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Sample records for regulates sonic hedgehog

  1. Sonic Hedgehog regulates thymic epithelial cell differentiation

    PubMed Central

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L.; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-01-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus. PMID

  2. Sonic Hedgehog regulates thymic epithelial cell differentiation.

    PubMed

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-04-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus.

  3. Regulation of Patched by Sonic Hedgehog in the Developing Neural Tube

    NASA Astrophysics Data System (ADS)

    Marigo, Valeria; Tabin, Clifford J.

    1996-09-01

    Ventral cell fates in the central nervous system are induced by Sonic hedgehog, a homolog of hedgehog, a secreted Drosophila protein. In the central nervous system, Sonic hedgehog has been identified as the signal inducing floor plate, motor neurons, and dopaminergic neurons. Sonic hedgehog is also involved in the induction of ventral cell type in the developing somites. ptc is a key gene in the Drosophila hedgehog signaling pathway where it is involved in transducing the hedgehog signal and is also a transcriptional target of the signal. PTC, a vertebrate homolog of this Drosophila gene, is genetically downstream of Sonic hedgehog (Shh) in the limb bud. We analyze PTC expression during chicken neural and somite development and find it expressed in all regions of these tissues known to be responsive to Sonic hedgehog signal. As in the limb bud, ectopic expression of Sonic hedgehog leads to ectopic induction of PTC in the neural tube and paraxial mesoderm. This conservation of regulation allows us to use PTC as a marker for Sonic hedgehog response. The pattern of PTC expression suggests that Sonic hedgehog may play an inductive role in more dorsal regions of the neural tube than have been previously demonstrated. Examination of the pattern of PTC expression also suggests that PTC may act in a negative feedback loop to attenuate hedgehog signaling.

  4. Sortilin regulates sorting and secretion of Sonic hedgehog.

    PubMed

    Campbell, Charles; Beug, Shawn; Nickerson, Philip E B; Peng, Jimmy; Mazerolle, Chantal; Bassett, Erin A; Ringuette, Randy; Jama, Fadumo A; Morales, Carlos; Christ, Annabel; Wallace, Valerie A

    2016-10-15

    Sonic Hedgehog (Shh) is a secreted morphogen that is an essential regulator of patterning and growth. The Shh full-length protein undergoes autocleavage in the endoplasmic reticulum to generate the biologically active N-terminal fragment (ShhN), which is destined for secretion. We identified sortilin (Sort1), a member of the VPS10P-domain receptor family, as a new Shh trafficking receptor. We demonstrate that Sort-Shh interact by performing coimmunoprecipitation and proximity ligation assays in transfected cells and that they colocalize at the Golgi. Sort1 overexpression causes re-distribution of ShhN and, to a lesser extent, of full-length Shh to the Golgi and reduces Shh secretion. We show loss of Sort1 can partially rescue Hedgehog-associated patterning defects in a mouse model that is deficient in Shh processing, and we show that Sort1 levels negatively regulate anterograde Shh transport in axons in vitro and Hedgehog-dependent axon-glial interactions in vivo Taken together, we conclude that Shh and Sort1 can interact at the level of the Golgi and that Sort1 directs Shh away from the pathways that promote its secretion.

  5. YAP regulates neuronal differentiation through Sonic hedgehog signaling pathway

    SciTech Connect

    Lin, Yi-Ting; Ding, Jing-Ya; Li, Ming-Yang; Yeh, Tien-Shun; Wang, Tsu-Wei; Yu, Jenn-Yah

    2012-09-10

    Tight regulation of cell numbers by controlling cell proliferation and apoptosis is important during development. Recently, the Hippo pathway has been shown to regulate tissue growth and organ size in Drosophila. In mammalian cells, it also affects cell proliferation and differentiation in various tissues, including the nervous system. Interplay of several signaling cascades, such as Notch, Wnt, and Sonic Hedgehog (Shh) pathways, control cell proliferation during neuronal differentiation. However, it remains unclear whether the Hippo pathway coordinates with other signaling cascades in regulating neuronal differentiation. Here, we used P19 cells, a mouse embryonic carcinoma cell line, as a model to study roles of YAP, a core component of the Hippo pathway, in neuronal differentiation. P19 cells can be induced to differentiate into neurons by expressing a neural bHLH transcription factor gene Ascl1. Our results showed that YAP promoted cell proliferation and inhibited neuronal differentiation. Expression of Yap activated Shh but not Wnt or Notch signaling activity during neuronal differentiation. Furthermore, expression of Yap increased the expression of Patched homolog 1 (Ptch1), a downstream target of the Shh signaling. Knockdown of Gli2, a transcription factor of the Shh pathway, promoted neuronal differentiation even when Yap was over-expressed. We further demonstrated that over-expression of Yap inhibited neuronal differentiation in primary mouse cortical progenitors and Gli2 knockdown rescued the differentiation defect in Yap over-expressing cells. In conclusion, our study reveals that Shh signaling acts downstream of YAP in regulating neuronal differentiation. -- Highlights: Black-Right-Pointing-Pointer YAP promotes cell proliferation and inhibits neuronal differentiation in P19 cells. Black-Right-Pointing-Pointer YAP promotes Sonic hedgehog signaling activity during neuronal differentiation. Black-Right-Pointing-Pointer Knockdown of Gli2 rescues the Yap

  6. Interaction of PACAP with Sonic hedgehog reveals complex regulation of the hedgehog pathway by PKA.

    PubMed

    Niewiadomski, Pawel; Zhujiang, Annie; Youssef, Mary; Waschek, James A

    2013-11-01

    Sonic hedgehog (Shh) signaling is essential for proliferation of cerebellar granule cell progenitors (cGCPs) and its aberrant activation causes a cerebellar cancer medulloblastoma. Pituitary adenylate cyclase activating polypeptide (PACAP) inhibits Shh-driven proliferation of cGCPs and acts as tumor suppressor in murine medulloblastoma. We show that PACAP blocks canonical Shh signaling by a mechanism that involves activation of protein kinase A (PKA) and inhibition of the translocation of the Shh-dependent transcription factor Gli2 into the primary cilium. PKA is shown to play an essential role in inhibiting gene transcription in the absence of Shh, but global PKA activity levels are found to be a poor predictor of the degree of Shh pathway activation. We propose that the core Shh pathway regulates a small compartmentalized pool of PKA in the vicinity of primary cilia. GPCRs that affect global PKA activity levels, such as the PACAP receptor, cooperate with the canonical Shh signal to regulate Gli protein phosphorylation by PKA. This interaction serves to fine-tune the transcriptional and physiological function of the Shh pathway.

  7. Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling

    PubMed Central

    Breunig, Joshua J.; Sarkisian, Matthew R.; Arellano, Jon I.; Morozov, Yury M.; Ayoub, Albert E.; Sojitra, Sonal; Wang, Baolin; Flavell, Richard A.; Rakic, Pasko; Town, Terrence

    2008-01-01

    Primary cilia are present on mammalian neurons and glia, but their function is largely unknown. We generated conditional homozygous mutant mice for a gene we termed Stumpy. Mutants lack cilia and have conspicuous abnormalities in postnatally developing brain regions, including a hypoplasic hippocampus characterized by a primary deficiency in neural stem cells known as astrocyte-like neural precursors (ALNPs). Previous studies suggested that primary cilia mediate sonic hedgehog (Shh) signaling. Here, we find that loss of ALNP cilia leads to abrogated Shh activity, increased cell cycle exit, and morphological abnormalities in ALNPs. Processing of Gli3, a mediator of Shh signaling, is also altered in the absence of cilia. Further, key mediators of the Shh pathway localize to ALNP cilia. Thus, selective targeting of Shh machinery to primary cilia confers to ALNPs the ability to differentially respond to Shh mitogenic signals compared to neighboring cells. Our data suggest these organelles are cellular “antennae” critically required to modulate ALNP behavior. PMID:18728187

  8. Sonic hedgehog elevates N-myc gene expression in neural stem cells.

    PubMed

    Liu, Dongsheng; Wang, Shouyu; Cui, Yan; Shen, Lun; Du, Yanping; Li, Guilin; Zhang, Bo; Wang, Renzhi

    2012-08-05

    Proliferation of neural stem cells is regulated by the secreted signaling molecule sonic hedgehog. In this study, neural stem cells were infected with recombinant adeno-associated virus expressing sonic hedgehog-N-enhanced green fluorescent protein. The results showed that overexpression of sonic hedgehog in neural stem cells induced the increased expression of Gli1 and N-myc, a target gene of sonic hedgehog. These findings suggest that N-myc is a direct downstream target of the sonic hedgehog signal pathway in neural stem cells. Sonic hedgehog and N-myc are important mediators of sonic hedgehog-induced proliferation of neural stem cells.

  9. Sonic hedgehog is a regulator of extracellular glutamate levels and epilepsy.

    PubMed

    Feng, Shengjie; Ma, Shaorong; Jia, Caixia; Su, Yujuan; Yang, Shenglian; Zhou, Kechun; Liu, Yani; Cheng, Ju; Lu, Dunguo; Fan, Liu; Wang, Yizheng

    2016-05-01

    Sonic hedgehog (Shh), both as a mitogen and as a morphogen, plays an important role in cell proliferation and differentiation during early development. Here, we show that Shh inhibits glutamate transporter activities in neurons, rapidly enhances extracellular glutamate levels, and affects the development of epilepsy. Shh is quickly released in response to epileptic, but not physiological, stimuli. Inhibition of neuronal glutamate transporters by Shh depends on heterotrimeric G protein subunit Gαi and enhances extracellular glutamate levels. Inhibiting Shh signaling greatly reduces epileptiform activities in both cell cultures and hippocampal slices. Moreover, pharmacological or genetic inhibition of Shh signaling markedly suppresses epileptic phenotypes in kindling or pilocarpine models. Our results suggest that Shh contributes to the development of epilepsy and suppression of its signaling prevents the development of the disease. Thus, Shh can act as a modulator of neuronal activity, rapidly regulating glutamate levels and promoting epilepsy.

  10. Sonic hedgehog processing and release are regulated by glypican heparan sulfate proteoglycans.

    PubMed

    Ortmann, Corinna; Pickhinke, Ute; Exner, Sebastian; Ohlig, Stefanie; Lawrence, Roger; Jboor, Hamodah; Dreier, Rita; Grobe, Kay

    2015-06-15

    All Hedgehog morphogens are released from producing cells, despite being synthesized as N- and C-terminally lipidated molecules, a modification that firmly tethers them to the cell membrane. We have previously shown that proteolytic removal of both lipidated peptides, called shedding, releases bioactive Sonic hedgehog (Shh) morphogens from the surface of transfected Bosc23 cells. Using in vivo knockdown together with in vitro cell culture studies, we now show that glypican heparan sulfate proteoglycans regulate this process, through their heparan sulfate chains, in a cell autonomous manner. Heparan sulfate specifically modifies Shh processing at the cell surface, and purified glycosaminoglycans enhance the proteolytic removal of N- and C-terminal Shh peptides under cell-free conditions. The most likely explanation for these observations is direct Shh processing in the extracellular compartment, suggesting that heparan sulfate acts as a scaffold or activator for Shh ligands and the factors required for their turnover. We also show that purified heparan sulfate isolated from specific cell types and tissues mediates the release of bioactive Shh from pancreatic cancer cells, revealing a previously unknown regulatory role for these versatile molecules in a pathological context.

  11. Cooperatively transcriptional and epigenetic regulation of sonic hedgehog overexpression drives malignant potential of breast cancer.

    PubMed

    Duan, Zhao-Heng; Wang, Hao-Chuan; Zhao, Dong-Mei; Ji, Xiao-Xin; Song, Min; Yang, Xiao-Jun; Cui, Wei

    2015-08-01

    Sonic hedgehog (Shh), a ligand of Hedgehog signaling pathway, is considered an important oncogene and an exciting potential therapeutic target in several cancers. Comprehensive understanding of the regulation mechanism of Shh in cancer cells is necessary to find an effective approach to selectively block its tumorigenic function. We and others previously demonstrated that nuclear factor-kappa B (NF-κB) activation and promoter hypomethylation contributed to the overexpression of Shh. However, the relationship between transcriptional and epigenetic regulation of Shh, and their roles in the malignant phenotype of cancer cells are still not clearly elucidated. In the present study, our data showed that the level of Shh was higher in breast cancer tissues with positive NF-κB nuclear staining and promoter hypomethylation. In addition, survival analysis revealed that Shh overexpression, but not hypomethylation and NF-κB nuclear staining, was a poor prognosis indicator for breast cancers. Moreover, in vitro data demonstrated that both NF-κB activation and hypomethylation in promoter region were positively associated with the overexpression of Shh. Mechanistically, the hypomethylation in Shh promoter could facilitate NF-κB binding to its site, and subsequently cooperate to induce transcription of Shh. Furthermore, the biological function data indicated that overexpressed Shh enhanced the self-renewal capacity and migration ability of breast cancer cells, which could be augmented by promoter demethylation and NF-κB activation. Overall, our findings reveal multiple and cooperative mechanisms of Shh upregulation in cancer cells, and the roles of Shh in tumor malignant behavior, thus suggesting a new strategy for therapeutic interventions to reduce Shh in tumors and improve patients' prognosis.

  12. [The role of sonic hedgehog pathway in skin carcinogenesis].

    PubMed

    Lesiak, Aleksandra; Sysa-Jedrzejowska, Anna; Narbutt, Joanna

    2010-08-01

    Non melanoma skin cancers (NMSC) involving basal (BCC)--and squamosus cell carcinomas (SCC) and are the most frequent skin cancers in Caucasians. Ultraviolet radiation is the main environmental risk factor for NMSC development. The aim of this paper is to review the latest opinions concerning the role of sonic hedgehog pathway in non-melanoma skin cancers development. Experimental data indicate that sonic hedgehog pathway might be involved in skin carcinogenesis. Under physiological conditions sonic hedgehog pathway is responsible for normal embryogenesis, regeneration of damaged tissues and for regulation of cell proliferation. It was revealed that UVR caused inactivated mutation in PATCHED gene encoding Ptch1 protein. These events lead to deregulation of sonic hedgehog pathway trough activation of Smo protein and Gli transcriptional factors what stimulates cell proliferation and in consequence NMSC development. Literature data indicate that understanding of molecular background of skin cancers might be a reason for introduction of new therapeutic approaches including sonic hedgehog pathway inhibitors.

  13. Sonic Hedgehog Promotes Neurite Outgrowth of Primary Cortical Neurons Through Up-Regulating BDNF Expression.

    PubMed

    He, Weiliang; Cui, Lili; Zhang, Cong; Zhang, Xiangjian; He, Junna; Xie, Yanzhao

    2016-04-01

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

  14. Signaling domain of Sonic Hedgehog as cannibalistic calcium-regulated zinc-peptidase.

    PubMed

    Rebollido-Rios, Rocio; Bandari, Shyam; Wilms, Christoph; Jakuschev, Stanislav; Vortkamp, Andrea; Grobe, Kay; Hoffmann, Daniel

    2014-07-01

    Sonic Hedgehog (Shh) is a representative of the evolutionary closely related class of Hedgehog proteins that have essential signaling functions in animal development. The N-terminal domain (ShhN) is also assigned to the group of LAS proteins (LAS = Lysostaphin type enzymes, D-Ala-D-Ala metalloproteases, Sonic Hedgehog), of which all members harbor a structurally well-defined Zn2+ center; however, it is remarkable that ShhN so far is the only LAS member without proven peptidase activity. Another unique feature of ShhN in the LAS group is a double-Ca2+ center close to the zinc. We have studied the effect of these calcium ions on ShhN structure, dynamics, and interactions. We find that the presence of calcium has a marked impact on ShhN properties, with the two calcium ions having different effects. The more strongly bound calcium ion significantly stabilizes the overall structure. Surprisingly, the binding of the second calcium ion switches the putative catalytic center from a state similar to LAS enzymes to a state that probably is catalytically inactive. We describe in detail the mechanics of the switch, including the effect on substrate co-ordinating residues and on the putative catalytic water molecule. The properties of the putative substrate binding site suggest that ShhN could degrade other ShhN molecules, e.g. by cleavage at highly conserved glycines in ShhN. To test experimentally the stability of ShhN against autodegradation, we compare two ShhN mutants in vitro: (1) a ShhN mutant unable to bind calcium but with putative catalytic center intact, and thus, according to our hypothesis, a constitutively active peptidase, and (2) a mutant carrying additionally mutation E177A, i.e., with the putative catalytically active residue knocked out. The in vitro results are consistent with ShhN being a cannibalistic zinc-peptidase. These experiments also reveal that the peptidase activity depends on pH.

  15. p63 sustains self-renewal of mammary cancer stem cells through regulation of Sonic Hedgehog signaling

    PubMed Central

    Memmi, Elisa Maria; Sanarico, Anna Giulia; Giacobbe, Arianna; Peschiaroli, Angelo; Frezza, Valentina; Cicalese, Angelo; Pisati, Federica; Tosoni, Daniela; Zhou, Huiqing; Tonon, Giovanni; Antonov, Alexey; Melino, Gerry; Pelicci, Pier Giuseppe; Bernassola, Francesca

    2015-01-01

    The predominant p63 isoform, ΔNp63, is a master regulator of normal epithelial stem cell (SC) maintenance. However, in vivo evidence of the regulation of cancer stem cell (CSC) properties by p63 is still limited. Here, we exploit the transgenic MMTV-ErbB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2) mouse model of carcinogenesis to dissect the role of p63 in the regulation of mammary CSC self-renewal and breast tumorigenesis. ErbB2 tumor cells enriched for SC-like properties display increased levels of ΔNp63 expression compared with normal mammary progenitors. Down-regulation of p63 in ErbB2 mammospheres markedly restricts self-renewal and expansion of CSCs, and this action is fully independent of p53. Furthermore, transplantation of ErbB2 progenitors expressing shRNAs against p63 into the mammary fat pads of syngeneic mice delays tumor growth in vivo. p63 knockdown in ErbB2 progenitors diminishes the expression of genes encoding components of the Sonic Hedgehog (Hh) signaling pathway, a driver of mammary SC self-renewal. Remarkably, p63 regulates the expression of Sonic Hedgehog (Shh), GLI family zinc finger 2 (Gli2), and Patched1 (Ptch1) genes by directly binding to their gene regulatory regions, and eventually contributes to pathway activation. Collectively, these studies highlight the importance of p63 in maintaining the self-renewal potential of mammary CSCs via a positive modulation of the Hh signaling pathway. PMID:25739959

  16. Mechanical stimulation promote the osteogenic differentiation of bone marrow stromal cells through epigenetic regulation of Sonic Hedgehog.

    PubMed

    Wang, Chuandong; Shan, Shengzhou; Wang, Chenglong; Wang, Jing; Li, Jiao; Hu, Guoli; Dai, Kerong; Li, Qingfeng; Zhang, Xiaoling

    2017-03-15

    Mechanical unloading leads to bone loss and disuse osteoporosis partly due to impaired osteoblastogenesis of bone marrow stromal cells (BMSCs). However, the underlying molecular mechanisms of this phenomenon are not fully understood. In this study, we demonstrated that cyclic mechanical stretch (CMS) promotes osteoblastogenesis of BMSCs both in vivo and in vitro. Besides, we found that Hedgehog (Hh) signaling pathway was activated in this process. Inhibition of which by either knockdown of Sonic hedgehog (Shh) or treating BMSCs with Hh inhibitors attenuated the osteogenic effect of CMS on BMSCs, suggesting that Hh signaling pathway acts as an endogenous mediator of mechanical stimuli on BMSCs. Furthermore, we demonstrated that Shh expression level was regulated by DNA methylation mechanism. Chromatin Immunoprecipitation (ChIP) assay showed that DNA methyltransferase 3b (Dnmt3b) binds to Shh gene promoter, leading to DNA hypermethylation in mechanical unloading BMSCs. However, mechanical stimulation down-regulates the protein level of Dnmt3b, results in DNA demethylation and Shh expression. More importantly, we found that inhibition of Dnmt3b partly rescued bone loss in HU mice by mechanical unloading. Our results demonstrate, for the first time, that mechanical stimulation regulates osteoblastic genes expression via direct regulation of Dnmt3b, and the therapeutic inhibition of Dnmt3b may be an efficient strategy for enhancing bone formation under mechanical unloading.

  17. Ski modulate the characteristics of pancreatic cancer stem cells via regulating sonic hedgehog signaling pathway.

    PubMed

    Song, Libin; Chen, Xiangyuan; Gao, Song; Zhang, Chenyue; Qu, Chao; Wang, Peng; Liu, Luming

    2016-10-12

    Evidence from in vitro and in vivo studies shows that Ski may act as both a tumor proliferation-promoting factor and a metastatic suppressor in human pancreatic cancer and also may be a therapeutic target of integrative therapies. At present, pancreatic cancer stem cells (CSCs) are responsible for tumor recurrence accompanied by resistance to conventional therapies. Sonic hedgehog (Shh) signaling pathway is found to be aberrantly activated in CSCs. The objectives of this study were to investigate the role of Ski in modulating pancreatic CSCs and to examine the molecular mechanisms involved in pancreatic cancer treatment both in vivo and in vitro. In in vitro study, the results showed that enhanced Ski expression could increase the expression of pluripotency maintaining markers, such as CD24, CD44, Sox-2, and Oct-4, and also components of Shh signaling pathway, such as Shh, Ptch-1, Smo, Gli-1, and Gli-2, whereas depletion of Ski to the contrary. Then, we investigated the underlying mechanism and found that inhibiting Gli-2 expression by short interfering RNA (siRNA) can decrease the effects of Ski on the maintenance of pancreatic CSCs, indicating that Ski mediates the pluripotency of pancreatic CSCs mainly through Shh pathway. The conclusion is that Ski may be an important factor in maintaining the stemness of pancreatic CSCs through modulating Shh pathway.

  18. Sonic hedgehog signaling regulates amygdalar neurogenesis and extinction of fear memory.

    PubMed

    Hung, Hui-Chi; Hsiao, Ya-Hsin; Gean, Po-Wu

    2015-10-01

    It is now recognized that neurogenesis occurs throughout life predominantly in the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricle. In the present study, we investigated the relationship between neurogenesis in the amygdala and extinction of fear memory. Mice received 15 tone-footshock pairings. Twenty-four hours after training, the mice were given 15 tone-alone trials (extinction training) once per day for 7 days. Two hours before extinction training, the mice were injected intraperitoneally with 5-bromo-3-deoxyuridine (BrdU). BrdU-positive and NeuN-positive cells were analyzed 52 days after the training. A group of mice that received tone-footshock pairings but no extinction training served as controls (FC+No-Ext). The number of BrdU(+)/NeuN(+) cells was significantly higher in the extinction (FC+Ext) than in the FC+No-Ext mice. Proliferation inhibitor methylazoxymethanol acetate (MAM) or DNA synthesis inhibitor cytosine arabinoside (Ara-C) reduced neurogenesis and retarded extinction. Silencing Sonic hedgehog (Shh) gene with short hairpin interfering RNA (shRNA) by means of a retrovirus expression system to knockdown Shh specifically in the mitotic neurons reduced neurogenesis and retarded extinction. By contrast, over-expression of Shh increased neurogenesis and facilitated extinction. These results suggest that amygdala neurogenesis and Shh signaling are involved in the extinction of fear memory.

  19. Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics.

    PubMed

    Moore, Bryn S; Stepanchick, Ann N; Tewson, Paul H; Hartle, Cassandra M; Zhang, Jin; Quinn, Anne Marie; Hughes, Thomas E; Mirshahi, Tooraj

    2016-11-15

    Protein kinase A (PKA) phosphorylates Gli proteins, acting as a negative regulator of the Hedgehog pathway. PKA was recently detected within the cilium, and PKA activity specifically in cilia regulates Gli processing. Using a cilia-targeted genetically encoded sensor, we found significant basal PKA activity. Using another targeted sensor, we measured basal ciliary cAMP that is fivefold higher than whole-cell cAMP. The elevated basal ciliary cAMP level is a result of adenylyl cyclase 5 and 6 activity that depends on ciliary phosphatidylinositol (3,4,5)-trisphosphate (PIP3), not stimulatory G protein (Gαs), signaling. Sonic Hedgehog (SHH) reduces ciliary cAMP levels, inhibits ciliary PKA activity, and increases Gli1. Remarkably, SHH regulation of ciliary cAMP and downstream signals is not dependent on inhibitory G protein (Gαi/o) signaling but rather Ca(2+) entry through a Gd(3+)-sensitive channel. Therefore, PIP3 sustains high basal cAMP that maintains PKA activity in cilia and Gli repression. SHH activates Gli by inhibiting cAMP through a G protein-independent mechanism that requires extracellular Ca(2+) entry.

  20. Generation of reactive astrocytes from NG2 cells is regulated by sonic hedgehog.

    PubMed

    Honsa, Pavel; Valny, Martin; Kriska, Jan; Matuskova, Hana; Harantova, Lenka; Kirdajova, Denisa; Valihrach, Lukas; Androvic, Peter; Kubista, Mikael; Anderova, Miroslava

    2016-09-01

    NG2 cells, a fourth glial cell type in the adult mammalian central nervous system, produce oligodendrocytes in the healthy nervous tissue, and display wide differentiation potential under pathological conditions, where they could give rise to reactive astrocytes. The factors that control the differentiation of NG2 cells after focal cerebral ischemia (FCI) are largely unknown. Here, we used transgenic Cspg4-cre/Esr1/ROSA26Sortm14(CAG-tdTomato) mice, in which tamoxifen administration triggers the expression of red fluorescent protein (tomato) specifically in NG2 cells and cells derived therefrom. Differentiation potential (in vitro and in vivo) of tomato-positive NG2 cells from control or postischemic brains was determined using the immunohistochemistry, single cell RT-qPCR and patch-clamp method. The ischemic injury was induced by middle cerebral artery occlusion, a model of FCI. Using genetic fate-mapping method, we identified sonic hedgehog (Shh) as an important factor that influences differentiation of NG2 cells into astrocytes in vitro. We also manipulated Shh signaling in the adult mouse brain after FCI. Shh signaling activation significantly increased the number of astrocytes derived from NG2 cells in the glial scar around the ischemic lesion, while Shh signaling inhibition caused the opposite effect. Since Shh signaling modifications did not change the proliferation rate of NG2 cells, we can conclude that Shh has a direct influence on the differentiation of NG2 cells and therefore, on the formation and composition of a glial scar, which consequently affects the degree of the brain damage. GLIA 2016;64:1518-1531.

  1. Sonic hedgehog signaling inhibition provides opportunities for targeted therapy by sulforaphane in regulating pancreatic cancer stem cell self-renewal.

    PubMed

    Rodova, Mariana; Fu, Junsheng; Watkins, Dara Nall; Srivastava, Rakesh K; Shankar, Sharmila

    2012-01-01

    Dysregulation of the sonic hedgehog (Shh) signaling pathway has been associated with cancer stem cells (CSC) and implicated in the initiation of pancreatic cancer. Pancreatic CSCs are rare tumor cells characterized by their ability to self-renew, and are responsible for tumor recurrence accompanied by resistance to current therapies. The lethality of these incurable, aggressive and invasive pancreatic tumors remains a daunting clinical challenge. Thus, the objective of this study was to investigate the role of Shh pathway in pancreatic cancer and to examine the molecular mechanisms by which sulforaphane (SFN), an active compound in cruciferous vegetables, inhibits self-renewal capacity of human pancreatic CSCs. Interestingly, we demonstrate here that Shh pathway is highly activated in pancreatic CSCs and plays important role in maintaining stemness by regulating the expression of stemness genes. Given the requirement for Hedgehog in pancreatic cancer, we investigated whether hedgehog blockade by SFN could target the stem cell population in pancreatic cancer. In an in vitro model, human pancreatic CSCs derived spheres were significantly inhibited on treatment with SFN, suggesting the clonogenic depletion of the CSCs. Interestingly, SFN inhibited the components of Shh pathway and Gli transcriptional activity. Interference of Shh-Gli signaling significantly blocked SFN-induced inhibitory effects demonstrating the requirement of an active pathway for the growth of pancreatic CSCs. SFN also inhibited downstream targets of Gli transcription by suppressing the expression of pluripotency maintaining factors (Nanog and Oct-4) as well as PDGFRα and Cyclin D1. Furthermore, SFN induced apoptosis by inhibition of BCL-2 and activation of caspases. Our data reveal the essential role of Shh-Gli signaling in controlling the characteristics of pancreatic CSCs. We propose that pancreatic cancer preventative effects of SFN may result from inhibition of the Shh pathway. Thus

  2. Sonic hedgehog regulates its own receptor on postcrossing commissural axons in a glypican1-dependent manner.

    PubMed

    Wilson, Nicole H; Stoeckli, Esther T

    2013-08-07

    Upon reaching their intermediate target, the floorplate, commissural axons acquire responsiveness to repulsive guidance cues, allowing the axons to exit the midline and adopt a contralateral, longitudinal trajectory. The molecular mechanisms that regulate this switch from attraction to repulsion remain poorly defined. Here, we show that the heparan sulfate proteoglycan Glypican1 (GPC1) is required as a coreceptor for the Shh-dependent induction of Hedgehog-interacting protein (Hhip) in commissural neurons. In turn, Hhip is required for postcrossing axons to respond to a repulsive anteroposterior Shh gradient. Thus, Shh is a cue with dual function. In precrossing axons it acts as an attractive guidance molecule in a transcription-independent manner. At the same time, Shh binds to GPC1 to induce the expression of its own receptor, Hhip, which mediates the repulsive response of postcrossing axons to Shh. Our study characterizes a molecular mechanism by which navigating axons switch their responsiveness at intermediate targets.

  3. Inhibitors of Hedgehog acyltransferase block Sonic Hedgehog signaling.

    PubMed

    Petrova, Elissaveta; Rios-Esteves, Jessica; Ouerfelli, Ouathek; Glickman, J Fraser; Resh, Marilyn D

    2013-04-01

    Inhibition of Sonic hedgehog (Shh) signaling is of great clinical interest. Here we exploit Hedgehog acyltransferase (Hhat)-mediated Shh palmitoylation, a modification critical for Shh signaling, as a new target for Shh pathway inhibition. A target-oriented high-throughput screen was used to identify small-molecule inhibitors of Hhat. In cells, these Hhat inhibitors specifically block Shh palmitoylation and inhibit autocrine and paracrine Shh signaling.

  4. Targeting sonic hedgehog signaling in neurological disorders.

    PubMed

    Patel, Sita Sharan; Tomar, Sunil; Sharma, Diksha; Mahindroo, Neeraj; Udayabanu, Malairaman

    2017-03-01

    Sonic hedgehog (Shh) signaling influences neurogenesis and neural patterning during the development of central nervous system. Dysregulation of Shh signaling in brain leads to neurological disorders like autism spectrum disorder, depression, dementia, stroke, Parkinson's diseases, Huntington's disease, locomotor deficit, epilepsy, demyelinating disease, neuropathies as well as brain tumors. The synthesis, processing and transport of Shh ligand as well as the localization of its receptors and signal transduction in the central nervous system has been carefully reviewed. Further, we summarize the regulation of small molecule modulators of Shh pathway with potential in neurological disorders. In conclusion, further studies are warranted to demonstrate the potential of positive and negative regulators of the Shh pathway in neurological disorders.

  5. Primary cilia and graded Sonic Hedgehog signaling.

    PubMed

    Sasai, Noriaki; Briscoe, James

    2012-01-01

    Cilia are evolutionary-conserved microtubule-containing organelles protruding from the surface of cells. They are classified into two types--primary and motile cilia. Primary cilia are nearly ubiquitous, at least in vertebrate cells, and it has become apparent that they play an essential role in the intracellular transduction of a range of stimuli. Most notable among these is Sonic Hedgehog. In this article we briefly summarize the structure and biogenesis of primary cilia. We discuss the evidence implicating cilia in the transduction of extrinsic signals. We focus on the involvement and molecular mechanism of cilia in signaling by Sonic Hedgehog in embryonic tissues, specifically the neural tube, and we discuss how cilia play an active role in the interpretation of gradients of Sonic Hedgehog (Shh) signaling.

  6. Chronic up-regulation of sonic hedgehog has little effect on postnatal craniofacial morphology of euploid and trisomic mice

    PubMed Central

    Singh, Nandini; Dutka, Tara; Reeves, Roger H.; Richtsmeier, Joan T.

    2015-01-01

    Background In Ts65Dn, a mouse model of Down syndrome (DS), brain and craniofacial abnormalities that parallel those in people with DS are linked to an attenuated cellular response to sonic hedgehog (SHH) signaling. If a similarly reduced response to SHH occurs in all trisomic cells, then chronic up-regulation of the pathway might have a positive effect on development in trisomic mice, resulting in amelioration of the craniofacial anomalies. Results We crossed Ts65Dn with Ptch1tm1Mps/+ mice and quantified the craniofacial morphology of Ts65Dn;Ptch+/− offspring to assess whether a chronic up-regulation of the SHH pathway rescued DS-related anomalies. Ts65Dn;Ptch1+/− mice experience a chronic increase in SHH in SHH-receptive cells due to haploinsufficiency of the pathway suppressor, Ptch1. Chronic up-regulation had minimal effect on craniofacial shape and did not correct facial abnormalities in Ts65Dn;Ptch+/− mice. We further compared effects of this chronic up-regulation of SHH to acute pathway stimulation in mice treated on the day of birth with a SHH pathway agonist, SAG. We found that SHH affects facial morphology differently based on chronic vs. acute postnatal pathway up-regulation. Conclusions Our findings have implications for understanding the function of SHH in craniofacial development and for the potential use of SHH-based agonists to treat DS-related abnormalities. PMID:26509735

  7. Tibial hemimelia-polydactyly-five-fingered hand syndrome associated with a 404 G>A mutation in a distant sonic hedgehog cis-regulator (ZRS): a case report.

    PubMed

    Cho, Tae-Joon; Baek, Goo Hyun; Lee, Hey-Ran; Moon, Hyuk Joo; Yoo, Won Joon; Choi, In Ho

    2013-05-01

    Tibial hemimelia-polydactyly-triphalangeal thumb syndrome is a distinct congenital limb anomaly complex, whose association with the 404 G>A mutation in a distant sonic hedgehog cis-regulator (ZRS) was suggested. The authors report a sporadic case of bilateral tibial hemimelia-preaxial polydactyly-five-fingered hands harboring the same mutation. This case further supports a causal relationship between this mutation and the phenotype.

  8. Wnt/β-Catenin and Sonic Hedgehog Pathways Interact in the Regulation of the Development of the Dorsal Mesenchymal Protrusion

    PubMed Central

    Briggs, Laura E.; Burns, Tara A.; Lockhart, Marie M.; Phelps, Aimee L.; Van den Hoff, Maurice J.B.; Wessels, Andy

    2016-01-01

    Background The dorsal mesenchymal protrusion (DMP) is a second heart field (SHF) derived tissue involved in cardiac septation. Molecular mechanisms controlling SHF/DMP development include the Bone Morphogenetic Protein and Wnt/β-catenin signaling pathways. Reduced expression of components in these pathways leads to inhibition of proliferation of the SHF/DMP precursor population and failure of the DMP to develop. While the Sonic Hedgehog (Shh) pathway has also been demonstrated to be critically important for SHF/DMP development and atrioventricular septation, its role in the regulation of SHF proliferation is contentious. Results Tissue-specific deletion of the Shh receptor Smoothened from the SHF resulted in compromised DMP formation and atrioventricular septal defects (AVSDs). Immunohistochemical analysis at critical stages of DMP development showed significant proliferation defect as well as reduction in levels of the Wnt/β-catenin pathway-intermediates β-catenin, Lef1, and Axin2. To determine whether the defects seen in the conditional Smoothened knock-out mouse could be attributed to reduced Wnt/β-catenin signaling, LiCl, a pharmacological activator of this Wnt/β-catenin pathway, was administered. This resulted in restoration of proliferation and partial rescue of the AVSD phenotype. Conclusions The data presented suggest that the Wnt/β-catenin pathway interact with the Shh pathway in the regulation of SHF/DMP-precursor proliferation and, hence, the development of the DMP. PMID:26297872

  9. LKB1 Regulates Cerebellar Development by Controlling Sonic Hedgehog-mediated Granule Cell Precursor Proliferation and Granule Cell Migration

    PubMed Central

    Men, Yuqin; Zhang, Aizhen; Li, Haixiang; Jin, Yecheng; Sun, Xiaoyang; Li, Huashun; Gao, Jiangang

    2015-01-01

    The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1Atoh1 CKO) to investigate the function of LKB1 in cerebellar development. The LKB1Atoh1 CKO mice displayed motor dysfunction. In the LKB1Atoh1 CKO cerebellum, the overall structure had a larger volume and morelobules. LKB1 inactivationled to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1Atoh1 CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development. PMID:26549569

  10. miR-326 Is Downstream of Sonic Hedgehog Signaling and Regulates the Expression of Gli2 and Smoothened

    PubMed Central

    Jiang, Zhihua; Cushing, Leah; Lü, Jining

    2014-01-01

    Sonic hedgehog (Shh) is expressed and secreted from the embryonic lung epithelium and acts on the adjacent mesenchymal cells via its receptor Patched (Ptch)/Smoothened (Smo) and transcriptional effectors Gli proteins. Genetic studies showed that the Shh pathway plays critical roles in mouse lung development. However, little is known about microRNAs (miRNAs) downstream of Shh in embryonic lungs. Here we profiled miRNAs in embryonic lung cultures treated with cyclopamine, a specific Smo antagonist or with Smo agonist by next-generation of sequencing. We then performed functional screening to examine whether some of these miRNAs can modulate the induction of Gli-responsive luciferase by Shh treatment. These analyses revealed that expression of miR-326 and its host gene, Arrestin β1, is selectively enriched in embryonic lung mesenchymal cells and is specifically influenced by Shh activity. Furthermore, functional analyses showed that miR-326 acts as a negative modulator for Shh signaling by directly targeting Smo and Gli2. Together, these findings suggest a novel miR-326–negative feedback loop in regulating the activity of Shh signaling. PMID:24617895

  11. miR-326 is downstream of Sonic hedgehog signaling and regulates the expression of Gli2 and smoothened.

    PubMed

    Jiang, Zhihua; Cushing, Leah; Ai, Xingbin; Lü, Jining

    2014-08-01

    Sonic hedgehog (Shh) is expressed and secreted from the embryonic lung epithelium and acts on the adjacent mesenchymal cells via its receptor Patched (Ptch)/Smoothened (Smo) and transcriptional effectors Gli proteins. Genetic studies showed that the Shh pathway plays critical roles in mouse lung development. However, little is known about microRNAs (miRNAs) downstream of Shh in embryonic lungs. Here we profiled miRNAs in embryonic lung cultures treated with cyclopamine, a specific Smo antagonist or with Smo agonist by next-generation of sequencing. We then performed functional screening to examine whether some of these miRNAs can modulate the induction of Gli-responsive luciferase by Shh treatment. These analyses revealed that expression of miR-326 and its host gene, Arrestin β1, is selectively enriched in embryonic lung mesenchymal cells and is specifically influenced by Shh activity. Furthermore, functional analyses showed that miR-326 acts as a negative modulator for Shh signaling by directly targeting Smo and Gli2. Together, these findings suggest a novel miR-326-negative feedback loop in regulating the activity of Shh signaling.

  12. Tbx1, a DiGeorge syndrome candidate gene, is regulated by sonic hedgehog during pharyngeal arch development.

    PubMed

    Garg, V; Yamagishi, C; Hu, T; Kathiriya, I S; Yamagishi, H; Srivastava, D

    2001-07-01

    Appropriate interactions between the epithelium and adjacent neural crest-derived mesenchyme are necessary for normal pharyngeal arch development. Disruption of pharyngeal arch development in humans underlies many of the craniofacial defects observed in the 22q11.2 deletion syndrome (del22q11), but the genes responsible remain unknown. Tbx1 is a T-box transcription factor that lies in the 22q11.2 locus. Tbx1 transcripts were found to be localized to the pharyngeal endoderm and the mesodermal core of the pharyngeal arches, but were not present in the neural crest-derived mesenchyme of the pharyngeal arches. Sonic hedgehog (Shh) is also expressed in the pharyngeal arches and is necessary for normal craniofacial development. We found that Tbx1 expression was dependent upon Shh signaling in mouse embryos, consistent with their overlapping expression in the pharyngeal arches. Furthermore, Shh was sufficient to induce Tbx1 expression when misexpressed in selected regions of chick embryos. These studies reveal a Shh-mediated pathway that regulates Tbx1 during pharyngeal arch development.

  13. Sonic hedgehog acts as a negative regulator of {beta}-catenin signaling in the adult tongue epithelium.

    PubMed

    Schneider, Fabian T; Schänzer, Anne; Czupalla, Cathrin J; Thom, Sonja; Engels, Knut; Schmidt, Mirko H H; Plate, Karl H; Liebner, Stefan

    2010-07-01

    Wnt/beta-catenin signaling has been implicated in taste papilla development; however, its role in epithelial maintenance and tumor progression in the adult tongue remains elusive. We show Wnt/beta-catenin pathway activation in reporter mice and by nuclear beta-catenin staining in the epithelium and taste papilla of adult mouse and human tongues. beta-Catenin activation in APC(min/+) mice, which carry a mutation in adenomatous poliposis coli (APC), up-regulates Sonic hedgehog (Shh) and Jagged-2 (JAG2) in the tongue epithelium without formation of squamous cell carcinoma (SCC). We demonstrate that Shh suppresses beta-catenin transcriptional activity in a signaling-dependent manner in vitro and in vivo. A similar regulation and function was observed for JAG2, suggesting that both pathways negatively regulate beta-catenin, thereby preventing SCC formation in the tongue. This was supported by reduced nuclear beta-catenin in the tongue epithelium of Patched(+/-) mice, exhibiting dominant active Shh signaling. At the invasive front of human tongue cancer, nuclear beta-catenin and Shh were increased, suggesting their participation in tumor progression. Interestingly, Shh but not JAG2 was able to reduce beta-catenin signaling in SCC cells, arguing for a partial loss of negative feedback on beta-catenin transcription in tongue cancer. We show for the first time that the putative Wnt/beta-catenin targets Shh and JAG2 control beta-catenin signaling in the adult tongue epithelium, a function that is partially lost in lingual SCC.

  14. Sonic Hedgehog Acts as a Negative Regulator of β-Catenin Signaling in the Adult Tongue Epithelium

    PubMed Central

    Schneider, Fabian T.; Schänzer, Anne; Czupalla, Cathrin J.; Thom, Sonja; Engels, Knut; Schmidt, Mirko H.H.; Plate, Karl H.; Liebner, Stefan

    2010-01-01

    Wnt/β-catenin signaling has been implicated in taste papilla development; however, its role in epithelial maintenance and tumor progression in the adult tongue remains elusive. We show Wnt/β-catenin pathway activation in reporter mice and by nuclear β-catenin staining in the epithelium and taste papilla of adult mouse and human tongues. β-Catenin activation in APCmin/+ mice, which carry a mutation in adenomatous poliposis coli (APC), up-regulates Sonic hedgehog (Shh) and Jagged-2 (JAG2) in the tongue epithelium without formation of squamous cell carcinoma (SCC). We demonstrate that Shh suppresses β-catenin transcriptional activity in a signaling-dependent manner in vitro and in vivo. A similar regulation and function was observed for JAG2, suggesting that both pathways negatively regulate β-catenin, thereby preventing SCC formation in the tongue. This was supported by reduced nuclear β-catenin in the tongue epithelium of Patched+/− mice, exhibiting dominant active Shh signaling. At the invasive front of human tongue cancer, nuclear β-catenin and Shh were increased, suggesting their participation in tumor progression. Interestingly, Shh but not JAG2 was able to reduce β-catenin signaling in SCC cells, arguing for a partial loss of negative feedback on β-catenin transcription in tongue cancer. We show for the first time that the putative Wnt/β-catenin targets Shh and JAG2 control β-catenin signaling in the adult tongue epithelium, a function that is partially lost in lingual SCC. PMID:20508033

  15. Polycomb-Mediated Repression and Sonic Hedgehog Signaling Interact to Regulate Merkel Cell Specification during Skin Development

    PubMed Central

    Bar, Carmit; Tsai, Pai-Chi; Valdes, Victor J.; Cohen, Idan; Santoriello, Francis J.; Zhao, Dejian; Hsu, Ya-Chieh; Ezhkova, Elena

    2016-01-01

    An increasing amount of evidence indicates that developmental programs are tightly regulated by the complex interplay between signaling pathways, as well as transcriptional and epigenetic processes. Here, we have uncovered coordination between transcriptional and morphogen cues to specify Merkel cells, poorly understood skin cells that mediate light touch sensations. In murine dorsal skin, Merkel cells are part of touch domes, which are skin structures consisting of specialized keratinocytes, Merkel cells, and afferent neurons, and are located exclusively around primary hair follicles. We show that the developing primary hair follicle functions as a niche required for Merkel cell specification. We find that intraepidermal Sonic hedgehog (Shh) signaling, initiated by the production of Shh ligand in the developing hair follicles, is required for Merkel cell specification. The importance of Shh for Merkel cell formation is further reinforced by the fact that Shh overexpression in embryonic epidermal progenitors leads to ectopic Merkel cells. Interestingly, Shh signaling is common to primary, secondary, and tertiary hair follicles, raising the possibility that there are restrictive mechanisms that regulate Merkel cell specification exclusively around primary hair follicles. Indeed, we find that loss of Polycomb repressive complex 2 (PRC2) in the epidermis results in the formation of ectopic Merkel cells that are associated with all hair types. We show that PRC2 loss expands the field of epidermal cells competent to differentiate into Merkel cells through the upregulation of key Merkel-differentiation genes, which are known PRC2 targets. Importantly, PRC2-mediated repression of the Merkel cell differentiation program requires inductive Shh signaling to form mature Merkel cells. Our study exemplifies how the interplay between epigenetic and morphogen cues regulates the complex patterning and formation of the mammalian skin structures. PMID:27414999

  16. An integrated approach identifies Nhlh1 and Insm1 as Sonic Hedgehog-regulated genes in developing cerebellum and medulloblastoma.

    PubMed

    De Smaele, Enrico; Fragomeli, Caterina; Ferretti, Elisabetta; Pelloni, Marianna; Po, Agnese; Canettieri, Gianluca; Coni, Sonia; Di Marcotullio, Lucia; Greco, Azzura; Moretti, Marta; Di Rocco, Concezio; Pazzaglia, Simona; Maroder, Marella; Screpanti, Isabella; Giannini, Giuseppe; Gulino, Alberto

    2008-01-01

    Medulloblastoma (MB) is the most common malignant brain tumor of childhood arising from deregulated cerebellar development. Sonic Hedgehog (Shh) pathway plays a critical role in cerebellar development and its aberrant expression has been identified in MB. Gene expression profiling of cerebella from 1- to 14-day-old mice unveiled a cluster of genes whose expression correlates with the levels of Hedgehog (HH) activity. From this cluster, we identified Insm1 and Nhlh1/NSCL1 as novel HH targets induced by Shh treatment in cultured cerebellar granule cell progenitors. Nhlh1 promoter was found to be bound and activated by Gli1 transcription factor. Remarkably, the expression of these genes is also upregulated in mouse and human HH-dependent MBs, suggesting that they may be either a part of the HH-induced tumorigenic process or a specific trait of HH-dependent tumor cells.

  17. The matricellular protein CCN1/Cyr61 is a critical regulator of Sonic Hedgehog in pancreatic carcinogenesis.

    PubMed

    Haque, Inamul; De, Archana; Majumder, Monami; Mehta, Smita; McGregor, Douglas; Banerjee, Sushanta K; Van Veldhuizen, Peter; Banerjee, Snigdha

    2012-11-09

    CCN1 is a matricellular protein and a member of the CCN family of growth factors. CCN1 is associated with the development of various cancers including pancreatic ductal adenocarcinoma (PDAC). Our recent studies found that CCN1 plays a critical role in pancreatic carcinogenesis through the induction of EMT and stemness. CCN1 mRNA and protein were detected in the early precursor lesions, and their expression intensified with disease progression. However, biochemical activity and the molecular targets of CCN1 in pancreatic cancer cells are unknown. Here we show that CCN1 regulates the Sonic Hedgehog (SHh) signaling pathway, which is associated with the PDAC progression and poor prognosis. SHh regulation by CCN1 in pancreatic cancer cells is mediated through the active Notch-1. Notably, active Notch-1is recruited by CCN1 in these cells via the inhibition of proteasomal degradation results in stabilization of the receptor. We find that CCN1-induced activation of SHh signaling might be necessary for CCN1-dependent in vitro pancreatic cancer cell migration and tumorigenicity of the side population of pancreatic cancer cells (cancer stem cells) in a xenograft in nude mice. Moreover, the functional role of CCN1 could be mediated through the interaction with the αvβ3 integrin receptor. These extensive studies propose that targeting CCN1 can provide a new treatment option for patients with pancreatic cancer since blocking CCN1 simultaneously blocks two critical pathways (i.e. SHh and Notch1) associated with the development of the disease as well as drug resistance.

  18. The Matricellular Protein CCN1/Cyr61 Is a Critical Regulator of Sonic Hedgehog in Pancreatic Carcinogenesis*

    PubMed Central

    Haque, Inamul; De, Archana; Majumder, Monami; Mehta, Smita; McGregor, Douglas; Banerjee, Sushanta K.; Van Veldhuizen, Peter; Banerjee, Snigdha

    2012-01-01

    CCN1 is a matricellular protein and a member of the CCN family of growth factors. CCN1 is associated with the development of various cancers including pancreatic ductal adenocarcinoma (PDAC). Our recent studies found that CCN1 plays a critical role in pancreatic carcinogenesis through the induction of EMT and stemness. CCN1 mRNA and protein were detected in the early precursor lesions, and their expression intensified with disease progression. However, biochemical activity and the molecular targets of CCN1 in pancreatic cancer cells are unknown. Here we show that CCN1 regulates the Sonic Hedgehog (SHh) signaling pathway, which is associated with the PDAC progression and poor prognosis. SHh regulation by CCN1 in pancreatic cancer cells is mediated through the active Notch-1. Notably, active Notch-1is recruited by CCN1 in these cells via the inhibition of proteasomal degradation results in stabilization of the receptor. We find that CCN1-induced activation of SHh signaling might be necessary for CCN1-dependent in vitro pancreatic cancer cell migration and tumorigenicity of the side population of pancreatic cancer cells (cancer stem cells) in a xenograft in nude mice. Moreover, the functional role of CCN1 could be mediated through the interaction with the αvβ3 integrin receptor. These extensive studies propose that targeting CCN1 can provide a new treatment option for patients with pancreatic cancer since blocking CCN1 simultaneously blocks two critical pathways (i.e. SHh and Notch1) associated with the development of the disease as well as drug resistance. PMID:23027863

  19. MYB Promotes Desmoplasia in Pancreatic Cancer through Direct Transcriptional Up-regulation and Cooperative Action of Sonic Hedgehog and Adrenomedullin.

    PubMed

    Bhardwaj, Arun; Srivastava, Sanjeev K; Singh, Seema; Tyagi, Nikhil; Arora, Sumit; Carter, James E; Khushman, Moh'd; Singh, Ajay P

    2016-07-29

    Extensive desmoplasia is a prominent pathological characteristic of pancreatic cancer (PC) that not only impacts tumor development, but therapeutic outcome as well. Recently, we demonstrated a novel role of MYB, an oncogenic transcription factor, in PC growth and metastasis. Here we studied its effect on pancreatic tumor histopathology and associated molecular and biological mechanisms. Tumor-xenografts derived from orthotopic-inoculation of MYB-overexpressing PC cells exhibited far-greater desmoplasia in histological analyses compared with those derived from MYB-silenced PC cells. These findings were further confirmed by immunostaining of tumor-xenograft sections with collagen-I, fibronectin (major extracellular-matrix proteins), and α-SMA (well-characterized marker of myofibroblasts or activated pancreatic stellate cells (PSCs)). Likewise, MYB-overexpressing PC cells provided significantly greater growth benefit to PSCs in a co-culture system as compared with the MYB-silenced cells. Interrogation of deep-sequencing data from MYB-overexpressing versus -silenced PC cells identified Sonic-hedgehog (SHH) and Adrenomedullin (ADM) as two differentially-expressed genes among others, which encode for secretory ligands involved in tumor-stromal cross-talk. In-silico analyses predicted putative MYB-binding sites in SHH and ADM promoters, which was later confirmed by chromatin-immunoprecipitation. A cooperative role of SHH and ADM in growth promotion of PSCs was confirmed in co-culture by using their specific-inhibitors and exogenous recombinant-proteins. Importantly, while SHH acted exclusively in a paracrine fashion on PSCs and influenced the growth of PC cells only indirectly, ADM could directly impact the growth of both PC cells and PSCs. In summary, we identified MYB as novel regulator of pancreatic tumor desmoplasia, which is suggestive of its diverse roles in PC pathobiology.

  20. Sonic Hedgehog Signaling in Limb Development

    PubMed Central

    Tickle, Cheryll; Towers, Matthew

    2017-01-01

    The gene encoding the secreted protein Sonic hedgehog (Shh) is expressed in the polarizing region (or zone of polarizing activity), a small group of mesenchyme cells at the posterior margin of the vertebrate limb bud. Detailed analyses have revealed that Shh has the properties of the long sought after polarizing region morphogen that specifies positional values across the antero-posterior axis (e.g., thumb to little finger axis) of the limb. Shh has also been shown to control the width of the limb bud by stimulating mesenchyme cell proliferation and by regulating the antero-posterior length of the apical ectodermal ridge, the signaling region required for limb bud outgrowth and the laying down of structures along the proximo-distal axis (e.g., shoulder to digits axis) of the limb. It has been shown that Shh signaling can specify antero-posterior positional values in limb buds in both a concentration- (paracrine) and time-dependent (autocrine) fashion. Currently there are several models for how Shh specifies positional values over time in the limb buds of chick and mouse embryos and how this is integrated with growth. Extensive work has elucidated downstream transcriptional targets of Shh signaling. Nevertheless, it remains unclear how antero-posterior positional values are encoded and then interpreted to give the particular structure appropriate to that position, for example, the type of digit. A distant cis-regulatory enhancer controls limb-bud-specific expression of Shh and the discovery of increasing numbers of interacting transcription factors indicate complex spatiotemporal regulation. Altered Shh signaling is implicated in clinical conditions with congenital limb defects and in the evolution of the morphological diversity of vertebrate limbs. PMID:28293554

  1. Sonic Hedgehog Signaling in Limb Development.

    PubMed

    Tickle, Cheryll; Towers, Matthew

    2017-01-01

    The gene encoding the secreted protein Sonic hedgehog (Shh) is expressed in the polarizing region (or zone of polarizing activity), a small group of mesenchyme cells at the posterior margin of the vertebrate limb bud. Detailed analyses have revealed that Shh has the properties of the long sought after polarizing region morphogen that specifies positional values across the antero-posterior axis (e.g., thumb to little finger axis) of the limb. Shh has also been shown to control the width of the limb bud by stimulating mesenchyme cell proliferation and by regulating the antero-posterior length of the apical ectodermal ridge, the signaling region required for limb bud outgrowth and the laying down of structures along the proximo-distal axis (e.g., shoulder to digits axis) of the limb. It has been shown that Shh signaling can specify antero-posterior positional values in limb buds in both a concentration- (paracrine) and time-dependent (autocrine) fashion. Currently there are several models for how Shh specifies positional values over time in the limb buds of chick and mouse embryos and how this is integrated with growth. Extensive work has elucidated downstream transcriptional targets of Shh signaling. Nevertheless, it remains unclear how antero-posterior positional values are encoded and then interpreted to give the particular structure appropriate to that position, for example, the type of digit. A distant cis-regulatory enhancer controls limb-bud-specific expression of Shh and the discovery of increasing numbers of interacting transcription factors indicate complex spatiotemporal regulation. Altered Shh signaling is implicated in clinical conditions with congenital limb defects and in the evolution of the morphological diversity of vertebrate limbs.

  2. The miR-17/92 polycistron is up-regulated in sonic hedgehog-driven medulloblastomas and induced by N-myc in sonic hedgehog-treated cerebellar neural precursors.

    PubMed

    Northcott, Paul A; Fernandez-L, Africa; Hagan, John P; Ellison, David W; Grajkowska, Wesia; Gillespie, Yancey; Grundy, Richard; Van Meter, Timothy; Rutka, James T; Croce, Carlo M; Kenney, Anna Marie; Taylor, Michael D

    2009-04-15

    Medulloblastoma is the most common malignant pediatric brain tumor, and mechanisms underlying its development are poorly understood. We identified recurrent amplification of the miR-17/92 polycistron proto-oncogene in 6% of pediatric medulloblastomas by high-resolution single-nucleotide polymorphism genotyping arrays and subsequent interphase fluorescence in situ hybridization on a human medulloblastoma tissue microarray. Profiling the expression of 427 mature microRNAs (miRNA) in a series of 90 primary human medulloblastomas revealed that components of the miR-17/92 polycistron are the most highly up-regulated miRNAs in medulloblastoma. Expression of miR-17/92 was highest in the subgroup of medulloblastomas associated with activation of the sonic hedgehog (Shh) signaling pathway compared with other subgroups of medulloblastoma. Medulloblastomas in which miR-17/92 was up-regulated also had elevated levels of MYC/MYCN expression. Consistent with its regulation by Shh, we observed that Shh treatment of primary cerebellar granule neuron precursors (CGNP), proposed cells of origin for the Shh-associated medulloblastomas, resulted in increased miR-17/92 expression. In CGNPs, the Shh effector N-myc, but not Gli1, induced miR-17/92 expression. Ectopic miR-17/92 expression in CGNPs synergized with exogenous Shh to increase proliferation and also enabled them to proliferate in the absence of Shh. We conclude that miR-17/92 is a positive effector of Shh-mediated proliferation and that aberrant expression/amplification of this miR confers a growth advantage to medulloblastomas.

  3. Sonic hedgehog patterning during cerebellar development.

    PubMed

    De Luca, Annarita; Cerrato, Valentina; Fucà, Elisa; Parmigiani, Elena; Buffo, Annalisa; Leto, Ketty

    2016-01-01

    The morphogenic factor sonic hedgehog (Shh) actively orchestrates many aspects of cerebellar development and maturation. During embryogenesis, Shh signaling is active in the ventricular germinal zone (VZ) and represents an essential signal for proliferation of VZ-derived progenitors. Later, Shh secreted by Purkinje cells sustains the amplification of postnatal neurogenic niches: the external granular layer and the prospective white matter, where excitatory granule cells and inhibitory interneurons are produced, respectively. Moreover, Shh signaling affects Bergmann glial differentiation and promotes cerebellar foliation during development. Here we review the most relevant functions of Shh during cerebellar ontogenesis, underlying its role in physiological and pathological conditions.

  4. Sonic hedgehog signaling in the postnatal brain.

    PubMed

    Álvarez-Buylla, Arturo; Ihrie, Rebecca A

    2014-09-01

    Sonic hedgehog (Shh) is a pleiotropic factor in the developing central nervous system (CNS), driving proliferation, specification, and axonal targeting in multiple sites within the forebrain, hindbrain, and spinal cord. Studies in embryonic CNS have shown how gradients of this morphogen are translated by neuroepithelial precursors to determine the types of neurons and glial cells they produce [1,2]. Shh also has a well-characterized role as a mitogen for specific progenitor cell types in neural development [3,4]. As we begin to appreciate that Shh continues to act in the adult brain, a central question is what functional role this ligand plays when major morphogenetic and proliferative processes are no longer in operation. A second fundamental question is whether similar signaling mechanisms operate in embryonic and adult CNS. In the two major germinal zones of the adult brain, Shh signaling modulates the self-renewal and specification of astrocyte-like primary progenitors, frequently referred to as neural stem cells (NSCs). It also may regulate the response of the mature brain to injury, as Shh signaling has been variously proposed to enhance or inhibit the development of a reactive astrocyte phenotype. The identity of cells producing the Shh ligand, and the conditions that trigger its release, are also areas of growing interest; both germinal zones in the adult brain contain Shh-responsive cells but do not autonomously produce this ligand. Here, we review recent findings revealing the function of this fascinating pathway in the postnatal and adult brain, and highlight ongoing areas of investigation into its actions long past the time when it shapes the developing brain.

  5. Sonic hedgehog: restricted expression and limb dysmorphologies.

    PubMed

    Hill, Robert E; Heaney, Simon J H; Lettice, Laura A

    2003-01-01

    Sonic hedgehog, SHH, is required for patterning the limb. The array of skeletal elements that compose the hands and feet, and the ordered arrangement of these bones to form the pattern of fingers and toes are dependent on SHH. The mechanism of action of SHH in the limb is not fully understood; however, an aspect that appears to be important is the localized, asymmetric expression of Shh. Shh is expressed in the posterior margin of the limb bud in a region defined as the zone of polarizing activity (ZPA). Analysis of mouse mutants which have polydactyly (extra toes) shows that asymmetric expression of Shh is lost due to the appearance of an ectopic domain of expression in the anterior limb margin. One such polydactylous mouse mutant, sasquatch (Ssq), maps to the corresponding chromosomal region of the human condition pre-axial polydactyly (PPD) and thus represents a model for this condition. The mutation responsible for Ssq is located 1 Mb away from the Shh gene; however, the mutation disrupts a long-range cis-acting regulator of Shh expression. By inference, human pre-axial polydactyly results from a similar disruption of Shh expression. Other human congenital abnormalities also map near the pre-axial polydactyly locus, suggesting a major chromosomal region for limb dysmorphologies. The distinct phenotypes range from loss of all bones of the hands and feet to syndactyly of the soft tissue and fusion of the digits. We discuss the role played by Shh expression in mouse mutant phenotypes and the human limb dysmorphologies.

  6. Sonic hedgehog: restricted expression and limb dysmorphologies

    PubMed Central

    Hill, Robert E; Heaney, Simon JH; Lettice, Laura A

    2003-01-01

    Sonic hedgehog, SHH, is required for patterning the limb. The array of skeletal elements that compose the hands and feet, and the ordered arrangement of these bones to form the pattern of fingers and toes are dependent on SHH. The mechanism of action of SHH in the limb is not fully understood; however, an aspect that appears to be important is the localized, asymmetric expression of Shh. Shh is expressed in the posterior margin of the limb bud in a region defined as the zone of polarizing activity (ZPA). Analysis of mouse mutants which have polydactyly (extra toes) shows that asymmetric expression of Shh is lost due to the appearance of an ectopic domain of expression in the anterior limb margin. One such polydactylous mouse mutant, sasquatch (Ssq), maps to the corresponding chromosomal region of the human condition pre-axial polydactyly (PPD) and thus represents a model for this condition. The mutation responsible for Ssq is located 1 Mb away from the Shh gene; however, the mutation disrupts a long-range cis-acting regulator of Shh expression. By inference, human pre-axial polydactyly results from a similar disruption of Shh expression. Other human congenital abnormalities also map near the pre-axial polydactyly locus, suggesting a major chromosomal region for limb dysmorphologies. The distinct phenotypes range from loss of all bones of the hands and feet to syndactyly of the soft tissue and fusion of the digits. We discuss the role played by Shh expression in mouse mutant phenotypes and the human limb dysmorphologies. PMID:12587915

  7. The regulation of tooth morphogenesis is associated with epithelial cell proliferation and the expression of Sonic hedgehog through epithelial-mesenchymal interactions

    SciTech Connect

    Ishida, Kentaro; Murofushi, Mayumi; Nakao, Kazuhisa; Morita, Ritsuko; Ogawa, Miho; Tsuji, Takashi

    2011-02-18

    Research highlights: {yields} Bioengineered teeth regulated the contact area of epithelium and mesenchyme. {yields} The crown width is regulated by the contact area of the epithelium and mesenchyme. {yields} This regulation is associated with cell proliferation and Sonic hedgehog expression. {yields} The cusp number is correlated with the crown width of the bioengineered tooth. {yields} Cell proliferation and Shh expression areas regulate the tooth morphogenesis. -- Abstract: Ectodermal organs, such as the tooth, salivary gland, hair, and mammary gland, develop through reciprocal epithelial-mesenchymal interactions. Tooth morphologies are defined by the crown width and tooth length (macro-morphologies), and by the number and locations of the cusp and roots (micro-morphologies). In our current study, we report that the crown width of a bioengineered molar tooth, which was reconstructed using dissociated epithelial and mesenchymal cells via an organ germ method, can be regulated by the contact area between epithelial and mesenchymal cell layers. We further show that this is associated with cell proliferation and Sonic hedgehog (Shh) expression in the inner enamel epithelium after the germ stage has formed a secondary enamel knot. We also demonstrate that the cusp number is significantly correlated with the crown width of the bioengineered tooth. These findings suggest that the tooth micro-morphology, i.e. the cusp formation, is regulated after the tooth width, or macro-morphology, is determined. These findings also suggest that the spatiotemporal patterning of cell proliferation and the Shh expression areas in the epithelium regulate the crown width and cusp formation of the developing tooth.

  8. Sonic Hedgehog Signaling and Hippocampal Neuroplasticity.

    PubMed

    Yao, Pamela J; Petralia, Ronald S; Mattson, Mark P

    2016-12-01

    Sonic hedgehog (Shh) is a secreted protein that controls the patterning of neural progenitor cells, and their neuronal and glial progeny, during development. Emerging findings suggest that Shh also has important roles in the formation and plasticity of neuronal circuits in the hippocampus, a brain region of fundamental importance in learning and memory. Shh mediates activity-dependent and injury-induced hippocampal neurogenesis. Activation of Shh receptors in the dendrites of hippocampal neurons engages a trans-neuronal signaling pathway that accelerates axon outgrowth and enhances glutamate release from presynaptic terminals. Impaired Shh signaling may contribute to the pathogenesis of several developmental and adult-onset neurological disorders that affect the hippocampus, suggesting a potential for therapeutic interventions that target Shh pathways.

  9. Anti-apoptotic role of sonic hedgehog on blood platelets.

    PubMed

    Kumari, Sharda; Chaurasia, Susheel N; Kumar, Kailash; Dash, Debabrata

    2014-12-01

    Sonic hedgehog (Shh) is an essential morphogen involved in vertebrate organogenesis. Perturbation of Hh signaling is associated with pathological consequences like tumor formation and chronic lung fibrosis. Platelets are highly sensitive circulating blood cells responsible for hemostasis, while hyperactivity of these cells lead to morbidities like ischemic heart diseases and stroke. Despite being terminally differentiated cells with life span of 10-12 days, platelets have recently been shown to respond to Wnt ligand, another developmental signal similar to Shh. In this study, we demonstrate that components of Shh signaling, Patched and Gli3, are expressed in human platelets consistent with existence of functional Hedgehog signaling in these cells. Shh had potent inhibitory effect on platelet apoptosis induced by ABT-737 or thrombin through attenuation of caspase-3 activity. The Shh-mediated pathway may thus represent a novel endogenous mechanism for regulating platelet activity and life span.

  10. Sonic hedgehog signaling in kidney fibrosis: a master communicator.

    PubMed

    Zhou, Dong; Tan, Roderick J; Liu, Youhua

    2016-09-01

    The hedgehog signaling cascade is an evolutionarily conserved pathway that regulates multiple aspects of embryonic development and plays a decisive role in tissue homeostasis. As the best studied member of three hedgehog ligands, sonic hedgehog (Shh) is known to be associated with kidney development and tissue repair after various insults. Recent studies uncover an intrinsic link between dysregulated Shh signaling and renal fibrogenesis. In various types of chronic kidney disease (CKD), Shh is upregulated specifically in renal tubular epithelium but targets interstitial fibroblasts, thereby mediating a dynamic epithelial- mesenchymal communication (EMC). Tubule-derived Shh acts as a growth factor for interstitial fibroblasts and controls a hierarchy of fibrosis-related genes, which lead to the excessive deposition of extracellular matrix in renal interstitium. In this review, we recapitulate the principle of Shh signaling, its activation and regulation in a variety of kidney diseases. We also discuss the potential mechanisms by which Shh promotes renal fibrosis and assess the efficacy of blocking this signaling in preclinical settings. Continuing these lines of investigations will provide novel opportunities for designing effective therapies to improve CKD prognosis in patients.

  11. Sonic Hedgehog pathway activity in prostate cancer

    PubMed Central

    BRAGINA, OLGA; NJUNKOVA, NATALJA; SERGEJEVA, SVETLANA; JÄRVEKÜLG, LILIAN; KOGERMAN, PRIIT

    2010-01-01

    Abnormal activation of the Sonic hedgehog (Shh) signaling pathway has been demonstrated in a number of human tumors, including prostate cancer. The study aimed to assess the activity of Shh pathway components (Shh, Gli1, Gli2 and Gli3), as well as the proliferation markers FoxA1 and Notch1 during cancer progression in the transgenic adenocarcinoma of the mouse prostate (TRAMP). We evaluated changes in respective proteins by immunohistochemistry at three time points (12, 17 and 21 weeks of age) in the tissue of TRAMP and C57Bl/6 mice. Moreover, the expression of mRNA of these proteins was assessed. The present study shows a significant age-dependent increase in the number of Shh, Gli1, Gli3 and FoxA1-positive prostate cells and a decrease in Gli2-positive cells in TRAMP. The study also supports the hypothesis that the development of prostate cancer and its metastasis is associated with activation of the Shh signaling pathway. PMID:22966302

  12. Probing extracellular Sonic hedgehog in neurons.

    PubMed

    Eitan, Erez; Petralia, Ronald S; Wang, Ya-Xian; Indig, Fred E; Mattson, Mark P; Yao, Pamela J

    2016-08-15

    The bioactivity of Sonic hedgehog (Shh) depends on specific lipid modifications; a palmitate at its N-terminus and a cholesterol at its C-terminus. This dual-lipid modification makes Shh molecules lipophilic, which prevents them from diffusing freely in extracellular space. Multiple lines of evidence indicate that Shh proteins are carried by various forms of extracellular vesicles (EVs). It also has been shown, for instance, that in some tissues Shh proteins are transported to neighboring cells directly via filopodia. We have previously reported that Shh proteins are expressed in hippocampal neurons. In this study we show that, in the hippocampus and cerebellum of postnatal day (P)2 rats, Shh is mostly found near or on the membrane surface of small neurites or filopodia. We also examined cultured hippocampal neurons where we observed noticeable and widespread Shh-immunolabeled vesicles located outside neurons. Through immunoelectron microscopy and biochemical analysis, we find Shh-containing EVs with a wide range of sizes. Unlike robust Shh activity in EVs isolated from cells overexpressing an N-terminal Shh fragment construct, we did not detect measurable Shh activity in EVs purified from the medium of cultured hippocampal neurons. These results suggest the complexity of the transcellular Shh signaling mechanisms in neurons.

  13. Proteoglycan interactions with Sonic Hedgehog specify mitogenic responses

    PubMed Central

    Chan, Jennifer A.; Balasubramanian, Srividya; Witt, Rochelle M.; Nazemi, Kellie J.; Choi, Yoojin; Pazyra-Murphy, Maria F.; Walsh, Carolyn O.; Thompson, Margaret; Segal, Rosalind A.

    2009-01-01

    SUMMARY Sonic Hedgehog (Shh) has dual roles in vertebrate development, as it promotes progenitor cell proliferation and induces tissue patterning. Here we show mitogenic and patterning functions of Shh can be uncoupled from one another. Using a genetic approach to selectively inhibit Shh-proteoglycan interactions in a mouse model, we show binding of Shh to proteoglycans is required for proliferation of neural stem/precursor cells but not for tissue patterning. Shh-proteoglycan interactions regulate both spatial and temporal features of Shh signaling. Proteoglycans localize Shh to specialized mitogenic niches and also act at the single cell level to regulate the duration of Shh signaling, thereby promoting a gene expression program important for cell division. As activation of the Shh pathway is a feature of diverse human cancers, selective stimulation of proliferation by Shh-proteoglycan interactions may also figure prominently in neoplastic growth. PMID:19287388

  14. Proteoglycan interactions with Sonic Hedgehog specify mitogenic responses.

    PubMed

    Chan, Jennifer A; Balasubramanian, Srividya; Witt, Rochelle M; Nazemi, Kellie J; Choi, Yoojin; Pazyra-Murphy, Maria F; Walsh, Carolyn O; Thompson, Margaret; Segal, Rosalind A

    2009-04-01

    Sonic Hedgehog (Shh) has dual roles in vertebrate development, promoting progenitor cell proliferation and inducing tissue patterning. We found that the mitogenic and patterning functions of Shh can be uncoupled from one another. Using a genetic approach to selectively inhibit Shh-proteoglycan interactions in a mouse model, we found that binding of Shh to proteoglycans was required for proliferation of neural stem/precursor cells, but not for tissue patterning. Shh-proteoglycan interactions regulated both spatial and temporal features of Shh signaling. Proteoglycans localized Shh to specialized mitogenic niches and also acted at the single-cell level to regulate the duration of Shh signaling, thereby promoting a gene expression program that is important for cell division. Because activation of the Shh pathway is a feature of diverse human cancers, selective stimulation of proliferation by Shh-proteoglycan interactions may also figure prominently in neoplastic growth.

  15. Application of sonic hedgehog to the developing chick limb.

    PubMed

    Tiecke, Eva; Tickle, Cheryll

    2007-01-01

    Here, we describe methods for applying Sonic hedgehog (Shh) to developing chick limbs. The Sonic hedgehog gene is expressed in the polarizing region, a signaling region at the posterior margin of the limb bud and application of Shh-expressing cells or Shh protein to early limb buds mimics polarizing region signaling. The polarizing region (or zone of polarizing activity) is involved in one of the best known cell-cell interactions in vertebrate embryos and is pivotal in controlling digit number and pattern. At later stages of limb development, the application of Shh protein to the regions between digit primordia can induce changes in digit morphogenesis.

  16. Sonic hedgehog signaling coordinates the proliferation and differentiation of neural stem/progenitor cells by regulating cell cycle kinetics during development of the neocortex.

    PubMed

    Komada, Munekazu

    2012-06-01

    Sonic hedgehog (Shh) acts as a morphogen in normal development of various vertebrate tissues and organs. Shh signaling is essential for patterning and cell-fate specification, particularly in the central nervous system. Shh signaling plays different roles depending on its concentration, area, and timing of exposure. During the development of the neocortex, a low level of Shh is expressed in the neural stem/progenitor cells as well as in mature neurons in the dorsal telencephalon. Shh signaling in neocortex development has been shown to regulate cell cycle kinetics of radial glial cells and intermediate progenitor cells, thereby maintaining the proliferation, survival and differentiation of neurons in the neocortex. During the development of the telencephalon, endogenous Shh signaling is involved in the transition of slow-cycling neural stem cells to fast-cycling neural progenitor cells. It seems that high-level Shh signaling in the ventral telencephalon is essential for ventral specification, while low-level Shh signaling in the dorsal telencephalon plays important roles in the fine-tuning of cell cycle kinetics. The Shh levels and multiple functions of Shh signaling are important for proper corticogenesis in the developing brain. The present paper discusses the roles of Shh signaling in the proliferation and differentiation of neural stem/progenitor cells.

  17. Sonic hedgehog signaling in the lung. From development to disease.

    PubMed

    Kugler, Matthias C; Joyner, Alexandra L; Loomis, Cynthia A; Munger, John S

    2015-01-01

    Over the past two decades, the secreted protein sonic hedgehog (SHH) has emerged as a critical morphogen during embryonic lung development, regulating the interaction between epithelial and mesenchymal cell populations in the airway and alveolar compartments. There is increasing evidence that the SHH pathway is active in adult lung diseases such as pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, and lung cancer, which raises two questions: (1) What role does SHH signaling play in these diseases? and (2) Is it a primary driver of the disease or a response (perhaps beneficial) to the primary disturbance? In this review we aim to fill the gap between the well-studied period of embryonic lung development and the adult diseased lung by reviewing the hedgehog (HH) pathway during the postnatal period and in adult uninjured and injured lungs. We elucidate the similarities and differences in the epithelial-mesenchymal interplay during the fibrosis response to injury in lung compared with other organs and present a critical appraisal of tools and agents available to evaluate HH signaling.

  18. MicroRNA-602 and microRNA-608 regulate sonic hedgehog expression via target sites in the coding region in human chondrocytes

    PubMed Central

    Akhtar, Nahid; Makki, Mohammad Shahidul; Haqqi, Tariq M.

    2015-01-01

    Objective Hedgehog(Hh) signaling has recently been associated with cartilage degradation in osteoarthritis(OA). As interleukin-1β(IL-1β) is a critical mediator of OA pathogenesis, here we determined whether IL-1β induces the expression of sonic hedgehog(SHH) and its regulation by microRNAs in human chondrocytes. Methods SHH protein expression in human OA-cartilage and in an animal model of OA was determined by immunohistochemistry and immunofluorescence respectively. Gene and protein expression in IL-1β or SHH-stimulated chondrocytes was determined by TaqMan assays and immunoblotting respectively. Effect of overexpression of miR-602 and miR-608 or their anatgomirs on SHH expression was evaluated by transient transfections of human chondrocytes and HEK-293 cells. Role of signaling pathways was evaluated using small molecule inhibitors. Binding of miRNAs with the putative “seed sequence” in the SHH mRNA was validated with a SHH luciferase reporter assay. Results Expression of SHH, PTCH-1, GLI-1, HHIP, MMP-13, and COL10A1 was high in damaged OAcartilage. Expression of SHH was inversely correlated with the expression of miR-608 in damaged cartilage and in IL-1β-stimulated chondrocytes. Transfection with miR-608 or miR-602 mimics inhibited the reporter activity and mutation of the miRNAs “seed sequences” abolished the repression of reporter activity. Overexpression of miR-602 or miR-608 inhibited the expression of SHH mRNA and protein and this was abrogated by antagomirs. Stimulation with SHH-protein up-regulated the MMP-13 expression and inhibition of Hh signaling blocked MMP-13 expression in OA chondrocytes. Conclusions miR-602 and miR-608 are important regulators of SHH expression in chondrocytes and their suppression by IL-1β may contribute to the enhanced expression of SHH and MMP-13 in OA. PMID:25385442

  19. Sonic hedgehog protein regulates fibroblast growth factor 8 expression in metanephric explant culture from BALB/c mice: Possible mechanisms associated with renal morphogenesis.

    PubMed

    Chen, Xing; Hou, Xiao-Ming; Fan, You-Fei; Jin, Yu-Ting; Wang, Yu-Lin

    2016-10-01

    The sonic hedgehog (SHH) morphogen regulates cell differentiation and controls a number of genes during renal morphogenesis. To date, the effects of SHH on fibroblast growth factors (Fgfs) in embryonic kidney development remain unclear. In the present study, explants of BALB/c mouse embryonic kidney tissues were used to investigate the role of exogenous SHH on Fgf8 and Fgf10 expression levels ex vivo. Ureteric bud branches and epithelial metanephric derivatives were used to determine the renal morphogenesis with Dolichos biflorus agglutinin or hematoxylin‑eosin staining. mRNA expression levels were determined using reverse transcription‑quantitative polymerase chain reaction, while the protein expression levels were examined using immunohistochemistry and western blot analysis. During the initial stages of metanephric development, low levels of SHH, Fgf8, and Fgf10 expression were observed, which were found to increase significantly during more advanced stages of metanephric development. In addition, exogenous SHH protein treatment increased the number of ureteric bud branches and enhanced the formation of nephrons. Exogenous SHH reduced the Fgf8 mRNA and protein expression levels, whereas cyclopamine (an SHH‑smoothened receptor inhibitor) interfered with SHH‑mediated downregulation of Fgf8 expression. By contrast, exogenous SHH protein was not found to modulate Fgf10 mRNA and protein expression levels. In conclusion, these results indicate that the modulatory effects of SHH on BALB/c mouse metanephric explant cultures may involve the regulation of Fgf8 expression but not Fgf10 expression, which provides evidence for the functional role of Fgf proteins in renal morphogenesis.

  20. Sonic hedgehog protein regulates fibroblast growth factor 8 expression in metanephric explant culture from BALB/c mice: Possible mechanisms associated with renal morphogenesis

    PubMed Central

    Chen, Xing; Hou, Xiao-Ming; Fan, You-Fei; Jin, Yu-Ting; Wang, Yu-Lin

    2016-01-01

    The sonic hedgehog (SHH) morphogen regulates cell differentiation and controls a number of genes during renal morphogenesis. To date, the effects of SHH on fibroblast growth factors (Fgfs) in embryonic kidney development remain unclear. In the present study, explants of BALB/c mouse embryonic kidney tissues were used to investigate the role of exogenous SHH on Fgf8 and Fgf10 expression levels ex vivo. Ureteric bud branches and epithelial metanephric derivatives were used to determine the renal morphogenesis with Dolichos biflorus agglutinin or hematoxylineosin staining. mRNA expression levels were determined using reverse transcription-quantitative polymerase chain reaction, while the protein expression levels were examined using immunohistochemistry and western blot analysis. During the initial stages of metanephric development, low levels of SHH, Fgf8, and Fgf10 expression were observed, which were found to increase significantly during more advanced stages of metanephric development. In addition, exogenous SHH protein treatment increased the number of ureteric bud branches and enhanced the formation of nephrons. Exogenous SHH reduced the Fgf8 mRNA and protein expression levels, whereas cyclopamine (an SHH-smoothened receptor inhibitor) interfered with SHH-mediated downregulation of Fgf8 expression. By contrast, exogenous SHH protein was not found to modulate Fgf10 mRNA and protein expression levels. In conclusion, these results indicate that the modulatory effects of SHH on BALB/c mouse metanephric explant cultures may involve the regulation of Fgf8 expression but not Fgf10 expression, which provides evidence for the functional role of Fgf proteins in renal morphogenesis. PMID:27510750

  1. Sonic hedgehog signaling in the development of the mouse hypothalamus

    PubMed Central

    Blaess, Sandra; Szabó, Nora; Haddad-Tóvolli, Roberta; Zhou, Xunlei; Álvarez-Bolado, Gonzalo

    2014-01-01

    The expression pattern of Sonic Hedgehog (Shh) in the developing hypothalamus changes over time. Shh is initially expressed in the prechordal mesoderm and later in the hypothalamic neuroepithelium—first medially, and then in two off-medial domains. This dynamic expression suggests that Shh might regulate several aspects of hypothalamic development. To gain insight into them, lineage tracing, (conditional) gene inactivation in mouse, in ovo loss- and gain-of-function approaches in chick and analysis of Shh expression regulation have been employed. We will focus on mouse studies and refer to chick and fish when appropriate to clarify. These studies show that Shh-expressing neuroepithelial cells serve as a signaling center for neighboring precursors, and give rise to most of the basal hypothalamus (tuberal and mammillary regions). Shh signaling is initially essential for hypothalamic induction. Later, Shh signaling from the neuroepithelium controls specification of the lateral hypothalamic area and growth-patterning coordination in the basal hypothalamus. To further elucidate the role of Shh in hypothalamic development, it will be essential to understand how Shh regulates the downstream Gli transcription factors. PMID:25610374

  2. Cloning and bioinformatical analysis of the N-terminus of the sonic hedgehog gene.

    PubMed

    Zhang, Yi; Zhao, Shu; Dong, Weiren; He, Suifen; Wang, Haihong; Zhang, Lihua; Tang, Yinjuan; Guo, Jiasong; Guo, Suiqun

    2013-01-25

    The sonic hedgehog protein not only plays a key role in early embryonic development, but also has essential effects on the adult nervous system, including neural stem cell proliferation, differentiation, migration and neuronal axon guidance. The N-terminal fragment of sonic hedgehog is the key functional element in this process. Therefore, this study aimed to clone and analyze the N-terminal fragment of the sonic hedgehog gene. Total RNA was extracted from the notochord of a Sprague-Dawley rat at embryonic day 9 and the N-terminal fragment of sonic hedgehog was amplified by nested reverse transcription-PCR. The N-terminal fragment of the sonic hedgehog gene was successfully cloned. The secondary and tertiary structures of the N-terminal fragment of the sonic hedgehog protein were predicted using Jpred and Phyre online.

  3. Role of Sonic Hedgehog Signaling in Oligodendrocyte Differentiation.

    PubMed

    Wang, Li-Chun; Almazan, Guillermina

    2016-12-01

    During development, the secreted molecule Sonic Hedgehog (Shh) is required for lineage specification and proliferation of oligodendrocyte progenitors (OLPs), which are the glia cells responsible for the myelination of axons in the central nervous system (CNS). Shh signaling has been implicated in controlling both the generation of oligodendrocytes (OLGs) during embryonic development and their production in adulthood. Although, some evidence points to a role of Shh signaling in OLG development, its involvement in OLG differentiation remains to be fully determined. The objective of this study was to assess whether Shh signaling is involved in OLG differentiation after neural stem cell commitment to the OLG lineage. To address these questions, we manipulated Shh signaling using cyclopamine, a potent inhibitor of Shh signaling activator Smoothened (Smo), alone or combined with the agonist SAG in OLG primary cultures and assessed expression of myelin-specific markers. We found that inactivation of Shh signaling caused a dose-dependent decrease in myelin basic protein (MBP) and myelin associated glycoprotein (MAG) in differentiating OLGs. Co-treatment of the cells with SAG reversed the inhibitory effect of cyclopamine on both myelin-specific protein levels and morphological changes associated with it. Further experiments are required to elucidate the molecular mechanism by which Shh signaling regulates OLG differentiation.

  4. Localization of Sonic hedgehog secreting and receiving cells in the developing and adult rat adrenal cortex.

    PubMed

    Guasti, Leonardo; Paul, Alex; Laufer, Ed; King, Peter

    2011-04-10

    Sonic hedgehog signaling was recently demonstrated to play an important role in murine adrenal cortex development. The organization of the rat adrenal differs from that of the mouse, with the zona glomerulosa and zona fasciculata separated by an undifferentiated zone in the rat, but not in the mouse. In the present study we aimed to determine the mRNA expression patterns of Sonic hedgehog and the hedgehog signaling pathway components Patched-1 and Gli1 in the developing and adult rat adrenal. Sonic hedgehog expression was detected at the periphery of the cortex in cells lacking CYP11B1 and CYP11B2 expression, while signal-receiving cells were localized in the overlying capsule mesenchyme. Using combined in situ hybridization and immunohistochemistry we found that the cells expressing Sonic hedgehog lie between the CYP11B2 and CYP11B1 layers, and thus Sonic hedgehog expression defines one cell population of the undifferentiated zone.

  5. The Sonic hedgehog gradient in the developing limb.

    PubMed

    Tickle, Cheryll; Barker, Heather

    2013-01-01

    A gradient of Sonic hedgehog (Shh) plays a major role in specifying the antero-posterior pattern of structures that develop in the distal part of the vertebrate limb, in particular, the antero-posterior pattern of the digits. Classical embryological experiments identified the polarizing region (or zone of polarizing activity, ZPA), a signaling region at the posterior margin of the early chick wing bud and, consistent with a model in which production of a diffusible morphogen specifies antero-posterior positional information, polarizing region signaling was shown to be dose dependent and long range. It is now well established that the vertebrate hedgehog gene, Sonic hedgehog (Shh), which encodes a secreted protein, is expressed in the polarizing region of the chick wing and that Shh signaling has the same characteristics as polarizing region signaling. Shh expression at the posterior of the early limb bud and the mechanism of Shh signal transduction are conserved among vertebrates including mammals. However, it is unlikely that a simple Shh gradient is responsible for digit pattern formation in mammalian limbs and there is still little understanding of how positional information specified by Shh signaling is encoded and translated into digit anatomy. Alterations in Shh signaling underlie some congenital limb abnormalities and also changes in timing and extent of Shh signaling appear to be related to the evolution of morphological diversity of vertebrate limbs.

  6. Targeting the Sonic Hedgehog Pathway in Keratocystic Odontogenic Tumor*

    PubMed Central

    Ren, Changchun; Amm, Hope M.; DeVilliers, Patricia; Wu, Yixin; Deatherage, Joseph R.; Liu, Zhongyu; MacDougall, Mary

    2012-01-01

    Keratocystic odontogenic tumors (KCOT) may occur sporadically or associated with the nevoid basal cell carcinoma syndrome. It is a benign aggressive tumor of odontogenic epithelial origin with a high rate of recurrence. A primary human keratocystic odontogenic tumor cell population, KCOT-1, has been established from a tumor explant culture. The KCOT-1 cells were characterized by growth rate, gene expression profiles of major tooth enamel matrix proteins (EMPs), amelogenin (AMELX), enamelin (ENAM), ameloblastin (AMBN), amelotin (AMTN), tumor-related proteins enamelysin (MMP-20), kallikrein-4 (KLK-4), and odontogenic ameloblast-associated protein (ODAM) using quantitative real-time reverse transcription-polymerase chain reaction. Cytokeratin 14 (CK14) was examined by immunohistochemistry. In addition, expression of the members of the sonic hedgehog (SHH) pathway, SHH, patched (PTCH-1), smoothened (SMO), GLI-1, and GLI-2 and of the NOTCH signaling pathway, NOTCH-1, NOTCH-2, NOTCH-3, JAG-2 (Jagged-2), and Delta-like-1 (DLL-1) were evaluated. KCOT-1 cells were treated with SMO antagonist cyclopamine. We found that cyclopamine significantly arrested the growth of KCOT-1 cells in a dose-dependent manner and that the effects of cyclopamine were abolished by adding SHH protein. The protein expression of the SHH pathway was down-regulated by cyclopamine, further confirming that cyclopamine inhibits the SHH signaling pathway; SHH down-regulation correlated with the down-regulation of the NOTCH signaling pathway as well. In conclusion, using an established KCOT-1 cell population, we characterized the gene expression profiles related to the EMPs, SHH, and NOTCH signaling pathway and confirmed that cyclopamine significantly arrested the growth of KCOT-1 cells and may be a viable agent as a novel therapeutic. PMID:22679015

  7. Sonic Hedgehog Activation Is Implicated in Diosgenin-Induced Megakaryocytic Differentiation of Human Erythroleukemia Cells

    PubMed Central

    Ghezali, Lamia; Liagre, Bertrand; Limami, Youness; Beneytout, Jean-Louis; Leger, David Yannick

    2014-01-01

    Differentiation therapy is a means to treat cancer and is induced by different agents with low toxicity and more specificity than traditional ones. Diosgenin, a plant steroid, is able to induce megakaryocytic differentiation or apoptosis in human HEL erythroleukemia cells in a dose-dependent manner. However, the exact mechanism by which diosgenin induces megakaryocytic differentiation has not been elucidated. In this study, we studied the involvement of Sonic Hedgehog in megakaryocytic differentiation induced by diosgenin in HEL cells. First, we showed that different elements of the Hedgehog pathway are expressed in our model by qRT-PCR. Then, we focused our interest on key elements in the Sonic Hedgehog pathway: Smoothened receptor, GLI transcription factor and the ligand Sonic Hedgehog. We showed that Smoothened and Sonic Hedgehog were overexpressed in disogenin-treated cells and that GLI transcription factors were activated. Then, we showed that SMO inhibition using siSMO or the GLI antagonist GANT-61, blocked megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, we demonstrated that Sonic Hedgehog pathway inhibition led to inhibition of ERK1/2 activation, a major physiological pathway involved in megakaryocytic differentiation. In conclusion, our study reports, for the first time, a crucial role for the Sonic Hedgehog pathway in diosgenin-induced megakaryocytic differentiation in HEL cells. PMID:24740159

  8. Sonic Hedgehog activation is implicated in diosgenin-induced megakaryocytic differentiation of human erythroleukemia cells.

    PubMed

    Ghezali, Lamia; Liagre, Bertrand; Limami, Youness; Beneytout, Jean-Louis; Leger, David Yannick

    2014-01-01

    Differentiation therapy is a means to treat cancer and is induced by different agents with low toxicity and more specificity than traditional ones. Diosgenin, a plant steroid, is able to induce megakaryocytic differentiation or apoptosis in human HEL erythroleukemia cells in a dose-dependent manner. However, the exact mechanism by which diosgenin induces megakaryocytic differentiation has not been elucidated. In this study, we studied the involvement of Sonic Hedgehog in megakaryocytic differentiation induced by diosgenin in HEL cells. First, we showed that different elements of the Hedgehog pathway are expressed in our model by qRT-PCR. Then, we focused our interest on key elements in the Sonic Hedgehog pathway: Smoothened receptor, GLI transcription factor and the ligand Sonic Hedgehog. We showed that Smoothened and Sonic Hedgehog were overexpressed in disogenin-treated cells and that GLI transcription factors were activated. Then, we showed that SMO inhibition using siSMO or the GLI antagonist GANT-61, blocked megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, we demonstrated that Sonic Hedgehog pathway inhibition led to inhibition of ERK1/2 activation, a major physiological pathway involved in megakaryocytic differentiation. In conclusion, our study reports, for the first time, a crucial role for the Sonic Hedgehog pathway in diosgenin-induced megakaryocytic differentiation in HEL cells.

  9. High frequency stimulation induces sonic hedgehog release from hippocampal neurons

    PubMed Central

    Su, Yujuan; Yuan, Yuan; Feng, Shengjie; Ma, Shaorong; Wang, Yizheng

    2017-01-01

    Sonic hedgehog (SHH) as a secreted protein is important for neuronal development in the central nervous system (CNS). However, the mechanism about SHH release remains largely unknown. Here, we showed that SHH was expressed mainly in the synaptic vesicles of hippocampus in both young postnatal and adult rats. High, but not low, frequency stimulation, induces SHH release from the neurons. Moreover, removal of extracellular Ca2+, application of tetrodotoxin (TTX), an inhibitor of voltage-dependent sodium channels, or downregulation of soluble n-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) proteins, all blocked SHH release from the neurons in response to HFS. Our findings suggest a novel mechanism to control SHH release from the hippocampal neurons. PMID:28262835

  10. Mechanism of inhibition of the tumor suppressor Patched by Sonic Hedgehog.

    PubMed

    Tukachinsky, Hanna; Petrov, Kostadin; Watanabe, Miyako; Salic, Adrian

    2016-10-04

    The Hedgehog cell-cell signaling pathway is crucial for animal development, and its misregulation is implicated in numerous birth defects and cancers. In unstimulated cells, pathway activity is inhibited by the tumor suppressor membrane protein, Patched. Hedgehog signaling is triggered by the secreted Hedgehog ligand, which binds and inhibits Patched, thus setting in motion the downstream events in signal transduction. Despite its critical importance, the mechanism by which Hedgehog antagonizes Patched has remained unknown. Here, we show that vertebrate Patched1 inhibition is caused by direct, palmitate-dependent interaction with the Sonic Hedgehog ligand. We find that a short palmitoylated N-terminal fragment of Sonic Hedgehog binds Patched1 and, strikingly, is sufficient to inhibit it and to activate signaling. The rest of Sonic Hedgehog confers high-affinity Patched1 binding and internalization through a distinct binding site, but, surprisingly, it is not absolutely required for signaling. The palmitate-dependent interaction with Patched1 is specifically impaired in a Sonic Hedgehog mutant causing human holoprosencephaly, the most frequent congenital brain malformation, explaining its drastically reduced potency. The palmitate-dependent interaction is also abolished in constitutively inhibited Patched1 point mutants causing the Gorlin cancer syndrome, suggesting that they might adopt a conformation distinct from the wild type. Our data demonstrate that Sonic Hedgehog signals via the palmitate-dependent arm of a two-pronged contact with Patched1. Furthermore, our results suggest that, during Hedgehog signaling, ligand binding inhibits Patched by trapping it in an inactive conformation, a mechanism that explains the dramatically reduced activity of oncogenic Patched1 mutants.

  11. Mechanism of inhibition of the tumor suppressor Patched by Sonic Hedgehog

    PubMed Central

    Tukachinsky, Hanna; Petrov, Kostadin; Watanabe, Miyako; Salic, Adrian

    2016-01-01

    The Hedgehog cell–cell signaling pathway is crucial for animal development, and its misregulation is implicated in numerous birth defects and cancers. In unstimulated cells, pathway activity is inhibited by the tumor suppressor membrane protein, Patched. Hedgehog signaling is triggered by the secreted Hedgehog ligand, which binds and inhibits Patched, thus setting in motion the downstream events in signal transduction. Despite its critical importance, the mechanism by which Hedgehog antagonizes Patched has remained unknown. Here, we show that vertebrate Patched1 inhibition is caused by direct, palmitate-dependent interaction with the Sonic Hedgehog ligand. We find that a short palmitoylated N-terminal fragment of Sonic Hedgehog binds Patched1 and, strikingly, is sufficient to inhibit it and to activate signaling. The rest of Sonic Hedgehog confers high-affinity Patched1 binding and internalization through a distinct binding site, but, surprisingly, it is not absolutely required for signaling. The palmitate-dependent interaction with Patched1 is specifically impaired in a Sonic Hedgehog mutant causing human holoprosencephaly, the most frequent congenital brain malformation, explaining its drastically reduced potency. The palmitate-dependent interaction is also abolished in constitutively inhibited Patched1 point mutants causing the Gorlin cancer syndrome, suggesting that they might adopt a conformation distinct from the wild type. Our data demonstrate that Sonic Hedgehog signals via the palmitate-dependent arm of a two-pronged contact with Patched1. Furthermore, our results suggest that, during Hedgehog signaling, ligand binding inhibits Patched by trapping it in an inactive conformation, a mechanism that explains the dramatically reduced activity of oncogenic Patched1 mutants. PMID:27647915

  12. Role of Sonic Hedgehog in idiopathic pulmonary fibrosis.

    PubMed

    Bolaños, Alfredo Lozano; Milla, Criselda Mendoza; Lira, José Cisneros; Ramírez, Remedios; Checa, Marco; Barrera, Lourdes; García-Alvarez, Jorge; Carbajal, Verónica; Becerril, Carina; Gaxiola, Miguel; Pardo, Annie; Selman, Moisés

    2012-12-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease of unknown etiology and uncertain pathogenic mechanisms. Recent studies indicate that the pathogenesis of the disease may involve the abnormal expression of certain developmental pathways. Here we evaluated the expression of Sonic Hedgehog (SHH), Patched-1, Smoothened, and transcription factors glioma-associated oncogene homolog (GLI)1 and GLI2 by RT-PCR, as well as their localization in IPF and normal lungs by immunohistochemistry. The effects of SHH on fibroblast proliferation, migration, collagen and fibronectin production, and apoptosis were analyzed by WST-1, Boyden chamber chemotaxis, RT-PCR, Sircol, and annexin V-propidium iodide binding assays, respectively. Our results showed that all the main components of the Sonic signaling pathway were overexpressed in IPF lungs. With the exception of Smoothened, they were also upregulated in IPF fibroblasts. SHH and GLI2 localized to epithelial cells, whereas Patched-1, Smoothened, and GLI1 were observed mainly in fibroblasts and inflammatory cells. No staining was detected in normal lungs. Recombinant SHH increased fibroblast proliferation (P < 0.05), collagen synthesis, (2.5 ± 0.2 vs. 4.5 ± 1.0 μg of collagen/ml; P < 0.05), fibronectin expression (2-3-fold over control), and migration (190.3 ± 12.4% over control, P < 0.05). No effect was observed on α-smooth muscle actin expression. SHH protected lung fibroblasts from TNF-α/IFN-γ/Fas-induced apoptosis (14.5 ± 3.2% vs. 37.3 ± 7.2%, P < 0.0001). This protection was accompanied by modifications in several apoptosis-related proteins, including increased expression of X-linked inhibitor of apoptosis. These findings indicate that the SHH pathway is activated in IPF lungs and that SHH may contribute to IPF pathogenesis by increasing the proliferation, migration, extracellular matrix production, and survival of fibroblasts.

  13. Sonic hedgehog in oral squamous cell carcinoma: An immunohistochemical study

    PubMed Central

    Srinath, Sahana; Iyengar, Asha R; Mysorekar, Vijaya

    2016-01-01

    Background: Recent studies have revealed the involvement of hedgehog (Hh) signaling component in proliferation and invasive behavior of many carcinomas. Aim: This study aims to identify the expression of sonic Hh (SHH) protein of SHH pathway in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC) using SHH (H-160) (Santa Cruz, sc-9042) which could have therapeutic implication in future. Materials and Methods: A total of 250 cases comprising 50 normal oral mucosa, 50 cases of oral epithelial dysplasia, 50 well, 50 moderate and 50 poorly differentiated OSCCs were included in the study. Immunohistochemical evaluation of SHH protein expression was conducted using monoclonal antibody. Interpretation of the expression was done by immunoreactive score of Remmele and Stegner (IRS) scoring method. Statistical Analysis: Chi-Square test was used to analyze the results. Results: The study showed that SHH signaling molecules are highly expressed in OSCC, and their expression was mainly in the cytoplasm of epithelial cells. Conclusion: The SHH signaling component is associated with the pathological parameter in OSCC and oral epithelial dysplasia. PMID:27721600

  14. Sonic hedgehog expression in a rat model of chronic pancreatitis

    PubMed Central

    Wang, Luo-Wei; Lin, Han; Lu, Yi; Xia, Wei; Gao, Jun; Li, Zhao-Shen

    2014-01-01

    AIM: To analyze the activation of sonic hedgehog (SHh) signaling pathways in a rat model of chronic pancreatitis. METHODS: Forty Wistar rats were randomly divided into 2 groups: experimental group and control group (20 rats in each group). Dibutyltin dichloride was infused into the tail vein of the rats to induce chronic pancreatitis in the experimental group. The same volume of ethanol and glycerol mixture was infused in the control group. The expression of Ptch, Smo and Gli were analyzed using immunohistochemistry, and real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Compared with the control group, significant histological changes in terms of the areas of abnormal architecture, glandular atrophy, fibrosis, pseudo tubular complexes, and edema were observed at week 4 in the experimental group. The expression of Ptch1, Smo and Gli1 in the pancreatic tissue increased significantly in the experimental group. Using RT-PCR, mRNA levels of Ptch, Smo and Gli in the experimental group increased significantly compared with the control group. CONCLUSION: The SHh signaling pathway is aberrantly activated in rats with chronic pancreatitis. The SHh signaling pathway plays an important role in the development of chronic pancreatitis. These results may be helpful in studies focusing on the relationship between chronic pancreatitis and pancreatic cancer. PMID:24782623

  15. Ontogenetic Expression of Sonic Hedgehog in the Chicken Subpallium

    PubMed Central

    Bardet, Sylvia M.; Ferran, José L. E.; Sanchez-Arrones, Luisa; Puelles, Luis

    2010-01-01

    Sonic hedgehog (SHH) is a secreted signaling factor that is implicated in the molecular patterning of the central nervous system (CNS), somites, and limbs in vertebrates. SHH has a crucial role in the generation of ventral cell types along the entire rostrocaudal axis of the neural tube. It is secreted early in development by the axial mesoderm (prechordal plate and notochord) and the overlying ventral neural tube. Recent studies clarified the impact of SHH signaling mechanisms on dorsoventral patterning of the spinal cord, but the corresponding phenomena in the rostral forebrain are slightly different and more complex. This notably involves separate Shh expression in the preoptic part of the forebrain alar plate, as well as in the hypothalamic floor and basal plates. The present work includes a detailed spatiotemporal description of the singular alar Shh expression pattern in the rostral preoptic forebrain of chick embryos, comparing it with FoxG1, Dlx5, Nkx2.1, and Nkx2.2 mRNA expression at diverse stages of development. As a result of this mapping, we report a subdivision of the preoptic region in dorsal and ventral zones; only the dorsal part shows Shh expression. The positive area impinges as well upon a median septocommissural preoptic domain. Our study strongly suggests tangential migration of Shh-positive cells from the preoptic region into other subpallial domains, particularly into the pallidal mantle and the intermediate septum. PMID:20700498

  16. Mesencephalic basolateral domain specification is dependent on Sonic Hedgehog

    PubMed Central

    Martinez-Lopez, Jesus E.; Moreno-Bravo, Juan A.; Madrigal, M. Pilar; Martinez, Salvador; Puelles, Eduardo

    2015-01-01

    In the study of central nervous system morphogenesis, the identification of new molecular markers allows us to identify domains along the antero-posterior and dorso-ventral (DV) axes. In the past years, the alar and basal plates of the midbrain have been divided into different domains. The precise location of the alar-basal boundary is still under discussion. We have identified Barhl1, Nhlh1 and Six3 as appropriate molecular markers to the adjacent domains of this transition. The description of their expression patterns and the contribution to the different mesencephalic populations corroborated their role in the specification of these domains. We studied the influence of Sonic Hedgehog on these markers and therefore on the specification of these territories. The lack of this morphogen produced severe alterations in the expression pattern of Barhl1 and Nhlh1 with consequent misspecification of the basolateral (BL) domain. Six3 expression was apparently unaffected, however its distribution changed leading to altered basal domains. In this study we confirmed the localization of the alar-basal boundary dorsal to the BL domain and demonstrated that the development of the BL domain highly depends on Shh. PMID:25741244

  17. Sonic Hedgehog Promotes Cementoblastic Differentiation via Activating the BMP Pathways.

    PubMed

    Bae, Won-Jung; Auh, Q-Schick; Lim, Hyun-Chang; Kim, Gyu-Tae; Kim, Hyun-Soo; Kim, Eun-Cheol

    2016-10-01

    Although sonic hedgehog (SHH), an essential molecule in embryogenesis and organogenesis, stimulates proliferation of human periodontal ligament (PDL) stem cells, the effects of recombinant human SHH (rh-SHH) on osteoblastic differentiation are unclear. To reveal the role of SHH in periodontal regeneration, expression of SHH in mouse periodontal tissues and its effects on the osteoblastic/cementoblastic differentiation in human cementoblasts were investigated. SHH is immunolocalized to differentiating cementoblasts, PDL cells, and osteoblasts of the developing mouse periodontium. Addition of rh-SHH increased cell growth, ALP activity, and mineralization nodule formation, and upregulated mRNA expression of osteoblastic and cementoblastic markers. The osteoblastic/cementoblastic differentiation of rh-SHH was abolished by the SHH inhibitor cyclopamine (Cy) and the BMP antagonist noggin. rh-SHH increased the expression of BMP-2 and -4 mRNA, as well as levels of phosphorylated Akt, ERK, p38, and JNK, and of MAPK and NF-κB activation, which were reversed by noggin, Cy, and BMP-2 siRNA. Collectively, this study is the first to demonstrate that SHH can promote cell growth and cell osteoblastic/cementoblastic differentiation via BMP pathway. Thus, SHH plays important roles in the development of periodontal tissue, and might represent a new therapeutic target for periodontitis and periodontal regeneration.

  18. Sonic Hedgehog Guides Axons via Zipcode Binding Protein 1-Mediated Local Translation.

    PubMed

    Lepelletier, Léa; Langlois, Sébastien D; Kent, Christopher B; Welshhans, Kristy; Morin, Steves; Bassell, Gary J; Yam, Patricia T; Charron, Frédéric

    2017-02-15

    Sonic hedgehog (Shh) attracts spinal cord commissural axons toward the floorplate. How Shh elicits changes in the growth cone cytoskeleton that drive growth cone turning is unknown. We find that the turning of rat commissural axons up a Shh gradient requires protein synthesis. In particular, Shh stimulation increases β-actin protein at the growth cone even when the cell bodies have been removed. Therefore, Shh induces the local translation of β-actin at the growth cone. We hypothesized that this requires zipcode binding protein 1 (ZBP1), an mRNA-binding protein that transports β-actin mRNA and releases it for local translation upon phosphorylation. We found that Shh stimulation increases phospho-ZBP1 levels in the growth cone. Disruption of ZBP1 phosphorylation in vitro abolished the turning of commissural axons toward a Shh gradient. Disruption of ZBP1 function in vivo in mouse and chick resulted in commissural axon guidance errors. Therefore, ZBP1 is required for Shh to guide commissural axons. This identifies ZBP1 as a new mediator of noncanonical Shh signaling in axon guidance.SIGNIFICANCE STATEMENT Sonic hedgehog (Shh) guides axons via a noncanonical signaling pathway that is distinct from the canonical Hedgehog signaling pathway that specifies cell fate and morphogenesis. Axon guidance is driven by changes in the growth cone in response to gradients of guidance molecules. Little is known about the molecular mechanism of how Shh orchestrates changes in the growth cone cytoskeleton that are required for growth cone turning. Here, we show that the guidance of axons by Shh requires protein synthesis. Zipcode binding protein 1 (ZBP1) is an mRNA-binding protein that regulates the local translation of proteins, including actin, in the growth cone. We demonstrate that ZBP1 is required for Shh-mediated axon guidance, identifying a new member of the noncanonical Shh signaling pathway.

  19. Coordinated regulation of dorsal bone morphogenetic protein 4 and ventral Sonic hedgehog signaling specifies the dorso-ventral polarity in the optic vesicle and governs ocular morphogenesis through fibroblast growth factor 8 upregulation.

    PubMed

    Kobayashi, Takuma; Yasuda, Kunio; Araki, Masasuke

    2010-05-01

    Dorsal and ventral specification in the early optic vesicle plays a crucial role in vertebrate ocular morphogenesis, and proper dorsal-ventral polarity in the optic vesicle ensures that distinct structures develop in separate domains within the eye primordium. The polarity is determined progressively during development by coordinated regulation of extraocular dorsal and ventral factors. In the present study, we cultured discrete portions of embryonic chick brains by preparing anterior cephalon, anterior dorsal cephalon and anterior ventral cephalon, and clearly demonstrate that bone morphogenetic protein 4 (BMP4) and Sonic hedgehog (Shh) constitute a dorsal-ventral signaling system together with fibroblast growth factor 8 (FGF8). BMP4 and Shh upregulate Tbx5 and Pax2, as reported previously, and at the same time Shh downregulates Tbx5, while BMP4 affects Pax2 expression to downregulate similarly. Shh induces Fgf8 expression in the ventral optic vesicle. This, in turn, determines the distinct boundary of the retinal pigmented epithelium and the neural retina by suppressing Mitf expression. The lens develops only when signals from both the dorsal and ventral regions come across together. Inverted deposition of Shh and BMP4 signals in organ-cultured optic vesicle completely re-organized ocular structures to be inverted. Based on these observations we propose a novel model in which the two signals govern the whole of ocular development when they encounter each other in the ocular morphogenic domain.

  20. Transcriptional responses of zebrafish embryos exposed to potential sonic hedgehog pathway interfering compounds deviate from expression profiles of cyclopamine.

    PubMed

    Büttner, Anita; Busch, Wibke; Klüver, Nils; Giannis, Athanassios; Scholz, Stefan

    2012-04-01

    The molecular responses of two small molecules, SANT-2 and GANT-61, potentially interfering with the sonic hedgehog pathway (Shh) have been studied in zebrafish embryos by microarray analysis. For both compounds and the positive reference cyclopamine previous reporter gene assays for the transcription factor Gli1 have indicated an inhibition of the hedgehog signaling pathway. In zebrafish embryos a typical phenotype (cyclopia) associated with Shh interference was only observed for cyclopamine. Furthermore, only cyclopamine led to the repression of genes specifically associated with hedgehog signaling and confirmed published microarray data. In contrast to these data hspb11 was additionally identified as the most pronounced down-regulated genes for exposure to cyclopamine. No or different effects on gene expression patterns were provoked by SANT-2 or GANT-61, respectively. Reasons for the discrepancies between cellular reporter and the zebrafish embryo assay and potential implications for the identification of compounds interfering with specific developmental pathways are discussed.

  1. Differential developmental strategies by Sonic hedgehog in thalamus and hypothalamus.

    PubMed

    Zhang, Yuanfeng; Alvarez-Bolado, Gonzalo

    2016-09-01

    The traditional concept of diencephalon (thalamus plus hypothalamus) and with it the entire traditional subdivision of the developing neural tube are being challenged by novel insights obtained by mapping the expression of key developmental genes. A model in which the hypothalamus is placed in the most rostral portion of the neural tube, followed caudally by a diencephalon formed by prethalamus, thalamus and pretectum has been proposed. The adult thalamus and hypothalamus are quite unlike each other in connectivity and functions. Here we review work on the role of the secreted morphogen protein Sonic hedgehog (Shh) in the developing diencephalon and hypothalamic region to show how different these two regions are also from this point of view. Shh from the prechordal plate (PCP) induces and patterns the hypothalamus but there is no evidence that this role is fulfilled by a morphogen gradient. Later, the hypothalamic primordium itself expresses Shh and a large part of the hypothalamus belongs to the Shh lineage, including the ventral domains. Neural Shh is necessary to complete the specification (lateral hypothalamus), differentiation and growth of the hypothalamus. Although Gli2A is the major effector of Shh in this region, hypothalamic specification also depends on the suppression of Gli3R by Shh secreted by the PCP as well as the neuroepithelium. The thalamus is patterned by an Shh morphogen gradient originated in the ZLI following similar mechanisms to those in the spinal cord. The thalamus itself does not belong to the Shh lineage. Gli2A is necessary for appropriate growth and specification of the thalamic nuclei, to the exception of the medial and intralaminar groups (limbic-related), whose development depends on Gli3R. Beyond specification and patterning, the scarce data available about cell sorting and aggregation in these two regions shows key differences between them as well. In summary, not only expression patterns but also developmental mechanisms support

  2. Sonic hedgehog signaling pathway mediates development of hepatocellular carcinoma.

    PubMed

    Cai, Heng; Li, Hongxing; Li, Jingmin; Li, Xiaoyan; Li, Yana; Shi, Yan; Wang, Dong

    2016-10-15

    Although abnormal activation of the sonic hedgehog (Shh) signaling pathway has been demonstrated in human hepatocellular carcinoma (HCC) patients and in most HCC cell lines, the mechanism by which the Shh pathway promotes the development of HCC remains uncertain. Using a liver cancer model induced by diethylnitrosamine (DEN) which mimics the process from liver injury, abnormal hepatocyte proliferation, and hepatocirrhosis to hepatocyte canceration, we investigated the abnormal activation of the Shh pathway by examining the expression of Shh, patched-1 (Ptch), smoothened (SMO), and glioma-associated oncogene-1 (Gli1) genes. During this process, the expression of CDK1 and cyclin B1 protein, which are two components of the M-phase promoting factor (MPF) controlling G2/M transition, was also examined to explore the potential relationship between Shh activation and cell cycle progression. We observed that the cells with Shh, Ptch, and Gli1 protein expression were mainly distributed in hyperplastic nodule, cancerous node, the epithelia of interlobular bile duct, and precancerous tissues. A gradually increasing tendency of the positive expression rate of Shh, Ptch, and Gli1 proteins in the process from the beginning normal tissue to the final cancer formation was revealed. The cyclin B1 and CDK1 expression level was higher in the DEN-induced rats as compared with normal rats, and their expression was mainly distributed in the portal area of the liver, hyperplastic nodule, cancerous node, and precancerous tissues. Our results suggested that the Shh signaling pathway is activated during liver carcinogenesis, and activated Shh signaling promotes the cell proliferation by facilitating the G2/M transition through increasing the expression of cyclin B1 and CDK1 protein, which eventually results in the development of liver cancer. Better understanding of the Shh signaling pathway in HCC may contribute to the development of novel therapeutic strategies in inhibiting cell

  3. Deletion of Atoh1 disrupts Sonic Hedgehog signaling in the developing cerebellum and prevents medulloblastoma.

    PubMed

    Flora, Adriano; Klisch, Tiemo J; Schuster, Gabriele; Zoghbi, Huda Y

    2009-12-04

    Granule neuron precursors (GNPs) are the most actively proliferating cells in the postnatal nervous system, and mutations in pathways that control the GNP cell cycle can result in medulloblastoma. The transcription factor Atoh1 has been suspected to contribute to GNP proliferation, but its role in normal and neoplastic postnatal cerebellar development remains unexplored. We show that Atoh1 regulates the signal transduction pathway of Sonic Hedgehog, an extracellular factor that is essential for GNP proliferation, and demonstrate that deletion of Atoh1 prevents cerebellar neoplasia in a mouse model of medulloblastoma. Our data shed light on the function of Atoh1 in postnatal cerebellar development and identify a new mechanism that can be targeted to regulate medulloblastoma formation.

  4. Regulation of sonic hedgehog-GLI1 downstream target genes PTCH1, Cyclin D2, Plakoglobin, PAX6 and NKX2.2 and their epigenetic status in medulloblastoma and astrocytoma

    PubMed Central

    2010-01-01

    Background The Sonic hedgehog (Shh) signaling pathway is critical for cell growth and differentiation. Impairment of this pathway can result in both birth defects and cancer. Despite its importance in cancer development, the Shh pathway has not been thoroughly investigated in tumorigenesis of brain tumors. In this study, we sought to understand the regulatory roles of GLI1, the immediate downstream activator of the Shh signaling pathway on its downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6 in medulloblastoma and astrocytic tumors. Methods We silenced GLI1 expression in medulloblastoma and astrocytic cell lines by transfection of siRNA against GLI1. Subsequently, we performed RT-PCR and quantitative real time RT-PCR (qRT-PCR) to assay the expression of downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6. We also attempted to correlate the pattern of expression of GLI1 and its regulated genes in 14 cell lines and 41 primary medulloblastoma and astrocytoma tumor samples. We also assessed the methylation status of the Cyclin D2 and PTCH1 promoters in these 14 cell lines and 58 primary tumor samples. Results Silencing expression of GLI1 resulted up-regulation of all target genes in the medulloblastoma cell line, while only PTCH1 was up-regulated in astrocytoma. We also observed methylation of the cyclin D2 promoter in a significant number of astrocytoma cell lines (63%) and primary astrocytoma tumor samples (32%), but not at all in any medulloblastoma samples. PTCH1 promoter methylation was less frequently observed than Cyclin D2 promoter methylation in astrocytomas, and not at all in medulloblastomas. Conclusions Our results demonstrate different regulatory mechanisms of Shh-GLI1 signaling. These differences vary according to the downstream target gene affected, the origin of the tissue, as well as epigenetic regulation of some of these genes. PMID:21059263

  5. A critical role for sonic hedgehog signaling in the early expansion of the developing brain.

    PubMed

    Britto, Joanne; Tannahill, David; Keynes, Roger

    2002-02-01

    The mechanisms that coordinate the three-dimensional shape of the vertebrate brain during development are largely unknown. We have found that sonic hedgehog (Shh) is crucial in driving the rapid, extensive expansion of the early vesicles of the developing midbrain and forebrain. Transient displacement of the notochord from the midbrain floor plate resulted in abnormal folding and overall collapse of the vesicles, accompanied by reduced cell proliferation and increased cell death in the midbrain. Simultaneously, expression of Shh decreased locally in the notochord and floor plate, whereas overt patterning and differentiation proceeded normally. Normal midbrain expansion was restored by implantation of Shh-secreting cells in a dose-dependent manner; conversely, expansion was retarded following antagonism of the Shh signaling pathway by cyclopamine. Our results indicate that Shh signaling from the ventral midline is essential for regulating brain morphogenesis during early development.

  6. Sonic Hedgehog Promotes Tumor Cell Survival by Inhibiting CDON Pro-Apoptotic Activity

    PubMed Central

    Rama, Nicolas; Delcros, Jean-Guy; Gadot, Nicolas; Scoazec, Jean-Yves; Krauss, Robert; Bernet, Agnès; Mehlen, Patrick

    2013-01-01

    The Hedgehog signaling is a determinant pathway for tumor progression. However, while inhibition of the Hedgehog canonical pathway—Patched–Smoothened–Gli—has proved efficient in human tumors with activating mutations in this pathway, recent clinical data have failed to show any benefit in other cancers, even though Sonic Hedgehog (SHH) expression is detected in these cancers. Cell-adhesion molecule-related/down-regulated by Oncogenes (CDON), a positive regulator of skeletal muscle development, was recently identified as a receptor for SHH. We show here that CDON behaves as a SHH dependence receptor: it actively triggers apoptosis in the absence of SHH. The pro-apoptotic activity of unbound CDON requires a proteolytic cleavage in its intracellular domain, allowing the recruitment and activation of caspase-9. We show that by inducing apoptosis in settings of SHH limitation, CDON expression constrains tumor progression, and as such, decreased CDON expression observed in a large fraction of human colorectal cancer is associated in mice with intestinal tumor progression. Reciprocally, we propose that the SHH expression, detected in human cancers and previously considered as a mechanism for activation of the canonical pathway in an autocrine or paracrine manner, actually provides a selective tumor growth advantage by blocking CDON-induced apoptosis. In support of this notion, we present the preclinical demonstration that interference with the SHH–CDON interaction triggers a CDON-dependent apoptosis in vitro and tumor growth inhibition in vivo. The latter observation qualifies CDON as a relevant alternative target for anticancer therapy in SHH-expressing tumors. PMID:23940460

  7. Expression and Role of Sonic Hedgehog in the Process of Fracture Healing with Aging.

    PubMed

    Matsumoto, Kenichi; Shimo, Tsuyoshi; Kurio, Naito; Okui, Tatsuo; Obata, Kyoichi; Masui, Masanori; Pang, Pai; Horikiri, Yuu; Sasaki, Akira

    2016-01-01

    Aging is one of the risk factors for delayed fracture healing. Sonic hedgehog (SHH) protein, an inducer of embryonic development, has been demonstrated to be activated in osteoblasts at the dynamic remodeling site of a bone fracture. Herein, we compared and examined the distribution patterns of SHH and the functional effect of SHH signaling on osteogenesis and osteoclastogenesis between young (5-week-old) and aged (60-week-old) mice during fracture healing. We found that SHH was expressed in bone marrow cells from the fractured site of the rib of young mice on day 5, but was barely detectable in the corresponding cells from the rib of aged mice. SHH was also detected in osteoblasts and bone marrow cells at the callus remodeling stage on days 14 and 28 in both young and aged mice. The number of alkaline phosphatase (ALP)-positive osteoblasts was significantly higher in young mice on days 5 and 14, whereas the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts was significantly higher in aged mice. SHH stimulated significantly more osteoblast formation in the young compared to old mice. SHH stimulated the osteoclast formation directly in the aged mice and suppressed the formation indirectly through osteoprotegerin expression in the young mice. Results indicate that an aged-related delay of fracture healing may contribute to the unbalanced bone formation and resorption, regulated by hedgehog signaling.

  8. Sonic Hedgehog: A Good Gene Gone Bad? Detection and Treatment of Genetic Abnormalities.

    ERIC Educational Resources Information Center

    Yaich, Lauren E.

    2001-01-01

    Presents a case of a baby born with the genetic condition holoprosencephaly in which students explore the "Sonic hedgehog" gene, signal transduction, and the ethics of body and tissue donation. Presents a two-part assignment that features students writing an informed consent document that explains the science behind this congenital abnormality,…

  9. Analysis of the Sonic Hedgehog signaling pathway in normal and abnormal bladder development.

    PubMed

    DeSouza, Kristin R; Saha, Monalee; Carpenter, Ashley R; Scott, Melissa; McHugh, Kirk M

    2013-01-01

    In this study, we examined the expression of Sonic Hedgehog, Patched, Gli1, Gli2, Gli3 and Myocardin in the developing bladders of male and female normal and megabladder (mgb-/-) mutant mice at embryonic days 12 through 16 by in situ hybridization. This analysis indicated that each member of the Sonic Hedgehog signaling pathway as well as Myocardin displayed distinct temporal and spatial patterns of expression during normal bladder development. In contrast, mgb-/- bladders showed both temporal and spatial changes in the expression of Patched, Gli1 and Gli3 as well as a complete lack of Myocardin expression. These changes occurred primarily in the outer mesenchyme of developing mgb-/- bladders consistent with the development of an amuscular bladder phenotype in these animals. These results provide the first comprehensive analysis of the Sonic Hedgehog signaling pathway during normal bladder development and provide strong evidence that this key signaling cascade is critical in establishing radial patterning in the developing bladder. In addition, the lack of detrusor smooth muscle development observed in mgb-/- mice is associated with bladder-specific temporospatial changes in Sonic Hedgehog signaling coupled with a lack of Myocardin expression that appears to result in altered patterning of the outer mesenchyme and poor initiation and differentiation of smooth muscle cells within this region of the developing bladder.

  10. Involvement of brain-derived neurotrophic factor and sonic hedgehog in the spinal cord plasticity after neurotoxic partial removal of lumbar motoneurons.

    PubMed

    Gulino, Rosario; Gulisano, Massimo

    2012-07-01

    Adult mammals could spontaneously achieve a partial sensory-motor recovery after spinal cord injury, by mechanisms including synaptic plasticity. We previously showed that this recovery is associated to the expression of synapsin-I, and that sonic hedgehog and Notch-1 could be also involved in plasticity. The role of brain-derived neurotrophic factor and glutamate receptors in regulating synaptic efficacy has been explored in the last decade but, although these mechanisms are now well-defined in the brain, the molecular mechanisms underlying the so called "spinal learning" are still less clear. Here, we measured the expression levels of choline acetyltransferase, synapsin-I, sonic hedgehog, Notch-1, glutamate receptor subunits (GluR1, GluR2, GluR4, NMDAR1) and brain-derived neurotrophic factor, in a motoneuron-depleted mouse spinal lesion model obtained by intramuscular injection of cholera toxin-B saporin. The lesion caused the down-regulation of the majority of analysed proteins. Moreover, we found that in lesioned but not in control spinal tissue, synapsin-I expression is associated to that of both brain-derived neurotrophic factor and sonic hedgehog, whereas GluR2 expression is linked to that of Shh. These results suggest that brain-derived neurotrophic factor and sonic hedgehog could collaborate in modulating synaptic plasticity after the removal of motoneurons, by a mechanism involving both pre- and post-synaptic processes. Interestingly, the involvement of sonic hedgehog showed here is novel, and offers new routes to address spinal cord plasticity and repair.

  11. Targeting of sonic hedgehog-Gli signaling: A potential therapeutic target for patients with breast cancer.

    PubMed

    Song, Lingqin; Wang, Weifeng; Liu, Di; Zhao, Yang; He, Jianjun; Wang, Xijing; Dai, Zhijun; Zhang, Huimin; Li, Xiao

    2016-08-01

    Breast cancer is the most common malignant cancer among women. The Hedgehog (Hh) signaling pathway serves a key role in malignant cancer cell growth and migration. However, little is known with regard to the specific function of the Hh signaling pathway in human breast cancer. The current study investigated the specific role of Hh signaling in the human breast cancer cell line MDA-MB-231. Expression of components of Shh-Gli signaling, as well as the Gli-responsive genes B-cell lymphoma 2 (Bcl-2) and cyclin D1, were investigated in MDA-MB-231 cells using western blotting. The effects of Shh-Gli signaling on MDA-MB-231 proliferation were analyzed by MTT assay. The role of E-cadherin in the epithelial-mesenchymal transition process was determined by western blot while matrix metalloproteinase (MMP)-9/MMP-2 secretion was studied by enzyme-linked immunosorbent assay. The results indicated that Shh-Gli signaling was activated in MDA-MB-231 cells, significantly enhancing cell viability. Overexpression of Gli positively regulated the transcription of Bcl-2 and cyclin D1 thereby regulating MDA-MB-231 cell proliferation and survival. Treatment of MDA-MB-231 cells with human sonic hedgehog, n-terminus for 72 h significantly reduced E-cadherin protein levels and enhanced secretion of MMP-9 and MMP-2. These findings suggest that Shh-Gli signaling is significantly activated in human breast cancer cells, and is accompanied by enhanced cell viability, proliferation and migration capacities.

  12. Targeting of sonic hedgehog-Gli signaling: A potential therapeutic target for patients with breast cancer

    PubMed Central

    Song, Lingqin; Wang, Weifeng; Liu, Di; Zhao, Yang; He, Jianjun; Wang, Xijing; Dai, Zhijun; Zhang, Huimin; Li, Xiao

    2016-01-01

    Breast cancer is the most common malignant cancer among women. The Hedgehog (Hh) signaling pathway serves a key role in malignant cancer cell growth and migration. However, little is known with regard to the specific function of the Hh signaling pathway in human breast cancer. The current study investigated the specific role of Hh signaling in the human breast cancer cell line MDA-MB-231. Expression of components of Shh-Gli signaling, as well as the Gli-responsive genes B-cell lymphoma 2 (Bcl-2) and cyclin D1, were investigated in MDA-MB-231 cells using western blotting. The effects of Shh-Gli signaling on MDA-MB-231 proliferation were analyzed by MTT assay. The role of E-cadherin in the epithelial-mesenchymal transition process was determined by western blot while matrix metalloproteinase (MMP)-9/MMP-2 secretion was studied by enzyme-linked immunosorbent assay. The results indicated that Shh-Gli signaling was activated in MDA-MB-231 cells, significantly enhancing cell viability. Overexpression of Gli positively regulated the transcription of Bcl-2 and cyclin D1 thereby regulating MDA-MB-231 cell proliferation and survival. Treatment of MDA-MB-231 cells with human sonic hedgehog, n-terminus for 72 h significantly reduced E-cadherin protein levels and enhanced secretion of MMP-9 and MMP-2. These findings suggest that Shh-Gli signaling is significantly activated in human breast cancer cells, and is accompanied by enhanced cell viability, proliferation and migration capacities. PMID:27446389

  13. Sonic hedgehog controls enteric nervous system development by patterning the extracellular matrix.

    PubMed

    Nagy, Nandor; Barad, Csilla; Graham, Hannah K; Hotta, Ryo; Cheng, Lily S; Fejszak, Nora; Goldstein, Allan M

    2016-01-15

    The enteric nervous system (ENS) develops from neural crest cells that migrate along the intestine, differentiate into neurons and glia, and pattern into two plexuses within the gut wall. Inductive interactions between epithelium and mesenchyme regulate gut development, but the influence of these interactions on ENS development is unknown. Epithelial-mesenchymal recombinations were constructed using avian hindgut mesenchyme and non-intestinal epithelium from the bursa of Fabricius. These recombinations led to abnormally large and ectopically positioned ganglia. We hypothesized that sonic hedgehog (Shh), a secreted intestinal epithelial protein not expressed in the bursa, mediates this effect. Inhibition of Shh signaling, by addition of cyclopamine or a function-blocking antibody, resulted in large, ectopic ganglia adjacent to the epithelium. Shh overexpression, achieved in ovo using Shh-encoding retrovirus and in organ culture using recombinant protein, led to intestinal aganglionosis. Shh strongly induced the expression of versican and collagen type IX, whereas cyclopamine reduced expression of these chondroitin sulfate proteoglycans that are known to be inhibitory to neural crest cell migration. Shh also inhibited enteric neural crest-derived cell (ENCC) proliferation, promoted neuronal differentiation, and reduced expression of Gdnf, a key regulator of ENS formation. Ptc1 and Ptc2 were not expressed by ENCCs, and migration of isolated ENCCs was not inhibited by Shh protein. These results suggest that epithelial-derived Shh acts indirectly on the developing ENS by regulating the composition of the intestinal microenvironment.

  14. Sonic hedgehog controls enteric nervous system development by patterning the extracellular matrix

    PubMed Central

    Nagy, Nandor; Barad, Csilla; Graham, Hannah K.; Hotta, Ryo; Cheng, Lily S.; Fejszak, Nora; Goldstein, Allan M.

    2016-01-01

    The enteric nervous system (ENS) develops from neural crest cells that migrate along the intestine, differentiate into neurons and glia, and pattern into two plexuses within the gut wall. Inductive interactions between epithelium and mesenchyme regulate gut development, but the influence of these interactions on ENS development is unknown. Epithelial-mesenchymal recombinations were constructed using avian hindgut mesenchyme and non-intestinal epithelium from the bursa of Fabricius. These recombinations led to abnormally large and ectopically positioned ganglia. We hypothesized that sonic hedgehog (Shh), a secreted intestinal epithelial protein not expressed in the bursa, mediates this effect. Inhibition of Shh signaling, by addition of cyclopamine or a function-blocking antibody, resulted in large, ectopic ganglia adjacent to the epithelium. Shh overexpression, achieved in ovo using Shh-encoding retrovirus and in organ culture using recombinant protein, led to intestinal aganglionosis. Shh strongly induced the expression of versican and collagen type IX, whereas cyclopamine reduced expression of these chondroitin sulfate proteoglycans that are known to be inhibitory to neural crest cell migration. Shh also inhibited enteric neural crest-derived cell (ENCC) proliferation, promoted neuronal differentiation, and reduced expression of Gdnf, a key regulator of ENS formation. Ptc1 and Ptc2 were not expressed by ENCCs, and migration of isolated ENCCs was not inhibited by Shh protein. These results suggest that epithelial-derived Shh acts indirectly on the developing ENS by regulating the composition of the intestinal microenvironment. PMID:26674309

  15. Sonic hedgehog functions upstream of disrupted-in-schizophrenia 1 (disc1): implications for mental illness

    PubMed Central

    Boyd, Penelope J.; Cunliffe, Vincent T.; Roy, Sudipto; Wood, Jonathan D.

    2015-01-01

    ABSTRACT DISRUPTED-IN-SCHIZOPHRENIA (DISC1) has been one of the most intensively studied genetic risk factors for mental illness since it was discovered through positional mapping of a translocation breakpoint in a large Scottish family where a balanced chromosomal translocation was found to segregate with schizophrenia and affective disorders. While the evidence for it being central to disease pathogenesis in the original Scottish family is compelling, recent genome-wide association studies have not found evidence for common variants at the DISC1 locus being associated with schizophrenia in the wider population. It may therefore be the case that DISC1 provides an indication of biological pathways that are central to mental health issues and functional studies have shown that it functions in multiple signalling pathways. However, there is little information regarding factors that function upstream of DISC1 to regulate its expression and function. We herein demonstrate that Sonic hedgehog (Shh) signalling promotes expression of disc1 in the zebrafish brain. Expression of disc1 is lost in smoothened mutants that have a complete loss of Shh signal transduction, and elevated in patched mutants which have constitutive activation of Shh signalling. We previously demonstrated that disc1 knockdown has a dramatic effect on the specification of oligodendrocyte precursor cells (OPC) in the hindbrain and Shh signalling is known to be essential for the specification of these cells. We show that disc1 is prominently expressed in olig2-positive midline progenitor cells that are absent in smo mutants, while cyclopamine treatment blocks disc1 expression in these cells and mimics the effect of disc1 knock down on OPC specification. Various features of a number of psychiatric conditions could potentially arise through aberrant Hedgehog signalling. We therefore suggest that altered Shh signalling may be an important neurodevelopmental factor in the pathobiology of mental illness. PMID

  16. Primary cilium and sonic hedgehog signaling during neural tube patterning: role of GPCRs and second messengers.

    PubMed

    Pal, Kasturi; Mukhopadhyay, Saikat

    2015-04-01

    The ventral neural tube in vertebrates is patterned by a gradient of sonic hedgehog (Shh) secreted from the notochord and floor plate. Forward genetic screens first pointed to the role of the primary cilium in ventral neural tube patterning. Further research has shown that most components of the Shh pathway localize to or shuttle through the primary cilium. In the absence of Shh, the bifunctional Gli transcription factors are proteolytically processed into repressor forms in a protein kinase A (PKA)- and cilium-dependent manner. Recent work suggests that the orphan G-protein-coupled receptor (GPCR) Gpr161 localizes to cilia, and functions as a negative regulator of Shh signaling by determining Gli processing via cAMP signaling. The primary cilium also functions as a signaling compartment for calcium in the Shh pathway. A better understanding of the role of the cilium as a signaling compartment, and the interplay of second messenger systems that regulate PKA activation and Gli amplification during signaling is critical for deciphering the role of Shh during development, neuronal differentiation, and tumorigenesis.

  17. Sonic Hedgehog, VACTERL, and Fanconi anemia: Pathogenetic connections and therapeutic implications.

    PubMed

    Lubinsky, Mark

    2015-11-01

    Three systems with VACTERL association findings- mutations of the Sonic Hedgehog (SHH) signaling pathway in mice, murine adriamycin teratogenicity, and human Fanconi anemia (FA) pathway mutations, may all involve a similar mechanism. SHH is up-regulated in irradiated cells, and DNA breaks common with radiation damage in the adriamycin and FA systems are plausible signals for such effects, which would affect development. Since FA related DNA breakage occurs throughout life, SHH disturbances may account for later FA related findings involving hematopoietic and malignancy issues. In support, androgen, a standard treatment for FA hematologic failure, down-regulates SHH, and common FA malignancies such as squamous cell carcinomas and acute myeloid leukemia have been linked to enhanced SHH function. This suggests that interventions lowering SHH levels may be useful therapeutically. Also supporting a connection between pre- and post- natal findings, the frequency and number of VACTERL anomalies with FA correlate with the severity and onset of hematopoietic and malignancy issues. In FA, radial anomalies are the most common of these defects, followed by renal findings, while vertebral and gastrointestinal anomalies are relatively uncommon, a pattern that differs from observations of the VACTERL association. Genes with more severe effects also show a greatly increased incidence of brain abnormalities, and a paucity of such findings with other FA genes suggests that brain development is relatively refractory to SHH related effects, accounting for the rarity of such findings with the association.

  18. Reduced pepsin A processing of sonic hedgehog in parietal cells precedes gastric atrophy and transformation.

    PubMed

    Zavros, Yana; Waghray, Meghna; Tessier, Arthur; Bai, Longchuan; Todisco, Andrea; L Gumucio, Deborah; Samuelson, Linda C; Dlugosz, Andrzej; Merchant, Juanita L

    2007-11-16

    Sonic hedgehog (Shh) is not only essential to the development of the gastrointestinal tract, but is also necessary to maintain the characteristic acid-secreting phenotype of the adult stomach. Gastrin is the only hormone capable of stimulating gastric acid and is thus required to maintain functional parietal cells. We have shown previously that gastrin-null mice display gastric atrophy and metaplasia prior to progression to distal, intestinal-type gastric cancer. Because reduced levels of Shh peptide correlate with gastric atrophy, we examined whether gastrin regulates Shh expression in parietal cells. We show here that gastrin stimulates Shh gene expression and acid-dependent processing of the 45-kDa Shh precursor to the 19-kDa secreted peptide in primary parietal cell cultures. This cleavage was blocked by the proton pump inhibitor omeprazole and mediated by the acid-activated protease pepsin A. Pepsin A was also the protease responsible for processing Shh in tissue extracts from human stomach. By contrast, extracts prepared from neoplastic gastric mucosa had reduced levels of pepsin A and did not process Shh. Therefore processing of Shh in the normal stomach is hormonally regulated, acid-dependent, and mediated by the aspartic protease pepsin A. Moreover parietal cell atrophy, a known pre-neoplastic lesion, correlates with loss of Shh processing.

  19. Exogenous Sonic hedgehog modulates the pool of GABAergic interneurons during cerebellar development.

    PubMed

    De Luca, A; Parmigiani, E; Tosatto, G; Martire, S; Hoshino, M; Buffo, A; Leto, K; Rossi, F

    2015-04-01

    All cerebellar GABAergic interneurons were derived from a common pool of precursor cells residing in the embryonic ventricular zone (VZ) and migrating in the prospective white matter (PWM) after birth, where both intrinsic and extrinsic factors contribute to regulate their amplification. Among the environmental factors, we focused on Sonic hedgehog (Shh), a morphogen well known to regulate neural progenitor cell proliferation. We asked if and how exogenous Shh treatment affects the lineage of cerebellar GABAergic interneurons. To address these issues, exogenous Shh was administered to embryonic and postnatal organotypic slices. We found that Shh is able to expand the pool of interneuron progenitors residing in the embryonic epithelium and in the postnatal PWM. In particular, Shh signalling pathway was highly mitogenic at early developmental stages of interneuron production, whereas its effect decreased after the first postnatal week. Gene expression analysis of sorted cells and in situ hybridization further showed that immature interneurons express both the Shh receptor patched and the Shh target gene Gli1. Thus, within the interneuron lineage, Shh might exert regulatory functions also in postmitotic cells. On the whole, our data enlighten the role of Shh during cerebellar maturation and further broaden our knowledge on the amplification mechanisms of the interneuron progenitor pool.

  20. Identification of sonic hedgehog as a candidate gene responsible for the polydactylous mouse mutant Sasquatch.

    PubMed

    Sharpe, J; Lettice, L; Hecksher-Sorensen, J; Fox, M; Hill, R; Krumlauf, R

    1999-01-28

    The mouse mutants of the hemimelia-luxate group (lx, lu, lst, Dh, Xt, and the more recently identified Hx, Xpl and Rim4; [1] [2] [3] [4] [5]) have in common preaxial polydactyly and longbone abnormalities. Associated with the duplication of digits are changes in the regulation of development of the anterior limb bud resulting in ectopic expression of signalling components such as Sonic hedgehog (Shh) and fibroblast growth factor-4 (Fgf4), but little is known about the molecular causes of this misregulation. We generated, by a transgene insertion event, a new member of this group of mutants, Sasquatch (Ssq), which disrupted aspects of both anteroposterior (AP) and dorsoventral (DV) patterning. The mutant displayed preaxial polydactyly in the hindlimbs of heterozygous embryos, and in both hindlimbs and forelimbs of homozygotes. The Shh, Fgf4, Fgf8, Hoxd12 and Hoxd13 genes were all ectopically expressed in the anterior region of affected limb buds. The insertion site was found to lie close to the Shh locus. Furthermore, expression from the transgene reporter has come under the control of a regulatory element that directs a pattern mirroring the endogenous expression pattern of Shh in limbs. In abnormal limbs, both Shh and the reporter were ectopically induced in the anterior region, whereas in normal limbs the reporter and Shh were restricted to the zone of polarising activity (ZPA). These data strongly suggest that Ssq is caused by direct interference with the cis regulation of the Shh gene.

  1. Curcumin Suppresses Lung Cancer Stem Cells via Inhibiting Wnt/β-catenin and Sonic Hedgehog Pathways.

    PubMed

    Zhu, Jian-Yun; Yang, Xue; Chen, Yue; Jiang, Ye; Wang, Shi-Jia; Li, Yuan; Wang, Xiao-Qian; Meng, Yu; Zhu, Ming-Ming; Ma, Xiao; Huang, Cong; Wu, Rui; Xie, Chun-Feng; Li, Xiao-Ting; Geng, Shan-Shan; Wu, Jie-Shu; Zhong, Cai-Yun; Han, Hong-Yu

    2017-02-15

    Cancer stem cells (CSCs) are highly implicated in the progression of human cancers. Thus, targeting CSCs may be a promising strategy for cancer therapy. Wnt/β-catenin and Sonic Hedgehog pathways play an important regulatory role in maintaining CSC characteristics. Natural compounds, such as curcumin, possess chemopreventive properties. However, the interventional effect of curcumin on lung CSCs has not been clarified. In the present study, tumorsphere formation assay was used to enrich lung CSCs from A549 and H1299 cells. We showed that the levels of lung CSC markers (CD133, CD44, ALDHA1, Nanog and Oct4) and the number of CD133-positive cells were significantly elevated in the sphere-forming cells. We further illustrated that curcumin efficiently abolished lung CSC traits, as evidenced by reduced tumorsphere formation, reduced number of CD133-positive cells, decreased expression levels of lung CSC markers, as well as proliferation inhibition and apoptosis induction. Moreover, we demonstrated that curcumin suppressed the activation of both Wnt/β-catenin and Sonic Hedgehog pathways. Taken together, our data suggested that curcumin exhibited its interventional effect on lung CSCs via inhibition of Wnt/β-catenin and Sonic Hedgehog pathways. These novel findings could provide new insights into the potential therapeutic application of curcumin in lung CSC elimination and cancer intervention. Copyright © 2017 John Wiley & Sons, Ltd.

  2. Cholesterol and its derivatives in Sonic Hedgehog signaling and Cancer

    PubMed Central

    Riobo, Natalia A.

    2012-01-01

    The connection between the Hedgehog pathway and cholesterol has been recognized since the early days that shaped our current understanding of this unique pathway. Cholesterol and related lipids are intricately linked to HH signaling: from the role of cholesterol in HH biosynthesis to the modulation of HH signal reception and transduction by other sterols, passing by the phylogenetic relationships among many components of the HH pathway that resemble or contain lipid-binding domains. Here I review the connections between HH signaling, cholesterol and its derivatives and analyze the potential implications for HH-dependent cancers. PMID:22832232

  3. Interaction of sonic hedgehog (SHH) pathway with cancer stem cell genes in gastric cancer.

    PubMed

    Samadani, Ali Akbar; Akhavan-Niaki, Haleh

    2015-03-01

    Gastric cancer may appear by frequent genetic or epigenetic changes in oncogenes, tumor suppressor or DNA mismatch repair genes. Molecular studies show the possibility of involvement of certain cancer pathways in gastric cancer. In this respect, DNA methylation is one of the most important epigenetic alterations in gastric cancer and identifying the signaling mechanism and also methylation of some genes that are involved in gastric cancer can help to improve treatment strategies. Relatively, there are many reported methylation alteration of genes in stem cells in all kinds of tumors with some of these genes having a key role in tumor development. Correspondingly, KLF5, CDX1/2, WNT1 and FEM1A are considerable genes in gastric cancer, although many researches and studies have illustrated that sonic hedgehog and expression of its signaling cascade proteins are related in gastric cancer. Relatively, modification in these genes causes many eclectic cancers such as rhabdomyosarcoma and diverse kinds of digestive system tumor development. Conspicuously, these master genes have a noticeable role in stem cell's growth regulation as well as other kinds of cancer such as breast cancer and leukemia. Hence, we concluded that research and studies on methylation and expression of these genes and also the investigation of molecular signaling in gastric cancer can acquire impressive conclusions in order to control and treat this common place and serious problem.

  4. Sonic Hedgehog Signaling Mediates Epithelial–Mesenchymal Communication and Promotes Renal Fibrosis

    PubMed Central

    Ding, Hong; Zhou, Dong; Hao, Sha; Zhou, Lili; He, Weichun; Nie, Jing; Hou, Fan Fan

    2012-01-01

    Sonic hedgehog (Shh) signaling is a developmental signal cascade that plays an essential role in regulating embryogenesis and tissue homeostasis. Here, we investigated the potential role of Shh signaling in renal interstitial fibrogenesis. Ureteral obstruction induced Shh, predominantly in the renal tubular epithelium of the fibrotic kidneys. Using Gli1lacZ knock-in mice, we identified renal interstitial fibroblasts as Shh-responding cells. In cultured renal fibroblasts, recombinant Shh protein activated Gli1 and induced α-smooth muscle actin (α-SMA), desmin, fibronectin, and collagen I expression, suggesting that Shh signaling promotes myofibroblast activation and matrix production. Blockade of Shh signaling with cyclopamine abolished the Shh-mediated induction of Gli1, Snail1, α-SMA, fibronectin, and collagen I. In vivo, the kidneys of Gli1-deficient mice were protected against the development of interstitial fibrosis after obstructive injury. In wild-type mice, cyclopamine did not affect renal Shh expression but did inhibit induction of Gli1, Snail1, and α-SMA. In addition, cyclopamine reduced matrix expression and mitigated fibrotic lesions. These results suggest that tubule-derived Shh mediates epithelial–mesenchymal communication by targeting interstitial fibroblasts after kidney injury. We conclude that Shh/Gli1 signaling plays a critical role in promoting fibroblast activation, production of extracellular matrix, and development of renal interstitial fibrosis. PMID:22302193

  5. Developmental hypothyroidism abolishes bilateral differences in sonic hedgehog gene control in the rat hippocampal dentate gyrus.

    PubMed

    Tanaka, Takeshi; Wang, Liyun; Kimura, Masayuki; Abe, Hajime; Mizukami, Sayaka; Yoshida, Toshinori; Shibutani, Makoto

    2015-03-01

    Both developmental and adult-stage hypothyroidism disrupt rat hippocampal neurogenesis. We previously showed that exposing mouse offspring to manganese permanently disrupts hippocampal neurogenesis and abolishes the asymmetric distribution of cells expressing Mid1, a molecule regulated by sonic hedgehog (Shh) signaling. The present study examined the involvement of Shh signaling on the disruption of hippocampal neurogenesis in rats with hypothyroidism. Pregnant rats were treated with methimazole (MMI) at 0 or 200 ppm in the drinking water from gestation day 10-21 days after delivery (developmental hypothyroidism). Adult male rats were treated with MMI in the same manner from postnatal day (PND) 46 to PND 77 (adult-stage hypothyroidism). Developmental hypothyroidism reduced the number of Mid1(+) cells within the subgranular zone of the dentate gyrus of offspring on PND 21, and consequently abolished the normal asymmetric predominance of Mid1(+) cells on the right side through the adult stage. In control animals, Shh was expressed in a subpopulation of hilar neurons, showing asymmetric distribution with left side predominance on PND 21; however, this asymmetry did not continue through the adult stage. Developmental hypothyroidism increased Shh(+) neurons bilaterally and abolished the asymmetric distribution pattern on PND 21. Adult hypothyroidism also disrupted the asymmetric distribution of Mid1(+) cells but did not affect the distribution of Shh(+) hilar neurons. The results suggest that the hippocampal neurogenesis disruption seen in hypothyroidism involves changes in asymmetric Shh(+) neuron distribution in developmental hypothyroidism and altered Mid1 expression in both developmental and adult-stage hypothyroidism.

  6. Interleukin-1β induces blood-brain barrier disruption by downregulating Sonic hedgehog in astrocytes.

    PubMed

    Wang, Yue; Jin, Shijie; Sonobe, Yoshifumi; Cheng, Yi; Horiuchi, Hiroshi; Parajuli, Bijay; Kawanokuchi, Jun; Mizuno, Tetsuya; Takeuchi, Hideyuki; Suzumura, Akio

    2014-01-01

    The blood-brain barrier (BBB) is composed of capillary endothelial cells, pericytes, and perivascular astrocytes, which regulate central nervous system homeostasis. Sonic hedgehog (SHH) released from astrocytes plays an important role in the maintenance of BBB integrity. BBB disruption and microglial activation are common pathological features of various neurologic diseases such as multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease. Interleukin-1β (IL-1β), a major pro-inflammatory cytokine released from activated microglia, increases BBB permeability. Here we show that IL-1β abolishes the protective effect of astrocytes on BBB integrity by suppressing astrocytic SHH production. Astrocyte conditioned media, SHH, or SHH signal agonist strengthened BBB integrity by upregulating tight junction proteins, whereas SHH signal inhibitor abrogated these effects. Moreover, IL-1β increased astrocytic production of pro-inflammatory chemokines such as CCL2, CCL20, and CXCL2, which induce immune cell migration and exacerbate BBB disruption and neuroinflammation. Our findings suggest that astrocytic SHH is a potential therapeutic target that could be used to restore disrupted BBB in patients with neurologic diseases.

  7. Mutations of the Sonic Hedgehog Pathway Underlie Hypothalamic Hamartoma with Gelastic Epilepsy.

    PubMed

    Hildebrand, Michael S; Griffin, Nicole G; Damiano, John A; Cops, Elisa J; Burgess, Rosemary; Ozturk, Ezgi; Jones, Nigel C; Leventer, Richard J; Freeman, Jeremy L; Harvey, A Simon; Sadleir, Lynette G; Scheffer, Ingrid E; Major, Heather; Darbro, Benjamin W; Allen, Andrew S; Goldstein, David B; Kerrigan, John F; Berkovic, Samuel F; Heinzen, Erin L

    2016-08-04

    Hypothalamic hamartoma (HH) with gelastic epilepsy is a well-recognized drug-resistant epilepsy syndrome of early life.(1) Surgical resection allows limited access to the small deep-seated lesions that cause the disease. Here, we report the results of a search for somatic mutations in paired hamartoma- and leukocyte-derived DNA samples from 38 individuals which we conducted by using whole-exome sequencing (WES), chromosomal microarray (CMA), and targeted resequencing (TRS) of candidate genes. Somatic mutations were identified in genes involving regulation of the sonic hedgehog (Shh) pathway in 14/38 individuals (37%). Three individuals had somatic mutations in PRKACA, which encodes a cAMP-dependent protein kinase that acts as a repressor protein in the Shh pathway, and four subjects had somatic mutations in GLI3, an Shh pathway gene associated with HH. In seven other individuals, we identified two recurrent and three single brain-tissue-specific, large copy-number or loss-of-heterozygosity (LOH) variants involving multiple Shh genes, as well as other genes without an obvious biological link to the Shh pathway. The Shh pathway genes in these large somatic lesions include the ligand itself (SHH and IHH), the receptor SMO, and several other Shh downstream pathway members, including CREBBP and GLI2. Taken together, our data implicate perturbation of the Shh pathway in at least 37% of individuals with the HH epilepsy syndrome, consistent with the concept of a developmental pathway brain disease.

  8. Targeting the Sonic Hedgehog Signaling Pathway: Review of Smoothened and GLI Inhibitors.

    PubMed

    Rimkus, Tadas K; Carpenter, Richard L; Qasem, Shadi; Chan, Michael; Lo, Hui-Wen

    2016-02-15

    The sonic hedgehog (Shh) signaling pathway is a major regulator of cell differentiation, cell proliferation, and tissue polarity. Aberrant activation of the Shh pathway has been shown in a variety of human cancers, including, basal cell carcinoma, malignant gliomas, medulloblastoma, leukemias, and cancers of the breast, lung, pancreas, and prostate. Tumorigenesis, tumor progression and therapeutic response have all been shown to be impacted by the Shh signaling pathway. Downstream effectors of the Shh pathway include smoothened (SMO) and glioma-associated oncogene homolog (GLI) family of zinc finger transcription factors. Both are regarded as important targets for cancer therapeutics. While most efforts have been devoted towards pharmacologically targeting SMO, developing GLI-targeted approach has its merit because of the fact that GLI proteins can be activated by both Shh ligand-dependent and -independent mechanisms. To date, two SMO inhibitors (LDE225/Sonidegib and GDC-0449/Vismodegib) have received FDA approval for treating basal cell carcinoma while many clinical trials are being conducted to evaluate the efficacy of this exciting class of targeted therapy in a variety of cancers. In this review, we provide an overview of the biology of the Shh pathway and then detail the current landscape of the Shh-SMO-GLI pathway inhibitors including those in preclinical studies and clinical trials.

  9. Targeting the Sonic Hedgehog Signaling Pathway: Review of Smoothened and GLI Inhibitors

    PubMed Central

    Rimkus, Tadas K.; Carpenter, Richard L.; Qasem, Shadi; Chan, Michael; Lo, Hui-Wen

    2016-01-01

    The sonic hedgehog (Shh) signaling pathway is a major regulator of cell differentiation, cell proliferation, and tissue polarity. Aberrant activation of the Shh pathway has been shown in a variety of human cancers, including, basal cell carcinoma, malignant gliomas, medulloblastoma, leukemias, and cancers of the breast, lung, pancreas, and prostate. Tumorigenesis, tumor progression and therapeutic response have all been shown to be impacted by the Shh signaling pathway. Downstream effectors of the Shh pathway include smoothened (SMO) and glioma-associated oncogene homolog (GLI) family of zinc finger transcription factors. Both are regarded as important targets for cancer therapeutics. While most efforts have been devoted towards pharmacologically targeting SMO, developing GLI-targeted approach has its merit because of the fact that GLI proteins can be activated by both Shh ligand-dependent and -independent mechanisms. To date, two SMO inhibitors (LDE225/Sonidegib and GDC-0449/Vismodegib) have received FDA approval for treating basal cell carcinoma while many clinical trials are being conducted to evaluate the efficacy of this exciting class of targeted therapy in a variety of cancers. In this review, we provide an overview of the biology of the Shh pathway and then detail the current landscape of the Shh-SMO-GLI pathway inhibitors including those in preclinical studies and clinical trials. PMID:26891329

  10. Oxidative damage and TGF-β differentially induce lung epithelial cell sonic hedgehog and tenascin-C expression: implications for the regulation of lung remodelling in idiopathic interstitial lung disease

    PubMed Central

    Fitch, Paul M; Howie, Sarah E M; Wallace, William A H

    2011-01-01

    Idiopathic interstitial lung diseases (iILDs) are characterized by inflammation, hyperplasia of Type-II alveolar epithelial cells (AECs) and lung remodelling often with progressive fibrosis. It remains unclear which signals initiate iILD and/or maintain the disease processes. Using real-time RT-PCR and immunohistochemistry on archival biopsies of three patterns of iILD (usual interstitial pneumonitis/UIP, non-specific interstitial pneumonitis/NSIP and cryptogenic organizing pneumonia/COP) we investigated whether hedgehog signalling (previously associated with lung damage and repair) was functional and whether the damage associated extracellular matrix protein tenascin-C was present in activated Type-II AECs in all three iILDs. Using tissue culture, protein and mRNA detection we also determined how two signals (oxidative damage and TGF-β) associated with iILD pathogenesis affected Sonic hedgehog (SHH) and tenascin-C production by a Type-II AEC cell line. We report that SHH pathway and tenascin-C mRNA and proteins were found in UIP, NSIP and COP. SHH signalling was most active at sites of immature organizing fibrous tissue (fibroblastic foci) in UIP. In vitro Type-II AECs constitutively secrete SHH but not tenascin-C. Oxidative injury stimulated SHH release whereas TGF-β inhibited it. TGF-β and oxidative damage both upregulated tenascin-C mRNA but only TGF-β induced synthesis and release of a distinct protein isoform. SHH signalling is active in Type-II AECs from three types of ILD and all three express tenascin-C. PMID:21039988

  11. Androgenic regulation of hedgehog signaling pathway components in prostate cancer cells.

    PubMed

    Chen, Mengqian; Tanner, Matthew; Levine, Alice C; Levina, Elina; Ohouo, Patrice; Buttyan, Ralph

    2009-01-01

    Hedgehog signaling is thought to play a role in several human cancers including prostate cancer. Although prostate cancer cells express many of the gene products involved in hedgehog signaling, these cells are refractory to the canonical signaling effects of exogenous hedgehog ligands or to activated Smoothened, the hedgehog-regulated mediator of Gli transcriptional activation. Here, we show that the expression of hedgehog ligands and some hedgehog target genes are regulated by androgen in the human prostate cancer cell line, LNCaP and its more metastatic variants (C4-2 and C4-2B). Androgen (R1881) strongly suppressed the expression of hedgehog ligands in these cells and their prolonged maintenance in androgen-deficient medium upregulated Sonic and Indian hedgehog mRNA and protein levels by up to 30,000-fold. Hedgehogs were released into the conditioned medium of androgen-deprived LNCaP cells and this medium was able to increase hedgehog target gene expression in hedgehog-responsive mouse fibroblasts (MC3T3-E1). Moreover, this activity was accompanied by increased expression of Gli target genes, Patched 1 and Gli2, in LNCaP that could be suppressed by cyclopamine, indicating that chronic androgen-deprivation also re-awakens the autocrine responsiveness of the cancer cells to hedgehog. In contrast to the suppressive effects of R1881 on hedgehog ligand and Gli2 expression, we found that Gli1 expression in LNCaP cells was induced by R1881. Given the ability of androgen to modulate the expression and release of hedgehog ligands and the activity of the autocrine hedgehog signaling pathway in these prostate cancer cells, our results imply that chronic androgen deprivation therapy (ADT) for prostate cancer might create a hedgehog signaling environment in the region of the tumor that could ultimately impact on the long term effectiveness of this treatment. This consideration supports the idea of clinically testing hedgehog-blocking drugs in conjunction with ADT in patients

  12. Increased expression of the sonic hedgehog and vascular endothelial growth factor with co-localization in varicocele veins.

    PubMed

    Wang, Shih-Ho; Yang, Wen-Kai; Lee, Jane-Dar

    2017-03-01

    Objectives Varicocele is characterized by dilatation and tortuosity of the internal spermatic vein. Sonic hedgehog plays an important role in angiogenesis and vascular remodeling under hypoxic stress. We studied the relationship and distribution of SHH and vascular endothelial growth factor in internal spermatic vein in patients diagnosed with varicocele. Methods Specimens of 1 cm were taken from the internal spermatic vein during left varicocele repair (N = 20). The control samples of ISV were obtained from eight male patients who underwent left inguinal herniorrhaphy. We analyzed the sonic hedgehog and vascular endothelial growth factor expression and distribution by immunoblotting, immunohistochemistry, immunofluorescent staining, and confocal laser scanning microscopy. The data were analyzed using the Student's t test. Results Immunoblotting showed higher expression of sonic hedgehog and vascular endothelial growth factor proteins in varicocele veins than in the control group ( P < 0.05) which located over muscle layer and endothelium was demonstrated by immunohistochemical staining. Both proteins with co-localization in the muscle layer and especially distributed in endothelium of varicocele veins were revealed under confocal microscopy. Conclusions These findings showed the upexpression of sonic hedgehog and vascular endothelial growth factor with co-localization in varicocele veins which imply that the reducing hypoxia or using sonic hedgehog antagonists may be helpful for this vascular disease.

  13. Noggin and Sonic hedgehog are involved in compensatory changes within the motoneuron-depleted mouse spinal cord.

    PubMed

    Gulino, Rosario; Gulisano, Massimo

    2013-09-15

    Sonic hedgehog and Noggin are morphogenetic factors involved in neural induction and ventralization of the neural tube, but recent findings suggest that they could participate in regeneration and functional recovery after injury. Here, in order to verify if these mechanisms could occur in the spinal cord and involve synaptic plasticity, we measured the expression levels of Sonic hedgehog, Noggin, Choline Acetyltransferase, Synapsin-I and Glutamate receptor subunits (GluR1, GluR2, GluR4), in a motoneuron-depleted mouse spinal cord lesion model obtained by intramuscular injection of Cholera toxin-B saporin. The lesion caused differential expression changes of the analyzed proteins. Moreover, motor performance was found correlated with Sonic hedgehog and Noggin expression in lesioned animals. The results also suggest that Sonic hedgehog could collaborate in modulating synaptic plasticity. Together, these findings confirm that the injured mammalian spinal cord has intrinsic potential for repair and that some proteins classically involved in development, such as Sonic hedgehog and Noggin could have important roles in regeneration and functional restoration, by mechanisms including synaptic plasticity.

  14. Expression of Sonic hedgehog during cell proliferation in the human cerebellum.

    PubMed

    Haldipur, Parthiv; Bharti, Upasna; Govindan, Subashika; Sarkar, Chitra; Iyengar, Soumya; Gressens, Pierre; Mani, Shyamala

    2012-05-01

    The regulation of cell proliferation in the external granular layer (EGL) of the developing cerebellum is important for its normal patterning. An important signal that regulates EGL cell proliferation is Sonic hedgehog (Shh). Shh is secreted by the Purkinje cells (PC) and has a mitogenic effect on the granule cell precursors of the EGL. Deregulation of Shh signaling has been associated with abnormal development, and been implicated in medulloblastomas, which are tumors that arise from the cerebellum. Given the importance of the Shh pathway in cerebellum development and disease, there has been no systematic study of its expression pattern during human cerebellum development. In this study, we describe the expression pattern of Shh, its receptor patched, smoothened, and its effectors that belong to the Gli family of transcription factors, during normal human cerebellum development from 10 weeks of gestational age, and in medulloblastomas that represents a case of abnormal cell proliferation in the cerebellum. This expression pattern is compared to equivalent stages in the normal development of cerebellum in mouse, as well as in tumors. Important differences between human and mouse that reflect differences in the normal developmental program between the 2 species are observed. First, in humans there appears to be a stage of Shh signaling within the EGL, when the PC are not yet the source of Shh. Second, unlike in the postnatal mouse cerebellum, expression of Shh in the PC in the postnatal human cerebellum is downregulated. Finally, medulloblastomas in the human but not in patched heterozygote mouse express Shh. These results highlight cross-species differences in the regulation of the Shh signaling pathway.

  15. Epithelial cells supply Sonic Hedgehog to the perinatal dentate gyrus via transport by platelets.

    PubMed

    Choe, Youngshik; Huynh, Trung; Pleasure, Samuel J

    2015-10-12

    Dentate neural stem cells produce neurons throughout life in mammals. Sonic hedgehog (Shh) is critical for maintenance of these cells; however, the perinatal source of Shh is enigmatic. In the present study, we examined the role of Shh expressed by hair follicles (HFs) that expand perinatally in temporal concordance with the proliferation of Shh-responding dentate stem cells. Specific inhibition of Shh from HFs or from epithelial sources in general hindered development of Shh-responding dentate stem cells. We also found that the blood-brain barrier (BBB) of the perinatal dentate gyrus (DG) is leaky with stem cells in the dentate exposed to blood-born factors. In attempting to identify how Shh might be transported in blood, we found that platelets contain epithelial Shh, provide Shh to the perinatal DG and that inhibition of platelet generation reduced hedgehog-responsive dentate stem cells.

  16. LRP2, an auxiliary receptor that controls sonic hedgehog signaling in development and disease.

    PubMed

    Christ, Annabel; Herzog, Katja; Willnow, Thomas E

    2016-05-01

    To fulfill their multiple roles in organ development and adult tissue homeostasis, hedgehog (HH) morphogens act through their receptor Patched (PTCH) on target cells. However, HH actions also require HH binding proteins, auxiliary cell surface receptors that agonize or antagonize morphogen signaling in a context-dependent manner. Here, we discuss recent findings on the LDL receptor-related protein 2 (LRP2), an exemplary HH binding protein that modulates sonic hedgehog activities in stem and progenitor cell niches in embryonic and adult tissues. LRP2 functions are crucial for developmental processes in a number of tissues, including the brain, the eye, and the heart, and defects in this receptor pathway are the cause of devastating congenital diseases in humans. Developmental Dynamics 245:569-579, 2016. © 2016 Wiley Periodicals, Inc.

  17. Estradiol triggers sonic-hedgehog-induced angiogenesis during peripheral nerve regeneration by downregulating hedgehog-interacting protein.

    PubMed

    Sekiguchi, Haruki; Ii, Masaaki; Jujo, Kentaro; Renault, Marie-Ange; Thorne, Tina; Clarke, Trevor; Ito, Aiko; Tanaka, Toshikazu; Klyachko, Ekaterina; Tabata, Yasuhiko; Hagiwara, Nobuhisa; Losordo, Douglas

    2012-04-01

    Both estradiol (E2) and Sonic Hedgehog (Shh) contribute to angiogenesis and nerve regeneration. Here, we investigated whether E2 improves the recovery of injured nerves by downregulating the Shh inhibitor hedgehog-interacting protein (HIP) and increasing Shh-induced angiogenesis. Mice were treated with local injections of E2 or placebo one week before nerve-crush injury; 28 days after injury, nerve conduction velocity, exercise duration, and vascularity were significantly greater in E2-treated mice than in placebo-treated mice. E2 treatment was also associated with higher mRNA levels of Shh, the Shh receptor Patched-1, and the Shh transcriptional target Gli1, but with lower levels of HIP. The E2-induced enhancement of nerve vascularity was abolished by the Shh inhibitor cyclopamine, and the effect of E2 treatment on Shh, Gli1, and HIP mRNA expression was abolished by the E2 inhibitor ICI. Gli-luciferase activity in human umbilical-vein endothelial cells (HUVECs) increased more after treatment with E2 and Shh than after treatment with E2 alone, and E2 treatment reduced HIP expression in HUVECs and Schwann cells without altering Shh expression. Collectively, these findings suggest that E2 improves nerve recovery, at least in part, by reducing HIP expression, which subsequently leads to an increase in Shh signaling and Shh-induced angiogenesis.

  18. The role of the sonic hedgehog signaling pathway in early brain injury after experimental subarachnoid hemorrhage in rats.

    PubMed

    Li, Tao; Zhang, Jie; Liu, Rong-Yao; Lian, Zhi-Gang; Chen, Xiao-Lin; Ma, Li; Sun, Hao-Min; Zhao, Yuan-Li

    2013-09-27

    Previous studies have demonstrated that the sonic hedgehog (Shh) pathway plays a neuro-protective role. However, whether the Shh pathway is induced by subarachnoid hemorrhage (SAH) has not been investigated. We sought to investigate Shh activation in the cortex in the early stage of SAH, and assessed the effect of cyclopamine (a specific inhibitor of the Shh pathway) on Shh pathway regulation and evaluated the impact of cyclopamine on SAH. We found that the Shh pathway was up-regulated in the cortex after SAH, and that blocking the Shh pathway increased cell apoptosis. Early brain damages, including brain edema, blood-brain barrier impairment, and cortical apoptosis were significantly aggravated following with cyclopamine treatment compared with vehicle treatment. Our results suggest that the Shh pathway should be activated in the brain after SAH, and plays a beneficial role in SAH development, possibly by inhibiting cerebral oxidative stress through induction of antioxidant and detoxifying enzymes.

  19. Novel association of VACTERL, neural tube defect and crossed renal ectopia: sonic hedgehog signaling: a point of coherence?

    PubMed

    Vaze, Dhananjay; Mahalik, Santosh; Rao, Katragadda L N

    2012-12-01

    The present case report describes two patients with a novel combination of VACTERL (vertebral, anorectal, cardiac, tracheoesophageal, renal, limb), neural tube defect and crossed renal ectopia. Though cases of VACTERL associated with crossed renal ectopia have been described, the present case report is the first to describe its combination with neural tube defect. The cases reported here are significant because central nervous system manifestations are scarce in VACTERL syndrome. The role of sonic hedgehog pathway has been proposed in VACTERL association and neural tube defects. Axial Sonic hedgehog signaling has also been implicated in the mediolateral positioning of the renal parenchyma. With this knowledge, the etiopathogenesis of this novel combination is discussed to highlight the role of sonic hedgehog signaling as a point of coherence.

  20. Role of Sonic Hedgehog (Shh) Signaling in Bladder Cancer Stemness and Tumorigenesis.

    PubMed

    Syed, Islam S; Pedram, Akbari; Farhat, Walid A

    2016-02-01

    Sonic hedgehog (Shh) signaling pathway has emerged as a critical component of bladder development, cancer initiation, and progression. While the role of Shh signaling in bladder development is well documented, its role in bladder cancer progression is uncertain. Additionally, epithelial-to-mesenchymal transition (EMT) has been identified to promote bladder cancer progression in the initial stages and also contribute to drug resistance in the later stage and ultimately metastasis. We speculate that epithelial-to-mesenchymal transitions (EMT) and Shh fuel the carcinogenesis process. This review presents the most recent studies focusing on the role of Shh signaling in bladder cancer progression.

  1. Targeting Sonic Hedgehog: a new way to mow down pancreatic cancer?

    PubMed

    Cengel, Keith A

    2004-02-01

    Despite continuing development of new therapies, the prognosis for patients with pancreatic cancer remains extremely poor. In part, this may relate to molecular abnormalities that stimulate pancreatic tumorigenesis and also contribute to reduced sensitivity to standard treatments such as chemotherapy and radiotherapy. Two recent reports in Nature suggest that Sonic Hedgehog (Shh) overexpression may contribute to pancreatic tumorigenesis and that cyclopamine, a specific inhibitor of Shh signaling, can reduce pancreatic cancer cell growth and viability. This discovery is exciting and suggests that targeting Shh signaling may be an effective novel approach to therapy in patients with this devastating disease.

  2. Bridging the gap: heparan sulfate and Scube2 assemble Sonic hedgehog release complexes at the surface of producing cells.

    PubMed

    Jakobs, P; Schulz, P; Ortmann, C; Schürmann, S; Exner, S; Rebollido-Rios, R; Dreier, R; Seidler, D G; Grobe, K

    2016-05-20

    Decision making in cellular ensembles requires the dynamic release of signaling molecules from the producing cells into the extracellular compartment. One important example of molecules that require regulated release in order to signal over several cell diameters is the Hedgehog (Hh) family, because all Hhs are synthesized as dual-lipidated proteins that firmly tether to the outer membrane leaflet of the cell that produces them. Factors for the release of the vertebrate Hh family member Sonic Hedgehog (Shh) include cell-surface sheddases that remove the lipidated terminal peptides, as well as the soluble glycoprotein Scube2 that cell-nonautonomously enhances this process. This raises the question of how soluble Scube2 is recruited to cell-bound Shh substrates to regulate their turnover. We hypothesized that heparan sulfate (HS) proteoglycans (HSPGs) on the producing cell surface may play this role. In this work, we confirm that HSPGs enrich Scube2 at the surface of Shh-producing cells and that Scube2-regulated proteolytic Shh processing and release depends on specific HS. This finding indicates that HSPGs act as cell-surface assembly and storage platforms for Shh substrates and for protein factors required for their release, making HSPGs critical decision makers for Scube2-dependent Shh signaling from the surface of producing cells.

  3. Bridging the gap: heparan sulfate and Scube2 assemble Sonic hedgehog release complexes at the surface of producing cells

    PubMed Central

    Jakobs, P.; Schulz, P.; Ortmann, C.; Schürmann, S.; Exner, S.; Rebollido-Rios, R.; Dreier, R.; Seidler, D. G.; Grobe, K.

    2016-01-01

    Decision making in cellular ensembles requires the dynamic release of signaling molecules from the producing cells into the extracellular compartment. One important example of molecules that require regulated release in order to signal over several cell diameters is the Hedgehog (Hh) family, because all Hhs are synthesized as dual-lipidated proteins that firmly tether to the outer membrane leaflet of the cell that produces them. Factors for the release of the vertebrate Hh family member Sonic Hedgehog (Shh) include cell-surface sheddases that remove the lipidated terminal peptides, as well as the soluble glycoprotein Scube2 that cell-nonautonomously enhances this process. This raises the question of how soluble Scube2 is recruited to cell-bound Shh substrates to regulate their turnover. We hypothesized that heparan sulfate (HS) proteoglycans (HSPGs) on the producing cell surface may play this role. In this work, we confirm that HSPGs enrich Scube2 at the surface of Shh-producing cells and that Scube2-regulated proteolytic Shh processing and release depends on specific HS. This finding indicates that HSPGs act as cell-surface assembly and storage platforms for Shh substrates and for protein factors required for their release, making HSPGs critical decision makers for Scube2-dependent Shh signaling from the surface of producing cells. PMID:27199253

  4. Progenitor cell proliferation in the retina is dependent on Notch-independent Sonic hedgehog/Hes1 activity

    PubMed Central

    Wall, Dana S.; Mears, Alan J.; McNeill, Brian; Mazerolle, Chantal; Thurig, Sherry; Wang, Yaping; Kageyama, Ryoichiro; Wallace, Valerie A.

    2009-01-01

    Sonic hedgehog (Shh) is an indispensable, extrinsic cue that regulates progenitor and stem cell behavior in the developing and adult mammalian central nervous system. Here, we investigate the link between the Shh signaling pathway and Hes1, a classical Notch target. We show that Shh-driven stabilization of Hes1 is independent of Notch signaling and requires the Shh effector Gli2. We identify Gli2 as a primary mediator of this response by showing that Gli2 is required for Hh (Hedgehog)-dependent up-regulation of Hes1. We also show using chromatin immunoprecipitation that Gli2 binds to the Hes1 promoter, which suggests that Hes1 is a Hh-dependent direct target of Gli2 signaling. Finally, we show that Shh stimulation of progenitor proliferation and cell diversification requires Gli2 and Hes1 activity. This paper is the first demonstration of the mechanistic and functional link between Shh, Gli, and Hes1 in the regulation of progenitor cell behavior. PMID:19124651

  5. Mesocortical Dopamine Phenotypes in Mice Lacking the Sonic Hedgehog Receptor Cdon.

    PubMed

    Verwey, Michael; Grant, Alanna; Meti, Nicholas; Adye-White, Lauren; Torres-Berrío, Angelica; Rioux, Veronique; Lévesque, Martin; Charron, Frederic; Flores, Cecilia

    2016-01-01

    Motivated behaviors and many psychopathologies typically involve changes in dopamine release from the projections of the ventral tegmental area (VTA) and/or the substantia nigra pars compacta (SNc). The morphogen Sonic Hedgehog (Shh) specifies fates of midbrain dopamine neurons, but VTA-specific effects of Shh signaling are also being uncovered. In this study, we assessed the role of the Shh receptor Cdon in the development of VTA and SNc dopamine neurons. We find that Cdon is expressed in the proliferating progenitor zone of the embryonic ventral midbrain and that the number of proliferating cells in this region is increased in mouse Cdon(-/-) embryos. Consistent with a role of Shh in the regulation of neuronal proliferation in this region, we find that the number of tyrosine hydroxylase (TH)-positive neurons is increased in the VTA of Cdon(-/-) mice at birth and that this effect endures into adulthood. In contrast, the number of TH-positive neurons in the SNc is not altered in Cdon(-/-) mice at either age. Moreover, adult Cdon(-/-) mice have a greater number of medial prefrontal cortex (mPFC) dopamine presynaptic sites, and increased baseline concentrations of dopamine and dopamine metabolites selectively in this region. Finally, consistent with increased dopamine function in the mPFC, we find that adult Cdon(-/-) mice fail to exhibit behavioral plasticity upon repeated amphetamine treatment. Based on these data, we suggest that Cdon plays an important role encoding the diversity of dopamine neurons in the midbrain, influencing both the development of the mesocortical dopamine pathway and behavioral outputs that involve this neural circuitry.

  6. Mesocortical Dopamine Phenotypes in Mice Lacking the Sonic Hedgehog Receptor Cdon

    PubMed Central

    Grant, Alanna; Meti, Nicholas; Adye-White, Lauren; Rioux, Veronique

    2016-01-01

    Abstract Motivated behaviors and many psychopathologies typically involve changes in dopamine release from the projections of the ventral tegmental area (VTA) and/or the substantia nigra pars compacta (SNc). The morphogen Sonic Hedgehog (Shh) specifies fates of midbrain dopamine neurons, but VTA-specific effects of Shh signaling are also being uncovered. In this study, we assessed the role of the Shh receptor Cdon in the development of VTA and SNc dopamine neurons. We find that Cdon is expressed in the proliferating progenitor zone of the embryonic ventral midbrain and that the number of proliferating cells in this region is increased in mouse Cdon−/− embryos. Consistent with a role of Shh in the regulation of neuronal proliferation in this region, we find that the number of tyrosine hydroxylase (TH)-positive neurons is increased in the VTA of Cdon−/− mice at birth and that this effect endures into adulthood. In contrast, the number of TH-positive neurons in the SNc is not altered in Cdon−/− mice at either age. Moreover, adult Cdon−/− mice have a greater number of medial prefrontal cortex (mPFC) dopamine presynaptic sites, and increased baseline concentrations of dopamine and dopamine metabolites selectively in this region. Finally, consistent with increased dopamine function in the mPFC, we find that adult Cdon−/− mice fail to exhibit behavioral plasticity upon repeated amphetamine treatment. Based on these data, we suggest that Cdon plays an important role encoding the diversity of dopamine neurons in the midbrain, influencing both the development of the mesocortical dopamine pathway and behavioral outputs that involve this neural circuitry. PMID:27419218

  7. Sonic Hedgehog modulates EGFR dependent proliferation of neural stem cells during late mouse embryogenesis through EGFR transactivation

    PubMed Central

    Reinchisi, Gisela; Parada, Margarita; Lois, Pablo; Oyanadel, Claudia; Shaughnessy, Ronan; Gonzalez, Alfonso; Palma, Verónica

    2013-01-01

    Sonic Hedgehog (Shh/GLI) and EGFR signaling pathways modulate Neural Stem Cell (NSC) proliferation. How these signals cooperate is therefore critical for understanding normal brain development and function. Here we report a novel acute effect of Shh signaling on EGFR function. We show that during late neocortex development, Shh mediates the activation of the ERK1/2 signaling pathway in Radial Glial cells (RGC) through EGFR transactivation. This process is dependent on metalloprotease activity and accounts for almost 50% of the EGFR-dependent mitogenic response of late NSCs. Furthermore, in HeLa cancer cells, a well-known model for studying the EGFR receptor function, Shh also induces cell proliferation involving EGFR activation, as reflected by EGFR internalization and ERK1/2 phosphorylation. These findings may have important implications for understanding the mechanisms that regulate NSC proliferation during neurogenesis and may lead to novel approaches to the treatment of tumors. PMID:24133411

  8. Sonic Hedgehog modulates EGFR dependent proliferation of neural stem cells during late mouse embryogenesis through EGFR transactivation.

    PubMed

    Reinchisi, Gisela; Parada, Margarita; Lois, Pablo; Oyanadel, Claudia; Shaughnessy, Ronan; Gonzalez, Alfonso; Palma, Verónica

    2013-01-01

    Sonic Hedgehog (Shh/GLI) and EGFR signaling pathways modulate Neural Stem Cell (NSC) proliferation. How these signals cooperate is therefore critical for understanding normal brain development and function. Here we report a novel acute effect of Shh signaling on EGFR function. We show that during late neocortex development, Shh mediates the activation of the ERK1/2 signaling pathway in Radial Glial cells (RGC) through EGFR transactivation. This process is dependent on metalloprotease activity and accounts for almost 50% of the EGFR-dependent mitogenic response of late NSCs. Furthermore, in HeLa cancer cells, a well-known model for studying the EGFR receptor function, Shh also induces cell proliferation involving EGFR activation, as reflected by EGFR internalization and ERK1/2 phosphorylation. These findings may have important implications for understanding the mechanisms that regulate NSC proliferation during neurogenesis and may lead to novel approaches to the treatment of tumors.

  9. Intrastriatal injection of sonic hedgehog reduces behavioral impairment in a rat model of Parkinson's disease.

    PubMed

    Tsuboi, Kyoko; Shults, Clifford W

    2002-01-01

    Sonic hedgehog (Shh), a member of hedgehog (hh) family of signaling molecules, is necessary for normal axial patterning and cellular differentiation in the developing central nervous system. Shh also promotes the survival of fetal dopaminergic neurons and protects cultures of fetal midbrain dopaminergic neurons from the toxic effects of N-methyl-4-phenylpyridinium (MPP(+)), a neurotoxin that selectively injures nigral dopaminergic neurons. The mRNA expression of Shh and its putative receptor in the adult brain indicates an important role of Shh in the mature nervous system in addition to its roles during embryogenesis. In this study we examined the behavioral and anatomical effects of intrastriatal injection of singly myristoylated wild-type human Sonic hedgehog N-terminal fragment (Shh-M) in a rat model of Parkinson's disease (PD). Five groups of rats received a series of four intrastriatal injections of Shh-M (180 ng, 540 ng, or 4.275 microg per injection), glial cell line-derived neurotrophic factor (GDNF) (1 microg/injection), or vehicle on days 1, 3, 5, and 8. On day 4, the animals received an intrastriatal injection of 15 microg 6-hydroxydopamine (6-OHDA) free base. Intrastriatal administration of Shh (180 ng/injection) twice before and after a single intrastriatal injection of 6-OHDA reduced apomorphine- and amphetamine-induced rotation and forelimb akinesia and partially preserved dopaminergic axons in the striatum. This is the first demonstration in vivo that Shh reduces behavioral deficits induced by intrastriatal 6-OHDA lesion and suggests that Shh may be useful in the treatment of disorders that affect the nigrostriatal system, such as PD.

  10. The G-protein Alpha Subunit Gsα Is A Tumor Suppressor In Sonic Hedgehog-driven Medulloblastoma

    PubMed Central

    He, Xuelian; Zhang, Liguo; Chen, Ying; Remke, Marc; Shih, David; Lu, Fanghui; Wang, Haibo; Deng, Yaqi; Yu, Yang; Xia, Yong; Wu, Xiaochong; Ramaswamy, Vijay; Hu, Tom; Wang, Fan; Zhou, Wenhao; Burns, Dennis K.; Kim, Se Hoon; Kool, Marcel; Pfister, Stefan M.; Weinstein, Lee S.; Pomeroy, Scott L.; Gilbertson, Richard J.; Rubin, Joshua B.; Hou, Yiping; Wechsler-Reya, Robert; Taylor, Michael D.; Lu, Q. Richard

    2014-01-01

    Medulloblastoma, the most common malignant childhood brain tumor, exhibits distinct molecular subtypes and cellular origins. Genetic alterations driving medulloblastoma initiation and progression remain poorly understood. Herein, we identify GNAS, encoding the G-protein Gsα, as a potent tumor suppressor gene that defines a subset of aggressive Sonic Hedgehog (Shh)-driven human medulloblastomas. Ablation of the single Gnas gene in anatomically-distinct progenitors is sufficient to induce Shh-associated medulloblastomas, which recapitulate their human counterparts. Gsα is highly enriched at the primary cilium of granule neuron precursors and suppresses Shh-signaling by regulating both the cAMP-dependent pathway and ciliary trafficking of Hedgehog pathway components. Elevation of a Gsα effector, cAMP, effectively inhibits tumor cell proliferation and progression in Gnas mutants. Thus, our gain- and loss-of-function studies identify a previously unrecognized tumor suppressor function for Gsα that acts as a molecular link across Shh-group medulloblastomas of disparate cellular and anatomical origins, illuminating G-protein modulation as a potential therapeutic avenue. PMID:25150496

  11. Sonic hedgehog lineage in the mouse hypothalamus: from progenitor domains to hypothalamic regions

    PubMed Central

    2012-01-01

    Background The hypothalamus is a brain region with essential functions for homeostasis and energy metabolism, and alterations of its development can contribute to pathological conditions in the adult, like hypertension, diabetes or obesity. However, due to the anatomical complexity of the hypothalamus, its development is not well understood. Sonic hedgehog (Shh) is a key developmental regulator gene expressed in a dynamic pattern in hypothalamic progenitor cells. To obtain insight into hypothalamic organization, we used genetic inducible fate mapping (GIFM) to map the lineages derived from Shh-expressing progenitor domains onto the four rostrocaudally arranged hypothalamic regions: preoptic, anterior, tuberal and mammillary. Results Shh-expressing progenitors labeled at an early stage (before embryonic day (E)9.5) contribute neurons and astrocytes to a large caudal area including the mammillary and posterior tuberal regions as well as tanycytes (specialized median eminence glia). Progenitors labeled at later stages (after E9.5) give rise to neurons and astrocytes of the entire tuberal region and in particular the ventromedial nucleus, but not to cells in the mammillary region and median eminence. At this stage, an additional Shh-expressing domain appears in the preoptic area and contributes mostly astrocytes to the hypothalamus. Shh-expressing progenitors do not contribute to the anterior region at any stage. Finally, we show a gradual shift from neurogenesis to gliogenesis, so that progenitors expressing Shh after E12.5 generate almost exclusively hypothalamic astrocytes. Conclusions We define a fate map of the hypothalamus, based on the dynamic expression of Shh in the hypothalamic progenitor zones. We provide evidence that the large neurogenic Shh-expressing progenitor domains of the ventral diencephalon are continuous with those of the midbrain. We demonstrate that the four classical transverse zones of the hypothalamus have clearly defined progenitor domains

  12. Sonic Hedgehog Pathway Contributes to Gastric Cancer Cell Growth and Proliferation

    PubMed Central

    Wan, Jianhua; Zhou, Ji; Zhao, Hailong; Wang, Mei; Wei, Zhuanqin; Gao, Hongyan

    2014-01-01

    Abstract The Sonic Hedgehog (Shh) signaling pathway is commonly activated in gastrointestinal cancer. However, our understanding of the Shh pathway in gastric cancer remains limited. Here we examined the effects of cyclopamine, a specific inhibitor of the Shh signaling pathway, on cell growth and proliferation in gastric primary cancer cells GAM-016 and the MKN-45 cell line. The results showed that the Shh signaling molecules SHH, PTCH, SMO, GLI1, and GLI2 were intact and activated in both types of cells. Furthermore, we observed that cyclopamine inhibited gastric cancer cell proliferation through cell cycle arrest and apoptosis. An in vivo study using NOD/SCID mouse xenografts demonstrated that cyclopamine significantly prevented tumor growth and development. Our study indicated that Shh signaling pathway could promote gastric cancer cell proliferation and tumor development, and blocking this pathway may be a potential strategy in gastric cancer treatment. PMID:24804165

  13. Sonic Hedgehog promotes proliferation of Notch-dependent monociliated choroid plexus tumour cells

    PubMed Central

    Li, Li; Grausam, Katie B.; Wang, Jun; Lun, Melody P.; Ohli, Jasmin; Lidov, Hart G. W.; Calicchio, Monica L.; Zeng, Erliang; Salisbury, Jeffrey L.; Wechsler-Reya, Robert J.; Lehtinen, Maria K.; Schüller, Ulrich; Zhao, Haotian

    2016-01-01

    Aberrant Notch signaling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly pediatric brain neoplasms. We developed animal models of CP tumours by inducing sustained expression of Notch1 that recapitulate properties of human CP tumours with aberrant NOTCH signaling. Whole transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate diffferentiation. A Shh-driven signaling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from mono-ciliated progenitors in the roof plate characterized by elevated Notch signaling. Abnormal SHH signaling and distinct ciliogenesis are detected in human CP tumours, suggesting SHH pathway and cilia differentiation as potential therapeutic avenues. PMID:26999738

  14. Activation of sonic hedgehog signaling enhances cell migration and invasion by induction of matrix metalloproteinase-2 and -9 via the phosphoinositide-3 kinase/AKT signaling pathway in glioblastoma.

    PubMed

    Chang, Liang; Zhao, Dan; Liu, Hui-Bin; Wang, Qiu-Shi; Zhang, Ping; Li, Chen-Long; Du, Wen-Zhong; Wang, Hong-Jun; Liu, Xing; Zhang, Zhi-Ren; Jiang, Chuan-Lu

    2015-11-01

    Aberrant hedgehog signaling contributes to the development of various malignancies, including glioblastoma (GBM). However, the potential mechanism of hedgehog signaling in GBM migration and invasion has remained to be elucidated. The present study showed that enhanced hedgehog signaling by recombinant human sonic hedgehog N‑terminal peptide (rhSHH) promoted the adhesion, invasion and migration of GBM cells, accompanied by increases in mRNA and protein levels of matrix metalloproteinase‑2 (MMP‑2) and MMP‑9. However, inhibition of hedgehog signaling with cyclopamine suppressed the adhesion, invasion and migration of GBM cells, accompanied by decreases in mRNA and protein levels of MMP‑2 and ‑9. Furthermore, it was found that MMP‑2- and MMP‑9-neutralizing antibodies or GAM6001 reversed the inductive effects of rhSHH on cell migration and invasion. In addition, enhanced hedgehog signaling by rhSHH increased AKT phosphorylation, whereas blockade of hedgehog signaling decreased AKT phosphorylations. Further experiments showed that LY294002, an inhibitor of phosphoinositide-3 kinase (PI3K), decreased rhSHH‑induced upregulation of MMP‑2 and ‑9. Finally, the protein expression of glioblastoma-associated oncogene 1 was positively correlated with levels of phosphorylated AKT as well as protein expressions of MMP‑2 and ‑9 in GBM tissue samples. In conclusion, the present study indicated that the hedgehog pathway regulates GBM-cell migration and invasion by increasing MMP-2 and MMP-9 production via the PI3K/AKT pathway.

  15. The Role of Sonic Hedgehog Signaling in Osteoclastogenesis and Jaw Bone Destruction

    PubMed Central

    Shimo, Tsuyoshi; Matsumoto, Kenichi; Takabatake, Kiyofumi; Aoyama, Eriko; Takebe, Yuichiro; Ibaragi, Soichiro; Okui, Tatsuo; Kurio, Naito; Takada, Hiroyuki; Obata, Kyoichi; Pang, Pai; Iwamoto, Masahiro; Nagatsuka, Hitoshi; Sasaki, Akira

    2016-01-01

    Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of its expression appear to correlate with disease progression and metastasis. However, the role of SHH in bone destruction associated with oral squamous cell carcinomas is still unclear. In this study we analyzed SHH expression and the role played by SHH signaling in gingival carcinoma-induced jawbone destruction. From an analysis of surgically resected lower gingival squamous cell carcinoma mandible samples, we found that SHH was highly expressed in tumor cells that had invaded the bone matrix. On the other hand, the hedgehog receptor Patched and the signaling molecule Gli-2 were highly expressed in the osteoclasts and the progenitor cells. SHH stimulated osteoclast formation and pit formation in the presence of the receptor activator for nuclear factor-κB ligand (RANKL) in CD11b+ mouse bone marrow cells. SHH upregulated phosphorylation of ERK1/2 and p38 MAPK, NFATc1, tartrate-resistant acid phosphatase (TRAP), and Cathepsin K expression in RAW264.7 cells. Our results suggest that tumor-derived SHH stimulated the osteoclast formation and bone resorption in the tumor jawbone microenvironment. PMID:27007126

  16. Regulation of Hedgehog signaling by ubiquitination

    PubMed Central

    Hsia, Elaine Y. C.; Gui, Yirui; Zheng, Xiaoyan

    2015-01-01

    The Hedgehog (Hh) signaling pathway plays crucial roles both in embryonic development and in adult stem cell function. The timing, duration and location of Hh signaling activity need to be tightly controlled. Abnormalities of Hh signal transduction lead to birth defects or malignant tumors. Recent data point to ubiquitination-related posttranslational modifications of several key Hh pathway components as an important mechanism of regulation of the Hh pathway. Here we review how ubiquitination regulates the localization, stability and activity of the key Hh signaling components. PMID:26366162

  17. Alteration of hedgehog signaling by chronic exposure to different pesticide formulations and unveiling the regenerative potential of recombinant sonic hedgehog in mouse model of bone marrow aplasia.

    PubMed

    Chaklader, Malay; Law, Sujata

    2015-03-01

    Chronic pesticide exposure-induced downregulation of hedgehog signaling and its subsequent degenerative effects on the mammalian hematopoietic system have not been investigated yet. However a number of concurrent studies have pointed out the positive correlation between chronic pesticide exposure induced bone marrow failure and immune suppression. Here, we have given an emphasis on the recapitulation of human marrow aplasia like condition in mice by chronic mixed pesticide exposures and simultaneously unravel the role of individual pesticides in the said event. Unlike the effect of mixed pesticide, individual pesticides differentially alter the hedgehog signaling in the bone marrow primitive hematopoietic compartment (Sca1 + compartment) and stromal compartment. Individually, hexaconazole disrupted hematopoietic as well as stromal hedgehog signaling activation through inhibiting SMO and facilitating PKC δ expression. On contrary, both chlorpyriphos and cypermethrin increased the sequestration and degradation of GLI1 by upregulating SU(FU) and βTrCP, respectively. However, cypermethrin-mediated inhibition of hedgehog signaling has partly shown to be circumvented by non-canonical activation of GLI1. Finally, we have tested the regenerative response of sonic hedgehog and shown that in vitro supplemented recombinant SHH protein augmented clonogenic stromal progenitors (CFU-F) as well as primitive multipotent hematopoietic clones including CFU-GEMM and CFU-GM of mixed pesticide-induced aplastic marrow. It is an indication of the marrow regeneration. Finally, our findings provide a gripping evidence that downregulated hedgehog signaling contribute to pesticide-mediated bone marrow aplasia but it could be recovered by proper supplementation of recombinant SHH along with hematopoietic base cocktail. Furthermore, SU(FU) and GLI1 can be exploited as future theradiagnostic markers for early marrow aplasia diagnosis.

  18. The sonic hedgehog signaling pathway stimulates anaplastic thyroid cancer cell motility and invasiveness by activating Akt and c-Met.

    PubMed

    Williamson, Ashley J; Doscas, Michelle E; Ye, Jin; Heiden, Katherine B; Xing, Mingzhao; Li, Yi; Prinz, Richard A; Xu, Xiulong

    2016-03-01

    The sonic hedgehog (Shh) pathway is highly activated in thyroid neoplasms and promotes thyroid cancer stem-like cell phenotype, but whether the Shh pathway regulates thyroid tumor cell motility and invasiveness remains unknown. Here, we report that the motility and invasiveness of two anaplastic thyroid tumor cell lines, KAT-18 and SW1736, were inhibited by two inhibitors of the Shh pathway (cyclopamine and GANT61). Consistently, the cell motility and invasiveness was decreased by Shh and Gli1 knockdown, and was increased by Gli1 overexpression in KAT-18 cells. Mechanistic studies revealed that Akt and c-Met phosphorylation was decreased by a Gli1 inhibitor and by Shh and Gli1 knockdown, but was increased by Gli1 overexpression. LY294002, a PI-3 kinase inhibitor, and a c-Met inhibitor inhibited the motility and invasiveness of Gli1-transfected KAT-18 cells more effectively than the vector-transfected cells. Knockdown of Snail, a transcription factor regulated by the Shh pathway, led to decreased cell motility and invasiveness in KAT-18 and SW1736 cells. However, key epithelial-to-mesenchymal transition (EMT) markers including E-cadherin and vimentin as well as Slug were not affected by cyclopamine and GANT61 in either SW1736 or WRO82, a well differentiated follicular thyroid carcinoma cell line. Our data suggest that the Shh pathway-stimulated thyroid tumor cell motility and invasiveness is largely mediated by AKT and c-Met activation with little involvement of EMT.

  19. KIF13B establishes a CAV1-enriched microdomain at the ciliary transition zone to promote Sonic hedgehog signalling

    PubMed Central

    Schou, Kenneth B.; Mogensen, Johanne B.; Morthorst, Stine K.; Nielsen, Brian S.; Aleliunaite, Aiste; Serra-Marques, Andrea; Fürstenberg, Nicoline; Saunier, Sophie; Bizet, Albane A.; Veland, Iben R.; Akhmanova, Anna; Christensen, Søren T.; Pedersen, Lotte B.

    2017-01-01

    Ciliary membrane composition is controlled by transition zone (TZ) proteins such as RPGRIP1, RPGRIPL and NPHP4, which are vital for balanced coordination of diverse signalling systems like the Sonic hedgehog (Shh) pathway. Activation of this pathway involves Shh-induced ciliary accumulation of Smoothened (SMO), which is disrupted by disease-causing mutations in TZ components. Here we identify kinesin-3 motor protein KIF13B as a novel member of the RPGRIP1N-C2 domain-containing protein family and show that KIF13B regulates TZ membrane composition and ciliary SMO accumulation. KIF13B is upregulated during ciliogenesis and is recruited to the ciliary base by NPHP4, which binds to two distinct sites in the KIF13B tail region, including an RPGRIP1N-C2 domain. KIF13B and NPHP4 are both essential for establishment of a CAV1 membrane microdomain at the TZ, which in turn is required for Shh-induced ciliary SMO accumulation. Thus KIF13B is a novel regulator of ciliary TZ configuration, membrane composition and Shh signalling. PMID:28134340

  20. Paracrine sonic hedgehog signaling contributes significantly to acquired steroidogenesis in the prostate tumor microenvironment.

    PubMed

    Lubik, Amy A; Nouri, Mannan; Truong, Sarah; Ghaffari, Mazyar; Adomat, Hans H; Corey, Eva; Cox, Michael E; Li, Na; Guns, Emma S; Yenki, Parvin; Pham, Steven; Buttyan, Ralph

    2017-01-15

    Despite the substantial benefit of androgen deprivation therapy (ADT) for metastatic prostate cancer, patients often progress to castration-resistant disease (CRPC) that is more difficult to treat. CRPC is associated with renewed androgen receptor activity in tumor cells and restoration of tumor androgen levels through acquired intratumoral steroidogenesis (AIS). Although prostate cancer (PCa) cells have been shown to have steroidogenic capability in vitro, we previously found that benign prostate stromal cells (PrSCs) can also synthesize testosterone (T) from an adrenal precursor, DHEA, when stimulated with a hedgehog (Hh) pathway agonist, SAG. Here, we show exposure of PrSCs to a different Smoothened (Smo) agonist, Ag1.5, or to conditioned medium from sonic hedgehog overexpressing LNCaP cells induces steroidogenic enzyme expression in PrSCs and significantly increases production of T and its precursor steroids in a Smo-dependent manner from 22-OH-cholesterol substrate. Hh agonist-/ligand-treated PrSCs produced androgens at a rate similar to or greater than that of PCa cell lines. Likewise, primary bone marrow stromal cells became more steroidogenic and produced T under the influence of Smo agonist. Treatment of mice bearing LNCaP xenografts with a Smo antagonist, TAK-441, delayed the onset of CRPC after castration and substantially reduced androgen levels in residual tumors. These outcomes support the idea that stromal cells in ADT-treated primary or metastatic prostate tumors can contribute to AIS as a consequence of a paracrine Hh signaling microenvironment. As such, Smo antagonists may be useful for targeting prostate tumor stromal cell-derived AIS and delaying the onset of CRPC after ADT.

  1. Smoothened regulation in response to Hedgehog stimulation

    PubMed Central

    Jiang, Kai; Jia, Jianhang

    2016-01-01

    The Hedgehog (Hh) signaling pathway play critical roles in embryonic development and adult tissue homeostasis. A critical step in Hh signal transduction is how Hh receptor Patched (Ptc) inhibits the atypical G protein-coupled receptor Smoothened (Smo) in the absence of Hh and how this inhibition is release by Hh stimulation. It is unlikely that Ptc inhibits Smo by direct interaction. Here we discuss how Hh regulates the phosphorylation and ubiquitination of Smo, leading to cell surface and ciliary accumulation of Smo in Drosophila and vertebrate cells, respectively. In addition, we discuss how PI(4)P phospholipid acts in between Ptc and Smo to regulate Smo phosphorylation and activation in response to Hh stimulation. PMID:26973699

  2. Reiterative pattern of sonic hedgehog expression in the catshark dentition reveals a phylogenetic template for jawed vertebrates.

    PubMed

    Smith, Moya M; Fraser, Gareth J; Chaplin, Natalie; Hobbs, Carl; Graham, Anthony

    2009-04-07

    For a dentition representing the most basal extant gnathostomes, that of the shark can provide us with key insights into the evolution of vertebrate dentitions. To detail the pattern of odontogenesis, we have profiled the expression of sonic hedgehog, a key regulator of tooth induction. We find in the catshark (Scyliorhinus canicula) that intense shh expression first occurs in a bilaterally symmetrical pattern restricted to broad regions in each half of the dentition in the embryo jaw. As in the mouse, there follows a changing temporal pattern of shh spatial restriction corresponding to epithelial bands of left and right dental fields, but also a subfield for symphyseal teeth. Then, intense shh expression is restricted to loci coincident with a temporal series of teeth in iterative jaw positions. The developmental expression of shh reveals previously undetected timing within epithelial stages of tooth formation. Each locus at alternate, even then odd, jaw positions establishes precise sequential timing for successive replacement within each tooth family. Shh appears first in the central cusp, iteratively along the jaw, then reiteratively within each tooth for secondary cusps. This progressive, sequential restriction of shh is shared by toothed gnathostomes and conserved through 500 million years of evolution.

  3. Spatial and temporal pattern for the dentition in the Australian lungfish revealed with sonic hedgehog expression profile.

    PubMed

    Smith, Moya M; Okabe, Masataka; Joss, Jean

    2009-02-22

    We report a temporal order of tooth addition in the Australian lungfish where timing of tooth induction is sequential in the same pattern as osteichthyans along the lower jaw. The order of tooth initiation in Neoceratodus starts from the midline tooth, together with left and right ones at jaw position 2, followed by 3 and then 1. This is the pattern order for dentary teeth of several teleosts and what we propose represents a stereotypic initiation pattern shared with all osteichthyans, including the living sister group to all tetrapods, the Australian lungfish. This is contrary to previous opinions that the lungfish dentition is otherwise derived and uniquely different. Sonic hedgehog (shh) expression is intensely focused on tooth positions at different times corresponding with their initiation order. This deployment of shh is required for lungfish tooth induction, as cyclopamine treatment results in complete loss of these teeth when applied before they develop. The temporal sequence of tooth initiation is possibly regulated by shh and is know to be required for dentition pattern in other osteichthyans, including cichlid fish and snakes. This reflects a shared developmental process with jawed vertebrates at the level of the tooth module but differs with the lack of replacement teeth.

  4. Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells

    PubMed Central

    Miura, Hirohito; Scott, Jennifer K.; Harada, Shuitsu; Barlow, Linda A.

    2014-01-01

    Background Taste buds contain ~60 elongate cells and several basal cells. Elongate cells comprise three functional taste cell types: I - glial cells, II - bitter/sweet/umami receptor cells, and III - sour detectors. Although taste cells are continuously renewed, lineage relationships among cell types are ill-defined. Basal cells have been proposed as taste bud stem cells, a subset of which express Sonic hedgehog (Shh). However, Shh+ basal cells turnover rapidly suggesting that Shh+ cells are precursors of some or all taste cell types. Results To fate map Shh-expressing cells, mice carrying ShhCreERT2 and a high (CAG-CAT-EGFP) or low (R26RLacZ) efficiency reporter allele were given tamoxifen to activate Cre in Shh+ cells. Using R26RLacZ, lineage-labeled cells occur singly within buds, supporting a post-mitotic state for Shh+ cells. Using either reporter, we show that Shh+ cells differentiate into all three taste cell types, in proportions reflecting cell type ratios in taste buds (I > II > III). Conclusions Shh+ cells are not stem cells, but are post-mitotic, immediate precursors of taste cells. Shh+ cells differentiate into each of the three taste cell types, and the choice of a specific taste cell fate is regulated to maintain the proper ratio within buds. PMID:24590958

  5. The effect of multivalent Sonic hedgehog on differentiation of human embryonic stem cells into dopaminergic and GABAergic neurons.

    PubMed

    Vazin, Tandis; Ashton, Randolph S; Conway, Anthony; Rode, Nikhil A; Lee, Susan M; Bravo, Verenice; Healy, Kevin E; Kane, Ravi S; Schaffer, David V

    2014-01-01

    Stem cell differentiation is regulated by complex repertoires of signaling ligands which often use multivalent interactions, where multiple ligands tethered to one entity interact with multiple cellular receptors to yield oligomeric complexes. One such ligand is Sonic hedgehog (Shh), whose posttranslational lipid modifications and assembly into multimers enhance its biological potency, potentially through receptor clustering. Investigations of Shh typically utilize recombinant, monomeric protein, and thus the impact of multivalency on ligand potency is unexplored. Among its many activities, Shh is required for ventralization of the midbrain and forebrain and is therefore critical for the development of midbrain dopaminergic (mDA) and forebrain gamma-aminobutyric acid (GABA) inhibitory neurons. We have designed multivalent biomaterials presenting Shh in defined spatial arrangements and investigated the role of Shh valency in ventral specification of human embryonic stem cells (hESCs) into these therapeutically relevant cell types. Multivalent Shh conjugates with optimal valencies, compared to the monomeric Shh, increased the percentages of neurons belonging to mDA or forebrain GABAergic fates from 33% to 60% or 52% to 86%, respectively. Thus, multivalent Shh bioconjugates can enhance neuronal lineage commitment of pluripotent stem cells and thereby facilitate efficient derivation of neurons that could be used to treat Parkinson's and epilepsy patients.

  6. Expression pattern of sonic hedgehog signaling and calcitonin gene-related peptide in the socket healing process after tooth extraction.

    PubMed

    Pang, Pai; Shimo, Tsuyoshi; Takada, Hiroyuki; Matsumoto, Kenichi; Yoshioka, Norie; Ibaragi, Soichiro; Sasaki, Akira

    2015-11-06

    Sonic Hedgehog (SHH), a neural development inducer, plays a significant role in the bone healing process. Calcitonin gene-related peptide (CGRP), a neuropeptide marker of sensory nerves, has been demonstrated to affect bone formation. The roles of SHH signaling and CGRP-positive sensory nerves in the alveolar bone formation process have been unknown. Here we examined the expression patterns of SHH signaling and CGRP in mouse socket by immunohistochemistry and immunofluorescence analysis. We found that the expression level of SHH peaked at day 3 and was then decreased at 5 days after tooth extraction. CGRP, PTCH1 and GLI2 were each expressed in a similar pattern with their highest expression levels at day 5 and day 7 after tooth extraction. CGRP and GLI2 were co-expressed in some inflammatory cells and bone forming cells. In some areas, CGRP-positive neurons expressed GLI2. In conclusion, SHH may affect alveolar bone healing by interacting with CGRP-positive sensory neurons and thus regulate the socket's healing process after tooth extraction.

  7. Sonic hedgehog signals to multiple prostate stromal stem cells that replenish distinct stromal subtypes during regeneration

    PubMed Central

    Peng, Yu-Ching; Levine, Charles M.; Zahid, Sarwar; Wilson, E. Lynette; Joyner, Alexandra L.

    2013-01-01

    The adult mouse prostate has a seemingly endless capacity for regeneration, and sonic hedgehog (SHH) signaling has been implicated in this stem cell-driven process. However, it is not clear whether SHH acts on the epithelium or stromal cells that secrete factors required for epithelial expansion. Because little is known about stromal stem cells compared with their epithelial counterparts, we used in vivo mouse genetics tools to characterize four prostate stromal subtypes and their stem cells. Using knockin reporter alleles, we uncovered that SHH signals from prostate basal epithelial cells to adjacent stromal cells. Furthermore, the SHH target gene Gli1 is preferentially expressed in subepithelial fibroblast-like cells, one of four prostate stromal subtypes and the subtype closest to the epithelial source of SHH. Using Genetic Inducible Fate Mapping to mark adult Gli1- or Smooth muscle actin-expressing cells and follow their fate during regeneration, we uncovered that Gli1-expressing cells exhibit long-term self-renewal capacity during multiple rounds of androgen-mediated regeneration after castration-induced involution, and depleted smooth muscle cells are mainly replenished by preexisting smooth muscle cells. Based on our Genetic Inducible Fate Mapping studies, we propose a model where SHH signals to multiple stromal stem cells, which are largely unipotent in vivo. PMID:24218555

  8. Anti-apoptotic role of the sonic hedgehog signaling pathway in the proliferation of ameloblastoma.

    PubMed

    Kanda, Shiori; Mitsuyasu, Takeshi; Nakao, Yu; Kawano, Shintaro; Goto, Yuichi; Matsubara, Ryota; Nakamura, Seiji

    2013-09-01

    Sonic hedgehog (SHH) signaling pathway is crucial to growth and patterning during organogenesis. Aberrant activation of the SHH signaling pathway can result in tumor formation. We examined the expression of SHH signaling molecules and investigated the involvement of the SHH pathway in the proliferation of ameloblastoma, the most common benign tumor of the jaws. We used immunohistochemistry on ameloblastoma specimens and immunocytochemistry and reverse transcription-PCR on the ameloblastoma cell line AM-1. We also used the inhibitors of SHH signaling, SHH neutralizing antibody and cyclopamine, to assess the effects of SHH on the proliferation of AM-1 cells. We detected expression of SHH, patched, GLI1, GLI2 and GLI3 in the ameloblastoma specimens and AM-1 cells. The proliferation of these cells was significantly inhibited in the presence of SHH neutralizing antibody or cyclopamine; this was confirmed by BrdU incorporation assays. Furthermore, in the presence of SHH neutralizing antibody, nuclear translocation of GLI1 and GLI2 was abolished, apoptosis was induced, BCL-2 expression decreased and BAX expression increased. Our results suggest that the SHH signaling pathway is constitutively active in ameloblastoma and plays an anti-apoptotic role in the proliferation of ameloblastoma cells through autocrine loop stimulation.

  9. Sonic hedgehog and neurotrophin-3 increase oligodendrocyte numbers and myelination after spinal cord injury

    PubMed Central

    Goodman, Ashley G.; Kukushliev, Todor V.; Hassani, Donna M.; Cummings, Brian J.; Anderson, Aileen J.; Shea, Lonnie D.

    2014-01-01

    Spinal cord injury (SCI) results in loss of sensory and motor function below the level of injury and has limited available therapies. Multiple channel bridges have been investigated as a means to create a permissive environment for regeneration, with channels supporting axonal growth through the injury. Bridges support robust axon growth with myelination of the axons, and herein we investigated the cell types that are myelinating the axons and whether trophic factors can enhance myelination. Lentivirus encoding for neurotrophin-3 (NT3), sonic hedgehog (SHH) and the combination of these factors was delivered from bridges implanted into a lateral hemisection defect at T9/T10 in mice, and the response of endogenous progenitor cells within the spinal cord was investigated. Relative to control, the localized sustained expression of these factors significantly increased growth of regenerating axons into the bridge and enhanced axon myelination 8 weeks after injury. SHH decreased Sox2+ cells and increased Olig2+ cells, whereas NT3 alone or in combination with SHH enhanced GFAP+ and Olig2+ cells relative to control. For delivery of lentivirus encoding for either factor, we identified cells at various stages of differentiation along the oligodendrocyte lineage (e.g., O4+, GalC+). Expression of NT3 enhanced myelination primarily by infiltrating Schwann cells, whereas SHH over-expression substantially increased myelination by oligodendrocytes. Gene delivery represents a promising tool to direct activation and differentiation of endogenous progenitor cells for applications in regenerative medicine. PMID:24873988

  10. The role of sonic hedgehog in normal and abnormal craniofacial morphogenesis.

    PubMed

    Hu, D; Helms, J A

    1999-11-01

    There is growing evidence that implicates a role for Sonic hedgehog (SHH) in morphogenesis of the craniofacial complex. Mutations in human and murine SHH cause midline patterning defects that are manifested in the head as holoprosencephaly and cyclopia. In addition, teratogens such as jervine, which inhibit the response of tissues to SHH, also produce cyclopia. Thus, the loss of SHH signaling during early stages of neural plate patterning has a profound influence of craniofacial morphogenesis. However, the severity of these defects precludes analyses of SHH function during later stages of craniofacial development. We have used an embryonic chick system to study the role of SHH during these later stages of craniofacial development. Using a combination of surgical and molecular experiments, we show here that SHH is essential for morphogenesis of the frontonasal and maxillary processes (FNP and MXPs), which give rise to the mid- and upper face. Transient loss of SHH signaling in the embryonic face inhibits growth of the primordia and results in defects analogous to hypotelorism and cleft lip/palate, characteristics of the mild forms of holoprosencephaly. In contrast, excess SHH leads to a mediolateral widening of the FNP and a widening between the eyes, a condition known as hypertelorism. In severe cases, this widening is accompanied by facial duplications. Collectively, these experiments demonstrate that SHH has multiple and profound effects on the entire spectrum of craniofacial development, and perturbations in SHH signaling are likely to underlie a number of human craniofacial anomalies.

  11. Sonic hedgehog initiates cochlear hair cell regeneration through downregulation of retinoblastoma protein

    SciTech Connect

    Lu, Na; Chen, Yan; Wang, Zhengmin; Chen, Guoling; Lin, Qin; Chen, Zheng-Yi; Li, Huawei

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer Shh activation in neonatal cochleae enhances sensory cell proliferation. Black-Right-Pointing-Pointer Proliferating supporting cells can transdifferentiate into hair cells. Black-Right-Pointing-Pointer Shh promotes proliferation by transiently modulating pRb activity. Black-Right-Pointing-Pointer Shh inhibits pRb by inhibiting transcription and increasing phosphorylation of pRb. -- Abstract: Cell cycle re-entry by cochlear supporting cells and/or hair cells is considered one of the best approaches for restoring hearing loss as a result of hair cell damage. To identify mechanisms that can be modulated to initiate cell cycle re-entry and hair cell regeneration, we studied the effect of activating the sonic hedgehog (Shh) pathway. We show that Shh signaling in postnatal rat cochleae damaged by neomycin leads to renewed proliferation of supporting cells and hair cells. Further, proliferating supporting cells are likely to transdifferentiate into hair cells. Shh treatment leads to inhibition of retinoblastoma protein (pRb) by increasing phosphorylated pRb and reducing retinoblastoma gene transcription. This results in upregulation of cyclins B1, D2, and D3, and CDK1. These results suggest that Shh signaling induces cell cycle re-entry in cochlear sensory epithelium and the production of new hair cells, in part by attenuating pRb function. This study provides an additional route to modulate pRb function with important implications in mammalian hair cell regeneration.

  12. WIP1 modulates responsiveness to Sonic Hedgehog signaling in neuronal precursor cells and medulloblastoma

    PubMed Central

    Wen, Jing; Lee, Juhyun; Malhotra, Anshu; Nahta, Rita; Arnold, Amanda R.; Buss, Meghan C.; Brown, Briana D.; Maier, Caroline; Kenney, Anna M.; Remke, Marc; Ramaswamy, Vijay; Taylor, Michael D.; Castellino, Robert C.

    2016-01-01

    High-level amplification of the protein phosphatase PPM1D (WIP1) is present in a subset of medulloblastomas (MBs) that have an expression profile consistent with active Sonic Hedgehog (SHH) signaling. We found that WIP1 overexpression increased expression of Shh target genes and cell proliferation in response to Shh stimulation in NIH3T3 and cerebellar granule neuron precursor (cGNP) cells in a p53-independent manner. Thus, we developed a mouse in which WIP1 is expressed in the developing brain under control of the Neurod2 promoter (ND2:WIP1). The external granule layer in early post-natal ND2:WIP1 mice exhibited increased proliferation and expression of Shh downstream targets. MB incidence increased and survival decreased when ND2:WIP1 mice were crossed with a Shh-activated MB mouse model. Conversely, Wip1 knock out significantly suppressed MB formation in two independent mouse models of Shh-activated MB. Furthermore, Wip1 knock-down or treatment with a WIP1 inhibitor suppressed the effects of Shh stimulation and potentiated the growth inhibitory effects of SHH pathway-inhibiting drugs in Shh-activated MB cells in vitro. This suggests an important cross-talk between SHH and WIP1 pathways that accelerates tumorigenesis and supports WIP1 inhibition as a potential treatment strategy for MB. PMID:27086929

  13. WIP1 modulates responsiveness to Sonic Hedgehog signaling in neuronal precursor cells and medulloblastoma.

    PubMed

    Wen, J; Lee, J; Malhotra, A; Nahta, R; Arnold, A R; Buss, M C; Brown, B D; Maier, C; Kenney, A M; Remke, M; Ramaswamy, V; Taylor, M D; Castellino, R C

    2016-10-20

    High-level amplification of the protein phosphatase PPM1D (WIP1) is present in a subset of medulloblastomas (MBs) that have an expression profile consistent with active Sonic Hedgehog (SHH) signaling. We found that WIP1 overexpression increased expression of Shh target genes and cell proliferation in response to Shh stimulation in NIH3T3 and cerebellar granule neuron precursor cells in a p53-independent manner. Thus, we developed a mouse in which WIP1 is expressed in the developing brain under control of the Neurod2 promoter (ND2:WIP1). The external granule layer (EGL) in early postnatal ND2:WIP1 mice exhibited increased proliferation and expression of Shh downstream targets. MB incidence increased and survival decreased when ND2:WIP1 mice were crossed with an Shh-activated MB mouse model. Conversely, Wip1 knockout significantly suppressed MB formation in two independent mouse models of Shh-activated MB. Furthermore, Wip1 knockdown or treatment with a WIP1 inhibitor suppressed the effects of Shh stimulation and potentiated the growth inhibitory effects of SHH pathway-inhibiting drugs in Shh-activated MB cells in vitro. This suggests an important cross-talk between SHH and WIP1 pathways that accelerates tumorigenesis and supports WIP1 inhibition as a potential treatment strategy for MB.

  14. Hair follicles' transit-amplifying cells govern concurrent dermal adipocyte production through Sonic Hedgehog.

    PubMed

    Zhang, Bing; Tsai, Pai-Chi; Gonzalez-Celeiro, Meryem; Chung, Oliver; Boumard, Benjamin; Perdigoto, Carolina N; Ezhkova, Elena; Hsu, Ya-Chieh

    2016-10-15

    Growth and regeneration of one tissue within an organ compels accommodative changes in the surrounding tissues. However, the molecular nature and operating logic governing these concurrent changes remain poorly defined. The dermal adipose layer expands concomitantly with hair follicle downgrowth, providing a paradigm for studying coordinated changes of surrounding lineages with a regenerating tissue. Here, we discover that hair follicle transit-amplifying cells (HF-TACs) play an essential role in orchestrating dermal adipogenesis through secreting Sonic Hedgehog (SHH). Depletion of Shh from HF-TACs abrogates both dermal adipogenesis and hair follicle growth. Using cell type-specific deletion of Smo, a gene required in SHH-receiving cells, we found that SHH does not act on hair follicles, adipocytes, endothelial cells, and hematopoietic cells for adipogenesis. Instead, SHH acts directly on adipocyte precursors, promoting their proliferation and their expression of a key adipogenic gene, peroxisome proliferator-activated receptor γ (Pparg), to induce dermal adipogenesis. Our study therefore uncovers a critical role for TACs in orchestrating the generation of both their own progeny and a neighboring lineage to achieve concomitant tissue production across lineages.

  15. May Sonic Hedgehog proteins be markers for malignancy in uterine smooth muscle tumors?

    PubMed

    Garcia, Natalia; Bozzini, Nilo; Baiocchi, Glauco; da Cunha, Isabela Werneck; Maciel, Gustavo Arantes; Soares Junior, José Maria; Soares, Fernando Augusto; Baracat, Edmund Chada; Carvalho, Katia Candido

    2016-04-01

    Several studies have demonstrated that the Sonic Hedgehog signaling pathway (SHH) plays an important role in tumorigenesis and cellular differentiation. We analyzed the protein expression of SHH pathway components and evaluated whether their profile could be useful for the diagnosis, prognosis, or prediction of the risk of malignancy for uterine smooth muscle tumors (USMTs). A total of 176 samples (20 myometrium, 119 variants of leiomyoma, and 37 leiomyosarcoma) were evaluated for the protein expression of the SHH signaling components, HHIP1 (SHH inhibitor), and BMP4 (SHH target) by immunohistochemistry. Western blot analysis was performed to verify the specificity of the antibodies. We grouped leiomyoma samples into conventional leiomyomas and unusual leiomyomas that comprise atypical, cellular, mitotically active leiomyomas and uterine smooth muscle tumors of uncertain malignant potential. Immunohistochemical analysis showed that SMO, SUFU, GLI1, GLI3, and BMP4 expression gradually increased depending on to the histologic tissue type. The protein expression of SMO, SUFU, and GLI1 was increased in unusual leiomyoma and leiomyosarcoma samples compared to normal myometrium. The inhibitor HHIP1 showed higher expression in myometrium, whereas only negative or basal expression of SMO, SUFU, GLI1, and GLI3 was detected in these samples. Strong expression of SHH was associated with poorer overall survival. Our data suggest that the expression of SHH proteins can be useful for evaluating the potential risk of malignancy for USMTs. Moreover, GLI1 and SMO may serve as future therapeutic targets for women with USMTs.

  16. Complete and sustained response of adult medulloblastoma to first-line sonic hedgehog inhibition with vismodegib.

    PubMed

    Lou, Emil; Schomaker, Matthew; Wilson, Jon D; Ahrens, Mary; Dolan, Michelle; Nelson, Andrew C

    2016-08-12

    Medulloblastoma is an aggressive primitive neuroectodermal tumor of the cerebellum that is rare in adults. Medulloblastomas fall into 4 prognostically significant molecular subgroups that are best defined by experimental gene expression profiles: the WNT pathway, sonic hedgehog (SHH) pathway, and subgroups 3 and 4 (non-SHH/WNT). Medulloblastoma of adults belong primarily to the SHH category. Vismodegib, an SHH-pathway inhibitor FDA-approved in 2012 for treatment of basal cell carcinoma, has been used successfully in the setting of chemorefractory medulloblastoma, but not as a first-line therapy. In this report, we describe a sustained response of an unresectable multifocal form of adult medulloblastoma to vismodegib. Molecular analysis in this case revealed mutations in TP53 and a cytogenetic abnormality, i17q, that is prevalent and most often associated with subgroup 4 rather than the SHH-activated form of medulloblastoma. Our findings indicate that vismodegib may also block alternate, non-canonical forms of downstream SHH pathway activation. These findings provide strong impetus for further investigation of vismodegib in clinical trials in the first-line setting for pediatric and adult forms of medulloblastoma.

  17. Ectopic expression of Sonic Hedgehog in a cryptorchid man with azoospermia: a case report.

    PubMed

    Zou, Shasha; Wang, Yanan; Chen, Tingting; Song, Pingping; Xin, Daiying; Ping, Ping; Huang, Yiran; Li, Zheng; Hu, Hongliang

    2014-04-01

    A 30-year-old man presented with a left undescended testis, right testicular deficiency and azoospermia. Testicular biopsy revealed an absence of spermatocytes and increased numbers of Leydig cells in the undescended testis. Additional comparative analyses were undertaken to explore Sonic Hedgehog (Shh) immunostaining in the testis of juvenile and adult mice, in the testis of the patient with cryptorchidism, and in archival testicular tissue from a patient with obstructive azoospermia and a patient with prostate cancer. Shh immunostaining was demonstrated in spermatocytes in juvenile and adult mouse testis and in the patients with obstructive azoospermia and prostate cancer, suggesting that Shh signalling is involved in normal spermatogenesis. In the patient with cryptorchidism, Shh immunostaining was localized to the Leydig cells, which suggests that Shh might be involved in the abnormal expansion of the Leydig cell population in the testis. These preliminary data on the appearance of Shh protein during normal spermatogenesis might provide the basis for further investigations to clarify the role of Shh signalling in spermatogenesis during normal and pathogenic testis development.

  18. Sonic hedgehog stimulates neurite outgrowth in a mechanical stretch model of reactive-astrogliosis.

    PubMed

    Berretta, Antonio; Gowing, Emma K; Jasoni, Christine L; Clarkson, Andrew N

    2016-02-23

    Although recovery following a stroke is limited, undamaged neurons under the right conditions can establish new connections and take on-board lost functions. Sonic hedgehog (Shh) signaling is integral for developmental axon growth, but its role after injury has not been fully examined. To investigate the effects of Shh on neuronal sprouting after injury, we used an in vitro model of glial scar, whereby cortical astrocytes were mechanically traumatized to mimic reactive astrogliosis observed after stroke. This mechanical trauma impaired neurite outgrowth from post-natal cortical neurons plated on top of reactive astrocytes. Addition of Shh to the media, however, resulted in a concentration-dependent increase in neurite outgrowth. This response was inhibited by cyclopamine and activated by oxysterol 20(S)-hydroxycholesterol, both of which modulate the activity of the Shh co-receptor Smoothened (Smo), demonstrating that Shh-mediated neurite outgrowth is Smo-dependent. In addition, neurite outgrowth was not associated with an increase in Gli-1 transcription, but could be inhibited by PP2, a selective inhibitor of Src family kinases. These results demonstrate that neurons exposed to the neurite growth inhibitory environment associated with a glial scar can be stimulated by Shh, with signaling occurring through a non-canonical pathway, to overcome this suppression and stimulate neurite outgrowth.

  19. Intrinsic facilitation of adult peripheral nerve regeneration by the Sonic hedgehog morphogen.

    PubMed

    Martinez, Jose A; Kobayashi, Masaki; Krishnan, Anand; Webber, Christine; Christie, Kimberly; Guo, GuiFang; Singh, Vandana; Zochodne, Douglas W

    2015-09-01

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

  20. Enhancement of Dopaminergic Differentiation in Proliferating Midbrain Neuroblasts by Sonic Hedgehog and Ascorbic Acid

    PubMed Central

    Volpicelli, Floriana; Consales, Claudia; Caiazzo, Massimiliano; Colucci-D'Amato, Luca; Perrone-Capano, Carla; di Porzio, Umberto

    2004-01-01

    We analyzed the molecular mechanisms involved in the acquisition and maturation of dopaminergic (DA) neurons generated in vitro from rat ventral mesencephalon (MES) cells in the presence of mitogens or specific signaling molecules. The addition of basic fibroblast growth factor (bFGF) to MES cells in serum-free medium stimulates the proliferation of neuroblasts but delays DA differentiation. Recombinant Sonic hedgehog (SHH) protein increases up to three fold the number of tyrosine hydroxylase (TH)-positive cells and their differentiation, an effect abolished by anti-SHH antibodies. The expanded cultures are rich in nestin-positive neurons, glial cells are rare, all TH+ neurons are DA, and all DA and GABAergic markers analyzed are expressed. Adding ascorbic acid to bFGF/SHH-treated cultures resulted in a further five- to seven-fold enhancement of viable DA neurons. This experimental system also provides a powerful tool to generate DA neurons from single embryos. Our strategy provides an enriched source of MES DA neurons that are useful for analyzing molecular mechanisms controlling their function and for experimental regenerative approaches in DA dysfunction. PMID:15303305

  1. Aberrant activation of Sonic hedgehog signaling in chronic cholecystitis and gallbladder carcinoma.

    PubMed

    Xie, Fang; Xu, Xiaoping; Xu, Angao; Liu, Cuiping; Liang, Fenfen; Xue, Minmin; Bai, Lan

    2014-03-01

    Sonic hedgehog (Shh) signaling has been extensively studied and is implicated in various inflammatory diseases and malignant tumors. We summarized the clinicopathological features and performed immunohistochemistry assays to examine expression of Shh signaling proteins in 10 normal mucosa, 32 gallbladder carcinoma (GBC), and 95 chronic cholecystitis (CC) specimens. The CC specimens were classified into three groups according to degree of inflammation. Compared with normal mucosa, CC, and GBC specimens exhibited increased expression of Shh. The immunoreactive score of Shh in the GBC group was higher than that in the mild to moderate CC groups but lower than that in the severe CC group (P < .05). Expression of Patched (Ptch) and Gli1 gradually increased from non-malignant cholecystitis to malignant tumors. Compared with CC specimens, GBC specimens showed higher cytoplasmic and membranous expression for Ptch (P < .05). Gli1 staining showed cytoplasmic expression of Gli1 in both CC (60% for mild, 77% for moderate, and 84% for severe) and GBC specimens (97%). Nuclear expression of Gli1 was detected in 16% of severe CC specimens with moderate to poor atypical hyperplasia, and in 62.5% of GBC specimens. Shh expression strongly correlated with expression of Ptch and Gli1. Furthermore, patients with strongly positive Gli1 staining had significantly lower survival rates than those with weakly positive staining. Our data indicate that the Shh signaling pathway is aberrantly activated in CC and GBC, and altered Shh signaling may be involved in the course of development from CC to gallbladder carcinogenesis.

  2. Temporal perturbations in sonic hedgehog signaling elicit the spectrum of holoprosencephaly phenotypes

    PubMed Central

    Cordero, Dwight; Marcucio, Ralph; Hu, Diane; Gaffield, William; Tapadia, Minal; Helms, Jill A.

    2004-01-01

    One of the most perplexing questions in clinical genetics is why patients with identical gene mutations oftentimes exhibit radically different clinical features. This inconsistency between genotype and phenotype is illustrated in the malformation spectrum of holoprosencephaly (HPE). Family members carrying identical mutations in sonic hedgehog (SHH) can exhibit a variety of facial features ranging from cyclopia to subtle midline asymmetries. Such intrafamilial variability may arise from environmental factors acting in conjunction with gene mutations that collectively reduce SHH activity below a critical threshold. We undertook a series of experiments to test the hypothesis that modifying the activity of the SHH signaling pathway at discrete periods of embryonic development could account for the phenotypic spectrum of HPE. Exposing avian embryos to cyclopamine during critical periods of craniofacial development recreated a continuum of HPE-related defects. The craniofacial malformations included hypotelorism, midfacial hypoplasia, and facial clefting and were not the result of excessive crest cell apoptosis. Rather, they resulted from molecular reprogramming of an organizing center whose activity controls outgrowth and patterning of the mid and upper face. Collectively, these data reveal one mechanism by which the variable expressivity of a disorder such as HPE can be produced through temporal disruption of a single molecular pathway. PMID:15314685

  3. "Atypical" regulation of Hedgehog-dependent cancers.

    PubMed

    Atwood, Scott X; Oro, Anthony E

    2014-02-10

    Growing evidence indicates targeting PKCι may be effective in treating Hedgehog-dependent cancers. In this issue of Cancer Cell, Justilien and colleagues present the surprising finding that PKCι promotes Hedgehog ligand production and lung squamous cell carcinoma growth through SOX2, rather than the canonical transcription factor GLI.

  4. Initiation and patterning of the snake dentition are dependent on Sonic hedgehog signaling.

    PubMed

    Buchtová, Marcela; Handrigan, Gregory R; Tucker, Abigail S; Lozanoff, Scott; Town, Liam; Fu, Katherine; Diewert, Virginia M; Wicking, Carol; Richman, Joy M

    2008-07-01

    Here we take the first look at cellular dynamics and molecular signaling in the developing snake dentition. We found that tooth formation differs from rodents in several respects. The majority of snake teeth bud off of a deep, ribbon-like dental lamina rather than as separate tooth germs. Prior to and after dental lamina ingrowth, we observe asymmetries in cell proliferation and extracellular matrix distribution suggesting that localized signaling by a secreted protein is involved. We cloned Sonic hedgehog from the African rock python Python sebae and traced its expression in the species as well as in two other snakes, the closely-related Python regius and the more derived corn snake Elaphe guttata (Colubridae). We found that expression of Shh is first confined to the odontogenic band and defines the position of the future dental lamina. Shh transcripts in pythons are progressively restricted to the oral epithelium on one side of the dental lamina and remain in this position throughout the prehatching period. Shh is expressed in the inner enamel epithelium and the stellate reticulum of the tooth anlagen, but is absent from the outer enamel epithelium and its derivative, the successional lamina. This suggests that signals other than Shh are responsible for replacement tooth formation. Functional studies using cyclopamine to block Hh signaling during odontogenesis prevented initiation and extension of the dental lamina into the mesenchyme, and also affected the directionality of this process. Further, blocking Hh signaling led to disruptions of the inner enamel epithelium. To explore the role of Shh in lamina extension, we looked at its expression in the premaxillary teeth, which form closer to the oral surface than elsewhere in the mouth. Oral ectodermal Shh expression in premaxillary teeth is lost soon after the teeth form reinforcing the idea that Shh is controlling the depth of the dental lamina. In summary, we have found diverse roles for Shh in patterning the

  5. Sonic hedgehog is a chemotactic neural crest cell guide that is perturbed by ethanol exposure.

    PubMed

    Tolosa, Ezequiel J; Fernández-Zapico, Martín E; Battiato, Natalia L; Rovasio, Roberto A

    2016-01-01

    Our aim was to understand the involvement of Sonic hedgehog (Shh) morphogen in the oriented distribution of neural crest cells (NCCs) toward the optic vesicle and to look for potential disorders of this guiding mechanism after ethanol exposure. In vitro directional analysis showed the chemotactic response of NCCs up Shh gradients and to notochord co-cultures (Shh source) or to their conditioned medium, a response inhibited by anti-Shh antibody, receptor inhibitor cyclopamine and anti-Smo morpholino (MO). Expression of the Ptch-Smo receptor complex on in vitro NCCs was also shown. In whole embryos, the expression of Shh mRNA and protein was seen in the ocular region, and of Ptch, Smo and Gli/Sufu system on cephalic NCCs. Anti-Smo MO or Ptch-mutated plasmid (Ptch1(Δloop2)) impaired cephalic NCC migration/distribution, with fewer cells invading the optic region and with higher cell density at the homolateral mesencephalic level. Beads embedded with cyclopamine (Smo-blocking) or Shh (ectopic signal) supported the role of Shh as an in vivo guide molecule for cephalic NCCs. Ethanol exposure perturbed in vitro and in vivo NCC migration. Early stage embryos treated with ethanol, in a model reproducing Fetal Alcohol Syndrome, showed later disruptions of craniofacial development associated with abnormal in situ expression of Shh morphogen. The results show the Shh/Ptch/Smo-dependent migration of NCCs toward the optic vesicle, with the support of specific inactivation with genetic and pharmacological tools. They also help to understand mechanisms of accurate distribution of embryonic cells and of their perturbation by a commonly consumed teratogen, and demonstrate, in addition to its other known developmental functions, a new biological activity of cellular guidance for Shh.

  6. Relation between sonic hedgehog pathway gene polymorphisms and basal cell carcinoma development in the Polish population.

    PubMed

    Lesiak, Aleksandra; Sobolewska-Sztychny, Dorota; Majak, Paweł; Sobjanek, Michał; Wodz, Karolina; Sygut, Karolina Przybyłowska-; Majsterek, Ireneusz; Wozniacka, Anna; Narbutt, Joanna

    2016-01-01

    In recent decades, increases have been observed in the incidence of nonmelanoma skin cancers, including basal cell carcinoma (BCC) and squamous cell carcinoma. BCC is the most common neoplasm in Caucasian populations. Sonic hedgehog (Shh) pathway impairment plays a key role in BCC pathogenesis, and there is evidence that Shh pathway genetic variations may predispose to BCC development. We genotyped 22 single-nucleotide polymorphisms (SNPs) in 4 Shh pathway genes: SHH, GLI, SMO, and PTCH. The study group consisted of 142 BCC patients and 142 age-matched, sex-matched healthy subjects (controls). SNPs were assessed using the PCR-RFLP method. The genotype distribution for the polymorphisms in the rs104894049 331 A/T SHH, rs104894040 349 T/C SHH, and rs41303402 385 G/A SMO genes differed significantly between the BCC patients and the controls. The presence of CC genotype in the SHH rs104894040 349 T/C polymorphism was linked to the highest risk of BCC development (OR 87.9, p < 0.001). Other genotypes, such as the TT in SHH rs104894049 331 A/T and the GG in SMO rs41303402 385 G/A also statistically raised the risk of BCC, but these associations were weaker. Other investigated polymorphisms showed no statistical differences between patients and controls. The results obtained testify to the importance of the SHH and SMO gene polymorphisms in skin cancerogenesis. These results mainly underline the potential role of SHH3 rs104894040 349 T/C gene polymorphism in the development of skin basal cell carcinomas in patients of Polish origin.

  7. Sonic hedgehog-mediated epithelial-mesenchymal transition in renal tubulointerstitial fibrosis.

    PubMed

    Bai, Yongheng; Lu, Hong; Lin, Chengcheng; Xu, Yaya; Hu, Dannü; Liang, Yong; Hong, Weilong; Chen, Bicheng

    2016-05-01

    The sonic hedgehog (SHH) signaling pathway plays a critical role in embryonic development, tissue regeneration and organogenesis. The activation of SHH signaling produces profibrogenic effects in various tissues, such as the liver and the biliary ducts. However, the role of SHH signaling in renal fibrogenesis remains to be elucidated. For this purpose, in the present study, we evaluated the hypothesis that activated SHH signaling promotes the acquisition of a myofibroblastic phenotype through the epithelial-mesenchymal transition (EMT), resulting in renal interstitial fibrosis (RIF). Kidney samples from rats subjected to unilateral or bilateral ureteral obstruction exhibited the enhanced expression of SHH-pathway proteins, mesenchymal markers and the decreased expression of epithelial markers. Overactive SHH signaling as well as tubular EMT and RIF in the obstructed kidneys were inhibited by recanalization of the ureter. In vitro, SHH signaling was activated during EMT induction and extracellular matrix (ECM) deposition was observed in transforming growth factor-β1 (TGF-β1)-treated renal tubular epithelial cells [RTECs; NRK-52E cell line]. Exogenous SHH activated SHH signaling and resulted in the upregulated expression of mesenchymal genes, the profibrogenic cytokine TGF-β1, and the downregulated expression of epithelial markers. The blockade of SHH signaling with cyclopamine abolished SHH-mediated EMT as well as the acquisition of a myofibroblastic phenotype, and decreased TGF-β1 expression and ECM production. Thus, taken together, these findings demonstrate that the activation of the SHH signaling pathway promotes the induction of EMT and renal tubulointerstitial fibrosis. The pharmacological inhibition of SHH signaling may potentially be of therapeutic value in the management of fibrotic kidney diseases.

  8. Assessment of the stromal contribution to Sonic Hedgehog-dependent pancreatic adenocarcinoma.

    PubMed

    Damhofer, Helene; Medema, Jan Paul; Veenstra, Veronique L; Badea, Liviu; Popescu, Irinel; Roelink, Henk; Bijlsma, Maarten F

    2013-12-01

    Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. It is typically detected at an advanced stage, at which the therapeutic options are very limited. One remarkable feature of PDAC that contributes to its resilience to treatment is the extreme stromal activation seen in these tumors. Often, the vast majority of tumor bulk consists of non-tumor cells that together provide a tumor-promoting environment. One of the signals that maintains and activates the stroma is the developmental protein Sonic Hedgehog (SHH). As the disease progresses, tumor cells produce increasing amounts of SHH, which activates the surrounding stroma to aid in tumor progression. To better understand this response and identify targets for inhibition, we aimed to elucidate the proteins that mediate the SHH-driven stromal response in PDAC. For this a novel mixed-species coculture model was set up in which the cancer cells are human, and the stroma is modeled by mouse fibroblasts. In conjunction with next-generation sequencing we were able to use the sequence difference between these species to genetically distinguish between the epithelial and stromal responses to SHH. The stromal SHH-dependent genes from this analysis were validated and their relevance for human disease was subsequently determined in two independent patient cohorts. In non-microdissected tissue from PDAC patients, in which a large amount of stroma is present, the targets were confirmed to associate with tumor stroma versus normal pancreatic tissue. Patient survival analysis and immunohistochemistry identified CDA, EDIL3, ITGB4, PLAUR and SPOCK1 as SHH-dependent stromal factors that are associated with poor prognosis in PDAC patients. Summarizing, the presented data provide insight into the role of the activated stroma in PDAC, and how SHH acts to mediate this response. In addition, the study has yielded several candidates that are interesting therapeutic targets for a disease for which treatment

  9. Sonic Hedgehog Signaling Affected by Promoter Hypermethylation Induces Aberrant Gli2 Expression in Spina Bifida.

    PubMed

    Lu, Xiao-Lin; Wang, Li; Chang, Shao-Yan; Shangguan, Shao-Fang; Wang, Zhen; Wu, Li-Hua; Zou, Ji-Zhen; Xiao, Ping; Li, Rui; Bao, Yi-Hua; Qiu, Z-Y; Zhang, Ting

    2016-10-01

    GLI2 is a key mediator of the sonic hedgehog (Shh) signaling pathway and plays an important role in neural tube development during vertebrate embryogenesis; however, the role of gli2 in human folate-related neural tube defects remains unclear. In this study, we compared methylation status and polymorphisms of gli2 between spina bifida patients and a control group to explore the underlying mechanisms related to folate deficiency in spina bifida. No single nucleotide polymorphism was found to be significantly different between the two groups, although gli2 methylation levels were significantly increased in spina bifida samples, accompanied by aberrant GLI2 expression. Moreover, a prominent negative correlation was found between the folate level in brain tissue and the gli2 methylation status (r = -0.41, P = 0.014), and gli2 hypermethylation increased the risk of spina bifida with an odds ratio of 12.45 (95 % confidence interval: 2.71-57.22, P = 0.001). In addition, we established a cell model to illustrate the effect of gli2 expression and the accessibility of chromatin affected by methylation. High gli2 and gli1 mRNA expression was detected in 5-Aza-treated cells, while gli2 hypermethylation resulted in chromatin inaccessibility and a reduced association with nuclear proteins containing transcriptional factors. More meaningful to the pathway, the effect gene of the Shh pathway, gli1, was found to have a reduced level of expression along with a decreased expression of gli2 in our cell model. Aberrant high methylation resulted in the low expression of gli2 in spina bifida, which was affected by the change in chromatin status and the capacity of transcription factor binding.

  10. The Role of the Sonic Hedgehog Pathway for Prostate Cancer Progression

    DTIC Science & Technology

    2007-02-01

    tomatidine , a structurally similar but non-specific compound for hedgehog signaling did not affect cell growth. In contrast, cell growth of HepG2 was...hedgehog signaling status (our unpublished observation), and was, thus, not used in this study. Tomatidine (2 mM in 0.5% FBS DMEM, Sigma Cat# T2909), a...inhibition was specific because addition of tomatidine , a structurally similar but non-specific compound for hedgehog signaling, did not affect cell growth

  11. The Role of the Sonic Hedgehog Pathway for Prostate Cancer Progression

    DTIC Science & Technology

    2005-02-01

    junctions so as not to detect genomic DNA and the primers and probe sequences were Additional material searched against the Celera database to confirm...searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send...activation of the hedgehog pathway occurs frequently in advanced human prostate cancer. We find that high levels of hedgehog target genes, PTCHI and hedgehog

  12. The Role of the Sonic Hedgehog Pathway for Prostate Cancer Progression

    DTIC Science & Technology

    2005-02-01

    junctions so as not to detect genomic DNA and the primers and probe sequences were Additional material searched against the Celera database to confirm...instructions. searching existing data sources, gathenng and maintaining the data needed, and completing and reviewing this collection of information. Send...activation of the hedgehog pathway occurs frequently in advanced human prostate cancer. We find that high levels of hedgehog target genes, PTCH1 and hedgehog

  13. Nicotine induces self-renewal of pancreatic cancer stem cells via neurotransmitter-driven activation of sonic hedgehog signalling.

    PubMed

    Al-Wadei, Mohammed H; Banerjee, Jheelam; Al-Wadei, Hussein A N; Schuller, Hildegard M

    2016-01-01

    A small subpopulation of pancreatic cancer cells with characteristics of stem cells drive tumour initiation, progression and metastasis. A better understanding of the regulation of cancer stem cells may lead to more effective cancer prevention and therapy. We have shown that the proliferation and migration of pancreatic cancer cell lines is activated by the nicotinic receptor-mediated release of stress neurotransmitters, responses reversed by γ-aminobutyric acid (GABA). However, the observed cancer inhibiting effects of GABA will only succeed clinically if GABA inhibits pancreatic cancer stem cells (PCSCs) in addition to the more differentiated cancer cells that comprise the majority of cancer tissues and cell lines. Using PCSCs isolated from two pancreatic cancer patients by cell sorting and by spheroid formation assay from pancreatic cancer cell line Panc-1, we tested the hypothesis that nicotine induces the self-renewal of PCSCs. Nicotinic acetylcholine receptors (nAChRs) α3, α4, α5 and α7 were expressed and chronic exposure to nicotine increased the protein expression of these receptors. Immunoassays showed that PCSCs produced the stress neurotransmitters epinephrine and norepinephrine and the inhibitory neurotransmitter GABA. Chronic nicotine significantly increased the production of stress neurotransmitters and sonic hedgehog (SHH) while inducing Gli1 protein and decreasing GABA. GABA treatment inhibited the induction of SHH and Gli1. Spheroid formation and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assays showed significant nicotine-induced increases in self renewal and cell proliferation, responses blocked by GABA. Our data suggest that nicotine increases the SHH-mediated malignant potential of PCSCs and that GABA prevents these effects.

  14. Hedgehog signaling regulates E-cadherin expression for the maintenance of the actin cytoskeleton and tight junctions

    PubMed Central

    Xiao, Chang; Ogle, Sally A.; Schumacher, Michael A.; Schilling, Neal; Tokhunts, Robert A.; Orr-Asman, Melissa A.; Miller, Marian L.; Robbins, David J.; Hollande, Frederic

    2010-01-01

    In the stomach, strictly regulated cell adherens junctions are crucial in determining epithelial cell differentiation. Sonic Hedgehog (Shh) regulates epithelial cell differentiation in the adult stomach. We sought to identify whether Shh plays a role in regulating adherens junction protein E-cadherin as a mechanism for epithelial cell differentiation. Mouse nontumorigenic gastric epithelial (IMGE-5) cells treated with Hedgehog signaling inhibitor cyclopamine and anti-Shh 5E1 antibody or transduced with short hairpin RNA against Skinny Hedgehog (IMGE-5Ski) were cultured. A mouse model expressing a parietal cell-specific deletion of Shh (HKCre/ShhKO) was used to identify further changes in adherens and tight junctions. Inhibition of Hedgehog signaling in IMGE-5 cells caused loss of E-cadherin expression accompanied by disruption of F-actin cortical expression and relocalization of zonula occludens-1 (ZO-1). Loss of E-cadherin was also associated with increased proliferation in IMGE-5Ski cells and increased expression of the mucous neck cell lineage marker MUC6. Compared with membrane-expressed E-cadherin and ZO-1 protein in controls, dissociation of E-cadherin/β-catenin and ZO-1/occludin protein complexes was observed in HKCre/ShhKO mice. In conclusion, we demonstrate that Hedgehog signaling regulates E-cadherin expression that is required for the maintenance of F-actin cortical expression and stability of tight junction protein ZO-1. PMID:20847300

  15. Hedgehog pathway regulators influence cervical cancer cell proliferation, survival and migration

    SciTech Connect

    Samarzija, Ivana; Beard, Peter

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer Unknown cellular mutations complement papillomavirus-induced carcinogenesis. Black-Right-Pointing-Pointer Hedgehog pathway components are expressed by cervical cancer cells. Black-Right-Pointing-Pointer Hedgehog pathway activators and inhibitors regulate cervical cancer cell biology. Black-Right-Pointing-Pointer Cell immortalization by papillomavirus and activation of Hedgehog are independent. -- Abstract: Human papillomavirus (HPV) infection is considered to be a primary hit that causes cervical cancer. However, infection with this agent, although needed, is not sufficient for a cancer to develop. Additional cellular changes are required to complement the action of HPV, but the precise nature of these changes is not clear. Here, we studied the function of the Hedgehog (Hh) signaling pathway in cervical cancer. The Hh pathway can have a role in a number of cancers, including those of liver, lung and digestive tract. We found that components of the Hh pathway are expressed in several cervical cancer cell lines, indicating that there could exists an autocrine Hh signaling loop in these cells. Inhibition of Hh signaling reduces proliferation and survival of the cervical cancer cells and induces their apoptosis as seen by the up-regulation of the pro-apoptotic protein cleaved caspase 3. Our results indicate that Hh signaling is not induced directly by HPV-encoded proteins but rather that Hh-activating mutations are selected in cells initially immortalized by HPV. Sonic Hedgehog (Shh) ligand induces proliferation and promotes migration of the cervical cancer cells studied. Together, these results indicate pro-survival and protective roles of an activated Hh signaling pathway in cervical cancer-derived cells, and suggest that inhibition of this pathway may be a therapeutic option in fighting cervical cancer.

  16. Cerebrospinal fluid from patients with amyotrophic lateral sclerosis inhibits sonic hedgehog function

    PubMed Central

    Drannik, Anna; Martin, Joan; Peterson, Randy; Ma, Xiaoxing; Jiang, Fan; Turnbull, John

    2017-01-01

    Sonic hedgehog (Shh) is a morphogen essential to the developing nervous system that continues to play an important role in adult life by contributing to cell proliferation and differentiation, maintaining blood-brain barrier integrity, and being cytoprotective against oxidative and excitotoxic stress, all features of importance in amyotrophic lateral sclerosis (ALS). ALS is a fatal disease characterized by selective loss of motor neurons due to poorly understood mechanisms. Evidence indicates that Shh might play an important role in ALS, and that Shh signaling might be also adversely affected in ALS. Since little is known about the functional status of Shh pathway in patients with ALS, we therefore sought to determine whether Shh protein levels or biological activity in cerebrospinal fluid (CSF) was less in ALS patients than controls, and whether these measures could be correlated with ALS disease severity and disease progression, and with other CSF analytes of biological interest in ALS. Comparing Shh levels in the CSF of normal controls (n = 13), neurological controls (n = 12), and ALS patients (n = 9) measured by ELISA, we found that CSF Shh levels were not different between controls and ALS patients. However, when assessing Shh biological activity in CSF using in vitro cell-based assays, which measure Shh activity as inducible Gli-driven luminescence, we found that in the presence of exogenous recombinant Shh or the Shh agonist, purmorphamine, the inducible activity of CSF was significantly augmented in the control groups as expected, but not in the ALS group, suggesting the presence of an inhibitor of Shh signaling in ALS CSF samples. Since purmorphamine acts on Smoothened, downstream of Shh and its receptor Patched, the inhibitory action is downstream of Smoothened. Our results also demonstrated that while the inhibitory effect of ALS CSF on Shh signaling did not correlate significantly with ALS disease characteristics, the levels of IL-1β and TNF-α did. In

  17. The buccohypophyseal canal is an ancestral vertebrate trait maintained by modulation in sonic hedgehog signaling

    PubMed Central

    2013-01-01

    Background The pituitary gland is formed by the juxtaposition of two tissues: neuroectoderm arising from the basal diencephalon, and oral epithelium, which invaginates towards the central nervous system from the roof of the mouth. The oral invagination that reaches the brain from the mouth is referred to as Rathke’s pouch, with the tip forming the adenohypophysis and the stalk disappearing after the earliest stages of development. In tetrapods, formation of the cranial base establishes a definitive barrier between the pituitary and oral cavity; however, numerous extinct and extant vertebrate species retain an open buccohypophyseal canal in adulthood, a vestige of the stalk of Rathke’s pouch. Little is currently known about the formation and function of this structure. Here we have investigated molecular mechanisms driving the formation of the buccohypophyseal canal and their evolutionary significance. Results We show that Rathke’s pouch is located at a boundary region delineated by endoderm, neural crest-derived oral mesenchyme and the anterior limit of the notochord, using CD1, R26R-Sox17-Cre and R26R-Wnt1-Cre mouse lines. As revealed by synchrotron X-ray microtomography after iodine staining in mouse embryos, the pouch has a lobulated three-dimensional structure that embraces the descending diencephalon during pituitary formation. Polarisfl/fl; Wnt1-Cre, Ofd1-/- and Kif3a-/- primary cilia mouse mutants have abnormal sonic hedgehog (Shh) signaling and all present with malformations of the anterior pituitary gland and midline structures of the anterior cranial base. Changes in the expressions of Shh downstream genes are confirmed in Gas1-/- mice. From an evolutionary perspective, persistence of the buccohypophyseal canal is a basal character for all vertebrates and its maintenance in several groups is related to a specific morphology of the midline that can be related to modulation in Shh signaling. Conclusion These results provide insight into a poorly

  18. Sonic Hedgehog Signaling Protects Human Hepatocellular Carcinoma Cells Against Ionizing Radiation in an Autocrine Manner

    SciTech Connect

    Chen, Yu-Jen; Lin, Chin-Ping; Hsu, Ming-Ling; Shieh, Hui-Ru; Chao, Nicholas K.; Chao, K.S. Clifford

    2011-07-01

    Purpose: Sonic hedgehog (Shh) signaling is critical to embryogenesis and resistance to chemotherapy. We aimed to examine the role of Shh signaling in the response to radiation of human hepatocellular carcinoma (HCC) cells. Methods and Materials: Response to ionizing radiation therapy (RT) was evaluated by clonogenic assay. Quantitative RT-polymerase chain reaction for patched-1 (PTCH-1) expression was performed. Cytosolic accumulation of Shh and nuclear translocation of Gli-1 were assessed by immunofluorescence. Gli-1 knockdown was done by RNA interference (RNAi). Immunoprecipitation was performed to detect Shh ligand in conditioned medium. Immunofluorescent stain for {gamma}-H2AX was used as an index of DNA double strand breaks (DSB). Expression of proteins related to DNA damage repair was assessed by Western blotting. Results: We found that Shh ligand could protect human HCC HA22T and Sk-Hep1 cells against RT. In HA22T cells, Shh ligand activated the Shh signaling with upregulation of Shh, PTCH-1, and Gli-1 expression. The nuclear translocation of Gli-1 further supports the activation of Gli-1. The radioprotection by Shh ligand was partly blocked by Shh antibody neutralization and was abolished by Gli-1 RNAi, suggesting a critical role of Shh signaling in radiation resistance. Furthermore, we noted that soluble factors secreted into conditioned medium, either constitutively or responding to radiation, by HA22T or Sk-Hep1 cells protected subsequent culturing cells against RT. Immunoprecipitation shows the presence of Shh peptide in conditioned medium. Intriguingly, antibody neutralization of Shh ligand or knockdown of Gli-1 reversed the radioprotective effect of conditioned medium. Furthermore, Shh ligand reduced the RT-induced phosphorylation of checkpoint kinase 1 and impaired the repair of DNA DSB. Conclusions: Activation of Shh signaling protects HCC cells against ionizing radiation in an autocrine manner. Impairment of DNA damage repair might involve

  19. Sonic hedgehog (Shh) signaling promotes tumorigenicity and stemness via activation of epithelial-to-mesenchymal transition (EMT) in bladder cancer.

    PubMed

    Islam, S S; Mokhtari, R B; Noman, A S; Uddin, M; Rahman, M Z; Azadi, M A; Zlotta, A; van der Kwast, T; Yeger, H; Farhat, W A

    2016-05-01

    Activation of the sonic hedgehog (Shh) signaling pathway controls tumorigenesis in a variety of cancers. Here, we show a role for Shh signaling in the promotion of epithelial-to-mesenchymal transition (EMT), tumorigenicity, and stemness in the bladder cancer. EMT induction was assessed by the decreased expression of E-cadherin and ZO-1 and increased expression of N-cadherin. The induced EMT was associated with increased cell motility, invasiveness, and clonogenicity. These progression relevant behaviors were attenuated by treatment with Hh inhibitors cyclopamine and GDC-0449, and after knockdown by Shh-siRNA, and led to reversal of the EMT phenotype. The results with HTB-9 were confirmed using a second bladder cancer cell line, BFTC905 (DM). In a xenograft mouse model TGF-β1 treated HTB-9 cells exhibited enhanced tumor growth. Although normal bladder epithelial cells could also undergo EMT and upregulate Shh with TGF-β1 they did not exhibit tumorigenicity. The TGF-β1 treated HTB-9 xenografts showed strong evidence for a switch to a more stem cell like phenotype, with functional activation of CD133, Sox2, Nanog, and Oct4. The bladder cancer specific stem cell markers CK5 and CK14 were upregulated in the TGF-β1 treated xenograft tumor samples, while CD44 remained unchanged in both treated and untreated tumors. Immunohistochemical analysis of 22 primary human bladder tumors indicated that Shh expression was positively correlated with tumor grade and stage. Elevated expression of Ki-67, Shh, Gli2, and N-cadherin were observed in the high grade and stage human bladder tumor samples, and conversely, the downregulation of these genes were observed in the low grade and stage tumor samples. Collectively, this study indicates that TGF-β1-induced Shh may regulate EMT and tumorigenicity in bladder cancer. Our studies reveal that the TGF-β1 induction of EMT and Shh is cell type context dependent. Thus, targeting the Shh pathway could be clinically beneficial in the

  20. Sonic hedgehog-expressing cells in the developing limb measure time by an intrinsic cell cycle clock.

    PubMed

    Chinnaiya, Kavitha; Tickle, Cheryll; Towers, Matthew

    2014-07-08

    How time is measured is an enduring issue in developmental biology. Classical models of somitogenesis and limb development implicated intrinsic cell cycle clocks, but their existence remains controversial. Here we show that an intrinsic cell cycle clock in polarizing region cells of the chick limb bud times the duration of Sonic hedgehog (Shh) expression, which encodes the morphogen specifying digit pattern across the antero-posterior axis (thumb to little finger). Timing by this clock starts when polarizing region cells fall out of range of retinoic acid signalling. We found that timing of Shh transcription by the cell cycle clock can be reset, thus revealing an embryonic form of self-renewal. In contrast, antero-posterior positional values cannot be reset, suggesting that this may be an important constraint on digit regeneration. Our findings provide the first evidence for an intrinsic cell cycle timer controlling duration and patterning activity of a major embryonic signalling centre.

  1. Peptide amphiphile nanofiber hydrogel delivery of sonic hedgehog protein to the cavernous nerve to promote regeneration and prevent erectile dysfunction.

    PubMed

    Choe, Shawn; Bond, Christopher W; Harrington, Daniel A; Stupp, Samuel I; McVary, Kevin T; Podlasek, Carol A

    2017-01-01

    Erectile dysfunction (ED) has high impact on quality of life in prostatectomy, diabetic and aging patients. An underlying mechanism is cavernous nerve (CN) injury, which causes ED in up to 80% of prostatectomy patients. We examine how sonic hedgehog (SHH) treatment with innovative peptide amphiphile nanofiber hydrogels (PA), promotes CN regeneration after injury. SHH and its receptors patched (PTCH1) and smoothened (SMO) are localized in PG neurons and glia. SMO undergoes anterograde transport to signal to downstream targets. With crush injury, PG neurons degenerate and undergo apoptosis. SHH protein decreases, SMO localization changes to the neuronal cell surface, and anterograde transport stops. With SHH treatment SHH is taken up at the injury site and undergoes retrograde transport to PG neurons, allowing SMO transport to occur, and neurons remain intact. SHH treatment prevents neuronal degeneration, maintains neuronal, glial and downstream target signaling, and is significant as a regenerative therapy.

  2. Serotonin Regulates Calcium Homeostasis in Lactation by Epigenetic Activation of Hedgehog Signaling

    PubMed Central

    Laporta, Jimena; Keil, Kimberly P.; Weaver, Samantha R.; Cronick, Callyssa M.; Prichard, Austin P.; Crenshaw, Thomas D.; Heyne, Galen W.; Vezina, Chad M.; Lipinski, Robert J.

    2014-01-01

    Calcium homeostasis during lactation is critical for maternal and neonatal health. We previously showed that nonneuronal/peripheral serotonin [5-hydroxytryptamine (5-HT)] causes the lactating mammary gland to synthesize and secrete PTHrP in an acute fashion. Here, using a mouse model, we found that genetic inactivation of tryptophan hydroxylase 1 (Tph1), which catalyzes the rate-limiting step in peripheral 5-HT synthesis, reduced circulating and mammary PTHrP expression, osteoclast activity, and maternal circulating calcium concentrations during the transition from pregnancy to lactation. Tph1 inactivation also reduced sonic hedgehog signaling in the mammary gland during lactation. Each of these deficiencies was rescued by daily injections of 5-hydroxy-L-tryptophan (an immediate precursor of 5-HT) to Tph1-deficient dams. We used immortalized mouse embryonic fibroblasts to demonstrate that 5-HT induces PTHrP through a sonic hedgehog-dependent signal transduction mechanism. We also found that 5-HT altered DNA methylation of the Shh gene locus, leading to transcriptional initiation at an alternate start site and formation of a variant transcript in mouse embryonic fibroblasts in vitro and in mammary tissue in vivo. These results support a new paradigm of 5-HT-mediated Shh regulation involving DNA methylation remodeling and promoter switching. In addition to having immediate implications for lactation biology, identification and characterization of a novel functional regulatory relationship between nonneuronal 5-HT, hedgehog signaling, and PTHrP offers new avenues for the study of these important factors in development and disease. PMID:25192038

  3. Brown adipocyte differentiation is regulated by hedgehog signaling during development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During development, brown fat tissue arises from mesenchymal precursor cells under the control of signaling networks that are not yet well understood. The Hedgehog (Hh) signaling pathway is one of the major signaling pathways that regulate mesenchymal cell fate. However, whether the Hh pathway contr...

  4. Sonic hedgehog stimulates the proliferation of rat gastric mucosal cells through ERK activation by elevating intracellular calcium concentration

    SciTech Connect

    Osawa, Hiroyuki; Ohnishi, Hirohide . E-mail: hohnishi@jichi.ac.jp; Takano, Koji; Noguti, Takasi; Mashima, Hirosato; Hoshino, Hiroko; Kita, Hiroto; Sato, Kiichi; Matsui, Hirofumi; Sugano, Kentaro

    2006-06-02

    Sonic Hedgehog (Shh), a member of hedgehog peptides family, is expressed in gastric gland epithelium. To elucidate Shh function to gastric mucosal cells, we examined the effect of Shh on the proliferation of a rat normal gastric mucosal cell line, RGM-1. RGM-1 cells express essential components of Shh receptor system, patched-1, and smoothened. Shh enhanced DNA synthesis in RGM-1 cells and elevated intracellular calcium concentration ([Ca{sup 2+}]{sub i}). In addition, Shh as well as calcium ionophore A32187 rapidly activated ERK. However, Shh failed to activate ERK under calcium-free culture condition. Pretreatment of cells with PD98059 attenuated the DNA synthesis promoted by Shh. Moreover, when cells were pretreated with cyclopamine, Shh could not elevate [Ca{sup 2+}]{sub i}, activate ERK or promote DNA synthesis. On the other hand, although Shh induced Gli-1 nuclear accumulation in RGM-1 cells, Shh activated ERK even in cells pretreated with actinomycin D. These results indicate that Shh promotes the proliferation of RGM-1 cells through an intracellular calcium- and ERK-dependent but transcription-independent pathway via Patched/Smoothened receptor system.

  5. The Role of the Sonic Hedgehog Pathway for Prostate Cancer Progression

    DTIC Science & Technology

    2006-02-01

    development of prostate cancer. Furthermore, Professor Leland Chung has started collaboration with my laboratory on the effects of hedgehog...activation in several types of human cancer, particularly in prostate cancer. Two long-term collaborations (with Drs. Hong Wu and Leland Chung

  6. YB-1 is elevated in medulloblastoma and drives proliferation in Sonic hedgehog - dependent cerebellar granule neuron progenitor cells and medulloblastoma cells

    PubMed Central

    Dey, Abhinav; Robitaille, Mélanie; Remke, Marc; Maier, Caroline; Malhotra, Anshu; Gregorieff, Alex; Wrana, Jeffrey L; Taylor, Michael D; Angers, Stéphane; Kenney, Anna Marie

    2016-01-01

    Post-natal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells-of-origin for the SHH-associated subgroup of medulloblastoma (MB), is driven by Sonic Hedgehog (Shh) and Insulin-like Growth Factor (IGF) in the developing cerebellum. Shh induces the oncogene Yes-associated protein (YAP), which drives IGF2 expression in CGNPs and mouse Shh-associated medulloblastomas. To determine how IGF2 expression is regulated downstream of YAP, we carried out an unbiased screen for transcriptional regulators bound to IGF2 promoters. We report that Y-box binding protein-1 (YB-1), an onco-protein regulating transcription and translation, binds to IGF2 promoter P3. We observed that YB-1 is up-regulated across human medulloblastoma subclasses as well as in other varieties of pediatric brain tumors. Utilizing the cerebellar progenitor model for the Shh-subgroup of MB in mice, we show for the first time that YB-1 is induced by Shh in CGNPs. Its expression is YAP-dependent and it is required for IGF2 expression in CGNPs. Finally, both gain-of function and loss-of-function experiments reveal that YB-1 activity is required for sustaining CGNP and medulloblastoma cell (MBC) proliferation. Collectively, our findings describe a novel role for YB-1 in driving proliferation in the developing cerebellum and medulloblastoma cells and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas. PMID:26725322

  7. Zfp423 Regulates Sonic Hedgehog Signaling via Primary Cilium Function

    PubMed Central

    Hamilton, Bruce A.

    2016-01-01

    Zfp423 encodes a 30-zinc finger transcription factor that intersects several canonical signaling pathways. Zfp423 mutations result in ciliopathy-related phenotypes, including agenesis of the cerebellar vermis in mice and Joubert syndrome (JBTS19) and nephronophthisis (NPHP14) in humans. Unlike most ciliopathy genes, Zfp423 encodes a nuclear protein and its developmental expression is complex, leading to alternative proposals for cellular mechanisms. Here we show that Zfp423 is expressed by cerebellar granule cell precursors, that loss of Zfp423 in these precursors leads to cell-intrinsic reduction in proliferation, loss of response to Shh, and primary cilia abnormalities that include diminished frequency of both Smoothened and IFT88 localization. Loss of Zfp423 alters expression of several genes encoding key cilium components, including increased expression of Tulp3. Tulp3 is a direct binding target of Zfp423 and reducing the overexpression of Tulp3 in Zfp423-deficient cells suppresses Smoothened translocation defects. These results define Zfp423 deficiency as a bona fide ciliopathy, acting upstream of Shh signaling, and indicate a mechanism intrinsic to granule cell precursors for the resulting cerebellar hypoplasia. PMID:27727273

  8. Sonic hedgehog released from scratch-injured astrocytes is a key signal necessary but not sufficient for the astrocyte de-differentiation.

    PubMed

    Yang, Hao; Feng, Guo-Dong; Olivera, Cathy; Jiao, Xi-Ying; Vitale, Angela; Gong, Ju; You, Si-Wei

    2012-09-01

    Recent studies demonstrated that mature atrocytes have the capacity for de-differentiating into neural stem/progenitor cells (NSPCs) in vitro and in vivo. However, it is still unknown what signals endow astroglial cells with a de-differentiation potential. Furthermore, the signaling molecules and underlying mechanism that confer astrocytes with the competence of NSPC phenotypes have not been completely elucidated. Here, we found that sonic hedgehog (Shh) production in astrocytes following mechanical injury was significantly elevated, and that incubation of astrocyes with the injured astrocyte conditioned medium (ACM) causes astrocytes to gradually lose their immunophenotypical profiles, and acquire NSPC characteristics, as demonstrated by down-regulation of typical astrocytic markers (GFAP and S100) and up-regulation of markers that are generally expressed in NSCs, (nestin, Sox2, and CD133). ACM treated astrocytes exhibit self-renewal capacity and multipotency similar to NSPCs. Concomitantly, in addition to Ptc, there was a significant up-regulation of the Shh downstream signal components Gli2 and Cyclin D1 which are involved in cell proliferation, dramatic changes in cell morphology, and the disruption of cell-cycle G1 arrest. Conversely, the depletion of Shh by administration of its neutralizing antibody (Shh n-Ab) effectively inhibited the de-differentiation process. Strikingly, Shh alone had little effect on astrocyte de-differentiation to NSPCs. These data above suggest that Shh is a key instructive molecule while other molecules secreted from insulted astrocytes may synergistically promote the de-differentiation event.

  9. YB-1 is elevated in medulloblastoma and drives proliferation in Sonic hedgehog-dependent cerebellar granule neuron progenitor cells and medulloblastoma cells.

    PubMed

    Dey, A; Robitaille, M; Remke, M; Maier, C; Malhotra, A; Gregorieff, A; Wrana, J L; Taylor, M D; Angers, S; Kenney, A M

    2016-08-11

    Postnatal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells of origin for the SHH-associated subgroup of medulloblastoma, is driven by Sonic hedgehog (Shh) and insulin-like growth factor (IGF) in the developing cerebellum. Shh induces the oncogene Yes-associated protein (YAP), which drives IGF2 expression in CGNPs and mouse Shh-associated medulloblastomas. To determine how IGF2 expression is regulated downstream of YAP, we carried out an unbiased screen for transcriptional regulators bound to IGF2 promoters. We report that Y-box binding protein-1 (YB-1), an onco-protein regulating transcription and translation, binds to IGF2 promoter P3. We observed that YB-1 is upregulated across human medulloblastoma subclasses as well as in other varieties of pediatric brain tumors. Utilizing the cerebellar progenitor model for the Shh subgroup of medulloblastoma in mice, we show for the first time that YB-1 is induced by Shh in CGNPs. Its expression is YAP-dependent and it is required for IGF2 expression in CGNPs. Finally, both gain-of function and loss-of-function experiments reveal that YB-1 activity is required for sustaining CGNP and medulloblastoma cell (MBC) proliferation. Collectively, our findings describe a novel role for YB-1 in driving proliferation in the developing cerebellum and MBCs and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas.

  10. Sonic hedgehog stimulates glycolysis and proliferation of breast cancer cells: Modulation of PFKFB3 activation

    SciTech Connect

    Ge, Xin; Lyu, Pengwei; Gu, Yuanting; Li, Lin; Li, Jingruo; Wang, Yan; Zhang, Linfeng; Fu, Chao; Cao, Zhang

    2015-08-28

    Sonic hesgehog (Shh) signaling has been reported to play an essential role in cancer progression. The mechanism of Shh involved in breast cancer carcinogenesis remains unclear. The present study sought to explore whether Shh signaling could regulate the glycolytic metabolism in breast cancers. Overexpression of the smoothed (Smo) and Gli-1 was found in human primary breast cancers. The expressions of Shh and Gli-1 correlated significantly with tumor size and tumor stage. In vitro, human recombinant Shh (rShh) triggered Smo and Gli-1 expression, promoted glucose utilization and lactate production, and accelerated cell proliferation in MCF-7 and MDA-MB-231 cells. Notably, rShh did not alter 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) expression but augmented PFKFB3 phosphorylation on ser{sup 461}, along with elevated fructose-2,6-bisphosphate (F2,6BP) generation by MCF-7 and MDA-MB-231 cells. This effect could be dampened by Smo siRNA but not by Gli-1 siRNA. In addition, our data showed the upregulated expressions of MAPK by rShh and elevatory PFKFB3 phosphorylation by p38/MAPK activated kinase (MK2). In conclusion, our study characterized a novel role of Shh in promoting glycolysis and proliferation of breast cancer cells via PFKFB3 phosphorylation, which was mediated by Smo and p38/MK2. - Highlights: • Overexpression of Smo and Gli-1 was found in human primary breast cancers. • Shh promoted glucose utilization, lactate production, and cell proliferation. • Shh did not alter PFKFB3 expression but augmented PFKFB3 phosphorylation on ser461. • Shh acts on PFKFB3 phosphorylation via Smo and p38 MAPK/MK2.

  11. Hedgehog signaling pathway regulates ovarian cancer invasion and migration via adhesion molecule CD24

    PubMed Central

    Zeng, Chunyan; Chen, Tingtao; Zhang, Yan; Chen, Qi

    2017-01-01

    Hedgehog (Hh) signalling plays an important role in cancer; however, its mechanism in ovarian cancer migration and invasion remains unclear. In the present study, we aimed to clarify the effect of the Hh signalling pathway on ovarian cancer migration and invasion through the regulation of CD24 expression, both in vitro and in vivo. Patients with ovarian cancer (n = 97) were recruited for this study. Evaluation of the explored the role parameters of patients indicated that CD24 expression was negatively associated with age, histological type and lymph node metastasis (p>0.05), but was positively associated with the clinical stage and pathological grading (p<0.05).The in vitro results indicated that the activator (sonic hedgehog, Shh) and inhibitor (GANT61) of Hh signalling significantly enhanced and reduced CD24 expression, respectively, at both the gene and protein levels (p<0.05).The addition of Shh significantly enhanced cellular migration and invasion of SKOV3 cells in vitro (p<0.05) Down regulation of CD24 using siRNA inhibited the tumour-promoting effects of Shh, and the in vivo results confirmed that GANT61 significantly inhibited CD24 expression and reduced tumour growth (p<0.01). In conclusion, the expression of CD24 can be regulated by Hh signalling, and downregulation of CD24 could play an important role in inhibiting ovarian cancer progression. PMID:28382140

  12. Regulation of Hedgehog Signalling Inside and Outside the Cell

    PubMed Central

    Ramsbottom, Simon A.; Pownall, Mary E.

    2016-01-01

    The hedgehog (Hh) signalling pathway is conserved throughout metazoans and plays an important regulatory role in both embryonic development and adult homeostasis. Many levels of regulation exist that control the release, reception, and interpretation of the hedgehog signal. The fatty nature of the Shh ligand means that it tends to associate tightly with the cell membrane, and yet it is known to act as a morphogen that diffuses to elicit pattern formation. Heparan sulfate proteoglycans (HSPGs) play a major role in the regulation of Hh distribution outside the cell. Inside the cell, the primary cilium provides an important hub for processing the Hh signal in vertebrates. This review will summarise the current understanding of how the Hh pathway is regulated from ligand production, release, and diffusion, through to signal reception and intracellular transduction. PMID:27547735

  13. Concerted actions of ameliorated colitis, aberrant crypt foci inhibition and 15-hydroxyprostaglandin dehydrogenase induction by sonic hedgehog inhibitor led to prevention of colitis-associated cancer.

    PubMed

    Kangwan, Napapan; Kim, Yoon-Jae; Han, Young-Min; Jeong, Migyeong; Park, Jong-Min; Hahm, Ki-Baik

    2016-03-15

    The sonic hedgehog (Shh) signaling has been known to contribute to carcinogenesis in organ, where hedgehog exerted organogenesis and in cancers, which are developed based on mutagenic inflammation. Therefore, colitis-associated cancer (CAC) can be a good model to prove whether Shh inhibitors can be applied to prevent, as the efforts to discover potent anti-inflammatory agent are active to prevent CAC. Here, under the hypothesis that Shh inhibitors can prevent CAC, mouse model was generated to develop CAC by azoxymethane (AOM)-initiated, dextran sodium sulfate-promoted carcinogenesis. Shh inhibitors, cerulenin and itraconazole were treated by oral gavage and the mice were sacrificed at early phase of 3 weeks and late phase of 16 weeks. Compared to control group, the number of aberrant crypt foci at 3 weeks and tumor incidence at 16 weeks were all significantly decreased with Shh inhibitor. Significant attenuations of macrophage infiltration accompanied with significant decreases of IL-6, COX-2, STAT3 and NF-κB as well as significant ameliorations of β-catenin nuclear translocation, cyclin D1 and CDK4 were imposed with Shh inhibitors. Especially, CAC was accompanied with significant cancellation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), but their levels were significantly preserved with Shh inhibitors. Among inflammatory mediators, significantly decreased levels of IL-6 and TNF-α, regulated with repressed NF-κb and STAT3, were prominent with Shh inhibitor, whereas significant inductions of apoptosis were noted with Shh inhibitors. In conclusion, Shh inhibitors significantly prevented CAC covering either ameliorating oncogenic inflammation or suppressing tumor proliferation, especially supported with significant inhibition of IL-6 and STAT3 signaling, 15-PGDH preservation and apoptosis induction.

  14. Characterization of the human oncogene SCL/TAL1 interrupting locus (Stil) mediated Sonic hedgehog (Shh) signaling transduction in proliferating mammalian dopaminergic neurons

    SciTech Connect

    Sun, Lei; Carr, Aprell L.; Li, Ping; Lee, Jessica; McGregor, Mary; Li, Lei

    2014-07-11

    Highlights: • Stil is a human oncogene that is conserved in vertebrate species. • Stil functions in the Shh pathway in mammalian cells. • The expression of Stil is required for mammalian dopaminergic cell proliferation. - Abstract: The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in all vertebrate species. In humans, the expression of Stil is involved in cancer cell survival, apoptosis and proliferation. In this research, we investigated the roles of Stil expression in cell proliferation of mammalian dopaminergic (DA) PC12 cells. Stil functions through the Sonic hedgehog (Shh) signal transduction pathway. Co-immunoprecipitation tests revealed that STIL interacts with Shh downstream components, which include SUFU and GLI1. By examining the expression of Stil, Gli1, CyclinD2 (cell-cycle marker) and PCNA (proliferating cell nuclear antigen), we found that up-regulation of Stil expression (transfection with overexpression plasmids) increased Shh signaling transduction and PC12 cell proliferation, whereas down-regulation of Stil expression (by shRNA) inhibited Shh signaling transduction, and thereby decreased PC12 cell proliferation. Transient transfection of PC12 cells with Stil knockdown or overexpression plasmids did not affect PC12 cell neural differentiation, further indicating the specific roles of Stil in cell proliferation. The results from this research suggest that Stil may serve as a bio-marker for neurological diseases involved in DA neurons, such as Parkinson’s disease.

  15. The teratogenic effects of prenatal ethanol exposure are exacerbated by Sonic Hedgehog or GLI2 haploinsufficiency in the mouse.

    PubMed

    Kietzman, Henry W; Everson, Joshua L; Sulik, Kathleen K; Lipinski, Robert J

    2014-01-01

    Disruption of the Hedgehog signaling pathway has been implicated as an important molecular mechanism in the pathogenesis of fetal alcohol syndrome. In severe cases, the abnormalities of the face and brain that result from prenatal ethanol exposure fall within the spectrum of holoprosencephaly. Single allele mutations in the Hh pathway genes Sonic Hedgehog (SHH) and GLI2 cause holoprosencephaly with extremely variable phenotypic penetrance in humans. Here, we tested whether mutations in these genes alter the frequency or severity of ethanol-induced dysmorphology in a mouse model. Timed pregnancies were established by mating Shh(+/-) or Gli2(+/-) male mice backcrossed to C57BL/6J strain, with wildtype females. On gestational day 7, dams were treated with two i.p. doses of 2.9 g/kg ethanol (or vehicle alone), administered four hrs apart. Fetuses were then genotyped and imaged, and the severity of facial dysmorphology was assessed. Following ethanol exposure, mean dysmorphology scores were increased by 3.2- and 6.6-fold in Shh(+/-) and Gli2(+/-) groups, respectively, relative to their wildtype littermates. Importantly, a cohort of heterozygous fetuses exhibited phenotypes not typically produced in this model but associated with severe holoprosencephaly, including exencephaly, median cleft lip, otocephaly, and proboscis. As expected, a correlation between the severity of facial dysmorphology and medial forebrain deficiency was observed in affected animals. While Shh(+/-) and Gli2(+/-) mice have been described as phenotypically normal, these results illustrate a functional haploinsufficiency of both genes in combination with ethanol exposure. By demonstrating an interaction between specific genetic and environmental risk factors, this study provides important insights into the multifactorial etiology and complex pathogenesis of fetal alcohol syndrome and holoprosencephaly.

  16. Targeting EGFR and sonic hedgehog pathways for locally advanced eyelid and periocular carcinomas

    PubMed Central

    Yin, Vivian T; Merritt, Helen; Esmaeli, Bita

    2014-01-01

    For patients with metastatic or locally advanced eyelid and periocular carcinoma not amenable to surgical excision, targeted therapies have shown efficacy with better tolerability compared to cytotoxic chemotherapy. Overexpression of epithelial growth factor receptor was found in squamous cell carcinomas. Vismodegib targets the mutation in the hedgehog pathway identified in basal cell carcinoma and basal cell nevus syndrome. Targeted therapies provide a novel and potentially effective treatment alternative for patients with eyelid carcinoma not amendable for surgery, including those with metastatic, locally advanced disease, advanced age, and significant comorbidities. High cost, need for long-term treatment, and toxicity are relative limitations. PMID:25232546

  17. Lycium barbarum Polysaccharides Protect against Trimethyltin Chloride-Induced Apoptosis via Sonic Hedgehog and PI3K/Akt Signaling Pathways in Mouse Neuro-2a Cells.

    PubMed

    Zhao, Wanyun; Pan, Xiaoqi; Li, Tao; Zhang, Changchun; Shi, Nian

    2016-01-01

    Trimethyltin chloride (TMT) is a classic neurotoxicant that can cause severe neurodegenerative diseases. Some signaling pathways involving cell death play pivotal roles in the central nervous system. In this study, the role of Sonic Hedgehog (Shh) and PI3K/Akt pathways in TMT-induced apoptosis and protective effect of Lycium barbarum polysaccharides (LBP) on mouse neuro-2a (N2a) cells were investigated. Results showed that TMT treatment significantly enhanced apoptosis, upregulated proapoptotic Bax, downregulated antiapoptotic Bcl-2 expression, and increased caspase-3 activity in a dose-dependent manner in N2a cells. TMT induced oxidative stress in cells, performing reactive oxygen species (ROS) and malondialdehyde (MDA) excessive generation, and superoxide dismutase (SOD) activity reduction. TMT significantly decreased phosphorylated glycogen synthase kinase-3β (GSK-3β) and inhibited Shh and PI3K/Akt pathways. However, the addition of LBP upregulated GSK-3β phosphorylation, activated Shh and PI3K/Akt pathways, and eventually reduced apoptosis and oxidative stress caused by TMT. The interaction between Shh and PI3K/Akt pathways was clarified by specific PI3K inhibitor LY294002 or Shh inhibitor GDC-0449. Moreover, LY294002 and GDC-0449 pretreatment both induced phosphorylated GSK-3β downregulation and significantly promoted apoptosis induced by TMT. These results suggest that LBP could reduce TMT-induced N2a cells apoptosis by regulating GSK-3β phosphorylation, Shh, and PI3K/Akt signaling pathways.

  18. A shared role for sonic hedgehog signalling in patterning chondrichthyan gill arch appendages and tetrapod limbs.

    PubMed

    Gillis, J Andrew; Hall, Brian K

    2016-04-15

    Chondrichthyans (sharks, skates, rays and holocephalans) possess paired appendages that project laterally from their gill arches, known as branchial rays. This led Carl Gegenbaur to propose that paired fins (and hence tetrapod limbs) originally evolved via transformation of gill arches. Tetrapod limbs are patterned by asonic hedgehog(Shh)-expressing signalling centre known as the zone of polarising activity, which establishes the anteroposterior axis of the limb bud and maintains proliferative expansion of limb endoskeletal progenitors. Here, we use loss-of-function, label-retention and fate-mapping approaches in the little skate to demonstrate that Shh secretion from a signalling centre in the developing gill arches establishes gill arch anteroposterior polarity and maintains the proliferative expansion of branchial ray endoskeletal progenitor cells. These findings highlight striking parallels in the axial patterning mechanisms employed by chondrichthyan branchial rays and paired fins/limbs, and provide mechanistic insight into the anatomical foundation of Gegenbaur's gill arch hypothesis.

  19. Sonic hedgehog-induced type 3 deiodinase blocks thyroid hormone action enhancing proliferation of normal and malignant keratinocytes.

    PubMed

    Dentice, Monica; Luongo, Cristina; Huang, Stephen; Ambrosio, Raffaele; Elefante, Antonia; Mirebeau-Prunier, Delphine; Zavacki, Ann Marie; Fenzi, Gianfranco; Grachtchouk, Marina; Hutchin, Mark; Dlugosz, Andrzej A; Bianco, Antonio C; Missero, Caterina; Larsen, P Reed; Salvatore, Domenico

    2007-09-04

    The Sonic hedgehog (Shh) pathway plays a critical role in hair follicle physiology and is constitutively active in basal cell carcinomas (BCCs), the most common human malignancy. Type 3 iodothyronine deiodinase (D3), the thyroid hormone-inactivating enzyme, is frequently expressed in proliferating and neoplastic cells, but its role in this context is unknown. Here we show that Shh, through Gli2, directly induces D3 in proliferating keratinocytes and in mouse and human BCCs. We demonstrate that Gli-induced D3 reduces intracellular active thyroid hormone, thus resulting in increased cyclin D1 and keratinocyte proliferation. D3 knockdown caused a 5-fold reduction in the growth of BCC xenografts in nude mice. Shh-induced thyroid hormone degradation via D3 synergizes with the Shh-mediated reduction of the type 2 deiodinase, the thyroxine-activating enzyme, and both effects are reversed by cAMP. This previously unrecognized functional cross-talk between Shh/Gli2 and thyroid hormone in keratinocytes is a pathway by which Shh produces its proliferative effects and offers a potential therapeutic approach to BCC.

  20. SBE6: a novel long-range enhancer involved in driving sonic hedgehog expression in neural progenitor cells

    PubMed Central

    Benabdallah, Nezha S.; Gautier, Philippe; Hekimoglu-Balkan, Betul; Lettice, Laura A.; Bhatia, Shipra

    2016-01-01

    The expression of genes with key roles in development is under very tight spatial and temporal control, mediated by enhancers. A classic example of this is the sonic hedgehog gene (Shh), which plays a pivotal role in the proliferation, differentiation and survival of neural progenitor cells both in vivo and in vitro. Shh expression in the brain is tightly controlled by several known enhancers that have been identified through genetic, genomic and functional assays. Using chromatin profiling during the differentiation of embryonic stem cells to neural progenitor cells, here we report the identification of a novel long-range enhancer for Shh—Shh-brain-enhancer-6 (SBE6)—that is located 100 kb upstream of Shh and that is required for the proper induction of Shh expression during this differentiation programme. This element is capable of driving expression in the vertebrate brain. Our study illustrates how a chromatin-focused approach, coupled to in vivo testing, can be used to identify new cell-type specific cis-regulatory elements, and points to yet further complexity in the control of Shh expression during embryonic brain development. PMID:27852806

  1. Inactivation of Sonic Hedgehog Signaling and Polydactyly in Limbs of Hereditary Multiple Malformation, a Novel Type of Talpid Mutant

    PubMed Central

    Matsubara, Yoshiyuki; Nakano, Mikiharu; Kawamura, Kazuki; Tsudzuki, Masaoki; Funahashi, Jun-Ichi; Agata, Kiyokazu; Matsuda, Yoichi; Kuroiwa, Atsushi; Suzuki, Takayuki

    2016-01-01

    Hereditary Multiple Malformation (HMM) is a naturally occurring, autosomal recessive, homozygous lethal mutation found in Japanese quail. Homozygote embryos (hmm−/−) show polydactyly similar to talpid2 and talpid3 mutants. Here we characterize the molecular profile of the hmm−/− limb bud and identify the cellular mechanisms that cause its polydactyly. The hmm−/− limb bud shows a severe lack of sonic hedgehog (SHH) signaling, and the autopod has 4 to 11 unidentifiable digits with syn-, poly-, and brachydactyly. The Zone of Polarizing Activity (ZPA) of the hmm−/− limb bud does not show polarizing activity regardless of the presence of SHH protein, indicating that either the secretion pathway of SHH is defective or the SHH protein is dysfunctional. Furthermore, mesenchymal cells in the hmm−/− limb bud do not respond to ZPA transplanted from the normal limb bud, suggesting that signal transduction downstream of SHH is also defective. Since primary cilia are present in the hmm−/− limb bud, the causal gene must be different from talpid2 and talpid3. In the hmm−/− limb bud, a high amount of GLI3A protein is expressed and GLI3 protein is localized to the nucleus. Our results suggest that the regulatory mechanism of GLI3 is disorganized in the hmm−/− limb bud. PMID:28083533

  2. SBE6: a novel long-range enhancer involved in driving sonic hedgehog expression in neural progenitor cells.

    PubMed

    Benabdallah, Nezha S; Gautier, Philippe; Hekimoglu-Balkan, Betul; Lettice, Laura A; Bhatia, Shipra; Bickmore, Wendy A

    2016-11-01

    The expression of genes with key roles in development is under very tight spatial and temporal control, mediated by enhancers. A classic example of this is the sonic hedgehog gene (Shh), which plays a pivotal role in the proliferation, differentiation and survival of neural progenitor cells both in vivo and in vitro. Shh expression in the brain is tightly controlled by several known enhancers that have been identified through genetic, genomic and functional assays. Using chromatin profiling during the differentiation of embryonic stem cells to neural progenitor cells, here we report the identification of a novel long-range enhancer for Shh-Shh-brain-enhancer-6 (SBE6)-that is located 100 kb upstream of Shh and that is required for the proper induction of Shh expression during this differentiation programme. This element is capable of driving expression in the vertebrate brain. Our study illustrates how a chromatin-focused approach, coupled to in vivo testing, can be used to identify new cell-type specific cis-regulatory elements, and points to yet further complexity in the control of Shh expression during embryonic brain development.

  3. Sonic hedgehog promotes neurite outgrowth of cortical neurons under oxidative stress: Involving of mitochondria and energy metabolism.

    PubMed

    He, Weiliang; Cui, Lili; Zhang, Cong; Zhang, Xiangjian; He, Junna; Xie, Yanzhao; Chen, Yanxia

    2017-01-01

    Oxidative stress has been demonstrated to be involved in the etiology of several neurobiological disorders. Sonic hedgehog (Shh), a secreted glycoprotein factor, has been implicated in promoting several aspects of brain remodeling process. Mitochondria may play an important role in controlling fundamental processes in neuroplasticity. However, little evidence is available about the effect and the potential mechanism of Shh on neurite outgrowth in primary cortical neurons under oxidative stress. Here, we revealed that Shh treatment significantly increased the viability of cortical neurons in a dose-dependent manner, which was damaged by hydrogen peroxide (H2O2). Shh alleviated the apoptosis rate of H2O2-induced neurons. Shh also increased neuritogenesis injuried by H2O2 in primary cortical neurons. Moreover, Shh reduced the generation of reactive oxygen species (ROS), increased the activities of SOD and and decreased the productions of MDA. In addition, Shh protected mitochondrial functions, elevated the cellular ATP levels and amelioratesd the impairment of mitochondrial complex II activities of cortical neurons induced by H2O2. In conclusion, all these results suggest that Shh acts as a prosurvival factor playing an essential role to neurite outgrowth of cortical neuron under H2O2 -induced oxidative stress, possibly through counteracting ROS release and preventing mitochondrial dysfunction and ATP as well as mitochondrial complex II activities against oxidative stress.

  4. CD44, Sonic Hedgehog, and Gli1 Expression Are Prognostic Biomarkers in Gastric Cancer Patients after Radical Resection.

    PubMed

    Jian-Hui, Chen; Er-Tao, Zhai; Si-Le, Chen; Hui, Wu; Kai-Ming, Wu; Xin-Hua, Zhang; Chuang-Qi, Chen; Shi-Rong, Cai; Yu-Long, He

    2016-01-01

    Aim. CD44 and Sonic Hedgehog (Shh) signaling are important for gastric cancer (GC). However, the clinical impact, survival, and recurrence outcome of CD44, Shh, and Gli1 expressions in GC patients following radical resection have not been elucidated. Patients and Methods. CD44, Shh, and Gli1 protein levels were quantified by immunohistochemistry (IHC). The association between CD44, Shh, and Gli1 expression and clinicopathological features or prognosis of GC patients was determined. The biomarker risk score was calculated by the IHC staining score of CD44, Shh, and Gli1 protein. Results. The IHC positive staining of CD44, Shh, and Gli1 proteins was correlated with larger tumour size, worse gross type and histological type, and advanced TNM stage, which also predicted shorter overall survival (OS) and disease-free survival (DFS) after radical resection. Multivariate analysis indicated the Gli1 protein and Gli1, CD44 proteins were predictive biomarkers for OS and DFS, respectively. If biomarker risk score was taken into analysis, it was the independent prognostic factor for OS and DFS. Conclusions. CD44 and Shh signaling are important biomarkers for tumour aggressiveness, survival, and recurrence in GC.

  5. Injury-stimulated Sonic hedgehog expression in microglia contributes to neuroinflammatory response in the MPTP model of Parkinson's disease.

    PubMed

    Lee, Jeong Hwi; Chung, Young Cheul; Bok, Eugene; Lee, Hankyu; Huh, Sue Hee; Lee, Ji Eun; Jin, Byung Kwan; Ko, Hyuk Wan

    2017-01-22

    Parkinson's disease (PD) is a progressive neurodegenerative disorder in which dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) region are selectively destroyed. Sonic hedgehog (Shh) has been well known to play a key role in a variety of processes such as embryogenesis, cell proliferation and protection, and tissue repair during inflammation. However, the evidences for the innate role of Shh in adult brain injury are presently lacking and studies have been needed to unveil the importance of Shh in the process of neurodegeneration. Here, we investigated the role of Shh in the pathologic progress of Parkinson's disease in MPTP-induced animal model system. Interestingly, we observed that Shh expression was gradually increased in MPTP affected SNpc region. Activated microglia exclusively expressed SHH in vivo and we could recapitulate Shh induction in activated cultured primary microglia cells. Using the SHH responsive Cre-loxP binary genetic reporter transgenic mouse system, we also found that most of the cell types except for oligodendrocyte in the SNpc region reacted to the SHH by MPTP injection. Taken together, activated microglia induced Shh expression and most neural cells except oligodendrocyte responded to microglia-derived SHH in MPTP-treated SN. These results suggest that SHH in activated microglia by MPTP-injection might be involved in the innate processes of recovery from neurotoxin induced injury in the PD animal model system.

  6. Inactivation of Sonic Hedgehog Signaling and Polydactyly in Limbs of Hereditary Multiple Malformation, a Novel Type of Talpid Mutant.

    PubMed

    Matsubara, Yoshiyuki; Nakano, Mikiharu; Kawamura, Kazuki; Tsudzuki, Masaoki; Funahashi, Jun-Ichi; Agata, Kiyokazu; Matsuda, Yoichi; Kuroiwa, Atsushi; Suzuki, Takayuki

    2016-01-01

    Hereditary Multiple Malformation (HMM) is a naturally occurring, autosomal recessive, homozygous lethal mutation found in Japanese quail. Homozygote embryos (hmm(-/-)) show polydactyly similar to talpid(2) and talpid(3) mutants. Here we characterize the molecular profile of the hmm(-/-) limb bud and identify the cellular mechanisms that cause its polydactyly. The hmm(-/-) limb bud shows a severe lack of sonic hedgehog (SHH) signaling, and the autopod has 4 to 11 unidentifiable digits with syn-, poly-, and brachydactyly. The Zone of Polarizing Activity (ZPA) of the hmm(-/-) limb bud does not show polarizing activity regardless of the presence of SHH protein, indicating that either the secretion pathway of SHH is defective or the SHH protein is dysfunctional. Furthermore, mesenchymal cells in the hmm(-/-) limb bud do not respond to ZPA transplanted from the normal limb bud, suggesting that signal transduction downstream of SHH is also defective. Since primary cilia are present in the hmm(-/-) limb bud, the causal gene must be different from talpid(2) and talpid(3). In the hmm(-/-) limb bud, a high amount of GLI3A protein is expressed and GLI3 protein is localized to the nucleus. Our results suggest that the regulatory mechanism of GLI3 is disorganized in the hmm(-/-) limb bud.

  7. Polydatin ameliorates renal ischemia/reperfusion injury by decreasing apoptosis and oxidative stress through activating sonic hedgehog signaling pathway.

    PubMed

    Meng, Qiu-Hong; Liu, Hong-Bao; Wang, Jian-Bo

    2016-10-01

    Polydatin, a glucoside of resveratrol, recently has been demonstrated possibly to exert its biological effects by targeting sonic hedgehog (Shh). However, whether Shh signaling pathway is involved in the therapeutic effects of polydatin for renal ischemia/reperfusion (I/R) injury has not been evaluated. Our results showed that I/R induced the secretion of Shh, upregulated Patched and Smoothened, and enhanced the nuclear translocation and target gene transcription of Glioblastoma 1 in renal I/R injury models, which were further upregulated after the administration of polydatin significantly and in turn exerted prominent nephroprotective effects against cell apoptosis and oxidative stress. The treatment with cyclopamine (a specific inhibitor of Smoothened) or 5E1 (an anti-Shh antibody) not only markedly inhibited the activation of the Shh pathway, but also dramatically suppressed the nephroprotective effects of polydatin above-mentioned. These results advance our knowledge that polydatin can provide protection for kidneys against I/R injury by enhancing antioxidant capacity and decreasing cell apoptosis through activating Shh signaling pathway.

  8. Resveratrol Pretreatment Decreases Ischemic Injury and Improves Neurological Function Via Sonic Hedgehog Signaling After Stroke in Rats.

    PubMed

    Yu, Pingping; Wang, Li; Tang, Fanren; Zeng, Li; Zhou, Luling; Song, Xiaosong; Jia, Wei; Chen, Jixiang; Yang, Qin

    2017-01-01

    Resveratrol has neuroprotective effects for ischemic cerebral stroke. However, its neuroprotective mechanism for stroke is less well understood. Beneficial actions of the activated Sonic hedgehog (Shh) signaling pathway in stroke, such as improving neurological function, promoting neurogenesis, anti-oxidative, anti-apoptotic, and pro-angiogenic effects, have been noted, but relatively little is known about the role of Shh signaling in resveratrol-reduced cerebral ischemic injury after stroke. The present study tests whether the Shh pathway mediates resveratrol to decrease cerebral ischemic injury and improve neurological function after stroke. We observed that resveratrol pretreatment significantly improved neurological function, decreased infarct volume, enhanced vitality, and reduced apoptosis of neurons in vivo and vitro after stroke. Meanwhile, expression levels of Shh, Ptc-1, Smo, and Gli-1 mRNAs were significantly upregulated and Gli-1 was relocated to the nucleus. Intriguingly, in vivo and in vitro inhibition of the Shh signaling pathway with cyclopamine, a Smo inhibitor, completely reversed the above effects of resveratrol. These results suggest that decreased cerebral ischemic injury and improved neurological function by resveratrol may be mediated by the Shh signaling pathway.

  9. Effect of methacrylic acid beads on the sonic hedgehog signaling pathway and macrophage polarization in a subcutaneous injection mouse model.

    PubMed

    Lisovsky, Alexandra; Zhang, David K Y; Sefton, Michael V

    2016-08-01

    Poly(methacrylic acid-co-methyl methacrylate) (MAA) beads promote a vascular regenerative response when used in diabetic wound healing. Previous studies reported that MAA beads modulated the expression of sonic hedgehog (Shh) and inflammation related genes in diabetic wounds. The aim of this work was to follow up on these observations in a subcutaneous injection model to study the host response in the absence of the confounding factors of diabetic wound healing. In this model, MAA beads improved vascularization in healthy mice of both sexes compared to control poly(methyl methacrylate) (MM) beads, with a stronger effect seen in males than females. MAA-induced vessels were perfusable, as evidenced from the CLARITY-processed images. In Shh-Cre-eGFP/Ptch1-LacZ non-diabetic transgenic mice, the increased vessel formation was accompanied by a higher density of cells expressing GFP (Shh) and β-Gal (patched 1, Ptch1) suggesting MAA enhanced the activation of the Shh pathway. Ptch1 is the Shh receptor and a target of the pathway. MAA beads also modulated the inflammatory cell infiltrate in CD1 mice: more neutrophils and more macrophages were noted with MAA relative to MM beads at days 1 and 7, respectively. In addition, MAA beads biased macrophages towards a MHCII-CD206+ ("M2") polarization state. This study suggests that the Shh pathway and an altered inflammatory response are two elements of the complex mechanism whereby MAA-based biomaterials effect vascular regeneration.

  10. Activation of sonic hedgehog signaling attenuates oxidized low-density lipoprotein-stimulated brain microvascular endothelial cells dysfunction in vitro.

    PubMed

    Jiang, Xiu-Long; Chen, Ting; Zhang, Xu

    2015-01-01

    The study was performed to investigate the role of sonic hedgehog (SHH) in the oxidized low-density lipoprotein (oxLDL)-induced blood-brain barrier (BBB) disruption. The primary mouse brain microvascular endothelial cells (MBMECs) were exposed to oxLDL. The results indicated that treatment of MBMECs with oxLDL decreased the cell viability, and oxidative stress was involved in oxLDL-induce MBMECs dysfunction with increasing intracellular ROS and MDA formation as well as decreasing NO release and eNOS mRNA expression. In addition, SHH signaling components, such as SHH, Smo and Gli1, mRNA and protein levels were significantly decreased after incubation with increasing concentrations of oxLDL. Treatment with oxLDL alone or SHH loss-of-function significantly increased the permeability of MBMECs, and overexpression of SHH attenuated oxLDL-induced elevation of permeability in MBMECs. Furthermore, SHH gain-of-function could reverse oxLDL-induced apoptosis through inhibition caspase3 and caspase8 levels in MBMECs. Taken together, these results demonstrated that the suppression of SHH in MBMECs might contribute to the oxLDL-induced disruption of endothelial barrier. However, the overexpression of SHH could reverse oxLDL-induced endothelial cells dysfunction in vitro.

  11. Sonic Hedgehog Controls the Phenotypic Fate and Therapeutic Efficacy of Grafted Neural Precursor Cells in a Model of Nigrostriatal Neurodegeneration.

    PubMed

    Madhavan, Lalitha; Daley, Brian F; Davidson, Beverly L; Boudreau, Ryan L; Lipton, Jack W; Cole-Strauss, Allyson; Steece-Collier, Kathy; Collier, Timothy J

    2015-01-01

    The expression of soluble growth and survival promoting factors by neural precursor cells (NPCs) is suggested to be a prominent mechanism underlying the protective and regenerative effects of these cells after transplantation. Nevertheless, how and to what extent specific NPC-expressed factors contribute to therapeutic effects is not well understood. Using RNA silencing, the current study investigated the roles of two donor NPC molecules, namely glial cell-line derived neurotrophic factor (GDNF) and sonic hedgehog (SHH), in the protection of substantia nigra dopamine neurons in rats treated with 6-hydroxydopamine (6-OHDA). Analyses indicate that as opposed to the knock-down of GDNF, SHH inhibition caused a profound decline in nigrostriatal neuroprotection. Further, SHH silencing also curbed endogenous neurogenesis and the migration of host brdU+/dcx+ neural precursors into the striatum, which was present in the animals receiving control or GDNF silenced NPCs. A change in graft phenotype, mainly reflected by a reduced proportion of undifferentiated nestin+ cells, as well as a significantly greater host microglial activity, suggested an important role for these processes in the attenuation of neuroprotection and neurogenesis upon SHH silencing. Overall these studies reveal core mechanisms fundamental to grafted NPC-based therapeutic effects, and delineate the particular contributions of two graft-expressed molecules, SHH and GDNF, in mediating midbrain dopamine neuron protection, and host plasticity after NPC transplantation.

  12. Microduplications encompassing the Sonic hedgehog limb enhancer ZRS are associated with Haas-type polysyndactyly and Laurin-Sandrow syndrome.

    PubMed

    Lohan, S; Spielmann, M; Doelken, S C; Flöttmann, R; Muhammad, F; Baig, S M; Wajid, M; Hülsemann, W; Habenicht, R; Kjaer, K W; Patil, S J; Girisha, K M; Abarca-Barriga, H H; Mundlos, S; Klopocki, E

    2014-10-01

    Laurin-Sandrow syndrome (LSS) is a rare autosomal dominant disorder characterized by polysyndactyly of hands and/or feet, mirror image duplication of the feet, nasal defects, and loss of identity between fibula and tibia. The genetic basis of LSS is currently unknown. LSS shows phenotypic overlap with Haas-type polysyndactyly (HTS) regarding the digital phenotype. Here we report on five unrelated families with overlapping microduplications encompassing the Sonic hedgehog (SHH) limb enhancer ZPA regulatory sequence (ZRS) on chromosome 7q36. Clinically, the patients show polysyndactyly phenotypes and various types of lower limb malformations ranging from syndactyly to mirror image polydactyly with duplications of the fibulae. We show that larger duplications of the ZRS region (>80 kb) are associated with HTS, whereas smaller duplications (<80 kb) result in the LSS phenotype. On the basis of our data, the latter can be clearly distinguished from HTS by the presence of mirror image polysyndactyly of the feet with duplication of the fibula. Our results expand the clinical phenotype of the ZRS-associated syndromes and suggest that smaller duplications (<80 kb) are associated with a more severe phenotype. In addition, we show that these small microduplications within the ZRS region are the underlying genetic cause of Laurin-Sandrow syndrome.

  13. Disruption of sonic hedgehog signaling in Ellis-van Creveld dwarfism confers protection against bipolar affective disorder.

    PubMed

    Ginns, E I; Galdzicka, M; Elston, R C; Song, Y E; Paul, S M; Egeland, J A

    2015-10-01

    Ellis-van Creveld syndrome, an autosomal recessively inherited chondrodysplastic dwarfism, is frequent among Old Order Amish of Pennsylvania. Decades of longitudinal research on bipolar affective disorder (BPAD) revealed cosegregation of high numbers of EvC and Bipolar I (BPI) cases in several large Amish families descending from the same pioneer. Despite the high prevalence of both disorders in these families, no EvC individual has ever been reported with BPI. The proximity of the EVC gene to our previously reported chromosome 4p16 BPAD locus with protective alleles, coupled with detailed clinical observations that EvC and BPI do not occur in the same individuals, led us to hypothesize that the genetic defect causing EvC in the Amish confers protection from BPI. This hypothesis is supported by a significant negative association of these two disorders when contrasted with absence of disease (P=0.029, Fisher's exact test, two-sided, verified by permutation to estimate the null distribution of the test statistic). As homozygous Amish EVC mutations causing EvC dwarfism do so by disrupting sonic hedgehog (Shh) signaling, our data implicate Shh signaling in the underlying pathophysiology of BPAD. Understanding how disrupted Shh signaling protects against BPI could uncover variants in the Shh pathway that cause or increase risk for this and related mood disorders.

  14. Recombinant EDA or Sonic Hedgehog rescue the branching defect in Ectodysplasin A pathway mutant salivary glands in vitro.

    PubMed

    Wells, K L; Mou, C; Headon, D J; Tucker, A S

    2010-10-01

    Hypohidrotic ectodermal dysplasia (HED) is characterized by defective ectodermal organ development. This includes the salivary glands (SGs), which have an important role in lubricating the oral cavity. In humans and mice, HED is caused by mutations in Ectodysplasin A (Eda) pathway genes. Various phenotypes of the mutant mouse Eda(Ta/Ta), which lacks the ligand Eda, can be rescued by maternal injection or in vitro culture supplementation with recombinant EDA. However, the response of the SGs to this treatment has not been investigated. Here, we show that the submandibular glands (SMGs) of Eda(Ta/Ta) mice exhibit impaired branching morphogenesis, and that supplementation of Eda(Ta/Ta) SMG explants with recombinant EDA rescues the defect. Supplementation of Edar(dlJ/dlJ) SMGs with recombinant Sonic hedgehog (Shh) also rescues the defect, whereas treatment with recombinant Fgf8 does not. This work is the first to test the ability of putative Eda target molecules to rescue Eda pathway mutant SMGs.

  15. Loss and Re-emergence of Legs in Snakes by Modular Evolution of Sonic hedgehog and HOXD Enhancers.

    PubMed

    Leal, Francisca; Cohn, Martin J

    2016-11-07

    Limb reduction and loss are hallmarks of snake evolution. Although advanced snakes are completely limbless, basal and intermediate snakes retain pelvic girdles and small rudiments of the femur. Moreover, legs may have re-emerged in extinct snake lineages [1-5], suggesting that the mechanisms of limb development were not completely lost in snakes. Here we report that hindlimb development arrests in python embryos as a result of mutations that abolish essential transcription factor binding sites in the limb-specific enhancer of Sonic hedgehog (SHH). Consequently, SHH transcription is weak and transient in python hindlimb buds, leading to early termination of a genetic circuit that drives limb outgrowth. Our results suggest that degenerate evolution of the SHH limb enhancer played a role in reduction of hindlimbs during snake evolution. By contrast, HOXD digit enhancers are conserved in pythons, and HOXD gene expression in the hindlimb buds progresses to the distal phase, forming an autopodial (digit) domain. Python hindlimb buds then develop transitory pre-chondrogenic condensations of the tibia, fibula, and footplate, raising the possibility that re-emergence of hindlimbs during snake evolution did not require de novo re-evolution of lost structures but instead could have resulted from persistence of embryonic legs. VIDEO ABSTRACT.

  16. Embryonic tongue morphogenesis in an organ culture model of mouse mandibular arches: blocking Sonic hedgehog signaling leads to microglossia.

    PubMed

    Torii, Daisuke; Soeno, Yuuichi; Fujita, Kazuya; Sato, Kaori; Aoba, Takaaki; Taya, Yuji

    2016-01-01

    Mouse tongue development is initiated with the formation of lateral lingual swellings just before fusion between the mediodorsal surfaces of the mandibular arches at around embryonic day 11.0. Here, we investigated the role of Sonic hedgehog (Shh) signaling in embryonic mouse tongue morphogenesis. For this, we used an organ culture model of the mandibular arches from mouse embryos at embryonic day 10.5. When the Shh signaling inhibitor jervine was added to the culture medium for 24-96 h, the formation of lateral lingual swellings and subsequent epithelial invagination into the mesenchyme were impaired markedly, leading to a hypoplastic tongue with an incomplete oral sulcus. Notably, jervine treatment reduced the proliferation of non-myogenic mesenchymal cells at the onset of forming the lateral lingual swellings, whereas it did not affect the proliferation and differentiation of a myogenic cell lineage, which created a cell community at the central circumferential region of the lateral lingual swellings as seen in vivo and in control cultures lacking the inhibitor. Thus, epithelium-derived Shh signaling stimulates the proliferation of non-myogenic mesenchymal cells essential for forming lateral lingual swellings and contributes to epithelial invagination into the mesenchyme during early tongue development.

  17. Manipulations of PKA in chick limb development reveal roles in digit patterning including a positive role in Sonic Hedgehog signaling.

    PubMed

    Tiecke, Eva; Turner, Roisin; Sanz-Ezquerro, Juan Jose; Warner, Anne; Tickle, Cheryll

    2007-05-01

    Sonic Hedgehog (Shh) signaling by the polarizing region, at the posterior of the vertebrate limb bud, is pivotal in determining digit number and identity. Shh establishes a gradient of the bifunctional transcriptional effector, Gli3, with high levels of full-length activator (Gli3A) in the posterior bud, where digits form, and high levels of shorter repressor (Gli3R) in the anterior. Repressor formation depends on protein kinase A (PKA), but in Drosophila, PKA also plays a role in activator function. Increasing PKA levels in chick limb development using Forskolin had no effect on posterior polarizing activity but weak polarizing activity, based on ligand-independent Shh signaling, was induced in anterior limb bud cells resulting in extra digits. Manipulating PKA activity levels directly with a retrovirus expressing activated PKA induced extra digits similar to those induced by Forskolin treatment suggesting that PKA may have a previously unrecognized positive role in Shh signaling in vertebrate limbs. Expressing dominant negative PKA also induced extra, sometimes multiple digits, from anterior limb bud demonstrating the negative role in Shh signaling. PKA levels in the limb bud are high posteriorly and low anteriorly, suggesting that PKA activity may influence the outcome of Shh signaling in normal development.

  18. Hedgehog signaling regulates gene expression in planarian glia

    PubMed Central

    Wang, Irving E; Lapan, Sylvain W; Scimone, M Lucila; Clandinin, Thomas R; Reddien, Peter W

    2016-01-01

    Hedgehog signaling is critical for vertebrate central nervous system (CNS) development, but its role in CNS biology in other organisms is poorly characterized. In the planarian Schmidtea mediterranea, hedgehog (hh) is expressed in medial cephalic ganglia neurons, suggesting a possible role in CNS maintenance or regeneration. We performed RNA sequencing of planarian brain tissue following RNAi of hh and patched (ptc), which encodes the Hh receptor. Two misregulated genes, intermediate filament-1 (if-1) and calamari (cali), were expressed in a previously unidentified non-neural CNS cell type. These cells expressed orthologs of astrocyte-associated genes involved in neurotransmitter uptake and metabolism, and extended processes enveloping regions of high synapse concentration. We propose that these cells are planarian glia. Planarian glia were distributed broadly, but only expressed if-1 and cali in the neuropil near hh+ neurons. Planarian glia and their regulation by Hedgehog signaling present a novel tractable system for dissection of glia biology. DOI: http://dx.doi.org/10.7554/eLife.16996.001 PMID:27612382

  19. Impact of Sonic Hedgehog Pathway Expression on Outcome in HPV Negative Head and Neck Carcinoma Patients after Surgery and Adjuvant Radiotherapy

    PubMed Central

    Enzenhofer, Elisabeth; Parzefall, Thomas; Haymerle, Georg; Schneider, Sven; Kadletz, Lorenz; Heiduschka, Gregor; Pammer, Johannes; Oberndorfer, Felicitas; Wrba, Fritz; Loader, Benjamin; Grasl, Matthäus Christoph; Perisanidis, Christos; Erovic, Boban M.

    2016-01-01

    Introduction HPV positive patients suffering from head and neck cancer benefit from intensified radiotherapy when applied as a primary as well as an adjuvant treatment strategy. However, HPV negative patients treated with surgery and adjuvant radiotherapy lack validated prognostic biomarkers. It is therefore important to define prognostic biomarkers in this particular patient population. Especially, ´high-risk groups´ need to be defined in order to adapt treatment protocols. Since dysregulation of the sonic hedgehog pathway plays an important role in carcinogenesis, we aimed to assess whether members of the sonic hedgehog-signaling pathway may act as prognostic factors in patients with HPV negative head and neck squamous cell carcinoma. Materials and Methods In this prospective study, pretreatment tumor biopsies of patients with head and neck squamous cell carcinoma were taken during panendoscopy (2005 to 2008). All patients were treated with surgery and postoperative radiotherapy. After assessment of HPV and p16 status, protein expression profiles of the Sonic hedgehog-signaling pathway were determined by immunohistochemistry and tissue microarray analyses in 36 HPV negative tumor biopsies. Expression profiles of Sonic hedgehog, Indian hedgehog, Patched, Smoothened, Gli-1, Gli-2 and Gli-3 were correlated with patients´ clinical data, local-control rate, disease-free as well as overall survival. Data from The Cancer Genome Atlas databank were used for external validation of our results. Results Gli-1 (p = 0.04) and Gli-2 (p = 0.02) overexpression was significantly linked to improved overall survival of HPV negative patients. Gli-2 (p = 0.04) overexpression correlated significantly with prolonged disease-free survival. Cox-multivariate analysis showed that overexpression of Gli-2 correlated independently (HR 0.40, 95% CI 0.16–0.95, p = 0.03) with increased overall survival. Discussion Gli-1 and Gli-2 overexpression represents a substantial prognostic factor for

  20. Modulation of Sonic hedgehog signaling and WW domain containing oxidoreductase WOX1 expression enhances radiosensitivity of human glioblastoma cells.

    PubMed

    Chiang, Ming-Fu; Chen, Hsin-Hong; Chi, Chih-Wen; Sze, Chun-I; Hsu, Ming-Ling; Shieh, Hui-Ru; Lin, Chin-Ping; Tsai, Jo-Ting; Chen, Yu-Jen

    2015-03-01

    WW domain containing oxidoreductase, designated WWOX, FOR or WOX1, is a known pro-apoptotic factor when ectopically expressed in various types of cancer cells, including glioblastoma multiforme (GBM). The activation of sonic hedgehog (Shh) signaling, especially paracrine Shh secretion in response to radiation, is associated with impairing the effective irradiation of cancer cells. Here, we examined the role of Shh signaling and WOX1 overexpression in the radiosensitivity of human GBM cells. Our results showed that ionizing irradiation (IR) increased the cytoplasmic Shh and nuclear Gli-1 content in GBM U373MG and U87MG cells. GBM cells with exogenous Shh treatment exhibited similar results. Pretreatment with Shh peptides protected U373MG and U87MG cells against IR in a dose-dependent manner. Cyclopamine, a Hedgehog/Smoothened (SMO) inhibitor, reversed the protective effect of Shh in U87MG cells. Cyclopamine increased Shh plus IR-induced H2AX, a marker of DNA double-strand breaks, in these cells. To verify the role of Shh signaling in the radiosensitivity of GBM cells, we tested the effect of the Gli family zinc finger 1 (Gli-1) inhibitor zerumbone and found that it could sensitize GBM cells to IR. We next examined the role of WOX1 in radiosensitivity. Overexpression of WOX1 enhanced the radiosensitivity of U87MG (possessing wild type p53 or WTp53) but not U373MG (harboring mutant p53 or MTp53) cells. Pretreatment with Shh peptides protected both WOX1-overexpressed U373MG and U87MG cells against IR and increased the cytoplasmic Shh and nuclear Gli-1 content. Zerumbone enhanced the radiosensitivity of WOX1-overexpressed U373MG and U87MG cells. In conclusion, overexpression of WOX1 preferentially sensitized human GBM cells possessing wild type p53 to radiation therapy. Blocking of Shh signaling may enhance radiosensitivity independently of the expression of p53 and WOX1. The crosstalk between Shh signaling and WOX1 expression in human glioblastoma warrants further

  1. Sonic hedgehog (Shh)/Gli modulates the spatial organization of neuroepithelial cell proliferation in the developing chick optic tectum

    PubMed Central

    2012-01-01

    Background Sonic hedgehog (Shh)/Gli pathway plays an important regulatory role on the neuroepithelial cells (NEc) proliferation in the dorsal regions of the developing vertebrate Central Nervous System. The aim of this paper was to analyze the effect of the Shh/Gli signaling pathway activation on the proliferation dynamics and/or the spatial organization of the NEc proliferation activity during early stages of the developing chick optic tectum (OT). In ovo pharmacological gain and loss of hedgehog function approaches were complemented with in vivo electroporation experiments in order to create ectopic sources of either Shh or Gli activator (GliA) proteins in the OT. NEc proliferating activity was analyzed at ED 4/4.5 by recording the spatial co-ordinates of the entire population of mitotic NEc (mNEc) located along OT dorsal-ventral sections. Several space signals (numerical sequences) were derived from the mNEc spatial co-ordinate records and analyzed by different standardized non-linear methods of signal analysis. Results In ovo pharmacologic treatment with cyclopamine resulted in dramatic failure in the OT expansion while the agonist purmorphamine produced the opposite result, a huge expansion of the OT vesicle. Besides, GliA and Shh misexpressions interfere with the formation of the intertectal fissure located along the dorsal midline. This morphogenetic alteration is accompanied by an increase in the mNEc density. There is a gradient in the response of NEcs to Shh and GliA: the increase in mNEc density is maximal near the dorsal regions and decrease towards the OT-tegmental boundary. Biomathematical analyses of the signals derived from the mNEc records show that both Shh and GliA electroporations change the proliferation dynamics and the spatial organization of the mNEc as revealed by the changes in the scaling index estimated by these methods. Conclusions The present results show that the Shh/Gli signaling pathway plays a critical role in the OT expansion and

  2. Is Sonic Hedgehog Involved in Human Fracture Healing? - A Prospective Study on Local and Systemic Concentrations of SHH

    PubMed Central

    Eipeldauer, Stefan; Thomas, Anita; Hoechtl-Lee, Leonard; Kecht, Mathias; Binder, Harald; Koettstorfer, Julia; Gregori, Markus; Sarahrudi, Kambiz

    2014-01-01

    Introduction Sonic Hedgehog (SHH) is a new signalling pathway in bone repair. Evidence exist that SHH pathway plays a significant role in vasculogenesis and limb development during embryogenesis. Some in vitro and animal studies has already proven its potential for bone regeneration. However, no data on the role of SHH in the human fracture healing have been published so far. Methods Seventy-five patients with long bone fractures were included into the study and divided in 2 groups. First group contained 69 patients with normal fracture healing. Four patients with impaired fracture healing formed the second group. 34 volunteers donated blood samples as control. Serum samples were collected over a period of 1 year following a standardized time schedule. In addition, SHH levels were measured in fracture haematoma and serum of 16 patients with bone fractures. Results Fracture haematoma and patients serum both contained lower SHH concentrations compared to control serum. The comparison between the patients' serum SHH level and the control serum revealed lower levels for the patients at all measurement time points. Significantly lower concentrations were observed at weeks 1 and 2 after fracture. SHH levels were slightly decreased in patients with impaired fracture healing without statistical significance. Conclusion This is the first study to report local and systemic concentration of SHH in human fracture healing and SHH serum levels in healthy adults. A significant reduction of the SHH levels during the inflammatory phase of fracture healing was found. SHH concentrations in fracture haematoma and serum were lower than the concentration in control serum for the rest of the healing period. Our findings indicate that there is no relevant involvement of SHH in human fracture healing. Fracture repair process seem to reduce the SHH level in human. Further studies are definitely needed to clarify the underlying mechanisms. PMID:25501422

  3. Prenatal cadmium exposure dysregulates sonic hedgehog and Wnt/beta-catenin signaling in the thymus resulting in altered thymocyte development

    SciTech Connect

    Hanson, Miranda L.; Brundage, Kathleen M.; Schafer, Rosana; Tou, Janet C.; Barnett, John B.

    2010-01-15

    Cadmium (Cd) is both an environmental pollutant and a component of cigarette smoke. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports in the literature of immunomodulatory effects of prenatal exposure to Cd. The sonic hedgehog (Shh) and Wnt/beta-catenin pathways are required for thymocyte maturation. Several studies have demonstrated that Cd exposure affects these pathways in different organ systems. This study was designed to investigate the effect of prenatal Cd exposure on thymocyte development, and to determine if these effects were linked to dysregulation of Shh and Wnt/beta-catenin pathways. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose (10 ppm) of Cd throughout pregnancy and effects on the thymus were assessed on the day of birth. Thymocyte phenotype was determined by flow cytometry. A Gli:luciferase reporter cell line was used to measure Shh signaling. Transcription of target genes and translation of key components of both signaling pathways were assessed using real-time RT-PCR and western blot, respectively. Prenatal Cd exposure increased the number of CD4{sup +} cells and a subpopulation of double-negative cells (DN; CD4{sup -}CD8{sup -}), DN4 (CD44{sup -}CD25{sup -}). Shh and Wnt/beta-catenin signaling were both decreased in the thymus. Target genes of Shh (Patched1 and Gli1) and Wnt/beta-catenin (c-fos, and c-myc) were affected differentially among thymocyte subpopulations. These findings suggest that prenatal exposure to Cd dysregulates two signaling pathways in the thymus, resulting in altered thymocyte development.

  4. The sonic hedgehog signaling pathway contributes to the development of salivary gland neoplasms regardless of perineural infiltration.

    PubMed

    Vidal, Manuela Torres Andion; Lourenço, Sílvia Vanessa; Soares, Fernando Augusto; Gurgel, Clarissa Araújo; Studart, Eduardo J B; Valverde, Ludmila de Faro; Araújo, Iguaracyra Barreto de Oliveira; Ramos, Eduardo Antônio Gonçalves; Xavier, Flávia Caló de Aquino; Dos Santos, Jean Nunes

    2016-07-01

    The pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) are common tumors arising from salivary glands whose histopathology is heterogeneous. The sonic hedgehog signaling pathway (Hh) and signal transducer and activator of transcription 3 (STAT3) play important roles in cell proliferation, favoring tumor growth. The aim of this investigation was to study components of the Hh pathway, as well as STAT3 in salivary gland neoplasms in an attempt to add information about the biological characteristics of these neoplasms. We used 9 cases of PA, 17 cases of ACC, and 20 cases of MEC. Using immunohistochemistry, SHH, GLI1, SUFU, HHIP, and STAT3 were investigated. For comparative purposes, MCM3 (cellular proliferation marker) was also included. In PA, there was high expression of cytoplasmic SHH and SUFU and low expression of STAT3 and MCM3. In the ACC, there was high expression of GLI1, HHIP, and STAT3 and low expression of SHH, SUFU, and MCM3. In the MEC, we observed high expression of SHH, GLI1, SUFU, and HHIP and low expression of STAT3 and MCM3. There was a statistically significant difference between SHH (p = 0.0064), STAT3 (p = 0.0003), and MCM3 (p = 0.0257) when all tumors were compared and a higher expression in parenchyma for all tumors when stroma and parenchyma were compared (p < 0.05). These findings suggests a possible role of Hh pathway in the development and maintenance of the cytoarchitectural pattern of PA, ACC, and MEC, as well as the participation of STAT3 in the development of ACC, irrespective perineural infiltration.

  5. Sonic hedgehog improves ischemia-induced neovascularization by enhancing endothelial progenitor cell function in type 1 diabetes.

    PubMed

    Qin, Yuan; He, Yan-Huan; Hou, Ning; Zhang, Gen-Shui; Cai, Yi; Zhang, Gui-Ping; Xiao, Qing; He, Li-Shan; Li, Su-Juan; Yi, Quan; Luo, Jian-Dong

    2016-03-05

    The Sonic hedgehog (Shh) pathway is downregulated in type 1 diabetes, and it has been reported that augmentation of this pathway may alleviate diabetic complications. However, the cellular mechanisms underlying these protective effects are poorly understood. Recent studies indicate that impaired function of endothelial progenitor cells (EPCs) may contribute to cardiovascular problems in diabetes. We hypothesized that impaired Shh signaling contribute to endothelial progenitor cell dysfunction and that activating the Shh signaling pathway may rescue EPC function and promote diabetic neovascularization. Adult male C57/B6 mice and streptozotocin (STZ)-induced type 1 diabetic mice were used. Gli1 and Ptc1 protein levels were reduced in EPCs from diabetic mice, indicating inhibition of the Shh signaling pathway. EPC migration, tube formation ability, and mobilization were impaired in diabetic mice compared with non-diabetic controls (p < 0.05 vs control), and all were improved by in vivo administration of the Shh pathway receptor agonist SAG (p < 0.05 vs diabetes). SAG significantly increased capillary density and blood perfusion in the ischemic hindlimbs of diabetic mice (p < 0.05 vs diabetes). The AKT activity was lower in EPCs from diabetic mice than those from non-diabetic controls (p < 0.05 vs control). This decreased AKT activity led to an increased GSK-3β activity and degradation of the Shh pathway transcription factor Gli1/Gli2. SAG significantly increased the activity of AKT in EPCs. Our data clearly demonstrate that an impaired Shh pathway mediated by the AKT/GSK-3β pathway can contribute to EPC dysfunction in diabetes and thus activating the Shh signaling pathway can restore both the number and function of EPCs and increase neovascularization in type 1 diabetic mice.

  6. Insight from Frogs: Sonic Hedgehog Gene Expression and a Re-evaluation of the Vertebrate Odontogenic Band.

    PubMed

    Grieco, Theresa M; Hlusko, Leslea J

    2016-08-01

    While the identification of conserved processes across multiple taxa leads to an understanding of fundamental developmental mechanisms, the ways in which different animals fail to conform to common developmental processes can elucidate how evolution modifies development to result in the vast array of morphologies seen today-the developmental mechanisms that lead to anatomical variation. Odontogenesis-how teeth are initiated and formed-is well suited to the examination of both developmental conservation and phenotypic diversity. We suggest here that the study of early tooth development, the period of odontogenic band development, reveals departures from conserved mechanisms that question the role of players in the developmental process. In the earliest stages of odontogenesis, Sonic hedgehog (Shh) gene expression is interpreted as critical evidence of tooth initiation prior to any histological indication. However, a detailed examination of studies of tooth development across a wide range of taxa reveals that several vertebrate species fail to conform to the expectations of the Shh Consensus Model, calling for a reconsideration of the assumed causality of epithelial Shh in tooth initiation. We present new Shh gene expression data for an amphibian, the frog Silurana (Xenopus) tropicalis. In these animals, craniofacial and odontogenic developmental processes are more disjunct, and thereby provide a natural test of the hypothesis that Shh is immediately required for subsequent tooth development. Our results suggest that Shh expression may actually be related to the formation of the mouth rather than a required precursor to subsequent tooth formation. Anat Rec, 299:1099-1109, 2016. © 2016 Wiley Periodicals, Inc.

  7. Sonic hedgehog and retinoic Acid induce bone marrow-derived stem cells to differentiate into glutamatergic neural cells.

    PubMed

    Yu, Zhenhai; Wu, Shixing; Liu, Zhen; Lin, Haiyan; Chen, Lei; Yuan, Xinli; Zhang, Zhiying; Liu, Fang; Zhang, Chuansen

    2015-01-01

    Studies have showed that transplanted stem cells in the inner ear won't regenerate to replace the damaged sensory hair cells. They can spontaneously differentiate into mesenchymal cells and fibrocytes in the damaged inner ear. Only mature sensory cells of MSCs-derived possess the great potency for cell transplantation in the treatment of sensorineural hearing loss. So, we try to establish an efficient generation of the glutamatergic sensory neural phenotype for the cell transplantation of the hearing loss. We isolated MSCs from femoral and tibial bones according to their adherence to culture dishes. After purification, proliferation, and passaged, cells became homogeneous in appearance, showing more uniformity and grew in a monolayer with a typical spindle-shape morphology. The cell surface markers were assessed using FACS to characterize the isolated cells. For neural induction to harvest the glutamatergic sensory neurons, passage 3 MSCs were incubated with preinduced medium for 24 hr, and neural-induced medium for an additional 14 days. The cells exhibit a typical neural shape. RT-PCR analysis indicated that the mRNA levels of the neural cell marker nestin, Tau, MAP-2, β-tubulin III, GluR-3, and GluR-4 were higher compared with primary MSCs. Immunohistochemistry and western-blotting proofed that nestin, MAP-2, β-tubulin III, and GluR-4 proteins indeed exhibit their expression difference in the induced cells compared to the MSCs. We show an efficient protocol by the combined applications of Sonic Hedgehog (Shh) and Retinoic Acid (RA) to induce MSCs to differentiate into the glutamatergic sensory neuron which were identified from the morphological, biochemical, and molecular characteristics.

  8. Duration of culture and sonic hedgehog signaling differentially specify PV versus SST cortical interneuron fates from embryonic stem cells.

    PubMed

    Tyson, Jennifer A; Goldberg, Ethan M; Maroof, Asif M; Xu, Qing; Petros, Timothy J; Anderson, Stewart A

    2015-04-01

    Medial ganglionic eminence (MGE)-derived GABAergic cortical interneurons (cINs) consist of multiple subtypes that are involved in many cortical functions. They also have a remarkable capacity to migrate, survive and integrate into cortical circuitry after transplantation into postnatal cortex. These features have engendered considerable interest in generating distinct subgroups of interneurons from pluripotent stem cells (PSCs) for the study of interneuron fate and function, and for the development of cell-based therapies. Although advances have been made, the capacity to generate highly enriched pools of subgroup fate-committed interneuron progenitors from PSCs has remained elusive. Previous studies have suggested that the two main MGE-derived interneuron subgroups--those expressing somatostatin (SST) and those expressing parvalbumin (PV)--are specified in the MGE from Nkx2.1-expressing progenitors at higher or lower levels of sonic hedgehog (Shh) signaling, respectively. To further explore the role of Shh and other factors in cIN fate determination, we generated a reporter line such that Nkx2.1-expressing progenitors express mCherry and postmitotic Lhx6-expressing MGE-derived interneurons express GFP. Manipulations of Shh exposure and time in culture influenced the subgroup fates of ESC-derived interneurons. Exposure to higher Shh levels, and collecting GFP-expressing precursors at 12 days in culture, resulted in the strongest enrichment for SST interneurons over those expressing PV, whereas the strongest enrichment for PV interneurons was produced by lower Shh and by collecting mCherry-expressing cells after 17 days in culture. These findings confirm that fate determination of cIN subgroups is crucially influenced by Shh signaling, and provide a system for the further study of interneuron fate and function.

  9. Pleiotropic functions of embryonic sonic hedgehog expression link jaw and taste bud amplification with eye loss during cavefish evolution.

    PubMed

    Yamamoto, Yoshiyuki; Byerly, Mardi S; Jackman, William R; Jeffery, William R

    2009-06-01

    This study addresses the role of sonic hedgehog (shh) in increasing oral-pharyngeal constructive traits (jaws and taste buds) at the expense of eyes in the blind cavefish Astyanax mexicanus. In cavefish embryos, eye primordia degenerate under the influence of hyperactive Shh signaling. In concert, cavefish show amplified jaw size and taste bud numbers as part of a change in feeding behavior. To determine whether pleiotropic effects of hyperactive Shh signaling link these regressive and constructive traits, shh expression was compared during late development of the surface-dwelling (surface fish) and cave-dwelling (cavefish) forms of Astyanax. After an initial expansion along the midline of early embryos, shh was elevated in the oral-pharyngeal region in cavefish and later was confined to taste buds. The results of shh inhibition and overexpression experiments indicate that Shh signaling has an important role in oral and taste bud development. Conditional overexpression of an injected shh transgene at specific times in development showed that taste bud amplification and eye degeneration are sensitive to shh overexpression during the same early developmental period, although taste buds are not formed until much later. Genetic crosses between cavefish and surface fish revealed an inverse relationship between eye size and jaw size/taste bud number, supporting a link between oral-pharyngeal constructive traits and eye degeneration. The results suggest that hyperactive Shh signaling increases oral and taste bud amplification in cavefish at the expense of eyes. Therefore, selection for constructive oral-pharyngeal traits may be responsible for eye loss during cavefish evolution via pleiotropic function of the Shh signaling pathway.

  10. AB300. SPR-27 Sonic hedgehog promotes sprouting of neurons in the pelvic ganglia and cavernous nerve during regeneration

    PubMed Central

    Dobbs, Ryan; Choe, Shawn; Harrington, Daniel A.; Stupp, Samuel I.; McVary, Kevin T.; Podlasek, Carol A.

    2016-01-01

    Objective We’ve shown in previous studies that sonic hedgehog (SHH) protein delivered by nanoparticle based peptide amphiphile (PA) hydrogels to the cavernous nerve (CN) at the time of crush injury (mimicking prostatectomy), are neuroprotective and promote CN regeneration in a rat model. The mechanism of how SHH promotes CN regeneration is unknown. We hypothesize that SHH promotes sprouting of CN axons, in order to enhance nerve regeneration. We examine this hypothesis in an in vitro organ culture model. Methods The caudal portion of the pelvic ganglia (innervates penis) and CN were dissected from adult Sprague Dawley rats (n=47) and placed in Matrigel in growth factor reduced medium and were grown for three to five days. Pelvic ganglia were exposed to Affi-Gel beads containing: (I) SHH protein; (II) 5e1 and cyclopamine SHH inhibitors; and (III) SHH protein delivered by PA. Additional pelvic ganglia/CN tissue underwent CN crush and were exposed to SHH protein or PBS/mouse serum albumin (MSA) protein. Sprouting was evaluated for number of sprouts and their length, and by immunohistochemical analysis for sprouting markers (GAP43 and nNOS). Results Sprouting of pelvic ganglia and CN axons was increased with SHH treatment. Sprouts were more abundant, longer in length, with larger arborization of sprouts, in comparison to controls. More sprouting was promoted with SHH treatment of CN injured nerves. The CN had similar sprouting potential at 4 and 9 days after crush injury. Localization of SHH delivery makes a difference in sprouting potential. Conclusions The mechanism of how SHH PA treatment promotes CN regeneration, involves enhanced sprouting of pelvic ganglia and CN neurons. Understanding the mechanism of SHH PA action on neuronal tissue is critical for translation to prostatectomy patients and to further enhance regeneration. Funding Source(s) NIH/NIDDK DK079184

  11. Suppression of hedgehog signaling regulates hepatic stellate cell activation and collagen secretion.

    PubMed

    Li, Tao; Leng, Xi-Sheng; Zhu, Ji-Ye; Wang, Gang

    2015-01-01

    Hepatic stellate cells (HSCs) play an important role in liver fibrosis. This study investigates the expression of hedgehog in HSC and the role of hedgehog signaling on activation and collagen secretion of HSC. Liver ex vivo perfusion with collagenase IV and density gradient centrifugation were used to isolate HSC. Expression of hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 in HSC were detected by RT-PCR. Hedgehog siRNA vectors targeting Ihh, Smo and Gli2 were constructed and transfected into HSC respectively. Suppression of hedgehog signaling were detected by SYBR Green fluorescence quantitative RT-PCR. Effects of hedgehog signaling inhibition on HSC activation and collagen I secretion were analyzed. Hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 were expressed in HSC. siRNA vectors targeting Ihh, Smo and Gli2 were successfully constructed and decreased target gene expression. Suppression of hedgehog signaling significantly decreased the expression of α-SMA in HSC (P<0.01). Collagen type I secretion of HSC were also significantly decreased (P<0.01). In summary, HSC activation and collagen secretion can be regulated by hedgehog signaling. Hedgehog may play a role in the pathogenesis of liver fibrosis.

  12. Inhibition of APP gamma-secretase restores Sonic Hedgehog signaling and neurogenesis in the Ts65Dn mouse model of Down syndrome.

    PubMed

    Giacomini, Andrea; Stagni, Fiorenza; Trazzi, Stefania; Guidi, Sandra; Emili, Marco; Brigham, Elizabeth; Ciani, Elisabetta; Bartesaghi, Renata

    2015-10-01

    Neurogenesis impairment starting from early developmental stages is a key determinant of intellectual disability in Down syndrome (DS). Previous evidence provided a causal relationship between neurogenesis impairment and malfunctioning of the mitogenic Sonic Hedgehog (Shh) pathway. In particular, excessive levels of AICD (amyloid precursor protein intracellular domain), a cleavage product of the trisomic gene APP (amyloid precursor protein) up-regulate transcription of Ptch1 (Patched1), the Shh receptor that keeps the pathway repressed. Since AICD results from APP cleavage by γ-secretase, the goal of the current study was to establish whether treatment with a γ-secretase inhibitor normalizes AICD levels and restores neurogenesis in trisomic neural precursor cells. We found that treatment with a selective γ-secretase inhibitor (ELND006; ELN) restores proliferation in neurospheres derived from the subventricular zone (SVZ) of the Ts65Dn mouse model of DS. This effect was accompanied by reduction of AICD and Ptch1 levels and was prevented by inhibition of the Shh pathway with cyclopamine. Treatment of Ts65Dn mice with ELN in the postnatal period P3-P15 restored neurogenesis in the SVZ and hippocampus, hippocampal granule cell number and synapse development, indicating a positive impact of treatment on brain development. In addition, in the hippocampus of treated Ts65Dn mice there was a reduction in the expression levels of various genes that are transcriptionally regulated by AICD, including APP, its origin substrate. Inhibitors of γ-secretase are currently envisaged as tools for the cure of Alzheimer's disease because they lower βamyloid levels. Current results provide novel evidence that γ-secretase inhibitors may represent a strategy for the rescue of neurogenesis defects in DS.

  13. Sonic hedgehog promotes proliferation and differentiation of adult muscle cells: Involvement of MAPK/ERK and PI3K/Akt pathways.

    PubMed

    Elia, Dafna; Madhala, Dorit; Ardon, Eti; Reshef, Ram; Halevy, Orna

    2007-09-01

    Sonic hedgehog (Shh) has been reported to act as a mitogen and survival factor for muscle satellite cells. However, its role in their differentiation remains ambiguous. Here, we provide evidence that Shh promotes the proliferation and differentiation of primary cultures of chicken adult myoblasts (also termed satellite cells) and mouse myogenic C2 cells. These effects are reversed by cyclopamine, a specific chemical inhibitor of the Shh pathway. In addition, we show that Shh and its downstream molecules are expressed in adult myoblast cultures and localize adjacent to Pax7 in muscle sections. These gene expressions are regulated during postnatal muscle growth in chicks. Most importantly, we report that Shh induces MAPK/ERK and phosphoinositide 3-kinase (PI3K)-dependent Akt phosphorylation and that activation of both signaling pathways is essential for Shh's signaling in muscle cells. However, the effect of Shh on Akt phosphorylation is more robust than that on MAPK/ERK, and data suggest that Shh influences these pathways in a manner similar to IGF-I. By exploiting specific chemical inhibitors of the MAPK/ERK and PI3K/Akt signaling pathways, UO126 and Ly294002, respectively, we demonstrate that Shh-induced Akt phosphorylation, but not that of MAPK/ERK, is required for its promotive effects on muscle cell proliferation and differentiation. Taken together, we suggest that Shh acts in an autocrinic manner in adult myoblasts, and provide first evidence of a role for PI3K/Akt in Shh signaling during myoblast differentiation.

  14. Sonic hedgehog increases the skin wound-healing ability of mouse embryonic stem cells through the microRNA 200 family

    PubMed Central

    Suh, Han Na; Han, Ho Jae

    2015-01-01

    Background and Purpose To use stem cell therapy effectively, it is important to enhance the therapeutic potential of stem cells with soluble factors. Although sonic hedgehog (shh) is important in maintaining the stem cell, the recovery effect of mouse embryonic stem cells (mESCs) with shh has not yet been elucidated. The present study investigated the effect of mESCs with shh in skin recovery in vivo as well as the related intracellular signal pathways in vitro. Experimental Approach The healing effect of mESCs with shh on skin wounds was examined in vivo in ICR mice. The involvement of Smads, the microRNA (miR)-200 family, zinc finger E-box-binding homeobox (ZEBs) and E-cadherin on shh-induced mESC migration and self-renewal was determined in vitro. Key Results The mESCs with shh increased re-epithelialization and VEGF expression in skin wounds. Shh-treated mESCs increased both secreted and intracellular levels of VEGF. Shh induced dephosphorylation of glycogen synthase kinase 3β through the Smoothened receptor and increased the phosphorylation of Smad1 and Smad2/3 in mESCs. Shh-induced decrease of the mmu-miR-141, -200c, -200a, -200b and -429 expression levels was significantly reversed by Smad4 siRNA. Shh increased nuclear expression of ZEB1/ZEB2 and decreased E-cadherin expression while increasing cell migration and skin wound healing. Both these effects were reversed by mmu-miR-141 and -200b mimics. Conclusions and Implications Mouse ESCs accelerated skin wound healing by shh through down-regulating E-cadherin, an effect dependent on mmu-miR-141 and -200b. Our data provides evidence for the effectiveness of shh in stem cell-based therapy in vivo. PMID:25257936

  15. The influence of metal-ion binding on the structure and surface composition of Sonic Hedgehog: a combined classical and hybrid QM/MM MD study.

    PubMed

    Hitzenberger, Manuel; Hofer, Thomas S

    2016-08-10

    In this work, the influence of the metal ions present in vertebrate Sonic Hedgehog was assessed by a series of molecular mechanics molecular dynamics simulations with differing ionic compositions. The obtained data suggest that Ca(ii) binding has a very distinct influence on the composition of the protein surface surrounding the binding site by shaping several ionic interactions with negatively charged sidechains that otherwise would be pointing towards the solvent, repelling potential ligands. Furthermore, the Ca(ii) ions play an important role in the stability of the loop regions where they are coordinated. In contrast, the removal of the Zn(ii) ion results in no noticeable destabilization of its chemical surrounding, however, it is shown that the destabilizing effect of removed Ca(ii) ions is amplified if Zn(ii) is absent as well. Furthermore, a quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulation of Sonic Hedgehog with special focus on the Zn(ii) binding site has been conducted. The results indicate that QM/MM in contrast to pure MM accurately reproduces structural features also found by experimental studies and therefore is able to provide credible predictions not only of the dynamical properties of the studied system but also of protein-ligand interactions at the metal ion binding site.

  16. Hedgehog pathway-regulated gene networks in cerebellum development and tumorigenesis.

    PubMed

    Lee, Eunice Y; Ji, Hongkai; Ouyang, Zhengqing; Zhou, Baiyu; Ma, Wenxiu; Vokes, Steven A; McMahon, Andrew P; Wong, Wing H; Scott, Matthew P

    2010-05-25

    Many genes initially identified for their roles in cell fate determination or signaling during development can have a significant impact on tumorigenesis. In the developing cerebellum, Sonic hedgehog (Shh) stimulates the proliferation of granule neuron precursor cells (GNPs) by activating the Gli transcription factors. Inappropriate activation of Shh target genes results in unrestrained cell division and eventually medulloblastoma, the most common pediatric brain malignancy. We find dramatic differences in the gene networks that are directly driven by the Gli1 transcription factor in GNPs and medulloblastoma. Gli1 binding location analysis revealed hundreds of genomic loci bound by Gli1 in normal and cancer cells. Only one third of the genes bound by Gli1 in GNPs were also bound in tumor cells. Correlation with gene expression levels indicated that 116 genes were preferentially transcribed in tumors, whereas 132 genes were target genes in both GNPs and medulloblastoma. Quantitative PCR and in situ hybridization for some putative target genes support their direct regulation by Gli. The results indicate that transformation of normal GNPs into deadly tumor cells is accompanied by a distinct set of Gli-regulated genes and may provide candidates for targeted therapies.

  17. Microparticles Carrying Sonic Hedgehog Favor Neovascularization through the Activation of Nitric Oxide Pathway in Mice

    PubMed Central

    Benameur, Tarek; Soleti, Raffaella; Porro, Chiara; Andriantsitohaina, Ramaroson; Martínez, Maria Carmen

    2010-01-01

    Background Microparticles (MPs) are vesicles released from plasma membrane upon cell activation and during apoptosis. Human T lymphocytes undergoing activation and apoptosis generate MPs bearing morphogen Shh (MPsShh+) that are able to regulate in vitro angiogenesis. Methodology/Principal Findings Here, we investigated the ability of MPsShh+ to modulate neovascularization in a model of mouse hind limb ischemia. Mice were treated in vivo for 21 days with vehicle, MPsShh+, MPsShh+ plus cyclopamine or cyclopamine alone, an inhibitor of Shh signalling. Laser doppler analysis revealed that the recovery of the blood flow was 1.4 fold higher in MPsShh+-treated mice than in controls, and this was associated with an activation of Shh pathway in muscles and an increase in NO production in both aorta and muscles. MPsShh+-mediated effects on flow recovery and NO production were completely prevented when Shh signalling was inhibited by cyclopamine. In aorta, MPsShh+ increased activation of eNOS/Akt pathway, and VEGF expression, being inhibited by cyclopamine. By contrast, in muscles, MPsShh+ enhanced eNOS expression and phosphorylation and decreased caveolin-1 expression, but cyclopamine prevented only the effects of MPsShh+ on eNOS pathway. Quantitative RT-PCR revealed that MPsShh+ treatment increased FGF5, FGF2, VEGF A and C mRNA levels and decreased those of α5-integrin, FLT-4, HGF, IGF-1, KDR, MCP-1, MT1-MMP, MMP-2, TGFβ1, TGFβ2, TSP-1 and VCAM-1, in ischemic muscles. Conclusions/Significance These findings suggest that MPsShh+ may contribute to reparative neovascularization after ischemic injury by regulating NO pathway and genes involved in angiogenesis. PMID:20856928

  18. Genetic interactions between the hedgehog co-receptors Gas1 and Boc regulate cell proliferation during murine palatogenesis

    PubMed Central

    Xavier, Guilherme M.; Seppala, Maisa; Papageorgiou, Spyridon N.; Fan, Chen-Ming; Cobourne, Martyn T.

    2016-01-01

    Abnormal regulation of Sonic hedgehog (Shh) signaling has been described in a variety of human cancers and developmental anomalies, which highlights the essential role of this signaling molecule in cell cycle regulation and embryonic development. Gas1 and Boc are membrane co-receptors for Shh, which demonstrate overlapping domains of expression in the early face. This study aims to investigate potential interactions between these co-receptors during formation of the secondary palate. Mice with targeted mutation in Gas1 and Boc were used to generate Gas1; Boc compound mutants. The expression of key Hedgehog signaling family members was examined in detail during palatogenesis via radioactive in situ hybridization. Morphometric analysis involved computational quantification of BrdU-labeling and cell packing; whilst TUNEL staining was used to assay cell death. Ablation of Boc in a Gas1 mutant background leads to reduced Shh activity in the palatal shelves and an increase in the penetrance and severity of cleft palate, associated with failed elevation, increased proliferation and reduced cell death. Our findings suggest a dual requirement for Boc and Gas1 during early development of the palate, mediating cell cycle regulation during growth and subsequent fusion of the palatal shelves. PMID:27811357

  19. Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals

    PubMed Central

    Fu, Lin; Wu, Hailong; Cheng, Steven Y; Gao, Daming; Zhang, Lei; Zhao, Yun

    2016-01-01

    Hedgehog signaling plays very important roles in development and cancers. Vertebrates have three transcriptional factors, Gli1, Gli2 and Gli3. Among them, Gli3 is a very special transcriptional factor which closely resembles Cubitus interruptus (Ci, in Drosophila) structurally and functionally as a ‘double agent’ for Shh target gene expression. Here we show that Gli3 full-length, but not the truncated form, can be methylated at K436 and K595. This methylation is specifically catalyzed by Set7, a lysine methyltransferase (KMT). Methylation at K436 and K595 respectively increases the stability and DNA binding ability of Gli3, resulting in an enhancement of Shh signaling activation. Furthermore, functional experiments indicate that the Gli3 methylation contributes to the tumor growth and metastasis in non-small cell lung cancer in vitro and in vivo. Therefore, we propose that Set7 mediated methylation is a novel PTM of Gli3, which positively regulates the transactivity of Gli3 and the activation of Shh signaling. DOI: http://dx.doi.org/10.7554/eLife.15690.001 PMID:27146893

  20. Autonomous and nonautonomous roles of Hedgehog signaling in regulating limb muscle formation

    PubMed Central

    Hu, Jimmy Kuang-Hsien; McGlinn, Edwina; Harfe, Brian D.; Kardon, Gabrielle; Tabin, Clifford J.

    2012-01-01

    Muscle progenitor cells migrate from the lateral somites into the developing vertebrate limb, where they undergo patterning and differentiation in response to local signals. Sonic hedgehog (Shh) is a secreted molecule made in the posterior limb bud that affects patterning and development of multiple tissues, including skeletal muscles. However, the cell-autonomous and non-cell-autonomous functions of Shh during limb muscle formation have remained unclear. We found that Shh affects the pattern of limb musculature non-cell-autonomously, acting through adjacent nonmuscle mesenchyme. However, Shh plays a cell-autonomous role in maintaining cell survival in the dermomyotome and initiating early activation of the myogenic program in the ventral limb. At later stages, Shh promotes slow muscle differentiation cell-autonomously. In addition, Shh signaling is required cell-autonomously to regulate directional muscle cell migration in the distal limb. We identify neuroepithelial cell transforming gene 1 (Net1) as a downstream target and effector of Shh signaling in that context. PMID:22987639

  1. Hedgehog-mediated regulation of PPARγ controls metabolic patterns in neural precursors and shh-driven medulloblastoma.

    PubMed

    Bhatia, Bobby; Potts, Chad R; Guldal, Cemile; Choi, SunPhil; Korshunov, Andrey; Pfister, Stefan; Kenney, Anna M; Nahlé, Zaher A

    2012-04-01

    Sonic hedgehog (Shh) signaling is critical during development and its aberration is common across the spectrum of human malignancies. In the cerebellum, excessive activity of the Shh signaling pathway is associated with the devastating pediatric brain tumor medulloblastoma. We previously demonstrated that exaggerated de novo lipid synthesis is a hallmark of Shh-driven medulloblastoma and that hedgehog signaling inactivates the Rb/E2F tumor suppressor complex to promote lipogenesis. Indeed, such Shh-mediated metabolic reprogramming fuels tumor progression, in an E2F1- and FASN-dependent manner. Here, we show that the nutrient sensor PPARγ is a key component of the Shh metabolic network, particularly its regulation of glycolysis. Our data show that in primary cerebellar granule neural precursors (CGNPs), proposed medulloblastoma cells-of-origin, Shh stimulation elicits a marked induction of PPARγ alongside major glycolytic markers. This is also documented in the actively proliferating Shh-responsive CGNPs in the developing cerebellum, and PPARγ expression is strikingly elevated in Shh-driven medulloblastoma in vivo. Importantly, pharmacological blockade of PPARγ and/or Rb inactivation inhibits CGNP proliferation, drives medulloblastoma cell death and extends survival of medulloblastoma-bearing animals in vivo. This coupling of mitogenic Shh signaling to a major nutrient sensor and metabolic transcriptional regulator define a novel mechanism through which Shh signaling engages the nutrient sensing machinery in brain cancer, controls the cell cycle, and regulates the glycolytic index. This also reveals a dominant role of Shh in the etiology of glucose metabolism in medulloblastoma and underscores the function of the Shh → E2F1 → PPARγ axis in altering substrate utilization patterns in brain cancers in favor of tumor growth. These findings emphasize the value of PPARγ downstream of Shh as a global therapeutic target in hedgehog-dependent and/or Rb

  2. Drosophila Vps36 regulates Smo trafficking in Hedgehog signaling.

    PubMed

    Yang, Xiaofeng; Mao, Feifei; Lv, Xiangdong; Zhang, Zhao; Fu, Lin; Lu, Yi; Wu, Wenqing; Zhou, Zhaocai; Zhang, Lei; Zhao, Yun

    2013-09-15

    The hedgehog (Hh) signaling pathway plays a very important role in metazoan development by controlling pattern formation. Malfunction of the Hh signaling pathway leads to numerous serious human diseases, including congenital disorders and cancers. The seven-transmembrane domain protein Smoothened (Smo) is a key transducer of the Hh signaling pathway, and mediates the graded Hh signal across the cell plasma membrane, thereby inducing the proper expression of downstream genes. Smo accumulation on the cell plasma membrane is regulated by its C-tail phosphorylation and the graded Hh signal. The inhibitory mechanism for Smo membrane accumulation in the absence of Hh, however, is still largely unknown. Here, we report that Vps36 of the ESCRT-II complex regulates Smo trafficking between the cytosol and plasma membrane by specifically recognizing the ubiquitin signal on Smo in the absence of Hh. Furthermore, in the absence of Hh, Smo is ubiquitylated on its cytoplasmic part, including its internal loops and C-tail. Taken together, our data suggest that the ESCRT-II complex, especially Vps36, has a special role in controlling Hh signaling by targeting the membrane protein Smo for its trafficking in the absence of Hh, thereby regulating Hh signaling activity.

  3. AMP-activated protein kinase-dependent autophagy mediated the protective effect of sonic hedgehog pathway on oxygen glucose deprivation-induced injury of cardiomyocytes.

    PubMed

    Xiao, Qing; Yang, Ya; Qin, Yuan; He, Yan-Hua; Chen, Kui-Xiang; Zhu, Jian-Wei; Zhang, Gui-Ping; Luo, Jian-Dong

    2015-02-13

    Sonic hedgehog (Shh) pathway has been reported to protect cardiomyocytes in myocardial infarction (MI), but the underlying mechanism is not clear. Here, we provide evidence that Shh pathway induces cardiomyocytes survival through AMP-activated protein kinase-dependent autophagy. Shh pathway agonist SAG increased the expression of LC3-II, and induced the formation of autophagosomes in cultured H9c2 cardiomyocytes under oxygen glucose deprivation (OGD) 1 h and 4 h. Moreover, SAG induced a profound AMP-activated protein kinase (AMPK) activation, and then directly phosphorylated and activated the downstream autophagy initiator Ulk1, independent of the autophagy suppressor mammalian target of rapamycin (mTOR) complex 1. Taken together, our results have shown that Shh activates AMPK-dependent autophagy in cardiomyocytes under OGD, suggesting a role of autophagy in Shh-induced cellular protection.

  4. An Nfic-hedgehog signaling cascade regulates tooth root development

    PubMed Central

    Liu, Yang; Feng, Jifan; Li, Jingyuan; Zhao, Hu; Ho, Thach-Vu; Chai, Yang

    2015-01-01

    Coordination between the Hertwig's epithelial root sheath (HERS) and apical papilla (AP) is crucial for proper tooth root development. The hedgehog (Hh) signaling pathway and Nfic are both involved in tooth root development; however, their relationship has yet to be elucidated. Here, we establish a timecourse of mouse molar root development by histological staining of sections, and we demonstrate that Hh signaling is active before and during root development in the AP and HERS using Gli1 reporter mice. The proper pattern of Hh signaling activity in the AP is crucial for the proliferation of dental mesenchymal cells, because either inhibition with Hh inhibitors or constitutive activation of Hh signaling activity in transgenic mice leads to decreased proliferation in the AP and shorter roots. Moreover, Hh activity is elevated in Nfic−/− mice, a root defect model, whereas RNA sequencing and in situ hybridization show that the Hh attenuator Hhip is downregulated. ChIP and RNAscope analyses suggest that Nfic binds to the promoter region of Hhip. Treatment of Nfic−/− mice with Hh inhibitor partially restores cell proliferation, AP growth and root development. Taken together, our results demonstrate that an Nfic-Hhip-Hh signaling pathway is crucial for apical papilla growth and proper root formation. This discovery provides insight into the molecular mechanisms regulating tooth root development. PMID:26293299

  5. Hedgehog signaling regulates dental papilla formation and tooth size during zebrafish odontogenesis

    PubMed Central

    Yu, Jeffrey C.; Fox, Zachary D.B.; Crimp, James L.; Littleford, Hana E.; Jowdry, Andrea L.; Jackman, William R.

    2015-01-01

    Background Intercellular communication by the hedgehog cell signaling pathway is necessary for tooth development throughout the vertebrates, but it remains unclear which specific developmental signals control cell behavior at different stages of odontogenesis. To address this issue, we have manipulated hedgehog activity during zebrafish tooth development and visualized the results using confocal microscopy. Results We first established that reporter lines for dlx2b, fli1, NF-κB, and prdm1a are markers for specific subsets of tooth germ tissues. We then blocked hedgehog signaling with cyclopamine and observed a reduction or elimination of the cranial neural crest derived dental papilla, which normally contains the cells that later give rise to dentin-producing odontoblasts. Upon further investigation we observed that the dental papilla begins to form and then regresses in the absence of hedgehog signaling, through a mechanism unrelated to cell proliferation or apoptosis. We also found evidence of an isometric reduction in tooth size that correlates with the time of earliest hedgehog inhibition. Conclusions We hypothesize that these results reveal a previously uncharacterized function of hedgehog signaling during tooth morphogenesis, regulating the number of cells in the dental papilla and thereby controlling tooth size. PMID:25645398

  6. Hedgehog-dependent E3-ligase Midline1 regulates ubiquitin-mediated proteasomal degradation of Pax6 during visual system development

    PubMed Central

    Pfirrmann, Thorsten; Jandt, Enrico; Ranft, Swantje; Lokapally, Ashwin; Neuhaus, Herbert; Perron, Muriel

    2016-01-01

    Pax6 is a key transcription factor involved in eye, brain, and pancreas development. Although pax6 is expressed in the whole prospective retinal field, subsequently its expression becomes restricted to the optic cup by reciprocal transcriptional repression of pax6 and pax2. However, it remains unclear how Pax6 protein is removed from the eyestalk territory on time. Here, we report that Mid1, a member of the RBCC/TRIM E3 ligase family, which was first identified in patients with the X-chromosome–linked Opitz BBB/G (OS) syndrome, interacts with Pax6. We found that the forming eyestalk is a major domain of mid1 expression, controlled by the morphogen Sonic hedgehog (Shh). Here, Mid1 regulates the ubiquitination and proteasomal degradation of Pax6 protein. Accordantly, when Mid1 levels are knocked down, Pax6 expression is expanded and eyes are enlarged. Our findings indicate that remaining or misaddressed Pax6 protein is cleared from the eyestalk region to properly set the border between the eyestalk territory and the retina via Mid1. Thus, we identified a posttranslational mechanism, regulated by Sonic hedgehog, which is important to suppress Pax6 activity and thus breaks pax6 autoregulation at defined steps during the formation of the visual system. PMID:27555585

  7. Identification of Small Molecules Which Induce Skeletal Muscle Differentiation in Embryonic Stem Cells via Activation of the Wnt and Inhibition of Smad2/3 and Sonic Hedgehog Pathways.

    PubMed

    Lee, Hyunwoo; Haller, Corinne; Manneville, Carole; Doll, Thierry; Fruh, Isabelle; Keller, Caroline Gubser; Richards, Shola M; Ibig-Rehm, Yvonne; Patoor, Maude; Goette, Marjo; Bouchez, Laure C; Mueller, Matthias

    2016-02-01

    The multilineage differentiation capacity of mouse and human embryonic stem (ES) cells offers a testing platform for small molecules that mediate mammalian lineage determination and cellular specialization. Here we report the identification of two small molecules which drives mouse 129 ES cell differentiation to skeletal muscle with high efficiency without any genetic modification. Mouse embryoid bodies (EBs) were used to screen a library of 1,000 small molecules to identify compounds capable of inducing high levels of Pax3 mRNA. Stimulation of EBs with SMIs (skeletal muscle inducer, SMI1 and SMI2) from the screen resulted in a high percentage of intensively twitching skeletal muscle fibers 3 weeks after induction. Gene expression profiling studies that were carried out for mode of actions analysis showed that SMIs activated genes regulated by the Wnt pathway and inhibited expression of Smad2/3 and Sonic Hedgehog (Shh) target genes. A combination of three small molecules known to modulate these three pathways acted similarly to the SMIs found here, driving ES cells from 129 as well as Balb/c and C57Bl/6 to skeletal muscle. Taken together, these data demonstrate that the SMI drives ES cells to skeletal muscle via concerted activation of the Wnt pathway, and inhibition of Smad2/3 signaling and Shh pathways. This provides important developmental biological information about skeletal muscle differentiation from embryonic stem cells and may lead to the development of new therapeutics for muscle disease.

  8. Testing chemotherapeutic agents in the feather follicle identifies a selective blockade of cell proliferation and a key role for sonic hedgehog signaling in chemotherapy-induced tissue damage.

    PubMed

    Xie, Guojiang; Wang, Hangwei; Yan, Zhipeng; Cai, Linyan; Zhou, Guixuan; He, Wanzhong; Paus, Ralf; Yue, Zhicao

    2015-03-01

    Chemotherapeutic agents induce complex tissue responses in vivo and damage normal organ functions. Here we use the feather follicle to investigate details of this damage response. We show that cyclophosphamide treatment, which causes chemotherapy-induced alopecia in mice and man, induces distinct defects in feather formation: feather branching is transiently and reversibly disrupted, thus leaving a morphological record of the impact of chemotherapeutic agents, whereas the rachis (feather axis) remains unperturbed. Similar defects are observed in feathers treated with 5-fluorouracil or taxol but not with doxorubicin or arabinofuranosyl cytidine (Ara-C). Selective blockade of cell proliferation was seen in the feather branching area, along with a downregulation of sonic hedgehog (Shh) transcription, but not in the equally proliferative rachis. Local delivery of the Shh inhibitor, cyclopamine, or Shh silencing both recapitulated this effect. In mouse hair follicles, those chemotherapeutic agents that disrupted feather formation also downregulated Shh gene expression and induced hair loss, whereas doxorubicin or Ara-C did not. Our results reveal a mechanism through which chemotherapeutic agents damage rapidly proliferating epithelial tissue, namely via the cell population-specific, Shh-dependent inhibition of proliferation. This mechanism may be targeted by future strategies to manage chemotherapy-induced tissue damage.

  9. Heparan Sulfate Proteoglycans Containing a Glypican 5 Core and 2-O-Sulfo-iduronic Acid Function as Sonic Hedgehog Co-receptors to Promote Proliferation*♦

    PubMed Central

    Witt, Rochelle M.; Hecht, Marie-Lyn; Pazyra-Murphy, Maria F.; Cohen, Samuel M.; Noti, Christian; van Kuppevelt, Toin H.; Fuller, Maria; Chan, Jennifer A.; Hopwood, John J.; Seeberger, Peter H.; Segal, Rosalind A.

    2013-01-01

    Sonic Hedgehog (Shh) signaling is crucial for growth, cell fate determination, and axonal guidance in the developing nervous system. Although the receptors Patched (Ptch1) and Smoothened (Smo) are required for Shh signaling, a number of distinct co-receptors contribute to these critical responses to Shh. Several membrane-embedded proteins such as Boc, Cdo, and Gas1 bind Shh and promote signaling. In addition, heparan sulfate proteoglycans (HSPGs) have also been implicated in the initiation of Shh responses. However, the attributes of HSPGs that function as co-receptors for Shh have not yet been defined. Here, we identify HSPGs containing a glypican 5 core protein and 2-O-sulfo-iduronic acid residues at the nonreducing ends of the glycans as co-receptors for Shh. These HSPG co-receptors are expressed by cerebellar granule cell precursors and promote Shh binding and signaling. At the subcellular level, these HSPG co-receptors are located adjacent to the primary cilia that act as Shh signaling organelles. Thus, Shh binds to HSPG co-receptors containing a glypican 5 core and 2-O-sulfo-iduronic acid to promote neural precursor proliferation. PMID:23867465

  10. Heparan sulfate proteoglycans containing a glypican 5 core and 2-O-sulfo-iduronic acid function as Sonic Hedgehog co-receptors to promote proliferation.

    PubMed

    Witt, Rochelle M; Hecht, Marie-Lyn; Pazyra-Murphy, Maria F; Cohen, Samuel M; Noti, Christian; van Kuppevelt, Toin H; Fuller, Maria; Chan, Jennifer A; Hopwood, John J; Seeberger, Peter H; Segal, Rosalind A

    2013-09-06

    Sonic Hedgehog (Shh) signaling is crucial for growth, cell fate determination, and axonal guidance in the developing nervous system. Although the receptors Patched (Ptch1) and Smoothened (Smo) are required for Shh signaling, a number of distinct co-receptors contribute to these critical responses to Shh. Several membrane-embedded proteins such as Boc, Cdo, and Gas1 bind Shh and promote signaling. In addition, heparan sulfate proteoglycans (HSPGs) have also been implicated in the initiation of Shh responses. However, the attributes of HSPGs that function as co-receptors for Shh have not yet been defined. Here, we identify HSPGs containing a glypican 5 core protein and 2-O-sulfo-iduronic acid residues at the nonreducing ends of the glycans as co-receptors for Shh. These HSPG co-receptors are expressed by cerebellar granule cell precursors and promote Shh binding and signaling. At the subcellular level, these HSPG co-receptors are located adjacent to the primary cilia that act as Shh signaling organelles. Thus, Shh binds to HSPG co-receptors containing a glypican 5 core and 2-O-sulfo-iduronic acid to promote neural precursor proliferation.

  11. Developmental hypothyroxinemia and hypothyroidism reduce proliferation of cerebellar granule neuron precursors in rat offspring by downregulation of the sonic hedgehog signaling pathway.

    PubMed

    Wang, Yuan; Wang, Yi; Dong, Jing; Wei, Wei; Song, Binbin; Min, Hui; Yu, Ye; Lei, Xibing; Zhao, Ming; Teng, Weiping; Chen, Jie

    2014-06-01

    Iodine deficiency (ID)-induced hypothyroxinemia and hypothyroidism during development result in dysfunction of the central nervous system, affecting psychomotor and motor function, although the underlying mechanisms causing these alterations are still unclear. Therefore, our aim is to study the effects of developmental hypothyroxinemia, caused by mild ID, and developmental hypothyroidism, caused by severe ID or methimazole (MMZ), on the proliferation of cerebellar granule neuron precursors (CGNPs), an excellent experimental model of cerebellar development and function. The sonic hedgehog (Shh) signaling pathway is essential for CGNP proliferation, and as such, its activation is also investigated here. A maternal hypothyroxinemia model was established in Wistar rats by administrating a mild ID diet, and two maternal hypothyroidism models were developed either by administrating a severe ID diet or MMZ water. Our results showed that hypothyroxinemia and hypothyroidism reduced proliferation of CGNPs on postnatal day (PN) 7, PN14, and PN21. Accordingly, the mean intensity of proliferating cell nuclear antigen and Ki67 nuclear antigen immunofluorescence was reduced in the mild ID, severe ID, and MMZ groups. Moreover, maternal hypothyroxinemia and hypothyroidism reduced expression of the Shh signaling pathway on PN7, PN14, and PN21. Our study supports the hypothesis that developmental hypothyroxinemia induced by mild ID, and hypothyroidism induced by severe ID or MMZ, reduce the proliferation of CGNPs, which may be ascribed to the downregulation of the Shh signaling pathway.

  12. Anti-apoptotic effects of Sonic hedgehog signalling through oxidative stress reduction in astrocytes co-cultured with excretory-secretory products of larval Angiostrongylus cantonensis.

    PubMed

    Chen, Kuang-Yao; Chiu, Cheng-Hsun; Wang, Lian-Chen

    2017-02-07

    Angiostrongylus cantonensis, the rat lungworm, is an important aetiologic agent of eosinophilic meningitis and meningoencephalitis in humans. Co-culturing astrocytes with soluble antigens of A. cantonensis activated the Sonic hedgehog (Shh) signalling pathway and inhibited the apoptosis of astrocytes via the activation of Bcl-2. This study was conducted to determine the roles of the Shh signalling pathway, apoptosis, and oxidative stress in astrocytes after treatment with excretory-secretory products (ESP) from A. cantonensis fifth-stage larvae. Although astrocyte viability was significantly decreased after ESP treatment, the expression of Shh signalling pathway related proteins (Shh, Ptch-1 and Gli-1) was significantly increased. However, apoptosis in astrocytes was significantly decreased after activation of the Shh signalling pathway. Moreover, superoxide and hydrogen superoxide levels in astrocytes were significantly reduced after the activation of Shh pathway signalling due to increasing levels of the antioxidants catalase and superoxide dismutase. These findings indicate that the anti-apoptotic effects of the Shh signalling pathway in the astrocytes of mice infected with A. cantonensis are due to reduced levels of oxidative stress caused by the activation of antioxidants.

  13. Mature salivary gland rests within sonic hedgehog-positive medulloblastoma: case report and insights into the molecular genetics and embryopathology of ectopic intracranial salivary gland analogs.

    PubMed

    Shammassian, Berje; Manjila, Sunil; Cox, Efrem; Onwuzulike, Kaine; Wang, Dehua; Rodgers, Mark; Stearns, Duncan; Selman, Warren R

    2016-12-01

    Intracranial ectopic salivary gland rests within dural-based lesions are reported very infrequently in the literature. The authors report the unique case of a 12-year-old boy with a cerebellar medulloblastoma positive for sonic hedgehog (Shh) that contained intraaxial mature ectopic salivary gland rests. The patient underwent clinical and radiological monitoring postoperatively, until he died of disseminated disease. An autopsy showed no evidence of salivary glands within disseminated lesions. The intraaxial presence of salivary gland rests and concomitant Shh positivity of the described tumor point to a disorder in differentiation as opposed to ectopic developmental foci, which are uniformly dural based in the described literature. The authors demonstrate the characteristic "papilionaceous" appearance of the salivary glands with mucicarmine stain and highlight the role of Shh signaling in explaining the intraaxial presence of seromucous gland analogs. This article reports the first intraaxial posterior fossa tumor with heterotopic salivary gland rests, and it provides molecular and embryopathological insights into the development of these lesions.

  14. Noggin-Mediated Retinal Induction Reveals a Novel Interplay Between Bone Morphogenetic Protein Inhibition, Transforming Growth Factor β, and Sonic Hedgehog Signaling.

    PubMed

    Messina, Andrea; Lan, Lei; Incitti, Tania; Bozza, Angela; Andreazzoli, Massimiliano; Vignali, Robert; Cremisi, Federico; Bozzi, Yuri; Casarosa, Simona

    2015-08-01

    It has long been known that the depletion of bone morphogenetic protein (BMP) is one of the key factors necessary for the development of anterior neuroectodermal structures. However, the precise molecular mechanisms that underlie forebrain regionalization are still not completely understood. Here, we show that Noggin1 is involved in the regionalization of anterior neural structures in a dose-dependent manner. Low doses of Noggin1 expand prosencephalic territories, while higher doses specify diencephalic and retinal regions at the expense of telencephalic areas. A similar dose-dependent mechanism determines the ability of Noggin1 to convert pluripotent cells in prosencephalic or diencephalic/retinal precursors, as shown by transplant experiments and molecular analyses. At a molecular level, the strong inhibition of BMP signaling exerted by high doses of Noggin1 reinforces the Nodal/transforming growth factor (TGF)β signaling pathway, leading to activation of Gli1 and Gli2 and subsequent activation of Sonic Hedgehog (SHH) signaling. We propose a new role for Noggin1 in determining specific anterior neural structures by the modulation of TGFβ and SHH signaling.

  15. Network modeling of TGFβ signaling in hepatocellular carcinoma epithelial-to-mesenchymal transition reveals joint sonic hedgehog and Wnt pathway activation.

    PubMed

    Steinway, Steven Nathaniel; Zañudo, Jorge G T; Ding, Wei; Rountree, Carl Bart; Feith, David J; Loughran, Thomas P; Albert, Reka

    2014-11-01

    Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by cancer cells to leave the primary tumor site, invade surrounding tissue, and establish distant metastases. A hallmark of EMT is the loss of E-cadherin expression, and one major signal for the induction of EMT is TGFβ, which is dysregulated in up to 40% of hepatocellular carcinoma (HCC). We have constructed an EMT network of 70 nodes and 135 edges by integrating the signaling pathways involved in developmental EMT and known dysregulations in invasive HCC. We then used discrete dynamic modeling to understand the dynamics of the EMT network driven by TGFβ. Our network model recapitulates known dysregulations during the induction of EMT and predicts the activation of the Wnt and Sonic hedgehog (SHH) signaling pathways during this process. We show, across multiple murine (P2E and P2M) and human HCC cell lines (Huh7, PLC/PRF/5, HLE, and HLF), that the TGFβ signaling axis is a conserved driver of mesenchymal phenotype HCC and confirm that Wnt and SHH signaling are induced in these cell lines. Furthermore, we identify by network analysis eight regulatory feedback motifs that stabilize the EMT process and show that these motifs involve cross-talk among multiple major pathways. Our model will be useful in identifying potential therapeutic targets for the suppression of EMT, invasion, and metastasis in HCC.

  16. Anti-apoptotic effects of Sonic hedgehog signalling through oxidative stress reduction in astrocytes co-cultured with excretory-secretory products of larval Angiostrongylus cantonensis

    PubMed Central

    Chen, Kuang-Yao; Chiu, Cheng-Hsun; Wang, Lian-Chen

    2017-01-01

    Angiostrongylus cantonensis, the rat lungworm, is an important aetiologic agent of eosinophilic meningitis and meningoencephalitis in humans. Co-culturing astrocytes with soluble antigens of A. cantonensis activated the Sonic hedgehog (Shh) signalling pathway and inhibited the apoptosis of astrocytes via the activation of Bcl-2. This study was conducted to determine the roles of the Shh signalling pathway, apoptosis, and oxidative stress in astrocytes after treatment with excretory-secretory products (ESP) from A. cantonensis fifth-stage larvae. Although astrocyte viability was significantly decreased after ESP treatment, the expression of Shh signalling pathway related proteins (Shh, Ptch-1 and Gli-1) was significantly increased. However, apoptosis in astrocytes was significantly decreased after activation of the Shh signalling pathway. Moreover, superoxide and hydrogen superoxide levels in astrocytes were significantly reduced after the activation of Shh pathway signalling due to increasing levels of the antioxidants catalase and superoxide dismutase. These findings indicate that the anti-apoptotic effects of the Shh signalling pathway in the astrocytes of mice infected with A. cantonensis are due to reduced levels of oxidative stress caused by the activation of antioxidants. PMID:28169282

  17. A variant in the sonic hedgehog regulatory sequence (ZRS) is associated with triphalangeal thumb and deregulates expression in the developing limb

    PubMed Central

    Furniss, Dominic; Lettice, Laura A.; Taylor, Indira B.; Critchley, Paul S.; Giele, Henk; Hill, Robert E.; Wilkie, Andrew O.M.

    2008-01-01

    A locus for triphalangeal thumb, variably associated with pre-axial polydactyly, was previously identified in the zone of polarizing activity regulatory sequence (ZRS), a long range limb-specific enhancer of the Sonic Hedgehog (SHH) gene at human chromosome 7q36.3. Here, we demonstrate that a 295T>C variant in the human ZRS, previously thought to represent a neutral polymorphism, acts as a dominant allele with reduced penetrance. We found this variant in three independently ascertained probands from southern England with triphalangeal thumb, demonstrated significant linkage of the phenotype to the variant (LOD = 4.1), and identified a shared microsatellite haplotype around the ZRS, suggesting that the probands share a common ancestor. An individual homozygous for the 295C allele presented with isolated bilateral triphalangeal thumb resembling the heterozygous phenotype, suggesting that the variant is largely dominant to the wild-type allele. As a functional test of the pathogenicity of the 295C allele, we utilized a mutated ZRS construct to demonstrate that it can drive ectopic anterior expression of a reporter gene in the developing mouse forelimb. We conclude that the 295T>C variant is in fact pathogenic and, in southern England, appears to be the most common cause of triphalangeal thumb. Depending on the dispersal of the founding mutation, it may play a wider role in the aetiology of this disorder. PMID:18463159

  18. Overexpression of sonic hedgehog in the triple negative breast cancer: clinicopathological characteristics of high burden breast cancer patients from Bangladesh.

    PubMed

    Noman, A S; Uddin, M; Rahman, M Z; Nayeem, M J; Alam, S S; Khatun, Z; Wahiduzzaman, M; Sultana, A; Rahman, M L; Ali, M Y; Barua, D; Ahmed, I; Islam, M S; Aboussekhra, A; Yeger, H; Farhat, W A; Islam, S S

    2016-01-05

    Dysregulation of Hedgehog (Hh) signaling pathway has been documented in mammary gland development and breast cancer (BC) progression. Despite the remarkable progress in therapeutic interventions, BC related mortality in Bangladesh increased in the last decade. Triple negative breast cancer (TNBC) still presents a critical therapeutic challenge. Thus effective targeted therapy is urgently needed. In this study, we report the clinicopathological characteristics and prognosis of BC patients from Bangladesh. Routine immunohistochemical analysis and high throughput RNA-Seq data from the TCGA library were used to analyze the expression pattern and association of high and low level of Shh expression in a collection of BC patients with a long-term follow-up. High levels of Shh were observed in a subset of BC tumors with poor prognostic pathological features. Higher level of Shh expression correlated with a significantly poorer overall survival of patients compared with patients whose tumors expressed a low level of Shh. These data support the contention that Shh could be a novel biomarker for breast cancer that is involved in mediating the aggressive phenotype of BC. We propose that BC patients exhibiting a higher level of Shh expression, representing a subset of BC patients, would be amenable to Shh targeted therapy.

  19. Positive and Negative Regulation of Muscle Cell Identity by Members of the hedgehog and TGF-β Gene Families

    PubMed Central

    Du, Shao Jun; Devoto, Stephen H.; Westerfield, Monte; Moon, Randall T.

    1997-01-01

    We have examined whether the development of embryonic muscle fiber type is regulated by competing influences between Hedgehog and TGF-β signals, as previously shown for development of neuronal cell identity in the neural tube. We found that ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite. Ectopic expression in the notochord of Dorsalin-1, a member of the TGF-β superfamily, inhibits the formation of muscle pioneer cells, demonstrating that TGF-β signals can antagonize the induction of muscle pioneer cells by Hedgehog. We propose that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-β signals exert competing positive and negative influences on the development of muscle pioneer cells. PMID:9314535

  20. Hedgehog antagonist REN(KCTD11) regulates proliferation and apoptosis of developing granule cell progenitors.

    PubMed

    Argenti, Beatrice; Gallo, Rita; Di Marcotullio, Lucia; Ferretti, Elisabetta; Napolitano, Maddalena; Canterini, Sonia; De Smaele, Enrico; Greco, Azzura; Fiorenza, Maria Teresa; Maroder, Marella; Screpanti, Isabella; Alesse, Edoardo; Gulino, Alberto

    2005-09-07

    During the early development of the cerebellum, a burst of granule cell progenitor (GCP) proliferation occurs in the outer external granule layer (EGL), which is sustained mainly by Purkinje cell-derived Sonic Hedgehog (Shh). Shh response is interrupted once GCPs move into the inner EGL, where granule progenitors withdraw proliferation and start differentiating and migrating toward the internal granule layer (IGL). Failure to interrupt Shh signals results in uncoordinated proliferation and differentiation of GCPs and eventually leads to malignancy (i.e., medulloblastoma). The Shh inhibitory mechanisms that are responsible for GCP growth arrest and differentiation remain unclear. Here we report that REN, a putative tumor suppressor frequently deleted in human medulloblastoma, is expressed to a higher extent in nonproliferating inner EGL and IGL granule cells than in highly proliferating outer EGL cells. Accordingly, upregulated REN expression occurs along GCP differentiation in vitro, and, in turn, REN overexpression promotes growth arrest and increases the proportion of p27/Kip1+ GCPs. REN also impairs both Gli2-dependent gene transcription and Shh-enhanced expression of the target Gli1 mRNA, thus antagonizing the Shh-induced effects on the proliferation and differentiation of cultured GCPs. Conversely, REN functional knock-down impairs Hedgehog antagonism and differentiation and sustains the proliferation of GCPs. Finally, REN enhances caspase-3 activation and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling apoptotic GCP numbers; therefore, the pattern of REN expression, its activity, and its antagonism on the Hedgehog pathway suggest that this gene may represent a restraint of Shh signaling at the outer to inner EGL GCP transitions. Medulloblastoma-associated REN loss of function might withdraw such a limiting signal for immature cell expansion, thus favoring tumorigenesis.

  1. Seminiferous cord formation is regulated by hedgehog signaling in the marsupial.

    PubMed

    Chung, Jin Wei; Pask, Andrew J; Renfree, Marilyn B

    2012-03-01

    The signaling molecule DHH, secreted by Sertoli cells, has essential regulatory functions in testicular differentiation. DHH is required for the differentiation of peritubular myoid cells that line the seminiferous cords and steroidogenic Leydig cells. The testicular cords in Dhh-null male mice lack a basal lamina and develop abnormally. To date, the DHH-signaling pathway has never been examined outside of any eutherian mammals. This study examined the effects of inhibition of DHH signaling in a marsupial mammal, the tammar wallaby, by culturing gonads in vitro in the presence of the hedgehog-signaling inhibitors cyclopamine and forskolin. Disruption of hedgehog signaling in the tammar testes caused highly disorganized cord formation. SOX9 protein remained strongly expressed in Sertoli cells, laminin distribution was highly fragmented, and germ cells were distributed around the cortical regions of treated testes in an ovarianlike morphology. This suggests that hedgehog signaling regulates cord formation in the tammar wallaby testis as it does in eutherian mammals. These data demonstrate that the hedgehog pathway has been highly conserved in mammals for at least 160 million years.

  2. Sonic hedgehog inhibitors prevent colitis-associated cancer via orchestrated mechanisms of IL-6/gp130 inhibition, 15-PGDH induction, Bcl-2 abrogation, and tumorsphere inhibition.

    PubMed

    Kangwan, Napapan; Kim, Yoon-Jae; Han, Young Min; Jeong, Migyeong; Park, Jong-Min; Go, Eun-Jin; Hahm, Ki-Baik

    2016-02-16

    Sonic hedgehog (SHH) signaling is essential in normal development of the gastrointestinal (GI) tract, whereas aberrantly activated SHH is implicated in GI cancers because it facilitates carcinogenesis by redirecting stem cells. Since colitis-associated cancer (CAC) is associated with inflammatory bowel diseases, in which SHH and IL-6 signaling, inflammation propagation, and cancer stem cell (CSC) activation have been implicated, we hypothesized that SHH inhibitors may prevent CAC by blocking the above SHH-related carcinogenic pathways. In the intestinal epithelial cells IEC-6 and colon cancer cells HCT-116, IL-6 expression and its signaling were assessed with SHH inhibitors and levels of other inflammatory mediators, proliferation, apoptosis, tumorsphere formation, and tumorigenesis were also measured. CAC was induced in C57BL/6 mice by administration of azoxymethane followed by dextran sodium sulfate administration. SHH inhibitors were administered by oral gavage and the mice were sacrificed at 16 weeks. TNF-α-stimulated IEC-6 cells exhibited increased levels of proinflammatory cytokines and enzymes, whereas SHH inhibitors suppressed TNF-α-induced inflammatory signaling, especially IL-6/IL-6R/gp130 signaling. SHH inhibitors significantly induced apoptosis, inhibited cell proliferation, suppressed tumorsphere formation, and reduced stemness factors. In the mouse model, SHH inhibitors significantly reduced tumor incidence and multiplicity, decreased the expression of IL-6, TNF-α, COX-2, STAT3, and NF-κB, and significantly induced apoptosis. In colosphere xenografts, SHH inhibitor significantly suppressed tumorigenesis by inhibiting tumorsphere formation. Taken together, our data suggest that administration of SHH inhibitors could be an effective strategy to prevent colitis-induced colorectal carcinogenesis, mainly by targeting IL-6 signaling, ablating CSCs, and suppressing oncogenic inflammation, achieving chemoquiescence ultimately.

  3. PI3K/AKT/mTOR and sonic hedgehog pathways cooperate together to inhibit human pancreatic cancer stem cell characteristics and tumor growth.

    PubMed

    Sharma, Narinder; Nanta, Rajesh; Sharma, Jay; Gunewardena, Sumedha; Singh, Karan P; Shankar, Sharmila; Srivastava, Rakesh K

    2015-10-13

    Cancer stem cells (CSCs) play major roles in cancer initiation, progression, and metastasis. It is evident from growing reports that PI3K/Akt/mTOR and Sonic Hedgehog (Shh) signaling pathways are aberrantly reactivated in pancreatic CSCs. Here, we examined the efficacy of combining NVP-LDE-225 (PI3K/mTOR inhibitor) and NVP-BEZ-235 (Smoothened inhibitor) on pancreatic CSCs characteristics, microRNA regulatory network, and tumor growth. NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting pancreatic CSC's characteristics and tumor growth in mice by acting at the level of Gli. Combination of NVP-LDE-225 and NVP-BEZ-235 inhibited self-renewal capacity of CSCs by suppressing the expression of pluripotency maintaining factors Nanog, Oct-4, Sox-2 and c-Myc, and transcription of Gli. NVP-LDE-225 co-operated with NVP-BEZ-235 to inhibit Lin28/Let7a/Kras axis in pancreatic CSCs. Furthermore, a superior interaction of these drugs was observed on spheroid formation by pancreatic CSCs isolated from Pankras/p53 mice. The combination of these drugs also showed superior effects on the expression of proteins involved in cell proliferation, survival and apoptosis. In addition, NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting EMT through modulation of cadherin, vimentin and transcription factors Snail, Slug and Zeb1. In conclusion, these data suggest that the combined inhibition of PI3K/Akt/mTOR and Shh pathways may be beneficial for the treatment of pancreatic cancer.

  4. The differentiation of amniotic fluid stem cells into sweat glandlike cells is enhanced by the presence of Sonic hedgehog in the conditioned medium.

    PubMed

    Liang, Hansi; Sun, Qing; Zhen, Yunfang; Li, Fang; Xu, YunYun; Liu, Yao; Zhang, Xueguang; Qin, Mingde

    2016-09-01

    After patients suffer severe full-thickness burn injuries, the current treatments cannot lead to the complete self-regeneration of the sweat gland structure and function. Therefore, it is important to identify new methods for acquiring sufficient functional sweat gland cells to restore skin function. In this study, we induced CD117+ human amniotic fluid stem (hAFS) cells to differentiate into sweat glandlike (hAFS-SG) cells based on the use of conditioned medium (CM) from the human sweat gland (hSG) cells. Real-time PCR and immunofluorescent staining were used to confirm the expression of the sweat gland-related genes Ectodysplasin-A (EDA), Ectodysplasin-A receptor (EDAR), keratin 8 (K8) and carcino-embryonic antigen (CEA). Transmission electron microscopy analysis shows that microvilli, the cellular structures that are typical for hSG cells, can also be observed on the membrane of the hAFS-SG cells. Our test for the calcium response to acetylcholine (Ach) proved that hAFS-SG cells have the potential to respond to Ach in a manner similar to normal sweat glands. A three-dimensional culture is an effective approach that stimulates the hAFS-SG cells to form tubular structures and drives hAFS-SG cells to mature into higher stage. We also found that epidermal growth factor enhances the efficiency of differentiation and that Sonic hedgehog is an important factor of the CM that influences sweat gland differentiation. Our study provides the basis for further investigations into novel methods of inducing stem cells to differentiate into sweat glandlike cells.

  5. Inhibitor of Differentiation-1 and Hypoxia-Inducible Factor-1 Mediate Sonic Hedgehog Induction by Amyloid Beta-Peptide in Rat Cortical Neurons.

    PubMed

    Hung, Yu-Hsing; Chang, Shih-Hsin; Huang, Chao-Tzu; Yin, Jiu-Haw; Hwang, Chi-Shin; Yang, Liang-Yo; Yang, Ding-I

    2016-03-01

    One major pathological hallmark of Alzheimer's disease (AD) is the accumulation of senile plaques mainly composed of neurotoxic amyloid beta-peptide (Aβ) in the patients' brains. Sonic hedgehog (SHH) is a morphogen critically involved in the embryonic development of the central nervous system (CNS). In the present study, we tested whether Aβ may induce SHH expression and explored its underlying mechanisms. We found that both Aβ25-35 and Aβ1-42 enhanced SHH expression in the primary cortical neurons derived from fetal rat brains. Immunohistochemistry revealed heightened expression of SHH in the cortex and hippocampus of aged (9 and 12 months old) AD transgenic mouse brains as compared to age-matched littermate controls. Chromatin immunoprecipitation (ChIP) assay demonstrated that Aβ25-35 enhanced binding of hypoxia-inducible factor-1 (HIF-1) to the promoter of the Shh gene in primary cortical cultures; consistently, Aβ25-35 induction of SHH was abolished by HIF-1α small interfering RNA (siRNA). Aβ25-35 also time-dependently induced inhibitor of differentiation-1 (Id1) that has been shown to stabilize HIF-1α; further, Aβ25-35-mediated induction of HIF-1α and SHH was both suppressed by Id1 siRNA. Pharmacological induction of HIF-1α by cobalt chloride and application of the cell-permeable recombinant Id1 proteins were both sufficient to induce SHH expression. Finally, both the SHH pathway inhibitor cyclopamine and its neutralizing antibody attenuated Aβ cytotoxicity, albeit to a minor extent. These results thus established a signaling cascade of "Aβ → Id1 → HIF-1 → SHH" in primary rat cortical cultures; furthermore, SHH may in part contribute to Aβ neurotoxicity.

  6. Comparison of cortical and white matter traumatic brain injury models reveals differential effects in the subventricular zone and divergent Sonic hedgehog signaling pathways in neuroblasts and oligodendrocyte progenitors.

    PubMed

    Mierzwa, Amanda J; Sullivan, Genevieve M; Beer, Laurel A; Ahn, Sohyun; Armstrong, Regina C

    2014-01-01

    The regenerative capacity of the central nervous system must be optimized to promote repair following traumatic brain injury (TBI) and may differ with the site and form of damage. Sonic hedgehog (Shh) maintains neural stem cells and promotes oligodendrogenesis. We examined whether Shh signaling contributes to neuroblast (doublecortin) or oligodendrocyte progenitor (neural/glial antigen 2 [NG2]) responses in two distinct TBI models. Shh-responsive cells were heritably labeled in vivo using Gli1-CreER(T2);R26-YFP bitransgenic mice with tamoxifen administration on Days 2 and 3 post-TBI. Injury to the cerebral cortex was produced with mild controlled cortical impact. Yellow fluorescent protein (YFP) cells decreased in cortical lesions. Total YFP cells increased in the subventricular zone (SVZ), indicating Shh pathway activation in SVZ cells, including doublecortin-labeled neuroblasts. The alternate TBI model produced traumatic axonal injury in the corpus callosum. YFP cells decreased within the SVZ and were rarely double labeled as NG2 progenitors. NG2 progenitors increased in the cortex, with a similar pattern in the corpus callosum. To further test the potential of NG2 progenitors to respond through Shh signaling, Smoothened agonist was microinjected into the corpus callosum to activate Shh signaling. YFP cells and NG2 progenitors increased in the SVZ but were not double labeled. This result indicates that either direct Smoothened activation in NG2 progenitors does not signal through Gli1 or that Smoothened agonist acts indirectly to increase NG2 progenitors. Therefore, in all conditions, neuroblasts exhibited differential Shh pathway utilization compared with oligodendrocyte progenitors. Notably, cortical versus white matter damage from TBI produced opposite responses of Shh-activated cells within the SVZ.

  7. Structural basis of SUFU-GLI interaction in human Hedgehog signalling regulation.

    PubMed

    Cherry, Amy L; Finta, Csaba; Karlström, Mikael; Jin, Qianren; Schwend, Thomas; Astorga-Wells, Juan; Zubarev, Roman A; Del Campo, Mark; Criswell, Angela R; de Sanctis, Daniele; Jovine, Luca; Toftgård, Rune

    2013-12-01

    Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

  8. Structural basis of SUFU–GLI interaction in human Hedgehog signalling regulation

    PubMed Central

    Cherry, Amy L.; Finta, Csaba; Karlström, Mikael; Jin, Qianren; Schwend, Thomas; Astorga-Wells, Juan; Zubarev, Roman A.; Del Campo, Mark; Criswell, Angela R.; de Sanctis, Daniele; Jovine, Luca; Toftgård, Rune

    2013-01-01

    Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU–GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU–GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics. PMID:24311597

  9. Structural basis of SUFU–GLI interaction in human Hedgehog signalling regulation

    SciTech Connect

    Cherry, Amy L.; Finta, Csaba; Karlström, Mikael; Jin, Qianren; Schwend, Thomas; Astorga-Wells, Juan; Zubarev, Roman A.; Del Campo, Mark; Criswell, Angela R.; Sanctis, Daniele de; Jovine, Luca Toftgård, Rune

    2013-12-01

    Crystal and small-angle X-ray scattering structures of full-length human SUFU alone and in complex with the conserved SYGHL motif from GLI transcription factors show major conformational changes associated with binding and reveal an intrinsically disordered region crucial for pathway activation. Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU–GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU–GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

  10. Inhibition of the CyclinD1 promoter in response to sonic hedgehog signaling pathway transduction is mediated by Gli1.

    PubMed

    Lin, Zhongxiao; Sheng, Hansong; You, Chaoguo; Cai, Ming; Zhang, Yiping; Yu, Li Sheng; Yu, Xiaoming; Lin, Jian; Zhang, Nu

    2017-01-01

    Medulloblastoma (MB) is the most common malignant tumor of the central nervous system in children. Accumulating evidence suggests a major role for the activation of the sonic hedgehog (SHH) signaling pathway in the development of MB cells; however, the mechanisms underlying the effect of this pathway on tumor survival and growth remain poorly understood. The Gli family zinc finger 1 (Gli1) transcription factor is considered as a mediator of the SHH signaling pathway in MB cells. Therefore, the present study investigated whether the SHH signaling pathway promotes the apoptosis of MB cells via downregulation of Gli1. GANT61, a novel Gli1 inhibitor, is known to have an in vitro activity against tumors. In the current study, Daoy cells were treated with different concentrations of GANT61 for 24 h, and the effect on cell proliferation was assayed by cell counting kit-8 assay. In addition, the cell cycle progression and apoptosis were assayed by flow cytometry analysis and hematoxylin-eosin (HE) staining. The effects of GANT61 treatment on SHH signaling pathway at the mRNA level were assayed by polymerase chain reaction (PCR). To further elucidate the inhibitory effects of GANT61 on the expression of Gli1 and CyclinD1, their protein levels were examined by western blot and immunofluorescence. The results indicated that GANT61 significantly inhibited the proliferation of Daoy cells in a dose-dependent manner, compared with the control group (P<0.05). HE staining revealed that cells had increasingly abnormal protuberance with increasing GANT61 concentration. Flow cytometry analysis also demonstrated that GANT61 induced G1/S arrest and apoptosis of Daoy cells in a dose-dependent manner (P<0.05). Gli1 and CyclinD1 mRNA expression levels were downregulated by GANT61 treatment (P<0.05); similarly, their protein levels were downregulated by GANT61 treatment in a dose-dependent manner (P<0.05). In conclusion, Gli1 expression was significantly associated with CyclinD1 expression

  11. Inhibition of the CyclinD1 promoter in response to sonic hedgehog signaling pathway transduction is mediated by Gli1

    PubMed Central

    Lin, Zhongxiao; Sheng, Hansong; You, Chaoguo; Cai, Ming; Zhang, Yiping; Yu, Li Sheng; Yu, Xiaoming; Lin, Jian; Zhang, Nu

    2017-01-01

    Medulloblastoma (MB) is the most common malignant tumor of the central nervous system in children. Accumulating evidence suggests a major role for the activation of the sonic hedgehog (SHH) signaling pathway in the development of MB cells; however, the mechanisms underlying the effect of this pathway on tumor survival and growth remain poorly understood. The Gli family zinc finger 1 (Gli1) transcription factor is considered as a mediator of the SHH signaling pathway in MB cells. Therefore, the present study investigated whether the SHH signaling pathway promotes the apoptosis of MB cells via downregulation of Gli1. GANT61, a novel Gli1 inhibitor, is known to have an in vitro activity against tumors. In the current study, Daoy cells were treated with different concentrations of GANT61 for 24 h, and the effect on cell proliferation was assayed by cell counting kit-8 assay. In addition, the cell cycle progression and apoptosis were assayed by flow cytometry analysis and hematoxylin-eosin (HE) staining. The effects of GANT61 treatment on SHH signaling pathway at the mRNA level were assayed by polymerase chain reaction (PCR). To further elucidate the inhibitory effects of GANT61 on the expression of Gli1 and CyclinD1, their protein levels were examined by western blot and immunofluorescence. The results indicated that GANT61 significantly inhibited the proliferation of Daoy cells in a dose-dependent manner, compared with the control group (P<0.05). HE staining revealed that cells had increasingly abnormal protuberance with increasing GANT61 concentration. Flow cytometry analysis also demonstrated that GANT61 induced G1/S arrest and apoptosis of Daoy cells in a dose-dependent manner (P<0.05). Gli1 and CyclinD1 mRNA expression levels were downregulated by GANT61 treatment (P<0.05); similarly, their protein levels were downregulated by GANT61 treatment in a dose-dependent manner (P<0.05). In conclusion, Gli1 expression was significantly associated with CyclinD1 expression

  12. Hedgehog-PKA Signaling and gnrh3 Regulate the Development of Zebrafish gnrh3 Neurons

    PubMed Central

    Kuo, Ming-Wei; Lou, Show-Wan; Chung, Bon-chu

    2014-01-01

    GnRH neurons secrete GnRH that controls the development of the reproduction system. Despite many studies, the signals controlling the development of GnRH neurons from its progenitors have not been fully established. To understand the development of GnRH neurons, we examined the development of gnrh3-expressing cells using a transgenic zebrafish line that expresses green fluorescent protein (GFP) and LacZ driven by the gnrh3 promoter. GFP and LacZ expression recapitulated that of gnrh3 in the olfactory region, olfactory bulb and telencephalon. Depletion of gnrh3 by morpholinos led to a reduction of GFP- and gnrh3-expressing cells, while over-expression of gnrh3 mRNA increased the number of these cells. This result indicates a positive feed-forward regulation of gnrh3 cells by gnrh3. The gnrh3 cells were absent in embryos that lack Hedgehog signaling, but their numbers were increased in embryos overexpressing shhb. We manipulated the amounts of kinase that antagonizes the Hedgehog signaling pathway, protein kinase A (PKA), by treating embryos with PKA activator forskolin or by injecting mRNAs encoding its constitutively active catalytic subunit (PKA*) and dominant negative regulatory subunit (PKI) into zebrafish embryos. PKA* misexpression or forskolin treatment decreased GFP cell numbers, while PKI misexpression led to ectopic production of GFP cells. Our data indicate that the Hedgehog-PKA pathway participates in the development of gnrh3-expressing neurons during embryogenesis. PMID:24879419

  13. Disturbed MEK/ERK signaling increases osteoclast activity via the Hedgehog-Gli pathway in postmenopausal osteoporosis.

    PubMed

    Li, Xiaojie; Jie, Qiang; Zhang, Hongyang; Zhao, Yantao; Lin, Yangjing; Du, Junjie; Shi, Jun; Wang, Long; Guo, Kai; Li, Yong; Wang, Chunhui; Gao, Bo; Huang, Qiang; Liu, Jian; Yang, Liu; Luo, Zhuojing

    2016-11-01

    Postmenopausal osteoporosis is a worldwide health problem and is characterized by increased and activated osteoclasts. However, the mechanism by which osteoclasts are dysregulated in postmenopausal osteoporosis is not fully understood. In this study, we found that the Hedgehog-Gli pathway was upregulated in postmenopausal osteoporotic osteoclasts and that 17β-estradiol both inhibited osteoclastogenesis and induced osteoclast apoptosis by downregulating Hedgehog-Gli signaling. Furthermore, we demonstrated that the Hedgehog-Gli pathway was negatively regulated by MEK/ERK signaling and that this effect was Sonic Hedgehog (SHH)-dependent and was partially blocked by an anti-SHH antibody. Moreover, we found that the stimulatory effect of Hedgehog signaling on osteoclastogenesis and the inhibitory effect on osteoclast apoptosis were dependent on the Gli family of transcription factors. The pathways and molecules that contribute to the regulation of osteoclastogenesis and apoptosis represent potential new strategies for designing molecular drugs for the treatment of postmenopausal osteoporosis.

  14. Planarian Hedgehog/Patched establishes anterior–posterior polarity by regulating Wnt signaling

    PubMed Central

    Yazawa, Shigenobu; Umesono, Yoshihiko; Hayashi, Tetsutaro; Tarui, Hiroshi; Agata, Kiyokazu

    2009-01-01

    Despite long-standing interest, the molecular mechanisms underlying the establishment of anterior–posterior (AP) polarity remain among the unsolved mysteries in metazoans. In the planarians (a family of flatworms), canonical Wnt/β-catenin signaling is required for posterior specification, as it is in many animals. However, the molecular mechanisms regulating the posterior-specific induction of Wnt genes according to the AP polarity have remained unclear. Here, we demonstrate that Hedgehog (Hh) signaling is responsible for the establishment of AP polarity via its regulation of the transcription of Wnt family genes during planarian regeneration. We found that RNAi gene knockdown of Dugesia japonica patched (Djptc) caused ectopic tail formation in the anterior blastema of body fragments, resulting in bipolar-tails regeneration. In contrast, RNAi of hedgehog (Djhh) and gli (Djgli) caused bipolar-heads regeneration. We show that Patched-mediated Hh signaling was crucial for posterior specification, which is established by regulating the transcription of Wnt genes via downstream Gli activity. Moreover, differentiated cells were responsible for the posterior specification of undifferentiated stem cells through Wnt/β-catenin signaling. Surprisingly, Djhh was expressed in neural cells all along the ventral nerve cords (along the AP axis), but not in the posterior blastema of body fragments, where the expression of Wnt genes was induced for posteriorization. We therefore propose that Hh signals direct head or tail regeneration according to the AP polarity, which is established by Hh signaling activity along the body's preexisting nervous system. PMID:20018728

  15. Hedgehog Signaling Regulates the Ciliary Transport of Odorant Receptors in Drosophila.

    PubMed

    Sanchez, Gonzalo M; Alkhori, Liza; Hatano, Eduardo; Schultz, Sebastian W; Kuzhandaivel, Anujaianthi; Jafari, Shadi; Granseth, Björn; Alenius, Mattias

    2016-01-26

    Hedgehog (Hh) signaling is a key regulatory pathway during development and also has a functional role in mature neurons. Here, we show that Hh signaling regulates the odor response in adult Drosophila olfactory sensory neurons (OSNs). We demonstrate that this is achieved by regulating odorant receptor (OR) transport to and within the primary cilium in OSN neurons. Regulation relies on ciliary localization of the Hh signal transducer Smoothened (Smo). We further demonstrate that the Hh- and Smo-dependent regulation of the kinesin-like protein Cos2 acts in parallel to the intraflagellar transport system (IFT) to localize ORs within the cilium compartment. These findings expand our knowledge of Hh signaling to encompass chemosensory modulation and receptor trafficking.

  16. Notochord-derived hedgehog is essential for tail regeneration in Xenopus tadpole

    PubMed Central

    2014-01-01

    Background Appendage regeneration in amphibians is regulated by the combinatorial actions of signaling molecules. The requirement of molecules secreted from specific tissues is reflected by the observation that the whole process of regeneration can be inhibited if a certain tissue is removed from the amputated stump. Interestingly, urodeles and anurans show different tissue dependencies during tail regeneration. The spinal cord is essential for tail regeneration in urodele but not in anuran larva, whereas the notochord but not the spinal cord is essential for tail regeneration in anuran tadpoles. Sonic hedgehog is one of the signaling molecules responsible for such phenomenon in axolotl, as hedgehog signaling is essential for overall tail regeneration and sonic hedgehog is exclusively expressed in the spinal cord. In order to know whether hedgehog signaling is involved in the molecular mechanism underlying the inconsistent tissue dependency for tail regeneration between anurans and urodeles, we investigated expression of hedgehog signal-related genes in the regenerating tail of Xenopus tadpole and examined the effect of the hedgehog signal inhibitor, cyclopamine, on the tail regeneration. Results In Xenopus, sonic hedgehog is expressed exclusively in the notochord but not in the spinal cord of the regenerate. Overall regeneration was severely impaired in cyclopamine-treated tadpoles. Notochord maturation in the regenerate, including cell alignment and vacuolation, and myofiber formation were inhibited. Proliferation of spinal cord cells in the neural ampulla and of mesenchymal cells was also impaired. Conclusion As in the axolotl, hedgehog signaling is required for multiple steps in tail regeneration in the Xenopus tadpole, although the location of the Shh source is quite different between the two species. This difference in Shh localization is the likely basis for the differing tissue requirement for tail regeneration between urodeles and anurans. PMID:24941877

  17. Genetic and epigenetic mechanisms regulating hedgehog expression in the Drosophila wing.

    PubMed

    Bejarano, Fernando; Milán, Marco

    2009-03-15

    Stable subdivision of Drosophila limbs into Anterior (A) and Posterior (P) compartments is a consequence of asymmetric signaling by Hedgehog (Hh) from P to A cells. The activity of the homeodomain protein Engrailed (En) in P cells has been reported to help to generate this asymmetry by inducing the expression of hedgehog and simultaneously repressing the expression of the essential downstream component of the Hh signaling pathway Cubitus interruptus (Ci). In A cells, Ci has a major role in the repression of hh. Here we have revised the genetic and epigenetic mechanisms involved in the regulation of hh in the P compartment. First, we present evidence that hh expression in P cells is a consequence of the repression of ci by the activity of En. Thus, in the absence of Ci and En activities, cells do express hh. We also present data supporting the maintenance of hh expression in P cells through epigenetic mechanisms, and a permissive role of Notch signaling in this process. Notch and Trithorax (TrxG) group of proteins exert their action through a previously defined hh Polycomb Responsive Element (PRE).

  18. The Kinesin-4 Protein KIF7 Regulates Mammalian Hedgehog Signaling by Organizing the Cilia Tip Compartment

    PubMed Central

    He, Mu; Subramanian, Radhika; Bangs, Fiona; Omelchenko, Tatiana; Liem, Karel F.; Kapoor, Tarun M.; Anderson, Kathryn V.

    2014-01-01

    Mammalian Hedgehog (Hh) signal transduction requires the primary cilium, a microtubule-based organelle, and the Gli/Sufu complexes that mediate Hh signaling are enriched at cilia tips. KIF7, a kinesin-4 family protein, is a conserved regulator of the Hh signaling pathway and a human ciliopathy protein. Here we show that KIF7 localizes to cilia tips, the site of microtubule plus-ends, where it limits cilia length and controls cilia structure. Purified recombinant KIF7 binds the plus-ends of growing microtubules in vitro, where it reduces the rate of microtubule growth and increases the frequency of microtubule catastrophe. KIF7 is not required for normal intraflagellar transport or for trafficking of Hh pathway proteins into cilia. Instead, a central function of KIF7 in the mammalian Hh pathway is to control cilia architecture and to create a single cilia tip compartment where Gli/Sufu activation can be correctly regulated. PMID:24952464

  19. Regulation of Smoothened Trafficking and Hedgehog Signaling by the SUMO Pathway.

    PubMed

    Ma, Guoqiang; Li, Shuang; Han, Yuhong; Li, Shuangxi; Yue, Tao; Wang, Bing; Jiang, Jin

    2016-11-21

    Hedgehog (Hh) signaling plays a central role in development and diseases. Hh activates its signal transducer and GPCR-family protein Smoothened (Smo) by inducing Smo phosphorylation, but whether Smo is activated through other post-translational modifications remains unexplored. Here we show that sumoylation acts in parallel with phosphorylation to promote Smo cell-surface expression and Hh signaling. We find that Hh stimulates Smo sumoylation by dissociating it from a desumoylation enzyme Ulp1. Sumoylation of Smo in turn recruits a deubiquitinase UBPY/USP8 to antagonize Smo ubiquitination and degradation, leading to its cell-surface accumulation and elevated Hh pathway activity. We also provide evidence that Shh stimulates sumoylation of mammalian Smo (mSmo) and that sumoylation promotes ciliary localization of mSmo and Shh pathway activity. Our findings reveal a conserved mechanism whereby the SUMO pathway promotes Hh signaling by regulating Smo subcellular localization and shed light on how sumoylation regulates membrane protein trafficking.

  20. Indian Hedgehog Signaling Regulates Transcription and Expression of Collagen Type X via Runx2/Smads Interactions*

    PubMed Central

    Amano, Katsuhiko; Densmore, Michael; Nishimura, Riko; Lanske, Beate

    2014-01-01

    Indian hedgehog (Ihh) is essential for chondrocyte differentiation and endochondral ossification and acts with parathyroid hormone-related peptide in a negative feedback loop to regulate early chondrocyte differentiation and entry to hypertrophic differentiation. Independent of this function, we and others recently reported independent Ihh functions to promote chondrocyte hypertrophy and matrix mineralization in vivo and in vitro. However, the molecular mechanisms for these actions and their functional significance are still unknown. We recently discovered that Ihh overexpression in chondrocytes stimulated the expression of late chondrocyte differentiation markers and induced matrix mineralization. Focusing on collagen type X (Col10α1) expression and transcription, we observed that hedgehog downstream transcription factors GLI-Krüppel family members (Gli) 1/2 increased COL10A1 promoter activity and identified a novel Gli1/2 response element in the 250-bp basic promoter. In addition, we found that Ihh induced Runx2 expression in chondrocytes without up-regulating other modulators of chondrocyte maturation such as Mef2c, Foxa2, and Foxa3. Runx2 promoted Col10α1 expression in cooperation with Ihh. Further analyses using promoter assays, immunofluorescence, and binding assays showed the interaction of Gli1/2 in a complex with Runx2/Smads induces chondrocyte differentiation. Finally, we could demonstrate that Ihh promotes in vitro matrix mineralization using similar molecular mechanisms. Our data provide an in vitro mechanism for Ihh signaling to positively regulate Col10α1 transcription. Thus, Ihh signaling could be an important player for not only early chondrocyte differentiation but maturation and calcification of chondrocytes. PMID:25028519

  1. Epigenetic regulation of human hedgehog interacting protein in glioma cell lines and primary tumor samples

    PubMed Central

    Shahi, Mehdi H.; Zazpe, Idoya; Afzal, Mohammad; Sinha, Subrata; Rebhun, Robert B.; Meléndez, Bárbara; Rey, Juan A.

    2016-01-01

    Glioma constitutes one of the most common groups of brain tumors, and its prognosis is influenced by different genetic and epigenetic modulations. In this study, we demonstrated low or no expression of hedgehog interacting protein (HHIP) in most of the cell lines and primary glioma tumor samples. We further proceeded to promoter methylation study of this gene in the same cell lines and primary tumor samples and found 87 % (7/8) HHIP methylation in glioblastoma cell lines and 75 % (33/44) in primary tumor samples. These methylation pattern correlates with low or unexpressed HHIP in both cell lines and primary tumor samples. Our results suggest the possibility of epigenetic regulation of this gene in glioma, similarly to medulloblastoma, gastric, hepatic, and pancreatic cancers. Also, HHIP might be a diagnostic or prognostic marker in glioma and help to the detection of these tumors in early stages of disease. PMID:25416442

  2. Hedgehog signaling and gastrointestinal cancer

    PubMed Central

    Saqui-Salces, Milena; Merchant, Juanita L.

    2017-01-01

    Hedgehog (Hh) signaling is critical for embryonic development and in differentiation, proliferation, and maintenance of multiple adult tissues. De-regulation of the Hh pathway is associated with birth defects and cancer. In the gastrointestinal tract, Hh ligands Sonic (Shh) and Indian (Ihh), as well as the receptor Patched (Ptch1), and transcription factors of Glioblastoma family (Gli) are all expressed during development. In the adult, Shh expression is restricted to the stomach and colon, while Ihh expression occurs throughout the luminal gastrointestinal tract, its expression being highest in the proximal duodenum. Several studies have demonstrated a requirement for Hh signaling during gastrointestinal tract development. However to date, the specific role of the Hh pathway in the adult stomach and intestine is not completely understood. The current review will place into context the implications of recent published data related to the biochemistry and cell biology of Hh signaling on the luminal gastrointestinal tract during development, normal physiology and subsequently carcinogenesis. PMID:20307590

  3. Interplay between menin and Dnmt1 reversibly regulates pancreatic cancer cell growth downstream of the Hedgehog signaling pathway.

    PubMed

    Cheng, Peng; Wang, Yun-Feng; Li, Gang; Yang, Sheng-sheng; Liu, Che; Hu, Hao; Jin, Gang; Hu, Xian-Gui

    2016-01-01

    Menin, the product of the Men1 gene, which is frequently mutated in pancreatic neuroendocrine tumors, acts as a chromatin-remodeling factor to modulate the transcription of cell cycle regulators by interacting with histone modification factors. However, the function of menin and its underlying mechanisms in pancreatic ductal adenocarcinoma remain unknown. Here, we found that menin inhibited pancreatic cancer cell growth in vitro and in vivo and that its expression was gradually lost during pancreatic carcinogenesis. Menin overexpression significantly activated the expression of the cyclin-dependent kinase (CDK) inhibitors p18 and p27, accompanied with a decrease in DNA methylation levels of p18 and p27 promoters. Mechanistically, we found that interaction of menin with DNA methyltransferase 1 (Dnmt1) competitively pulled down Dnmt1 from p18 and p27 promoters, leading to the downregulation of DNA methylation levels. Moreover, menin expression was suppressed by Dnmt1 downstream of the Hedgehog signaling pathway, and menin overexpression strongly antagonized the promotion effect of hedgehog signaling on pancreatic cancer cell proliferation. Taken together, the interaction between menin and Dnmt1 reversibly regulates pancreatic cancer cell growth downstream of Hedgehog pathways with complex mutual modulation networks, suggesting that the Hedgehog/Dnmt1/menin axis is a potential molecular target for pancreatic cancer therapy.

  4. Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration.

    PubMed

    Zhu, Zhong Xin; Sun, Cong Cong; Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui; Zheng, Jia Yong; Zhou, Xuan; Cong, Wei Tao; Li, Xiao Kun; Jin, Li Tai

    2017-03-28

    Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy.

  5. SOX18 Is a Novel Target Gene of Hedgehog Signaling in Cervical Carcinoma Cell Lines

    PubMed Central

    Popovic, Jelena; Schwirtlich, Marija; Rankovic, Branislava; Stevanovic, Milena

    2015-01-01

    Although there is much evidence showing functional relationship between Hedgehog pathway, in particular Sonic hedgehog, and SOX transcription factors during embryonic development, scarce data are available regarding their crosstalk in cancer cells. SOX18 protein plays an important role in promoting tumor angiogenesis and therefore emerged as a promising potential target in antiangiogenic tumor therapy. Recently it became evident that expression of SOX18 gene in tumors is not restricted to endothelium of accompanying blood and lymphatic vessels, but in tumor cells as well.In this paper we have identified human SOX18 gene as a novel target gene of Hedgehog signaling in cervical carcinoma cell lines. We have presented data showing that expression of SOX18 gene is regulated by GLI1 and GLI2 transcription factors, final effectors of Hedgehog signaling, and that modulation of Hedgehog signaling activity in considerably influence SOX18 expression. We consider important that Hedgehog pathway inhibitors reduced SOX18 expression, thus showing, for the first time, possibility for manipulationwith SOX18 gene expression. In addition, we analyzed the role of SOX18 in malignant potential of cervical carcinoma cell line, and showed that its overexpression has no influence on cells proliferation and viability, but substantially promotes migration and invasion of cells in vitro. Pro-migratory effect of SOX18 suggests its role in promoting malignant spreading, possibly in response to Hedgehog activation. PMID:26588701

  6. Sox4 regulates choroid fissure closure by limiting Hedgehog signaling during ocular morphogenesis

    PubMed Central

    Wen, Wen; Pillai-Kastoori, Lakshmi; Wilson, Stephen G.; Morris, Ann C.

    2015-01-01

    SoxC transcription factors play critical roles in many developmental processes, including neurogenesis, cardiac formation, and skeletal differentiation. In vitro and in vivo loss-of-function studies have suggested that SoxC genes are required for oculogenesis, however the mechanism was poorly understood. Here, we have explored the function of the SoxC factor Sox4 during zebrafish eye development. We show that sox4a and sox4b are expressed in the forebrain and periocular mesenchyme adjacent to the optic stalk during early eye development. Knockdown of sox4 in zebrafish resulted in coloboma, a structural malformation of the eye that is a significant cause of pediatric visual impairment in humans, in which the choroid fissure fails to close. Sox4 morphants displayed altered proximo-distal patterning of the optic vesicle, including expanded pax2 expression in the optic stalk, as well as ectopic cell proliferation in the retina. We show that the abnormal ocular morphogenesis observed in Sox4-deficient zebrafish is caused by elevated Hedgehog (Hh) signaling, and this is due to increased expression of the Hh pathway ligand Indian hedgehog b (ihhb). Consistent with these results, coloboma in sox4 morphants could be rescued by pharmacological treatment with the Hh inhibitor cyclopamine, or by co-knockdown of ihhb. Conversely, overexpression of sox4 reduced Hh signaling and ihhb expression, resulting in cyclopia. Finally, we demonstrate that sox4 and sox11 have overlapping, but not completely redundant, functions in regulating ocular morphogenesis. Taken together, our data demonstrate that Sox4 is required to limit the extent of Hh signaling during eye development, and suggest that mutations in SoxC factors could contribute to the development of coloboma. PMID:25557621

  7. Regulation of Smoothened Phosphorylation and High-Level Hedgehog Signaling Activity by a Plasma Membrane Associated Kinase.

    PubMed

    Li, Shuangxi; Li, Shuang; Han, Yuhong; Tong, Chao; Wang, Bing; Chen, Yongbin; Jiang, Jin

    2016-06-01

    Hedgehog (Hh) signaling controls embryonic development and adult tissue homeostasis through the G protein coupled receptor (GPCR)-family protein Smoothened (Smo). Upon stimulation, Smo accumulates on the cell surface in Drosophila or primary cilia in vertebrates, which is thought to be essential for its activation and function, but the underlying mechanisms remain poorly understood. Here we show that Hh stimulates the binding of Smo to a plasma membrane-associated kinase Gilgamesh (Gish)/CK1γ and that Gish fine-tunes Hh pathway activity by phosphorylating a Ser/Thr cluster (CL-II) in the juxtamembrane region of Smo carboxyl-terminal intracellular tail (C-tail). We find that CL-II phosphorylation is promoted by protein kinase A (PKA)-mediated phosphorylation of Smo C-tail and depends on cell surface localization of both Gish and Smo. Consistent with CL-II being critical for high-threshold Hh target gene expression, its phosphorylation appears to require higher levels of Hh or longer exposure to the same level of Hh than PKA-site phosphorylation on Smo. Furthermore, we find that vertebrate CK1γ is localized at the primary cilium to promote Smo phosphorylation and Sonic hedgehog (Shh) pathway activation. Our study reveals a conserved mechanism whereby Hh induces a change in Smo subcellular localization to promote its association with and activation by a plasma membrane localized kinase, and provides new insight into how Hh morphogen progressively activates Smo.

  8. Hedgehog-regulated atypical PKC promotes phosphorylation and activation of Smoothened and Cubitus interruptus in Drosophila

    PubMed Central

    Jiang, Kai; Liu, Yajuan; Fan, Junkai; Epperly, Garretson; Gao, Tianyan; Jiang, Jin; Jia, Jianhang

    2014-01-01

    Smoothened (Smo) is essential for transduction of the Hedgehog (Hh) signal in both insects and vertebrates. Cell surface/cilium accumulation of Smo is thought to play an important role in Hh signaling, but how the localization of Smo is controlled remains poorly understood. In this study, we demonstrate that atypical PKC (aPKC) regulates Smo phosphorylation and basolateral accumulation in Drosophila wings. Inactivation of aPKC by either RNAi or a mutation inhibits Smo basolateral accumulation and attenuates Hh target gene expression. In contrast, expression of constitutively active aPKC elevates basolateral accumulation of Smo and promotes Hh signaling. The aPKC-mediated phosphorylation of Smo at Ser680 promotes Ser683 phosphorylation by casein kinase 1 (CK1), and these phosphorylation events elevate Smo activity in vivo. Moreover, aPKC has an additional positive role in Hh signaling by regulating the activity of Cubitus interruptus (Ci) through phosphorylation of the Zn finger DNA-binding domain. Finally, the expression of aPKC is up-regulated by Hh signaling in a Ci-dependent manner. Our findings indicate a direct involvement of aPKC in Hh signaling beyond its role in cell polarity. PMID:25349414

  9. SUMO regulates the activity of Smoothened and Costal-2 in Drosophila Hedgehog signaling

    PubMed Central

    Zhang, Jie; Liu, Yajuan; Jiang, Kai; Jia, Jianhang

    2017-01-01

    In Hedgehog (Hh) signaling, the GPCR-family protein Smoothened (Smo) acts as a signal transducer that is regulated by phosphorylation and ubiquitination, which ultimately change the cell surface accumulation of Smo. However, it is not clear whether Smo is regulated by other post-translational modifications, such as sumoylation. Here, we demonstrate that knockdown of the small ubiquitin-related modifier (SUMO) pathway components Ubc9 (a SUMO-conjugating enzyme E2), PIAS (a SUMO-protein ligase E3), and Smt3 (the SUMO isoform in Drosophila) by RNAi prevents Smo accumulation and alters Smo activity in the wing. We further show that Hh-induced-sumoylation stabilizes Smo, whereas desumoylation by Ulp1 destabilizes Smo in a phosphorylation independent manner. Mechanistically, we discover that excessive Krz, the Drosophila β-arrestin 2, inhibits Smo sumoylation and prevents Smo accumulation through Krz regulatory domain. Krz likely facilitates the interaction between Smo and Ulp1 because knockdown of Krz by RNAi attenuates Smo-Ulp1 interaction. Finally, we provide evidence that Cos2 is also sumoylated, which counteracts its inhibitory role on Smo accumulation in the wing. Taken together, we have uncovered a novel mechanism for Smo activation by sumoylation that is regulated by Hh and Smo interacting proteins. PMID:28195188

  10. Nek2A/SuFu feedback loop regulates Gli-mediated Hedgehog signaling pathway

    PubMed Central

    Zhou, Fen; Huang, Dengliang; Li, Yong; Hu, Guanghui; Rao, Hai; Lu, Quqin; Luo, Shiwen; Wang, Yao

    2017-01-01

    Suppressor of Fused (SuFu), one of the most conserved components of the Hedgehog (Hh) signaling, binds Gli transcription factors and impedes activation of target gene expression in mammalian cells. Despite the central importance of SuFu in the Hh pathway, little is known about SuFu regulation. In a previous study, we identified NIMA-related expressed kinase 2A (Nek2A) as a SuFu-interacting protein. Here, we show that Nek2A stabilizes SuFu through impairing ubiquitin/proteasome degradation of SuFu. In addition, Nek2A negatively regulates target genes of Hh signaling as well as Gli2 transcriptional activity. In turn, inhibition of Hh signaling by GANT61 diminishes mRNA and protein levels of Nek2A, and Hh agonist promotes transcription of NEK2A gene. Chromatin immunoprecipitation assays revealed that Gli1 and Gli2 directly bind to the promoter regions of NEK2A gene and induced its transcription. Thus, we uncovered one of the mechanisms by which Nek2A acts as a modulator of the Hh signaling pathway in the context of a novel negative-feedback loop, which may offer new insights into Gli-mediated Hh signaling regulation in development and human diseases. PMID:28035348

  11. Regulation of mammalian Gli proteins by Costal 2 and PKA in Drosophila reveals Hedgehog pathway conservation.

    PubMed

    Marks, Steven A; Kalderon, Daniel

    2011-06-01

    Hedgehog (Hh) signaling activates full-length Ci/Gli family transcription factors and prevents Ci/Gli proteolytic processing to repressor forms. In the absence of Hh, Ci/Gli processing is initiated by direct Pka phosphorylation. Despite those fundamental similarities between Drosophila and mammalian Hh pathways, the differential reliance on cilia and some key signal transduction components had suggested a major divergence in the mechanisms that regulate Ci/Gli protein activities, including the role of the kinesin-family protein Costal 2 (Cos2), which directs Ci processing in Drosophila. Here, we show that Cos2 binds to three regions of Gli1, just as for Ci, and that Cos2 functions to silence mammalian Gli1 in Drosophila in a Hh-regulated manner. Cos2 and the mammalian kinesin Kif7 can also direct Gli3 and Ci processing in fly, underscoring a fundamental conserved role for Cos2 family proteins in Hh signaling. We also show that direct PKA phosphorylation regulates the activity, rather than the proteolysis of Gli in Drosophilia, and we provide evidence for an analogous action of PKA on Ci.

  12. Transcriptional regulation of WNT2B based on the balance of Hedgehog, Notch, BMP and WNT signals.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2009-05-01

    We cloned and characterized human WNT2B in 1996, and then others cloned and characterized mouse, chicken, and zebrafish WNT2B orthologs. WNT2B is expressed in several types of human cancer, such as basal cell carcinoma, gastric cancer, breast cancer, head/neck squamous cell carcinoma, cervical cancer and leukemia. WNT2B is one of canonical WNTs transducing signals through Frizzled (FZD) and LRP5/LRP6 receptors to beta-catenin-TCF/LEF signaling cascade. Here, refined integrative genomic analyses on WNT2B orthologs were carried out to elucidate its transcriptional mechanisms. GLI-, double FOX-, HES/HEY-, bHLH-, and Sp1-binding sites within mammalian WNT2B promoter were well conserved. Because GLI1, FOXA2, FOXC2, FOXE1, FOXF1 and FOXL1 are direct target genes of Hedgehog-GLI2 signaling cascade, Hedgehog signals should induce WNT2B upregulation through GLI family members as well as FOX family members. Notch, BMP and Hedgehog signals inhibit WNT2B expression via HES/HEY-binding to N-box, whereas BMP and WNT signals inhibit bHLH transcription factor-induced WNT2B expression via ID1, ID2, ID3, MSX1 or MSX2. Together these facts indicate that Hedgehog signals and bHLH transcription factors are involved in WNT2B upregulation, which is counteracted by BMP, WNT and Notch signals. Mesenchymal BMP induces IHH expression in gastrointestinal epithelial cells, and then epithelial Hedgehog induces WNT2B and BMP4 expression in mesenchymal cells. NF-kappaB signals induce SHH upregulation, and WNT2B is upregulated in inflammatory bowel disease (IBD). BMP-IHH and inflammation-SHH signaling loops are involved in WNT2B up-regulation during embryogenesis, adult tissue homeostasis, and carcinogenesis.

  13. Hedgehog signaling pathway regulated the target genes for adipogenesis in silkworm Bombyx mori.

    PubMed

    Liang, Shuang; Chen, Rui-Ting; Zhang, Deng-Pan; Xin, Hu-Hu; Lu, Yan; Wang, Mei-Xian; Miao, Yun-Gen

    2015-10-01

    Hedgehog (Hh) signals regulate invertebrate and vertebrate development, yet the role of the pathway in adipose development remains poorly understood. In this report, we found that Hh pathway components are expressed in the fat body of silkworm larvae. Functional analysis of these components in a BmN cell line model revealed that activation of the Hh gene stimulated transcription of Hh pathway components, but inhibited the expression of the adipose marker gene AP2. Conversely, specific RNA interference-mediated knockdown of Hh resulted in increased AP2 expression. This further showed the regulation of Hh signal on the adipose marker gene. In silkworm larval models, enhanced adipocyte differentiation and an increase in adipocyte cell size were observed in silkworms that had been treated with a specific Hh signaling pathway antagonist, cyclopamine. The fat-body-specific Hh blockade tests were consistent with Hh signaling inhibiting silkworm adipogenesis. Our results indicate that the role of Hh signaling in inhibiting fat formation is conserved in vertebrates and invertebrates.

  14. Regulation of Articular Chondrocyte Proliferation and Differentiation by Indian Hedgehog and Parathyroid Hormone-related Protein

    PubMed Central

    Chen, Xuesong; Macica, Carolyn; Nasiri, Ali; Broadus, Arthur E.

    2008-01-01

    Objective The chondrocytes of the epiphyseal growth zone are regulated by the Indian hedgehog (Ihh)-parathyroid hormone-related protein (PTHrP) axis. In weight-bearing joints, this growth zone comes to be subdivided by the secondary ossification center into distinct articular and growth cartilage structures. Here, we explored the cells of origin, localization, regulation of expression, and putative functions of Ihh and PTHrP in articular cartilage in the mouse. Methods We assessed Ihh and PTHrP expression in an allelic PTHrP-lacZ knockin mouse and several versions of PTHrP-null mice. Selected joints were unloaded surgically to examine load-induction of PTHrP and Ihh. Results The embryonic growth zone appears to serve as the source of PTHrP-expressing proliferative chondrocytes that populate both the forming articular cartilage and growth plate structures. In articular cartilage, these cells take the form of articular chondrocytes in the mid-zone. In PTHrP-knockout mice, mineralizing chondrocytes encroach upon developing articular cartilage but appear to be prevented from mineralizing the joint space by Ihh-driven surface chondrocyte proliferation. In growing and adult mice, PTHrP expression in articular chondrocytes is load-induced, and unloading is associated with rapid changes in PTHrP expression and articular chondrocyte differentiation. Conclusion We conclude that the PTHrP-Ihh axis participates in the maintenance of articular cartilage. Dysregulation of this system might contribute to the pathogenesis of arthritis. PMID:19035497

  15. Pancreatic Mesenchyme Regulates Islet Cellular Composition in a Patched/Hedgehog-Dependent Manner

    PubMed Central

    Hibsher, Daniel; Epshtein, Alona; Oren, Nufar; Landsman, Limor

    2016-01-01

    Pancreas development requires restrained Hedgehog (Hh) signaling activation. While deregulated Hh signaling in the pancreatic mesenchyme has been long suggested to be detrimental for proper organogenesis, this association was not directly shown. Here, we analyzed the contribution of mesenchymal Hh signaling to pancreas development. To increase Hh signaling in the pancreatic mesenchyme of mouse embryos, we deleted Patched1 (Ptch1) in these cells. Our findings indicate that deregulated Hh signaling in mesenchymal cells was sufficient to impair pancreas development, affecting both endocrine and exocrine cells. Notably, transgenic embryos displayed disrupted islet cellular composition and morphology, with a reduced β-cell portion. Our results indicate that the cell-specific growth rates of α- and β-cell populations, found during normal development, require regulated mesenchymal Hh signaling. In addition, we detected hyperplasia of mesenchymal cells upon elevated Hh signaling, accompanied by them acquiring smooth-muscle like phenotype. By specifically manipulating mesenchymal cells, our findings provide direct evidence for the non-autonomous roles of the Hh pathway in pancreatic epithelium development. To conclude, we directly show that regulated mesenchymal Hh signaling is required for pancreas organogenesis and establishment of its proper cellular composition. PMID:27892540

  16. Gli2 Acetylation at Lysine 757 Regulates Hedgehog-Dependent Transcriptional Output by Preventing Its Promoter Occupancy

    PubMed Central

    D'Amico, Davide; Di Magno, Laura; Infante, Paola; De Smaele, Enrico; Giannini, Giuseppe; Di Marcotullio, Lucia; Screpanti, Isabella; Gulino, Alberto; Canettieri, Gianluca

    2013-01-01

    The morphogenic Hedgehog (Hh) signaling regulates postnatal cerebellar development and its aberrant activation leads to medulloblastoma. The transcription factors Gli1 and Gli2 are the activators of Hh pathway and their function is finely controlled by different covalent modifications, such as phosphorylation and ubiquitination. We show here that Gli2 is endogenously acetylated and that this modification represents a key regulatory step for Hedgehog signaling. The histone acetyltransferase (HAT) coactivator p300, but not other HATs, acetylates Gli2 at the conserved lysine K757 thus inhibiting Hh target gene expression. By generating a specific anti acetyl-Gli2(Lys757) antisera we demonstrated that Gli2 acetylation is readily detectable at endogenous levels and is attenuated by Hh agonists. Moreover, Gli2 K757R mutant activity is higher than wild type Gli2 and is no longer enhanced by Hh agonists, indicating that acetylation represents an additional level of control for signal dependent activation. Consistently, in sections of developing mouse cerebella Gli2 acetylation correlates with the activation status of Hedgehog signaling. Mechanistically, acetylation at K757 prevents Gli2 entry into chromatin. Together, these data illustrate a novel mechanism of regulation of the Hh signaling whereby, in concert with Gli1, Gli2 acetylation functions as a key transcriptional checkpoint in the control of morphogen-dependent processes. PMID:23762415

  17. Lizard tail regeneration: regulation of two distinct cartilage regions by Indian hedgehog.

    PubMed

    Lozito, Thomas P; Tuan, Rocky S

    2015-03-15

    Lizards capable of caudal autotomy exhibit the remarkable ability to "drop" and then regenerate their tails. However, the regenerated lizard tail (RLT) is known as an "imperfect replicate" due to several key anatomical differences compared to the original tail. Most striking of these "imperfections" concerns the skeleton; instead of the vertebrae of the original tail, the skeleton of the RLT takes the form of an unsegmented cartilage tube (CT). Here we have performed the first detailed staging of skeletal development of the RLT CT, identifying two distinct mineralization events. CTs isolated from RLTs of various ages were analyzed by micro-computed tomography to characterize mineralization, and to correlate skeletal development with expression of endochondral ossification markers evaluated by histology and immunohistochemistry. During early tail regeneration, shortly after CT formation, the extreme proximal CT in direct contact with the most terminal vertebra of the original tail develops a growth plate-like region that undergoes endochondral ossification. Proximal CT chondrocytes enlarge, express hypertrophic markers, including Indian hedgehog (Ihh), apoptose, and are replaced by bone. During later stages of tail regeneration, the distal CT mineralizes without endochondral ossification. The sub-perichondrium of the distal CT expresses Ihh, and the perichondrium directly calcifies without cartilage growth plate formation. The calcified CT perichondrium also contains a population of stem/progenitor cells that forms new cartilage in response to TGF-β stimulation. Treatment with the Ihh inhibitor cyclopamine inhibited both proximal CT ossification and distal CT calcification. Thus, while the two mineralization events are spatially, temporally, and mechanistically very different, they both involve Ihh. Taken together, these results suggest that Ihh regulates CT mineralization during two distinct stages of lizard tail regeneration.

  18. Expression of Sonic Hedgehog (SHH) in human lung cancer and the impact of YangZheng XiaoJi on SHH-mediated biological function of lung cancer cells and tumor growth.

    PubMed

    Jiang, Wen G; Ye, Lin; Ruge, Fiona; Sun, Ping-Hui; Sanders, Andrew J; Ji, Ki; Lane, Jane; Zhang, Lijian; Satherley, Lucy; Weeks, Hoi P; Zhi, Xiuyi; Gao, Yong; Wei, Cong; Wu, Yiling; Mason, Malcolm D

    2015-03-01

    Sonic Hedgehog (SHH) is a protein that is aberrantly expressed in various human tumors. SHH and its signaling molecules have been indicated as potential therapeutic targets. In the present study, we evaluated the expression of SHH transcript in human non-small cell lung cancer (NSCLC) tissues and investigated the impact of inhibiting SHH together with a traditional Chinese medicine formula, YangZheng XiaoJi (YZXJ), on the function and growth of lung cancer cells. Human NSCLC tissues had significantly higher levels of the SHH transcript compared matched normal lung tissues (n=83). TNM2 tumors and tumors with pleural invasion had higher levels than TNM1 and non-invasive tumors. High SHH levels were associated with a shorter overall survival (OS) of the patients. A SHH inhibitor, cyclopamine, and YZXJ alone or in combination had a marked inhibitory effect on cellular invasion and cellular migration of human lung cancer cells, A549 and SKMES1. YangZheng XiaoJi and its combination with cyclopamine also significantly reduced the growth of lung tumors in vivo together with a reduction of SHH and smoothened (Smo) proteins in the lung tumors. The present study provides evidence that blocking SHH by way of small inhibitor and by YangZheng XiaoJi has a profound influence on lung cancer cells as seen by in vitro invasion and cell migration and in vivo tumor growth. Together with the aberrant expression of SHH in NSCLC tumors in the patients, it is suggested that SHH is a potential target for therapies for NSCLC.

  19. The conserved barH-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by Sonic Hedgehog.

    PubMed

    Juraver-Geslin, Hugo A; Gómez-Skarmeta, José Luis; Durand, Béatrice C

    2014-12-01

    In this study, we investigated the gene regulatory network that governs formation of the Zona limitans intrathalamica (ZLI), a signaling center that secretes Sonic Hedgehog (Shh) to control the growth and regionalization of the caudal forebrain. Using loss- and gain-of-function, explants and grafting experiments in amphibians, we demonstrate that barhl2 acts downstream of otx2 and together with the iroquois (irx)-3 gene in establishment of the ZLI compartment initiated by Shh influence. We find that the presumptive (pre)-ZLI domain expresses barhl2, otx2 and irx3, whereas the thalamus territory caudally bordering the pre-ZLI expresses barhl2, otx2 and irx1/2 and early on irx3. We demonstrate that Barhl2 activity is required for determination of the ZLI and thalamus fates and that within the p2 alar plate the ratio of Irx3 to Irx1/2 contributes to ZLI specification and size determination. We show that when continuously exposed to Shh, neuroepithelial cells coexpressing barhl2, otx2 and irx3 acquire two characteristics of the ZLI compartment-the competence to express shh and the ability to segregate from anterior neural plate cells. In contrast, neuroepithelial cells expressing barhl2, otx2 and irx1/2, are not competent to express shh. Noteworthy in explants, under Shh influence, ZLI-like cells segregate from thalamic-like cells. Our study establishes that Barhl2 activity plays a key role in p2 alar plate patterning, specifically ZLI formation, and provides new insights on establishment of the signaling center of the caudal forebrain.

  20. Targeting Breast Cancer Recurrence via Hedgehog-mediated Sensitization of Breast Cancer Stem Cells

    DTIC Science & Technology

    2011-07-01

    established a treatment model in which the Smoothened agonist was sufficient to partially rescue the Sonic Hedgehog knockout suggesting that similar... Hedgehog -mediated Sensitization of Breast Cancer Stem Cells PRINCIPAL INVESTIGATOR: David J. Robbins, Ph.D...June 2010 – 14 June 2011 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting Breast Cancer Recurrence via Hedgehog -mediated Sensitization of

  1. Intestinal cell kinase, a protein associated with endocrine-cerebro-osteodysplasia syndrome, is a key regulator of cilia length and Hedgehog signaling.

    PubMed

    Moon, Heejung; Song, Jieun; Shin, Jeong-Oh; Lee, Hankyu; Kim, Hong-Kyung; Eggenschwiller, Jonathan T; Bok, Jinwoong; Ko, Hyuk Wan

    2014-06-10

    Endocrine-cerebro-osteodysplasia (ECO) syndrome is a recessive genetic disorder associated with multiple congenital defects in endocrine, cerebral, and skeletal systems that is caused by a missense mutation in the mitogen-activated protein kinase-like intestinal cell kinase (ICK) gene. In algae and invertebrates, ICK homologs are involved in flagellar formation and ciliogenesis, respectively. However, it is not clear whether this role of ICK is conserved in mammals and how a lack of functional ICK results in the characteristic phenotypes of human ECO syndrome. Here, we generated Ick knockout mice to elucidate the precise role of ICK in mammalian development and to examine the pathological mechanisms of ECO syndrome. Ick null mouse embryos displayed cleft palate, hydrocephalus, polydactyly, and delayed skeletal development, closely resembling ECO syndrome phenotypes. In cultured cells, down-regulation of Ick or overexpression of kinase-dead or ECO syndrome mutant ICK resulted in an elongation of primary cilia and abnormal Sonic hedgehog (Shh) signaling. Wild-type ICK proteins were generally localized in the proximal region of cilia near the basal bodies, whereas kinase-dead ICK mutant proteins accumulated in the distal part of bulged ciliary tips. Consistent with these observations in cultured cells, Ick knockout mouse embryos displayed elongated cilia and reduced Shh signaling during limb digit patterning. Taken together, these results indicate that ICK plays a crucial role in controlling ciliary length and that ciliary defects caused by a lack of functional ICK leads to abnormal Shh signaling, resulting in congenital disorders such as ECO syndrome.

  2. The role of nutraceuticals in the regulation of Wnt and Hedgehog signaling in cancer

    PubMed Central

    Li, Yiwei; Wang, Zhiwei; Kong, Dejuan

    2010-01-01

    Multiple cellular signaling pathways have been involved in the processes of cancer cell invasion and metastasis. Among many signaling pathways, Wnt and Hedgehog (Hh) signaling pathways are critically involved in embryonic development, in the biology of cancer stem cells (CSCs) and in the acquisition of epithelial to mesenchymal transition (EMT), and thus this article will remain focused on Wnt and Hh signaling. Since CSCs and EMT are also known to be responsible for cancer cell invasion and metastasis, the Wnt and Hedgehog signaling pathways are also intimately associated with cancer invasion and metastasis. Emerging evidence suggests the beneficial role of chemopreventive agents commonly known as nutraceutical in cancer. Among many such agents, soy isoflavones, curcumin, green tea polyphenols, 3,3′-diindolylmethane, resveratrol, lycopene, vitamin D, etc. have been found to prevent, reverse, or delay the carcinogenic process. Interestingly, these agents have also shown to prevent or delay the progression of cancer, which could in part be due to their ability to attack CSCs or EMT-type cells by attenuating the Wnt and Hedgehog signaling pathways. In this review, we summarize the current state of our knowledge on the role of Wnt and Hedgehog signaling pathways, and their targeted inactivation by chemopreventive agents (nutraceuticals) for the prevention of tumor progression and/or treatment of human malignancies. PMID:20711635

  3. Zebrafish blowout provides genetic evidence for Patched1-mediated negative regulation of Hedgehog signaling within the proximal optic vesicle of the vertebrate eye.

    PubMed

    Lee, Jiwoon; Willer, Jason R; Willer, Gregory B; Smith, Kierann; Gregg, Ronald G; Gross, Jeffrey M

    2008-07-01

    In this study, we have characterized the ocular defects in the recessive zebrafish mutant blowout that presents with a variably penetrant coloboma phenotype. blowout mutants develop unilateral or bilateral colobomas and as a result, the retina and retinal pigmented epithelium are not contained within the optic cup. Colobomas result from defects in optic stalk morphogenesis whereby the optic stalk extends into the retina and impedes the lateral edges of the choroid fissure from meeting and fusing. The expression domain of the proximal optic vesicle marker pax2a is expanded in blowout at the expense of the distal optic vesicle marker pax6, suggesting that the initial patterning of the optic vesicle into proximal and distal territories is disrupted in blowout. Later aspects of distal optic cup formation (i.e. retina development) are normal in blowout mutants, however. Positional cloning of blowout identified a nonsense mutation in patched1, a negative regulator of the Hedgehog pathway, as the underlying cause of the blowout phenotype. Expanded domains of expression of the Hedgehog target genes patched1 and patched2 were observed in blowout, consistent with a loss of Patched1 function and upregulation of Hedgehog pathway activity. Moreover, colobomas in blowout could be suppressed by pharmacologically inhibiting the Hedgehog pathway with cyclopamine, and maximal rescue occurred when embryos were exposed to cyclopamine between 5.5 and 13 hours post-fertilization. These observations highlight the critical role that Hedgehog pathway activity plays in mediating patterning of the proximal/distal axis of the optic vesicle during the early phases of eye development and they provide genetic confirmation for the integral role that patched1-mediated negative regulation of Hedgehog signaling plays during vertebrate eye development.

  4. Human hedgehog interacting protein expression and promoter methylation in medulloblastoma cell lines and primary tumor samples

    PubMed Central

    Shahi, Mehdi H.; Afzal, Mohammad; Sinha, Subrata; Eberhart, Charles G.; Rey, Juan A.; Fan, Xing

    2015-01-01

    Medulloblastoma is the most common pediatric brain tumor and its development is affected by genetic and epigenetic factors. In this study we found there is low or no expression of the hedgehog interacting protein (HHIP), a negative regulator of the sonic hedgehog pathway, in most medulloblastoma cell lines and primary samples explored. We proceeded to promoter methylation assays of this gene by MCA-Meth, and found that HHIP was hypermethylated in all medulloblastoma cell lines, but only in 2 out of 14 (14%) primary tumor samples. Methylation correlated with low or unexpressed HHIP in cell lines but not in primary tumor samples. These results suggest the possibility of epigenetic regulation of HHIP in medulloblastoma, similarly to gastric, hepatic and pancreatic cancer. However, HHIP seems to be not only under regulation of promoter methylation, but under other factors involved in the control of its low levels of expression in medulloblastoma. PMID:20853133

  5. Habenular Neurogenesis in Zebrafish Is Regulated by a Hedgehog, Pax6 Proneural Gene Cascade

    PubMed Central

    Naye, François; Peers, Bernard; Roussigné, Myriam; Blader, Patrick

    2016-01-01

    The habenulae are highly conserved nuclei in the dorsal diencephalon that connect the forebrain to the midbrain and hindbrain. These nuclei have been implicated in a broad variety of behaviours in humans, primates, rodents and zebrafish. Despite this, the molecular mechanisms that control the genesis and differentiation of neural progenitors in the habenulae remain relatively unknown. We have previously shown that, in zebrafish, the timing of habenular neurogenesis is left-right asymmetric and that in the absence of Nodal signalling this asymmetry is lost. Here, we show that habenular neurogenesis requires the homeobox transcription factor Pax6a and the redundant action of two proneural bHLH factors, Neurog1 and Neurod4. We present evidence that Hedgehog signalling is required for the expression of pax6a, which is in turn necessary for the expression of neurog1 and neurod4. Finally, we demonstrate by pharmacological inhibition that Hedgehog signalling is required continuously during habenular neurogenesis and by cell transplantation experiments that pathway activation is required cell autonomously. Our data sheds light on the mechanism underlying habenular development that may provide insights into how Nodal signalling imposes asymmetry on the timing of habenular neurogenesis. PMID:27387288

  6. Sonic Hedgehog Signaling Promotes Tumor Growth

    DTIC Science & Technology

    2006-02-01

    UGSM-2 cells were determined to be tetraploid by comparison to ploidy number of known diploid cells: freshly isolated splenocytes from the spleen of a...propagated continuously without evi- dence of crisis. Immortalized mouse cells are typically tetraploid and these cells remained stably tetraploid for... tetraploid (Fig. 1B). Recent studies revealed that INK4a/ MEFs can acquire chromosomal rearrangements at high passage [29]. To assess tumorigenicity

  7. Sonic Hedgehog Signaling Promotes Tumor Growth

    DTIC Science & Technology

    2007-02-01

    phase. PloidyAnalysis UGSM-2 cells were determined to be tetraploid by comparison to ploidy number of known diploid cells: freshly isolated splenocytes...continuously without evi- dence of crisis. Immortalized mouse cells are typically tetraploid and these cells remained stably tetraploid for over 100...line, UGSM-2, was selected for use in subsequent experi- ments. Like the parent mixed cell population, UGSM-2 cellswere found to be stably tetraploid

  8. Hedgehog signaling regulates drug sensitivity by targeting ABC transporters ABCB1 and ABCG2 in epithelial ovarian cancer.

    PubMed

    Chen, Yi; Bieber, Marcia M; Teng, Nelson N H

    2014-08-01

    A major challenge of successful chemotherapy in ovarian cancer is overcoming intrinsic or acquired multi-drug resistance caused by active drug efflux mediated by ATP-binding cassette (ABC) transporters. Regulation of these transporters in ovarian cancer is poorly understood. We have found that abnormal expression of the hedgehog (Hh) signaling pathway transcription factor Gli1 is involved in the regulation of ABC transporters ABCB1 and ABCG2 in ovarian cancer. Hh is a known regulator of cancer cell proliferation and differentiation in several other types of invasive and metastatic malignancies. Our work has demonstrated that Gli1 is abnormally activated in a portion of ovarian cancers. Inhibition of Gli1 expression decreases ABCB1 and ABCG2 gene expression levels and enhances the response of ovarian cancer cells to certain chemotherapeutic drugs. The underlying mechanism is a direct association of Gli1 with a specific consensus sequence located in the promoter region of ABCB1 and ABCG2 genes. This study provides new understanding of ABC gene regulation by Hh signaling pathway, which may lead to the identification of new markers to detect and to anticipate ovarian cancer chemotherapy drug sensitivity.

  9. Graded hedgehog and fibroblast growth factor signaling independently regulate pituitary cell fates and help establish the pars distalis and pars intermedia of the zebrafish adenohypophysis.

    PubMed

    Guner, Burcu; Ozacar, A Tuba; Thomas, Jeanne E; Karlstrom, Rolf O

    2008-09-01

    The vertebrate adenohypophysis forms as a placode at the anterior margin of the neural plate, requiring both hedgehog (Hh) and fibroblast growth factor (Fgf) mediated cell-cell signaling for induction and survival of endocrine cell types. Using small molecule inhibitors to modulate signaling levels during zebrafish development we show that graded Hh and Fgf signaling independently help establish the two subdomains of the adenohypophysis, the anteriorly located pars distalis (PD) and the posterior pars intermedia (PI). High levels of Hh signaling are required for formation of the PD and differentiation of anterior endocrine cell types, whereas lower levels of Hh signaling are required for formation of the PI and differentiation of posterior endocrine cell types. In contrast, high Fgf signaling levels are required for formation of the PI and posterior endocrine cell differentiation, whereas anterior regions require lower levels of Fgf signaling. Based on live observations and marker analyses, we show that the PD forms first at the midline closest to the central nervous system source of Sonic hedgehog. In contrast the PI appears to form from more lateral/posterior cells close to a central nervous system source of Fgf3. Together our data show that graded Hh and Fgf signaling independently direct induction of the PD and PI and help establish endocrine cell fates along the anterior/posterior axis of the zebrafish adenohypophysis. These data suggest that there are distinct origins and signaling requirements for the PD and PI.

  10. Sox11 Is Required to Maintain Proper Levels of Hedgehog Signaling during Vertebrate Ocular Morphogenesis

    PubMed Central

    Pillai-Kastoori, Lakshmi; Wen, Wen; Wilson, Stephen G.; Strachan, Erin; Lo-Castro, Adriana; Fichera, Marco; Musumeci, Sebastiano A.; Lehmann, Ordan J.; Morris, Ann C.

    2014-01-01

    Ocular coloboma is a sight-threatening malformation caused by failure of the choroid fissure to close during morphogenesis of the eye, and is frequently associated with additional anomalies, including microphthalmia and cataracts. Although Hedgehog signaling is known to play a critical role in choroid fissure closure, genetic regulation of this pathway remains poorly understood. Here, we show that the transcription factor Sox11 is required to maintain specific levels of Hedgehog signaling during ocular development. Sox11-deficient zebrafish embryos displayed delayed and abnormal lens formation, coloboma, and a specific reduction in rod photoreceptors, all of which could be rescued by treatment with the Hedgehog pathway inhibitor cyclopamine. We further demonstrate that the elevated Hedgehog signaling in Sox11-deficient zebrafish was caused by a large increase in shha transcription; indeed, suppressing Shha expression rescued the ocular phenotypes of sox11 morphants. Conversely, over-expression of sox11 induced cyclopia, a phenotype consistent with reduced levels of Sonic hedgehog. We screened DNA samples from 79 patients with microphthalmia, anophthalmia, or coloboma (MAC) and identified two novel heterozygous SOX11 variants in individuals with coloboma. In contrast to wild type human SOX11 mRNA, mRNA containing either variant failed to rescue the lens and coloboma phenotypes of Sox11-deficient zebrafish, and both exhibited significantly reduced transactivation ability in a luciferase reporter assay. Moreover, decreased gene dosage from a segmental deletion encompassing the SOX11 locus resulted in microphthalmia and related ocular phenotypes. Therefore, our study reveals a novel role for Sox11 in controlling Hedgehog signaling, and suggests that SOX11 variants contribute to pediatric eye disorders. PMID:25010521

  11. The ESCRT machinery regulates the secretion and long-range activity of Hedgehog.

    PubMed

    Matusek, Tamás; Wendler, Franz; Polès, Sophie; Pizette, Sandrine; D'Angelo, Gisela; Fürthauer, Maximilian; Thérond, Pascal P

    2014-12-04

    The conserved family of Hedgehog (Hh) proteins acts as short- and long-range secreted morphogens, controlling tissue patterning and differentiation during embryonic development. Mature Hh carries hydrophobic palmitic acid and cholesterol modifications essential for its extracellular spreading. Various extracellular transportation mechanisms for Hh have been suggested, but the pathways actually used for Hh secretion and transport in vivo remain unclear. Here we show that Hh secretion in Drosophila wing imaginal discs is dependent on the endosomal sorting complex required for transport (ESCRT). In vivo the reduction of ESCRT activity in cells producing Hh leads to a retention of Hh at the external cell surface. Furthermore, we show that ESCRT activity in Hh-producing cells is required for long-range signalling. We also provide evidence that pools of Hh and ESCRT proteins are secreted together into the extracellular space in vivo and can subsequently be detected together at the surface of receiving cells. These findings uncover a new function for ESCRT proteins in controlling morphogen activity and reveal a new mechanism for the transport of secreted Hh across the tissue by extracellular vesicles, which is necessary for long-range target induction.

  12. Regulation mechanisms of the hedgehog pathway in pancreatic cancer: a review.

    PubMed

    Honselmann, Kim Christin; Pross, Moritz; Jung, Carlo Maria Felix; Wellner, Ulrich Friedrich; Deichmann, Steffen; Keck, Tobias; Bausch, Dirk

    2015-01-31

    Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of death from cancer. Its 5-year survival rate is less than 5%. This poor prognosis is mostly due to the cancer's early invasion and metastasis formation, leading to an initial diagnosis at an advanced incurable stage in the majority of patients. The only potentially curative treatment is radical surgical resection. The effect of current chemotherapeutics or radiotherapy is limited. Novel therapeutic strategies are therefore much needed. One of the hallmarks of PDAC is its abundant desmoplastic (stromal) reaction. The Hedgehog (Hh) signaling pathway is critical for embryologic development of the pancreas. Aberrant Hh signaling promotes pancreatic carcinogenesis, the maintenance of the tumor microenvironment and stromal growth. The canonical Hh-pathway in the tumor stroma has been targeted widely but has not yet lead to hopeful clinical results. Targeting both the tumor and its surrounding stroma through Hh pathway inhibition by also targeting non-canonical pathways as apparent in the tumor cell may therefore be a novel treatment strategy for PDAC.

  13. Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila.

    PubMed

    Li, Shuang; Chen, Yongbin; Shi, Qing; Yue, Tao; Wang, Bing; Jiang, Jin

    2012-01-01

    Hedgehog transduces signal by promoting cell surface expression of the seven-transmembrane protein Smoothened (Smo) in Drosophila, but the underlying mechanism remains unknown. Here we demonstrate that Smo is downregulated by ubiquitin-mediated endocytosis and degradation, and that Hh increases Smo cell surface expression by inhibiting its ubiquitination. We find that Smo is ubiquitinated at multiple Lysine residues including those in its autoinhibitory domain (SAID), leading to endocytosis and degradation of Smo by both lysosome- and proteasome-dependent mechanisms. Hh inhibits Smo ubiquitination via PKA/CK1-mediated phosphorylation of SAID, leading to Smo cell surface accumulation. Inactivation of the ubiquitin activating enzyme Uba1 or perturbation of multiple components of the endocytic machinery leads to Smo accumulation and Hh pathway activation. In addition, we find that the non-visual β-arrestin Kurtz (Krz) interacts with Smo and acts in parallel with ubiquitination to downregulate Smo. Finally, we show that Smo ubiquitination is counteracted by the deubiquitinating enzyme UBPY/USP8. Gain and loss of UBPY lead to reciprocal changes in Smo cell surface expression. Taken together, our results suggest that ubiquitination plays a key role in the downregulation of Smo to keep Hh pathway activity off in the absence of the ligand, and that Hh-induced phosphorylation promotes Smo cell surface accumulation by inhibiting its ubiquitination, which contributes to Hh pathway activation.

  14. Regulation of Ci-SCFSlimb binding, Ci proteolysis and Hedgehog pathway activity by Ci phosphorylation

    PubMed Central

    Smelkinson, Margery G.; Zhou, Qianhe; Kalderon, Daniel

    2007-01-01

    SUMMARY Hedgehog (Hh) proteins signal by inhibiting the proteolytic processing of Ci/Gli family transcription factors and by increasing Ci/Gli specific activity. In the absence of Hh, phosphorylation of Ci/Gli triggers binding to SCF ubiquitin ligase complexes and consequent proteolysis. Here we define the principal SCFSlimb binding site in Ci as an extended variant of a canonical Slimb/β-TRCP binding motif that can be created by PKA-priming of five successive CK1 sites. GSK3 enhances binding primarily through a nearby region of Ci, which may contact an SCF component other than Slimb. Studies of Ci variants with altered CK1 and GSK3 sites suggest that the large number of phosphorylation sites that direct SCFSlimb binding confers a Hh response that is both sensitive and graded, and that in the Drosophila wing disc, morphogenetic responses involve changes in both the level and specific activity of Ci. We also show that when Ci proteolysis is compromised, its specific activity is limited principally by Su(fu) and not by Cos2 cytoplasmic tethering or PKA phosphorylation. PMID:17925225

  15. A Comparison of Ci/Gli Activity as Regulated by Sufu in Drosophila and Mammalian Hedgehog Response

    PubMed Central

    Zhang, Chi; Guo, Yurong; Beachy, Philip A.

    2015-01-01

    Suppressor of fused (Su(fu)/Sufu), one of the most conserved components of the Hedgehog (Hh) signaling pathway, binds Ci/Gli transcription factors and impedes activation of target gene expression. In Drosophila, the Su(fu) mutation has a minimal phenotype, and we show here that Ci transcriptional activity in large part is regulated independently of Su(fu) by other pathway components. Mutant mice lacking Sufu in contrast show excessive pathway activity and die as embryos with patterning defects. Here we show that in cultured cells Hh stimulation can augment transcriptional activity of a Gli2 variant lacking Sufu interaction and, surprisingly, that regulation of Hh pathway targets is nearly normal in the neural tube of Sufu-/- mutant embryos that also lack Gli1 function. Some degree of Hh-induced transcriptional activation of Ci/Gli thus can occur independently of Sufu in both flies and mammals. We further note that Sufu loss can also reduce Hh induction of high-threshold neural tube fates, such as floor plate, suggesting a possible positive pathway role for Sufu. PMID:26271100

  16. Protein-bound Polysaccharide-K Inhibits Hedgehog Signaling Through Down-regulation of MAML3 and RBPJ Transcription Under Hypoxia, Suppressing the Malignant Phenotype in Pancreatic Cancer.

    PubMed

    Yamasaki, Akio; Onishi, Hideya; Imaizumi, Akira; Kawamoto, Makoto; Fujimura, Akiko; Oyama, Yasuhiro; Katano, Mitsuo

    2016-08-01

    Hedgehog signaling is activated in pancreatic cancer and could be a therapeutic target. We previously demonstrated that recombination signal binding protein for immunoglobulin-kappa-J region (RBPJ) and mastermind-like 3 (MAML3) contribute to the hypoxia-induced up-regulation of Smoothened (SMO) transcription. We have also shown that protein-bound polysaccharide-K (PSK) could be effective for refractory pancreatic cancer that down-regulates SMO transcription under hypoxia. In this study, we evaluated whether the anticancer mechanism of PSK involves inhibiting RBPJ and MAML3 expression under hypoxia. PSK reduced SMO, MAML3 and RBPJ expression in pancreatic cancer cells under hypoxia. PSK also blocked RBPJ-induced invasiveness under hypoxia by inhibiting matrix metalloproteinase expression. Lastly, we showed that PSK attenuated RBPJ-induced proliferation both in vitro and in vivo. These results suggest that PSK suppresses Hedgehog signaling through down-regulation of MAML3 and RBPJ transcription under hypoxia, inhibiting the induction of a malignant phenotype in pancreatic cancer. Our results may lead to development of new treatments for refractory pancreatic cancer using PSK as a Hedgehog inhibitor.

  17. Two lamprey Hedgehog genes share non-coding regulatory sequences and expression patterns with gnathostome Hedgehogs.

    PubMed

    Kano, Shungo; Xiao, Jin-Hua; Osório, Joana; Ekker, Marc; Hadzhiev, Yavor; Müller, Ferenc; Casane, Didier; Magdelenat, Ghislaine; Rétaux, Sylvie

    2010-10-13

    Hedgehog (Hh) genes play major roles in animal development and studies of their evolution, expression and function point to major differences among chordates. Here we focused on Hh genes in lampreys in order to characterize the evolution of Hh signalling at the emergence of vertebrates. Screening of a cosmid library of the river lamprey Lampetra fluviatilis and searching the preliminary genome assembly of the sea lamprey Petromyzon marinus indicate that lampreys have two Hh genes, named Hha and Hhb. Phylogenetic analyses suggest that Hha and Hhb are lamprey-specific paralogs closely related to Sonic/Indian Hh genes. Expression analysis indicates that Hha and Hhb are expressed in a Sonic Hh-like pattern. The two transcripts are expressed in largely overlapping but not identical domains in the lamprey embryonic brain, including a newly-described expression domain in the nasohypophyseal placode. Global alignments of genomic sequences and local alignment with known gnathostome regulatory motifs show that lamprey Hhs share conserved non-coding elements (CNE) with gnathostome Hhs albeit with sequences that have significantly diverged and dispersed. Functional assays using zebrafish embryos demonstrate gnathostome-like midline enhancer activity for CNEs contained in intron2. We conclude that lamprey Hh genes are gnathostome Shh-like in terms of expression and regulation. In addition, they show some lamprey-specific features, including duplication and structural (but not functional) changes in the intronic/regulatory sequences.

  18. Development of anticancer agents targeting the Hedgehog signaling.

    PubMed

    Zhang, Xiangqian; Tian, Ye; Yang, Yanling; Hao, Jijun

    2017-03-17

    Hedgehog signaling is an evolutionarily conserved pathway which is essential in embryonic and postnatal development as well as adult organ homeostasis. Abnormal regulation of Hedgehog signaling is implicated in many diseases including cancer. Consequently, substantial efforts have made in the past to develop potential therapeutic agents that specifically target the Hedgehog signaling for cancer treatment. Here, we review the therapeutic agents for inhibition of the Hedgehog signaling and their clinical advances in cancer treatment.

  19. A switch from low to high Shh activity regulates establishment of limb progenitors and signaling centers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The patterning and growth of the embryonic vertebrate limb is dependent on Sonic hedgehog (Shh), a morphogen that regulates the activity of Gli transcription factors. However, "Shh" expression is not observed during the first 12 hours of limb development. During this phase, the limb bud is prepatter...

  20. A Polyamine Twist on Hedgehog Signaling.

    PubMed

    Zhao, Xuesong; Segal, Rosalind A

    2015-10-12

    The Hedgehog pathway plays important roles in embryonic development and oncogenesis, but how it affects metabolism is less clear. D'Amico et al. (2015) now demonstrate that the Hedgehog pathway regulates translation of ornithine decarboxylase, thereby enhancing polyamine biosynthesis and cell proliferation in neural precursor cells and in brain tumors.

  1. sm"FISH"ing for Hedgehog.

    PubMed

    Drummond, Michael L; Atwood, Scott X

    2017-01-01

    Patched (Ptch) receptors are critical negative regulators of Hedgehog signaling, where Ptch1 loss causes basal cell carcinoma and Ptch1;Ptch2 loss disrupts skin and hair follicle development. Adolphe et al. use single molecule fluorescent in situ hybridization to show quantitatively that Ptch receptors create a Hedgehog signaling gradient that may specify hair follicle development.

  2. COP9-Hedgehog axis regulates the function of the germline stem cell progeny differentiation niche in the Drosophila ovary.

    PubMed

    Lu, Tinglin; Wang, Su; Gao, Yuan; Mao, Ying; Yang, Zhihao; Liu, Luping; Song, Xiaoqing; Ni, Jianquan; Xie, Ting

    2015-12-15

    Both stem cell self-renewal and lineage differentiation are controlled extrinsically as well as intrinsically. Germline stem cells (GSCs) in the Drosophila ovary provide an attractive model in which to study both stem cell self-renewal and lineage differentiation at the molecular and cellular level. Recently, we have proposed that escort cells (ECs) form a differentiation niche to control GSC lineage specification extrinsically. However, it remains poorly understood how the maintenance and function of the differentiation niche are regulated at the molecular level. Here, this study reveals a new role of COP9 in the differentiation niche to modulate autocrine Hedgehog (Hh) signaling, thereby promoting GSC lineage differentiation. COP9, which is a highly conserved protein complex composed of eight CSN subunits, catalyzes the removal of Nedd8 protein modification from target proteins. Our genetic results have demonstrated that all the COP9 components and the hh pathway components, including hh itself, are required in ECs to promote GSC progeny differentiation. Interestingly, COP9 is required in ECs to maintain Hh signaling activity, and activating Hh signaling in ECs can partially bypass the requirement for COP9 in GSC progeny differentiation. Finally, both COP9 and Hh signaling in ECs promote GSC progeny differentiation partly by preventing BMP signaling and maintaining cellular processes. Therefore, this study has demonstrated that the COP9-Hh signaling axis operates in the differentiation niche to promote GSC progeny differentiation partly by maintaining EC cellular processes and preventing BMP signaling. This provides new insight into how the function of the differentiation niche is regulated at the molecular level.

  3. Cytoneme-mediated delivery of hedgehog regulates the expression of bone morphogenetic proteins to maintain germline stem cells in Drosophila.

    PubMed

    Rojas-Ríos, Patricia; Guerrero, Isabel; González-Reyes, Acaimo

    2012-01-01

    Stem cells reside in specialised microenvironments, or niches, which often contain support cells that control stem cell maintenance and proliferation. Hedgehog (Hh) proteins mediate homeostasis in several adult niches, but a detailed understanding of Hh signalling in stem cell regulation is lacking. Studying the Drosophila female germline stem cell (GSC) niche, we show that Hh acts as a critical juxtacrine signal to maintain the normal GSC population of the ovary. Hh production in cap cells, a type of niche support cells, is regulated by the Engrailed transcription factor. Hh is then secreted to a second, adjacent population of niche cells, the escort cells, where it activates transcription of the GSC essential factors Decapentaplegic (Dpp) and Glass bottom boat (Gbb). In wild-type niches, Hh protein decorates short filopodia that originate in the support cap cells and that are functionally relevant, as they are required to transduce the Hh pathway in the escort cells and to maintain a normal population of GSCs. These filopodia, reminiscent of wing disc cytonemes, grow several fold in length if Hh signalling is impaired within the niche. Because these long cytonemes project directionally towards the signalling-deficient region, cap cells sense and react to the strength of Hh pathway transduction in the niche. Thus, the GSC niche responds to insufficient Hh signalling by increasing the range of Hh spreading. Although the signal(s) perceived by the cap cells and the receptor(s) involved are still unknown, our results emphasise the integration of signals necessary to maintain a functional niche and the plasticity of cellular niches to respond to challenging physiological conditions.

  4. Novel neutralizing hedgehog antibody MEDI-5304 exhibits antitumor activity by inhibiting paracrine hedgehog signaling.

    PubMed

    Michaud, Neil R; Wang, Youzhen; McEachern, Kristen A; Jordan, Jerold J; Mazzola, Anne Marie; Hernandez, Axel; Jalla, Sanjoo; Chesebrough, Jon W; Hynes, Mark J; Belmonte, Matthew A; Wang, Lidong; Kang, Jaspal S; Jovanovic, Jelena; Laing, Naomi; Jenkins, David W; Hurt, Elaine; Liang, Meina; Frantz, Christopher; Hollingsworth, Robert E; Simeone, Diane M; Blakey, David C; Bedian, Vahe

    2014-02-01

    The hedgehog pathway has been implicated in the tumorigenesis, tumor progression, and metastasis of numerous human cancers. We generated the first fully human hedgehog antibody MEDI-5304 and characterized its antitumor activity and preclinical toxicology. MEDI-5304 bound sonic hedgehog (SHH) and Indian hedgehog (IHH) with low picomolar affinity and neutralized SHH and IHH activity in cellular mGLI1 reporter assays. The antibody inhibited transcription of hedgehog target genes and osteoblast differentiation of C3H10T1/2 cells. We evaluated the activity of MEDI-5304 in vivo in model systems that allowed us to evaluate two primary hypotheses of hedgehog function in human cancer, paracrine signaling between tumor and stromal cells and cancer stem cell (CSC) self-renewal. MEDI-5304 displayed robust pharmacodynamic effects in stromal cells that translated to antitumor efficacy as a single agent in an HT-29/MEF coimplantation model of paracrine hedgehog signaling. MEDI-5304 also improved responses to carboplatin in the HT-29/MEF model. The antibody, however, had no effect as a single agent or in combination with gemcitabine on the CSC frequency or growth of several primary pancreatic cancer explant models. These findings support the conclusion that hedgehog contributes to tumor biology via paracrine tumor-stromal signaling but not via CSC maintenance or propagation. Finally, the only safety study finding associated with MEDI-5304 was ondontodysplasia in rats. Thus, MEDI-5304 represents a potent dual hedgehog inhibitor suitable for continued development to evaluate efficacy and safety in human patients with tumors harboring elevated levels of SHH or IHH.

  5. Hedgehog is a positive regulator of FGF signalling during embryonic tracheal cell migration.

    PubMed

    Butí, Elisenda; Mesquita, Duarte; Araújo, Sofia J

    2014-01-01

    Cell migration is a widespread and complex process that is crucial for morphogenesis and for the underlying invasion and metastasis of human cancers. During migration, cells are steered toward target sites by guidance molecules that induce cell direction and movement through complex intracellular mechanisms. The spatio-temporal regulation of the expression of these guidance molecules is of extreme importance for both normal morphogenesis and human disease. One way to achieve this precise regulation is by combinatorial inputs of different transcription factors. Here we used Drosophila melanogaster mutants with migration defects in the ganglionic branches of the tracheal system to further clarify guidance regulation during cell migration. By studying the cellular consequences of overactivated Hh signalling, using ptc mutants, we found that Hh positively regulates Bnl/FGF levels during embryonic stages. Our results show that Hh modulates cell migration non-autonomously in the tissues surrounding the action of its activity. We further demonstrate that the Hh signalling pathway regulates bnl expression via Stripe (Sr), a zinc-finger transcription factor with homology to the Early Growth Response (EGR) family of vertebrate transcription factors. We propose that Hh modulates embryonic cell migration by participating in the spatio-temporal regulation of bnl expression in a permissive mode. By doing so, we provide a molecular link between the activation of Hh signalling and increased chemotactic responses during cell migration.

  6. Nkx factors specifically regulate expression of Hedgehog receptor isoforms in early embryonic development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: NK homeobox family members are tissue-specific transcription factors that regulate developmental genes. Homozygous disruption of Nkx3.2 produces severe developmental defects of the axial skeleton, skull, spleen, and stomach. Murine mutation of Nkx2.5 results in death at E9 with defects i...

  7. Low-level Ga-Al-As laser irradiation enhances osteoblast proliferation through activation of Hedgehog signaling pathway

    NASA Astrophysics Data System (ADS)

    Li, Qiushi; Qu, Zhou; Chen, Yingxin; Liu, Shujie; Zhou, Yanmin

    2014-12-01

    Low-level laser irradiation has been reported to promote bone formation, but the molecular mechanism is still unclear. Hedgehog signaling pathway has been reported to play an important role in promoting bone formation. The aim of the present study was to examine whether low-level Ga-Al-As laser (808 nm) irradiation could have an effect on Hedgehog signaling pathway during osteoblast proliferation in vitro. Mouse osteoblastic cell line MC3T3-E1 was cultured in vitro. The cultures after laser irradiation (3.75J/cm2) were treated with recombinant N-terminals Sonic Hedgehog (N-Shh)or Hedgehog inhibitor cyclopamine (cy). The experiment was divided into 4 group, group 1:laser irradiation, group 2: laser irradiation and N-Shh, group 3: laser irradiation and cy, group 4:control with no laser irradiation. On day 1,2 and 3,cell proliferation was determined by cell counting, Cell Counting Kit-8.On 12 h and 24 h, cell cycle was detected by flow cytometry. Proliferation activity of laser irradiation and N-Shh group was remarkably increased compared with those of laser irradiation group. Proliferation activity of laser irradiation and cy group was remarkably decreased compared with those of laser irradiation group, however proliferation activity of laser irradiation and cy group was remarkably increased compared with those of control group. These results suggest that low-level Ga-Al-As laser irradiation activate Hedgehog signaling pathway during osteoblast proliferation in vitro. Hedgehog signaling pathway is one of the signaling pathways by which low-level Ga-Al-As laser irradiation regulates osteoblast proliferation.

  8. Hedgehog signaling is a potent regulator of liver lipid metabolism and reveals a GLI-code associated with steatosis.

    PubMed

    Matz-Soja, Madlen; Rennert, Christiane; Schönefeld, Kristin; Aleithe, Susanne; Boettger, Jan; Schmidt-Heck, Wolfgang; Weiss, Thomas S; Hovhannisyan, Amalya; Zellmer, Sebastian; Klöting, Nora; Schulz, Angela; Kratzsch, Jürgen; Guthke, Reinhardt; Gebhardt, Rolf

    2016-05-17

    Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries and is increasing in prevalence. The pathomechanisms, however, are poorly understood. This study assessed the unexpected role of the Hedgehog pathway in adult liver lipid metabolism. Using transgenic mice with conditional hepatocyte-specific deletion of Smoothened in adult mice, we showed that hepatocellular inhibition of Hedgehog signaling leads to steatosis by altering the abundance of the transcription factors GLI1 and GLI3. This steatotic 'Gli-code' caused the modulation of a complex network of lipogenic transcription factors and enzymes, including SREBP1 and PNPLA3, as demonstrated by microarray analysis and siRNA experiments and could be confirmed in other steatotic mouse models as well as in steatotic human livers. Conversely, activation of the Hedgehog pathway reversed the "Gli-code" and mitigated hepatic steatosis. Collectively, our results reveal that dysfunctions in the Hedgehog pathway play an important role in hepatic steatosis and beyond.

  9. Hedgehog signaling is a potent regulator of liver lipid metabolism and reveals a GLI-code associated with steatosis

    PubMed Central

    Matz-Soja, Madlen; Rennert, Christiane; Schönefeld, Kristin; Aleithe, Susanne; Boettger, Jan; Schmidt-Heck, Wolfgang; Weiss, Thomas S; Hovhannisyan, Amalya; Zellmer, Sebastian; Klöting, Nora; Schulz, Angela; Kratzsch, Jürgen; Guthke, Reinhardt; Gebhardt, Rolf

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries and is increasing in prevalence. The pathomechanisms, however, are poorly understood. This study assessed the unexpected role of the Hedgehog pathway in adult liver lipid metabolism. Using transgenic mice with conditional hepatocyte-specific deletion of Smoothened in adult mice, we showed that hepatocellular inhibition of Hedgehog signaling leads to steatosis by altering the abundance of the transcription factors GLI1 and GLI3. This steatotic 'Gli-code' caused the modulation of a complex network of lipogenic transcription factors and enzymes, including SREBP1 and PNPLA3, as demonstrated by microarray analysis and siRNA experiments and could be confirmed in other steatotic mouse models as well as in steatotic human livers. Conversely, activation of the Hedgehog pathway reversed the "Gli-code" and mitigated hepatic steatosis. Collectively, our results reveal that dysfunctions in the Hedgehog pathway play an important role in hepatic steatosis and beyond. DOI: http://dx.doi.org/10.7554/eLife.13308.001 PMID:27185526

  10. A new role for Hedgehogs in juxtacrine signaling.

    PubMed

    Pettigrew, Christopher A; Asp, Eva; Emerson, Charles P

    2014-02-01

    The Hedgehog pathway plays important roles in embryonic development, adult stem cell maintenance and tumorigenesis. In mammals these effects are mediated by Sonic, Desert and Indian Hedgehog (Shh, Dhh and Ihh). Shh undergoes autocatalytic cleavage and dual lipidation prior to secretion and forming a response gradient. Post-translational processing and secretion of Dhh and Ihh ligands has not previously been investigated. This study reports on the synthesis, processing, secretion and signaling activities of SHH, IHH and DHH preproteins expressed in cultured cells, providing unexpected evidence that DHH does not undergo substantial autoprocessing or secretion, and does not function in paracrine signaling. Rather, DHH functions as a juxtacrine signaling ligand to activate a cell contact-mediated HH signaling response, consistent with its localised signaling in vivo. Further, the LnCAP prostate cancer cell, when induced to express endogenous DHH and SHH, is active only in juxtacrine signaling. Domain swap studies reveal that the C-terminal domain of HH regulates its processing and secretion. These findings establish a new regulatory role for HHs in cell-mediated juxtacrine signaling in development and cancer.

  11. Oestrogen receptor-alpha regulates non-canonical Hedgehog-signalling in the mammary gland

    PubMed Central

    Okolowsky, Nadia; Furth, Priscilla A.; Hamel, Paul A.

    2014-01-01

    Mesenchymal dysplasia (mes) mice harbour a truncation in the C-terminal region of the Hh-ligand receptor, Patched-1 (mPtch1). While the mes variant of mPtch1 binds to Hh-ligands with an affinity similar to that of wild type mPtch1 and appears to normally regulate canonical Hh-signalling via smoothened, the mes mutation causes, among other non-lethal defects, a block to mammary ductal elongation at puberty. We demonstrated previously Hh-signalling induces the activation of Erk1/2 and c-src independently of its control of smo activity. Furthermore, mammary epithelial cell-directed expression of an activated allele of c-src rescued the block to ductal elongation in mes mice, albeit with delayed kinetics. Given that this rescue was accompanied by an induction in estrogen receptor-alpha (ERα) expression and that complex regulatory interactions between ERα and c-src are required for normal mammary gland development, it was hypothesized that expression of ERα would also overcome the block to mammary ductal elongation at puberty in the mes mouse. We demonstrate here that conditional expression of ERα in luminal mammary epithelial cells on the mes background facilitates ductal morphogenesis with kinetics similar to that of the MMTV-c-srcAct mice. We demonstrate further that Erk1/2 is activated in primary mammary epithelial cells by Shh-ligand and that this activation is blocked by the inhibitor of c-src, PP2, is partially blocked by the ERα inhibitor, ICI 182780 but is not blocked by the smo-inhibitor, SANT-1. These data reveal an apparent Hh-signalling cascade operating through c-src and ERα that is required for mammary gland morphogenesis at puberty. PMID:24769368

  12. The Hedgehog Inhibitor Cyclopamine Reduces β-Catenin-Tcf Transcriptional Activity, Induces E-Cadherin Expression, and Reduces Invasion in Colorectal Cancer Cells

    PubMed Central

    Qualtrough, David; Rees, Phil; Speight, Beverley; Williams, Ann C.; Paraskeva, Christos

    2015-01-01

    Colorectal cancer is a major global health problem resulting in over 600,000 deaths world-wide every year with the majority of these due to metastatic disease. Wnt signalling, and more specifically β-catenin-related transcription, has been shown to drive both tumorigenesis and the metastatic process in colorectal neoplasia, yet its complex interactions with other key signalling pathways, such as hedgehog, remain to be elucidated. We have previously shown that the Hedgehog (HH) signalling pathway is active in cells from colorectal tumours, and that inhibition of the pathway with cyclopamine induces apoptosis. We now show that cyclopamine treatment reduces β-catenin related transcription in colorectal cancer cell lines, and that this effect can be reversed by addition of Sonic Hedgehog protein. We also show that cyclopamine concomitantly induces expression of the tumour suppressor and prognostic indicator E-cadherin. Consistent with a role for HH in regulating the invasive potential we show that cyclopamine reduces the expression of transcription factors (Slug, Snail and Twist) associated with the epithelial-mesenchymal transition and reduces the invasiveness of colorectal cancer cells in vitro. Taken together, these data show that pharmacological inhibition of the hedgehog pathway has therapeutic potential in the treatment of colorectal cancer. PMID:26393651

  13. Hedgehog signaling pathway in small bovine ovarian follicles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The hedgehog signaling pathway is involved in the regulation of cell proliferation, differentiation, and turnover in a variety of mammalian embryonic and adult tissues including bovine ovarian granulosa and theca cells. Binding of hedgehog to the patch receptor derepresses smoothened resulting in t...

  14. Outfoxing the Hedgehog

    ERIC Educational Resources Information Center

    Barbieri, Richard

    2011-01-01

    Jim Collins's "Good to Great" has attained near-scriptural status in organizations, including nonprofits, which Collins says constitute a third of his readers. The pivot point in "Good to Great" is the Hedgehog Concept. The "Hedgehog Concept" (HC), this author claims, is dangerous for schools because it distorts the nature of education. As Collins…

  15. Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer.

    PubMed

    Watkins, D Neil; Berman, David M; Burkholder, Scott G; Wang, Baolin; Beachy, Philip A; Baylin, Stephen B

    2003-03-20

    Embryonic signalling pathways regulate progenitor cell fates in mammalian epithelial development and cancer. Prompted by the requirement for sonic hedgehog (Shh) signalling in lung development, we investigated a role for this pathway in regeneration and carcinogenesis of airway epithelium. Here we demonstrate extensive activation of the hedgehog (Hh) pathway within the airway epithelium during repair of acute airway injury. This mode of Hh signalling is characterized by the elaboration and reception of the Shh signal within the epithelial compartment, and immediately precedes neuroendocrine differentiation. We reveal a similar pattern of Hh signalling in airway development during normal differentiation of pulmonary neuroendocrine precursor cells, and in a subset of small-cell lung cancer (SCLC), a highly aggressive and frequently lethal human tumour with primitive neuroendocrine features. These tumours maintain their malignant phenotype in vitro and in vivo through ligand-dependent Hh pathway activation. We propose that some types of SCLC might recapitulate a critical, Hh-regulated event in airway epithelial differentiation. This requirement for Hh pathway activation identifies a common lethal malignancy that may respond to pharmacological blockade of the Hh signalling pathway.

  16. The energy sensor AMPK regulates Hedgehog signaling in human cells through a unique Gli1 metabolic checkpoint

    PubMed Central

    Di Magno, Laura; Basile, Alessio; Coni, Sonia; Manni, Simona; Sdruscia, Giulia; D'Amico, Davide; Antonucci, Laura; Infante, Paola; De Smaele, Enrico; Cucchi, Danilo; Ferretti, Elisabetta; Di Marcotullio, Lucia; Screpanti, Isabella; Canettieri, Gianluca

    2016-01-01

    Hedgehog signaling controls proliferation of cerebellar granule cell precursors (GCPs) and its aberrant activation is a leading cause of Medulloblastoma, the most frequent pediatric brain tumor. We show here that the energy sensor AMPK inhibits Hh signaling by phosphorylating a single residue of human Gli1 that is not conserved in other species. Studies with selective agonists and genetic deletion have revealed that AMPK activation inhibits canonical Hh signaling in human, but not in mouse cells. Indeed we show that AMPK phosphorylates Gli1 at the unique residue Ser408, which is conserved only in primates but not in other species. Once phosphorylated, Gli1 is targeted for proteasomal degradation. Notably, we show that selective AMPK activation inhibits Gli1-driven proliferation and that this effect is linked to Ser408 phosphorylation, which represents a key metabolic checkpoint for Hh signaling. Collectively, this data unveil a novel mechanism of inhibition of Gli1 function, which is exclusive for human cells and may be exploited for the treatment of Medulloblastoma or other Gli1 driven tumors. PMID:26843621

  17. Prognostic value of hedgehog signaling pathway in patients with colon cancer.

    PubMed

    Xu, Meihua; Li, Xinhua; Liu, Ting; Leng, Aimin; Zhang, Guiying

    2012-06-01

    Hedgehog signaling pathway plays an important role in normal mammalian gastrointestinal development and is implicated in the oncogenesis of various tumors. However, its correlation with progression and prognosis of colon cancer has not been well documented. This study was designed to investigate expression patterns of related proteins in hedgehog signaling pathway in colon cancer to elucidate its prognostic value in this tumor. Using human colon cancer and their corresponding non-diseased colon from 228 patients' biopsies, the expression of sonic hedgehog, its receptor Patched, and downstream transcription factor Gli1 was investigated by immunohistochemical staining to assess their association with the clinicopathological characteristics of colon cancer. Disease-free survival and overall survival were examined by Kaplan-Meier estimates and the log-rank test. Prognostic factors were determined by multivariate Cox analysis. One hundred and thirty-eight patients (59.6%) had sonic hedgehog-positive tumors and that the disease-free survival (43.5 vs. 73.3%, P < 0.001), and overall survival rates (50.7 vs. 88.9%, P < 0.001) of patients with sonic hedgehog-positive tumors were much lower than those of patients with sonic hedgehog-negative tumors. In addition, 163 patients (71.5%) had Patched-positive tumors, and the disease-free survival (41.7 vs. 76.9%, P < 0.001) and overall survival rates (55.2 vs. 80.0%, P = 0.002) of patients with Patched-positive tumors were also lower than those of patients with Patched-negative tumors. Moreover, positive Gli1 expression had a bad effect on the disease-free survival (41.9 vs. 73.2%, P < 0.001) and overall survival rate of patients with colon cancer (50.0 vs. 89.3%, P < 0.001). In a multivariate analysis, sonic hedgehog, Patched, and Gli1 status were indicators for poor disease-free survival and overall survival. These results have shown that the increasing expression of sonic hedgehog, Patched, and Gli1 are indicators for a poor

  18. Oestrogen receptor-α contributes to the regulation of the hedgehog signalling pathway in ERα-positive gastric cancer

    PubMed Central

    Kameda, C; Nakamura, M; Tanaka, H; Yamasaki, A; Kubo, M; Tanaka, M; Onishi, H; Katano, M

    2010-01-01

    Background: Oestrogen receptor-alpha (ERα) is highly expressed in diffuse-type gastric cancer and oestrogen increases the proliferation of ERα-positive gastric cancer. However, a detailed mechanism by which oestrogen increases the proliferation of these cells is still unclear. Methods: We used 17-β-oestradiol (E2) as a stimulator against the ERα pathway. Pure anti-oestrogen drug ICI 182 780 (ICI) and small interfering RNA against ERα (ERα siRNA) were used as inhibitors. Cyclopamine (Cyc) was used as the hedgehog (Hh) pathway inhibitor. Two human ERα-positive gastric cancer cells were used as target cells. Effects of the stimulator and inhibitor on E2-induced cell proliferation were also examined. Results: In ERα-positive cells, E2 increased not only cell proliferation but also one of the ligands of the Hh pathway, Shh expression. 17-β-Oestradiol-induced cell proliferation was suppressed by ICI, ERα siRNA or Cyc. The increased expression of Shh induced by E2 was suppressed by ICI and ERα siRNA but not by Cyc. Furthermore, recombinant Shh activated the Hh pathway and increased cell proliferation, whereas anti-Shh antibody suppressed E2-induced cell proliferation. When a relationship between ERα and Shh expressions was analysed using surgically resected gastric cancer specimens, a positive correlation was found, suggesting a linkage between the ERα and Hh pathways. Conclusion: Our data indicate that activation of the ERα pathway promotes cell proliferation by activating the Hh pathway in a ligand-dependent manner through Shh induction of ERα-positive gastric cancer. PMID:20087349

  19. Metastable nematic hedgehogs

    NASA Astrophysics Data System (ADS)

    Rosso, Riccardo; Virga, Epifanio G.

    1996-07-01

    For nematic liquid crystals, we study the local stability of a radial hedgehog against biaxial perturbations. Our analysis employs the Landau - de Gennes functional to describe the free energy stored in a ball, whose radius is a parameter of the model. We find that a radial hedgehog may be either unstable or metastable, depending on the values of the elastic constants. For unstable hedgehogs, we give an explicit expression for the radius of the ball within which the instability manifests itself: it can be interpreted as the size of the biaxial core of the defect; it is of the same order of magnitude as the radius of the disclination ring predicted by Penzenstadler and Trebin's model. The metastable hedgehogs predicted by our model are the major novelty of the paper. They tell us that we may also expect truly uniaxial point defects, whose core contains no biaxial structure.

  20. LKB1 suppresses proliferation and invasion of prostate cancer through hedgehog signaling pathway.

    PubMed

    Xu, Peiyuan; Cai, Fei; Liu, Xiaofei; Guo, Lele

    2014-01-01

    Activation of the hedgehog (Hh) signaling pathway has been implicated in the development of many human malignancies. Hh signaling target genes, such as patched (PTCH), smoothened (SMO) and sonic hedgehog (SHH), are markers of Hh signaling activation in most Hh-associated tumors. The protein kinase LKB1 has been shown to slow proliferation and induce cell-cycle arrest in many cell lines. However, the function of LKB1 in prostate cancer development remains largely unclear. In this study, the expression of LKB1 in human prostate cancer tissue samples and prostate cancer cell lines was detected, and the effects of LKB1 on prostate cancer cell proliferation and invasion were evaluated. Moreover, the influence of LKB1 on target genes of the Hh signaling pathway was analyzed. The results indicated that knockdown of LKB1 expression by RNA interference promoted cell proliferation, colony formation and invasion. Meanwhile, we observed that LKB1 siRNA increased the expression of factors related to Hh signaling reporter activity in prostate cancer cells, including PTCH, SMO and SHH. These findings suggest that LKB1 is a putative tumor suppressor gene in prostate cancer, and that LKB1 is negatively correlated with the expression of Hh signaling related transcription factors. Our results suggest that LKB1 may inhibit tumorigenesis by regulating the Hh signaling pathway in certain cancers.

  1. Hedgehog associated to microparticles inhibits adipocyte differentiation via a non-canonical pathway

    PubMed Central

    Fleury, Audrey; Hoch, Lucile; Martinez, M. Carmen; Faure, Hélène; Taddei, Maurizio; Petricci, Elena; Manetti, Fabrizio; Girard, Nicolas; Mann, André; Jacques, Caroline; Larghero, Jérôme; Ruat, Martial; Andriantsitohaina, Ramaroson; Le Lay, Soazig

    2016-01-01

    Hedgehog (Hh) is a critical regulator of adipogenesis. Extracellular vesicles are natural Hh carriers, as illustrated by activated/apoptotic lymphocytes specifically shedding microparticles (MP) bearing the morphogen (MPHh+). We show that MPHh+ inhibit adipocyte differentiation and orientate mesenchymal stem cells towards a pro-osteogenic program. Despite a Smoothened (Smo)-dependency, MPHh+ anti-adipogenic effects do not activate a canonical Hh signalling pathway in contrast to those elicited either by the Smo agonist SAG or recombinant Sonic Hedgehog. The Smo agonist GSA-10 recapitulates many of the hallmarks of MPHh+ anti-adipogenic effects. The adipogenesis blockade induced by MPHh+ and GSA-10 was abolished by the Smo antagonist LDE225. We further elucidate a Smo/Lkb1/Ampk axis as the non-canonical Hh pathway used by MPHh+ and GSA-10 to inhibit adipocyte differentiation. Our results highlight for the first time the ability of Hh-enriched MP to signal via a non-canonical pathway opening new perspectives to modulate fat development. PMID:27010359

  2. Vertebrate Hedgehog is secreted on two types of extracellular vesicles with different signaling properties

    PubMed Central

    Vyas, Neha; Walvekar, Ankita; Tate, Dhananjay; Lakshmanan, Vairavan; Bansal, Dhiru; Cicero, Alessandra Lo; Raposo, Graca; Palakodeti, Dasaradhi; Dhawan, Jyotsna

    2014-01-01

    Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tissues. Multiple proposed mechanisms to regulate Hh dispersion includes lipoprotein particles and exosomes. Here we report that vertebrate Sonic Hedgehog (Shh) is secreted on two types of extracellular-vesicles/exosomes, from human cell lines and primary chick notochord cells. Although largely overlapping in size as estimated from electron micrographs, the two exosomal fractions exhibited distinct protein and RNA composition. We have probed the functional properties of these vesicles using cell-based assays of Hh-elicited gene expression. Our results suggest that while both Shh-containing exo-vesicular fractions can activate an ectopic Gli-luciferase construct, only exosomes co-expressing Integrins can activate endogenous Shh target genes HNF3β and Olig2 during the differentiation of mouse ES cells to ventral neuronal progenitors. Taken together, our results demonstrate that primary vertebrate cells secrete Shh in distinct vesicular forms, and support a model where packaging of Shh along with other signaling proteins such as Integrins on exosomes modulates target gene activation. The existence of distinct classes of Shh-containing exosomes also suggests a previously unappreciated complexity for fine-tuning of Shh-mediated gradients and pattern formation. PMID:25483805

  3. Vertebrate Hedgehog is secreted on two types of extracellular vesicles with different signaling properties.

    PubMed

    Vyas, Neha; Walvekar, Ankita; Tate, Dhananjay; Lakshmanan, Vairavan; Bansal, Dhiru; Lo Cicero, Alessandra; Raposo, Graca; Palakodeti, Dasaradhi; Dhawan, Jyotsna

    2014-12-08

    Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tissues. Multiple proposed mechanisms to regulate Hh dispersion includes lipoprotein particles and exosomes. Here we report that vertebrate Sonic Hedgehog (Shh) is secreted on two types of extracellular-vesicles/exosomes, from human cell lines and primary chick notochord cells. Although largely overlapping in size as estimated from electron micrographs, the two exosomal fractions exhibited distinct protein and RNA composition. We have probed the functional properties of these vesicles using cell-based assays of Hh-elicited gene expression. Our results suggest that while both Shh-containing exo-vesicular fractions can activate an ectopic Gli-luciferase construct, only exosomes co-expressing Integrins can activate endogenous Shh target genes HNF3β and Olig2 during the differentiation of mouse ES cells to ventral neuronal progenitors. Taken together, our results demonstrate that primary vertebrate cells secrete Shh in distinct vesicular forms, and support a model where packaging of Shh along with other signaling proteins such as Integrins on exosomes modulates target gene activation. The existence of distinct classes of Shh-containing exosomes also suggests a previously unappreciated complexity for fine-tuning of Shh-mediated gradients and pattern formation.

  4. Hedgehog inhibition causes complete loss of limb outgrowth and transformation of digit identity in Xenopus tropicalis.

    PubMed

    Stopper, Geffrey F; Richards-Hrdlicka, Kathryn L; Wagner, Günter P

    2016-03-01

    The study of the tetrapod limb has contributed greatly to our understanding of developmental pathways and how changes to these pathways affect the evolution of morphology. Most of our understanding of tetrapod limb development comes from research on amniotes, with far less known about mechanisms of limb development in amphibians. To better understand the mechanisms of limb development in anuran amphibians, we used cyclopamine to inhibit Hedgehog signaling at various stages of development in the western clawed frog, Xenopus tropicalis, and observed resulting morphologies. We also analyzed gene expression changes resulting from similar experiments in Xenopus laevis. Inhibition of Hedgehog signaling in X. tropicalis results in limb abnormalities including reduced digit number, missing skeletal elements, and complete absence of limbs. In addition, posterior digits assume an anterior identity by developing claws that are usually only found on anterior digits, confirming Sonic hedgehog's role in digit identity determination. Thus, Sonic hedgehog appears to play mechanistically separable roles in digit number specification and digit identity specification as in other studied tetrapods. The complete limb loss observed in response to reduced Hedgehog signaling in X. tropicalis, however, is striking, as this functional role for Hedgehog signaling has not been found in any other tetrapod. This changed mechanism may represent a substantial developmental constraint to digit number evolution in frogs. J. Exp. Zool. (Mol. Dev. Evol.) 9999B:XX-XX, 2016. © 2016 Wiley Periodicals, Inc.

  5. The hedgehog system in ovarian follicles of cattle selected for twin ovulations and births: evidence of a link between the IGF and hedgehog systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The hedgehog system is involved in the regulation of ovarian function in drosophila, but its role in regulating ovarian follicular function in mammals is unclear. Therefore, gene expression of Indian hedgehog ligand (Ihh), its type 1 receptor (patched 1; Patch1), and IGF type 2 receptor (IGF2R) were...

  6. The hedgehog system in ovarian follicles of cattle selected for twin ovulations and births: evidence of a link between the IGF and hedgehog systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hedgehog signaling is involved in regulation of ovarian function in Drosophila but its role in regulating mammalian ovarian folliculogenesis is less clear. Therefore, gene expression of Indian hedgehog (IHH) and its type 1 receptor, patched 1 (PTCH1), were quantified in bovine granulosa (GC) or the...

  7. Use of a Conditional Ubr5 Mutant Allele to Investigate the Role of an N-End Rule Ubiquitin-Protein Ligase in Hedgehog Signalling and Embryonic Limb Development

    PubMed Central

    Kinsella, Elaine; Dora, Natalie; Mellis, David; Lettice, Laura; Deveney, Paul

    2016-01-01

    Hedgehog (Hh) signalling is a potent regulator of cell fate and function. While much is known about the events within a Hh-stimulated cell, far less is known about the regulation of Hh-ligand production. Drosophila Hyperplastic Discs (Hyd), a ubiquitin-protein ligase, represents one of the few non-transcription factors that independently regulates both hh mRNA expression and pathway activity. Using a murine embryonic stem cell system, we revealed that shRNAi of the mammalian homologue of hyd, Ubr5, effectively prevented retinoic-acid-induced Sonic hedgehog (Shh) expression. We next investigated the UBR5:Hh signalling relationship in vivo by generating and validating a mouse bearing a conditional Ubr5 loss-of-function allele. Conditionally deleting Ubr5 in the early embryonic limb-bud mesenchyme resulted in a transient decrease in Indian hedgehog ligand expression and decreased Hh pathway activity, around E13.5. Although Ubr5-deficient limbs and digits were, on average, shorter than control limbs, the effects were not statistically significant. Hence, while loss of UBR5 perturbed Hedgehog signalling in the developing limb, there were no obvious morphological defects. In summary, we report the first conditional Ubr5 mutant mouse and provide evidence for a role for UBR5 in influencing Hh signalling, but are uncertain to whether the effects on Hedgehog signaling were direct (cell autonomous) or indirect (non-cell-autonomous). Elaboration of the cellular/molecular mechanism(s) involved may help our understanding on diseases and developmental disorders associated with aberrant Hh signalling. PMID:27299863

  8. Hedgehog nanopackages ready for dispatch.

    PubMed

    Vincent, Jean-Paul

    2008-06-27

    Hedgehog proteins are intercellular long-range signaling molecules that spread within tissues and activate gene expression during development. Vyas et al. (2008) propose that Hedgehog forms nanometer-sized oligomers that localize in proteoglycan-rich clusters at the surface of cells expressing Hedgehog. This nanoscale organization and enrichment in clusters ensures that Hedgehog is able to spread and activate signaling over many cell diameters.

  9. Sex and hedgehog: roles of genes in the hedgehog signaling pathway in mammalian sexual differentiation.

    PubMed

    Franco, Heather L; Yao, Humphrey H-C

    2012-01-01

    The chromosome status of the mammalian embryo initiates a multistage process of sexual development in which the bipotential reproductive system establishes itself as either male or female. These events are governed by intricate cell-cell and interorgan communication that is regulated by multiple signaling pathways. The hedgehog signaling pathway was originally identified for its key role in the development of Drosophila, but is now recognized as a critical developmental regulator in many species, including humans. In addition to its developmental roles, the hedgehog signaling pathway also modulates adult organ function, and misregulation of this pathway often leads to diseases, such as cancer. The hedgehog signaling pathway acts through its morphogenetic ligands that signal from ligand-producing cells to target cells over a specified distance. The target cells then respond in a graded manner based on the concentration of the ligands that they are exposed to. Through this unique mechanism of action, the hedgehog signaling pathway elicits cell fate determination, epithelial-mesenchymal interactions, and cellular homeostasis. Here, we review current findings on the roles of hedgehog signaling in the sexually dimorphic development of the reproductive organs with an emphasis on mammals and comparative evidence in other species.

  10. Regulation of DNA damage following termination of Hedgehog (HH) survival signaling at the level of the GLI genes in human colon cancer.

    PubMed

    Agyeman, Akwasi; Mazumdar, Tapati; Houghton, Janet A

    2012-08-01

    Transcriptional regulation of the Hedgehog (HH) signaling response is mediated by GLI genes (GLI1, GLI2) downstream of SMO, that are also activated by oncogenic signaling pathways. We have demonstrated the importance of targeting GLI downstream of SMO in the induction of cell death in human colon carcinoma cells. In HT29 cells inhibition of GLI1/GLI2 by the small molecule inhibitor GANT61 induced DNA double strand breaks (DSBs) and activation of ATM, MDC1 and NBS1; γH2AX and MDC1, NBS1 and MDC1 co-localized in nuclear foci. Early activation of ATM was decreased by 24 hr, when p-NBS1(Ser343), activated by ATM, was significantly reduced in cell extracts. Bound γH2AX was detected in isolated chromatin fractions or nuclei during DNA damage but not during DNA repair. MDC1 was tightly bound to chromatin at 32 hr as cells accumulated in early S-phase prior to becoming subG1, and during DNA repair. Limited binding of NBS1 was detected at all times during DNA damage but was strongly bound during DNA repair. Transient overexpression of NBS1 protected HT29 cells from GANT61-induced cell death, while knockdown of H2AX by H2AXshRNA delayed DNA damage signaling. Data demonstrate following GLI1/GLI2 inhibition: 1) induction of DNA damage in cells that are also resistant to SMO inhibitors, 2) dynamic interactions between γH2AX, MDC1 and NBS1 in single cell nuclei and in isolated chromatin fractions, 3) expression and chromatin binding properties of key mediator proteins that mark DNA damage or DNA repair, and 4) the importance of NBS1 in the DNA damage response mechanism.

  11. Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

    PubMed

    Sugiyama, Y; Sasajima, J; Mizukami, Y; Koizumi, K; Kawamoto, T; Ono, Y; Karasaki, H; Tanabe, H; Fujiya, M; Kohgo, Y

    2016-06-01

    The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

  12. Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling

    SciTech Connect

    Huang, Pengxiang; Nedelcu, Daniel; Watanabe, Miyako; Jao, Cindy; Kim, Youngchang; Liu, Jing; Salic, Adrian

    2016-08-01

    In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol.

  13. Reciprocal interplay between thyroid hormone and microRNA-21 regulates hedgehog pathway–driven skin tumorigenesis

    PubMed Central

    Di Girolamo, Daniela; Ambrosio, Raffaele; De Stefano, Maria A.; Mancino, Giuseppina; Porcelli, Tommaso; Luongo, Cristina; Di Cicco, Emery; Scalia, Giulia; Vecchio, Luigi Del; Colao, Annamaria; Dlugosz, Andrzej A.; Missero, Caterina; Salvatore, Domenico

    2016-01-01

    The thyroid hormone–inactivating (TH-inactivating) enzyme type 3 iodothyronine deiodinase (D3) is an oncofetal protein that is rarely expressed in adult life but has been shown to be reactivated in the context of proliferation and neoplasms. D3 terminates TH action within the tumor microenvironment, thereby enhancing cancer cell proliferation. However, the pathological role of D3 and the contribution of TH metabolism in cancer have yet to be fully explored. Here, we describe a reciprocal regulation between TH action and the cancer-associated microRNA-21 (miR21) in basal cell carcinoma (BCC) skin tumors. We found that, besides being negatively regulated by TH at the transcriptional level, miR21 attenuates the TH signal by increasing D3 levels. The ability of miR21 to positively regulate D3 was mediated by the tumor suppressor gene GRHL3, a hitherto unrecognized D3 transcriptional inhibitor. Finally, in a BCC mouse model, keratinocyte-specific D3 depletion markedly reduced tumor growth. Together, our results establish TH action as a critical hub of multiple oncogenic pathways and provide functional and mechanistic evidence of the involvement of TH metabolism in BCC tumorigenesis. Moreover, our results identify a miR21/GRHL3/D3 axis that reduces TH in the tumor microenvironment and has potential to be targeted as a therapeutic approach to BCC. PMID:27159391

  14. Ultrasonic/Sonic Anchor

    NASA Technical Reports Server (NTRS)

    Bar-Cohen, Yoseph; Sherrit, Stewart

    2009-01-01

    The ultrasonic/sonic anchor (U/S anchor) is an anchoring device that drills a hole for itself in rock, concrete, or other similar material. The U/S anchor is a recent addition to a series of related devices, the first of which were reported in "Ultrasonic/Sonic Drill/Corers With Integrated Sensors"

  15. Greased hedgehogs: new links between hedgehog signaling and cholesterol metabolism.

    PubMed

    Breitling, Rainer

    2007-11-01

    The close link between signaling by the developmental regulators of the Hedgehog family and cholesterol biochemistry has been known for some time. The morphogen is covalently attached to cholesterol in a peculiar autocatalytic reaction and embryonal disruption of cholesterol synthesis leads to malformations that mimic Hh signaling defects. Recently, it was furthermore shown that secreted Hh could hitchhike on lipoprotein particles to establish its morphogenic gradient in the developing embryo. Additionally, there is new evidence that the Hh-receptor Patched transmits the Hh signal by modulating the secretion of an inhibitory sterol molecule from the receiving cells. Here we present some of the most recent discoveries on the Hh-sterol link and discuss their implications from a systems design perspective. We predict that a robust functioning of the Hh pathway will require the involvement of more sterol metabolites, and these should be the subject of future research.

  16. Taxane-induced hedgehog signaling is linked to expansion of breast cancer stem-like populations after chemotherapy.

    PubMed

    Sims-Mourtada, Jennifer; Opdenaker, Lynn M; Davis, Joshua; Arnold, Kimberly M; Flynn, Daniel

    2015-11-01

    Recurrence of breast cancer after chemotherapy is thought to arise from resistant breast cancer stem cells which are eventually able to repopulate the tumor. The Hedgehog (HH) signaling pathway has been shown to regulate the proliferation and survival of breast cancer stem cells, and has been shown to promote resistance to chemotherapy through the activation of multi-drug resistance and pro survival pathways. Here we report that exposure of heterogenous breast cancer cell lines to docetaxel (DOC) resulted in release of Sonic Hedgehog ligand (SHH) and activation of the HH pathway as evidenced by increased expression and nuclear translocation of the downstream effector Gli-1 at 4-24 h after DOC treatment. This activation had little effect on the bulk of the tumor cell population as inhibition of HH signaling failed to increase apoptosis in response to DOC. However, HH pathway activation was required for clonogenic growth of cell lines after DOC. Increases in stemness markers as well as mammosphere formation were observed after treatment with DOC suggesting an increase in the breast cancer stem cell populations. These increases were similar to that of cell lines cultured in the presence of recombinant SHH and could be eliminated by co-treatment with HH inhibitors. These results suggest that HH pathway activation induced by DOC treatment does not have a chemosensitizing effect on the heterogeneous tumor population, but may be required for survival and expansion of breast cancer stem cells after chemotherapy.

  17. Sonic spectrometer and treatment system

    DOEpatents

    Slomka, B.J.

    1997-06-03

    A novel system and method is developed for treating an object with sonic waveforms. A traveling broad-band sonic waveform containing a broad-band of sonic frequencies is radiated at the object. A traveling reflected sonic waveform containing sonic frequencies reflected by the object is received in response to the traveling broad-band sonic waveform. A traveling transmitted sonic waveform containing sonic frequencies transmitted through the object is also received in response to the traveling broad-band sonic waveform. In a resonance mode, the frequency spectra of the broad-band and reflected sonic waveforms is analyzed so as to select one or more sonic frequencies that cause the object to resonate. An electrical resonance treatment sonic waveform containing the sonic frequencies that cause the object to resonate is then radiated at the object so as to treat the object. In an absorption mode, the frequency spectra of the electrical broad-band, reflected, and transmitted sonic waveforms is compared so as to select one or more sonic frequencies that are absorbed by the object. An electrical absorption treatment sonic waveform containing the sonic frequencies that are absorbed by the object is then radiated at the object so as to treat the object. 1 fig.

  18. Sonic spectrometer and treatment system

    DOEpatents

    Slomka, Bogdan J.

    1997-06-03

    A novel system and method for treating an object with sonic waveforms. A traveling broad-band sonic waveform containing a broad-band of sonic frequencies is radiated at the object. A traveling reflected sonic waveform containing sonic frequencies reflected by the object is received in response to the traveling broad-band sonic waveform. A traveling transmitted sonic waveform containing sonic frequencies transmitted through the object is also received in response to the traveling broad-band sonic waveform. In a resonance mode, the frequency spectra of the broad-band and reflected sonic waveforms is analyzed so as to select one or more sonic frequencies that cause the object to resonate. An electrical resonance treatment sonic waveform containing the sonic frequencies that cause the object to resonate is then radiated at the object so as to treat the object. In an absorption mode, the frequency spectra of the electrical broad-band, reflected, and transmitted sonic waveforms is compared so as to select one or more sonic frequencies that are absorbed by the object. An electrical absorption treatment sonic waveform containing the sonic frequencies that are absorbed by the object is then radiated at the object so as to treat the object.

  19. Glucocorticoid hedgehog agonists in neurogenesis.

    PubMed

    Wang, Jiangbo; Barak, Larry S; Mook, Robert A; Chen, Wei

    2011-01-01

    The process of neurogenesis in mammals, which is prolific and widespread at birth, gradually slows with aging and in humans becomes restricted to areas including the cerebellum and hippocampus. It has been reported that exposure to glucocorticoids can impair neurogenesis in both adults and children. Glucocorticoids are known to bind with high affinity to intracellular receptors. Glucocorticoid blood levels are normally regulated by environmental stresses, but because of their clinical utility, exogenous glucocorticoids are frequently administered in drug formulations. Consequently, concerns have arisen about the consequences of glucocorticoid use on neurogenesis and health, especially in the pediatric population. In this article, we will review recent findings that a select number of related glucocorticoids, halcinonide, fluticasone propionate, clobetasol propionate, and fluocinonide, also bind the hedgehog pathway receptor Smoothened. We will discuss their pharmacology and also a most surprising result; that this select group of compounds, which includes FDA approved drugs, unlike typical glucocorticoids such as dexamethasone, stimulate stem cell growth, and thus enhance neurogenesis.

  20. Targeting hedgehog in hematologic malignancy.

    PubMed

    Irvine, David A; Copland, Mhairi

    2012-03-08

    The Hedgehog pathway is a critical mediator of embryonic patterning and organ development, including hematopoiesis. It influences stem cell fate, differentiation, proliferation, and apoptosis in responsive tissues. In adult organisms, hedgehog pathway activity is required for aspects of tissue maintenance and regeneration; however, there is increasing awareness that abnormal hedgehog signaling is associated with malignancy. Hedgehog signaling is critical for early hematopoietic development, but there is controversy over its role in normal hematopoiesis in adult organisms where it may be dispensable. Conversely, hedgehog signaling appears to be an important survival and proliferation signal for a spectrum of hematologic malignancies. Furthermore, hedgehog signaling may be critical for the maintenance and expansion of leukemic stem cells and therefore provides a possible mechanism to selectively target these primitive cell subpopulations, which are resistant to conventional chemotherapy. Indeed, phase 1 clinical trials of hedgehog pathway inhibitors are currently underway to test this hypothesis in myeloid leukemias. This review covers: (1) the hedgehog pathway and its role in normal and malignant hematopoiesis, (2) the recent development of clinical grade small molecule inhibitors of the pathway, and (3) the potential utility of hedgehog pathway inhibition as a therapeutic strategy in hemato-oncology.

  1. The Insecticide Synergist Piperonyl Butoxide Inhibits Hedgehog Signaling: Assessing Chemical Risks

    PubMed Central

    Chen, Wei

    2012-01-01

    The spread of chemicals, including insecticides, into the environment often raises public health concerns, as exemplified by a recent epidemiologic study associating in utero piperonyl butoxide (PBO) exposure with delayed mental development. The insecticide synergist PBO is listed among the top 10 chemicals detected in indoor dust; a systematic assessment of risks from PBO exposure, as for many toxicants unfortunately, may be underdeveloped when important biological targets that can cause toxicity are unknown. Hedgehog/Smoothened signaling is critical in neurological development. This study was designed to use novel high-throughput in vitro drug screening technology to identify modulators of Hedgehog signaling in environmental chemicals to assist the assessment of their potential risks. A directed library of 1408 environmental toxicants was screened for Hedgehog/Smoothened antagonist activity using a high-content assay that evaluated the interaction between Smoothened and βarrestin2 green fluorescent protein. PBO was identified as a Hedgehog/Smoothened antagonist capable of inhibiting Hedgehog signaling. We found that PBO bound Smoothened and blocked Smoothened overexpression–induced Gli-luciferase reporter activity but had no effect on Gli-1 downstream transcriptional factor–induced Gli activity. PBO inhibited Sonic Hedgehog ligand–induced Gli signaling and mouse cerebellar granular precursor cell proliferation. Moreover, PBO disrupted zebrafish development. Our findings demonstrate the value of high-throughput target-based screening strategies that can successfully evaluate large numbers of environmental toxicants and identify key targets and unknown biological activity that is helpful in properly assessing potential risks. PMID:22552772

  2. Sonic boom acceptability studies

    NASA Technical Reports Server (NTRS)

    Shepherd, Kevin P.; Sullivan, Brenda M.; Leatherwood, Jack D.; Mccurdy, David A.

    1992-01-01

    The determination of the magnitude of sonic boom exposure which would be acceptable to the general population requires, as a starting point, a method to assess and compare individual sonic booms. There is no consensus within the scientific and regulatory communities regarding an appropriate sonic boom assessment metric. Loudness, being a fundamental and well-understood attribute of human hearing was chosen as a means of comparing sonic booms of differing shapes and amplitudes. The figure illustrates the basic steps which yield a calculated value of loudness. Based upon the aircraft configuration and its operating conditions, the sonic boom pressure signature which reaches the ground is calculated. This pressure-time history is transformed to the frequency domain and converted into a one-third octave band spectrum. The essence of the loudness method is to account for the frequency response and integration characteristics of the auditory system. The result of the calculation procedure is a numerical description (perceived level, dB) which represents the loudness of the sonic boom waveform.

  3. The Hedgehog Receptor Patched Is Involved in Cholesterol Transport

    PubMed Central

    Bidet, Michel; Joubert, Olivier; Lacombe, Benoit; Ciantar, Marine; Nehmé, Rony; Mollat, Patrick; Brétillon, Lionel; Faure, Hélène; Bittman, Robert; Ruat, Martial; Mus-Veteau, Isabelle

    2011-01-01

    Background Sonic hedgehog (Shh) signaling plays a crucial role in growth and patterning during embryonic development, and also in stem cell maintenance and tissue regeneration in adults. Aberrant Shh pathway activation is involved in the development of many tumors, and one of the most affected Shh signaling steps found in these tumors is the regulation of the signaling receptor Smoothened by the Shh receptor Patched. In the present work, we investigated Patched activity and the mechanism by which Patched inhibits Smoothened. Methodology/Principal Findings Using the well-known Shh-responding cell line of mouse fibroblasts NIH 3T3, we first observed that enhancement of the intracellular cholesterol concentration induces Smoothened enrichment in the plasma membrane, which is a crucial step for the signaling activation. We found that binding of Shh protein to its receptor Patched, which involves Patched internalization, increases the intracellular concentration of cholesterol and decreases the efflux of a fluorescent cholesterol derivative (BODIPY-cholesterol) from these cells. Treatment of fibroblasts with cyclopamine, an antagonist of Shh signaling, inhibits Patched expression and reduces BODIPY-cholesterol efflux, while treatment with the Shh pathway agonist SAG enhances Patched protein expression and BODIPY-cholesterol efflux. We also show that over-expression of human Patched in the yeast S. cerevisiae results in a significant boost of BODIPY-cholesterol efflux. Furthermore, we demonstrate that purified Patched binds to cholesterol, and that the interaction of Shh with Patched inhibits the binding of Patched to cholesterol. Conclusion/Significance Our results suggest that Patched may contribute to cholesterol efflux from cells, and to modulation of the intracellular cholesterol concentration. This activity is likely responsible for the inhibition of the enrichment of Smoothened in the plasma membrane, which is an important step in Shh pathway activation. PMID

  4. Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

    PubMed Central

    Maun, Henry R.; Wen, Xiaohui; Lingel, Andreas; de Sauvage, Frederic J.; Lazarus, Robert A.; Scales, Suzie J.; Hymowitz, Sarah G.

    2010-01-01

    Proper hedgehog (Hh) signaling is crucial for embryogenesis and tissue regeneration. Dysregulation of this pathway is associated with several types of cancer. The monoclonal antibody 5E1 is a Hh pathway inhibitor that has been extensively used to elucidate vertebrate Hh biology due to its ability to block binding of the three mammalian Hh homologs to the receptor, Patched1 (Ptc1). Here, we engineered a murine:human chimeric 5E1 (ch5E1) with similar Hh-binding properties to the original murine antibody. Using biochemical, biophysical, and x-ray crystallographic studies, we show that, like the regulatory receptors Cdon and Hedgehog-interacting protein (Hhip), ch5E1 binding to Sonic hedgehog (Shh) is enhanced by calcium ions. In the presence of calcium and zinc ions, the ch5E1 binding affinity increases 10–20-fold to tighter than 1 nm primarily because of a decrease in the dissociation rate. The co-crystal structure of Shh bound to the Fab fragment of ch5E1 reveals that 5E1 binds at the pseudo-active site groove of Shh with an epitope that largely overlaps with the binding site of its natural receptor antagonist Hhip. Unlike Hhip, the side chains of 5E1 do not directly coordinate the Zn2+ cation in the pseudo-active site, despite the modest zinc-dependent increase in 5E1 affinity for Shh. Furthermore, to our knowledge, the ch5E1 Fab-Shh complex represents the first structure of an inhibitor antibody bound to a metalloprotease fold. PMID:20504762

  5. Characterization of Hedgehog Acyltransferase Inhibitors Identifies a Small Molecule Probe for Hedgehog Signaling by Cancer Cells.

    PubMed

    Rodgers, Ursula R; Lanyon-Hogg, Thomas; Masumoto, Naoko; Ritzefeld, Markus; Burke, Rosemary; Blagg, Julian; Magee, Anthony I; Tate, Edward W

    2016-12-16

    The Sonic Hedgehog (Shh) signaling pathway plays a critical role during embryonic development and cancer progression. N-terminal palmitoylation of Shh by Hedgehog acyltransferase (Hhat) is essential for efficient signaling, raising interest in Hhat as a novel drug target. A recently identified series of dihydrothienopyridines has been proposed to function via this mode of action; however, the lead compound in this series (RUSKI-43) was subsequently shown to possess cytotoxic activity unrelated to canonical Shh signaling. To identify a selective chemical probe for cellular studies, we profiled three RUSKI compounds in orthogonal cell-based assays. We found that RUSKI-43 exhibits off-target cytotoxicity, masking its effect on Hhat-dependent signaling, hence results obtained with this compound in cells should be treated with caution. In contrast, RUSKI-201 showed no off-target cytotoxicity, and quantitative whole-proteome palmitoylation profiling with a bioorthogonal alkyne-palmitate reporter demonstrated specific inhibition of Hhat in cells. RUSKI-201 is the first selective Hhat chemical probe in cells and should be used in future studies of Hhat catalytic function.

  6. Characterization of Hedgehog Acyltransferase Inhibitors Identifies a Small Molecule Probe for Hedgehog Signaling by Cancer Cells

    PubMed Central

    2016-01-01

    The Sonic Hedgehog (Shh) signaling pathway plays a critical role during embryonic development and cancer progression. N-terminal palmitoylation of Shh by Hedgehog acyltransferase (Hhat) is essential for efficient signaling, raising interest in Hhat as a novel drug target. A recently identified series of dihydrothienopyridines has been proposed to function via this mode of action; however, the lead compound in this series (RUSKI-43) was subsequently shown to possess cytotoxic activity unrelated to canonical Shh signaling. To identify a selective chemical probe for cellular studies, we profiled three RUSKI compounds in orthogonal cell-based assays. We found that RUSKI-43 exhibits off-target cytotoxicity, masking its effect on Hhat-dependent signaling, hence results obtained with this compound in cells should be treated with caution. In contrast, RUSKI-201 showed no off-target cytotoxicity, and quantitative whole-proteome palmitoylation profiling with a bioorthogonal alkyne-palmitate reporter demonstrated specific inhibition of Hhat in cells. RUSKI-201 is the first selective Hhat chemical probe in cells and should be used in future studies of Hhat catalytic function. PMID:27779865

  7. Aberrations and therapeutics involving the developmental pathway Hedgehog in pancreatic cancer.

    PubMed

    Kelleher, Fergal C; McDermott, Raymond

    2012-01-01

    To conduct a systematic review of the role that the hedgehog signaling pathway has in pancreatic cancer tumorigenesis. A PubMed search from 2000 to 2010 and literature-based references were sourced. It was found that in 2009 a genetic analysis of pancreatic cancers discovered that a core set of 12 cellular signaling pathways including hedgehog were genetically altered in 67-100% of cases. Second, in vitro and in vivo studies of treatment with cyclopamine (a naturally occurring antagonist of the hedgehog signaling pathway component; Smoothened) have shown that inhibition of hedgehog can abrogate pancreatic cancer metastasis. Third, experimental evidence has demonstrated that sonic hedgehog (Shh) is correlated with desmoplasia in pancreatic cancer. This is important because targeting the Shh pathway potentially may facilitate chemotherapeutic drug delivery as pancreatic cancers tend to have a dense fibrotic stroma that extrinsically compressed the tumor vasculature leading to a hypoperfusing intratumoral circulation. It is probable that patients with locally advanced pancreatic cancer will derive the greatest benefit from treatment with Smoothened antagonists. Fourth, it has been found that ligand-dependent activation by hedgehog occurs in the tumor stromal microenvironment in pancreatic cancer, a paracrine effect on tumorigenesis. Finally, in pancreatic cancer, cells with the CD44+CD24+ESA+ immunophenotype select a population enriched for cancer initiating stem cells. Shh is increased 46-fold in CD44+CD24+ESA+ cells compared with normal pancreatic epithelial cells. Medications that destruct pancreatic cancer initiating stem cells are a potentially novel strategy in cancer treatment. In conclusion, aberrant hedgehog signaling occurs in pancreatic cancer tumorigenesis and therapeutics that target the transmembrane receptor Smoothened abrogate hedgehog signaling and may improve the outcomes of patients with pancreatic cancer.

  8. Epicardial regeneration is guided by cardiac outflow tract and Hedgehog signalling.

    PubMed

    Wang, Jinhu; Cao, Jingli; Dickson, Amy L; Poss, Kenneth D

    2015-06-11

    In response to cardiac damage, a mesothelial tissue layer enveloping the heart called the epicardium is activated to proliferate and accumulate at the injury site. Recent studies have implicated the epicardium in multiple aspects of cardiac repair: as a source of paracrine signals for cardiomyocyte survival or proliferation; a supply of perivascular cells and possibly other cell types such as cardiomyocytes; and as a mediator of inflammation. However, the biology and dynamism of the adult epicardium is poorly understood. To investigate this, we created a transgenic line to ablate the epicardial cell population in adult zebrafish. Here we find that genetic depletion of the epicardium after myocardial loss inhibits cardiomyocyte proliferation and delays muscle regeneration. The epicardium vigorously regenerates after its ablation, through proliferation and migration of spared epicardial cells as a sheet to cover the exposed ventricular surface in a wave from the chamber base towards its apex. By reconstituting epicardial regeneration ex vivo, we show that extirpation of the bulbous arteriosus-a distinct, smooth-muscle-rich tissue structure that distributes outflow from the ventricle-prevents epicardial regeneration. Conversely, experimental repositioning of the bulbous arteriosus by tissue recombination initiates epicardial regeneration and can govern its direction. Hedgehog (Hh) ligand is expressed in the bulbous arteriosus, and treatment with a Hh signalling antagonist arrests epicardial regeneration and blunts the epicardial response to muscle injury. Transplantation of Sonic hedgehog (Shh)-soaked beads at the ventricular base stimulates epicardial regeneration after bulbous arteriosus removal, indicating that Hh signalling can substitute for the influence of the outflow tract. Thus, the ventricular epicardium has pronounced regenerative capacity, regulated by the neighbouring cardiac outflow tract and Hh signalling. These findings extend our understanding of

  9. The Hedgehog signalling pathway mediates drug response of MCF-7 mammosphere cells in breast cancer patients.

    PubMed

    He, Miao; Fu, Yingzi; Yan, Yuanyuan; Xiao, Qinghuan; Wu, Huizhe; Yao, Weifan; Zhao, Haishan; Zhao, Lin; Jiang, Qian; Yu, Zhaojin; Jin, Feng; Mi, Xiaoyi; Wang, Enhua; Cui, Zeshi; Fu, Liwu; Chen, Jianju; Wei, Minjie

    2015-11-01

    BCSCs (breast cancer stem cells) have been shown to be resistant to chemotherapy. However, the mechanisms underlying BCSC-mediated chemoresistance remain poorly understood. The Hh (Hedgehog) pathway is important in the stemness maintenance of CSCs. Nonetheless, it is unknown whether the Hh pathway is involved in BCSC-mediated chemoresistance. In the present study, we cultured breast cancer MCF-7 cells in suspension in serum-free medium to obtain BCSC-enriched MCF-7 MS (MCF-7 mammosphere) cells. We showed that MCF-7 MS cells are sensitive to salinomycin, but not paclitaxel, distinct from parent MCF-7 cells. The expression of the critical components of Hh pathway, i.e., PTCH (Patched), SMO (Smoothened), Gli1 and Gli2, was significantly up-regulated in MCF-7 MS cells; salinomycin, but not paclitaxel, treatment caused a remarkable decrease in expression of those genes in MCF-7 MS cells, but not in MCF-7 cells. Salinomycin, but not paclitaxel, increased apoptosis, decreased the migration capacity of MCF-7 MS cells, accompanied by a decreased expression of c-Myc, Bcl-2 and Snail, the target genes of the Hh pathway. The salinomycin-induced cytotoxic effect could be blocked by Shh (Sonic Hedgehog)-mediated Hh signalling activation. Inhibition of the Hh pathway by cyclopamine could sensitize MCF-7 MS cells to paclitaxel. In addition, salinomycin, but not paclitaxel, significantly reduced the tumour growth, accompanied by decreased expression of PTCH, SMO, Gli1 and Gli2 in xenograft tumours. Furthermore, the expression of SMO and Gli1 was positively correlated with the expression of CD44+ / CD24-, and the expression of SMO and Gli1 in CD44+ / CD24- tissues was associated with a significantly shorter OS (overall survival) and DFS (disease-free survival) in breast cancer patients receiving chemotherapy.

  10. The Mode of Hedgehog Binding to Ihog Homologues is Not Conserved Across Different Phyla

    SciTech Connect

    McLellan, J.; Zheng, X; Hauk, G; Ghirlando, R; Beachy, P; Leahy, D

    2008-01-01

    Hedgehog (Hh) proteins specify tissue pattern in metazoan embryos by forming gradients that emanate from discrete sites of expression and elicit concentration-dependent cellular differentiation or proliferation responses1, 2. Cellular responses to Hh and the movement of Hh through tissues are both precisely regulated, and abnormal Hh signalling has been implicated in human birth defects and cancer3, 4, 5, 6, 7. Hh signalling is mediated by its amino-terminal domain (HhN), which is dually lipidated and secreted as part of a multivalent lipoprotein particle8, 9, 10. Reception of the HhN signal is modulated by several cell-surface proteins on responding cells, including Patched (Ptc), Smoothened (Smo), Ihog (known as CDO or CDON in mammals) and the vertebrate-specific proteins Hip (also known as Hhip) and Gas1 (ref. 11). Drosophila Ihog and its vertebrate homologues CDO and BOC contain multiple immunoglobulin and fibronectin type III (FNIII) repeats, and the first FNIII repeat of Ihog binds Drosophila HhN in a heparin-dependent manner12, 13. Surprisingly, pull-down experiments suggest that a mammalian Sonic hedgehog N-terminal domain (ShhN) binds a non-orthologous FNIII repeat of CDO12, 14. Here we report biochemical, biophysical and X-ray structural studies of a complex between ShhN and the third FNIII repeat of CDO. We show that the ShhN-CDO interaction is completely unlike the HhN-Ihog interaction and requires calcium, which binds at a previously undetected site on ShhN. This site is conserved in nearly all Hh proteins and is a hotspot for mediating interactions between ShhN and CDO, Ptc, Hip and Gas1. Mutations in vertebrate Hh proteins causing holoprosencephaly and brachydactyly type A1 map to this calcium-binding site and disrupt interactions with these partners.

  11. Maintenance of Taste Organs Is Strictly Dependent on Epithelial Hedgehog/GLI Signaling

    PubMed Central

    Mistretta, Charlotte M.

    2016-01-01

    For homeostasis, lingual taste papilla organs require regulation of epithelial cell survival and renewal, with sustained innervation and stromal interactions. To investigate a role for Hedgehog/GLI signaling in adult taste organs we used a panel of conditional mouse models to manipulate GLI activity within epithelial cells of the fungiform and circumvallate papillae. Hedgehog signaling suppression rapidly led to taste bud loss, papilla disruption, and decreased proliferation in domains of papilla epithelium that contribute to taste cells. Hedgehog responding cells were eliminated from the epithelium but retained in the papilla stromal core. Despite papilla disruption and loss of taste buds that are a major source of Hedgehog ligand, innervation to taste papillae was maintained, and not misdirected, even after prolonged GLI blockade. Further, vimentin-positive fibroblasts remained in the papilla core. However, retained innervation and stromal cells were not sufficient to maintain taste bud cells in the context of compromised epithelial Hedgehog signaling. Importantly taste organ disruption after GLI blockade was reversible in papillae that retained some taste bud cell remnants where reactivation of Hedgehog signaling led to regeneration of papilla epithelium and taste buds. Therefore, taste bud progenitors were either retained during epithelial GLI blockade or readily repopulated during recovery, and were poised to regenerate taste buds once Hedgehog signaling was restored, with innervation and papilla connective tissue elements in place. Our data argue that Hedgehog signaling is essential for adult tongue tissue maintenance and that taste papilla epithelial cells represent the key targets for physiologic Hedgehog-dependent regulation of taste organ homeostasis. Because disruption of GLI transcriptional activity in taste papilla epithelium is sufficient to drive taste organ loss, similar to pharmacologic Hedgehog pathway inhibition, the findings suggest that taste

  12. Loss of Pin1 Suppresses Hedgehog-Driven Medulloblastoma Tumorigenesis.

    PubMed

    Xu, Tao; Zhang, Honglai; Park, Sung-Soo; Venneti, Sriram; Kuick, Rork; Ha, Kimberly; Michael, Lowell Evan; Santi, Mariarita; Uchida, Chiyoko; Uchida, Takafumi; Srinivasan, Ashok; Olson, James M; Dlugosz, Andrzej A; Camelo-Piragua, Sandra; Rual, Jean-François

    2017-03-01

    Medulloblastoma is the most common malignant brain tumor in children. Therapeutic approaches to medulloblastoma (combination of surgery, radiotherapy, and chemotherapy) have led to significant improvements, but these are achieved at a high cost to quality of life. Alternative therapeutic approaches are needed. Genetic mutations leading to the activation of the Hedgehog pathway drive tumorigenesis in ~30% of medulloblastoma. In a yeast two-hybrid proteomic screen, we discovered a novel interaction between GLI1, a key transcription factor for the mediation of Hedgehog signals, and PIN1, a peptidylprolyl cis/trans isomerase that regulates the postphosphorylation fate of its targets. The GLI1/PIN1 interaction was validated by reciprocal pulldowns using epitope-tagged proteins in HEK293T cells as well as by co-immunoprecipiations of the endogenous proteins in a medulloblastoma cell line. Our results support a molecular model in which PIN1 promotes GLI1 protein abundance, thus contributing to the positive regulation of Hedgehog signals. Most importantly, in vivo functional analyses of Pin1 in the GFAP-tTA;TRE-SmoA1 mouse model of Hedgehog-driven medulloblastoma demonstrate that the loss of Pin1 impairs tumor development and dramatically increases survival. In summary, the discovery of the GLI1/PIN1 interaction uncovers PIN1 as a novel therapeutic target in Hedgehog-driven medulloblastoma tumorigenesis.

  13. Transcriptional activation of hedgehog pathway components in aggressive hemangioma.

    PubMed

    Wendling-Keim, Danielle S; Wanie, Lynn; Grantzow, Rainer; Kappler, Roland

    2017-03-31

    Infantile hemangioma is a vascular neoplasm and is one of the most common tumors diagnosed in young children. Although most hemangiomas are harmless and involute spontaneously, some show severe progression, leading to serious complications, such as high output cardiac failure, ulcerations, compression of the trachea or deprivation amblyopia, depending on their size and localization. However, the pathogenesis and cause of hemangioma are largely unknown to date. The goal of this study was to identify markers that could predict hemangiomas with aggressive growth and severe progression that would benefit from early intervention. By using a PCR-based screening approach, we first confirmed that previously known markers of hemangioma, namely FGF2 and GLUT1, are highly expressed in hemangioma. Nevertheless, these genes did not show any differential expression between severely progressing tumors and mild tumors. However, transcriptional upregulation of several Hedgehog signaling components, comprising the ligand Sonic Hedgehog (SHH),the transcription factor GLI2 and its target gene FOXA2 were detected in extremely aggressive hemangioma specimens during the proliferation phase. Notably, GLI2 was even overexpressed in involuting hemangiomas if they showed an aggressive growth pattern. In conclusion, our data suggest that overexpression of the Hedgehog components SHH, GLI2 and FOXA2 might be used as markers of an aggressive hemangioma that would benefit from too early intervention, while FGF2 and GLUT1 are more general markers of hemangiomas. This article is protected by copyright. All rights reserved.

  14. Sonic levitation apparatus

    NASA Technical Reports Server (NTRS)

    Dunn, S. A.; Pomplum, A. R.; Paquette, E. G.; Ethridge, E. C.; Johnson, J. L. (Inventor)

    1984-01-01

    A sonic levitation apparatus is disclosed which includes a sonic transducer which generates acoustical energy responsive to the level of an electrical amplifier. A duct communicates with an acoustical chamber to deliver an oscillatory motion of air to a plenum section which contains a collimated hole structure having a plurality of parallel orifices. The collimated hole structure converts the motion of the air to a pulsed. Unidirectional stream providing enough force to levitate a material specimen. Particular application to the production of microballoons in low gravity environment is discussed.

  15. Ultrasonic/Sonic Jackhammer

    NASA Technical Reports Server (NTRS)

    Bar-Cohen, Yoseph; Sherrit, Stewart; Herz, Jack

    2005-01-01

    An ultrasonic/sonic jackhammer (USJ) is the latest in a series of related devices. Each of these devices cuts into a brittle material by means of hammering and chiseling actions of a tool bit excited with a combination of ultrasonic and sonic vibrations. A small-scale prototype of the USJ has been demonstrated. A fully developed, full-scale version of the USJ would be used for cutting through concrete, rocks, hard asphalt, and other materials to which conventional pneumatic jackhammers are applied, but the USJ would offer several advantages over conventional pneumatic jackhammers.

  16. Antiferromagnetic hedgehogs with superconducting cores

    SciTech Connect

    Goldbart, P.M.; Sheehy, D.E.

    1998-09-01

    Excitations of the antiferromagnetic state that resemble antiferromagnetic hedgehogs at large distances but are predominantly superconducting inside a core region are discussed within the context of Zhang{close_quote}s SO(5)-symmetry-based approach to the physics of high-temperature superconducting materials. Nonsingular, in contrast with their hedgehog cousins in pure antiferromagnetism, these texture excitations are what hedgehogs become when the antiferromagnetic order parameter is permitted to {open_quotes}escape{close_quotes} into superconducting directions. The structure of such excitations is determined in a simple setting, and a number of their experimental implications are examined. {copyright} {ital 1998} {ital The American Physical Society}

  17. Antiferromagnetic hedgehogs with superconducting cores

    NASA Astrophysics Data System (ADS)

    Goldbart, Paul M.; Sheehy, Daniel E.

    1998-09-01

    Excitations of the antiferromagnetic state that resemble antiferromagnetic hedgehogs at large distances but are predominantly superconducting inside a core region are discussed within the context of Zhang's SO(5)-symmetry-based approach to the physics of high-temperature superconducting materials. Nonsingular, in contrast with their hedgehog cousins in pure antiferromagnetism, these texture excitations are what hedgehogs become when the antiferromagnetic order parameter is permitted to ``escape'' into superconducting directions. The structure of such excitations is determined in a simple setting, and a number of their experimental implications are examined.

  18. The hedgehog signaling network, mammary stem cells, and breast cancer: connections and controversies.

    PubMed

    Lewis, M T; Visbal, A P

    2006-01-01

    Several signal transduction networks have been implicated in the regulation of mammary epithelial stem cell self-renewal and maintenance (Kalirai and Clarke 2006; Liu et al. 2005). These signaling networks include those of the Wnt, Notch, TGFO, EGF, FGF, IGF, and most recently, the Hedgehog (Hh) families of secreted ligands. However, we currently know very little about the cellular and molecular mechanisms by which these signaling pathways function to regulate normal epithelial stem/progenitor cells. What is clear is that the regulatory signaling networks thought to control normal stem/progenitor cell self-renewal and maintenance are, with the current sole exception of the hedgehog network, well-documented to have contributory roles in mammary cancer development and disease progression when misregulated. In this review, genetic regulation of mammary gland development by hedgehog network genes is outlined, highlighting a developing controversy as to whether activated hedgehog signaling regulates normal regenerative mammary epithelial stem cells or, indeed, whether activated hedgehog signaling functions at all in ductal development. In addition, the question of whether inappropriate hedgehog network activation influences breast cancer development is addressed, with emphasis on the prospects for using hedgehog signaling antagonists clinically for breast cancer treatment or prevention.

  19. Small molecule inhibitors of the hedgehog signaling pathway for the treatment of cancer.

    PubMed

    Yun, Jeong In; Kim, Hyoung Rae; Park, Haeil; Kim, Sang Kyum; Lee, Jongkook

    2012-08-01

    Over the past decade, the Hedgehog signaling pathway has attracted considerable interest because the pathway plays important roles in the tumorigenesis of several types of cancer as well as developmental processes. It has also been observed that Hedgehog signaling regulates the proliferation and self-renewal of cancer stem cells. A great number of Hedgehog pathway inhibitors have been discovered through small molecule screens and subsequent medicinal chemistry efforts. Among the inhibitors, several Smo antagonists have reached the clinical trial phase. It has been proved that the inhibition of Hedgehog signaling with Smo antagonists is beneficial to cancer patients with basal cell carcinoma and medulloblastoma. In this review, we provide an overview of Hedgehog pathway inhibitors with focusing on the preclinical and/or clinical efficacy and molecular mechanisms of these inhibitors.

  20. Deciphering the role of hedgehog signaling in pancreatic cancer.

    PubMed

    Gu, Dongsheng; Schlotman, Kelly E; Xie, Jingwu

    2016-09-01

    Pancreatic cancer, mostly pancreatic ductal adenocarcinoma (PDAC), is a leading cause of cancer-related death in the US, with a dismal median survival of 6 months. Thus, there is an urgent unmet need to identify ways to diagnose and to treat this deadly cancer. Although a number of genetic changes have been identified in pancreatic cancer, their mechanisms of action in tumor development, progression and metastasis are not completely understood. Hedgehog signaling, which plays a major role in embryonic development and stem cell regulation, is known to be activated in pancreatic cancer; however, specific inhibitors targeting the smoothened molecule failed to improve the condition of pancreatic cancer patients in clinical trials. Furthermore, results regarding the role of Hh signaling in pancreatic cancer are controversial with some reporting tumor promoting activities whereas others tumor suppressive actions. In this review, we will summarize what we know about hedgehog signaling in pancreatic cancer, and try to explain the contradicting roles of hedgehog signaling as well as the reason(s) behind the failed clinical trials. In addition to the canonical hedgehog signaling, we will also discuss several non-canonical hedgehog signaling mechanisms.

  1. Hedgehog signaling in the normal and neoplastic mammary gland.

    PubMed

    Visbal, Adriana P; Lewis, Michael T

    2010-09-01

    The hedgehog signal transduction network is a critical regulator of metazoan development. Inappropriate activation of this network is implicated in several different cancers, including breast. Genetic evidence in mice as well as molecular biological studies in human cells clearly indicate that activated signaling can lead to mammary hyperplasia and, in some cases, tumor formation. However, the exact role(s) activated hedgehog signaling plays in the development or progression of breast cancer also remain unclear. In this review, we have discussed recent data regarding the mechanism(s) by which the hedgehog network may signal in the mammary gland, as well as the data implicating activated signaling as a contributing factor to breast cancer development. Finally, we provide a brief update on the available hedgehog signaling inhibitors with respect to ongoing clinical trials, some of which will include locally advanced or metastatic breast cancers. Given the growing intensity with which the hedgehog signaling network is being studied in the normal and neoplastic mammary gland, a more complete understanding of this network should allow more effective targeting of its activities in breast cancer treatment or prevention.

  2. Yin-Yang strands of PCAF/Hedgehog axis in cancer control.

    PubMed

    Infante, Paola; Canettieri, Gianluca; Gulino, Alberto; Di Marcotullio, Lucia

    2014-08-01

    PCAF (p300/CBP associated factor) harbors acetyltransferase and a recently identified ubiquitylation activity that regulates gene expression in response to genotoxic stress or mitogenic signals. We highlight the dual role of PCAF in the control of Hedgehog signaling, a master regulator of tissue development, stemness, and tumorigenesis. By promoting histone acetylation at Hedgehog/GLI1 target gene promoters or direct ubiquitylation and proteolysis of GLI1, the PCAF/GLI1 axis stands as a promising therapeutic target for Hedgehog-dependent tumors.

  3. Patched targeting peptides for imaging and treatment of hedgehog positive breast tumors.

    PubMed

    Smith, Daniel; Kong, Fanlin; Yang, David; Larson, Richard; Sims-Mourtada, Jennifer; Woodward, Wendy A

    2014-01-01

    High tumor hedgehog expression is correlated with poor prognosis in invasive ductal carcinoma. Peptides which bind the patched receptor have recently been reported to have a growth inhibitory effect in tumors with activated hedgehog signaling. We sought to examine growth inhibition with these peptides in breast cancer cells and use these peptides as molecular imaging probes to follow changes in hedgehog expression after chemotherapy. Significant growth inhibition was observed in breast cancer cell lines treated with PTCH-blocking peptides. Significant in vitro uptake was observed with both FITC- and (99m)Tc-EC-peptide conjugates. In vivo imaging studies displayed greater accumulation of (99m)Tc-labeled peptides within tumors as compared to adjacent muscle tissue. Patched receptor expression increased after treatment and this correlated with an increase in tumor radiotracer uptake. These studies suggest that peptides which bind the sonic hedgehog docking site in patched receptor correlate with patched expression and can be used to image patched in vivo. Further, our data suggest that radiolabeled peptides may enable us to examine the activity of the hedgehog signaling pathway and to evaluate response to anti-cancer therapies.

  4. Hedgehogs in the dowser state.

    PubMed

    Pieranski, Pawel; Čopar, Simon; Godinho, Maria Helena; Dazza, Mallory

    2016-12-01

    We show how to easily generate point defects called hedgehogs, in the so-called quasi-planar texture --the dowser state-- of a nematic layer confined between surfaces with homeotropic anchoring conditions. We point out that the dowser texture can be preserved infinitely in spite of its higher energy with respect to the homogeneous homeotropic texture. For topological reasons the dowser state in a squeezed droplet must contain at least one hedgehog. We submitted this hedgehog to a rotating magnetic field and controlled the continuous evolution, transitioning continuously between radial, hyperbolic and circular hedgehogs, which, just as in previous experiments by Lavrentovich et al., are topologically equivalent states. The dynamics of this transformation is shown to be directly sensitive to energy costs of different geometric configurations of the hedgehog defect and therefore can be used as a rough probe for elastic constants; knowing the principal elastic constants K1,2,3, one can retrieve information about the K24 constant. We propose also a method of generation of hedgehog pairs by application of a Poiseuille flow to a dowser state wound by a rotating magnetic field.

  5. Homeodomains, Hedgehogs, and Happiness.

    PubMed

    Scott, Matthew P

    2016-01-01

    Developmental biologists have had a spectacular quarter century of discoveries, building on many decades of work earlier, discovering molecular, cellular, and genetic mechanisms that underlie the magical process by which an egg becomes a plant or animal. Among the discoveries were homeodomains, DNA-binding domains that allow transcription factors to recognize their target genes, and the Hedgehog signaling pathway, which is used in many organs and tissues for communication among cells. The experience of unveiling the mechanisms and molecules connected to both of these findings has been remarkable, joyful, difficult, and a time of great teamwork and collaboration within and between laboratory groups. More than ever it is possible to discern the evolutionary processes, and their mechanisms, that led to the diversity of life on earth. A huge amount of work remains to be done to obtain a broad understanding of what happened and how development works.

  6. A sesquiterpene lactone from Siegesbeckia glabrescens suppresses Hedgehog/Gli-mediated transcription in pancreatic cancer cells

    PubMed Central

    Lee, Hwa Jin; Wu, Qian; Li, Hua; Bae, Gyu-Un; Kim, An Keun; Ryu, Jae-Ha

    2016-01-01

    Pancreatic cancer is aggressive and therefore difficult to treat; however, continued efforts have been made with the aim of developing an effective therapy against the disease. The Hedgehog (Hh) signaling pathway is reportedly involved in the proliferation and survival of pancreatic cancer cells. The transcription factor glioma-associated oncogene (Gli) is a key component of the Hh signaling pathway and the primary effector of pancreatic cancer development. Inhibiting Gli is a proven therapeutic strategy for this disease. The present study examined the regulation of Gli and the expression of its target genes to identify an inhibitor of the Sonic Hh (Shh) pathway. A germacranolide sesquiterpene lactone (GSL) was isolated from Siegesbeckia glabrescens as an inhibitor of Gli-mediated transcription. The results demonstrated that GSL inhibited Shh-induced osteoblast differentiation and Gli homolog 1 (Gli1)-mediated transcriptional activity in mesenchymal C3H10T1/2 stem cells. Furthermore, GSL suppressed Gli-mediated transcriptional activity in human pancreatic cancer PANC-1 and AsPC-1 cells, which resulted in reduced cancer cell proliferation and downregulated expression of the Gli-target genes, Gli1 and cyclin D1. A sesquiterpene lactone from S. glabrescens may therefore serve as a candidate for the treatment of Hh/Gli-dependent pancreatic cancer. PMID:27698879

  7. Pilot Clinical Trial of Hedgehog Pathway Inhibitor GDC-0449 (Vismodegib) in Combination with Gemcitabine in Patients with Metastatic Pancreatic Adenocarcinoma

    PubMed Central

    Abel, Ethan V.; Griffith, Kent A.; Greenson, Joel K.; Takebe, Naoko; Khan, Gazala N.; Blau, John L.; Craig, Ronald; Balis, Ulysses G.; Zalupski, Mark M.; Simeone, Diane M.

    2014-01-01

    Background The hedgehog (HH) signaling pathway is a key regulator in tumorigenesis of pancreatic adenocarcinoma (PDA) and is up-regulated in PDA cancer stem cells (CSCs). GDC-0449 is an oral small-molecule inhibitor of HH pathway. This study assessed the effect of GDC-0449-mediated HH inhibition in paired biopsies, followed by combined treatment with gemcitabine, in patients with metastatic PDA. Methods Twenty-five patients were enrolled of which 23 underwent core biopsies at baseline and following 3 weeks of GDC-0449. On day 29, 23 patients started weekly gemcitabine while continuing GDC-0449. We evaluated GLI1 and PTCH1 inhibition, change in CSCs, Ki-67, fibrosis, and assessed tumor response, survival and toxicity. Results On pre-treatment biopsy, 75% of patients had elevated sonic hedgehog (SHH) expression. On post-treatment biopsy, GLI1 and PTCH1 decreased in 95.6% and 82.6% of 23 patients, fibrosis decreased in 45.4% of 22 and Ki-67 in 52.9% of 17 evaluable patients. No significant changes were detected in CSCs pre- and post-biopsy. The median progression-free and overall survival for all treated patients was 2.8 and 5.3 months. The response and disease control rate was 21.7% and 65.2%. No significant correlation was noted between CSCs, fibrosis, SHH, Ki-67, GLI1, PTCH1 (baseline values, or relative change on post-treatment biopsy) and survival. Grade >3 adverse events were noted in 56% of patients. Conclusion We show that GDC-0449 for 3 weeks leads to down-modulation of GLI1 and PTCH1, without significant changes in CSCs compared to baseline. GDC-0449 and gemcitabine was not superior to gemcitabine alone in the treatment of metastatic pancreatic cancer. PMID:25278454

  8. Fragmentation of DNA by sonication.

    PubMed

    Sambrook, Joseph; Russell, David W

    2006-09-01

    INTRODUCTIONDNA fragmentation is often necessary prior to library construction or subcloning for DNA sequencing. This protocol describes a method for DNA fragmentation by sonication. During sonication, DNA samples are subjected to hydrodynamic shearing by exposure to brief periods of sonication. DNA that has been sonicated for excessive periods of time is extremely difficult to clone. Most sonicators will not shear DNA to a size of less than 300-500 bp, and it is tempting to continue sonication until the entire DNA population has been reduced in size. However, the yield of subclones is usually greater if sonication is stopped when the fragments of the target DNA first reach a size of ~700 bp.

  9. Sonic rhinoplasty: innovative applications.

    PubMed

    Pribitkin, Edmund; Greywoode, Jewel D

    2013-04-01

    Sonic rhinoplasty involves the use of the Sonopet ultrasonic bone aspirator (Stryker, Inc., Kalamazoo, MI, USA) to precisely sculpt the nasal bones without damage to the surrounding nasal cartilage, soft tissue, and mucosa. By employing ultrasonic waves to emulsify and remove bone under concurrent irrigation and suction, sonic rhinoplasty improves upon the conventional osteotome, drill, rasp, and powered rasp techniques that may be associated with decreased visualization, heat generation, mechanical chatter, and a lack of surgical precision with attendant soft tissue injury. We have applied this technology to bony dorsal hump and nasal spine removal, deepening of the glabellar angle and reshaping of irregular nasal contours, septoplasty, turbinate reduction, and the correction of bony asymmetries.

  10. Ultrasonic/Sonic Jackhammer

    NASA Technical Reports Server (NTRS)

    Bar-Cohen, Yoseph (Inventor); Sherrit, Stewart (Inventor); Herz, Jack L. (Inventor)

    2014-01-01

    The invention provides a novel jackhammer that utilizes ultrasonic and/or sonic vibrations as source of power. It is easy to operate and does not require extensive training, requiring substantially less physical capabilities from the user and thereby increasing the pool of potential operators. An important safety benefit is that it does not fracture resilient or compliant materials such as cable channels and conduits, tubing, plumbing, cabling and other embedded fixtures that may be encountered along the impact path. While the ultrasonic/sonic jackhammer of the invention is able to cut concrete and asphalt, it generates little back-propagated shocks or vibrations onto the mounting fixture, and can be operated from an automatic platform or robotic system. PNEUMATICS; ULTRASONICS; IMPACTORS; DRILLING; HAMMERS BRITTLE MATERIALS; DRILL BITS; PROTOTYPES; VIBRATION

  11. Tumor-Derived Suppressor of Fused Mutations Reveal Hedgehog Pathway Interactions.

    PubMed

    Urman, Nicole M; Mirza, Amar; Atwood, Scott X; Whitson, Ramon J; Sarin, Kavita Y; Tang, Jean Y; Oro, Anthony E

    2016-01-01

    The Hedgehog pathway is a potent regulator of cellular growth and plays a central role in the development of many cancers including basal cell carcinoma (BCC). The majority of BCCs arise from mutations in the Patched receptor resulting in constitutive activation of the Hedgehog pathway. Secondary driver mutations promote BCC oncogenesis and occur frequently due to the high mutational burden resulting from sun exposure of the skin. Here, we uncover novel secondary mutations in Suppressor of Fused (SUFU), the major negative regulator of the Hedgehog pathway. SUFU normally binds to a Hedgehog transcriptional activator, GLI1, in order to prevent it from initiating transcription of Hedgehog target genes. We sequenced tumor-normal pairs from patients with early sporadic BCCs. This resulted in the discovery of nine mutations in SUFU, which were functionally investigated to determine whether they help drive BCC formation. Our results show that four of the SUFU mutations inappropriately activate the Hedgehog pathway, suggesting they may act as driver mutations for BCC development. Indeed, all four of the loss of function SUFU variants were found to disrupt its binding to GLI, leading to constitutive pathway activation. Our results from functional characterization of these mutations shed light on SUFU's role in Hedgehog signaling, tumor progression, and highlight a way in which BCCs can arise.

  12. Tumor-Derived Suppressor of Fused Mutations Reveal Hedgehog Pathway Interactions

    PubMed Central

    Urman, Nicole M.; Mirza, Amar; Atwood, Scott X.; Whitson, Ramon J.; Sarin, Kavita Y.; Tang, Jean Y.; Oro, Anthony E.

    2016-01-01

    The Hedgehog pathway is a potent regulator of cellular growth and plays a central role in the development of many cancers including basal cell carcinoma (BCC). The majority of BCCs arise from mutations in the Patched receptor resulting in constitutive activation of the Hedgehog pathway. Secondary driver mutations promote BCC oncogenesis and occur frequently due to the high mutational burden resulting from sun exposure of the skin. Here, we uncover novel secondary mutations in Suppressor of Fused (SUFU), the major negative regulator of the Hedgehog pathway. SUFU normally binds to a Hedgehog transcriptional activator, GLI1, in order to prevent it from initiating transcription of Hedgehog target genes. We sequenced tumor-normal pairs from patients with early sporadic BCCs. This resulted in the discovery of nine mutations in SUFU, which were functionally investigated to determine whether they help drive BCC formation. Our results show that four of the SUFU mutations inappropriately activate the Hedgehog pathway, suggesting they may act as driver mutations for BCC development. Indeed, all four of the loss of function SUFU variants were found to disrupt its binding to GLI, leading to constitutive pathway activation. Our results from functional characterization of these mutations shed light on SUFU’s role in Hedgehog signaling, tumor progression, and highlight a way in which BCCs can arise. PMID:28030567

  13. Hedgehog signaling is synergistically enhanced by nutritional deprivation and ligand stimulation in human fibroblasts of Gorlin syndrome.

    PubMed

    Mizuochi, Hiromi; Fujii, Katsunori; Shiohama, Tadashi; Uchikawa, Hideki; Shimojo, Naoki

    2015-02-13

    Hedgehog signaling is a pivotal developmental pathway that comprises hedgehog, PTCH1, SMO, and GLI proteins. Mutations in PTCH1 are responsible for Gorlin syndrome, which is characterized by developmental defects and tumorigenicity. Although the hedgehog pathway has been investigated extensively in Drosophila and mice, its functional roles have not yet been determined in human cells. In order to elucidate the mechanism by which transduction of the hedgehog signal is regulated in human tissues, we employed human fibroblasts derived from three Gorlin syndrome patients and normal controls. We investigated GLI1 transcription, downstream of hedgehog signaling, to assess native signal transduction, and then treated fibroblasts with a recombinant human hedgehog protein with or without serum deprivation. We also examined the transcriptional levels of hedgehog-related genes under these conditions. The expression of GLI1 mRNA was significantly higher in Gorlin syndrome-derived fibroblasts than in control cells. Hedgehog stimulation and nutritional deprivation synergistically enhanced GLI1 transcription levels, and this was blocked more efficiently by vismodegib, a SMO inhibitor, than by the natural compound, cyclopamine. Messenger RNA profiling revealed the increased expression of Wnt signaling and morphogenetic molecules in these fibroblasts. These results indicated that the hedgehog stimulation and nutritional deprivation synergistically activated the hedgehog signaling pathway in Gorlin syndrome fibroblasts, and this was associated with increments in the transcription levels of hedgehog-related genes such as those involved in Wnt signaling. These fibroblasts may become a significant tool for predicting the efficacies of hedgehog molecular-targeted therapies such as vismodegib.

  14. Hedgehog Signaling Antagonist GDC-0449 (Vismodegib) Inhibits Pancreatic Cancer Stem Cell Characteristics: Molecular Mechanisms

    PubMed Central

    Singh, Brahma N.; Fu, Junsheng; Srivastava, Rakesh K.; Shankar, Sharmila

    2011-01-01

    Background Recent evidence from in vitro and in vivo studies has demonstrated that aberrant reactivation of the Sonic Hedgehog (SHH) signaling pathway regulates genes that promote cellular proliferation in various human cancer stem cells (CSCs). Therefore, the chemotherapeutic agents that inhibit activation of Gli transcription factors have emerged as promising novel therapeutic drugs for pancreatic cancer. GDC-0449 (Vismodegib), orally administrable molecule belonging to the 2-arylpyridine class, inhibits SHH signaling pathway by blocking the activities of Smoothened. The objectives of this study were to examine the molecular mechanisms by which GDC-0449 regulates human pancreatic CSC characteristics in vitro. Methodology/Principal Findings GDC-0499 inhibited cell viability and induced apoptosis in three pancreatic cancer cell lines and pancreatic CSCs. This inhibitor also suppressed cell viability, Gli-DNA binding and transcriptional activities, and induced apoptosis through caspase-3 activation and PARP cleavage in pancreatic CSCs. GDC-0449-induced apoptosis in CSCs showed increased Fas expression and decreased expression of PDGFRα. Furthermore, Bcl-2 was down-regulated whereas TRAIL-R1/DR4 and TRAIL-R2/DR5 expression was increased following the treatment of CSCs with GDC-0449. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GDC-0449-treated pancreatic CSCs. Thus, activated Gli genes repress DRs and Fas expressions, up-regulate the expressions of Bcl-2 and PDGFRα and facilitate cell survival. Conclusions/Significance These data suggest that GDC-0499 can be used for the management of pancreatic cancer by targeting pancreatic CSCs. PMID:22087285

  15. Hedgehog signaling stimulates the formation of proliferating Müller glia-derived progenitor cells in the chick retina.

    PubMed

    Todd, Levi; Fischer, Andy J

    2015-08-01

    Müller glia can be stimulated to de-differentiate and become proliferating progenitor cells that regenerate neurons in the retina. The signaling pathways that regulate the formation of proliferating Müller glia-derived progenitor cells (MGPCs) are beginning to be revealed. The purpose of this study was to investigate whether Hedgehog (Hh) signaling influences the formation of MGPCs in the chick retina. We find that Hh signaling is increased in damaged retinas where MGPCs are known to form. Sonic Hedgehog (Shh) is normally present in the axons of ganglion cells, but becomes associated with Müller glia and MGPCs following retinal damage. Activation of Hh signaling with recombinant human SHH (rhShh) or smoothened agonist (SAG) increased levels of Ptch1, Gli1, Gli2, Gli3, Hes1 and Hes5, and stimulated the formation of proliferating MGPCs in damaged retinas. In undamaged retinas, SAG or rhShh had no apparent effect upon the Müller glia. However, SAG combined with FGF2 potentiated the formation of MGPCs, whereas SAG combined with IGF1 stimulated the nuclear migration of Müller glia, but not the formation of MGPCs. Conversely, inhibition of Hh signaling with KAAD-cyclopamine, Gli antagonists or antibody to Shh reduced numbers of proliferating MGPCs in damaged and FGF2-treated retinas. Hh signaling potentiates Pax6, Klf4 and cFos expression in Müller glia during the formation of MGPCs. We find that FGF2/MAPK signaling recruits Hh signaling into the signaling network that drives the formation of proliferating MGPCs. Our findings implicate Hh signaling as a key component of the network of signaling pathways that promote the de-differentiation of Müller glia and proliferation of MGPCs.

  16. Sonic Boom Modeling Technical Challenge

    NASA Technical Reports Server (NTRS)

    Sullivan, Brenda M.

    2007-01-01

    This viewgraph presentation reviews the technical challenges in modeling sonic booms. The goal of this program is to develop knowledge, capabilities and technologies to enable overland supersonic flight. The specific objectives of the modeling are: (1) Develop and validate sonic boom propagation model through realistic atmospheres, including effects of turbulence (2) Develop methods enabling prediction of response of and acoustic transmission into structures impacted by sonic booms (3) Develop and validate psychoacoustic model of human response to sonic booms under both indoor and outdoor listening conditions, using simulators.

  17. Sonication standard laboratory module

    DOEpatents

    Beugelsdijk, Tony; Hollen, Robert M.; Erkkila, Tracy H.; Bronisz, Lawrence E.; Roybal, Jeffrey E.; Clark, Michael Leon

    1999-01-01

    A standard laboratory module for automatically producing a solution of cominants from a soil sample. A sonication tip agitates a solution containing the soil sample in a beaker while a stepper motor rotates the sample. An aspirator tube, connected to a vacuum, draws the upper layer of solution from the beaker through a filter and into another beaker. This beaker can thereafter be removed for analysis of the solution. The standard laboratory module encloses an embedded controller providing process control, status feedback information and maintenance procedures for the equipment and operations within the standard laboratory module.

  18. Pancreas development is promoted by cyclopamine, a hedgehog signaling inhibitor.

    PubMed

    Kim, S K; Melton, D A

    1998-10-27

    Exposure to cyclopamine, a steroid alkaloid that blocks Sonic hedgehog (Shh) signaling, promotes pancreatic expansion in embryonic chicks. Heterotopic development of pancreatic endocrine and exocrine structures occurs in regions adjacent to the pancreas including stomach and duodenum, and insulin-producing islets in the pancreas are enlarged. The homeodomain transcription factor PDX1, required for pancreas development, is expressed broadly in the posterior foregut but pancreas development normally initiates only in a restricted region of PDX1-expressing posterior foregut where endodermal Shh expression is repressed. The results suggests that cyclopamine expands the endodermal region where Shh signaling does not occur, resulting in pancreatic differentiation in a larger region of PDX1-expressing foregut endoderm. Cyclopamine reveals the capacity of a broad region of the posterior embryonic foregut to form pancreatic cells and provides a means for expanding embryonic pancreas development.

  19. B4GALT family mediates the multidrug resistance of human leukemia cells by regulating the hedgehog pathway and the expression of p-glycoprotein and multidrug resistance-associated protein 1

    PubMed Central

    Zhou, H; Ma, H; Wei, W; Ji, D; Song, X; Sun, J; Zhang, J; Jia, L

    2013-01-01

    β-1, 4-Galactosyltransferase gene (B4GALT) family consists of seven members, which encode corresponding enzymes known as type II membrane-bound glycoproteins. These enzymes catalyze the biosynthesis of different glycoconjugates and saccharide structures, and have been recognized to be involved in various diseases. In this study, we sought to determine the expressional profiles of B4GALT family in four pairs of parental and chemoresistant human leukemia cell lines and in bone marrow mononuclear cells (BMMC) of leukemia patients with multidrug resistance (MDR). The results revealed that B4GALT1 and B4GALT5 were highly expressed in four MDR cells and patients, altered levels of B4GALT1 and B4GALT5 were responsible for changed drug-resistant phenotype of HL60 and HL60/adriamycin-resistant cells. Further data showed that manipulation of these two gene expression led to increased or decreased activity of hedgehog (Hh) signaling and proportionally mutative expression of p-glycoprotein (P-gp) and MDR-associated protein 1 (MRP1) that are both known to be related to MDR. Thus, we propose that B4GALT1 and B4GALT5, two members of B4GALT gene family, are involved in the development of MDR of human leukemia cells, probably by regulating the activity of Hh signaling and the expression of P-gp and MRP1. PMID:23744354

  20. MicroRNA125b-mediated Hedgehog signaling influences liver regeneration by chorionic plate-derived mesenchymal stem cells.

    PubMed

    Hyun, Jeongeun; Wang, Sihyung; Kim, Jieun; Kim, Gi Jin; Jung, Youngmi

    2015-09-15

    Although chorionic plate-derived mesenchymal stem cells (CP-MSCs) were shown to promote liver regeneration, the mechanisms underlying the effect remain unclear. Hedgehog (Hh) signaling orchestrates tissue reconstruction in damaged liver. MSCs release microRNAs mediating various cellular responses. Hence, we hypothesized that microRNAs from CP-MSCs regulated Hh signaling, which influenced liver regeneration. Livers were obtained from carbon tetrachloride (CCl4)-treated rats transplanted with human CP-MSCs (Tx) or saline (non-Tx). Sonic Hh, one of Hh ligands, increased in CCl4-treated liver, whereas it decreased in CP-MSC-treated liver with CCl4. The expression of Hh-target genes was significantly downregulated in the Tx. Reduced expansion of progenitors and regressed fibrosis were observed in the liver of the Tx rats. CP-MSCs suppressed the expression of Hh and profibrotic genes in co-cultured LX2 (human hepatic stellate cell) with CP-MSCs. MicroRNA-125b targeting smo was retained in exosomes of CP-MSCs. CP-MSCs with microRNA-125b inhibitor failed to attenuate the expression of Hh signaling and profibrotic genes in the activated HSCs. Therefore, these results demonstrated that microRNA-125b from CP-MSCs suppressed the activation of Hh signaling, which promoted the reduced fibrosis, suggesting that microRNA-mediated regulation of Hh signaling contributed to liver regeneration by CP-MSCs.

  1. Synergism between Hedgehog-GLI and EGFR signaling in Hedgehog-responsive human medulloblastoma cells induces downregulation of canonical Hedgehog-target genes and stabilized expression of GLI1.

    PubMed

    Götschel, Frank; Berg, Daniela; Gruber, Wolfgang; Bender, Christian; Eberl, Markus; Friedel, Myriam; Sonntag, Johanna; Rüngeler, Elena; Hache, Hendrik; Wierling, Christoph; Nietfeld, Wilfried; Lehrach, Hans; Frischauf, Annemarie; Schwartz-Albiez, Reinhard; Aberger, Fritz; Korf, Ulrike

    2013-01-01

    Aberrant activation of Hedgehog (HH) signaling has been identified as a key etiologic factor in many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling depend profoundly on interactions with other pathways, such as epidermal growth factor receptor-mediated signaling, which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. Our experimental data demonstrated that the Daoy human medulloblastoma cell line possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic HH or Smoothened agonist induced expression of GLI1 protein and simultaneously prevented the processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed at the transcriptomic and proteomic levels. The Daoy cells responded to the HH/EGF co-treatment by downregulating GLI1, PTCH, and HHIP at the transcript level; this was also observed when Amphiregulin (AREG) was used instead of EGF. We identified a novel crosstalk mechanism whereby EGFR signaling silences proteins acting as negative regulators of HH signaling, as AKT- and ERK-signaling independent process. EGFR/HH signaling maintained high GLI1 protein levels which contrasted the GLI1 downregulation on the transcript level. Conversely, a high-level synergism was also observed, due to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile. This suggests that there are more wide-spread, yet context-dependent interactions, between HH/GLI and growth factor receptor signaling in human malignancies.

  2. Synergism between Hedgehog-GLI and EGFR Signaling in Hedgehog-Responsive Human Medulloblastoma Cells Induces Downregulation of Canonical Hedgehog-Target Genes and Stabilized Expression of GLI1

    PubMed Central

    Götschel, Frank; Berg, Daniela; Gruber, Wolfgang; Bender, Christian; Eberl, Markus; Friedel, Myriam; Sonntag, Johanna; Rüngeler, Elena; Hache, Hendrik; Wierling, Christoph; Nietfeld, Wilfried; Lehrach, Hans; Frischauf, Annemarie; Schwartz-Albiez, Reinhard; Aberger, Fritz; Korf, Ulrike

    2013-01-01

    Aberrant activation of Hedgehog (HH) signaling has been identified as a key etiologic factor in many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling depend profoundly on interactions with other pathways, such as epidermal growth factor receptor-mediated signaling, which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. Our experimental data demonstrated that the Daoy human medulloblastoma cell line possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic HH or Smoothened agonist induced expression of GLI1 protein and simultaneously prevented the processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed at the transcriptomic and proteomic levels. The Daoy cells responded to the HH/EGF co-treatment by downregulating GLI1, PTCH, and HHIP at the transcript level; this was also observed when Amphiregulin (AREG) was used instead of EGF. We identified a novel crosstalk mechanism whereby EGFR signaling silences proteins acting as negative regulators of HH signaling, as AKT- and ERK-signaling independent process. EGFR/HH signaling maintained high GLI1 protein levels which contrasted the GLI1 downregulation on the transcript level. Conversely, a high-level synergism was also observed, due to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile. This suggests that there are more wide-spread, yet context-dependent interactions, between HH/GLI and growth factor receptor signaling in human malignancies. PMID:23762360

  3. Arsenic inhibits hedgehog signaling during P19 cell differentiation

    SciTech Connect

    Liu, Jui Tung; Bain, Lisa J.

    2014-12-15

    Arsenic is a toxicant found in ground water around the world, and human exposure mainly comes from drinking water or from crops grown in areas containing arsenic in soils or water. Epidemiological studies have shown that arsenic exposure during development decreased intellectual function, reduced birth weight, and altered locomotor activity, while in vitro studies have shown that arsenite decreased muscle and neuronal cell differentiation. The sonic hedgehog (Shh) signaling pathway plays an important role during the differentiation of both neurons and skeletal muscle. The purpose of this study was to investigate whether arsenic can disrupt Shh signaling in P19 mouse embryonic stem cells, leading to changes muscle and neuronal cell differentiation. P19 embryonic stem cells were exposed to 0, 0.25, or 0.5 μM of sodium arsenite for up to 9 days during cell differentiation. We found that arsenite exposure significantly reduced transcript levels of genes in the Shh pathway in both a time and dose-dependent manner. This included the Shh ligand, which was decreased 2- to 3-fold, the Gli2 transcription factor, which was decreased 2- to 3-fold, and its downstream target gene Ascl1, which was decreased 5-fold. GLI2 protein levels and transcriptional activity were also reduced. However, arsenic did not alter GLI2 primary cilium accumulation or nuclear translocation. Moreover, additional extracellular SHH rescued the inhibitory effects of arsenic on cellular differentiation due to an increase in GLI binding activity. Taken together, we conclude that arsenic exposure affected Shh signaling, ultimately decreasing the expression of the Gli2 transcription factor. These results suggest a mechanism by which arsenic disrupts cell differentiation. - Highlights: • Arsenic exposure decreases sonic hedgehog pathway-related gene expression. • Arsenic decreases GLI2 protein levels and transcriptional activity in P19 cells. • Arsenic exposure does not alter the levels of SHH

  4. Microchip sonic spray ionization.

    PubMed

    Pól, Jaroslav; Kauppila, Tiina J; Haapala, Markus; Saarela, Ville; Franssila, Sami; Ketola, Raimo A; Kotiaho, Tapio; Kostiainen, Risto

    2007-05-01

    The first microchip version of sonic spray ionization (SSI) as an atmospheric pressure ionization source for mass spectrometry (MS) is presented. The microchip used for SSI has recently been developed in our laboratory, and it has been used before as an atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) source. Now the ionization is achieved simply by applying high (sonic) speed nebulizer gas, without heat, corona discharge, or high voltage. The microchip SSI was applied to the analysis of tetra-N-butylammonium, verapamil, testosterone, angiotensin I, and ibuprofen. The limits of detection were in the range of 15 nM to 4 microM. The technique was found to be highly dependent on the position of the chip toward the mass spectrometer inlet, and on the gas and the sample solution flow rates. The microchip SSI provided dynamic linearity following a pattern similar to that used with electrospray, good quantitative repeatability (RSD=16%), and long-term signal stability.

  5. Hedgehog Signaling during Appendage Development and Regeneration

    PubMed Central

    Singh, Bhairab N.; Koyano-Nakagawa, Naoko; Donaldson, Andrew; Weaver, Cyprian V.; Garry, Mary G.; Garry, Daniel J.

    2015-01-01

    Regulatory networks that govern embryonic development have been well defined. While a common hypothesis supports the notion that the embryonic regulatory cascades are reexpressed following injury and tissue regeneration, the mechanistic regulatory pathways that mediate the regenerative response in higher organisms remain undefined. Relative to mammals, lower vertebrates, including zebrafish and newts, have a tremendous regenerative capacity to repair and regenerate a number of organs including: appendages, retina, heart, jaw and nervous system. Elucidation of the pathways that govern regeneration in these lower organisms may provide cues that will enhance the capacity for the regeneration of mammalian organs. Signaling pathways, such as the hedgehog pathway, have been shown to play critical functions during development and during regeneration in lower organisms. These signaling pathways have been shown to modulate multiple processes including cellular origin, positional identity and cellular maturation. The present review will focus on the cellular and molecular regulation of the hedgehog (HH) signaling pathway and its interaction with other signaling factors during appendage development and regeneration. PMID:26110318

  6. Evolutionary analyses of hedgehog and Hoxd-10 genes in fish species closely related to the zebrafish

    PubMed Central

    Zardoya, Rafael; Abouheif, Ehab; Meyer, Axel

    1996-01-01

    The study of development has relied primarily on the isolation of mutations in genes with specific functions in development and on the comparison of their expression patterns in normal and mutant phenotypes. Comparative evolutionary analyses can complement these approaches. Phylogenetic analyses of Sonic hedgehog (Shh) and Hoxd-10 genes from 18 cyprinid fish species closely related to the zebrafish provide novel insights into the functional constraints acting on Shh. Our results confirm and extend those gained from expression and crystalline structure analyses of this gene. Unexpectedly, exon 1 of Shh is found to be almost invariant even in third codon positions among these morphologically divergent species suggesting that this exon encodes for a functionally important domain of the hedgehog protein. This is surprising because the main functional domain of Shh had been thought to be that encoded by exon 2. Comparisons of Shh and Hoxd-10 gene sequences and of resulting gene trees document higher evolutionary constraints on the former than on the latter. This might be indicative of more general evolutionary patterns in networks of developmental regulatory genes interacting in a hierarchical fashion. The presence of four members of the hedgehog gene family in cyprinid fishes was documented and their homologies to known hedgehog genes in other vertebrates were established. PMID:8917540

  7. Genetics Home Reference: Weyers acrofacial dysostosis

    MedlinePlus

    ... genes are thought to help regulate the Sonic Hedgehog signaling pathway. This pathway plays roles in cell ... condition. Studies suggest that they interfere with Sonic Hedgehog signaling in the developing embryo, disrupting the formation ...

  8. Genetics Home Reference: Ellis-van Creveld syndrome

    MedlinePlus

    ... genes are thought to help regulate the Sonic Hedgehog signaling pathway. This pathway plays roles in cell ... condition. Studies suggest that they prevent normal Sonic Hedgehog signaling in the developing embryo, disrupting the formation ...

  9. Comparative genomics of the Hedgehog loci in chordates and the origins of Shh regulatory novelties.

    PubMed

    Irimia, Manuel; Royo, Jose L; Burguera, Demian; Maeso, Ignacio; Gómez-Skarmeta, José L; Garcia-Fernandez, Jordi

    2012-01-01

    The origin and evolution of the complex regulatory landscapes of some vertebrate developmental genes, often spanning hundreds of Kbp and including neighboring genes, remain poorly understood. The Sonic Hedgehog (Shh) genomic regulatory block (GRB) is one of the best functionally characterized examples, with several discrete enhancers reported within its introns, vast upstream gene-free region and neighboring genes (Lmbr1 and Rnf32). To investigate the origin and evolution of this GRB, we sequenced and characterized the Hedgehog (Hh) loci from three invertebrate chordate amphioxus species, which share several early expression domains with Shh. Using phylogenetic footprinting within and between chordate lineages, and reporter assays in zebrafish probing >30 Kbp of amphioxus Hh, we report large sequence and functional divergence between both groups. In addition, we show that the linkage of Shh to Lmbr1 and Rnf32, necessary for the unique gnatostomate-specific Shh limb expression, is a vertebrate novelty occurred between the two whole-genome duplications.

  10. Quiet Sonic Booms: A NASA and Industry Progress Report

    NASA Technical Reports Server (NTRS)

    Larson, David Nils; Martin, Roy; Haering, Edward A.

    2011-01-01

    The purpose of this Oral Presentation is to present a progress report on NASA and Industry efforts related to Quiet Sonic Boom Program activities. This presentation will review changes in aircraft shaping to produce quiet supersonic booms and associated supersonic flight test methods and results. In addition, new flight test profiles have been recently developed that have allowed for the generation of sonic booms of varying intensity. These new flight test profiles have allowed for ground testing of the response of various building structures to sonic booms and the associated public acceptability to various sonic boom intensities. The new flight test profiles and associated ground measurement test methods will be reviewed. Finally, this Oral Presentation will review the International Regulatory requirements that would be involved to change aviation regulation and allow for overland quiet supersonic flight.

  11. Confidence Intervals for Laboratory Sonic Boom Annoyance Tests

    NASA Technical Reports Server (NTRS)

    Rathsam, Jonathan; Christian, Andrew

    2016-01-01

    Commercial supersonic flight is currently forbidden over land because sonic booms have historically caused unacceptable annoyance levels in overflown communities. NASA is providing data and expertise to noise regulators as they consider relaxing the ban for future quiet supersonic aircraft. One deliverable NASA will provide is a predictive model for indoor annoyance to aid in setting an acceptable quiet sonic boom threshold. A laboratory study was conducted to determine how indoor vibrations caused by sonic booms affect annoyance judgments. The test method required finding the point of subjective equality (PSE) between sonic boom signals that cause vibrations and signals not causing vibrations played at various amplitudes. This presentation focuses on a few statistical techniques for estimating the interval around the PSE. The techniques examined are the Delta Method, Parametric and Nonparametric Bootstrapping, and Bayesian Posterior Estimation.

  12. Polo-Like Kinase 2 Is a Mediator of Hedgehog Survival Signaling in Cholangiocarcinoma

    PubMed Central

    Fingas, Christian D.; Mertens, Joachim C.; Razumilava, Nataliya; Sydor, Svenja; Bronk, Steven F.; Christensen, John D.; Rizvi, Sumera H.; Canbay, Ali; Treckmann, Jürgen W.; Paul, Andreas; Sirica, Alphonse E.; Gores, Gregory J.

    2013-01-01

    Cholangiocarcinoma (CCA) cells paradoxically express the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and thus rely on potent survival signals to circumvent cell death by TRAIL. Hedgehog (Hh) signaling is an important survival pathway in CCA. Herein, we further examine the mechanisms whereby Hh signaling mediates apoptosis resistance in CCA, revealing a pivotal role for the cell division regulating serine/threonine kinase polo-like kinase 2 (PLK2). We employed 50 human CCA samples (25 intrahepatic and 25 extrahepatic CCA) as well as human KMCH-1, Mz-CHA-1, and HUCCT-1 CCA cells for these studies. In vivo experiments were conducted using a syngeneic rat orthotopic CCA model. In human samples, polo-like kinase (PLK)1/2/3-immunoreactive cancer cells were present in the preponderance of intra- and extrahepatic CCA specimens. Inhibition of Hh signaling by cyclopamine reduced PLK2, but not PLK1 or PLK3, messenger RNA and protein expression in vehicle-treated and sonic Hh–treated CCA cells, confirming our previous microarray study. PLK2 regulation by Hh signaling appears to be direct, because the Hh transcription factors, glioma-associated oncogene 1 and 2, bind to the PLK2 promotor. Moreover, inhibition of PLK2 by the PLK inhibitor, BI 6727 (volasertib), or PLK2 knockdown was proapoptotic in CCA cells. BI 6727 administration or PLK2 knockdown decreased cellular protein levels of antiapoptotic myeloid cell leukemia 1 (Mcl-1), an effect reversed by the proteasome inhibitor, MG-132. Finally, BI 6727 administration reduced Mcl-1 protein expression in CCA cells, resulting in CCA cell apoptosis and tumor suppression in vivo. Conclusion PLK2 appears to be an important mediator of Hh survival signaling. These results suggest PLK inhibitors to be of therapeutic value for treatment of human CCA. PMID:23703673

  13. Targeting the hedgehog-glioma-associated oncogene homolog pathway inhibits bleomycin-induced lung fibrosis in mice.

    PubMed

    Moshai, Elika Farrokhi; Wémeau-Stervinou, Lidwine; Cigna, Natacha; Brayer, Stephanie; Sommé, Joëlle Marchal; Crestani, Bruno; Mailleux, Arnaud A

    2014-07-01

    Idiopathic pulmonary fibrosis has been associated with the reactivation of developmental pathways, notably the Hedgehog-Glioma-associated oncogene homolog (GLI) pathway. In this study, we determined whether the Hedgehog pathway was activated in bleomycin-induced lung injury in mice, and whether targeting the Hedgehog-Gli pathway could decrease bleomycin-induced lung fibrosis. After intratracheal injection of bleomycin on Day 0, C57Bl6 mice received GDC-0449 (an inhibitor of Smoothened, the transducer of the pathway), or 2,2'-[[Dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,N dimethylbenzenamine (GANT61; an inhibitor of GLI transcription factors in the nucleus), from Day 7 to Day 13. At Day 14, whole-lung homogenates were obtained for morphological analysis, assessment of cell apoptosis and proliferation, collagen quantification, and evaluation of profibrotic (transforming growth factor-β, connective tissue growth factor, plasminogen activator inhibitor 1, vascular endothelial growth factor-A) and proinflammatory mediators (IL-1β) expression. We showed that the Hedgehog pathway was activated in bleomycin-induced lung fibrosis on Day 14 after injury, with an increased lung expression of the ligand, Sonic Hedgehog, and with increased messenger RNA expression and nuclear localization of GLI1 and GLI2. Inhibition of Smoothened with GDC-0449 did not influence the development of bleomycin-induced lung fibrosis. By contrast, the inhibition of GLI activity with GANT61 decreased lung fibrosis and lung collagen accumulation, and promoted an antifibrotic and anti-inflammatory environment. Our results identify the hedgehog-Gli pathway as a profibrotic pathway in experimental fibrosis. Inhibition of the Hedgehog-Gli pathway at the level of GLI transcriptional activity could be a therapeutic option in fibrotic lung diseases.

  14. Loudness of shaped sonic booms

    NASA Astrophysics Data System (ADS)

    Shepherd, Kevin P.; Sullivan, Brenda M.

    A loudness model is adopted to study the feasibility of designing and operating a supersonic transport to produce minimized sonic booms. The loudness contours in this technique extend to a lower frequency (1 Hz) and thus are appropriate for sonic booms that contain significant low frequency energy. Input to the loudness calculation procedure is the power spectral density of the pressure-time signature. Calculations of loudness, for both indoor and outdoor conditions, demonstrate that shaped sonic booms are potentially more acceptable than N-waves possessing the same peak overpressure.

  15. Mammary gland tumors in captive African hedgehogs.

    PubMed

    Raymond, J T; Gerner, M

    2000-04-01

    From December 1995 to July 1999, eight mammary gland tumors were diagnosed in eight adult captive female African hedgehogs (Atelerix albiventris). The tumors presented as single or multiple subcutaneous masses along the cranial or caudal abdomen that varied in size for each hedgehog. Histologically, seven of eight (88%) mammary gland tumors were malignant. Tumors were classified as solid (4 cases), tubular (2 cases), and papillary (2 cases). Seven tumors had infiltrated into the surrounding stroma and three tumors had histologic evidence of neoplastic vascular invasion. Three hedgehogs had concurrent neoplasms. These are believed to be the first reported cases of mammary gland tumors in African hedgehogs.

  16. Vibration Penalty Estimates for Indoor Annoyance Caused by Sonic Boom

    NASA Technical Reports Server (NTRS)

    Rathsam, Jonathan; Klos, Jacob

    2016-01-01

    Commercial supersonic flight is currently forbidden over land because sonic booms have historically caused unacceptable annoyance levels in overflown communities. NASA is providing data and expertise to noise regulators as they consider relaxing the ban for future quiet supersonic aircraft. One key objective is a predictive model for indoor annoyance based on factors such as noise and indoor vibration levels. The current study quantified the increment in indoor sonic boom annoyance when sonic booms can be felt directly through structural vibrations in addition to being heard. A shaker mounted below each chair in the sonic boom simulator emulated vibrations transmitting through the structure to that chair. The vibration amplitudes were determined from numeric models of a large range of residential structures excited by the same sonic boom waveforms used in the experiment. The analysis yielded vibration penalties, which are the increments in sound level needed to increase annoyance as much as the vibration does. For sonic booms at acoustic levels from 75 to 84 dB Perceived Level, vibration signals with lower amplitudes (+1 sigma) yielded penalties from 0 to 5 dB, and vibration signals with higher amplitudes (+3 sigma) yielded penalties from 6 to 10 dB.

  17. REN(KCTD11) is a suppressor of Hedgehog signaling and is deleted in human medulloblastoma.

    PubMed

    Di Marcotullio, Lucia; Ferretti, Elisabetta; De Smaele, Enrico; Argenti, Beatrice; Mincione, Claudia; Zazzeroni, Francesca; Gallo, Rita; Masuelli, Laura; Napolitano, Maddalena; Maroder, Marella; Modesti, Andrea; Giangaspero, Felice; Screpanti, Isabella; Alesse, Edoardo; Gulino, Alberto

    2004-07-20

    Hedgehog signaling is suggested to be a major oncogenic pathway in medulloblastoma, which arises from aberrant development of cerebellar granule progenitors. Allelic loss of chromosome 17p has also been described as the most frequent genetic defect in this human neoplasia. This observation raises the question of a possible interplay between 17p deletion and the Hedgehog tumorigenic pathway. Here, we identify the human orthologue of mouse REN(KCTD11), previously reported to be expressed in differentiating and low proliferating neuroblasts. Human REN(KCTD11) maps to 17p13.2 and displays allelic deletion as well as significantly reduced expression in medulloblastoma. REN(KCTD11) inhibits medulloblastoma cell proliferation and colony formation in vitro and suppresses xenograft tumor growth in vivo. REN(KCTD11) seems to inhibit medulloblastoma growth by negatively regulating the Hedgehog pathway because it antagonizes the Gli-mediated transactivation of Hedgehog target genes, by affecting Gli1 nuclear transfer, and its growth inhibitory activity is impaired by Gli1 inactivation. Therefore, we identify REN(KCTD11) as a suppressor of Hedgehog signaling and suggest that its inactivation might lead to a deregulation of the tumor-promoting Hedgehog pathway in medulloblastoma.

  18. RENKCTD11 is a suppressor of Hedgehog signaling and is deleted in human medulloblastoma

    PubMed Central

    Di Marcotullio, Lucia; Ferretti, Elisabetta; De Smaele, Enrico; Argenti, Beatrice; Mincione, Claudia; Zazzeroni, Francesca; Gallo, Rita; Masuelli, Laura; Napolitano, Maddalena; Maroder, Marella; Modesti, Andrea; Giangaspero, Felice; Screpanti, Isabella; Alesse, Edoardo; Gulino, Alberto

    2004-01-01

    Hedgehog signaling is suggested to be a major oncogenic pathway in medulloblastoma, which arises from aberrant development of cerebellar granule progenitors. Allelic loss of chromosome 17p has also been described as the most frequent genetic defect in this human neoplasia. This observation raises the question of a possible interplay between 17p deletion and the Hedgehog tumorigenic pathway. Here, we identify the human orthologue of mouse RENKCTD11, previously reported to be expressed in differentiating and low proliferating neuroblasts. Human RENKCTD11 maps to 17p13.2 and displays allelic deletion as well as significantly reduced expression in medulloblastoma. RENKCTD11 inhibits medulloblastoma cell proliferation and colony formation in vitro and suppresses xenograft tumor growth in vivo. RENKCTD11 seems to inhibit medulloblastoma growth by negatively regulating the Hedgehog pathway because it antagonizes the Gli-mediated transactivation of Hedgehog target genes, by affecting Gli1 nuclear transfer, and its growth inhibitory activity is impaired by Gli1 inactivation. Therefore, we identify RENKCTD11 as a suppressor of Hedgehog signaling and suggest that its inactivation might lead to a deregulation of the tumor-promoting Hedgehog pathway in medulloblastoma. PMID:15249678

  19. The Sonic Altimeter for Aircraft

    NASA Technical Reports Server (NTRS)

    Draper, C S

    1937-01-01

    Discussed here are results already achieved with sonic altimeters in light of the theoretical possibilities of such instruments. From the information gained in this investigation, a procedure is outlined to determine whether or not a further development program is justified by the value of the sonic altimeter as an aircraft instrument. The information available in the literature is reviewed and condensed into a summary of sonic altimeter developments. Various methods of receiving the echo and timing the interval between the signal and the echo are considered. A theoretical discussion is given of sonic altimeter errors due to uncertainties in timing, variations in sound velocity, aircraft speed, location of the sending and receiving units, and inclinations of the flight path with respect to the ground surface. Plots are included which summarize the results in each case. An analysis is given of the effect of an inclined flight path on the frequency of the echo. A brief study of the acoustical phases of the sonic altimeter problem is carried through. The results of this analysis are used to predict approximately the maximum operating altitudes of a reasonably designed sonic altimeter under very good and very bad conditions. A final comparison is made between the estimated and experimental maximum operating altitudes which shows good agreement where quantitative information is available.

  20. Hedgehog Signaling Overcomes an EZH2-Dependent Epigenetic Barrier to Promote Cholangiocyte Expansion

    PubMed Central

    Lu, Jie; Almada, Luciana L.; Lomberk, Gwen; Fernandez-Zapico, Martin E.; Urrutia, Raul; Huebert, Robert C.

    2016-01-01

    Background & Aims Developmental morphogens play an important role in coordinating the ductular reaction and portal fibrosis occurring in the setting of cholangiopathies. However, little is known about how membrane signaling events in ductular reactive cells (DRCs) are transduced into nuclear transcriptional changes to drive cholangiocyte maturation and matrix deposition. Therefore, the aim of this study was to investigate potential mechanistic links between cell signaling events and epigenetic regulators in DRCs. Methods Using directed differentiation of induced pluripotent stem cells (iPSC), isolated DRCs, and in vivo models, we examine the mechanisms whereby sonic hedgehog (Shh) overcomes an epigenetic barrier in biliary precursors and promotes both cholangiocyte maturation and deposition of fibronectin (FN). Results We demonstrate, for the first time, that Gli1 influences the differentiation state and fibrogenic capacity of iPSC-derived hepatic progenitors and isolated DRCs. We outline a novel pathway wherein Shh-mediated Gli1 binding in key cholangiocyte gene promoters overcomes an epigenetic barrier conferred by the polycomb protein, enhancer of zeste homolog 2 (EZH2) and initiates the transcriptional program of cholangiocyte maturation. We also define previously unknown functional Gli1 binding sites in the promoters of cytokeratin (CK)7, CK19, and FN. Our in vivo results show that EZH2 KO mice fed the choline-deficient, ethanolamine supplemented (CDE) diet have an exaggerated cholangiocyte expansion associated with more robust ductular reaction and increased peri-portal fibrosis. Conclusion We conclude that Shh/Gli1 signaling plays an integral role in cholangiocyte maturation in vitro by overcoming an EZH2-dependent epigenetic barrier and this mechanism also promotes biliary expansion in vivo. PMID:27936185

  1. Paracrine Hedgehog signaling in stomach and intestine: new roles for Hedgehog in gastrointestinal patterning

    PubMed Central

    Kolterud, Åsa; Grosse, Ann S.; Zacharias, William J.; Walton, Katherine D.; Kretovich, Katherine E.; Madison, Blair; Waghray, Meghna; Ferris, Jennifer E.; Hu, Chunbo; Merchant, Juanita L.; Dlugosz, Andrzej; Kottmann, Andreas H.; Gumucio, Deborah L.

    2009-01-01

    Background & Aims Hedgehog signaling is critical in gastrointestinal patterning. Mice deficient in Hedgehog signaling exhibit abnormalities that mirror deformities seen in the human VACTERL (vertebral, anal, cardiac, tracheal, esophageal, renal, limb) association. However, the direction of Hedgehog signal flow is controversial and the cellular targets of Hedgehog signaling change with time during development. We profiled cellular Hedgehog response patterns from embryonic day 10.5 (E10.5) to adult in murine antrum, pyloric region, small intestine and colon. Methods Hedgehog signaling was profiled using Hedgehog pathway reporter mice and in situ hybridization. Cellular targets were identified by immunostaining. Ihh-overexpressing transgenic animals were generated and analyzed. Results Hedgehog signaling is strictly paracrine from antrum to colon throughout embryonic and adult life. Novel findings include: mesothelial cells of the serosa transduce Hedgehog signals in fetal life; the hindgut epithelium expresses Ptch but not Gli1 at E10.5; the two layers of the muscularis externa respond differently to Hedgehog signals; organogenesis of the pyloric sphincter is associated with robust Hedgehog signaling; dramatically different Hedgehog responses characterize stomach and intestine at E16; after birth, the muscularis mucosa and villus smooth muscle (SM) consist primarily of Hedgehog responsive cells and Hh levels actively modulate villus core SM. Conclusions These studies reveal a previously unrecognized association of paracrine Hedgehog signaling with several gastrointestinal patterning events involving the serosa, pylorus and villus smooth muscle. The results may have implications for several human anomalies and could potentially expand the spectrum of the human VACTERL association. PMID:19445942

  2. Cytologic diagnosis of diseases of hedgehogs.

    PubMed

    Juan-Sallés, Carles; Garner, Michael M

    2007-01-01

    This article focuses on neoplastic diseases because they may be the most frequent disease processes in captive hedgehogs according to the literature and authors' case files and the most common cases submitted for cytologic diagnosis in these species, particularly the African hedgehog (Atelerix albiventris).

  3. Hedgehog Signaling in Pancreatic Fibrosis and Cancer.

    PubMed

    Bai, Yongyu; Bai, Yongheng; Dong, Jiaojiao; Li, Qiang; Jin, Yuepeng; Chen, Bicheng; Zhou, Mengtao

    2016-03-01

    The hedgehog signaling pathway was first discovered in the 1980s. It is a stem cell-related pathway that plays a crucial role in embryonic development, tissue regeneration, and organogenesis. Aberrant activation of hedgehog signaling leads to pathological consequences, including a variety of human tumors such as pancreatic cancer. Multiple lines of evidence indicate that blockade of this pathway with several small-molecule inhibitors can inhibit the development of pancreatic neoplasm. In addition, activated hedgehog signaling has been reported to be involved in fibrogenesis in many tissues, including the pancreas. Therefore, new therapeutic targets based on hedgehog signaling have attracted a great deal of attention to alleviate pancreatic diseases. In this review, we briefly discuss the recent advances in hedgehog signaling in pancreatic fibrogenesis and carcinogenesis and highlight new insights on their potential relationship with respect to the development of novel targeted therapies.

  4. Hedgehog Signaling in Pancreatic Fibrosis and Cancer

    PubMed Central

    Bai, Yongyu; Bai, Yongheng; Dong, Jiaojiao; Li, Qiang; Jin, Yuepeng; Chen, Bicheng; Zhou, Mengtao

    2016-01-01

    Abstract The hedgehog signaling pathway was first discovered in the 1980s. It is a stem cell-related pathway that plays a crucial role in embryonic development, tissue regeneration, and organogenesis. Aberrant activation of hedgehog signaling leads to pathological consequences, including a variety of human tumors such as pancreatic cancer. Multiple lines of evidence indicate that blockade of this pathway with several small-molecule inhibitors can inhibit the development of pancreatic neoplasm. In addition, activated hedgehog signaling has been reported to be involved in fibrogenesis in many tissues, including the pancreas. Therefore, new therapeutic targets based on hedgehog signaling have attracted a great deal of attention to alleviate pancreatic diseases. In this review, we briefly discuss the recent advances in hedgehog signaling in pancreatic fibrogenesis and carcinogenesis and highlight new insights on their potential relationship with respect to the development of novel targeted therapies. PMID:26962810

  5. Cardiac assessment of African hedgehogs (Atelerix albiventris).

    PubMed

    Black, Peter A; Marshall, Cecilia; Seyfried, Alice W; Bartin, Anne M

    2011-03-01

    Cardiomyopathy is a common finding in captive African hedgehogs (Atelerix albiventris) at postmortem exam. To date, treatment attempts have been mostly empirical and unrewarding. The objective of this study was to determine reference cardiac values for captive African hedgehogs based on echocardiogram, electrocardiogram (ECG), and radiographs. Adult African hedgehogs with no clinical signs of cardiac disease (n = 13) were selected. Each animal was anesthetized with isoflurane via facemask and an echocardiogram, ECG, and radiographs were performed. Standard measurements were taken and the descriptive statistics performed. Values were comparable to limited data available in other hedgehog species and other similar-sized exotic species. Two animals were removed from consideration of reference values due to valvular defects that were considered significant. These data are the first establishing cardiac parameters in normal African hedgehogs using radiographic cardiac measurement, echocardiogram, and ECG. Evaluating animals with possible cardiomyopathy may allow for earlier diagnosis and more successful treatment.

  6. Release and transportation of Hedgehog molecules.

    PubMed

    Thérond, Pascal P

    2012-04-01

    Secretion of the Hedgehog morphogen induces different cell fates over the short and long ranges during developmental patterning. Mature Hedgehog carries hydrophobic palmitic acid and cholesterol modifications essential for its correct spread. The long-range activity of Hedgehog raises questions about how a dually lipidated protein can spread in the hydrophilic environment of the extracellular space. There is compelling experimental evidence in favour of the existence of several different carriers for Hedgehog transportation, via very different routes. This suggests that different accessory proteins and cellular machineries may be involved in the specific release of Hedgehog. I suggest that Hh carriers may work in parallel within a given cell and that developmental context may condition the choice of Hh carrier in secreting cells.

  7. Macrophage-derived Hedgehog Ligands Promotes Fibrogenic and Angiogenic Responses in Human Schistosomiasis mansoni

    PubMed Central

    Pereira, Thiago A.; Xie, Guanhua; Choi, Steve S.; Syn, Wing-Kin; Voieta, Izabela; Lu, Jiuyi; Chan, Isaac S.; Swiderska, Marzena; Amaral, Kirsten B.; Antunes, Carlos Maurício; Secor, William E.; Witek, Rafal P.; Lambertucci, José Roberto; Pereira, Fausto L.; Diehl, Anna Mae

    2012-01-01

    Background Schistosomiasis mansoni is a major cause of portal fibrosis and portal hypertension. The Hedgehog pathway regulates fibrogenic repair in some types of liver injury. Aims Determine if Hedgehog-pathway activation occurs during fibrosis progression in schistosomiasis and to determine if macrophage-related mechanisms are involved. Methods Immunohistochemistry was used to characterize the cells that generate and respond to Hedgehog ligands in 28 liver biopsies from patients with different grades of schistosomiasis fibrosis staged by ultrasound. Cultured macrophages (RAW264.7 and primary rat Kupffer cells) and primary rat liver sinusoidal endothelial cells (LSEC) were treated with schistosome egg antigen (SEA) and evaluated by qRT-PCR. Inhibition of the Hedgehog-pathway was used to investigate its role in alternative activation of macrophages (M2) and vascular tube formation. Results Patients with schistosomiasis expressed more ligands (Shh and Ihh) and target genes (Patched and Gli2) than healthy individuals. Activated LSEC and myofibroblasts were Hedgehog-responsive (Gli2(+)) and accumulated in parallel with fibrosis stage (p<0.05). Double IHC for Ihh/CD68 showed that Ihh(+) cells were macrophages. In vitro studies demonstrated that SEA stimulated macrophages to express Ihh and Shh mRNA (p<0.05). Conditioned media from such macrophages induced luciferase production by Shh-LightII cells (p<0.001) and Hedgehog inhibitors blocked this effect (p<0.001). SEA-treated macrophages also up-regulated their own expression of M2 markers, and Hh-pathway inhibitors abrogated this response (p<0.01). Inhibition of the Hedgehog pathway in LSEC blocked SEA-induced migration and tube formation. Conclusion SEA stimulates liver macrophages to produce Hh-ligands, which promote alternative activation of macrophages, fibrogenesis, and vascular remodeling in schistosomiasis. PMID:23121638

  8. Crosstalk between Wnt/β-catenin and Hedgehog/Gli signaling pathways in colon cancer and implications for therapy.

    PubMed

    Song, Li; Li, Zhuo-Yu; Liu, Wei-Ping; Zhao, Mei-Rong

    2015-01-01

    Wnt/β-catenin and Hedgehog/Gli signalings play key roles in multiple biogenesis such as embryonic development and tissue homeostasis. Dysregulations of these 2 pathways are frequently found in most cancers, particularly in colon cancer. Their crosstalk has been increasingly appreciated as an important mechanism in regulating colon cancer progression. Our studies into the link between Wnt/β-catenin and Hedgehog/Gli signalings in colon cancer revealed several possible crosstalk points and suggested potential therapeutic strategies for colon cancer.

  9. Hedgehog Signal Transduction: Key Players, Oncogenic Drivers, and Cancer Therapy.

    PubMed

    Pak, Ekaterina; Segal, Rosalind A

    2016-08-22

    The Hedgehog (Hh) signaling pathway governs complex developmental processes, including proliferation and patterning within diverse tissues. These activities rely on a tightly regulated transduction system that converts graded Hh input signals into specific levels of pathway activity. Uncontrolled activation of Hh signaling drives tumor initiation and maintenance. However, recent entry of pathway-specific inhibitors into the clinic reveals mixed patient responses and thus prompts further exploration of pathway activation and inhibition. In this review, we share emerging insights into regulated and oncogenic Hh signaling, supplemented with updates on the development and use of Hh pathway-targeted therapies.

  10. Inhibition of hedgehog signaling depresses self-renewal of pancreatic cancer stem cells and reverses chemoresistance.

    PubMed

    Huang, Feng-Ting; Zhuan-Sun, Yong-Xun; Zhuang, Yan-Yan; Wei, Shu-Li; Tang, Jian; Chen, Wen-Bo; Zhang, Shi-Neng

    2012-11-01

    Pancreatic cancer stem cells play a crucial role in tumorigenesis and chemoresistance. The Hedgehog signaling pathway is a key regulator in pancreatic tumorigenesis and drug resistance. To identify pancreatic cancer stem cells, tumorspheres derived from the PANC-1 pancreatic cancer cell line were cultured under a floating-culture system. PANC-1 tumorspheres possessed properties of self-renewal, differentiation, higher tumorigenesis and chemoresistance. It was observed that Hedgehog pathway is active in PANC-1 tumorspheres as shown by expression of hedgehog components Smo, Gil 1 and Gli 2, detected by quantitative RT-PCR and western blotting. After cyclopamine-mediated blockade of hedgehog, a decrease in proliferation of PANC-1 tumorspheres and G0/G1 transition were observed, as well as a decreased expression of Bmi-1 in PANC-1 tumorspheres. Cyclopamine reversed chemoresistance to gemcitabine, resulting in decreased expression of ABCG2 in PANC-1 tumorspheres. Taken together, our data indicate that PANC-1 tumorspheres have 'stemness' potential, and hedgehog signaling pathway plays an important role in the regulation of self-renewal and reversal of chemoresistance in cancer stem cells in pancreatic adenocarcinoma.

  11. Functional Interaction between HEXIM and Hedgehog Signaling during Drosophila Wing Development

    PubMed Central

    Nguyen, Duy; Fayol, Olivier; Buisine, Nicolas; Lecorre, Pierrette; Uguen, Patricia

    2016-01-01

    Studying the dynamic of gene regulatory networks is essential in order to understand the specific signals and factors that govern cell proliferation and differentiation during development. This also has direct implication in human health and cancer biology. The general transcriptional elongation regulator P-TEFb regulates the transcriptional status of many developmental genes. Its biological activity is controlled by an inhibitory complex composed of HEXIM and the 7SK snRNA. Here, we examine the function of HEXIM during Drosophila development. Our key finding is that HEXIM affects the Hedgehog signaling pathway. HEXIM knockdown flies display strong phenotypes and organ failures. In the wing imaginal disc, HEXIM knockdown initially induces ectopic expression of Hedgehog (Hh) and its transcriptional effector Cubitus interuptus (Ci). In turn, deregulated Hedgehog signaling provokes apoptosis, which is continuously compensated by apoptosis-induced cell proliferation. Thus, the HEXIM knockdown mutant phenotype does not result from the apoptotic ablation of imaginal disc; but rather from the failure of dividing cells to commit to a proper developmental program due to Hedgehog signaling defects. Furthermore, we show that ci is a genetic suppressor of hexim. Thus, HEXIM ensures the integrity of Hedgehog signaling in wing imaginal disc, by a yet unknown mechanism. To our knowledge, this is the first time that the physiological function of HEXIM has been addressed in such details in vivo. PMID:27176767

  12. Hedgehog signaling in bone regulates whole-body energy metabolism through a bone-adipose endocrine relay mediated by PTHrP and adiponectin.

    PubMed

    Zhang, Xu; Cheng, Qianni; Wang, Yixiang; Leung, Po Sing; Mak, Kinglun Kingston

    2017-02-01

    Bone plays a role in energy metabolism, but the interplay between bone and other organs in this process is not completely understood. Here, we show that upregulated Hh signaling in bones results in increased whole-body energy expenditure, white adipose tissue (WAT) browning, hypoglycemia and skeletal muscle atrophy. We found that Hh signaling induces PTHrP secretion from bones and causes WAT browning. Injection of PTHrP-neutralizing antibody attenuates WAT browning and improves the circulating blood glucose level while high-fat diet treatment only rescues hypoglycemia. Furthermore, bone-derived PTHrP stimulates adiponectin secretion in WAT and results in systemic increase of fatty acid oxidation and glucose uptake. Mechanistically, PTHrP activates both PKA/cAMP and Akt/Foxo pathways for Ucp1 expression in WAT. PTHrP couples adiponectin actions to activate the AMPK pathway in the skeletal muscles and liver, respectively, for fatty acid oxidation. Our findings establish a new bone-adipose hormonal relay that regulates whole-body energy metabolism.

  13. Hedgehog signaling controls mesenchymal growth in the developing mammalian digestive tract.

    PubMed

    Mao, Junhao; Kim, Byeong-Moo; Rajurkar, Mihir; Shivdasani, Ramesh A; McMahon, Andrew P

    2010-05-01

    Homeostasis of the vertebrate digestive tract requires interactions between an endodermal epithelium and mesenchymal cells derived from the splanchnic mesoderm. Signaling between these two tissue layers is also crucial for patterning and growth of the developing gut. From early developmental stages, sonic hedgehog (Shh) and indian hedgehog (Ihh) are secreted by the endoderm of the mammalian gut, indicative of a developmental role. Further, misregulated hedgehog (Hh) signaling is implicated in both congenital defects and cancers arising from the gastrointestinal tract. In the mouse, only limited gastrointestinal anomalies arise following removal of either Shh or Ihh. However, given the considerable overlap in their endodermal expression domains, a functional redundancy between these signals might mask a more extensive role for Hh signaling in development of the mammalian gut. To address this possibility, we adopted a conditional approach to remove both Shh and Ihh functions from early mouse gut endoderm. Analysis of compound mutants indicates that continuous Hh signaling is dispensable for regional patterning of the gut tube, but is essential for growth of the underlying mesenchyme. Additional in vitro analysis, together with genetic gain-of-function studies, further demonstrate that Hh proteins act as paracrine mitogens to promote the expansion of adjacent mesenchymal progenitors, including those of the smooth muscle compartment. Together, these studies provide new insights into tissue interactions underlying mammalian gastrointestinal organogenesis and disease.

  14. Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase.

    PubMed

    Lanyon-Hogg, Thomas; Masumoto, Naoko; Bodakh, George; Konitsiotis, Antonio D; Thinon, Emmanuelle; Rodgers, Ursula R; Owens, Raymond J; Magee, Anthony I; Tate, Edward W

    2015-12-01

    Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click-ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click-ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click-ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.

  15. Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase

    PubMed Central

    Lanyon-Hogg, Thomas; Masumoto, Naoko; Bodakh, George; Konitsiotis, Antonio D.; Thinon, Emmanuelle; Rodgers, Ursula R.; Owens, Raymond J.; Magee, Anthony I.; Tate, Edward W.

    2015-01-01

    Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click–ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click–ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click–ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation. PMID:26334609

  16. Cardiomyopathy in captive African hedgehogs (Atelerix albiventris).

    PubMed

    Raymond, J T; Garner, M M

    2000-09-01

    From 1994 to 1999, 16 captive African hedgehogs (Atelerix albiventris), from among 42 necropsy cases, were diagnosed with cardiomyopathy. The incidence of cardiomyopathy in this study population was 38%. Fourteen of 16 hedgehogs with cardiomyopathy were males and all hedgehogs were adult (>1 year old). Nine hedgehogs exhibited 1 or more of the following clinical signs before death: heart murmur, lethargy, icterus, moist rales, anorexia, dyspnea, dehydration, and weight loss. The remaining 7 hedgehogs died without premonitory clinical signs. Gross findings were cardiomegaly (6 cases), hepatomegaly (5 cases), pulmonary edema (5 cases), pulmonary congestion (4 cases), hydrothorax (3 cases), pulmonary infarct (1 case), renal infarcts (1 case), ascites (1 case), and 5 cases showed no changes. Histologic lesions were found mainly within the left ventricular myocardium and consisted primarily of myodegeneration, myonecrosis, atrophy, hypertrophy, and disarray of myofibers. All hedgehogs with cardiomyopathy had myocardial fibrosis, myocardial edema, or both. Other common histopathologic findings were acute and chronic passive congestion of the lungs, acute passive congestion of the liver, renal tubular necrosis, vascular thrombosis, splenic extramedullary hematopoiesis, and hepatic lipidosis. This is the first report of cardiomyopathy in African hedgehogs.

  17. Fiber optic probe augmented sonic scaling versus conventional sonic scaling.

    PubMed

    Johnson, G K; Reinhardt, R A; Tussing, G J; Krejci, R F

    1989-03-01

    Several factors, including access and visualization problems, make total deposit removal during scaling and root planing procedures extremely difficult. This study examined the effectiveness of a mode of therapy designed to improve access and visualization for sonic scaling compared to closed sonic instrumentation. Teeth with moderate to deep probing depths in six patients scheduled to receive immediate dentures were divided into three experimental groups: Group I, sonic scaling with access augmented by interdental papilla reflection and fiber optic illumination/transillumination (34 surfaces); Group II, closed sonic scaling (34 surfaces); and Group III, untreated controls (35 surfaces). Immediately after treatment the experimental teeth were extracted, stained with toluidine blue, and interproximal areas evaluated for remaining accretions with a microscope-digitizing pad-computer system. Group I had a significantly lower percentage (P less than 0.01) of remaining subgingival accretion coverage than Group II (1.30 +/- 0.25% vs 6.35 +/- 1.08%), and both Group I and II demonstrated significantly (P less than 0.01) fewer deposits than the control surfaces (46.61 +/- 4.32%). These findings suggest that minimal tissue reflection and fiber optic illumination/transillumination are beneficial adjuncts to deposit removal in moderate to deep periodontal pockets.

  18. Anomalous dispersions of `hedgehog' particles

    NASA Astrophysics Data System (ADS)

    Bahng, Joong Hwan; Yeom, Bongjun; Wang, Yichun; Tung, Siu On; Hoff, J. Damon; Kotov, Nicholas

    2015-01-01

    Hydrophobic particles in water and hydrophilic particles in oil aggregate, but can form colloidal dispersions if their surfaces are chemically camouflaged with surfactants, organic tethers, adsorbed polymers or other particles that impart affinity for the solvent and increase interparticle repulsion. A different strategy for modulating the interaction between a solid and a liquid uses surface corrugation, which gives rise to unique wetting behaviour. Here we show that this topographical effect can also be used to disperse particles in a wide range of solvents without recourse to chemicals to camouflage the particles' surfaces: we produce micrometre-sized particles that are coated with stiff, nanoscale spikes and exhibit long-term colloidal stability in both hydrophilic and hydrophobic media. We find that these `hedgehog' particles do not interpenetrate each other with their spikes, which markedly decreases the contact area between the particles and, therefore, the attractive forces between them. The trapping of air in aqueous dispersions, solvent autoionization at highly developed interfaces, and long-range electrostatic repulsion in organic media also contribute to the colloidal stability of our particles. The unusual dispersion behaviour of our hedgehog particles, overturning the notion that like dissolves like, might help to mitigate adverse environmental effects of the use of surfactants and volatile organic solvents, and deepens our understanding of interparticle interactions and nanoscale colloidal chemistry.

  19. Astrocytoma in an African hedgehog (Atelerix albiventris) suspected wobbly hedgehog syndrome.

    PubMed

    Nakata, Makoto; Miwa, Yasutsugu; Itou, Takuya; Uchida, Kazuyuki; Nakayama, Hiroyuki; Sakai, Takeo

    2011-10-01

    A 28-month-old African hedgehog was referred to our hospital with progressive tetraparesis. On the first presentation, the hedgehog was suspected as having wobbly hedgehog syndrome (WHS) and the animal was treated with medication and rehabilitation. The animal died 22 days after onset. Pathological examination revealed that the animal was involved in astrocytoma between the medulla oblongata and the spinal cord (C1). This report indicates that a primary central nervous system tumor should be considered as one of the differential diagnoses for hedgehogs presenting with progressive paresis, together with WHS.

  20. Sonicated Diagnostic Immunoblot for Bartonellosis

    PubMed Central

    Mallqui, Vania; Speelmon, Emily C.; Verástegui, Manuela; Maguiña-Vargas, Ciro; Pinell-Salles, Paula; Lavarello, Rosa; Delgado, Jose; Kosek, Margaret; Romero, Sofia; Arana, Yanina; Gilman, Robert H.

    2000-01-01

    Two simple Bartonella bacilliformis immunoblot preparation methods were developed. Antigen was prepared by two different methods: sonication of whole organisms or glycine extraction. Both methods were then tested for sensitivity and specificity. Well-defined control sera were utilized in the development of these diagnostic immunoblots, and possible cross-reactions were thoroughly examined. Sera investigated for cross-reaction with these diagnostic antigens were drawn from patients with brucellosis, chlamydiosis, Q fever, and cat scratch disease, all of whom were from regions where bartonellosis is not endemic. While both immunoblots yielded reasonable sensitivity and high specificity, we recommend the use of the sonicated immunoblot, which has a higher sensitivity when used to detect acute disease and produces fewer cross-reactions. The sonicated immunoblot reported here is 94% sensitive to chronic bartonellosis and 70% sensitive to acute bartonellosis. In a healthy group, it is 100% specific. This immunoblot preparation requires a simple sonication protocol for the harvesting of B. bacilliformis antigens and is well suited for use in regions of endemicity. PMID:10618267

  1. Matryoshka locally resonant sonic crystal.

    PubMed

    Elford, Daniel P; Chalmers, Luke; Kusmartsev, Feodor V; Swallowe, Gerry M

    2011-11-01

    The results of numerical modeling of sonic crystals with resonant array elements are reported. The investigated resonant elements include plain slotted cylinders as well as their various combinations, in particular, Russian doll or Matryoshka configurations. The acoustic band structure and transmission characteristics of such systems have been computed with the use of finite element methods. The general concept of a locally resonant sonic crystal is proposed that utilizes acoustic resonances to form additional band gaps that are decoupled from Bragg gaps. An existence of a separate attenuation mechanism associated with the resonant elements that increases performance in the lower frequency regime has been identified. The results show a formation of broad band gaps positioned significantly below the first Bragg frequency. For low frequency broadband attenuation, a most optimal configuration is the Matryoshka sonic crystal, where each scattering unit is composed of multiple concentric slotted cylinders. This system forms numerous gaps in the lower frequency regime, below Bragg bands, while maintaining a reduced crystal size viable for noise barrier technology. The finding opens alternative perspectives for the construction of sound barriers in the low frequency range usually inaccessible by traditional means including conventional sonic crystals.

  2. Matryoshka locally resonant sonic crystal

    NASA Astrophysics Data System (ADS)

    Elford, Daniel P.; Chalmers, Luke; Kusmartsev, Feodor V.; Swallowe, Gerry M.

    The results of numerical modelling of sonic crystals with resonant array elements are reported. The investigated resonant elements include plain slotted cylinders as well as various their combinations, in particular, Russian doll or Matryoshka configurations. The acoustic band structure and transmission characteristics of such systems have been computed with the use of finite element methods. The general concept of a locally resonant sonic crystal is proposed, which utilises acoustic resonances to form additional band gaps that are decoupled from Bragg gaps. An existence of a separate attenuation mechanism associated with the resonant elements, which increases performance in the lower frequency regime has been identified. The results show a formation of broad band gaps positioned significantly below the first Bragg frequency. For low frequency broadband attenuation a most optimal configuration is the Matryoshka sonic crystal, where each scattering unit is composed of multiple concentric slotted cylinders. This system forms numerous gaps in the lower frequency regime, below Bragg bands, whilst maintaining a reduced crystal size viable for noise barrier technology. The finding opens new perspectives for construction of sound barriers in the low frequency range usually inaccessible by traditional means including conventional sonic crystals.

  3. Real Time Sonic Boom Display

    NASA Technical Reports Server (NTRS)

    Haering, Ed

    2014-01-01

    This presentation will provide general information about sonic boom mitigation technology to the public in order to supply information to potential partners and licensees. The technology is a combination of flight data, atmospheric data and terrain information implemented into a control room real time display for flight planning. This research is currently being performed and as such, any results and conclusions are ongoing.

  4. The Sonic Pathfinder: An Evaluation.

    ERIC Educational Resources Information Center

    Dodds, Allan G.; And Others

    1984-01-01

    An objective evaluation of the Sonic Pathfinder, a new ultrasonic mobility aid, showed that use of the aid changes mobility in many ways. Reduced perception of environmental sounds was not reflected in performance. The majority of users traveled slowly and exhibited less than optimal strategies. (Author/CL)

  5. Detergent-solubilized Patched purified from Sf9 cells fails to interact strongly with cognate Hedgehog or Ihog homologs.

    PubMed

    Cleveland, Thomas E; McCabe, Jacqueline M; Leahy, Daniel J

    2014-12-01

    Patched (Ptc) is a twelve-pass transmembrane protein that functions as a receptor for the Hedgehog (Hh) family of morphogens. In addition to Ptc, several accessory proteins including the CDO/Ihog family of co-receptors are necessary for proper Hh signaling. Structures of Hh proteins bound to members of the CDO/Ihog family are known, but the nature of the full Hh receptor complex is not well understood. We have expressed the Drosophila Patched and Mouse Patched-1 proteins in Sf9 cells and find that Sonic Hedgehog will bind to Mouse Patched-1 in isolated Sf9 cell membranes but that purified, detergent-solubilized Ptc proteins do not interact strongly with cognate Hh and CDO/Ihog homologs. These results may reflect a nonnative conformation of detergent-solubilized Ptc or that an additional factor or factors lost during purification are required for high-affinity Ptc binding to Hh.

  6. Evolutionary Genomics and Adaptive Evolution of the Hedgehog Gene Family (Shh, Ihh and Dhh) in Vertebrates

    PubMed Central

    Pereira, Joana; Johnson, Warren E.; O’Brien, Stephen J.; Jarvis, Erich D.; Zhang, Guojie; Gilbert, M. Thomas P.; Vasconcelos, Vitor; Antunes, Agostinho

    2014-01-01

    The Hedgehog (Hh) gene family codes for a class of secreted proteins composed of two active domains that act as signalling molecules during embryo development, namely for the development of the nervous and skeletal systems and the formation of the testis cord. While only one Hh gene is found typically in invertebrate genomes, most vertebrates species have three (Sonic hedgehog – Shh; Indian hedgehog – Ihh; and Desert hedgehog – Dhh), each with different expression patterns and functions, which likely helped promote the increasing complexity of vertebrates and their successful diversification. In this study, we used comparative genomic and adaptive evolutionary analyses to characterize the evolution of the Hh genes in vertebrates following the two major whole genome duplication (WGD) events. To overcome the lack of Hh-coding sequences on avian publicly available databases, we used an extensive dataset of 45 avian and three non-avian reptilian genomes to show that birds have all three Hh paralogs. We find suggestions that following the WGD events, vertebrate Hh paralogous genes evolved independently within similar linkage groups and under different evolutionary rates, especially within the catalytic domain. The structural regions around the ion-binding site were identified to be under positive selection in the signaling domain. These findings contrast with those observed in invertebrates, where different lineages that experienced gene duplication retained similar selective constraints in the Hh orthologs. Our results provide new insights on the evolutionary history of the Hh gene family, the functional roles of these paralogs in vertebrate species, and on the location of mutational hotspots. PMID:25549322

  7. Evolutionary genomics and adaptive evolution of the Hedgehog gene family (Shh, Ihh and Dhh) in vertebrates.

    PubMed

    Pereira, Joana; Johnson, Warren E; O'Brien, Stephen J; Jarvis, Erich D; Zhang, Guojie; Gilbert, M Thomas P; Vasconcelos, Vitor; Antunes, Agostinho

    2014-01-01

    The Hedgehog (Hh) gene family codes for a class of secreted proteins composed of two active domains that act as signalling molecules during embryo development, namely for the development of the nervous and skeletal systems and the formation of the testis cord. While only one Hh gene is found typically in invertebrate genomes, most vertebrates species have three (Sonic hedgehog--Shh; Indian hedgehog--Ihh; and Desert hedgehog--Dhh), each with different expression patterns and functions, which likely helped promote the increasing complexity of vertebrates and their successful diversification. In this study, we used comparative genomic and adaptive evolutionary analyses to characterize the evolution of the Hh genes in vertebrates following the two major whole genome duplication (WGD) events. To overcome the lack of Hh-coding sequences on avian publicly available databases, we used an extensive dataset of 45 avian and three non-avian reptilian genomes to show that birds have all three Hh paralogs. We find suggestions that following the WGD events, vertebrate Hh paralogous genes evolved independently within similar linkage groups and under different evolutionary rates, especially within the catalytic domain. The structural regions around the ion-binding site were identified to be under positive selection in the signaling domain. These findings contrast with those observed in invertebrates, where different lineages that experienced gene duplication retained similar selective constraints in the Hh orthologs. Our results provide new insights on the evolutionary history of the Hh gene family, the functional roles of these paralogs in vertebrate species, and on the location of mutational hotspots.

  8. Twist transition of nematic hyperbolic hedgehogs.

    PubMed

    James, Richard; Fukuda, Jun-ichi

    2014-04-01

    Stability of an idealized hyperbolic hedgehog in a nematic liquid crystal against a twist transition is investigated by extending the methodology of Rüdinger and Stark [Liq. Cryst. 26, 753 (1999)], where the hedgehog is confined between two concentric spheres. In the ideal hyperbolic-hedgehog the molecular orientation is assumed to rotate proportionally with respect to the inclination angle, θ (and in the opposite sense). However, when splay, k11, and bend, k33, moduli differ this proportionality is lost and the liquid crystal deforms relative to the ideal with bend and splay. Although slight, these deformations are shown to significantly shift the transition if k11/k33 is small. By increasing the degree of confinement the twist transition can be inhibited, a characteristic both hyperbolic and radial hedgehogs have in common. The twist transition of a hyperbolic defect that accompanies a particle is found to be well predicted by the earlier stability analysis of a thick shell.

  9. Ectoparasites of hedgehogs (Erinaceus concolor) from Turkey.

    PubMed

    Girisgin, Ahmet Onur; Senlik, Bayram; Aydin, Levent; Cirak, Veli Y

    2015-01-01

    Hedgehogs are small, nocturnal, spiny-coated animals that have been growing in popularity as exotic pets. However, these animals are host to a wide variety of viruses, bacteria, fungi and parasites, some of which are of zoonotic character. Thus, because hedgehogs have a potential role to transmit zoonoses including arthropod-borne diseases, we examined them for their ectoparasites. The study was carried out on hedgehogs found dead mainly due to road casualties in the Bursa province of Turkey. The ectoparasites were collected by both insecticide spraying of the body and inspection on a white paper carefully. Totally three species of ticks (Rhipicephalus sanguineus, Hyalomma aegyptium, Haemophysalis parvo) and one flea species (Archeopsylla erinacei) were detected. The prevalence of mixed infestation with both ticks and fleas was 45.5%. Haemaphysalis parva was reported for the first time from hedgehogs (Erinaceus concolor) in Turkey. The occurrence of ectoparasites and their potential role as vectors of certain zoonotic diseases are briefly discussed.

  10. Improved Approximate Profile Function of Hedgehog Skyrmion

    NASA Astrophysics Data System (ADS)

    Ji, Yong-Lin; Jia, Duo-Jie; Xi, Guo-Zhu; Liu, Feng

    The profile function for Skyrme model is investigated in Hedgehog ansatz. An improved analytical solution to the Hedgehog Skyrmion is obtained by using tentative function method. It is found that ensuing calculated static energy is smaller than that in Ref. 13, and the isoscalar electric mean square radius √ {< r2>}I=0 and the isoscalar magnetic mean square radius √ {< r2>}M,I=0 well agree with experiment results.

  11. The primary cilia, a 'Rab-id' transit system for hedgehog signaling.

    PubMed

    Oro, Anthony E

    2007-12-01

    Intense focus has been centered around how the primary cilia transduces the hedgehog (Hh) signal from smoothened (Smo) to the Gli transcription factors. New data indicate that ligand and signaling lipids help regulate small GTPase-dependent accumulation and activity of signaling components.

  12. Hedgehogs and retinal ganglion cells: organizers of the mammalian retina.

    PubMed

    Dakubo, Gabriel D; Wallace, Valerie A

    2004-03-01

    The mature vertebrate retina develops from a population of multipotential neural progenitor cells that give rise to all of the retinal neurons and one glial cell type. Retinal histogenesis is regulated, in part, by cell extrinsic cues. A growing number of studies now implicate signaling by members of the Hedgehog (Hh) family of morphogens in vertebrate retinal development. In this review we will discuss the role of Hh signals from retinal ganglion cells (RGCs), the projection neurons of the retina, on proliferation, differentiation and lamination in the neural retina.

  13. High Speed Research Program Sonic Fatigue

    NASA Technical Reports Server (NTRS)

    Rizzi, Stephen A. (Technical Monitor); Beier, Theodor H.; Heaton, Paul

    2005-01-01

    The objective of this sonic fatigue summary is to provide major findings and technical results of studies, initiated in 1994, to assess sonic fatigue behavior of structure that is being considered for the High Speed Civil Transport (HSCT). High Speed Research (HSR) program objectives in the area of sonic fatigue were to predict inlet, exhaust and boundary layer acoustic loads; measure high cycle fatigue data for materials developed during the HSR program; develop advanced sonic fatigue calculation methods to reduce required conservatism in airframe designs; develop damping techniques for sonic fatigue reduction where weight effective; develop wing and fuselage sonic fatigue design requirements; and perform sonic fatigue analyses on HSCT structural concepts to provide guidance to design teams. All goals were partially achieved, but none were completed due to the premature conclusion of the HSR program. A summary of major program findings and recommendations for continued effort are included in the report.

  14. GSK-3 is a master regulator of neural progenitor homeostasis

    PubMed Central

    Kim, Woo-Yang; Wang, Xinshuo; Wu, Yaohong; Doble, Bradley W; Patel, Satish; Woodgett, James R; Snider, William D

    2016-01-01

    The development of the brain requires the exquisite coordination of progenitor proliferation and differentiation to achieve complex circuit assembly. It has been suggested that glycogen synthase kinase 3 (GSK-3) acts as an integrating molecule for multiple proliferation and differentiation signals because of its essential role in the RTK, Wnt and Shh signaling pathways. We created conditional mutations that deleted both the α and β forms of GSK-3 in mouse neural progenitors. GSK-3 deletion resulted in massive hyperproliferation of neural progenitors along the entire neuraxis. Generation of both intermediate neural progenitors and postmitotic neurons was markedly suppressed. These effects were associated with the dysregulation of β-catenin, Sonic Hedgehog, Notch and fibroblast growth factor signaling. Our results indicate that GSK-3 signaling is an essential mediator of homeostatic controls that regulate neural progenitors during mammalian brain development. PMID:19801986

  15. Review of sonic fatigue technology

    NASA Technical Reports Server (NTRS)

    Clarkson, B. L.

    1994-01-01

    From the early-1960s until the mid-1980s, there was very little theoretical development for sonic fatigue prediction. Design nomographs based on simple theoretical models and results of specially designed tests were developed for most common aircraft structures. The use of advanced composites in the 1980s, however, generated an increased interest in development of more sophisticated theoretical models because of the possibilities for a much wider range of structural designs. The purpose of this report is to review sonic fatigue technology and, in particular, to assess recent developments. It also suggests a plan for a coordinated program of theoretical and experimental work to meet the anticipated needs of future aerospace vehicles.

  16. Georgia Tech sonic boom simulator

    NASA Astrophysics Data System (ADS)

    Ahuja, Krish K.

    1992-04-01

    To examine the building and human response to sonic boom in the range 3 Hz to 30 Hz, Georgia Institute of Technology is building a special acoustic driver system to simulate sonic boom. To support the NASA LaRC program on building and human response, this simulator's capability has been extended to an upper frequency of 4 KHz. A residential test house was made available by Georgia Tech for these tests. At the time of preparation of this document, most of the acoustic drivers and the associated electronics have been built and assembled. The system has, however, not been fully tested. The following pages provide an overview of the progress to date. The acoustic driver systems, and the principle of their operation together with the test house are described. Future plans are also summarized.

  17. Georgia Tech sonic boom simulator

    NASA Technical Reports Server (NTRS)

    Ahuja, Krish K.

    1992-01-01

    To examine the building and human response to sonic boom in the range 3 Hz to 30 Hz, Georgia Institute of Technology is building a special acoustic driver system to simulate sonic boom. To support the NASA LaRC program on building and human response, this simulator's capability has been extended to an upper frequency of 4 KHz. A residential test house was made available by Georgia Tech for these tests. At the time of preparation of this document, most of the acoustic drivers and the associated electronics have been built and assembled. The system has, however, not been fully tested. The following pages provide an overview of the progress to date. The acoustic driver systems, and the principle of their operation together with the test house are described. Future plans are also summarized.

  18. Counterexamples to the Sonic Criterion

    NASA Astrophysics Data System (ADS)

    Elling, Volker

    2009-12-01

    We consider self-similar (pseudo-steady) shock reflection at an oblique wall. There are three parameters: wall corner angle, Mach number, angle of incident shock. Ever since Ernst Mach discovered the irregular reflection named after him, researchers have sought to predict precisely for which parameters the reflection is regular. Three conflicting proposals—the detachment, sonic and von Neumann criteria—have been studied extensively without a clear result. We demonstrate that the sonic criterion is not correct. We consider polytropic potential flow and prove that there is an open nonempty set of parameters that admit a global regular reflection with a reflected shock that is transonic. We also provide a clear physical reason: the flow type (sub- or supersonic) is not decisive; instead the reflected shock type (weak or strong) determines whether structural perturbations decay towards the reflection point.

  19. 46,XY Gonadal Dysgenesis due to a Homozygous Mutation in Desert Hedgehog (DHH) Identified by Exome Sequencing

    PubMed Central

    Merz, Hartmut; Birnbaum, Wiebke; Marshall, Louise; Schröder, Tatjana; Reiz, Benedikt; Kavran, Jennifer M.; Bäumer, Tobias; Capetian, Philipp; Hiort, Olaf

    2015-01-01

    Background: 46,XY disorders of sex development (DSD) comprise a heterogeneous group of congenital conditions. Mutations in a variety of genes can affect gonadal development or androgen biosynthesis/action and thereby influence the development of the internal and external genital organs. Objective: The objective of the study was to identify the genetic cause in two 46,XY sisters of a consanguineous family with DSD and gonadal tumor formation. Methods: We used a next-generation sequencing approach by exome sequencing. Electrophysiological and high-resolution ultrasound examination of peripheral nerves as well as histopathological examination of the gonads were performed. Results: We identified a novel homozygous R124Q mutation in the desert hedgehog gene (DHH), which alters a conserved residue among the three mammalian Hedgehog ligands sonic hedgehog, Indian hedgehog, and desert hedgehog. No other relevant mutations in DSD-related genes were encountered. The gonads of one patient showed partial gonadal dysgenesis with loss of Leydig cells in tubular areas with seminoma in situ and a hyperplasia of Leydig cell-like cells expressing CYP17A1 in more dysgenetic parts of the gonad. In addition, both patients suffer from a polyneuropathy. High-resolution ultrasound revealed a structural change of peripheral nerve structure that fits well to a minifascicle formation of peripheral nerves. Conclusion: Mutations in DHH play a role in 46,XY gonadal dysgenesis and are associated with seminoma formation and a neuropathy with minifascicle formation. Gonadal dysgenesis in these cases may be due to impairment of Sertoli cell-Leydig cell interaction during gonadal development. PMID:25927242

  20. [Flea bites caused by Archaeopsylla erinacei, the hedgehog flea].

    PubMed

    Bork, K; Honomichl, K; Hoede, N

    1987-11-01

    A hedgehog flea was the cause of multiple flea bites in a 48-year-old patient. The main host of the hedgehog flea is the European hedgehog, but the flea was also found in different furry mammals, such as polecats, brown rats and foxes. It was not previously known that Archaeopsylla erinacei attacks man.

  1. Disruption of KIF3A in patient-derived glioblastoma cells: effects on ciliogenesis, hedgehog sensitivity, and tumorigenesis

    PubMed Central

    Hoang-Minh, Lan B.; Deleyrolle, Loic P.; Siebzehnrubl, Dorit; Ugartemendia, George; Futch, Hunter; Griffith, Benjamin; Breunig, Joshua J.; De Leon, Gabriel; Mitchell, Duane A.; Semple-Rowland, Susan; Reynolds, Brent A.; Sarkisian, Matthew R.

    2016-01-01

    KIF3A, a component of the kinesin-2 motor, is necessary for the progression of diverse tumor types. This is partly due to its role in regulating ciliogenesis and cell responsiveness to sonic hedgehog (SHH). Notably, primary cilia have been detected in human glioblastoma multiforme (GBM) tumor biopsies and derived cell lines. Here, we asked whether disrupting KIF3A in GBM cells affected ciliogenesis, in vitro growth and responsiveness to SHH, or tumorigenic behavior in vivo. We used a lentiviral vector to create three patient-derived GBM cell lines expressing a dominant negative, motorless form of Kif3a (dnKif3a). In all unmodified lines, we found that most GBM cells were capable of producing ciliated progeny and that dnKif3a expression in these cells ablated ciliogenesis. Interestingly, unmodified and dnKif3a-expressing cell lines displayed differential sensitivities and pathway activation to SHH and variable tumor-associated survival following mouse xenografts. In one cell line, SHH-induced cell proliferation was prevented in vitro by either expressing dnKif3a or inhibiting SMO signaling using cyclopamine, and the survival times of mice implanted with dnKif3a-expressing cells were increased. In a second line, expression of dnKif3a increased the cells' baseline proliferation while, surprisingly, sensitizing them to SHH-induced cell death. The survival times of mice implanted with these dnKif3a-expressing cells were decreased. Finally, expression of dnKif3a in a third cell line had no effect on cell proliferation, SHH sensitivity, or mouse survival times. These findings indicate that KIF3A is essential for GBM cell ciliogenesis, but its role in modulating GBM cell behavior is highly variable. PMID:26760767

  2. Disruption of KIF3A in patient-derived glioblastoma cells: effects on ciliogenesis, hedgehog sensitivity, and tumorigenesis.

    PubMed

    Hoang-Minh, Lan B; Deleyrolle, Loic P; Siebzehnrubl, Dorit; Ugartemendia, George; Futch, Hunter; Griffith, Benjamin; Breunig, Joshua J; De Leon, Gabriel; Mitchell, Duane A; Semple-Rowland, Susan; Reynolds, Brent A; Sarkisian, Matthew R

    2016-02-09

    KIF3A, a component of the kinesin-2 motor, is necessary for the progression of diverse tumor types. This is partly due to its role in regulating ciliogenesis and cell responsiveness to sonic hedgehog (SHH). Notably, primary cilia have been detected in human glioblastoma multiforme (GBM) tumor biopsies and derived cell lines. Here, we asked whether disrupting KIF3A in GBM cells affected ciliogenesis, in vitro growth and responsiveness to SHH, or tumorigenic behavior in vivo. We used a lentiviral vector to create three patient-derived GBM cell lines expressing a dominant negative, motorless form of Kif3a (dnKif3a). In all unmodified lines, we found that most GBM cells were capable of producing ciliated progeny and that dnKif3a expression in these cells ablated ciliogenesis. Interestingly, unmodified and dnKif3a-expressing cell lines displayed differential sensitivities and pathway activation to SHH and variable tumor-associated survival following mouse xenografts. In one cell line, SHH-induced cell proliferation was prevented in vitro by either expressing dn