-26b-5p and miR-361-3p have been found to be down-regulated in tumor tissues. In addition to malignant conditions, Shh-interacting ncRNAs can affect tissue regeneration and development of neurodegenerative disorders. XIST, LOC101930370, lncRNA-Hh, circBCBM1, SNHG6, LINC‐PINT, TUG1 and LINC01426 are among long non-coding RNAs/circular RNAs that interact with Shh pathway. Moreover, miR-424, miR-26a, miR-1471, miR-125a, miR-210, miR-130a-5p, miR-199b, miR-155, let-7, miR-30c, miR-326, miR-26b-5p, miR-9, miR-132, miR-146a and miR-425-5p are among Shh-interacting miRNAs. The current review summarizes the interactions between ncRNAs and Shh in these contexts. [ABSTRACT FROM AUTHOR]

Liver cancer has the second highest worldwide cancer mortality rate and has limited therapeutic options. We analyzed 363 hepatocellular carcinoma (HCC) cases by whole-exome sequencing and DNA copy number analyses, and we analyzed 196 HCC cases by DNA methylation, RNA, miRNA, and proteomic expression also. DNA sequencing and mutation analysis identified significantly mutated genes, including LZTR1, EEF1A1, SF3B1, and SMARCA4. Significant alterations by mutation or downregulation by hypermethylation in genes likely to result in HCC metabolic reprogramming (ALB, APOB, and CPS1) were observed. Integrative molecular HCC subtyping incorporating unsupervised clustering of five data platforms identified three subtypes, one of which was associated with poorer prognosis in three HCC cohorts. Integrated analyses enabled development of a p53 target gene expression signature correlating with poor survival. Potential therapeutic targets for which inhibitors exist include WNT signaling, MDM4, MET, VEGFA, MCL1, IDH1, TERT, and immune checkpoint proteins CTLA-4, PD-1, and PD-L1.

Background Clinically, myocardial fibrosis is one of the most common complications caused by chronic kidney disease (CKD). However, the potential mechanisms of CKD‐induced myocardial fibrosis have not been clarified. Methods and Results In our in vivo study, a rat model of CKD with 5/6 nephrectomy was established. The CKD model was treated with the glioma 1 (Gli‐1) inhibitor GANT‐61, and myocardial fibrosis and serum intact fibroblast growth factor 23 levels were assessed 16 weeks after nephrectomy. Finally, we found that Gli‐1 and Smoothened in the Sonic Hedgehog (Shh) signaling pathway were activated and that collagen‐1 and collagen‐3, which constitute the fibrotic index, were expressed in CKD myocardial tissue. After administering the Gli‐1 inhibitor GANT‐61, the degree of myocardial fibrosis was reduced, and Gli‐1 expression was also inhibited. We also measured blood pressure, cardiac biomarkers, and other indicators in rats and performed hematoxylin‐eosin staining of myocardial tissue. Furthermore, in vitro studies showed that intact fibroblast growth factor 23 promoted cardiac fibroblast proliferation and transdifferentiation into myofibroblasts by activating the Shh signaling pathway, thereby promoting cardiac fibrosis, as manifested by increased expression of the Shh, Patch 1, and Gli‐1 mRNAs and Shh, Smoothened, and Gli‐1 proteins in the Shh signaling pathway. The protein and mRNA levels of other fibrosis indicators, such as α‐smooth muscle actin, which are also markers of transdifferentiation, collagen‐1, and collagen‐3, were increased. Conclusions On the basis of these results, intact fibroblast growth factor 23 promotes CKD‐induced myocardial fibrosis by activating the Shh signaling pathway.

Sonic hedgehog (SHH) medulloblastoma originates from the cerebellar granule neuron progenitor (CGNP) lineage, which depends on Hedgehog signaling for its perinatal expansion. Whereas SHH tumors exhibit overall deregulation of this pathway, they also show patient age-specific aberrations. To investigate whether the developmental stage of the CGNP can account for these age-specific lesions, we analyzed developing murine CGNP transcriptomes and observed highly dynamic gene expression as a function of age. Cross-species comparison with human SHH medulloblastoma showed partial maintenance of these expression patterns, and highlighted low primary cilium expression as hallmark of infant medulloblastoma and early embryonic CGNPs. This coincided with reduced responsiveness to upstream SHH pathway component Smoothened, whereas sensitivity to downstream components SUFU and GLI family proteins was retained. Together, these findings can explain the preference for SUFU mutations in infant medulloblastoma and suggest that drugs targeting the downstream SHH pathway will be most appropriate for infant patients. [ABSTRACT FROM AUTHOR]

Autism is a multifactorial neurodevelopmental condition; it demonstrates some main characteristics, such as impaired social relationships and increased repetitive behavior. The initiation of autism spectrum disorder is mostly triggered during brain development by the deregulation of signaling pathways. Sonic hedgehog (SHH) signaling is one such mechanism that influences neurogenesis and neural processes during the development of the central nervous system. SMO-SHH signaling is also an important part of a broad variety of neurological processes, including neuronal cell differentiation, proliferation, and survival. Dysregulation of SMO-SHH signaling leads to many physiological changes that lead to neurological disorders such as ASD and contribute to cognitive decline. The aberrant downregulation of SMO-SHH signals contributes to the proteolytic cleavage of GLI (glioma-associated homolog) into GLI3 (repressor), which increases oxidative stress, neuronal excitotoxicity, neuroinflammation, and apoptosis by suppressing target gene expression. We outlined in this review that SMO-SHH deregulation plays a crucial role in the pathogenesis of autism and addresses the current status of SMO-SHH pathway modulators. Additionally, a greater understanding of the SHH signaling pathway is an effort to improve successful treatment for autism and other neurological disorders. [ABSTRACT FROM AUTHOR]

Abstract Background The regulator of cullins-1 (ROC1) is an essential subunit in the cullin-RING ligase (CRL) protein complex and has been shown to be critical in bladder cancer cell survival and progression. This study aimed to explore the molecular mechanism of ROC1 action in the malignant progression of bladder cancer. Methods This study utilized ex vivo, in vitro, and in vivo nude mouse experiments to assess the underlying mechanisms of ROC1 in bladder cancer cells. The expression of the components of the sonic hedgehog (SHH) pathway was determined by western blot analysis. ROC1 expression in human tumors was evaluated by immunohistochemistry. Results ROC1 overexpression promoted the growth of bladder cancer cells, whereas knockdown of ROC1 expression had the opposite effect in bladder cancer cells. Mechanistically, ROC1 was able to target suppressor of fused homolog (SUFU) for ubiquitin-dependent degradation, allowing Gli2 release from the SUFU complex to activate the SHH pathway. Furthermore, knockdown of SUFU expression partially rescued the ROC1 knockdown-suppressed SHH activity as well as cancer cell growth inhibition. In ex vivo experiments, tissue microarray analysis of human bladder cancer specimens revealed a positive association of ROC1 expression with the SHH pathway activity. Conclusion This study demonstrated that dysregulation of the ROC1–SUFU–GLI2 axis plays an important role in bladder cancer progression and that targeting ROC1 expression is warranted in further investigations as a novel strategy for the future control of bladder cancer.

The discovery by Altman and coworkers of adult-born microneurons in the olfactory bulb and dentate gyrus has triggered a long stream of studies and many attempts to harness adult neurogenesis, promote regeneration after injury, and contrast cognitive decline in the elderly. Likewise, the discovery of postnatal neurogenesis in the cerebellum has provided the framework for many subsequent molecular studies, including investigations of developmental processes and the assessment of GC progenitor (GCP) clonal expansion in the context of human disease. Here, I will briefly discuss some of the discoveries made in the field of cerebellar development over the years building upon the findings of Altman and his colleagues, touching upon signaling pathways that regulate granule cell neurogenesis and their involvement in developmental and neoplastic disorders of the cerebellum. [ABSTRACT FROM AUTHOR]

The sonic hedgehog (SHH) pathway is an important signaling pathway for mammalian ovarian folliculogenesis and oocyte maturation. A previous study demonstrated that low-quality porcine cumulus–oocyte complexes (COCs) have low developmental competence, with lower SHH signaling protein expression before and after in vitro maturation (IVM) than high-quality COCs. However, there is no reported evidence on the restorative effects of SHH protein supplementation during the IVM of low-quality porcine COCs. Therefore, this study investigated the effects of SHH protein supplementation on the IVM of low-quality porcine COCs, as assessed by brilliant cresyl blue (BCB) staining. To examine this, we designed four groups: (i) BCB− (low-quality), (ii) BCB− + SHH, (iii) BCB+ (high-quality), and (iv) BCB+ + SHH. While the supplementation of SHH protein with high-quality COCs had no effect, supplementation with low-quality COCs significantly improved cumulus cell expansion, metaphase II rate, and subsequent embryo development following parthenogenetic activation. Our results provide the first evidence that the low developmental competence of low-quality porcine COCs can be improved by supplementation with the SHH protein. These results indicate that an active SHH signaling pathway is required for the acquisition of developmental competence in porcine COCs.

Sonic hedgehog (Shh) signaling regulates multiple morphogenetic processes during embryonic neurogenesis and craniofacial skeletal development. Gpr161 is a known negative regulator of Shh signaling. Nullizygous Gpr161 mice are embryonic lethal, presenting with structural defects involving the neural tube and the craniofacies. However, the lineage specific role of Gpr161 in later embryonic development has not been thoroughly investigated. We studied the Wnt1-Cre lineage specific role of Gpr161 during mouse embryonic development. We observed three major gross morphological phenotypes in Gpr161 cKO (Gpr161 f/f; Wnt1-Cre) fetuses; protrusive tectum defect, encephalocele, and craniofacial skeletal defect. The overall midbrain tissues were expanded and cell proliferation in ventricular zones of midbrain was increased in Gpr161 cKO fetuses, suggesting that protrusive tectal defects in Gpr161 cKO are secondary to the increased proliferation of midbrain neural progenitor cells. Shh signaling activity as well as upstream Wnt signaling activity were increased in midbrain tissues of Gpr161 cKO fetuses. RNA sequencing further suggested that genes in the Shh, Wnt, Fgf and Notch signaling pathways were differentially regulated in the midbrain of Gpr161 cKO fetuses. Finally, we determined that cranial neural crest derived craniofacial bone formation was significantly inhibited in Gpr161 cKO fetuses, which partly explains the development of encephalocele. Our results suggest that Gpr161 plays a distinct role in midbrain development and in the formation of the craniofacial skeleton during mouse embryogenesis. [ABSTRACT FROM AUTHOR]

Stromal androgen-receptor (AR) action is essential for prostate development, morphogenesis and regeneration. However, mechanisms underlying how stromal AR maintains the cell niche in support of pubertal prostatic epithelial growth are unknown. Here, using advanced mouse genetic tools, we demonstrate that selective deletion of stromal AR expression in prepubescent Shh-responsive Gli1-expressing cells significantly impedes pubertal prostate epithelial growth and development. Single-cell transcriptomic analyses showed that AR loss in these prepubescent Gli1- expressing cells dysregulates androgen signaling-initiated stromalepithelial paracrine interactions, leading to growth retardation of pubertal prostate epithelia and significant development defects. Specifically, AR loss elevates Shh-signaling activation in both prostatic stromal and adjacent epithelial cells, directly inhibiting prostatic epithelial growth. Single-cell trajectory analyses further identified aberrant differentiation fates of prostatic epithelial cells directly altered by stromal AR deletion. In vivo recombination of AR-deficient stromal Gli1-lineage cells with wild-type prostatic epithelial cells failed to develop normal prostatic epithelia. These data demonstrate previously unidentified mechanisms underlying how stromal AR-signaling facilitates Shh-mediated cell niches in pubertal prostatic epithelial growth and development. [ABSTRACT FROM AUTHOR]

Objectives: Sonic hedgehog (SHH) signaling is important in bladder development. Mice with defective hedgehog signaling develop bladder anomalies. Clinically, urinary tract malformations are reported in human fetuses and infants with mutations of SHH and related signaling pathway genes. Information on the expression of SHH and associated signaling genes in normal human bladder development is fragmentary. This study determined the temporal and spatial expression patterns of SHH signaling pathway components in human fetal bladders by immunohistochemistry (IHC).Material and Methods: Twenty-four bladder specimens from 16 male and 8 female human fetuses aged 12- to 36-week (wk) were obtained from the First Affiliated Hospital of Xi'an Jiaotong University. The tissue slides were processed for IHC staining with SHH, Patched1 (PTC-1), Patched2 (PTC-2), Smoothened (SMO), GLI1 and proliferating cell nuclear antigen (PCNA). The expression levels of each gene were analyzed by semi-quantitative histological scoring system.Results: High intensity of SHH and SMO expression was detected in developing bladder urothelial cells, with no staining in lamina propria (LP), but with minimal expression of SMO in differentiating smooth muscle (SM) layers. The spatial distribution pattern of PTC1 and GLI1 was more complex with minimal expression in the LP layer, moderate expression in the SM layer, and high expression in the urothelium. PTC2 expression was mainly localized in the urothelium and LP, but no expression in the SM layer. All of the SHH signaling components were detected in fetal bladder tissues throughout the development, with expression peaks at 12- and 23-wk, coinciding with high cell proliferation as indicated by PCNA staining in the cell nuclei of urothelium and SM.Conclusions: The autocrine SHH signaling in the developing urothelium, and paracrine SHH signaling in the developing smooth muscle layer, mediated by SMO, PTC-1 and GLI1 were demonstrated during human bladder development. Expression of SHH signaling components peaked at 12-and 23-wk. The first expression peak at 12-wk may relate to urothelium growth, SM induction, and dilation of the bladder cavity. The second expression peaked at 23-wk may relate to urothelium and SM layer differentiation.

Sonic hedgehog (Shh) signaling regulates multiple morphogenetic processes during embryonic neurogenesis and craniofacial skeletal development. Gpr161 is a known negative regulator of Shh signaling. Nullizygous Gpr161 mice are embryonic lethal, presenting with structural defects involving the neural tube and the craniofacies. However, the lineage specific role of Gpr161 in later embryonic development has not been thoroughly investigated. We studied the Wnt1-Cre lineage specific role of Gpr161 during mouse embryonic development. We observed three major gross morphological phenotypes in Gpr161 cKO (Gpr161 f/f; Wnt1-Cre) fetuses; protrusive tectum defect, encephalocele, and craniofacial skeletal defect. The overall midbrain tissues were expanded and cell proliferation in ventricular zones of midbrain was increased in Gpr161 cKO fetuses, suggesting that protrusive tectal defects in Gpr161 cKO are secondary to the increased proliferation of midbrain neural progenitor cells. Shh signaling activity as well as upstream Wnt signaling activity were increased in midbrain tissues of Gpr161 cKO fetuses. RNA sequencing further suggested that genes in the Shh, Wnt, Fgf and Notch signaling pathways were differentially regulated in the midbrain of Gpr161 cKO fetuses. Finally, we determined that cranial neural crest derived craniofacial bone formation was significantly inhibited in Gpr161 cKO fetuses, which partly explains the development of encephalocele. Our results suggest that Gpr161 plays a distinct role in midbrain development and in the formation of the craniofacial skeleton during mouse embryogenesis.

Cancer stem cells (CSCs) represent a small subset of slowly dividing cells with tumor-initiating ability. They can self-renew and differentiate into all the distinct cell populations within a tumor. CSCs are naturally resistant to chemotherapy or radiotherapy. CSCs, thus, can repopulate a tumor after therapy and are responsible for recurrence of disease. Stemness manifests itself through, among other things, the expression of stem cell markers, the ability to induce sphere formation and tumor growth in vivo, and resistance to chemotherapeutics and irradiation. Stemness is maintained by keeping levels of reactive oxygen species (ROS) low, which is achieved by enhanced activity of antioxidant pathways. Here, cellular sources of ROS, antioxidant pathways employed by CSCs, and underlying mechanisms to overcome resistance are discussed.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive type of malignant tumor with a poor prognosis and high mortality. Aberrant activation of hedgehog signaling plays a crucial role in the maintenance and progression of PDA. Here, we report that the dietary bioflavonoid quercetin has therapeutic potential for PDA by targeting sonic hedgehog (SHH) signaling. The effects of quercetin on the proliferation, apoptosis, migration, and invasion of pancreatic cancer cells (PCCs) and tumor growth and metastasis in PDA xenograft mouse models were evaluated. Additionally, SHH signaling activity was determined. Quercetin significantly inhibited PCC proliferation by downregulating c-Myc expression. In addition, quercetin suppressed epithelial-mesenchymal transition (EMT) by reducing TGF-β1 level, which resulted in inhibition of PCC migration and invasion. Moreover, quercetin induced PCC apoptosis through mitochondrial and death receptor pathways. In nude mouse models, PDA growth and metastasis were reduced by quercetin treatment. Mechanically, quercetin exerts its therapeutic effects on PDA by decreasing SHH activity. Interestingly, quercetin-induced SHH inactivation is mainly dependent on Gli2, but not Gli1. Enhance SHH activity by recombinant Shh protein abolished the quercetin-mediated inhibition of PCC proliferation, migration, and invasion. Furthermore, Shh activated TGF-β1/Smad2/3 signaling and promoted EMT by inducing the expression of Zeb2 and Snail1 that eventually resulted in a partial reversal of quercetin-mediated inhibition of PCC migration and invasion. We conclude that quercetin inhibited the growth, migration, and invasion and induced apoptosis of PCCs by antagonizing SHH and TGF-β/Smad signaling pathways. Thus, quercetin may be a potential candidate for PDA treatment. [ABSTRACT FROM AUTHOR]

Background: Nevoid basal cell carcinoma syndrome (NBCCS) is a neurocutaneous disease, characterized by tumorigenesis and developmental anomalies due to aberrant sonic hedgehog (Shh) signaling. Patients with NBCCS typically appear calm and carefree, suggesting that a specific personality in these patients may be associated with an enhanced hedgehog pathway. Our study aimed to determine the personality type in these patients. Methods: We enrolled 14 mentally normal patients with genetically confirmed NBCCS (seven males and seven females; mean age = 25.2 years) and 20 controls (10 males and 10 females; mean age = 27.9 years). The patients were assessed with the Japanese version of the Temperament and Character Inventory, based on the seven‐dimensional model of temperament and character, and their clinical symptoms were evaluated. The amygdala volumes of six patients with NBCCS were measured using magnetic resonance imaging with image‐processing software. Results: Patients with NBCCS scored significantly lower on harm avoidance (0.89) than controls (1.00; P = 0.0084). Moreover, patients with NBCCS and developmental malformations such as rib anomalies, who may have experienced Shh signaling enhancement from the prenatal period, scored significantly lower on harm avoidance (0.80 [P = 0.0031]). The left amygdala volume was also significantly reduced in patients with NBCCS (P = 0.0426). Conclusions: Patients with NBCCS who experienced increased Shh signaling from the prenatal period showed significantly lower harm avoidance related to serotonin. The left amygdala volume was significantly reduced in these patients. Our results indicate that Shh signaling may influence the human personality. [ABSTRACT FROM AUTHOR]

Carbon tetrachloride (CCl4) exposure can induce hepatic ductular reactions. To date, however, the related mechanism remains largely unknown. Sonic hedgehog (Shh) and Yes‐associated protein (Yap) signaling are correlated with liver injury and regeneration. Herein, we investigated the role of Shh and Yap signaling in the fate of ductular reaction cells in CCl4‐treated livers and the possible mechanisms. Wild‐type and Shh‐EGFP‐Cre male mice were exposed to CCl4 (2 mL/kg), and then treated with or without the Shh signaling inhibitor Gant61. The level of liver injury, proliferation of ductular reaction cells, and expression levels of mRNA and protein related to the Shh and Yap signaling components were assessed. Results showed that CCl4 treatment induced liver injury and promoted activation and proliferation of ductular reaction cells. In addition, CCl4 induced the expression of Shh ligands in hepatocytes, accompanied by activation of Shh and Yap1 signaling in the liver. Furthermore, administration of Gant61 ameliorated liver regeneration, inhibited hepatic ductular reactions, and decreased Shh and Yap1 signaling activity. Thus, Shh‐Yap1 signaling appears to play an integral role in the proliferation of ductular reaction cells in CCl4‐induced liver injury. This study should improve our understanding of the mechanism of CCl4‐induced liver injury and ductular reactions and provide support for future investigations on liver disease therapy. [ABSTRACT FROM AUTHOR]

Background: The regulator of cullins-1 (ROC1) is an essential subunit in the cullin-RING ligase (CRL) protein complex and has been shown to be critical in bladder cancer cell survival and progression. This study aimed to explore the molecular mechanism of ROC1 action in the malignant progression of bladder cancer. Methods: This study utilized ex vivo, in vitro, and in vivo nude mouse experiments to assess the underlying mechanisms of ROC1 in bladder cancer cells. The expression of the components of the sonic hedgehog (SHH) pathway was determined by western blot analysis. ROC1 expression in human tumors was evaluated by immunohistochemistry. Results: ROC1 overexpression promoted the growth of bladder cancer cells, whereas knockdown of ROC1 expression had the opposite effect in bladder cancer cells. Mechanistically, ROC1 was able to target suppressor of fused homolog (SUFU) for ubiquitin-dependent degradation, allowing Gli2 release from the SUFU complex to activate the SHH pathway. Furthermore, knockdown of SUFU expression partially rescued the ROC1 knockdown-suppressed SHH activity as well as cancer cell growth inhibition. In ex vivo experiments, tissue microarray analysis of human bladder cancer specimens revealed a positive association of ROC1 expression with the SHH pathway activity. Conclusion: This study demonstrated that dysregulation of the ROC1–SUFU–GLI2 axis plays an important role in bladder cancer progression and that targeting ROC1 expression is warranted in further investigations as a novel strategy for the future control of bladder cancer. [ABSTRACT FROM AUTHOR]

Androgens/androgen receptor (AR)-mediated signaling pathways are essential for prostate development, morphogenesis and regeneration. Specifically, stromal AR signaling has been shown to be essential for prostatic initiation. However, the molecular mechanisms underlying AR-initiated mesenchymal-epithelial interactions in prostate development remain unclear. Here, using a newly generated mouse model,we have directlyaddressed the fate and role of genetically marked AR-expressing cells during embryonic prostate development. Androgen signaling-initiated signaling pathways were identified in mesenchymal niche populations at single-cell transcriptomic resolution. The dynamic cell-signaling networks regulated by stromal AR were additionally characterized in relation to prostatic epithelial bud formation. Pseudotime analyses further revealed the differentiation trajectory and fate of AR-expressing cells in both prostatic mesenchymal and epithelial cell populations. Specifically, the cellular properties of Zeb1-expressing progenitors were assessed. Selective deletion of AR signaling in a subpopulation of mesenchymal rather than epithelial cells dysregulated the expression of the master regulators and significantly impaired prostatic bud formation. These data provide novel, high-resolution evidence demonstrating the important role of mesenchymal androgen signaling in the cellular niche controlling prostate early development by initiating dynamic mesenchyme-epithelia cell interactions. [ABSTRACT FROM AUTHOR]

Purmorphamine (PUR), an agonist of the Smoothened (Smo) receptor, has been shown to function as a neuroprotectant in acute experimental ischemic stroke. Its role in hypoxic-ischemic (HI) brain injury in neonatal mice remains unknown. Here we show that PUR attenuated acute brain injury, with a decrease in Bax/Bcl-2 ratio as well as inhibition of caspase-3 activation. These beneficial effects of PUR were associated with suppressing neuro-inflammation and oxidative stress. PUR exerted long-term protective effects upon tissue loss and improved neurobehavioral outcomes as determined at 14 and 28 days post-HI insult. Moreover, PUR increased synaptophysin (Syn) and postsynaptic density (PSD) protein 95 expression in HI-treated mice and attenuated synaptic loss. PUR upregulated the expression of Shh pathway mediators, while suppression of the Shh signaling pathway with cyclopamine (Cyc) reversed these beneficial effects of PUR on HI insult. Our study suggests a therapeutic potential for short-term PUR administration in HI-induced injury as a result of its capacity to exert multiple protective actions upon acute brain injury, long-term memory deficits, and impaired synapses. Moreover, we provide evidence indicating that one of the mechanisms underlying these beneficial effects of PUR involves activation of the Shh signaling pathway.

ARL13B is a regulatory GTPase highly enriched in cilia. Complete loss of Arl13b disrupts cilia architecture, protein trafficking and Sonic hedgehog signaling. To determine whether ARL13B is required within cilia, we knocked in a cilia-excluded variant of ARL13B (V358A) and showed it retains all known biochemical function. We found that ARL13BV358A protein was expressed but could not be detected in cilia, even when retrograde ciliary transport was blocked. We showed Arl13bV358A/V358A mice are viable and fertile with normal Shh signal transduction. However, in contrast to wild type cilia, Arl13bV358A/V358A cells displayed short cilia and lacked ciliary ARL3 and INPP5E. These data indicate that ARL13B’s role within cilia can be uncoupled from its function outside of cilia. Furthermore, these data imply that the cilia defects upon complete absence of ARL13B do not underlie the alterations in Shh transduction, which is unexpected given the requirement of cilia for Shh transduction.

Background/Aims: Injuries of the brain and spinal cord result in the formation of glial (reactive gliosis) and fibrotic (formed by fibroblasts) scars. Recent studies have shown that the fibrotic scar was much more important for hindering regeneration after brain or spinal cord injury than the astrocytic scar. However, it has been given much less attention for effects and mechanism of fibroblasts during formation of the fibrotic scar. Resveratrol may be a potential anti-scarring agent in burn-related scarring and keloid fibroblasts. However, it is unclear whether and how resveratrol affects formation of the fibrotic scar after brain or spinal cord injury. Earlier studies have shown that the activated Shh signaling has anti-apoptosis, anti-oxidation, anti-inflammation properties. Moreover, resveratrol can activate the Shh signaling. However, it is unclear how resveratrol activates the Shh signaling. Resveratrol is a activator of Sirt1. It is unknown whether resveratrol activates the Shh signaling via Sirt1. Methods: NIH3T3 cells, a fibroblast cell line, were used as model cells and treated with drugs. Cell viability was assessed by Cell Counting Kit 8. The expressions and activity of Shh signaling pathway proteins were evaluated by immunocytochemistry and Western blotting. Transcriptional activity of Gli-1 was detected with Dual-Luciferase Reporter Gene Assay Kit. Results: Resveratrol, Sirt1 agonist STR1720 and recombinant mouse Shh protein, an activator of hedgehog signaling, enhanced the viability of NIH3T3 cells, promoted Smo to translocated to the primary cilia and Gli-1 entered into the nuclei from cytoplasm, and upregulated expressions of Shh, Ptc-1, Smo, and Gli-1 proteins, which can be reversed by Smo antagonist cyclopamine and Sirt1 antagonist Sirtinol. Additionally, resveratrol increased transcriptional activity of Gli-1. Conclusion: We indicate in the first time that it may be mediated by Sirt1 for resveratrol activating the Shh signaling to enhance viability of NIH3T3 cells, and Sirt1 may be a regulator for upstream of the Shh signaling pathway.This study provides a basis for further investigating effects and mechanism of resveratrol during the formation of fibrous scar after brain or spinal cord injury.

In addition to their hypoglycemic effect, sodium-glucose cotransporter 2 (SGLT2) inhibitors have many other benefits. In the present study, we examine the anticancer effect of the SGLT2 inhibitor empagliflozin using cervical carcinoma models. In vivo antitumor activities of empagliflozin were observed in a nude mouse model. Empagliflozin intervention and downregulation of Sonic Hedgehog Signaling Molecule (Shh) inhibited the migration and promoted the apoptosis of cervical cancer cells in nude mice. Compared with the control group, the empagliflozin treatment group had an increased level of AMP-activated protein kinase (AMPK) and decreased levels of Forkhead Box A1 (FOXA1) and SHH in tumor tissue. In vitro experiments also showed that empagliflozin (50 μM) inhibited the migration of cervical cancer cells and induced their apoptosis by activating the AMPK/FOXA1 pathway and inhibiting the expression of SHH. Kaplan-Meier survival analysis was used to determine the relationship between SHH expression and total survival time. The results showed that in cervical cancer patients, high SHH expression resulted in unfavorable overall survival. The downregulation of SHH with small interfering RNA (siRNA) inhibited the migration and invasion and promoted the apoptosis of HeLa cells. These findings show that empagliflozin has a potential therapeutic effect on cervical cancer. This effect was related to the activation of the AMPK pathway and the inhibition of SHH expression. [ABSTRACT FROM AUTHOR]

Purmorphamine (PUR), an agonist of the Smoothened (Smo) receptor, has been shown to function as a neuroprotectant in acute experimental ischemic stroke. Its role in hypoxic-ischemic (HI) brain injury in neonatal mice remains unknown. Here we show that PUR attenuated acute brain injury, with a decrease in Bax/Bcl-2 ratio as well as inhibition of caspase-3 activation. These beneficial effects of PUR were associated with suppressing neuro-inflammation and oxidative stress. PUR exerted long-term protective effects upon tissue loss and improved neurobehavioral outcomes as determined at 14 and 28 days post-HI insult. Moreover, PUR increased synaptophysin (Syn) and postsynaptic density (PSD) protein 95 expression in HI-treated mice and attenuated synaptic loss. PUR upregulated the expression of Shh pathway mediators, while suppression of the Shh signaling pathway with cyclopamine (Cyc) reversed these beneficial effects of PUR on HI insult. Our study suggests a therapeutic potential for short-term PUR administration in HI-induced injury as a result of its capacity to exert multiple protective actions upon acute brain injury, long-term memory deficits, and impaired synapses. Moreover, we provide evidence indicating that one of the mechanisms underlying these beneficial effects of PUR involves activation of the Shh signaling pathway. [ABSTRACT FROM AUTHOR]

Approximately 2 million individuals experience a traumatic brain injury (TBI) every year in the United States. Secondary injury begins within minutes after TBI, with alterations in cellular function and chemical signaling that contribute to excitotoxicity. Post-traumatic seizures (PTS) are experienced in an increasing number of TBI individuals that also display resistance to traditional anti-seizure medications (ASMs). Sonic hedgehog (Shh) is a signaling pathway that is upregulated following central nervous system damage in zebrafish and aids injury-induced regeneration. Using a modified Marmarou weight drop on adult zebrafish, we examined PTS following TBI and Shh modulation. We found that inhibiting Shh signaling by cyclopamine significantly increased PTS in TBI fish, prolonged the timeframe PTS was observed, and decreased survival across all TBI severities. Shh-inhibited TBI fish failed to respond to traditional ASMs, but were attenuated when treated with CNQX, which blocks ionotropic glutamate receptors. We found that the Smoothened agonist, purmorphamine, increased Eaat2a expression in undamaged brains compared to untreated controls, and purmorphamine treatment reduced glutamate excitotoxicity following TBI. Similarly, purmorphamine reduced PTS, edema, and cognitive deficits in TBI fish, while these pathologies were increased and/or prolonged in cyclopamine-treated TBI fish. However, the increased severity of TBI phenotypes with cyclopamine was reduced by cotreating fish with ceftriaxone, which induces Eaat2a expression. Collectively, these data suggest that Shh signaling induces Eaat2a expression and plays a role in regulating TBI-induced glutamate excitotoxicity and TBI sequelae.

Background/Aims: Neurite outgrowth and synaptogenesis are critical steps for functional recovery after stroke. Resveratrol promotes neurite outgrowth and synaptogenesis, but the underlying mechanism is not well understood, although the Sonic hedgehog (Shh) signaling pathway may be involved. Given that resveratrol activates sirtuin (Sirt)1, the present study examined whether this is mediated by Shh signaling. Methods: Primary cortical neuron cultures were pretreated with drugs before oxygen-glucose deprivation/reoxygenation (OGD/R). Cell viability and apoptosis were evaluated with Cell Counting Kit 8 and by terminal deoxynucleotidyl transferase dUTP nick end labeling, respectively. Neurite outgrowth and synaptogenesis were assessed by immunocytochemistry and western blotting, which was also used to examine the expression of Sirt1 and Shh signaling proteins. Results: Resveratrol and the Smoothened (Smo) agonist purmophamine, which activates Shh signaling, increased viability, reduced apoptosis, and stimulated neurite outgrowth after OGD/R injury. Moreover, the expression of growth-associated protein(GAP)-43, synaptophysin, Shh, Patched (Ptc)-1, Smo, glioma-associated oncogene homolog (Gli)-1, and Sirt1 were upregulated under these conditions. These effects were reversed by treatment with the Smo inhibitor cyclopamine, whereas the Sirt1 inhibitor sirtinol reduced the levels of Shh, Ptc-1, Smo, and Gli-1. Conclusions: Resveratrol reduces neuronal injury following OGD/R injury and enhances neurite outgrowth and synaptogenesis by activating Shh signaling, which in turn induces Sirt1.

Cancer stem cells (CSC) are rare immortal cells within a tumor that are able to initiate tumor progression, development, and resistance. Advances studies show that, like normal stem cells, CSCs can be both self‐renewed and given rise to many cell types, therefore form tumors. A number of cell surface markers, such as CD44, CD24, and CD133 are frequently used to identify CSCs. CD133, a transmembrane glycoprotein, either alone or in collaboration with other markers, has been mainly considered to identify CSCs from different solid tumors. However, the exactness of CD133 as a cancer stem cell biomarker has not been approved yet. The clinical importance of CD133 is as a CSC marker in many cancers. Also, it contributes to shorter survival, tumor progression, and tumor recurrence. The expression of CD133 is controlled by many extracellular or intracellular factors, such as tumor microenvironment, epigenetic factors, signaling pathways, and miRNAs. In this study, it was attempted to determine: 1) CD133 function; 2) the role of CD133 in cancer; 3) CD133 regulation; 4) the therapeutic role of CD133 in cancers. [ABSTRACT FROM AUTHOR]

Epithelial-mesenchymal transition (EMT) provides a valuable source of fibroblasts that produce extracellular matrix in airway walls. The Sonic hedgehog (SHH) signaling pathway plays an essential role in regulating tissue turnover and homeostasis. SHH is strikingly upregulated in the bronchial epithelia during asthma. Snail1 is a major target of SHH signaling, which regulates EMT and fibroblast motility. The present study was designed to ascertain whether the combination of house dust mite (HDM) and transforming growth factor β1 (TGF-β1) could induce EMT via the SHH signaling pathway in human bronchial epithelial cells (HBECs). HBEC cultures were treated with HDM/TGF-β1 for different periods of time. The involvement of SHH signaling and EMT biomarkers was evaluated by quantitative real-time PCR, western blotting and immunofluorescence staining. Small-interfering RNA (siRNA) for glioma-associated antigen-1 (Gli1) or cyclopamine was used to inhibit SHH signaling in HBECs. HBECs stimulated by HDM/TGF-β1 exhibited morphological features of EMT. E-cadherin (an epithelial marker) was decreased after a 72-h exposure to HDM/TGF-β1 compared to that in the control cells, and the expression of type I collagen and FSP1 (mesenchymal markers) was increased. HDM/TGF-β1 activated the SHH signaling pathway in HBECs, which led to Gli1 nuclear translocation and the transcriptional activation of Snail1 expression. Moreover, gene silencing or the pharmacological inhibition of Gli1 ameliorated EMT. In summary, these findings suggest that HDM/TGF-β1 may induce EMT in HBECs via an SHH signaling mechanism. Inhibition of SHH signaling may be a novel therapeutic method for preventing airway remodeling in asthma. [ABSTRACT FROM AUTHOR]

Genistein, a soy derived isoflavanoid compound, exerts anticancer effects in various cancers. Nasopharyngeal cancer stem cells (NCSCs) are a small subpopulation of cancer cells which are responsible for initiation, progression, metastasis, and recurrence of nasopharyngeal cancer. The present study aimed to investigate the suppressive effects of genistein on NCSCs and its underlying mechanism. NCSCs were enriched from human nasopharyngeal cancer cell lines CNE2 and HONE1 through tumorsphere-forming assay. It was shown that genistein inhibited the tumorsphere formation capacity, decreased the number of EpCAM+ cells, downregulated the expression of NCSCs markers, suppressed cell proliferation, and induced apoptosis of NCSCs. Genistein suppressed the activity of Sonic hedgehog (SHH) signaling, which was important for the maintenance of NCSCs, while activation of SHH signaling by purmorphamine diminished the inhibitory effects of genistein on NCSCs. Our data suggested that genistein inhibited NCSCs through the suppression of SHH signaling. These findings support the use of genistein for targeting NCSCs. [ABSTRACT FROM AUTHOR]

Liver fibrosis is a continuous wound-healing process, which is due to excessive deposition of extracellular matrix (ECM) caused by activated Hepatic Stellate Cells (HSCs). Geniposide (GP) is a naturally occurring iridoid glucoside and is extracted from Gardenia jasminoides Ellis. GP has long been speculated to play a vital role in the treatment of hyperlipidemia and fatty liver. Emerging evidence has demonstrated that GP may be importantly associated with the pathophysiology of liver fibrosis. Nevertheless, the fundamental mechanism is yet uncertain. This studywas designed to explore the possible mechanism for the inhibitory effect of GP on CCl 4 -induced mice liver fibrosis. In line with several clinical reports, GP significantly reduced the levels of hyaluronic acid (HA), laminin (LN), hydroxyproline (HYP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Meanwhile, liver fibrosis was significantly alleviated by GP as indicated by decreased α-smooth muscle actin (α-SMA) expression and type I collagen alpha-1 (Col I α1) deposition. In addition, GP could reduce the cell viability (IC50 = 77.11 and 42.88 μM at 24 and 48 h respectively) and cause G2/M cell arrest of the activated HSC-T6 cells. Noteworthy, GP prominently suppressed the Sonic hedgehog (Shh) signaling pathway and it might inhibit the activation and proliferation of HSC-T6 cells through Shh signaling pathway. Taken together, the current research indicate that GP has a promising antifibrotic effect which could be partially attributed to its suppression of Shh signaling pathway. Hence GP could be employed as a promising therapeutic strategy for the treatment of clinical hepatic injury. Unlabelled Image • The inhibitor role of GP on CCl 4 -induced liver fibrosis. • Inhibition role of GP on the activation and proliferation of activated HSC-T6 cells. • GP suppressed the Shh signaling pathway. [ABSTRACT FROM AUTHOR]

Numb is an intracellular protein with multiple functions. The two prevalent isoforms, Numb p66 and Numb p72, are regulators of differentiation and proliferation in neuronal development. Additionally, Numb functions as cell fate determinant of stem cells and cancer stem cells and its abnormal expression has been described in several types of cancer. Involvement of deregulated Numb expression has been described in the malignant childhood brain tumor medulloblastoma, while Numb isoforms in these tumors and in cancer stem-like cells derived from them, have not been studied to date. Here we show that medulloblastoma stem-like cells and cerebellar neuronal stem cells (NSCs) express Numb p66 where its expression tampers stemness features. Furthermore, medulloblastoma samples evaluated in this study express decreased levels of Numb p66 while overexpressed Numb p72 compared with normal tissues. Our results uncover different roles for the two major Numb isoforms examined in medulloblastoma and a critical role for Numb p66 in regulating stem-like cells and NSCs maintenance.

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.

Background/Aims: Injuries of the brain and spinal cord result in the formation of glial (reactive gliosis) and fibrotic (formed by fibroblasts) scars. Recent studies have shown that the fibrotic scar was much more important for hindering regeneration after brain or spinal cord injury than the astrocytic scar. However, it has been given much less attention for effects and mechanism of fibroblasts during formation of the fibrotic scar. Resveratrol may be a potential anti-scarring agent in burn-related scarring and keloid fibroblasts. However, it is unclear whether and how resveratrol affects formation of the fibrotic scar after brain or spinal cord injury. Earlier studies have shown that the activated Shh signaling has anti-apoptosis, anti-oxidation, anti-inflammation properties. Moreover, resveratrol can activate the Shh signaling. However, it is unclear how resveratrol activates the Shh signaling. Resveratrol is a activator of Sirt1. It is unknown whether resveratrol activates the Shh signaling via Sirt1. Methods: NIH3T3 cells, a fibroblast cell line, were used as model cells and treated with drugs. Cell viability was assessed by Cell Counting Kit 8. The expressions and activity of Shh signaling pathway proteins were evaluated by immunocytochemistry and Western blotting. Transcriptional activity of Gli-1 was detected with Dual-Luciferase Reporter Gene Assay Kit. Results: Resveratrol, Sirt1 agonist STR1720 and recombinant mouse Shh protein, an activator of hedgehog signaling, enhanced the viability of NIH3T3 cells, promoted Smo to translocated to the primary cilia and Gli-1 entered into the nuclei from cytoplasm, and upregulated expressions of Shh, Ptc-1, Smo, and Gli-1 proteins, which can be reversed by Smo antagonist cyclopamine and Sirt1 antagonist Sirtinol. Additionally, resveratrol increased transcriptional activity of Gli-1. Conclusion: We indicate in the first time that it may be mediated by Sirt1 for resveratrol activating the Shh signaling to enhance viability of NIH3T3 cells, and Sirt1 may be a regulator for upstream of the Shh signaling pathway.This study provides a basis for further investigating effects and mechanism of resveratrol during the formation of fibrous scar after brain or spinal cord injury. [ABSTRACT FROM AUTHOR]

In the mouse neural tube, sonic hedgehog (Shh) secreted from the floor plate (FP) and the notochord (NC) regulates ventral patterning of the neural tube, and later is essential for the generation of oligodendrocyte precursor cells (OPCs). During early development, the NC is adjacent to the neural tube and induces ventral domains in it, including the FP. In the later stage of development, during gliogenesis in the spinal cord, the pMN domain receives strong Shh signaling input. While this is considered to be essential for the generation of OPCs, the actual role of this strong input in OPC generation remains unclear. Here we studied OPC generation in bromi mutant mice which show abnormal ciliary structure. Shh signaling occurs within cilia and has been reported to be weak in bromi mutants. At E11.5, accumulation of Patched1 mRNA, a Shh signaling reporter, is observed in the pMN domain of wild type but not bromi mutants, whereas expression of Gli1 mRNA, another Shh reporter, disappeared. Thus, Shh signaling input to the pMN domain at E12.5 was reduced in bromi mutant mice. In these mutants, induction of the FP structure was delayed and its size was reduced compared to wild type mice. Furthermore, while the p3 and pMN domains were induced, the length of the Nkx2.2-positive region and the number of Olig2-positive cells decreased. The number of OPCs was also significantly decreased in the E12.5 and E14.5 bromi mutant spinal cord. In contrast, motor neuron (MN) production, detected by HB9 expression, significantly increased. It is likely that the transition from MN production to OPC generation in the pMN domain is impaired in bromi mutant mice. These results suggest that strong Shh input to the pMN domain is not required for OPC generation but is essential for producing a sufficient number of OPCs. [ABSTRACT FROM AUTHOR]

Supplemental digital content is available in the text. Objectives: The aim of this study was to investigate the distribution of primary cilia on secretory cells in normal fallopian tube (FT) and serous tubal intraepithelial carcinoma (STIC). Methods: Fallopian tube tissue samples were obtained from 4 females undergoing prophylactic hysterectomies and 6 patients diagnosed with STIC. A mogp -TAg transgenic mouse STIC sample was also compared with a wild-type mouse FT sample. Serous tubal intraepithelial carcinoma was identified by hematoxylin and eosin staining and confirmed by positive Ki-67 and p53 immunohistochemical staining of tissue sections. We assessed the relative distribution of primary cilia on secretory cells and motile cilia on multiple ciliated cells by immunofluorescence and immunohistochemical staining. Ciliary function was assessed by immunofluorescence staining of specific ciliary marker proteins and responsiveness to Sonic Hedgehog signaling. Results: Primary cilia are widespread on secretory cells in the ampulla, isthmus, and in particular, the fimbriae of human FT where they may appear to mediate ciliary-mediated Sonic Hedgehog signaling. A statistically significant reduction in the number of primary cilia on secretory cells was observed in human STIC samples compared with normal controls (P < 0.0002, Student t test), supported by similar findings in a mouse STIC sample. Immunohistochemical staining for dynein axonemal heavy chain 5 discriminated multiple motile cilia from primary cilia in human FT. Conclusions: Primary cilia are widespread on secretory cells in the ampulla, isthmus, and in particular, the fimbriae of the human FT but are significantly reduced in both human and mouse STIC samples. Immunohistochemical staining for ciliary proteins may have clinical utility for early detection of STIC. [ABSTRACT FROM AUTHOR]

The beneficial effects of resveratrol on in vitro maturation (IVM) have been explained mainly by indirect antioxidant effects and limited information is available on the underlying mechanism by which resveratrol acts directly on porcine cumulus oocyte complexes (COCs). Recently, several studies reported that sonic hedgehog (SHH) signaling mediates resveratrol to exert its biological activities. Furthermore, SHH is an important signaling molecule for follicle development, oocyte maturation, and embryo development. Therefore, to elucidate the relationship between resveratrol and SHH signaling, we designed three groups: (i) control; (ii) resveratrol; and (iii) resveratrol with cyclopamine (SHH signaling inhibitor). We evaluated the effects of these agents on cumulus expansion, oocyte maturation, embryo development after parthenogenetic activation, expression levels of mRNAs in cumulus cells, oocytes and blastocysts, and protein expression in COCs. Resveratrol significantly increased the proportion of COCs exhibiting complete cumulus expansion (degree 4), oocyte nuclear maturation, cleavage and blastocyst formation rates and total cell numbers, which were blocked in the presence of cyclopamine. At the same time, a significant increase in the expression levels of mRNAs related to cumulus expansion, oocyte maturation and SHH signaling‐related mRNAs and proteins from the resveratrol treatment group was also inhibited by simultaneous addition of cyclopamine. In conclusion, our results indicate that SHH signaling mediates resveratrol to improve porcine cumulus expansion, oocyte maturation, and subsequent embryo development. [ABSTRACT FROM AUTHOR]