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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. Dendrosomatic Sonic Hedgehog Signaling in Hippocampal Neurons Regulates Axon Elongation

    PubMed Central

    Petralia, Ronald S.; Ott, Carolyn; Wang, Ya-Xian; Lippincott-Schwartz, Jennifer; Mattson, Mark P.

    2015-01-01

    The presence of Sonic Hedgehog (Shh) and its signaling components in the neurons of the hippocampus raises a question about what role the Shh signaling pathway may play in these neurons. We show here that activation of the Shh signaling pathway stimulates axon elongation in rat hippocampal neurons. This Shh-induced effect depends on the pathway transducer Smoothened (Smo) and the transcription factor Gli1. The axon itself does not respond directly to Shh; instead, the Shh signal transduction originates from the somatodendritic region of the neurons and occurs in neurons with and without detectable primary cilia. Upon Shh stimulation, Smo localization to dendrites increases significantly. Shh pathway activation results in increased levels of profilin1 (Pfn1), an actin-binding protein. Mutations in Pfn1's actin-binding sites or reduction of Pfn1 eliminate the Shh-induced axon elongation. These findings indicate that Shh can regulate axon growth, which may be critical for development of hippocampal neurons. SIGNIFICANCE STATEMENT Although numerous signaling mechanisms have been identified that act directly on axons to regulate their outgrowth, it is not known whether signals transduced in dendrites may also affect axon outgrowth. We describe here a transcellular signaling pathway in embryonic hippocampal neurons in which activation of Sonic Hedgehog (Shh) receptors in dendrites stimulates axon growth. The pathway involves the dendritic-membrane-associated Shh signal transducer Smoothened (Smo) and the transcription factor Gli, which induces the expression of the gene encoding the actin-binding protein profilin 1. Our findings suggest scenarios in which stimulation of Shh in dendrites results in accelerated outgrowth of the axon, which therefore reaches its presumptive postsynaptic target cell more quickly. By this mechanism, Shh may play critical roles in the development of hippocampal neuronal circuits. PMID:26658865

  7. 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.

  8. 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

  9. Sonic hedgehog and the molecular regulation of mouse neural tube closure.

    PubMed

    Ybot-Gonzalez, Patricia; Cogram, Patricia; Gerrelli, Dianne; Copp, Andrew J

    2002-05-01

    Neural tube closure is a fundamental embryonic event whose molecular regulation is poorly understood. As mouse neurulation progresses along the spinal axis, there is a shift from midline neural plate bending to dorsolateral bending. Here, we show that midline bending is not essential for spinal closure since, in its absence, the neural tube can close by a 'default' mechanism involving dorsolateral bending, even at upper spinal levels. Midline and dorsolateral bending are regulated by mutually antagonistic signals from the notochord and surface ectoderm. Notochordal signaling induces midline bending and simultaneously inhibits dorsolateral bending. Sonic hedgehog is both necessary and sufficient to inhibit dorsolateral bending, but is neither necessary nor sufficient to induce midline bending, which seems likely to be regulated by another notochordal factor. Attachment of surface ectoderm cells to the neural plate is required for dorsolateral bending, which ensures neural tube closure in the absence of sonic hedgehog signaling.

  10. Epithelial heparan sulfate regulates Sonic Hedgehog signaling in lung development.

    PubMed

    He, Hua; Huang, Meina; Sun, Shenfei; Wu, Yihui; Lin, Xinhua

    2017-08-01

    The tree-like structure of the mammalian lung is generated from branching morphogenesis, a reiterative process that is precisely regulated by numerous factors. How the cell surface and extra cellular matrix (ECM) molecules regulate this process is still poorly understood. Herein, we show that epithelial deletion of Heparan Sulfate (HS) synthetase Ext1 resulted in expanded branching tips and reduced branching number, associated with several mesenchymal developmental defects. We further demonstrate an expanded Fgf10 expression and increased FGF signaling activity in Ext1 mutant lungs, suggesting a cell non-autonomous mechanism. Consistent with this, we observed reduced levels of SHH signaling which is responsible for suppressing Fgf10 expression. Moreover, reactivating SHH signaling in mutant lungs rescued the tip dilation phenotype and attenuated FGF signaling. Importantly, the reduced SHH signaling activity did not appear to be caused by decreased Shh expression or protein stability; instead, biologically active form of SHH proteins were reduced in both the Ext1 mutant epithelium and surrounding wild type mesenchymal cells. Together, our study highlights the epithelial HS as a key player for dictating SHH signaling critical for lung morphogenesis.

  11. Epithelial heparan sulfate regulates Sonic Hedgehog signaling in lung development

    PubMed Central

    Sun, Shenfei; Wu, Yihui; Lin, Xinhua

    2017-01-01

    The tree-like structure of the mammalian lung is generated from branching morphogenesis, a reiterative process that is precisely regulated by numerous factors. How the cell surface and extra cellular matrix (ECM) molecules regulate this process is still poorly understood. Herein, we show that epithelial deletion of Heparan Sulfate (HS) synthetase Ext1 resulted in expanded branching tips and reduced branching number, associated with several mesenchymal developmental defects. We further demonstrate an expanded Fgf10 expression and increased FGF signaling activity in Ext1 mutant lungs, suggesting a cell non-autonomous mechanism. Consistent with this, we observed reduced levels of SHH signaling which is responsible for suppressing Fgf10 expression. Moreover, reactivating SHH signaling in mutant lungs rescued the tip dilation phenotype and attenuated FGF signaling. Importantly, the reduced SHH signaling activity did not appear to be caused by decreased Shh expression or protein stability; instead, biologically active form of SHH proteins were reduced in both the Ext1 mutant epithelium and surrounding wild type mesenchymal cells. Together, our study highlights the epithelial HS as a key player for dictating SHH signaling critical for lung morphogenesis. PMID:28859094

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. [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.

  17. 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.

  18. 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.

  19. 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

  20. 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. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. De-regulation of the sonic hedgehog pathway in the InsGas mouse model of gastric carcinogenesis

    PubMed Central

    El-Zaatari, M; Tobias, A; Grabowska, A M; Kumari, R; Scotting, P J; Kaye, P; Atherton, J; Clarke, P A; Powe, D G; Watson, S A

    2007-01-01

    This study investigated sonic hedgehog (Shh) signalling in gastric metaplasia in the insulin-gastrin (InsGas) hypergastrinaemic mouse +/− Helicobacter felis (H. felis) infection. Sonic hedgehog gene and protein expression was reduced in pre-metaplastic lesions from non-infected mice (90% gene reduction, P<0.01) compared to normal mucosa. Sonic hedgehog was reactivated in gastric metaplasia of H. felis-infected mice (3.5-fold increase, P<0.01) compared to pre-metaplastic lesions. Additionally, the Shh target gene, glioma-associated oncogene (Gli)-1, was significantly reduced in the gastric glands of InsGas mice (75% reduction, P<0.05) and reactivated with H. felis infection (P<0.05, base of glands, P<0.01 stroma of metaplastic glands). The ability of H. felis to activate the Shh pathway was investigated by measuring the effect of target cytokine, interleukin-8 (IL-8), on Shh expression in AGS and MGLVA1 cells, which was shown to induce Shh expression at physiological concentrations. H. felis induced the expression of NF-κB in inflammatory infiltrates in vivo, and the expression of the IL-8 mouse homologue, protein KC, in inflammatory infiltrates and metaplastic lesions. Sonic hedgehog pathway reactivation was paralleled with an increase in proliferation of metaplastic lesions (15.75 vs 4.39% in infected vs non-infected mice, respectively, P<0.001). Furthermore, Shh overexpression increased the growth rate of the gastric cancer cell line, AGS. The antiapoptotic protein, bcl-2, was expressed in the stroma of infected mice, along with a second Shh target gene, patched-1 (P=0.0001, stroma of metaplastic gland). This study provides evidence suggesting reactivation of Shh signalling from pre-metaplastic to advanced metaplastic lesions of the stomach and outlines the importance of the Shh pathway as a potential chemoprophylactic target for gastric carcinogenesis. PMID:17505514

  13. 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

  14. Sonic hedgehog regulation of Foxf2 promotes cranial neural crest mesenchyme proliferation and is disrupted in cleft lip morphogenesis.

    PubMed

    Everson, Joshua L; Fink, Dustin M; Yoon, Joon Won; Leslie, Elizabeth J; Kietzman, Henry W; Ansen-Wilson, Lydia J; Chung, Hannah M; Walterhouse, David O; Marazita, Mary L; Lipinski, Robert J

    2017-06-01

    Cleft lip is one of the most common human birth defects, yet our understanding of the mechanisms that regulate lip morphogenesis is limited. Here, we show in mice that sonic hedgehog (Shh)-induced proliferation of cranial neural crest cell (cNCC) mesenchyme is required for upper lip closure. Gene expression profiling revealed a subset of Forkhead box (Fox) genes that are regulated by Shh signaling during lip morphogenesis. During cleft pathogenesis, reduced proliferation in the medial nasal process mesenchyme paralleled the domain of reduced Foxf2 and Gli1 expression. SHH ligand induction of Foxf2 expression was dependent upon Shh pathway effectors in cNCCs, while a functional GLI-binding site was identified downstream of Foxf2 Consistent with the cellular mechanism demonstrated for cleft lip pathogenesis, we found that either SHH ligand addition or FOXF2 overexpression is sufficient to induce cNCC proliferation. Finally, analysis of a large multi-ethnic human population with cleft lip identified clusters of single-nucleotide polymorphisms in FOXF2 These data suggest that direct targeting of Foxf2 by Shh signaling drives cNCC mesenchyme proliferation during upper lip morphogenesis, and that disruption of this sequence results in cleft lip. © 2017. Published by The Company of Biologists Ltd.

  15. 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

  16. 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.

  17. 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

  18. 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.

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

    PubMed

    Perdigoto, Carolina N; Dauber, Katherine L; Bar, Carmit; Tsai, Pai-Chi; Valdes, Victor J; Cohen, Idan; Santoriello, Francis J; Zhao, Dejian; Zheng, Deyou; Hsu, Ya-Chieh; Ezhkova, Elena

    2016-07-01

    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.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. Recruitment of the Sonic hedgehog signalling cascade in electroconvulsive seizure-mediated regulation of adult rat hippocampal neurogenesis

    PubMed Central

    Banerjee, Sunayana B.; Rajendran, Rajeev; Dias, Brian G.; Ladiwala, Uma; Tole, Shubha; Vaidya, Vidita A.

    2007-01-01

    Electroconvulsive seizure (ECS) induces structural remodelling in the adult mammalian brain, including an increase in adult hippocampal neurogenesis. The molecular mechanisms that underlie this increase in the proliferation of adult hippocampal progenitors are at present not well understood. We hypothesized that ECS may recruit the Sonic hedgehog (Shh) pathway to mediate its effects on adult hippocampal neurogenesis, as Shh is known to enhance the proliferation of neuronal progenitors and is expressed in the adult basal forebrain, a region that sends robust projections to the hippocampus. Here we demonstrate that the ECS-induced increase in proliferation of adult hippocampal progenitors was completely blocked in animals treated with cyclopamine, a pharmacological inhibitor of Shh signalling. Our results suggest that both acute and chronic ECS enhance Shh signalling in the adult hippocampus, as we observed a robust upregulation of Patched (Ptc) mRNA, a component of the Shh receptor complex and a downstream transcriptional target of Shh signalling. This increase was rapid and restricted to the dentate gyrus, where the adult hippocampal progenitors reside. In addition, both acute and chronic ECS decreased Smoothened (Smo) mRNA, the other component of the Shh receptor complex, selectively within the dentate gyrus. However, ECS did not appear to influence Shh expression within the basal forebrain, the site from which it has been suggested to be anterogradely transported to the hippocampus. Together, our findings demonstrate that ECS regulates the Shh signalling cascade and indicate that the Shh pathway may be an important mechanism through which ECS enhances adult hippocampal neurogenesis. PMID:16197497

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. A Sonic hedgehog coreceptor, BOC regulates neuronal differentiation and neurite outgrowth via interaction with ABL and JNK activation.

    PubMed

    Vuong, Tuan Anh; Leem, Young-Eun; Kim, Bok-Geon; Cho, Hana; Lee, Sang-Jin; Bae, Gyu-Un; Kang, Jong-Sun

    2017-01-01

    Neurite outgrowth is a critical step for neurogenesis and remodeling synaptic circuitry during neuronal development and regeneration. An immunoglobulin superfamily member, BOC functions as Sonic hedgehog (Shh) coreceptor in canonical and noncanonical Shh signaling in neuronal development and axon outgrowth/guidance. However signaling mechanisms responsible for BOC action during these processes remain unknown. In our previous studies, a multiprotein complex containing BOC and a closely related protein CDO promotes myogenic differentiation through activation of multiple signaling pathways, including non-receptor tyrosine kinase ABL. Given that ABL and Jun. N-terminal kinase (JNK) are implicated in actin cytoskeletal dynamics required for neurogenesis, we investigated the relationship between BOC, ABL and JNK during neuronal differentiation. Here, we demonstrate that BOC and ABL are induced in P19 embryonal carcinoma (EC) cells and cortical neural progenitor cells (NPCs) during neuronal differentiation. BOC-depleted EC cells or Boc(-/-) NPCs exhibit impaired neuronal differentiation with shorter neurite formation. BOC interacts with ABL through its putative SH2 binding domain and seems to be phosphorylated in an ABL activity-dependent manner. Unlike wildtype BOC, ABL-binding defective BOC mutants exhibit impaired JNK activation and neuronal differentiation. Finally, Shh treatment enhances JNK activation which is diminished by BOC depletion. These data suggest that BOC interacts with ABL and activates JNK thereby promoting neuronal differentiation and neurite outgrowth. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. Function of Brg1 Chromatin Remodeling Factor in Sonic Hedgehog-Dependent Medulloblastoma Initiation and Maintenance

    DTIC Science & Technology

    2014-10-01

    Remodeling Factor in Sonic Hedgehog -Dependent Medulloblastoma Initiation and Maintenance PRINCIPAL INVESTIGATOR: Xuanming Shi CONTRACTING...Function of Brg1 Chromatin Remodeling Factor in Sonic Hedgehog -Dependent 5b. GRANT NUMBER W81XWH-12-1-0527 Medulloblastoma Initiation and Maintenance...medulloblastoma. 15. SUBJECT TERMS Medulloblastoma, Sonic Hedgehog , Chromatin remodeling, BAF complex, Brg1, mouse model of shh-subtype medulloblastoma

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. Sonic hedgehog-Dependent Induction of MicroRNA 31 and MicroRNA 150 Regulates Mycobacterium bovis BCG-Driven Toll-Like Receptor 2 Signaling

    PubMed Central

    Ghorpade, Devram Sampat; Holla, Sahana; Kaveri, Srini V.; Bayry, Jagadeesh; Patil, Shripad A.

    2013-01-01

    Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-α) secretion by macrophages was essential for robust SHH activation, as TNF-α−/− macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-α or blockade of TNF-α receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions. PMID:23166298

  8. 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

  9. 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.

  10. 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

  11. Metformin suppresses sonic hedgehog expression in pancreatic cancer cells.

    PubMed

    Nakamura, Masafumi; Ogo, Ayako; Yamura, Masahiro; Yamaguchi, Yoshiyuki; Nakashima, Hiroshi

    2014-04-01

    Metformin use has previously been associated with decreased cancer risk. The Hedgehog signaling pathway is a well-characterized early and late mediator of pancreatic cancer oncogenesis. The aim of the present study was to clarify the effect of metformin on factors involved in Hedgehog signaling. BxPC3 human pancreatic cancer cells were treated with metformin, and Sonic hedgehog (Shh) mRNA and protein levels were examined by real time reverse transcription-polymerase chain reaction, immunohistochemistry and immunoblotting, respectively. The effect of metformin on Shh levels was also examined in three other cancer cell lines. Shh protein and mRNA expression was suppressed by metformin in BxPC3 cells. This phenomenon was further confirmed in three other cancer cell lines. Shh mRNA expression was inhibited by metformin in a concentration-dependent manner in two cancer cell lines. Metformin reduces the expression of Shh in several cancer cell lines including pancreatic cancer cell.

  12. 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.

  13. Sonic hedgehog acts at multiple stages during pancreatic tumorigenesis

    PubMed Central

    Morton, Jennifer P.; Mongeau, Michelle E.; Klimstra, David S.; Morris, John P.; Lee, Yie Chia; Kawaguchi, Yoshiya; Wright, Christopher V. E.; Hebrok, Matthias; Lewis, Brian C.

    2007-01-01

    Activation of sonic hedgehog (Shh) signaling occurs in the majority of pancreatic ductal adenocarcinomas. Here we investigate the mechanisms by which Shh contributes to pancreatic tumorigenesis. We find that Shh expression enhances proliferation of pancreatic duct epithelial cells, potentially through the transcriptional regulation of the cell cycle regulators cyclin D1 and p21. We further show that Shh protects pancreatic duct epithelial cells from apoptosis through the activation of phosphatidylinositol 3-kinase signaling and the stabilization of Bcl-2 and Bcl-XL. Significantly, Shh also cooperates with activated K-Ras to promote pancreatic tumor development. Finally, Shh signaling enhances K-Ras-induced pancreatic tumorigenesis by reducing the dependence of tumor cells on the sustained activation of the MAPK and phosphatidylinositol 3-kinase/Akt/mTOR signaling pathways. Thus, our data suggest that Shh signaling contributes to tumor initiation in the pancreas through at least two mechanisms and additionally enhances tumor cell resistance to therapeutic intervention. Collectively, our findings demonstrate crucial roles for Shh signaling in multiple stages of pancreatic carcinogenesis. PMID:17372229

  14. 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.

  15. 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.

  16. 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.

  17. Paradox of sonic hedgehog (SHH) transcriptional regulation: Alternative transcription initiation overrides the effect of downstream promoter DNA methylation.

    PubMed

    ten Haaf, Anette; Franken, Laura; Heymann, Caroline; von Serenyi, Sonja; Cornelissen, Christian; de Hoon, Joep P J; Veeck, Jürgen; Lüscher, Bernhard; Knüchel, Ruth; Dahl, Edgar

    2011-04-01

    Recently, DNA methylation has been suggested as a potential mechanism involved in the transcriptional regulation of SHH gene expression in cancer. However, detailed analyses on the underlying transcriptional mechanisms of SHH expression have not been presented so far and were therefore the focus of this study. We found that the genomic region of SHH contains two different transcriptional start sites and four CpG islands spread from the 5' promoter region to the 3' end of the SHH gene. Based on this CpG island topology we analyzed the influence of DNA methylation within the promoter region as well as in exon 2 and exon 3 on SHH mRNA expression in a large set (n = 14) of benign and malignant human cell lines, and further elucidated the functionality of the two identified SHH transcription initiation sites. Methylation-specific PCR (MSP) clearly showed that SHH is expressed independently of DNA methylation within exon 2 and exon 3 of its genomic region, while methylation of the promoter region is able to abrogate SHH expression. Most interesting, we found activation of the upstream SHH promoter in several breast cancer cell lines when the downstream SHH promoter is methylated. These observations lead us to propose a transcriptional model for the SHH gene, in which combined mechanisms of DNA methylation and alternative promoter usage coordinate the transcriptional activity of this important developmental gene.

  18. 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

  19. Sonic Hedgehog Signalling Pathway and Ameloblastoma – A Review

    PubMed Central

    Mishra, Pallavi; Bandyopadhyay, Alokenath; Kumar, Harish; Mohiddin, Gouse

    2015-01-01

    Ameloblastoma is a benign but aggressive odontogenic neoplasm arising from odontogenic epithelium. Many theories have been proposed to explain the pathogenesis of ameloblatoma. Numerous signalling pathways have been implicated to be associated in the development and progression of this neoplasm. Studies have found association of various signalling molecules of Sonic Hedgehog Pathway, namely SHH, PTCH1, SMO, Gli 1, Gli 2, Gli 3, with ameloblastoma. Knowledge about this pathway will help us to understand the nature and behaviour of this neoplasm. This will open the door towards new treatment modalities. PMID:26674664

  20. Sonic Hedgehog-GLI Family Zinc Finger 1 Signaling Pathway Promotes the Growth and Migration of Pancreatic Cancer Cells by Regulating the Transcription of Eukaryotic Translation Initiation Factor 5A2.

    PubMed

    Xu, Xuanfu; Liu, Hua; Zhang, Hui; Dai, Weiqi; Guo, Chuanyong; Xie, Chuangao; Wei, Shumei; He, Shengli; Xu, Xiaorong

    2015-11-01

    The Hh (hedgehog) signaling pathway is still waiting for further studies because its downstream molecular mechanism remains elusive. Because EIF5A2 (eukaryotic translation initiation factor 5A2) gene was up-regulated upon Gli1 (GLI family zinc finger 1) in pancreatic cancer (PC) cells, we speculated that this pathway might promote tumor progression through regulating EIF5A2. We investigated regulation effect of Hh signaling pathway to EIF5A2 gene transcription by Gli1 knockdown or overexpression in PC cell lines first. Then, the regulation mechanism of Gli1 to EIF5A2 gene was studied at transcription level. Finally, we studied cancer-promoting effects of Gli1-dependent EIF5A2 in PC cells. The data showed that Gli1 up-regulated expression of EIF5A2 by promoting transcription via cis-acting elements in PC cells. Moreover, vimentin gene was up-regulated significantly by sonic hedgehog (SHh)/Gli1 expression increasing, and E-cadherin was significantly reduced. The EIF5A2 knockdown partially reversed cell proliferation and migration induced by artificial SHh overexpression and inhibited epithelial mesenchymal transition process in PC cells with SHh overexpression (P < 0.05). Our data establish a novel transcription mechanism of Gli1 to EIF5A2 gene in cis-regulatory manner in PC cells. Thus, EIF5A2 oncogene effect could be incorporated into cancer-promoting molecular network upon Hh signaling pathway.

  1. 14-3-3 proteins regulate a cell-intrinsic switch from sonic hedgehog-mediated commissural axon attraction to repulsion after midline crossing.

    PubMed

    Yam, Patricia T; Kent, Christopher B; Morin, Steves; Farmer, W Todd; Alchini, Ricardo; Lepelletier, Léa; Colman, David R; Tessier-Lavigne, Marc; Fournier, Alyson E; Charron, Frédéric

    2012-11-21

    Axons must switch responsiveness to guidance cues during development for correct pathfinding. Sonic Hedgehog (Shh) attracts spinal cord commissural axons ventrally toward the floorplate. We show that after crossing the floorplate, commissural axons switch their response to Shh from attraction to repulsion, so that they are repelled anteriorly by a posterior-high/anterior-low Shh gradient along the longitudinal axis. This switch is recapitulated in vitro with dissociated commissural neurons as they age, indicating that the switch is intrinsic and time dependent. 14-3-3 protein inhibition converted Shh-mediated repulsion of aged dissociated neurons to attraction and prevented the correct anterior turn of postcrossing commissural axons in vivo, an effect mediated through PKA. Conversely, overexpression of 14-3-3 proteins was sufficient to drive the switch from Shh-mediated attraction to repulsion both in vitro and in vivo. Therefore, we identify a 14-3-3 protein-dependent mechanism for a cell-intrinsic temporal switch in the polarity of axon turning responses. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. 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

  3. Sonic hedgehog expression during early tooth development in Suncus murinus.

    PubMed

    Miyado, Mami; Ogi, Hidenao; Yamada, Gen; Kitoh, Junzo; Jogahara, Takamichi; Oda, Sen-Ichi; Sato, Iwao; Miyado, Kenji; Sunohara, Masataka

    2007-11-16

    Tooth development is a highly organized process characterized by reciprocal interactions between epithelium and mesenchyme. However, the expression patterns and functions of molecules involved in mouse tooth development are unclear from the viewpoint of explaining human dental malformations and anomalies. Here, we show the expression of sonic hedgehog (Shh), a potent initiator of morphogenesis, during the early stages of tooth development in Suncus murinus. Initially, symmetrical, elongated expression of suncus Shh (sShh) was observed in the thin layer of dental epithelial cells along the mesial-distal axis of both jaws. As the dental epithelium continued to develop, sShh was strictly restricted to the predicted leading parts of the growing, invaginating epithelium corresponding to tooth primordia and enamel knots. We propose that some aspects of Shh function in tooth development are widely conserved in mammalian phylogeny.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. Pseudo-active sites of protease domains: HGF/Met and Sonic hedgehog signaling in cancer.

    PubMed

    Maun, Henry R; Kirchhofer, Daniel; Lazarus, Robert A

    2010-08-01

    Proteases represent a large class of enzymes with crucial biological functions. Although targeting various relevant proteases for therapeutic intervention has been widely investigated, structurally related proteins lacking proteolytic activity (pseudo-proteases) have received relatively little attention. Two distinct clinically relevant cancer pathways that contain signaling proteins with pseudo-protease domains include the Met and Hedgehog (Hh) pathways. The receptor tyrosine kinase Met pathway is driven by hepatocyte growth factor (HGF), a plasminogen-related ligand that binds Met and activates intracellular pathways resulting in cell proliferation, angiogenesis, motility and survival. HGF is a disulfide-linked alpha/beta-heterodimer having a trypsin serine protease-like beta-chain. The Hh pathway is driven by Sonic hedgehog (Shh), which has a Zn(2+) metalloprotease fold and binds Patched1 (Ptc1), which de-represses Smoothened and ultimately activates Gli-dependent transcription. Although HGF and Shh differ in structure and function, the pseudo-catalytic sites of both HGF and Shh are crucial for signal transduction. For HGF, this region binds the Met beta-propeller domain, which leads to Met dimerization and signaling. For Hh, this region binds to the antagonist receptor Hedgehog-interacting protein (Hhip) and most probably to Ptc1 as well. Thus, for both HGF and Hh pathways, targeting ligand pseudo-active sites represents a new strategy for regulation.

  15. 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. Copyright © 2017 the authors 0270-6474/17/371685-11$15.00/0.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. Design and Evolution of a Macrocyclic Peptide Inhibitor of the Sonic Hedgehog/Patched Interaction.

    PubMed

    Owens, Andrew E; de Paola, Ivan; Hansen, William A; Liu, Yi-Wen; Khare, Sagar D; Fasan, Rudi

    2017-09-13

    The hedgehog (Hh) signaling pathway plays a central role during embryonic development, and its aberrant activation has been implicated in the development and progression of several human cancers. Major efforts toward the identification of chemical modulators of the hedgehog pathway have yielded several antagonists of the GPCR-like smoothened receptor. In contrast, potent inhibitors of the sonic hedgehog/patched interaction, the most upstream event in ligand-induced activation of this signaling pathway, have been elusive. To address this gap, a genetically encoded cyclic peptide was designed based on the sonic hedgehog (Shh)-binding loop of hedgehog-interacting protein (HHIP) and subjected to multiple rounds of affinity maturation through the screening of macrocyclic peptide libraries produced in E. coli cells. Using this approach, an optimized macrocyclic peptide inhibitor (HL2-m5) was obtained that binds Shh with a KD of 170 nM, which corresponds to a 120-fold affinity improvement compared to the parent molecule. Importantly, HL2-m5 is able to effectively suppress Shh-mediated hedgehog signaling and Gli-controlled gene transcription in living cells (IC50 = 230 nM), providing the most potent inhibitor of the sonic hedgehog/patched interaction reported to date. This first-in-class macrocyclic peptide modulator of the hedgehog pathway is expected to provide a valuable probe for investigating and targeting ligand-dependent hedgehog pathway activation in cancer and other pathologies. This work also introduces a general strategy for the development of cyclopeptide inhibitors of protein-protein interactions.

  1. 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

  2. Sonic hedgehog pathway activation increases mitochondrial abundance and activity in hippocampal neurons

    PubMed Central

    Yao, Pamela J.; Manor, Uri; Petralia, Ronald S.; Brose, Rebecca D.; Wu, Ryan T. Y.; Ott, Carolyn; Wang, Ya-Xian; Charnoff, Ari; Lippincott-Schwartz, Jennifer; Mattson, Mark P.

    2017-01-01

    Mitochondria are essential organelles whose biogenesis, structure, and function are regulated by many signaling pathways. We present evidence that, in hippocampal neurons, activation of the Sonic hedgehog (Shh) signaling pathway affects multiple aspects of mitochondria. Mitochondrial mass was increased significantly in neurons treated with Shh. Using biochemical and fluorescence imaging analyses, we show that Shh signaling activity reduces mitochondrial fission and promotes mitochondrial elongation, at least in part, via suppression of the mitochondrial fission protein dynamin-like GTPase Drp1. Mitochondria from Shh-treated neurons were more electron-dense, as revealed by electron microscopy, and had higher membrane potential and respiratory activity. We further show that Shh protects neurons against a variety of stresses, including the mitochondrial poison rotenone, amyloid β-peptide, hydrogen peroxide, and high levels of glutamate. Collectively our data suggest a link between Shh pathway activity and the physiological properties of mitochondria in hippocampal neurons. PMID:27932496

  3. Sonic hedgehog acts cell-autonomously on muscle precursor cells to generate limb muscle diversity

    PubMed Central

    Anderson, Claire; Williams, Victoria C.; Moyon, Benjamin; Daubas, Philippe; Tajbakhsh, Shahragim; Buckingham, Margaret E.; Shiroishi, Toshihiko; Hughes, Simon M.; Borycki, Anne-Gaëlle

    2012-01-01

    How muscle diversity is generated in the vertebrate body is poorly understood. In the limb, dorsal and ventral muscle masses constitute the first myogenic diversification, as each gives rise to distinct muscles. Myogenesis initiates after muscle precursor cells (MPCs) have migrated from the somites to the limb bud and populated the prospective muscle masses. Here, we show that Sonic hedgehog (Shh) from the zone of polarizing activity (ZPA) drives myogenesis specifically within the ventral muscle mass. Shh directly induces ventral MPCs to initiate Myf5 transcription and myogenesis through essential Gli-binding sites located in the Myf5 limb enhancer. In the absence of Shh signaling, myogenesis is delayed, MPCs fail to migrate distally, and ventral paw muscles fail to form. Thus, Shh production in the limb ZPA is essential for the spatiotemporal control of myogenesis and coordinates muscle and skeletal development by acting directly to regulate the formation of specific ventral muscles. PMID:22987640

  4. Sonic hedgehog promotes stem-cell potential of Mueller glia in the mammalian retina

    SciTech Connect

    Wan Jin; Zheng Hua; Xiao Honglei; She Zhenjue; Zhou Guomin

    2007-11-16

    Mueller glia have been demonstrated to display stem-cell properties after retinal damage. Here, we report this potential can be regulated by Sonic hedgehog (Shh) signaling. Shh can stimulate proliferation of Mueller glia through its receptor and target gene expressed on them, furthermore, Shh-treated Mueller glia are induced to dedifferentiate by expressing progenitor-specific markers, and then adopt cell fate of rod photoreceptor. Inhibition of signaling by cyclopamine inhibits proliferation and dedifferentiation. Intraocular injection of Shh promotes Mueller glia activation in the photoreceptor-damaged retina, Shh also enhances neurogenic potential by producing more rhodopsin-positive photoreceptors from Mueller glia-derived cells. Together, these results provide evidences that Mueller glia act as potential stem cells in mammalian retina, Shh may have therapeutic effects on these cells for promoting the regeneration of retinal neurons.

  5. The Hedgehog signaling pathway in ovarian teratoma is stimulated by Sonic Hedgehog which induces internalization of Patched.

    PubMed

    Sabol, Maja; Car, Diana; Musani, Vesna; Ozretic, Petar; Oreskovic, Slavko; Weber, Igor; Levanat, Sonja

    2012-10-01

    The Hedgehog-Gli (Hh-Gli) signaling pathway was examined in ovarian dermoids, which show characteristics of both tumors and developmental malformations. Dermoids are classified as mature teratomas that present differentiation into various tissues, mostly epidermal elements such as glands, multilayered epithelium, hair follicles and occasionally bone and cartilage. Their development is attributed to aberrant meiosis of germinal cells within the ovary. We showed activation of the Hh-Gli signaling in ovarian dermoid primary cultures. Cyclopamine treatment slows down cell proliferation, while the Sonic Hedgehog (Shh) protein stimulates cell proliferation and induces internalization of the Patched (Ptch) protein, which accumulates in the form of granules in the cytoplasm, colocalized with the Shh protein. Cyclopamine treatment decreases Gli1 localization in the nucleus compared to non-treated cells. Based on our observations, the mechanism of Hedgehog activation in the ovarian dermoids could be the ligand-dependent autocrine pathway, which can also be stimulated by paracrine signals.

  6. 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.

  7. 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,…

  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. Zinc-dependent structural stability of human Sonic hedgehog.

    PubMed

    Day, E S; Wen, D; Garber, E A; Hong, J; Avedissian, L S; Rayhorn, P; Shen, W; Zeng, C; Bailey, V R; Reilly, J O; Roden, J A; Moore, C B; Williams, K P; Galdes, A; Whitty, A; Baker, D P

    1999-11-09

    The role of the zinc site in the N-terminal fragment of human Sonic hedgehog (ShhN) was explored by comparing the biophysical and functional properties of wild-type ShhN with those of mutants in which the zinc-coordinating residues H140, D147, and H182, or E176 which interacts with the metal ion via a bridging water molecule, were mutated to alanine. The wild-type and E176A mutant proteins retained 1 mol of zinc/mol of protein after extensive dialysis, whereas the H140A and D147A mutants retained only 0.03 and 0.05 mol of zinc/mol of protein, respectively. Assay of the wild-type and mutant proteins in two activity assays indicated that the wild-type and E176A mutant proteins had similar activity, whereas the H140A and D147A mutants were significantly less active. These assays also indicated that the H140A and D147A mutants were susceptible to proteolysis. CD, fluorescence, and (1)H NMR spectra of the H140A, D147A, and E176A mutants measured at 20 or 25 degrees C were very similar to those observed for wild-type ShhN. However, CD measurements at 37 degrees C showed evidence of some structural differences in the H140A and D147A mutants. Guanidine hydrochloride (GuHCl) denaturation studies revealed that the loss of zinc from the H140A and D147A mutants destabilized the folded proteins by approximately 3.5 kcal/mol, comparable to the effect of removing zinc from wild-type ShhN by treatment with EDTA. Thermal melting curves of wild-type ShhN gave a single unfolding transition with a midpoint T(m) of approximately 59 degrees C, whereas both the H140A and D147A mutants displayed two distinct transitions with T(m) values of 37-38 and 52-54 degrees C, similar to that observed for EDTA-treated wild-type ShhN. Addition of zinc to the H140A and D147A mutants resulted in a partial restoration of stability against thermal and GuHCl denaturation. The ability of these mutants to bind zinc was confirmed using a fluorescence-based binding assay that indicated that they bound zinc

  11. Identification of a family of fatty acid-speciated Sonic Hedgehog proteins, whose members display differential biological properties

    PubMed Central

    Houel, Stephane; Rodgriguez-Blanco, Jezabel; Singh, Samer; Schilling, Neal; J.Capobianco, Anthony; Ahn, Natalie G.; Robbins, David J.

    2015-01-01

    SUMMARY Hedgehog (HH) proteins are proteolytically processed into a biologically active form, which is covalently modified by cholesterol and palmitate. However, most studies of HH biogenesis have characterized protein from cells in which HH is over-expressed. We purified Sonic Hedgehog (SHH) from cells expressing physiologically relevant levels, and showed that it was more potent than SHH isolated from over-expressing cells. Furthermore, the SHH in our preparations were modified with a diverse spectrum of fatty acids on their amino-termini, and this spectrum of fatty acids varied dramatically depending on the growth conditions of the cells. The fatty acid composition of SHH affected its trafficking to lipid rafts, as well as its potency. Our results suggest that HH proteins exist as a family of diverse lipid-speciated proteins, which might be altered in different physiological and pathological contexts to regulate distinct properties of HH proteins. PMID:25732819

  12. Identification of a family of fatty-acid-speciated sonic hedgehog proteins, whose members display differential biological properties.

    PubMed

    Long, Jun; Tokhunts, Robert; Old, William M; Houel, Stephane; Rodgriguez-Blanco, Jezabel; Singh, Samer; Schilling, Neal; J Capobianco, Anthony; Ahn, Natalie G; Robbins, David J

    2015-03-03

    Hedgehog (HH) proteins are proteolytically processed into a biologically active form that is covalently modified by cholesterol and palmitate. However, most studies of HH biogenesis have characterized protein from cells in which HH is overexpressed. We purified Sonic Hedgehog (SHH) from cells expressing physiologically relevant levels and showed that it was more potent than SHH isolated from overexpressing cells. Furthermore, the SHH in our preparations was modified with a diverse spectrum of fatty acids on its amino termini, and this spectrum of fatty acids varied dramatically depending on the growth conditions of the cells. The fatty acid composition of SHH affected its trafficking to lipid rafts as well as its potency. Our results suggest that HH proteins exist as a family of diverse lipid-speciated proteins that might be altered in different physiological and pathological contexts in order to regulate distinct properties of HH proteins. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  13. 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.

  14. 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

  15. Alcohol Reduces Arterial Remodeling by Inhibiting Sonic Hedgehog-Stimulated Sca1(+) Progenitor Stem Cell Expansion.

    PubMed

    Fitzpatrick, Emma; Han, Xu; Liu, Weimin; Corcoran, Eoin; Burtenshaw, Denise; Alshamrani, Maryam; Morrow, David; Helt, Jay-Christian; Cahill, Paul A; Redmond, Eileen M

    2017-09-18

    Cell and molecular mechanisms mediating the cardiovascular effects of alcohol are not fully understood. Our aim was to determine the effect of moderate Ethanol (EtOH) on Sonic Hedgehog (SHh) signaling in regulating possible Sca1(+) progenitor stem cell involvement during pathologic arterial remodeling. Partial ligation or sham-operation of the left carotid artery was performed in transgenic Sca1-eGFP mice gavaged with or without 'daily moderate' EtOH. The EtOH group had reduced adventitial thickening and less neo-intimal formation, compared to ligated controls. There was expansion of eGFP expressing (i.e., Sca1(+) ) cells in remodeled vessels post-ligation (14d), especially in the neo-intima. Ethanol treatment reduced the number of Sca1(+) cells in ligated vessel cross-sections concomitant with diminished remodeling, compared to control ligated vessels. Moreover, EtOH attenuated SHh signaling in injured carotids as determined by immunohistochemical analysis of the target genes patched 1 (Ptch1) and Gli2, and RT-PCR of whole vessel Gli2 mRNA levels. Intraperitoneal injection of ligated Sca1 - eGFP mice with the SHh signaling inhibitor cyclopamine diminished hedgehog target gene expression, reduced the number of Sca1(+) cells, and ameliorated carotid remodeling. EtOH treatment of purified Sca1(+) adventitial progenitor stem cells in vitro inhibited SHh signaling, and their rSHh-induced differentiation to vascular smooth muscle cells. EtOH reduces SHh - responsive Sca1(+) progenitor cell myogenic differentiation/expansion in vitro and during arterial remodeling in response to ligation injury in vivo. Regulation of vascular Sca1(+) progenitor cells in this way may be an important novel mechanism contributing to alcohol's cardiovascular protective effects. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. 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.

  17. 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

  18. 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.

  19. Quiescent Sox2+ Cells Drive Hierarchical Growth and Relapse in Sonic Hedgehog Subgroup Medulloblastoma

    PubMed Central

    Vanner, Robert J.; Remke, Marc; Gallo, Marco; Selvadurai, Hayden J.; Coutinho, Fiona; Lee, Lilian; Kushida, Michelle; Head, Renee; Morrissy, Sorana; Zhu, Xueming; Aviv, Tzvi; Voisin, Veronique; Clarke, Ian D.; Li, Yisu; Mungall, Andrew J.; Moore, Richard A.; Ma, Yussanne; Jones, Steven J.M.; Marra, Marco A.; Malkin, David; Northcott, Paul A.; Kool, Marcel; Pfister, Stefan M.; Bader, Gary; Hochedlinger, Konrad; Korshunov, Andrey; Taylor, Michael D.; Dirks, Peter B.

    2015-01-01

    SUMMARY Functional heterogeneity within tumors presents a significant therapeutic challenge. Here we show that quiescent, therapy-resistant Sox2+ cells propagate sonic hedgehog subgroup medulloblastoma by a mechanism that mirrors a neurogenic program. Rare Sox2+ cells produce rapidly cycling doublecortin+ progenitors that, together with their postmitotic progeny expressing NeuN, comprise tumor bulk. Sox2+ cells are enriched following anti-mitotic chemotherapy and Smoothened inhibition, creating a reservoir for tumor regrowth. Lineage traces from Sox2+ cells increase following treatment, suggesting that this population is responsible for relapse. Targeting Sox2+ cells with the antineoplastic mithramycin abrogated tumor growth. Addressing functional heterogeneity and eliminating Sox2+ cells presents a promising therapeutic paradigm for treatment of sonic hedgehog subgroup medulloblastoma. PMID:24954133

  20. Quiescent sox2(+) cells drive hierarchical growth and relapse in sonic hedgehog subgroup medulloblastoma.

    PubMed

    Vanner, Robert J; Remke, Marc; Gallo, Marco; Selvadurai, Hayden J; Coutinho, Fiona; Lee, Lilian; Kushida, Michelle; Head, Renee; Morrissy, Sorana; Zhu, Xueming; Aviv, Tzvi; Voisin, Veronique; Clarke, Ian D; Li, Yisu; Mungall, Andrew J; Moore, Richard A; Ma, Yussanne; Jones, Steven J M; Marra, Marco A; Malkin, David; Northcott, Paul A; Kool, Marcel; Pfister, Stefan M; Bader, Gary; Hochedlinger, Konrad; Korshunov, Andrey; Taylor, Michael D; Dirks, Peter B

    2014-07-14

    Functional heterogeneity within tumors presents a significant therapeutic challenge. Here we show that quiescent, therapy-resistant Sox2(+) cells propagate sonic hedgehog subgroup medulloblastoma by a mechanism that mirrors a neurogenic program. Rare Sox2(+) cells produce rapidly cycling doublecortin(+) progenitors that, together with their postmitotic progeny expressing NeuN, comprise tumor bulk. Sox2(+) cells are enriched following anti-mitotic chemotherapy and Smoothened inhibition, creating a reservoir for tumor regrowth. Lineage traces from Sox2(+) cells increase following treatment, suggesting that this population is responsible for relapse. Targeting Sox2(+) cells with the antineoplastic mithramycin abrogated tumor growth. Addressing functional heterogeneity and eliminating Sox2(+) cells presents a promising therapeutic paradigm for treatment of sonic hedgehog subgroup medulloblastoma. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. 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

  2. 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.

  3. 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.

  4. Immunohistochemical analysis of Sonic hedgehog signalling in normal human urinary tract development

    PubMed Central

    Jenkins, Dagan; Winyard, Paul J D; Woolf, Adrian S

    2007-01-01

    Studies of mouse mutants have demonstrated that Sonic hedgehog (SHH) signalling has a functional role in morphogenesis and differentiation at multiple sites within the forming urinary tract, and urinary tract malformations have been reported in humans with mutations that disrupt SHH signalling. However, there is only strikingly sparse and fragmentary information about the expression of SHH and associated signalling genes in normal human urinary tract development. We used immunohistochemistry to demonstrate that SHH protein was localised in distinct urinary tract epithelia in developing normal humans, in the urothelium of the nascent bladder and in kidney medullary collecting ducts. The expression patterns of the SHH-transducing proteins Patched (PTCH) and Smoothened (SMO) were consistent with long-range paracrine signalling associated with detrusor smooth muscle differentiation in the urogenital sinus. In the developing kidney, SHH and PTCH were expressed in epithelia of the collecting system between 16–26 weeks – surprisingly, SMO was not detected. Analysis of cell proliferation and Cyclin B1 immunohistochemistry at 26 weeks, as compared with a 28 week sample in which SHH expression was down-regulated, was consistent with the idea that SHH and PTCH might influence medullary collecting duct growth by regulating the subcellular localisation of Cyclin B1 independently of SMO. Collectively, these descriptive results generate new hypotheses regarding SHH signal transduction in human urinary tract development and help to explain the varied urinary tract malformation phenotypes noted in individuals with mutations in the SHH pathway. PMID:17850284

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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

    2017-04-01

    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. Copyright © 2017 John Wiley & Sons, Ltd.

  10. 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.

  11. Sonic hedgehog pathway activation increases mitochondrial abundance and activity in hippocampal neurons.

    PubMed

    Yao, Pamela J; Manor, Uri; Petralia, Ronald S; Brose, Rebecca D; Wu, Ryan T Y; Ott, Carolyn; Wang, Ya-Xian; Charnoff, Ari; Lippincott-Schwartz, Jennifer; Mattson, Mark P

    2017-02-01

    Mitochondria are essential organelles whose biogenesis, structure, and function are regulated by many signaling pathways. We present evidence that, in hippocampal neurons, activation of the Sonic hedgehog (Shh) signaling pathway affects multiple aspects of mitochondria. Mitochondrial mass was increased significantly in neurons treated with Shh. Using biochemical and fluorescence imaging analyses, we show that Shh signaling activity reduces mitochondrial fission and promotes mitochondrial elongation, at least in part, via suppression of the mitochondrial fission protein dynamin-like GTPase Drp1. Mitochondria from Shh-treated neurons were more electron-dense, as revealed by electron microscopy, and had higher membrane potential and respiratory activity. We further show that Shh protects neurons against a variety of stresses, including the mitochondrial poison rotenone, amyloid β-peptide, hydrogen peroxide, and high levels of glutamate. Collectively our data suggest a link between Shh pathway activity and the physiological properties of mitochondria in hippocampal neurons. © 2017 Yao et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Diminished Sonic hedgehog signaling and lack of floor plate differentiation in Gli2 mutant mice.

    PubMed

    Ding, Q; Motoyama, J; Gasca, S; Mo, R; Sasaki, H; Rossant, J; Hui, C C

    1998-07-01

    Floor plate cells at the midline of the neural tube are specified by high-level activity of Sonic hedgehog (Shh) secreted by notochord, whereas motor neurons are thought to be specified by a lower level activity of Shh secreted in turn by floor plate cells. In Drosophila, the Gli zinc finger protein Cubitus interruptus functions as a transcription factor activating Hedgehog-responsive genes. We report that the expression of known Shh-responsive genes such as Ptc and Gli1 is downregulated in mutant mice lacking Gli2 function. Gli2 mutants fail to develop a floor plate yet still develop motor neurons, which occupy the ventral midline of the neural tube. Our results imply that Gli2 is required to mediate high level but not low level Shh activity and show that the development of motor neurons can occur in the absence of floor plate induction.

  6. Development of stratum intermedium and its role as a Sonic hedgehog-signaling structure during odontogenesis.

    PubMed

    Koyama, E; Wu, C; Shimo, T; Iwamoto, M; Ohmori, T; Kurisu, K; Ookura, T; Bashir, M M; Abrams, W R; Tucker, T; Pacifici, M

    2001-10-01

    Stratum intermedium is a transient and subtle epithelial structure closely associated with inner dental epithelium in tooth germs. Little is known about its development and roles. To facilitate analysis, we used bovine tooth germs, predicting that they may contain a more conspicuous stratum intermedium. Indeed, early bell stage bovine tooth germs already displayed an obvious stratum intermedium with a typical multilayered organization and flanking the enamel knot. Strikingly, with further development, the cuspally located stratum intermedium underwent thinning and involution, whereas a multilayered stratum intermedium formed at successive sites along the cusp-to-cervix axis of odontogenesis. In situ hybridization and immunohistochemistry showed that stratum intermedium produces the signaling molecule Sonic hedgehog (Shh). Maximal Shh expression was invariably seen in its thickest multilayered portions. Shh was also produced by inner dental epithelium; expression was not constant but varied with development and cytodifferentiation of ameloblasts along the cusp-to-cervix axis. Interestingly, maximal Shh expression in inner dental epithelium did not coincide with that in stratum intermedium. Both stratum intermedium and inner dental epithelium expressed the Shh receptor Patched2 (Ptch2), an indication of autocrine signaling loops. Shh protein, but not RNA, was present in underlying dental mesenchyme, probably resulting from gradual diffusion from epithelial layers and reflecting paracrine loops of action. To analyze the regulation of Shh expression, epithelial and mesenchymal layers were separated and maintained in organ culture. Shh expression decreased over time, but was maintained in unoperated specimens. Our data show for the first time that stratum intermedium is a highly regulated and Shh-expressing structure. Given its dynamic and apparently interactive properties, stratum intermedium may help orchestrate progression of odontogenesis from cusp to cervix

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. The Acid-Secreting Parietal Cell as an Endocrine Source of Sonic Hedgehog During Gastric Repair

    PubMed Central

    Engevik, Amy C.; Feng, Rui; Yang, Li

    2013-01-01

    Sonic Hedgehog (Shh) has been shown to regulate wound healing in various tissues. Despite its known function in tissue regeneration, the role of Shh secreted from the gastric epithelium during tissue repair in the stomach remains unknown. Here we tested the hypothesis that Shh secreted from the acid-secreting parietal cell is a fundamental circulating factor that drives gastric repair. A mouse model expressing a parietal cell-specific deletion of Shh (PC-ShhKO) was generated using animals bearing loxP sites flanking exon 2 of the Shh gene (Shhflx/flx) and mice expressing a Cre transgene under the control of the H+,K+-ATPase β-subunit promoter. Shhflx/flx, the H+,K+-ATPase β-subunit promoter, and C57BL/6 mice served as controls. Ulcers were induced via acetic acid injury. At 1, 2, 3, 4, 5, and 7 days after the ulcer induction, gastric tissue and blood samples were collected. Parabiosis experiments were used to establish the effect of circulating Shh on ulcer repair. Control mice exhibited an increased expression of Shh in the gastric tissue and plasma that correlated with the repair of injury within 7 days after surgery. PC-ShhKO mice showed a loss of ulcer repair and reduced Shh tissue and plasma concentrations. In a parabiosis experiment whereby a control mouse was paired with a PC-ShhKO littermate and both animals subjected to gastric injury, a significant increase in the circulating Shh was measured in both parabionts. Elevated circulating Shh concentrations correlated with the repair of gastric ulcers in the PC-ShhKO parabionts. Therefore, the acid-secreting parietal cell within the stomach acts as an endocrine source of Shh during repair. PMID:24092639

  15. 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

  16. 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.

  17. Expression of the sonic hedgehog pathway molecules in synchronous follicular adenoma and papillary carcinoma of the thyroid gland in predicting malignancy.

    PubMed

    Nelson, Kirbylee K; Gattuso, Paolo; Xu, Xiulong; Prinz, Richard A

    2010-10-01

    Recent studies have shown that the Sonic Hedgehog pathway plays an important role in tumorigenesis and cancer proliferation. The Sonic Hedgehog pathway is required for normal thyroid gland development, but when activated as a result of gene mutation or overexpression, it may stimulate thyroid tumor cell proliferation. This study determines whether 3 molecules, Patched, Smoothened, and Sonic Hedgehog, involved in the Sonic Hedgehog pathway are overexpressed equally in synchronous follicular thyroid adenoma and papillary thyroid carcinoma. Eighteen patients with synchronous follicular thyroid adenoma and papillary thyroid carcinoma underwent thyroidectomy. Immunohistochemistry was performed on the paraffin-embedded tissue to detect expression of Patched, Smoothened, and Sonic Hedgehog in both tumor types. The expression in these neoplasms was graded by 2 observers. Five patients had insufficient tumor tissue and were removed from the analysis. Patched expression was detected in 5 of 13 (38%) follicular adenomas and 5 of 12 (42%) papillary carcinomas. Smoothened was expressed in 4 of 13 (31%) follicular adenomas and 3 of 13 (23%) papillary carcinomas. Sonic Hedgehog was expressed in 4 of 13 (31%) follicular adenomas and 11 of 13 (85%) papillary carcinomas. Expression of the 3 molecules involved in the Sonic Hedgehog pathway was similar in follicular thyroid adenoma, but Sonic Hedgehog expression was a more sensitive indicator of malignancy in papillary thyroid carcinoma. The Sonic Hedgehog molecule may become a diagnostic marker when the cytologic or histologic features are not characteristic of a papillary carcinoma. Greater understanding of the Sonic Hedgehog pathway may provide molecular methods for preventing or treating papillary thyroid carcinoma. Copyright © 2010 Mosby, Inc. All rights reserved.

  18. 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.

  19. 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

  20. Sonic hedgehog pathway dysregulation in skin basal-cell carcinoma of a Polish population.

    PubMed

    Lesiak, Aleksandra; Sobolewska-Sztychny, Dorota; Danilewicz, Marian; Rogowski-Tylman, Michal; Sysa-Jedrzejowska, Anna; Sobjanek, Michal; Olejniczak-Staruch, Irmina; Narbutt, Joanna

    2013-01-01

    Sonic hedgehog (Shh) pathway impairment plays a key role in the pathogenesis of basal-cell carcinomas (BCC), the most frequent skin tumor among Caucasians. Shh, Smo, and Gli2 family proteins are necessary for adequate and controlled cell proliferation. The aim of this study was to evaluate Shh, Smo, and Smo expression in BCC skin biopsies taken from sun-exposed areas. 41 BCC skin biopsies and 22 healthy skin specimens (the control group) taken from the same areas served as material for the study. All specimens were immunohistochemically stained with monoclonal antibodies directed against the chosen proteins. Shh and Smo expression (cytoplasmic pattern) were recorded semiquantitatively using a four-grade score (0-3). Gli2 expression (nuclear pattern) was determined using an image analysis system (semiautomatic function). The immunoexpression of the Shh and Smo proteins significantly increased in the BCC group, as compared with the normal controls (for Shh, the mean intensity was 1.67 in BCC vs. 1.17 in the control group, p < 0.001; for Smo, the mean intensity was 1.46 in BCC vs. 0.99 in the control group, p < 0.001). The staining for Gli2 in the BCC group was completely negative, but indicated the presence of Gli2 in the control patients (1.15 Gli2+ cells/100 cells). Sonic hedgehog pathway dysregulation may play an important role in skin cancerogenesis leading to BCC development.

  1. Helicobacter pylori-induced Sonic Hedgehog expression is regulated by NFκB pathway activation: the use of a novel in vitro model to study epithelial response to infection.

    PubMed

    Schumacher, Michael A; Feng, Rui; Aihara, Eitaro; Engevik, Amy C; Montrose, Marshall H; Ottemann, Karen M; Zavros, Yana

    2015-02-01

    Helicobacter pylori (H. pylori) infection leads to acute induction of Sonic Hedgehog (Shh) in the stomach that is associated with the initiation of gastritis. The mechanism by which H. pylori induces Shh is unknown. Shh is a target gene of transcription factor Nuclear Factor-κB (NFκB). We hypothesize that NFκB mediates H. pylori-induced Shh. To visualize Shh ligand expression in response to H. pylori infection in vivo, we used a mouse model that expresses Shh fused to green fluorescent protein (Shh::GFP mice) in place of wild-type Shh. In vitro, changes in Shh expression were measured in response to H. pylori infection using 3-dimensional epithelial cell cultures grown from whole dissociated gastric glands (organoids). Organoids were generated from stomachs collected from the fundic region of control and mice expressing a parietal cell-specific deletion of Shh (PC-Shh(KO) mice). Within 2 days of infection, H. pylori induced Shh expression within parietal cells of Shh::GFP mice. Organoids expressed all major gastric cell markers, including parietal cell marker H(+) ,K(+) -ATPase and Shh. H. pylori infection of gastric organoids induced Shh expression; a response that was blocked by inhibiting NFκB signaling and correlated with IκB degradation. H. pylori infection of PC-Shh(KO) mouse-derived organoids did not result in the induction of Shh expression. Gastric organoids allow for the study of the interaction between H. pylori and the differentiated gastric epithelium independent of the host immune response. H. pylori induces Shh expression from the parietal cells, a response mediated via activation of NFκB signaling. © 2014 John Wiley & Sons Ltd.

  2. 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.

  3. 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

  4. 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

  5. Association analysis between the tag SNP for sonic hedgehog rs9333613 polymorphism and male sexual orientation.

    PubMed

    Wang, Binbin; Zhou, Sirui; Hong, Fuchang; Wang, Jing; Liu, Xiaoli; Cai, Yumao; Wang, Feng; Feng, Tiejian; Ma, Xu

    2012-01-01

    Male sexual orientation has been proposed to have genetic components, but previously suggested candidate genes have all received negative results. The human sonic hedgehog (SHH) gene is located in the 7q36 region, which was linked to male sexual orientation in a previous genome-wide association study. SHH is known to play an important role in embryo patterning, and there is evidence connecting it to sexual orientation. In this study, we performed an association analysis of the SHH tag single nucleotide polymorphism rs9333613 in 361 subjects and 319 Chinese male controls. We find a significant difference in genotype and allele distribution between identified homosexuals and heterosexual control subjects, suggesting that the SHH gene could potentially be associated with male sexual orientation.

  6. 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

  7. 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

  8. Controlled Delivery of Sonic Hedgehog Morphogen and Its Potential for Cardiac Repair

    PubMed Central

    Johnson, Noah Ray; Wang, Yadong

    2013-01-01

    The morphogen Sonic hedgehog (Shh) holds great promise for repair or regeneration of tissues suffering ischemic injury, however clinical translation is limited by its short half-life in the body. Here, we describe a coacervate delivery system which incorporates Shh, protects it from degradation, and sustains its release for at least 3 weeks. Shh released from the coacervate stimulates cardiac fibroblasts to upregulate the expression of multiple trophic factors including VEGF, SDF-1α, IGF-1, and Shh itself, for at least 48 hours. Shh coacervate also demonstrates cytoprotective effects for cardiomyocytes in a hydrogen peroxide-induced oxidative stress environment. In each of these studies the bioactivity of the Shh coacervate is enhanced compared to free Shh. These results warrant further investigation of the in vivo efficacy of Shh coacervate for cardiac repair. PMID:23690982

  9. 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.

  10. 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

  11. Using mechanistic Bayesian networks to identify downstream targets of the sonic hedgehog pathway.

    PubMed

    Shah, Abhik; Tenzen, Toyoaki; McMahon, Andrew P; Woolf, Peter J

    2009-12-18

    The topology of a biological pathway provides clues as to how a pathway operates, but rationally using this topology information with observed gene expression data remains a challenge. We introduce a new general-purpose analytic method called Mechanistic Bayesian Networks (MBNs) that allows for the integration of gene expression data and known constraints within a signal or regulatory pathway to predict new downstream pathway targets. The MBN framework is implemented in an open-source Bayesian network learning package, the Python Environment for Bayesian Learning (PEBL). We demonstrate how MBNs can be used by modeling the early steps of the sonic hedgehog pathway using gene expression data from different developmental stages and genetic backgrounds in mouse. Using the MBN approach we are able to automatically identify many of the known downstream targets of the hedgehog pathway such as Gas1 and Gli1, along with a short list of likely targets such as Mig12. The MBN approach shown here can easily be extended to other pathways and data types to yield a more mechanistic framework for learning genetic regulatory models.

  12. Comparative Analysis of Mutational Profile of Sonic hedgehog Gene in Gallbladder Cancer.

    PubMed

    Dixit, Ruhi; Pandey, Manoj; Tripathi, Sunil Kumar; Dwivedi, Amit Nandan Dhar; Shukla, Vijay Kumar

    2017-03-01

    Gallbladder cancer has high incidence in northeastern India; mortality too is high as the disease is often diagnosed late. Numerous studies have shown the role of sonic hedgehog (shh) in different cancers, an important ligand of the hedgehog signaling pathway. This study was carried out to evaluate the shh gene mutations in gallbladder cancer patients. PCR-SSCP was performed for shh gene in 50 samples each of gallbladder cancer, cholelithiasis, and control. The samples showing aberration in banding pattern were sequenced. Variation in banding pattern was observed in 20% gallbladder cancer cases, 10% in cholelithiasis, and none of the control (χ (2) = 11.111; p < 0.05). Sequencing results revealed seven novel point mutations in GBC cases. These novel mutations were found to be associated with histopathology (p < 0.05) and stage (p < 0.05) of gallbladder cancer. This study reveals several novel individual and repetitive mutations of shh gene in GBC and cholelithiasis samples that may be used as diagnostic markers for gallbladder carcinogenesis.

  13. Sonic hedgehog functions as a mitogen during bell stage of odontogenesis.

    PubMed

    Wu, Changshan; Shimo, Tsuyoshi; Liu, Mufei; Pacifici, Maurizio; Koyama, Eiki

    2003-01-01

    Epithelial-mesenchymal interactions are required for tissue growth and gene expression patterns during odontogenesis. We showed previously that Sonic hedgehog (SHH) is detectable in both dental epithelium and mesenchyme, while Shh transcripts are present in dental epithelium only, suggesting that SHH functions as an autocrine signal in epithelium and a paracrine signal in mesenchyme. This hypothesis was tested here. We found by in situ hybridization that the SHH autocrine receptor Ptch-2 is indeed expressed in dental epithelium whereas the paracrine receptor Ptc is expressed in mesenchyme. Bovine bell stage tooth germs were microsurgically separated into epithelial and mesenchymal portions and the resulting tissue fragments were organ-cultured. In epithelium fragments cultured by themselves, gene expression of Shh and Gli-1 (a putative transcriptional mediator of hedgehog signaling) was significantly decreased in both inner dental epithelium and stratum intermedium layers; this was accompanied by a sharp drop in epithelial cell proliferation. However, in companion control tissue fragments containing both epithelium and mesenchyme, Shh and Gli-1 expression as well as cell proliferation were maintained. Treatment of dental epithelial or mesenchymal cell populations in monolayer cultures with exogenous recombinant SHH stimulated cell proliferation. Together, the data provide clear evidence that Shh is synthesized by dental epithelium, reaches the underlying mesenchyme, and appears to act as an autocrine mitogen for epithelial cells and a paracrine mitogen for mesenchymal cells, thus exerting crucial functions in tooth germ growth, morphogenesis, and tissue-tissue interactions of bell stage of odontogenesis.

  14. Holoprosencephaly in RSH/Smith-Lemli-Opitz syndrome: Does abnormal cholesterol metabolism affect the function of sonic hedgehog?

    SciTech Connect

    Kelley, R.I.; Roessler, E.; Muenke, M.

    1996-12-30

    The RAH/Smith-Lemli-Opitz syndrome (RAH/SLOS) is an autosomal recessive malformation syndrome associated with increased levels of 7-dehydrocholesterol (7-DHC) and a defect of cholesterol biosynthesis at the level of 3{beta}-hydroxy-steroid-{Delta}{sup 7}-reductase (7-DHC reductase). Because rats exposed to inhibitors of 7-DHC reductase during development have a high frequency of holoprosencephaly (HPE), we have undertaken a search for biochemical evidence of RSH/SLOS and other possible defects of sterol metabolism among patients with various forms of HPE. We describe 4 patients, one with semilobar HPE and three others with less complete forms of the HPE sequence, in whom we have made a biochemical diagnosis of RAH/SLOS. The clinical and biochemical spectrum of these and other patients with RAH/SLOS suggests a role of abnormal sterol metabolism in the pathogenesis of their malformations. The association of HPE and RAH/SLOS is discussed in light of the recent discoveries that mutations in the embryonic patterning gene, Sonic Hedgehog (SHH), can cause HPE in humans and that the sonic hedgehog protein product undergoes autoproteolysis to form a cholesterol-modified active product. These clinical, biochemical, and molecular studies suggest that HPE and other malformations in SLOS may be caused by incomplete or abnormal modification of the sonic hedgehog protein and, possibly, other patterning proteins of the hedgehog class, a hypothesis testable in somatic cell systems. 37 refs., 1 fig.

  15. 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

  16. 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.

  17. 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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:26859575

  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. Transposon mutagenesis with coat color genotyping identifies an essential role for Skor2 in sonic hedgehog signaling and cerebellum development

    PubMed Central

    Wang, Baiping; Harrison, Wilbur; Overbeek, Paul A.; Zheng, Hui

    2011-01-01

    Correct development of the cerebellum requires coordinated sonic hedgehog (Shh) signaling from Purkinje to granule cells. How Shh expression is regulated in Purkinje cells is poorly understood. Using a novel tyrosinase minigene-tagged Sleeping Beauty transposon-mediated mutagenesis, which allows for coat color-based genotyping, we created mice in which the Ski/Sno family transcriptional co-repressor 2 (Skor2) gene is deleted. Loss of Skor2 leads to defective Purkinje cell development, a severe reduction of granule cell proliferation and a malformed cerebellum. Skor2 is specifically expressed in Purkinje cells in the brain, where it is required for proper expression of Shh. Skor2 overexpression suppresses BMP signaling in an HDAC-dependent manner and stimulates Shh promoter activity, suggesting that Skor2 represses BMP signaling to activate Shh expression. Our study identifies an essential function for Skor2 as a novel transcriptional regulator in Purkinje cells that acts upstream of Shh during cerebellum development. PMID:21937600

  1. 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.

  2. 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

  3. 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.

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

    PubMed Central

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

    2008-01-01

    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. PMID:19004755

  8. 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.

  9. Sonic Hedgehog Produced by Bone Marrow-Derived Mesenchymal Stromal Cells Supports Cell Survival in Myelodysplastic Syndrome

    PubMed Central

    Zou, Jixue; Hong, Yan; Tong, Yin; Wei, Ju; Qin, Youwen; Shao, Shan; Wang, Chun; Zhou, Kun

    2015-01-01

    The role of marrow microenvironment in the pathogenesis of myelodysplastic syndrome (MDS) remains controversial. Therefore, we studied the influence of bone marrow-derived mesenchymal stromal cells (BMSCs) from patients with different risk types of MDS on the survival of the MDS cell lines SKM-1 and MUTZ-1. We first demonstrated that the expression of Sonic hedgehog (Shh), smoothened (Smo), and glioma-associated oncogene homolog 1 (Gli1) was increased in MDS patients (n = 23); the increase in expression was positively correlated with the presence of high-risk factors. The Shh signaling inhibitor, cyclopamine, inhibited high-risk MDS BMSC-induced survival of SKM-1 and MUTZ-1 cells, suggesting a role for Shh signaling in MDS cell survival. Furthermore, cyclopamine-mediated inhibition of Shh signaling in SKM-1 and MUTZ-1 cells resulted in decreased DNMT1 expression and cell survival; however, exogenous Shh peptide had the opposite effect, suggesting that Shh signaling could regulate the expression of DNMT1, thereby modulating cell survival in MDS. In addition, the apoptosis of SKM-1 and MUTZ-1 cell increased significantly when cultured with cyclopamine and a demethylation agent, 5-Aza-2′-deoxycytidine. These findings suggest that Shh signaling from BMSCs is important in the pathogenesis of MDS and could play a role in disease progression by modulating methylation. PMID:25861282

  10. Variant allele frequency enrichment analysis in vitro reveals sonic hedgehog pathway to impede sustained temozolomide response in GBM.

    PubMed

    Biswas, Nidhan K; Chandra, Vikas; Sarkar-Roy, Neeta; Das, Tapojyoti; Bhattacharya, Rabindra N; Tripathy, Laxmi N; Basu, Sunandan K; Kumar, Shantanu; Das, Subrata; Chatterjee, Ankita; Mukherjee, Ankur; Basu, Pryiadarshi; Maitra, Arindam; Chattopadhyay, Ansuman; Basu, Analabha; Dhara, Surajit

    2015-01-21

    Neoplastic cells of Glioblastoma multiforme (GBM) may or may not show sustained response to temozolomide (TMZ) chemotherapy. We hypothesize that TMZ chemotherapy response in GBM is predetermined in its neoplastic clones via a specific set of mutations that alter relevant pathways. We describe exome-wide enrichment of variant allele frequencies (VAFs) in neurospheres displaying contrasting phenotypes of sustained versus reversible TMZ-responses in vitro. Enrichment of VAFs was found on genes ST5, RP6KA1 and PRKDC in cells showing sustained TMZ-effect whereas on genes FREM2, AASDH and STK36, in cells showing reversible TMZ-effect. Ingenuity pathway analysis (IPA) revealed that these genes alter cell-cycle, G2/M-checkpoint-regulation and NHEJ pathways in sustained TMZ-effect cells whereas the lysine-II&V/phenylalanine degradation and sonic hedgehog (Hh) pathways in reversible TMZ-effect cells. Next, we validated the likely involvement of the Hh-pathway in TMZ-response on additional GBM neurospheres as well as on GBM patients, by extracting RNA-sequencing-based gene expression data from the TCGA-GBM database. Finally, we demonstrated TMZ-sensitization of a TMZ non-responder neurosphere in vitro by treating them with the FDA-approved pharmacological Hh-pathway inhibitor vismodegib. Altogether, our results indicate that the Hh-pathway impedes sustained TMZ-response in GBM and could be a potential therapeutic target to enhance TMZ-response in this malignancy.

  11. Sonic hedgehog maintains cellular and neurochemical homeostasis in the adult nigrostriatal circuit.

    PubMed

    Gonzalez-Reyes, Luis E; Verbitsky, Miguel; Blesa, Javier; Jackson-Lewis, Vernice; Paredes, Daniel; Tillack, Karsten; Phani, Sudarshan; Kramer, Edgar R; Przedborski, Serge; Kottmann, Andreas H

    2012-07-26

    Non cell-autonomous processes are thought to play critical roles in the cellular maintenance of the healthy and diseased brain but mechanistic details remain unclear. We report that the interruption of a non cell-autonomous mode of sonic hedgehog (Shh) signaling originating from dopaminergic neurons causes progressive, adult-onset degeneration of dopaminergic, cholinergic, and fast spiking GABAergic neurons of the mesostriatal circuit, imbalance of cholinergic and dopaminergic neurotransmission, and motor deficits reminiscent of Parkinson's disease. Variable Shh signaling results in graded inhibition of muscarinic autoreceptor- and glial cell line-derived neurotrophic factor (GDNF)-expression in the striatum. Reciprocally, graded signals that emanate from striatal cholinergic neurons and engage the canonical GDNF receptor Ret inhibit Shh expression in dopaminergic neurons. Thus, we discovered a mechanism for neuronal subtype specific and reciprocal communication that is essential for neurochemical and structural homeostasis in the nigrostriatal circuit. These results provide integrative insights into non cell-autonomous processes likely at play in neurodegenerative conditions such as Parkinson's disease. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Sonic Hedgehog Maintains Cellular and Neurochemical Homeostasis in the Adult Nigrostriatal Circuit

    PubMed Central

    Gonzalez-Reyes, Luis E.; Verbitsky, Miguel; Blesa, Javier; Jackson-Lewis, Vernice; Paredes, Daniel; Tillack, Karsten; Phani, Sudarshan; Kramer, Edgar R.; Przedborski, Serge; Kottmann, Andreas H.

    2012-01-01

    SUMMARY Non cell-autonomous processes are thought to play critical roles in the cellular maintenance of the healthy and diseased brain but mechanistic details remain unclear. We report that the interruption of a non-cell autonomous mode of sonic hedgehog (Shh) signaling originating from dopaminergic neurons causes progressive, adult-onset degeneration of dopaminergic, cholinergic, and fast spiking GABAergic neurons of the mesostriatal circuit, imbalance of cholinergic and dopaminergic neurotransmission, and motor deficits reminiscent of Parkinson’s disease. Variable Shh signaling results in graded inhibition of muscarinic auto-receptor- and GDNF- expression in the striatum. Reciprocally, graded signals that emanate from striatal cholinergic neurons and engage the canonical GDNF receptor Ret inhibit Shh expression in dopaminergic neurons. Thus, we discovered a novel mechanism for neuronal subtype specific and reciprocal communication that is essential for neurochemical and structural homeostasis in the nigrostriatal circuit. These results provide integrative insights into non cell-autonomous processes likely at play in neurodegenerative conditions such as Parkinson’s disease. PMID:22841315

  13. Ectopic sonic hedgehog signaling impairs telencephalic dorsal midline development: implication for human holoprosencephaly.

    PubMed

    Huang, Xi; Litingtung, Ying; Chiang, Chin

    2007-06-15

    Holoprosencephaly (HPE) is the most common developmental anomaly of the human forebrain, and in its severe form, the cerebral hemispheres fail to completely separate into two distinct halves. Although disruption of ventral forebrain induction is thought to underlie most HPE cases, a subset of HPE patients exhibits preferential dysgenesis of forebrain dorsal midline structures with unknown etiology. In this study, we show that Sonic hedgehog (Shh) lacking cholesterol moiety in one allele (ShhN/+) in mice can elicit ectopic Shh signaling in early telencephalon to induce ventral progenitor marker expression in the cortical region and impair telencephalic dorsal midline development. Prolonged ectopic ShhN signaling impaired Bmp and Wnt signaling from the dorsal patterning center through upregulation of Fgf8, leading to augmented cell proliferation, decreased cell death and impaired roof plate morphogenesis. Accordingly, ShhN/+ mutant telencephalic dorsal midline structures, including cortical hem, hippocampus and choroid plexus, either failed to form or were hypoplastic. Strikingly, ShhN/+ mutants displayed a spectrum of phenotypic features such as failure of anterior cerebral hemisphere to divide, hydrocephalus and cleft palate which have been observed in a human patient with milder HPE predicted to produce SHHN protein due to a truncation mutation in one SHH allele. We propose that elevated ectopic Shh signaling can impair dorsal telencephalic midline morphogenesis, and lead to non-cleavage of midline structures mimicking human HPE with dorsal midline defects.

  14. 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. Copyright © 2015. Published by Elsevier Inc.

  15. 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.

  16. 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

  17. Correlations Between the Morphology of Sonic Hedgehog Expression Domains and Embryonic Craniofacial Shape

    PubMed Central

    Xu, Qiuping; Jamniczky, Heather; Hu, Diane; Green, Rebecca M.; Marcucio, Ralph S.; Hallgrimsson, Benedikt; Mio, Washington

    2015-01-01

    Quantitative analysis of gene expression domains and investigation of relationships between gene expression and developmental and phenotypic outcomes are central to advancing our understanding of the genotype-phenotype map. Gene expression domains typically have smooth but irregular shapes lacking homologous landmarks, making it difficult to analyze shape variation with the tools of landmark-based geometric morphometrics. In addition, 3D image acquisition and processing introduce many artifacts that further exacerbate the problem. To overcome these difficulties, this paper presents a method that combines optical projection tomography scanning, a shape regularization technique and a landmark-free approach to quantify variation in the morphology of Sonic hedgehog expression domains in the frontonasal ectodermal zone (FEZ) of avians and investigate relationships with embryonic craniofacial shape. The model reveals axes in FEZ and embryonic-head morphospaces along which variation exhibits a sharp linear relationship at high statistical significance. The technique should be applicable to analyses of other 3D biological structures that can be modeled as smooth surfaces and have ill-defined shape. PMID:26321772

  18. Correlations Between the Morphology of Sonic Hedgehog Expression Domains and Embryonic Craniofacial Shape.

    PubMed

    Xu, Qiuping; Jamniczky, Heather; Hu, Diane; Green, Rebecca M; Marcucio, Ralph S; Hallgrimsson, Benedikt; Mio, Washington

    2015-09-01

    Quantitative analysis of gene expression domains and investigation of relationships between gene expression and developmental and phenotypic outcomes are central to advancing our understanding of the genotype-phenotype map. Gene expression domains typically have smooth but irregular shapes lacking homologous landmarks, making it difficult to analyze shape variation with the tools of landmark-based geometric morphometrics. In addition, 3D image acquisition and processing introduce many artifacts that further exacerbate the problem. To overcome these difficulties, this paper presents a method that combines optical projection tomography scanning, a shape regularization technique and a landmark-free approach to quantify variation in the morphology of Sonic hedgehog expression domains in the frontonasal ectodermal zone (FEZ) of avians and investigate relationships with embryonic craniofacial shape. The model reveals axes in FEZ and embryonic-head morphospaces along which variation exhibits a sharp linear relationship at high statistical significance. The technique should be applicable to analyses of other 3D biological structures that can be modeled as smooth surfaces and have ill-defined shape.

  19. 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.

  20. ATOH1 Promotes Leptomeningeal Dissemination and Metastasis of Sonic Hedgehog Subgroup Medulloblastomas.

    PubMed

    Grausam, Katie B; Dooyema, Samuel D R; Bihannic, Laure; Premathilake, Hasitha; Morrissy, A Sorana; Forget, Antoine; Schaefer, Amanda M; Gundelach, Justin H; Macura, Slobodan; Maher, Diane M; Wang, Xin; Heglin, Alex H; Ge, Xijin; Zeng, Erliang; Puget, Stephanie; Chandrasekar, Indra; Surendran, Kameswaran; Bram, Richard J; Schüller, Ulrich; Talyor, Michael D; Ayrault, Olivier; Zhao, Haotian

    2017-07-15

    Medulloblastoma arising from the cerebellum is the most common pediatric brain malignancy, with leptomeningeal metastases often present at diagnosis and recurrence associated with poor clinical outcome. In this study, we used mouse medulloblastoma models to explore the relationship of tumor pathophysiology and dysregulated expression of the NOTCH pathway transcription factor ATOH1, which is present in aggressive medulloblastoma subtypes driven by aberrant Sonic Hedgehog/Patched (SHH/PTCH) signaling. In experiments with conditional ATOH1 mouse mutants crossed to Ptch1(+/-) mice, which develop SHH-driven medulloblastoma, animals with Atoh1 transgene expression developed highly penetrant medulloblastoma at a young age with extensive leptomeningeal disease and metastasis to the spinal cord and brain, resembling xenografts of human SHH medulloblastoma. Metastatic tumors retained abnormal SHH signaling like tumor xenografts. Conversely, ATOH1 expression was detected consistently in recurrent and metastatic SHH medulloblastoma. Chromatin immunoprecipitation sequencing and gene expression profiling identified candidate ATOH1 targets in tumor cells involved in development and tumorigenesis. Among these targets specific to metastatic tumors, there was an enrichment in those implicated in extracellular matrix remodeling activity, cytoskeletal network and interaction with microenvironment, indicating a shift in transcriptomic and epigenomic landscapes during metastasis. Treatment with bone morphogenetic protein or SHH pathway inhibitors decreased tumor cell proliferation and suppressed metastatic tumor growth, respectively. Our work reveals a dynamic ATOH1-driven molecular cascade underlying medulloblastoma metastasis that offers possible therapeutic opportunities. Cancer Res; 77(14); 3766-77. ©2017 AACR. ©2017 American Association for Cancer Research.

  1. 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.

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

    PubMed Central

    KANDA, SHIORI; MITSUYASU, TAKESHI; NAKAO, YU; KAWANO, SHINTARO; GOTO, YUICHI; MATSUBARA, RYOTA; NAKAMURA, SEIJI

    2013-01-01

    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. PMID:23835807

  3. 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.

  4. 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

  5. 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

  6. Crosstalk between Notch and Sonic hedgehog signaling in a mouse model of amyotrophic lateral sclerosis

    PubMed Central

    Ma, Xiaoxing; Drannik, Anna; Jiang, Fan; Peterson, Randy

    2017-01-01

    The developmental morphogen Sonic hedgehog (Shh) may continue to play a sustaining role in adult motor neurons, of potential relevance to motor neuron diseases including amyotrophic lateral sclerosis. The Shh signaling pathway is incompletely understood and interactions with other signaling pathways are possible. We focus here on Notch, and first show that there is an almost linear reduction in light output from a Gli reporter in Shh Light II cells in the presence of increasing concentrations of the Notch inhibitor DAPT (r2=0.982). Second, in the spinal cord of mutant superoxide dismutase mice, but not control mice, a key marker of Notch signaling changes with age. Before the onset of clinical signs, the Notch intracellular domain is expressed predominantly in motor neurons, but by 125 days of age, Notch intracellular domain expression is markedly reduced in motor neurons and increased in neighboring astroglia. Third, there is a parallel reduction in Gli protein expression in mutant superoxide dismutase mouse spinal motor neurons, consistent with the observed reduction in Notch signaling and also a redistribution of Gli away from the nucleus. Thus, there is a reduction in motor neuronal Notch signaling and associated changes in Shh signaling, occurring coincidentally with disease expression, that may contribute toward the dysfunction and death of motor neurons in amyotrophic lateral sclerosis. PMID:27984541

  7. Sonic Hedgehog Initiates Cochlear Hair Cell Regeneration through Downregulation of Retinoblastoma Protein

    PubMed Central

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

    2013-01-01

    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. PMID:23211596

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Sonic Hedgehog Mutations Identified in Holoprosencephaly Patients Can Act in a Dominant Negative Manner

    PubMed Central

    Singh, Samer; Tokhunts, Robert; Baubet, Valerie; Goetz, John A.; Huang, Zhen Jane; Schilling, Neal S.; Black, Kendall E.; MacKenzie, Todd A.; Dahmane, Nadia; Robbins, David J.

    2009-01-01

    Sonic Hedgehog (SHH) plays an important instructional role in vertebrate development, as exemplified by the numerous developmental disorders that occur when the SHH pathway is disrupted. Mutations in the SHH gene are the most common cause of sporadic and inherited Holoprosencephaly (HPE), a developmental disorder that is characterized by defective prosencephalon development. SHH HPE mutations provide a unique opportunity to better understand SHH biogenesis and signaling, and to decipher its role in the development of HPE. Here, we analyzed a panel of SHH HPE missense mutations that encode changes in the amino-terminal active domain of SHH. Our results show that SHH HPE mutations affect SHH biogenesis and signaling at multiple steps, which broadly results in low levels of protein expression, defective processing of SHH into its active form and protein with reduced activity. Additionally, we found that some inactive SHH proteins were able to modulate the activity of wt SHH in a dominant negative manner, both in vitro and in vivo. These findings show for the first time the susceptibility of SHH driven developmental processes to perturbations by low-activity forms of SHH. In conclusion, we demonstrate that SHH mutations found in HPE patients affect distinct steps of SHH biogenesis to attenuate SHH activity to different levels, and suggest that these variable levels of SHH activity might contribute to some of the phenotypic variation found in HPE patients. PMID:19057928

  15. 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

  16. Transient Overexpression of Sonic Hedgehog Alters the Architecture and Mechanical Properties of Trabecular Bone

    PubMed Central

    Kiuru, Maija; Solomon, Jason; Ghali, Bassem; van der Meulen, Marjolein; Crystal, Ronald G; Hidaka, Chisa

    2009-01-01

    Bone formation and remodeling involve coordinated interactions between osteoblasts and osteoclasts through signaling networks involving a variety of molecular pathways. We hypothesized that overexpression of Sonic hedgehog (Shh), a morphogen with a crucial role in skeletal development, would stimulate osteoblastogenesis and bone formation in adult animals in vivo. Systemic administration of adenovirus expressing the N-terminal form of Shh into adult mice resulted in a primary increase in osteoblasts and their precursors. Surprisingly, however, this was associated with altered trabecular morphology, decreased bone volume, and decreased compressive strength in the vertebrae. Whereas no change was detected in the number of osteoclast precursors, bone marrow stromal cells from Shh-treated mice showed enhanced osteoclastogenic potential in vitro. These effects were mediated by the PTH/PTH-related protein (PTHrP) pathway as evidenced by increased sensitivity to PTH stimulation and upregulation of the PTH/PTHrP receptor (PPR). Together, these data show that Shh has stimulatory effects on osteoprogenitors and osteoblasts in adult animals in vivo, which results in bone remodeling and reduced bone strength because of a secondary increase in osteoclastogenesis. PMID:19338448

  17. 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

  18. Regeneration of the Cavernous Nerve by Sonic Hedgehog Using Aligned Peptide Amphiphile Nanofibers

    PubMed Central

    Angeloni, Nicholas L.; Bond, Christopher W.; Tang, Yi; Harrington, Daniel A.; Zhang, Shuming; Stupp, Samuel I.; McKenna, Kevin E.; Podlasek, Carol A.

    2010-01-01

    Efforts to regenerate the cavernous nerve (CN), which provides innervation to the penis, have been minimally successful, with little translation into improved clinical outcomes. We propose that, Sonic hedgehog (SHH), is critical to maintain CN integrity, and that SHH delivered to the CN by novel peptide amphiphile (PA) nanofibers, will promote CN regeneration, restore physiological function, and prevent penile morphology changes that result in erectile dysfunction (ED). We performed localization studies, inhibition of SHH signaling in the CN, and treatment of crushed CNs with SHH protein via linear PA gels, which are an innovative extended release method of delivery. Morphological, functional and molecular analysis revealed that SHH protein is essential to maintain CN architecture, and that SHH treatment promoted CN regeneration, suppressed penile apoptosis and caused a 58% improvement in erectile function in less than half the time reported in the literature. These studies show that SHH has substantial clinical application to regenerate the CN in prostatectomy and diabetic patients, that this methodology has broad application to regenerate any peripheral nerve that SHH is necessary for maintenance of its structure, and that this nanotechnology method of protein delivery may have wide spread application as an in vivo delivery tool in many organs. PMID:20971506

  19. "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.

  20. Immunohistochemical expression of Sonic hedgehog in intraductal papillary mucinous tumor of the pancreas.

    PubMed

    Jang, Kee-Taek; Lee, Kyu Taek; Lee, Jong Gyun; Choi, Seoung Ho; Heo, Jin Seok; Choi, Dong Wook; Ahn, Geunghwan

    2007-09-01

    Aberrant expression of Sonic hedgehog (Shh) has been reported in many human cancers including ductal carcinoma of the pancreas. The intraductal papillary mucinous tumor (IPMT) has been considered as one of the precursor lesions of invasive ductal carcinoma of the pancreas. Shh expression in pancreatic IPMT has not been reported. We investigated an immunohistochemical (IHC) expression of Shh in 55 cases of pancreatic IPMT. We analyzed the IHC expression of Shh in the following histologic grades of tumor: adenoma (AD), moderate dysplasia (MD), noninvasive carcinoma (NIC), and invasive carcinoma (IC), and with the following histologic subtype classification: intestinal, pancreatobiliary, null, and unclassifiable type. IHC Shh expression was noted in 6 (46.2%) of 13 AD, 5 (35.7%) of 14 MD, 12 (80%) of 15 NIC, and 11 (84.6%) of 13 IC. Shh expression was significantly increased in malignant IPMT (NIC+IC) compared with nonmalignant IPMT (AD+MD) (82.1% vs. 40.7%, P=0.0005). IHC Shh expression was found in 11 (68.8%) of 16 intestinal types, 13 (92.8%) of 14 pancreatobiliary types, 8 (38.1%) of 21 null types, and 2 (50%) of 4 unclassifiable types. Intestinal and pancreatobiliary subtypes showed a high expression of Shh compared with the null and unclassifiable type of IPMT. All 3 cases of node metastasis showed IHC Shh expression in tumor cells of metastatic lymph nodes. Therefore, Shh expression may have a critical role in the late stage of carcinogenesis of IPMT, and may impact metastatic progression to the lymph nodes in malignant IPMT.

  1. 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

  2. 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.

  3. 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.

  4. Sonic hedgehog is cytoprotective against oxidative challenge in a cellular model of amyotrophic lateral sclerosis.

    PubMed

    Peterson, Randy; Turnbull, John

    2012-05-01

    We have previously demonstrated that primary cilia on spinal motor neurons are reduced in G93A SOD1 (mSOD) mice, a mouse model of amyotrophic lateral sclerosis (ALS). Sonic hedgehog (Shh) signaling involves the primary cilium and Shh has been shown to be cytoprotective in models of other neurodegenerative diseases. Thus, the Shh signaling pathway may bear further study in ALS. Accordingly, we established that interference with the Shh pathway (with the Shh antagonist cyclopamine or with miRNA 3245p) sensitized HT22 cells, while augmentation of the Shh pathway (with Shh or the Shh agonist purmorphamine) protected cells against hydrogen peroxide (H₂O₂) challenge. We ectopically expressed mSOD, human wild-type SOD1 (wtSOD), or an empty vector in HT22 cells. Compared to empty vector, wtSOD decreased cell death and mSOD increased cell death in response to H₂O₂ challenge. Treatment with cyclopamine or miRNA 3245p sensitized all three transfections to H₂O₂ challenge. Treatment with recombinant human Shh or purmorphamine decreased cell death after H₂O₂ challenge, an effect more pronounced in mSOD cells. Compared with empty vector, overexpression of wtSOD increased Shh and Gli transcript levels and increased activity in a Gli-responsive reporter assay. Overexpression of mSOD did not change Shh transcript levels, but decreased Gli transcript levels, especially Gli3, and reduced activity in a Gli reporter assay. These results suggest that overexpression of mSOD but not wtSOD reduces signaling in the Shh pathway and renders mSOD cells more susceptible to H₂O₂ challenge, and that treatment with Shh or Shh agonists is cytoprotective to mSOD cells. Shh or Shh agonists merit further consideration as potential therapy in ALS.

  5. 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.

  6. 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.

  7. 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

  8. The Timing of Sonic Hedgehog and Gli1 Expression Segregates Midbrain Dopamine Neurons

    PubMed Central

    Hayes, Lindsay; Zhang, Zhiwei; Albert, Paul; Zervas, Mark; Ahn, Sohyun

    2011-01-01

    The ventral midbrain (vMb) is organized into distinct anatomical domains and contains cohorts of functionally distinct subtypes of midbrain dopamine (mDA) neurons. We tested the hypothesis that genetic history and timing of gene expression within mDA neuron progenitors impart spatial diversity. Using Genetic Inducible Fate Mapping to mark the Sonic hedgehog (Shh) and Gli1 lineages at varying embryonic stages, we performed a quantitative and qualitative comparison of the two lineages’ contribution to the mDA neuron domains. Dynamic changes in Shh and Gli1 expression in the vMb primordia delineated their spatial contribution to the E12.5 vMb: Both lineages first contributed to the medial domain, but subsequently the Gli1 lineage exclusively contributed to the lateral vMb while the Shh lineage expanded more broadly across the vMb. The contribution of both lineages to the differentiated mDA neuron domain was initially biased anteriorly and became more uniform across the anterior/posterior vMb throughout development. Our findings demonstrate that the early Shh and Gli1 lineages specify mDA neurons of the substantia nigra pars compacta while the late Shh and Gli1 lineages maintain their progenitor state longer in the posterior vMb to extend the production of mDA neurons in the ventral tegmental area. Together, our study demonstrates that the timing of gene expression along with the genetic lineage (Shh or Gli1) within the neural progenitors segregate mDA neurons into distinct spatial domains. PMID:21713771

  9. 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

  10. 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

  11. Sonic hedgehog (SHH) signaling improves the angiogenic potential of Wharton's jelly-derived mesenchymal stem cells (WJ-MSC).

    PubMed

    Zavala, Gabriela; Prieto, Catalina P; Villanueva, Andrea A; Palma, Verónica

    2017-09-29

    Wharton's jelly-derived mesenchymal stem cells (WJ-MSC) show remarkable therapeutic potential to repair tissue upon injury via paracrine signaling by secreting diverse trophic factors that promote angiogenesis. However, the mechanisms and signaling pathways that regulate the induction of these specific factors are still mostly unknown. Emerging evidence suggests that Sonic hedgehog (SHH) plays a central role in angiogenesis and tissue maintenance. However, its contribution to the angiogenic potential of MSC has not been fully addressed. The aim of this work was to characterize the expression of the SHH pathway components in WJ-MSC primary cultures and to evaluate their angiogenic responsiveness to SHH signaling. Primary cell cultures obtained from human umbilical cords were treated with pharmacological modulators of the SHH pathway. We evaluated the modulation of diverse trophic factors in cell lysates, conditioned medium, and functional in vitro assays. In addition, we determined the angiogenic potential of the SHH pathway in the chicken chorioallantoic membrane, an in vivo model. Our results show that WJ-MSC express components of the canonical SHH pathway and are activated by its signaling. In fact, we provide evidence of basal autocrine/paracrine SHH signaling in WJ-MSC. SHH pathway stimulation promotes the secretion of angiogenic factors such as activin A, angiogenin, angiopoietin 1, granulocyte-macrophage colony-stimulating factor, matrix metallometallopeptidase -9, and urokinase-type plasminogen activator, enhancing the pro-angiogenic capabilities of WJ-MSC both in vitro and in vivo. WJ-MSC are a cell population responsive to SHH pathway stimulation. Basal SHH signaling is in part responsible for the angiogenic inductive properties of WJ-MSC. Overall, exogenous activation of the SHH pathway enhances the angiogenic properties of WJ-MSC, making this cell population an ideal target for treating tissue injury.

  12. The sonic hedgehog signaling pathway is reactivated in human renal cell carcinoma and plays orchestral role in tumor growth

    PubMed Central

    2009-01-01

    Background Human clear cell renal cell carcinoma (CRCC) remains resistant to therapies. Recent advances in Hypoxia Inducible Factors (HIF) molecular network led to targeted therapies, but unfortunately with only limited clinical significance. Elucidating the molecular processes involved in kidney tumorigenesis and resistance is central to the development of improved therapies, not only for kidney cancer but for many, if not all, cancer types. The oncogenic PI3K/Akt, NF-kB and MAPK pathways are critical for tumorigenesis. The sonic hedgehog (SHH) signaling pathway is crucial to normal development. Results By quantitative RT-PCR and immunoblot, we report that the SHH signaling pathway is constitutively reactivated in tumors independently of the von Hippel-Lindau (VHL) tumor suppressor gene expression which is inactivated in the majority of CRCC. The inhibition of the SHH signaling pathway by the specific inhibitor cyclopamine abolished CRCC cell growth as assessed by cell counting, BrdU incorporation studies, fluorescence-activated cell sorting and β-galactosidase staining. Importantly, inhibition of the SHH pathway induced tumor regression in nude mice through inhibition of cell proliferation and neo-vascularization, and induction of apoptosis but not senescence assessed by in vivo studies, immunoblot and immunohistochemistry. Gli1, cyclin D1, Pax2, Lim1, VEGF, and TGF-β were exclusively expressed in tumors and were shown to be regulated by SHH, as evidenced by immunoblot after SHH inhibition. Using specific inhibitors and immunoblot, the activation of the oncogenic PI3K/Akt, NF-kB and MAPK pathways was decreased by SHH inhibition. Conclusions These findings support targeting SHH for the treatment of CRCC and pave the way for innovative and additional investigations in a broad range of cancers. PMID:20015350

  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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. 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.

  15. 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

  16. GLI1, a crucial mediator of sonic hedgehog signaling in prostate cancer, functions as a negative modulator for androgen receptor

    SciTech Connect

    Chen, Guangchun; Goto, Yutaka; Sakamoto, Ryuichi; Tanaka, Kimitaka; Matsubara, Eri; Nakamura, Masafumi; Zheng, Hong; Lu, Jian; Takayanagi, Ryoichi; Nomura, Masatoshi

    2011-01-21

    Research highlights: {yields} GLI1, which play a central role in sonic hedgehog signaling in prostate cancer, can act as a co-repressor to substantially block androgen receptor-mediated transactivation. {yields} GLI1 directly interacts with AR. {yields} SHH-GLI pathway might be one of determinants governing the transition of prostate cancer from an androgen-dependent to an androgen-independent state. -- Abstract: Sonic hedgehog (SHH) signaling, acting in a combinatorial manner with androgen signaling, is essential for prostate patterning and development. Recently, elevated activation of SHH signaling has been shown to play important roles in proliferation, progression and metastasis of prostate cancer. In this report, we demonstrate for the first time, that GLI1, which has been shown to play a central role in SHH signaling in prostate cancer, can act as a co-repressor to substantially block androgen receptor (AR)-mediated transactivation, at least in part, by directly interacting with AR. Our observations suggest that the SHH-GLI pathway might be one of determinants governing the transition of prostate cancer from an androgen-dependent to an androgen-independent state by compensating, or even superseding androgen signaling.

  17. 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

  18. 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

  19. 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

  20. Primary cilia are critical for Sonic hedgehog-mediated dopaminergic neurogenesis in the embryonic midbrain.

    PubMed

    Gazea, Mary; Tasouri, Evangelia; Tolve, Marianna; Bosch, Viktoria; Kabanova, Anna; Gojak, Christian; Kurtulmus, Bahtiyar; Novikov, Orna; Spatz, Joachim; Pereira, Gislene; Hübner, Wolfgang; Brodski, Claude; Tucker, Kerry L; Blaess, Sandra

    2016-01-01

    Midbrain dopaminergic (mDA) neurons modulate various motor and cognitive functions, and their dysfunction or degeneration has been implicated in several psychiatric diseases. Both Sonic Hedgehog (Shh) and Wnt signaling pathways have been shown to be essential for normal development of mDA neurons. Primary cilia are critical for the development of a number of structures in the brain by serving as a hub for essential developmental signaling cascades, but their role in the generation of mDA neurons has not been examined. We analyzed mutant mouse lines deficient in the intraflagellar transport protein IFT88, which is critical for primary cilia function. Conditional inactivation of Ift88 in the midbrain after E9.0 results in progressive loss of primary cilia, a decreased size of the mDA progenitor domain, and a reduction in mDA neurons. We identified Shh signaling as the primary cause of these defects, since conditional inactivation of the Shh signaling pathway after E9.0, through genetic ablation of Gli2 and Gli3 in the midbrain, results in a phenotype basically identical to the one seen in Ift88 conditional mutants. Moreover, the expansion of the mDA progenitor domain observed when Shh signaling is constitutively activated does not occur in absence of Ift88. In contrast, clusters of Shh-responding progenitors are maintained in the ventral midbrain of the hypomorphic Ift88 mouse mutant, cobblestone. Despite the residual Shh signaling, the integrity of the mDA progenitor domain is severely disturbed, and consequently very few mDA neurons are generated in cobblestone mutants. Our results identify for the first time a crucial role of primary cilia in the induction of mDA progenitors, define a narrow time window in which Shh-mediated signaling is dependent upon normal primary cilia function for this purpose, and suggest that later Wnt signaling-dependent events act independently of primary cilia. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor

    PubMed Central

    Seidel, Kerstin; Ahn, Christina P.; Lyons, David; Nee, Alexander; Ting, Kevin; Brownell, Isaac; Cao, Tim; Carano, Richard A. D.; Curran, Tom; Schober, Markus; Fuchs, Elaine; Joyner, Alexandra; Martin, Gail R.; de Sauvage, Frederic J.; Klein, Ophir D.

    2010-01-01

    In many organ systems such as the skin, gastrointestinal tract and hematopoietic system, homeostasis is dependent on the continuous generation of differentiated progeny from stem cells. The rodent incisor, unlike human teeth, grows throughout the life of the animal and provides a prime example of an organ that rapidly deteriorates if newly differentiated cells cease to form from adult stem cells. Hedgehog (Hh) signaling has been proposed to regulate self-renewal, survival, proliferation and/or differentiation of stem cells in several systems, but to date there is little evidence supporting a role for Hh signaling in adult stem cells. We used in vivo genetic lineage tracing to identify Hh-responsive stem cells in the mouse incisor and we show that sonic hedgehog (SHH), which is produced by the differentiating progeny of the stem cells, signals to several regions of the incisor. Using a hedgehog pathway inhibitor (HPI), we demonstrate that Hh signaling is not required for stem cell survival but is essential for the generation of ameloblasts, one of the major differentiated cell types in the tooth, from the stem cells. These results therefore reveal the existence of a positive-feedback loop in which differentiating progeny produce the signal that in turn allows them to be generated from stem cells. PMID:20978073

  2. 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

  3. Peptide Amphiphile Nanofiber Delivery of Sonic Hedgehog Protein to Reduce Smooth Muscle Apoptosis in the Penis after Cavernous Nerve Resection

    PubMed Central

    Bond, Christopher W.; Angeloni, Nicholas L.; Harrington, Daniel A.; Stupp, Samuel I.; McKenna, Kevin E.; Podlasek, Carol A.

    2011-01-01

    Introduction Erectile dysfunction (ED) is a serious medical condition that affects 16–82% of prostate cancer patients treated by radical prostatectomy and current treatments are ineffective in 50–60% of prostatectomy patients. The reduced efficacy of treatments makes novel therapeutic approaches to treat ED essential. The secreted protein Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle and apoptosis that is decreased in cavernous nerve (CN) injury and diabetic ED models. Past studies using Affi-Gel beads have shown SHH protein to be effective in suppressing apoptosis caused by CN injury. Aim We hypothesize that SHH protein delivered via novel peptide amphiphile (PA) nanofibers will be effective in suppressing CN injury-induced apoptosis. Methods Adult Sprague Dawley rats (n = 50) were used to optimize PA injection in vivo. PA with SHH protein (n = 16) or bovine serum albumin (BSA) (control, n = 14) was injected into adult rats that underwent bilateral CN cut. Rats were sacrificed at 2, 4, and 7 days. Alexa Fluor-labeled SHH protein was used to determine the target of SHH signaling (n = 3). Main Outcome Measures Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and semi-quantitative immunohistochemical analysis for SHH protein and cluster differentiation protein three (CD3) were performed. Results SHH-PA caused a 25% and 16% reduction in apoptosis at 4 and 7 days after CN injury and a 9.3% and 19% increase in SHH protein at 4 and 7 days after CN injury. CD3 protein was not observed in SHH-PA-treated penis. In vitro, 73% of SHH protein diffused from PA within 6 days. Labeled SHH was observed in smooth muscle. Conclusions PA technology is effective in delivering SHH protein to the penis and SHH is effective in suppressing CN injury-induced apoptosis. These results suggest substantial translational potential of this methodology and show that only a short duration of SHH treatment is required to impact the apoptotic index

  4. 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.

  5. 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.

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

    PubMed Central

    Chinnaiya, Kavitha; Tickle, Cheryll; Towers, Matthew

    2014-01-01

    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. PMID:25001275

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

    USDA-ARS?s Scientific Manuscript database

    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...

  8. 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.

  9. Inhibition of Sonic hedgehog signaling leads to posterior digit loss in Ambystoma mexicanum: parallels to natural digit reduction in urodeles.

    PubMed

    Stopper, Geffrey F; Wagner, Günter P

    2007-01-01

    Molecular mechanisms patterning the tetrapod limb, including anterior-posterior axis determination involving Sonic hedgehog (Shh), have received much attention, particularly in amniotes. Anterior-posterior patterning in urodele amphibians differs radically from that of amniotes in that it shows a pronounced anterior-to-posterior sequence of digit development. In contrast, amniotes develop their digits almost simultaneously with a slight posterior-to-anterior polarity. Here we use cyclopamine, an inhibitor of the Hedgehog signaling pathway, to investigate the role of Shh in anterior-posterior patterning in the urodele limb. Inhibition of Shh signal transduction affects digit number long before their morphological appearance. In accordance with the apparently derived order of digit development in urodeles, exposure reproducibly removes digits in a posterior-to-anterior sequence, the inverse of their developmental sequence. This pattern of digit loss mimics the order of digit loss in natural variation. We suggest that variation in Shh expression and/or signal transmission may explain natural variation in digit number in urodeles.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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.

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

    DTIC Science & Technology

    2007-02-01

    lung cancer cell lines ( A549 , H82, H187, H196, H209, H460 , H661, H1299, BEAS2-B and BZR-T33) were purchased from ATCC and...our findings in the tumors, we examined eight lung cancer cell lines for Su(Fu) expression and found one cell line NCI -H209 with no detectable Su(Fu...hedgehog signaling activation in a small subset of lung cancers . Using a cell line with no detectable Su(Fu)

  2. 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

  3. 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.

  4. 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.

  5. Heterochronic Shift in Hox-Mediated Activation of Sonic hedgehog Leads to Morphological Changes during Fin Development

    PubMed Central

    Suda, Natsuno; Tamura, Mika; Ochi, Haruki; Tanaka, Mikiko

    2009-01-01

    We explored the molecular mechanisms of morphological transformations of vertebrate paired fin/limb evolution by comparative gene expression profiling and functional analyses. In this study, we focused on the temporal differences of the onset of Sonic hedgehog (Shh) expression in paired appendages among different vertebrates. In limb buds of chick and mouse, Shh expression is activated as soon as there is a morphological bud, concomitant with Hoxd10 expression. In dogfish (Scyliorhinus canicula), however, we found that Shh was transcribed late in fin development, concomitant with Hoxd13 expression. We utilized zebrafish as a model to determine whether quantitative changes in hox expression alter the timing of shh expression in pectoral fins of zebrafish embryos. We found that the temporal shift of Shh activity altered the size of endoskeletal elements in paired fins of zebrafish and dogfish. Thus, a threshold level of hox expression determines the onset of shh expression, and the subsequent heterochronic shift of Shh activity can affect the size of the fin endoskeleton. This process may have facilitated major morphological changes in paired appendages during vertebrate limb evolution. PMID:19365553

  6. 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.

  7. 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.

  8. A mouse model for embryonal tumors with multilayered rosettes uncovers the therapeutic potential of Sonic-hedgehog inhibitors.

    PubMed

    Neumann, Julia E; Wefers, Annika K; Lambo, Sander; Bianchi, Edoardo; Bockstaller, Marie; Dorostkar, Mario M; Meister, Valerie; Schindler, Pia; Korshunov, Andrey; von Hoff, Katja; Nowak, Johannes; Warmuth-Metz, Monika; Schneider, Marlon R; Renner-Müller, Ingrid; Merk, Daniel J; Shakarami, Mehdi; Sharma, Tanvi; Chavez, Lukas; Glass, Rainer; Chan, Jennifer A; Taketo, M Mark; Neumann, Philipp; Kool, Marcel; Schüller, Ulrich

    2017-10-01

    Embryonal tumors with multilayered rosettes (ETMRs) have recently been described as a new entity of rare pediatric brain tumors with a fatal outcome. We show here that ETMRs are characterized by a parallel activation of Shh and Wnt signaling. Co-activation of these pathways in mouse neural precursors is sufficient to induce ETMR-like tumors in vivo that resemble their human counterparts on the basis of histology and global gene-expression analyses, and that point to apical radial glia cells as the possible tumor cell of origin. Overexpression of LIN28A, which is a hallmark of human ETMRs, augments Sonic-hedgehog (Shh) and Wnt signaling in these precursor cells through the downregulation of let7-miRNA, and LIN28A/let7a interaction with the Shh pathway was detected at the level of Gli mRNA. Finally, human ETMR cells that were transplanted into immunocompromised host mice were responsive to the SHH inhibitor arsenic trioxide (ATO). Our work provides a novel mouse model in which to study this tumor type, demonstrates the driving role of Wnt and Shh activation in the growth of ETMRs and proposes downstream inhibition of Shh signaling as a therapeutic option for patients with ETMRs.

  9. Cell fate specification in the lingual epithelium is controlled by antagonistic activities of Sonic hedgehog and retinoic acid

    PubMed Central

    Neben, Cynthia L.; Harfe, Brian D.; Linde, Anders

    2017-01-01

    The interaction between signaling pathways is a central question in the study of organogenesis. Using the developing murine tongue as a model, we uncovered unknown relationships between Sonic hedgehog (SHH) and retinoic acid (RA) signaling. Genetic loss of SHH signaling leads to enhanced RA activity subsequent to loss of SHH-dependent expression of Cyp26a1 and Cyp26c1. This causes a cell identity switch, prompting the epithelium of the tongue to form heterotopic minor salivary glands and to overproduce oversized taste buds. At developmental stages during which Wnt10b expression normally ceases and Shh becomes confined to taste bud cells, loss of SHH inputs causes the lingual epithelium to undergo an ectopic and anachronic expression of Shh and Wnt10b in the basal layer, specifying de novo taste placode induction. Surprisingly, in the absence of SHH signaling, lingual epithelial cells adopted a Merkel cell fate, but this was not caused by enhanced RA signaling. We show that RA promotes, whereas SHH, acting strictly within the lingual epithelium, inhibits taste placode and lingual gland formation by thwarting RA activity. These findings reveal key functions for SHH and RA in cell fate specification in the lingual epithelium and aid in deciphering the molecular mechanisms that assign cell identity. PMID:28715412

  10. 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.

  11. 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.

  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. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Induction of mirror-image supernumerary jaws in chicken mandibular mesenchyme by Sonic Hedgehog-producing cells.

    PubMed

    Brito, José M; Teillet, Marie-Aimée; Le Douarin, Nicole M

    2008-07-01

    Previous studies have shown that Sonic Hedgehog (Shh) signaling is crucial for the development of the first branchial arch (BA1) into a lower-jaw in avian and mammalian embryos. We have already shown that if Shh expression is precociously inhibited in pharyngeal endoderm, neural crest cells migrate to BA1 but fail to survive, and Meckel's cartilage and associated structures do not develop. This phenotype can be rescued by addition of an exogenous source of Shh. To decipher the role of Shh, we explored the consequences of providing an extra source of Shh to the presumptive BA1 territory. Grafting quail fibroblasts engineered to produce Shh (QT6-Shh), at the 5- to 8-somite stage, resulted in the induction of mirror-image extra lower jaws, caudolateral to the normal one. It turns out that the oral opening epithelium, in which Shh, Fgf8 and Bmp4 are expressed in a definite pattern, functions as an organizing center for lower-jaw development. In our experimental design, the extra source of Shh activates Fgf8, Bmp4 and Shh genes in caudal BA1 ectoderm in a spatial pattern similar to that of the oral epithelium, and regularly leads to the formation of two extra lower-jaw-organizing centers with opposite rostrocaudal polarities. These results emphasize the similarities between the developmental processes of the limb and mandibular buds, and show that in both cases Shh-producing cells create a zone of polarizing activity for the structures deriving from them.

  4. 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

  5. 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

  6. Sonic hedgehog (SHH) and glioblastoma-2 (Gli-2) expressions are associated with poor jaundice-free survival in biliary atresia.

    PubMed

    Jung, Hae Yoen; Jing, Jin; Lee, Kyoung Bun; Jang, Ja-June

    2015-03-01

    Biliary atresia (BA) causes biliary obstruction in neonates. Although the Kasai operation can successfully treat certain BA cases, many patients exhibit recurrent jaundice and secondary biliary cirrhosis requiring liver transplantation. Consequently, studies of the prognostic factors of the Kasai operation are needed. Accordingly, sonic hedgehog (SHH) pathway expression at the extrahepatic bile duct (EHBD), an important bile duct repair mechanism, will be investigated via immunohistochemistry in patients with BA to examine the association with post-Kasai operation prognosis. Fifty-seven EHBD specimens were obtained during Kasai operations from 1992 to 2009. The SHH, patched (PTCH), and glioblastoma-2 (Gli-2) immunohistochemical staining results were analyzed quantitatively. Overall, 57.9% of patients had bile flow normalization after the Kasai operation; 43.1% did not. High preoperative serum total bilirubin, direct bilirubin, and aspartate aminotransferase levels were associated with sustained jaundice post-Kasai operation, as was an age ≥65days at the time of surgery (all p<0.05). High Gli-2 and SHH expression rates were significantly associated with early post-Kasai operation jaundice relapse. Strong Gli-2 and SHH expression in the EHBD might be a poor prognostic factor in Kasai operation-treated patients with BA. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. 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.

  8. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. 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

  10. 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

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

    PubMed Central

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

    2015-01-01

    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

  12. 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

  13. 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

  14. Genes targeted by the Hedgehog-signaling pathway can be regulated by Estrogen related receptor β.

    PubMed

    Lu, Yuan; Li, Jilong; Cheng, Jianlin; Lubahn, Dennis B

    2015-11-23

    Nuclear receptor family member, Estrogen related receptor β, and the Hedgehog signal transduction pathway are both reported to relate to tumorigenesis and induced pluripotent stem cell reprogramming. We hypothesize that Estrogen related receptor β can modulate the Hedgehog signaling pathway and affect Hedgehog driven downstream gene expression. We established an estrogen related receptor β-expressing Hedgehog-responsive NIH3T3 cell line by Esrrb transfection, and performed mRNA profiling using RNA-Seq after Hedgehog ligand conditioned medium treatment. Esrrb expression altered 171 genes, while Hedgehog signaling activation alone altered 339 genes. Additionally, estrogen related receptor β expression in combination with Hedgehog signaling activation affects a group of 109 Hedgehog responsive mRNAs, including Hsd11b1, Ogn, Smoc2, Igf1, Pdcd4, Igfbp4, Stmn1, Hp, Hoxd8, Top2a, Tubb4b, Sfrp2, Saa3, Prl2c3 and Dpt. We conclude that Estrogen related receptor β is capable of interacting with Hh-signaling downstream targets. Our results suggest a new level of regulation of Hedgehog signaling by Estrogen related receptor β, and indicate modulation of Estrogen related receptor β can be a new strategy to regulate various functions driven by the Hedgehog signaling pathway.

  15. 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.

  16. 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

  17. Improved smell function with increased nasal mucus sonic hedgehog in hyposmic patients after treatment with oral theophylline.

    PubMed

    Henkin, Robert I; Hosein, Suzanna; Stateman, William A; Knöppel, Alexandra B; Abdelmeguid, Mona

    We previously demonstrated the presence of sonic hedgehog (Shh) in nasal mucus in normal subjects and in patients with smell loss (hyposmia). Nasal mucus Shh levels were found significantly diminished in untreated hyposmic patients of multiple etiologies. Since treatment with oral theophylline has been previously associated with improvement in smell function we wished to study if such treatment increased nasal mucus Shh as well as improved smell function in patients with hyposmia. Forty-four patients with hyposmia of several etiologies were evaluated for changes in hyposmia by subjective measurements of smell, taste and flavor perception and by olfactometry. Measurements of nasal mucus Shh were made in relationship to each set of sensory measurements. Patients were treated with oral theophylline at doses of 200-800mg for periods of 2-10months with sensory function, nasal mucus Shh and serum theophylline levels evaluated at these time intervals. Nasal mucus Shh measurements were made with a sensitive spectrophotometric ELISA assay and theophylline with a fluorometric assay. There was consistent, significant improvement in subjective responses in smell, taste and flavor perception and in olfactometry associated with increased nasal mucus Shh and serum theophylline after theophylline treatment. Improvement in smell function and in nasal mucus Shh was positively correlated in a dose-response relationship after treatment with oral theophylline. Results are consistent with a successful role for theophylline in improvement of smell function in hyposmic patients of multiple etiologies associated with increased nasal mucus Shh which can act as a biochemical marker for smell function. Copyright © 2016. Published by Elsevier Inc.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. FOREBRAIN AND HINDBRAIN DEVELOPMENT IN ZEBRAFISH IS SENSITIVE TO ETHANOL EXPOSURE INVOLVING AGRIN, FGF AND SONIC HEDGEHOG FUNCTION

    PubMed Central

    Zhang, Chengjin; Ojiaku, Princess; Cole, Gregory J.

    2014-01-01

    BACKGROUND Ethanol is a teratogen that affects numerous developmental processes in the nervous system, which includes development and survival of GABAergic and glutamatergic neurons. Possible molecular mechanisms accounting for ethanol’s effects on nervous system development include perturbed fibroblast growth factor (Fgf) and Sonic hedgehog (Shh) signaling. In zebrafish, forebrain GABAergic neuron development is dependent on Fgf19 and Shh signaling. The present study was conducted to test the hypothesis that ethanol affects GABAergic and glutamatergic neuron development by disrupting Fgf, Shh, and agrin function. METHODS Zebrafish embryos were exposed to varying concentrations of ethanol during a range of developmental stages, in the absence or presence of morpholino oligonucleotides (MOs) that disrupt agrin or Shh function. In situ hybridization was employed to analyze glutamic acid decarboxylase (GAD1) gene expression, as well as markers of glutamatergic neurons. RESULTS Acute ethanol exposure results in marked reduction in GAD1 gene expression in forebrain and hindbrain, and reduction of glutamatergic neuronal markers in hindbrain. Subthreshold ethanol exposure, combined with agrin or Shh MO treatment, produces a similar diminution in expression of markers for GABAergic and glutamatergic neurons. Consistent with the ethanol effects on Fgf and Shh pathways, Fgf19, Fgf8 or Shh mRNA overexpression rescues ethanol-induced decreases in GAD1 and atonal1a gene expression. CONCLUSIONS These studies demonstrate that GABAergic and glutamatergic neuron development in zebrafish forebrain or cerebellum is sensitive to ethanol exposure, and provides additional evidence that a signaling pathway involving agrin, Fgfs and Shh may be a critical target of ethanol exposure during zebrafish embryogenesis. PMID:23184466

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

    PubMed Central

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

    2015-01-01

    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. PMID:25804737

  7. Sonic Hedgehog Gene Delivery to the Rodent Heart Promotes Angiogenesis via iNOS/Netrin-1/PKC Pathway

    PubMed Central

    Ahmed, Rafeeq P. H.; Haider, Khawaja Husnain; Shujia, Jiang; Afzal, Muhammad Rizwan; Ashraf, Muhammad

    2010-01-01

    Background We hypothesized that genetic modification of mesenchymal stem cells (MSCs) with Sonic Hedgehog (Shh) transgene, a morphogen during embryonic development and embryonic and adult stem cell growth, improved their survival and angiogenic potential in the ischemic heart via iNOS/netrin/PKC pathway. Methods/Principal Findings MSCs from young Fisher-344 rat bone marrow were purified and transfected with pCMV Shh plasmid (ShhMSCs). Immunofluorescence, RT-PCR and Western blotting showed higher expression of Shh in ShhMSCs which also led to increased expression of angiogenic and pro-survival growth factors in ShhMSCs. Significantly improved migration and tube formation was seen in ShhMSCs as compared to empty vector transfected MSCs (EmpMSCs). Significant upregulation of netrin-1 and iNOS was observed in ShhMSCs in PI3K independent but PKC dependent manner. For in vivo studies, acute myocardial infarction model was developed in Fisher-344 rats. The animals were grouped to receive 70 µl basal DMEM without cells (group-1) or containing 1×106 EmpMSCs (group-2) and ShhMSCs (group-3). Group-4 received recombinant netrin-1 protein injection into the infarcted heart. FISH and sry-quantification revealed improved survival of ShhMSCs post engraftment. Histological studies combined with fluorescent microspheres showed increased density of functionally competent blood vessels in group-3 and group-4. Echocardiography showed significantly preserved heart function indices post engraftment with ShhMSCs in group-3 animals. Conclusions/Significance Reprogramming of stem cells with Shh maximizes their survival and angiogenic potential in the heart via iNOS/netrin-1/PKC signaling. PMID:20052412

  8. Sonic Hedgehog gene delivery to the rodent heart promotes angiogenesis via iNOS/netrin-1/PKC pathway.

    PubMed

    Ahmed, Rafeeq P H; Haider, Khawaja Husnain; Shujia, Jiang; Afzal, Muhammad Rizwan; Ashraf, Muhammad

    2010-01-05

    We hypothesized that genetic modification of mesenchymal stem cells (MSCs) with Sonic Hedgehog (Shh) transgene, a morphogen during embryonic development and embryonic and adult stem cell growth, improved their survival and angiogenic potential in the ischemic heart via iNOS/netrin/PKC pathway. MSCs from young Fisher-344 rat bone marrow were purified and transfected with pCMV Shh plasmid ((Shh)MSCs). Immunofluorescence, RT-PCR and Western blotting showed higher expression of Shh in (Shh)MSCs which also led to increased expression of angiogenic and pro-survival growth factors in (Shh)MSCs. Significantly improved migration and tube formation was seen in (Shh)MSCs as compared to empty vector transfected MSCs ((Emp)MSCs). Significant upregulation of netrin-1 and iNOS was observed in (Shh)MSCs in PI3K independent but PKC dependent manner. For in vivo studies, acute myocardial infarction model was developed in Fisher-344 rats. The animals were grouped to receive 70 microl basal DMEM without cells (group-1) or containing 1x10(6) (Emp)MSCs (group-2) and (Shh)MSCs (group-3). Group-4 received recombinant netrin-1 protein injection into the infarcted heart. FISH and sry-quantification revealed improved survival of (Shh)MSCs post engraftment. Histological studies combined with fluorescent microspheres showed increased density of functionally competent blood vessels in group-3 and group-4. Echocardiography showed significantly preserved heart function indices post engraftment with (Shh)MSCs in group-3 animals. Reprogramming of stem cells with Shh maximizes their survival and angiogenic potential in the heart via iNOS/netrin-1/PKC signaling.

  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. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26254735

  12. 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.

  13. 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.

  14. The Sonic Hedgehog Signaling Pathway Induces Myopic Development by Activating Matrix Metalloproteinase (MMP)-2 in Guinea Pigs

    PubMed Central

    Chen, Minjie; Qian, Yishan; Dai, Jinhui; Chu, Renyuan

    2014-01-01

    Purpose To investigate whether the Sonic hedgehog (Shh) signaling induces myopic development by increasing the expression of matrix metalloproteinase (MMP)-2 in guinea pigs. Methods A translucent diffuser was glued onto the right eye to induce form-deprivation myopia (FDM) in 10 guinea pigs. Four guinea pigs were served as a control group. The other 100 guinea pigs were subdivided into 5 groups (20 per group) and received a 10 µl intravitreal injection every 2 days for 4 times. Two groups were injected with 20 or 50 µg/ml Shh amino-terminal peptide (Shh-N) into the right eye and 0.1% bovine serum albumin into the other. FDM was induced in the right eyes of the three cyclopamine-treated groups and both eyes were injected with 50, 100, or 200 µg/ml cyclopamine. Retinoscopic refraction and eye dimensions were assessed on Day 14 of treatment. MMP-2 protein expression was determined in both scleras by western blotting. Results Both concentrations of Shh-N stimulated myopic development and axial growth as compared with control eyes. Myopia and axial elongation were significantly greater in the 50 µg/ml than in the 20 µg/ml Shh-N group (P<0.001 and P = 0.0019, respectively). All three doses of cyclopamine significantly attenuated myopic development compared with the FDM group (P<0.0001). Cyclopamine at 100 or 200 µg/ml significantly reduced axial elongation compared with the FDM group (P = 0.044 and P = 0.001, respectively). FDM-induced myopia and axial elongation were significantly greater in the 50 µg/ml than in the 200 µg/ml cyclopamine group (P<0.0001 and P = 0.008, respectively). MMP-2 expression was significantly greater in Shh-N–treated eyes than in the control eyes, and was lower in the cyclopamine plus FDM groups than in the FDM group. Conclusions The Shh signaling pathway induces myopic development by activating MMP-2 in guinea pigs. PMID:24810957

  15. The sonic hedgehog signaling pathway induces myopic development by activating matrix metalloproteinase (MMP)-2 in Guinea pigs.

    PubMed

    Chen, Minjie; Qian, Yishan; Dai, Jinhui; Chu, Renyuan

    2014-01-01

    To investigate whether the Sonic hedgehog (Shh) signaling induces myopic development by increasing the expression of matrix metalloproteinase (MMP)-2 in guinea pigs. A translucent diffuser was glued onto the right eye to induce form-deprivation myopia (FDM) in 10 guinea pigs. Four guinea pigs were served as a control group. The other 100 guinea pigs were subdivided into 5 groups (20 per group) and received a 10 µl intravitreal injection every 2 days for 4 times. Two groups were injected with 20 or 50 µg/ml Shh amino-terminal peptide (Shh-N) into the right eye and 0.1% bovine serum albumin into the other. FDM was induced in the right eyes of the three cyclopamine-treated groups and both eyes were injected with 50, 100, or 200 µg/ml cyclopamine. Retinoscopic refraction and eye dimensions were assessed on Day 14 of treatment. MMP-2 protein expression was determined in both scleras by western blotting. Both concentrations of Shh-N stimulated myopic development and axial growth as compared with control eyes. Myopia and axial elongation were significantly greater in the 50 µg/ml than in the 20 µg/ml Shh-N group (P<0.001 and P = 0.0019, respectively). All three doses of cyclopamine significantly attenuated myopic development compared with the FDM group (P<0.0001). Cyclopamine at 100 or 200 µg/ml significantly reduced axial elongation compared with the FDM group (P = 0.044 and P = 0.001, respectively). FDM-induced myopia and axial elongation were significantly greater in the 50 µg/ml than in the 200 µg/ml cyclopamine group (P<0.0001 and P = 0.008, respectively). MMP-2 expression was significantly greater in Shh-N-treated eyes than in the control eyes, and was lower in the cyclopamine plus FDM groups than in the FDM group. The Shh signaling pathway induces myopic development by activating MMP-2 in guinea pigs.

  16. 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

  17. 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

  18. Neuropilins are positive regulators of Hedgehog signal transduction

    PubMed Central

    Hillman, R. Tyler; Feng, Brian Y.; Ni, Jun; Woo, Wei-Meng; Milenkovic, Ljiljana; Hayden Gephart, Melanie G.; Teruel, Mary N.; Oro, Anthony E.; Chen, James K.; Scott, Matthew P.

    2011-01-01

    The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. Here we show that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development. Using cell lines lacking key Hh pathway components, we show that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish, as we show that morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance our knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental signaling system. PMID:22051878

  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. Hedgehog signaling in prostate epithelial-mesenchymal growth regulation

    PubMed Central

    Peng, Yu-Ching; Joyner, Alexandra L.

    2015-01-01

    The prostate gland plays an important role in male reproduction, and is also an organ prone to diseases such as benign prostatic hyperplasia (BPH) and prostate cancer. The prostate consists of ducts with an inner layer of epithelium surrounded by stroma. Reciprocal signaling between these two cell compartments is instrumental to normal prostatic development, homeostasis, regeneration, as well as tumor formation. Hedgehog (HH) signaling is a master regulator in numerous developmental processes. In many organs, HH plays a key role in epithelial-mesenchymal signaling that regulates organ growth and tissue differentiation, and abnormal HH signaling has been implicated in the progression of various epithelial carcinomas. In this review, we focus on recent studies exploring the multipotency of endogenous postnatal and adult epithelial and stromal stem cells and studies addressing the role of HH in prostate development and cancer. We discuss the implications of the results for a new understanding of prostate development and disease. Insight into the cellular and molecular mechanisms underlying epithelial-mesenchymal growth regulation should provide a basis for devising innovative therapies to combat diseases of the prostate. PMID:25641695

  4. 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.

  5. Active Sonic Hedgehog Signaling between Androgen Independent Human Prostate Cancer Cells and Normal/Benign but Not Cancer-Associated Prostate Stromal Cells

    PubMed Central

    Shigemura, Katsumi; Huang, Wen-Chin; Li, Xiangyan; Zhau, Haiyen E.; Zhu, Guodong; Gotoh, Akinobu; Fujisawa, Masato; Xie, Jingwu; Marshall, Fray F.; Chung, Leland W. K.

    2012-01-01

    BACKGROUND Sonic hedgehog (Shh) signaling plays a pivotal role in stromal-epithelial interaction during normal development but its role in tumor-stromal interaction during carcinogenic progression is less well defined. Since hormone refractory prostate cancer with bone metastasis is difficult to treat, it is crucial to investigate how androgen independent (AI) human prostate cancer cells communicate with their associated stroma. METHODS Shh and its target transcription factor, Gli1 mRNA, were assessed by RT-PCR and/or quantitative RT-PCR in co-cultured cell recombinants comprised of AI C4-2 either with NPF (prostate fibroblasts from normal/benign prostate gland) or CPF cancer-associated stromal fibroblasts) under Shh/cyclopamine (a hedgehog signaling inhibitor) treatment. Human bone marrow stromal (HS27A) cells were used as controls. In vivo investigation was performed by checking serum PSA and immunohistochemical staining for the apoptosis-associated M30 gene in mice bearing chimeric C4-2/NPF tumors. RESULTS CONCLUSIONS Based on co-culture and chimeric tumor models, active Shh-mediated signaling was demonstrated between AI prostate cancer and NPF in a paracrine- and tumor progression-dependent manner. Our study suggests that drugs like cyclopamine that interfere with Shh signaling could be beneficial in preventing AI progression in prostate cancer cells. PMID:21520153

  6. 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

  7. Hedgehog Signaling Regulates Bladder Cancer Growth And Tumorigenicity

    PubMed Central

    Fei, Dennis Liang; Sanchez-Mejias, Avencia; Wang, Zhiqiang; Flaveny, Colin; Long, Jun; Singh, Samer; Rodriguez-Blanco, Jezabel; Tokhunts, Robert; Giambelli, Camilla; Briegel, Karoline J.; Schulz, Wolfgang A.; Gandolfi, A. Jay; Karagas, Margaret; Zimmers, Teresa A.; Jorda, Merce; Bejarano, Pablo; Capobianco, Anthony J.; Robbins, David J.

    2012-01-01

    The role of HEDGEHOG (HH) signaling in bladder cancer remains controversial. The gene encoding the HH receptor and negative regulator PATCHED1 (PTCH1) resides on a region of chromosome 9q, one copy of which is frequently lost in bladder cancer. Inconsistent with PTCH1 functioning as a classic tumor suppressor gene, loss-of-function mutations in the remaining copy of PTCH1 are not commonly found. Here, we provide direct evidence for a critical role of HH signaling in bladder carcinogenesis. We show that transformed human urothelial cells and many urothelial carcinoma (UC) cell lines exhibit constitutive HH signaling, which is required for their growth and tumorigenic properties. Surprisingly, rather than originating from loss of PTCH1, the constitutive HH activity observed in UC cell lines was HH ligand-dependent. Consistent with this finding, increased levels of HH and the HH target gene product GLI1 were found in resected human primary bladder tumors. Furthermore, based on the difference in intrinsic HH dependence of UC cell lines, a gene expression signature was identified that correlated with bladder cancer progression. Our findings therefore indicate that therapeutic targeting of the HH signaling pathway may be beneficial in the clinical management of bladder cancer. PMID:22815529

  8. 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

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

    PubMed

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

    2015-04-01

    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. 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. 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. © 2015 Wiley Periodicals, Inc.

  10. 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

  11. 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. © 2015 AlphaMed Press.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. BMP-2 and sonic hedgehog have contrary effects on adipocyte-like differentiation of C3H10T1/2 cells.

    PubMed

    Zehentner, B K; Leser, U; Burtscher, H

    2000-05-01

    The signaling pathways of bone morphogenic protein 2 (BMP-2) and Sonic hedgehog (Shh) are related during embryogenesis. Both proteins have been implicated as important components during osteogenic differentiation; e.g., considering their in vitro effects in the pluripotent C3H10T/1/2 cell system. Also, BMP-2 has been frequently reported to stimulate adipogenesis as well as osteogenesis in these cells. We investigated the relative potencies of Shh and BMP-2 with regard to adipogenesis. We performed differentiation experiments by stimulating C3H10T1/2 cells with BMP-2, Shh, or a combination. We monitored adipocyte-like differentiation via gene expression analysis and cytologic staining. An adipocytic phenotype was observed in BMP-2-treated cells, as shown by upregulation of two adipocytic marker mRNAs, PPAR-gamma and aP2, and by staining of lipid-filled cell vesicles with Oil Red O. In contrast, no adipocyte-like differentiation could be detected either after treatment with Shh or after exposure to a combination of Shh and BMP-2. Our results demonstrate for the first time that Shh and BMP-2 have contrary effects on adipocyte-like differentiation. Whereas BMP-2 promotes the adipocytic lineage, Shh suppresses the expression of the BMP-2-induced fat-cell phenotype.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. Hedgehog signaling in the stomach.

    PubMed

    Konstantinou, Daniel; Bertaux-Skeirik, Nina; Zavros, Yana

    2016-12-01

    The Hedgehog (Hh) signaling pathway not only plays a key part in controlling embryonic development, but in the adult stomach governs important cellular events such as epithelial cell differentiation, proliferation, gastric disease, and regeneration. In particular, Sonic Hedgehog (Shh) signaling has been well studied for its role in gastric physiology and pathophysiology. Shh is secreted from the gastric parietal cells and contributes to the regeneration of the epithelium in response to injury, or the development of gastritis during Helicobacter pylori infection. Dysregulation of the Shh signaling pathway leads to the disruption of gastric differentiation, loss of gastric acid secretion and the development of cancer. In this chapter, we will review the most recent findings that reveal the role of Shh as a regulator of gastric physiology, regeneration, and disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Temporal-spatial changes in Sonic Hedgehog expression and signaling reveal different potentials of ventral mesencephalic progenitors to populate distinct ventral midbrain nuclei

    PubMed Central

    2011-01-01

    Background The ventral midbrain contains a diverse array of neurons, including dopaminergic neurons of the ventral tegmental area (VTA) and substantia nigra (SN) and neurons of the red nucleus (RN). Dopaminergic and RN neurons have been shown to arise from ventral mesencephalic precursors that express Sonic Hedgehog (Shh). However, Shh expression, which is initially confined to the mesencephalic ventral midline, expands laterally and is then downregulated in the ventral midline. In contrast, expression of the Hedgehog target gene Gli1 initiates in the ventral midline prior to Shh expression, but after the onset of Shh expression it is expressed in precursors lateral to Shh-positive cells. Given these dynamic gene expression patterns, Shh and Gli1 expression could delineate different progenitor populations at distinct embryonic time points. Results We employed genetic inducible fate mapping (GIFM) to investigate whether precursors that express Shh (Shh-GIFM) or transduce Shh signaling (Gli1-GIFM) at different time points give rise to different ventral midbrain cell types. We find that precursors restricted to the ventral midline are labeled at embryonic day (E)7.5 with Gli1-GIFM, and with Shh-GIFM at E8.5. These precursors give rise to all subtypes of midbrain dopaminergic neurons and the anterior RN. A broader domain of progenitors that includes the ventral midline is marked with Gli1-GIFM at E8.5 and with Shh-GIFM at E9.5; these fate-mapped cells also contribute to all midbrain dopaminergic subtypes and to the entire RN. In contrast, a lateral progenitor domain that is labeled with Gli1-GIFM at E9.5 and with Shh-GIFM at E11.5 has a markedly reduced potential to give rise to the RN and to SN dopaminergic neurons, and preferentially gives rise to the ventral-medial VTA. In addition, cells derived from Shh- and Gli1-expressing progenitors located outside of the ventral midline give rise to astrocytes. Conclusions We define a ventral midbrain precursor map based on

  6. 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

  7. 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.

  8. 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.

  9. Hedgehog signaling regulates FOXA2 in esophageal embryogenesis and Barrett’s metaplasia

    PubMed Central

    Wang, David H.; Tiwari, Anjana; Kim, Monica E.; Clemons, Nicholas J.; Regmi, Nanda L.; Hodges, William A.; Berman, David M.; Montgomery, Elizabeth A.; Watkins, D. Neil; Zhang, Xi; Zhang, Qiuyang; Jie, Chunfa; Spechler, Stuart J.; Souza, Rhonda F.

    2014-01-01

    Metaplasia can result when injury reactivates latent developmental signaling pathways that determine cell phenotype. Barrett’s esophagus is a squamous-to-columnar epithelial metaplasia caused by reflux esophagitis. Hedgehog (Hh) signaling is active in columnar-lined, embryonic esophagus and inactive in squamous-lined, adult esophagus. We showed previously that Hh signaling is reactivated in Barrett’s metaplasia and overexpression of Sonic hedgehog (SHH) in mouse esophageal squamous epithelium leads to a columnar phenotype. Here, our objective was to identify Hh target genes involved in Barrett’s pathogenesis. By microarray analysis, we found that the transcription factor Foxa2 is more highly expressed in murine embryonic esophagus compared with postnatal esophagus. Conditional activation of Shh in mouse esophageal epithelium induced FOXA2, while FOXA2 expression was reduced in Shh knockout embryos, establishing Foxa2 as an esophageal Hh target gene. Evaluation of patient samples revealed FOXA2 expression in Barrett’s metaplasia, dysplasia, and adenocarcinoma but not in esophageal squamous epithelium or squamous cell carcinoma. In esophageal squamous cell lines, Hh signaling upregulated FOXA2, which induced expression of MUC2, an intestinal mucin found in Barrett’s esophagus, and the MUC2-processing protein AGR2. Together, these data indicate that Hh signaling induces expression of genes that determine an intestinal phenotype in esophageal squamous epithelial cells and may contribute to the development of Barrett’s metaplasia. PMID:25083987

  10. 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.

  11. 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.

  12. 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 Central

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

    2016-01-01

    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. PMID:26716648

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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).

  7. 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

  8. 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

  9. 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

  10. Hedgehog signaling establishes precursors for germline stem cell niches by regulating cell adhesion

    PubMed Central

    Lin, Kun-Yang; Kao, Shih-Han

    2017-01-01

    Stem cells require different types of supporting cells, or niches, to control stem cell maintenance and differentiation. However, little is known about how those niches are formed. We report that in the development of the Drosophila melanogaster ovary, the Hedgehog (Hh) gradient sets differential cell affinity for somatic gonadal precursors to specify stromal intermingled cells, which contributes to both germline stem cell maintenance and differentiation niches in the adult. We also report that Traffic Jam (an orthologue of a large Maf transcription factor in mammals) is a novel transcriptional target of Hh signaling to control cell–cell adhesion by negative regulation of E-cadherin expression. Our results demonstrate the role of Hh signaling in niche establishment by segregating somatic cell lineages for differentiation. PMID:28363970

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. Deubiquitination of Ci/Gli by Usp7/HAUSP regulates Hedgehog Signaling

    PubMed Central

    Zhou, Zizhang; Yao, Xia; Li, Shuang; Xiong, Yue; Dong, Xiaohua; Zhao, Yun; Jiang, Jin; Zhang, Qing

    2015-01-01

    SUMMARY Hedgehog (Hh) signaling plays essential roles in animal development and tissue homeostasis, and its misregulation causes congenital diseases and cancers. Regulation of the ubiquitin/proteasome-mediated proteolysis of Ci/Gli transcription factors is central to Hh signaling, but whether deubiquitinase is involved in this process remains unknown. Here, we show that Hh stimulates the binding of an ubiquitin-specific protease Usp7 to Ci, which positively regulates Hh signaling activity through inhibiting Ci ubiquitination and degradation mediated by both Slimb-Cul1 and Hib-Cul3 E3 ligases. Furthermore, we find that Usp7 forms a complex with GMP-synthetase (GMPS) to promote Hh pathway activity. Finally, we show that the mammalian counterpart of Usp7, HAUSP, positively regulates Hh signaling by modulating Gli ubiquitination and stability. Our findings reveal a conserved mechanism by which Ci/Gli is stabilized by a deubiquitination enzyme and identify Usp7/HUASP as a critical regulator of Hh signaling and potential therapeutic target for Hh-related cancers. PMID:26120032

  17. Deubiquitination of Ci/Gli by Usp7/HAUSP Regulates Hedgehog Signaling.

    PubMed

    Zhou, Zizhang; Yao, Xia; Li, Shuang; Xiong, Yue; Dong, Xiaohua; Zhao, Yun; Jiang, Jin; Zhang, Qing

    2015-07-06

    Hedgehog (Hh) signaling plays essential roles in animal development and tissue homeostasis, and its misregulation causes congenital diseases and cancers. Regulation of the ubiquitin/proteasome-mediated proteolysis of Ci/Gli transcription factors is central to Hh signaling, but whether deubiquitinase is involved in this process remains unknown. Here, we show that Hh stimulates the binding of a ubiquitin-specific protease Usp7 to Ci, which positively regulates Hh signaling activity through inhibiting Ci ubiquitination and degradation mediated by both Slimb-Cul1 and Hib-Cul3 E3 ligases. Furthermore, we find that Usp7 forms a complex with GMP-synthetase (GMPS) to promote Hh pathway activity. Finally, we show that the mammalian counterpart of Usp7, HAUSP, positively regulates Hh signaling by modulating Gli ubiquitination and stability. Our findings reveal a conserved mechanism by which Ci/Gli is stabilized by a deubiquitination enzyme and identify Usp7/HUASP as a critical regulator of Hh signaling and potential therapeutic target for Hh-related cancers. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved

    PubMed Central

    Chen, Miao-Hsueh; Wilson, Christopher W.; Li, Ya-Jun; Law, Kelvin King Lo; Lu, Chi-Sheng; Gacayan, Rhodora; Zhang, Xiaoyun; Hui, Chi-chung; Chuang, Pao-Tien

    2009-01-01

    A central question in Hedgehog (Hh) signaling is how evolutionarily conserved components of the pathway might use the primary cilium in mammals but not fly. We focus on Suppressor of fused (Sufu), a major Hh regulator in mammals, and reveal that Sufu controls protein levels of full-length Gli transcription factors, thus affecting the production of Gli activators and repressors essential for graded Hh responses. Surprisingly, despite ciliary localization of most Hh pathway components, regulation of Gli protein levels by Sufu is cilium-independent. We propose that Sufu-dependent processes in Hh signaling are evolutionarily conserved. Consistent with this, Sufu regulates Gli protein levels by antagonizing the activity of Spop, a conserved Gli-degrading factor. Furthermore, addition of zebrafish or fly Sufu restores Gli protein function in Sufu-deficient mammalian cells. In contrast, fly Smo is unable to translocate to the primary cilium and activate the mammalian Hh pathway. We also uncover a novel positive role of Sufu in regulating Hh signaling, resulting from its control of both Gli activator and repressor function. Taken together, these studies delineate important aspects of cilium-dependent and cilium-independent Hh signal transduction and provide significant mechanistic insight into Hh signaling in diverse species. PMID:19684112

  19. A Genome-Wide RNAi Screen Identifies Regulators of Cholesterol-Modified Hedgehog Secretion in Drosophila

    PubMed Central

    Ruel, Laurent; Lacas-Gervais, Sandra; Schaub, Sébastien; Thérond, Pascal

    2012-01-01

    Hedgehog (Hh) proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion. PMID:22432040

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

    PubMed Central

    Tong, Chao; Wang, Bing; Chen, Yongbin; Jiang, Jin

    2016-01-01

    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. PMID:27280464

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. Secreted HHIP1 interacts with heparan sulfate and regulates Hedgehog ligand localization and function

    PubMed Central

    Holtz, Alexander M.; Griffiths, Samuel C.; Davis, Samantha J.; Bishop, Benjamin; Siebold, Christian

    2015-01-01

    Vertebrate Hedgehog (HH) signaling is controlled by several ligand-binding antagonists including Patched-1 (PTCH1), PTCH2, and HH-interacting protein 1 (HHIP1), whose collective action is essential for proper HH pathway activity. However, the molecular mechanisms used by these inhibitors remain poorly understood. In this paper, we investigated the mechanisms underlying HHIP1 antagonism of HH signaling. Strikingly, we found evidence that HHIP1 non–cell-autonomously inhibits HH-dependent neural progenitor patterning and proliferation. Furthermore, this non–cell-autonomous antagonism of HH signaling results from the secretion of HHIP1 that is modulated by cell type–specific interactions with heparan sulfate (HS). These interactions are mediated by an HS-binding motif in the cysteine-rich domain of HHIP1 that is required for its localization to the neuroepithelial basement membrane (BM) to effectively antagonize HH pathway function. Our data also suggest that endogenous, secreted HHIP1 localization to HS-containing BMs regulates HH ligand distribution. Overall, the secreted activity of HHIP1 represents a novel mechanism to regulate HH ligand localization and function during embryogenesis. PMID:26056142

  8. 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. © 2014 Institute of Zoology, Chinese Academy of Sciences.

  9. 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

  10. 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

  11. Spatially restricted Hedgehog signaling regulates HGF-induced branching of the adult prostate

    PubMed Central

    Lim, Agnes; Shin, Kunyoo; Zhao, Chen; Kawano, Sally; Beachy, Philip A.

    2014-01-01

    Branching morphogenesis is thought to be governed by epithelial-stromal interactions, but the mechanisms underlying specification of branch location remain largely unknown. Prompted by the striking absence of Hedgehog (Hh) response at the sites of nascent buds in regenerating tubules of the adult prostate, we investigated the role of Hh signaling in adult prostate branching morphogenesis. We find that pathway activity is localized to stromal cells, and that its attenuation by genetic or pharmacologic manipulation leads to increased branching. Decreased pathway activity correlates with increased stromal production of Hepatocyte growth factor (Hgf), and we show that Hgf induces epithelial tubule branching. Regulation of Hgf expression by Hh signaling is indirect, mediated by Hh-induced expression of microRNAs miR-26a and miR-26b, which in turn down-regulate expression of Hgf. Prostate tubule branching thus may be initiated from regions of low Hh pathway activity, with implications for the prostatic hyperplasia commonly observed in late adulthood. PMID:25362352

  12. 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

  13. Hedgehog controls neural stem cells through p53-independent regulation of Nanog

    PubMed Central

    Po, Agnese; Ferretti, Elisabetta; Miele, Evelina; De Smaele, Enrico; Paganelli, Arianna; Canettieri, Gianluca; Coni, Sonia; Di Marcotullio, Lucia; Biffoni, Mauro; Massimi, Luca; Di Rocco, Concezio; Screpanti, Isabella; Gulino, Alberto

    2010-01-01

    Hedgehog (Hh) pathway has a pivotal function in development and tumorigenesis, processes sustained by stem cells (SCs). The transcription factor Nanog controls stemness acting as a key determinant of both embryonic SC self-renewal and differentiated somatic cells reprogramming to pluripotency, in concert with the loss of the oncosuppressor p53. How Nanog is regulated by microenvironmental signals in postnatal SC niches has been poorly investigated. Here, we show that Nanog is highly expressed in SCs from postnatal cerebellum and medulloblastoma, and acts as a critical mediator of Hh-driven self-renewal. Indeed, the downstream effectors of Hh activity, Gli1 and Gli2, bind to Nanog-specific cis-regulatory sequences both in mouse and human SCs. Loss of p53, a key event promoting cell stemness, activates Hh signalling, thereby contributing to Nanog upregulation. Conversely, Hh downregulates p53 but does not require p53 to control Nanog. Our data reveal a mechanism for the function of Hh in the control of stemness that represents a crucial component of an integrated circuitry determining cell fate decision and involved in the maintenance of cancer SCs. PMID:20581804

  14. Hedgehog Signaling Regulates Epithelial-Mesenchymal Transition in Pancreatic Cancer Stem-Like Cells

    PubMed Central

    Wang, Feng; Ma, Ling; Zhang, Zhengkui; Liu, Xiaoran; Gao, Hongqiao; Zhuang, Yan; Yang, Pei; Kornmann, Marko; Tian, Xiaodong; Yang, Yinmo

    2016-01-01

    Hedgehog (Hh) signaling is crucially involved in tumorigenesis. This study aimed to assess the role of Hh signaling in the regulation of epithelial-mesenchymal transition (EMT), stemness properties and chemoresistance of human pancreatic Panc-1 cancer stem cells (CSCs). Panc-1 cells were transfected with recombinant lentiviral vectors to silence SMO and serum-free floating-culture system was used to isolate Panc-1 tumorspheres. The expression of CSC and EMT markers was detected by flow cytometry, real-time RT-PCR and Western blot analysis. Malignant behaviors of Panc-1 CSC were evaluated by tumorigenicity assays and nude mouse lung metastasis model. We found that tumorspheres derived from pancreatic cancer cell line Panc-1 possessed self-renewal, differentiation and stemness properties. Hh pathway and EMT were active in Panc-1 tumorspheres. Inhibition of Hh signaling by SMO knockdown inhibited self-renewal, EMT, invasion, chemoresistance, pulmonary metastasis, tumorigenesis of pancreatic CSCs. In conclusion, Hh signaling contributes to the maintenance of stem-like properties and chemoresistance of pancreatic CSC and promotes the tumorigenesis and metastasis of pancreatic cancer. Hh pathway is a potential molecular target for the development of therapeutic strategies for pancreatic CSCs. PMID:26918054

  15. Hedgehog Zoonoses

    PubMed Central

    Riley, Patricia Y.

    2005-01-01

    Exotic pets, including hedgehogs, have become popular in recent years among pet owners, especially in North America. Such animals can carry and introduce zoonotic agents, a fact well illustrated by the recent outbreak of monkeypox in pet prairie dogs. We reviewed known and potential zoonotic diseases that could be carried and transmitted by pet hedgehogs or when rescuing and caring for wild-caught hedgehogs. PMID:15705314

  16. Tectonic, a novel regulator of the Hedgehog pathway required for both activation and inhibition

    PubMed Central

    Reiter, Jeremy F.; Skarnes, William C.

    2006-01-01

    We report the identification of a novel protein that participates in Hedgehog-mediated patterning of the neural tube. This protein, named Tectonic, is the founding member of a previously undescribed family of evolutionarily conserved secreted and transmembrane proteins. During neural tube development, mouse Tectonic is required for formation of the most ventral cell types and for full Hedgehog (Hh) pathway activation. Epistasis analyses reveal that Tectonic modulates Hh signal transduction downstream of Smoothened (Smo) and Rab23. Interestingly, characterization of Tectonic Shh and Tectonic Smo double mutants indicates that Tectonic plays an additional role in repressing Hh pathway activity. PMID:16357211

  17. Tectonic, a novel regulator of the Hedgehog pathway required for both activation and inhibition.

    PubMed

    Reiter, Jeremy F; Skarnes, William C

    2006-01-01

    We report the identification of a novel protein that participates in Hedgehog-mediated patterning of the neural tube. This protein, named Tectonic, is the founding member of a previously undescribed family of evolutionarily conserved secreted and transmembrane proteins. During neural tube development, mouse Tectonic is required for formation of the most ventral cell types and for full Hedgehog (Hh) pathway activation. Epistasis analyses reveal that Tectonic modulates Hh signal transduction downstream of Smoothened (Smo) and Rab23. Interestingly, characterization of Tectonic Shh and Tectonic Smo double mutants indicates that Tectonic plays an additional role in repressing Hh pathway activity.

  18. 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. Copyright © 2015 Elsevier Inc. All

  19. 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.

  20. 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.

  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. The cell-surface proteins Dally-like and Ihog differentially regulate Hedgehog signaling strength and range during development

    PubMed Central

    Yan, Dong; Wu, Yihui; Yang, Yongfei; Belenkaya, Tatyana Y.; Tang, Xiaofang; Lin, Xinhua

    2010-01-01

    Hedgehog (Hh) acts as a morphogen in various developmental contexts to specify distinct cell fates in a concentration-dependent manner. Hh signaling is regulated by two conserved cell-surface proteins: Ig/fibronectin superfamily member Interference hedgehog (Ihog) and Dally-like (Dlp), a glypican that comprises a core protein and heparan sulfate glycosaminoglycan (GAG) chains. Here, we show in Drosophila that the Dlp core protein can interact with Hh and is essential for its function in Hh signaling. In wing discs, overexpression of Dlp increases short-range Hh signaling while reducing long-range signaling. By contrast, Ihog has biphasic activity in Hh signaling in cultured cells: low levels of Ihog increase Hh signaling, whereas high levels decrease it. In wing discs, overexpression of Ihog represses high-threshold targets, while extending the range of low-threshold targets, thus showing opposite effects to Dlp. We further show that Ihog and its family member Boi are required to maintain Hh on the cell surface. Finally, Ihog and Dlp have complementary expression patterns in discs. These data led us to propose that Dlp acts as a signaling co-receptor. However, Ihog might not act as a classic co-receptor; rather, it may act as an exchange factor by retaining Hh on the cell surface, but also compete with the receptor for Hh binding. PMID:20501592

  4. 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.

  5. Zebrafish blowout provides genetic evidence for Patched1 mediated negative regulation of Hedgehog signaling within the proximal optic vesicle of the vertebrate eye

    PubMed Central

    Lee, Jiwoon; Willer, Jason R.; Willer, Gregory B.; Smith, Kierann; Gregg, Ronald G.; Gross, Jeffrey M.

    2008-01-01

    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. PMID:18479681

  6. 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

  7. 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

  8. Spatial Activation of TORC1 Is Regulated by Hedgehog and E2F1 Signaling in the Drosophila Eye.

    PubMed

    Kim, Wonho; Jang, Yoon-Gu; Yang, Jinsung; Chung, Jongkyeong

    2017-08-21

    Target of rapamycin complex 1 (TORC1) regulates cell growth in response to nutrients and growth factors. Although TORC1 signaling has been thoroughly studied at the cellular level, the regulation of TORC1 in multicellular tissues and organs has remained elusive. Here we found that TORC1 is selectively activated in the second mitotic wave (SMW), the terminal synchronous cell division, of the developing Drosophila eye. We demonstrated that Hedgehog (Hh) signaling regulates TORC1 through E2F1 and the cyclin D/Cdk4 complex in the SMW, and this regulation is independent from insulin and amino acid signaling pathways. TORC1 is necessary for the proper G1/S transition of the cells, and the activation of TORC1 rescues the cell-cycle defect of Hh signaling-deficient cells in the SMW. Based on this evolutionarily conserved regulation of TORC1 by Hh signaling, we propose that Hh-dependent developmental signaling pathways spatially regulate TORC1 activity in multicellular organisms. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. 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

  10. 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

  11. Smoothened Regulates Activator and Repressor Functions of Hedgehog Signaling via Two Distinct Mechanisms*

    PubMed Central

    Ogden, Stacey K.; Casso, David J.; Ascano, Manuel; Yore, Mark M.; Kornberg, Thomas B.; Robbins, David J.

    2013-01-01

    The secreted protein Hedgehog (Hh) plays an important role in metazoan development and as a survival factor for many human tumors. In both cases, Hh signaling proceeds through the activation of the seven-transmembrane protein Smoothened (Smo), which is thought to convert the Gli family of transcription factors from transcriptional repressors to transcriptional activators. Here, we provide evidence that Smo signals to the Hh signaling complex, which consists of the kinesin-related protein Costal2 (Cos2), the protein kinase Fused (Fu), and the Drosophila Gli homolog cubitus interruptus (Ci), in two distinct manners. We show that many of the commonly observed molecular events following Hh signaling are not transmitted in a linear fashion but instead are activated through two signals that bifurcate at Smo to independently affect activator and repressor pools of Ci. PMID:16423832

  12. 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.

  13. 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.

  14. 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

  15. DOSAGE-DEPENDENT REGULATION OF PANCREATIC CANCER GROWTH AND ANGIOGENESIS BY HEDGEHOG SIGNALING

    PubMed Central

    Mathew, Esha; Zhang, Yaqing; Holtz, Alexander M.; Kane, Kevin T.; Song, Jane Y.; Allen, Benjamin L.; Pasca di Magliano, Marina

    2014-01-01

    Summary Pancreatic cancer, a hypovascular and highly desmoplastic cancer, is characterized by tumor expression of Hedgehog (HH) ligands which signal to fibroblasts in the surrounding stroma that in turn promote tumor survival and growth. However, the mechanisms and consequences of stromal HH pathway activation are not well understood. Here we show that the HH co-receptors GAS1, BOC, and CDON are expressed in cancer-associated fibroblasts. Deletion of two co-receptors (Gas1 and Boc) in fibroblasts reduces HH-responsiveness. Strikingly, these fibroblasts promote greater tumor growth in vivo that correlates with increased tumor-associated vascularity. In contrast, deletion of all three co-receptors (Gas1, Boc and Cdon) results in the near complete abrogation of HH signaling and a corresponding failure to promote tumorigenesis and angiogenesis. Collectively, these data identify a novel role for HH-dosage in pancreatic cancer promotion and may explain the clinical failure of HH pathway blockade as a therapeutic approach in pancreatic cancer. PMID:25310976

  16. 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

  17. 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.

  18. Osr1 functions downstream of Hedgehog pathway to regulate foregut development.

    PubMed

    Han, Lu; Xu, Jingyue; Grigg, Emily; Slack, Megan; Chaturvedi, Praneet; Jiang, Rulang; Zorn, Aaron M

    2017-07-01

    During early fetal development, paracrine Hedgehog (HH) ligands secreted from the foregut epithelium activate Gli transcription factors in the surrounding mesenchyme to coordinate formation of the respiratory system, digestive track and the cardiovascular network. Although disruptions to this process can lead to devastating congenital defects, the underlying mechanisms and downstream targets, are poorly understood. We show that the zinc finger transcription factor Osr1 is a novel HH target as Osr1 expression in the foregut mesenchyme depends on HH signaling and the effector of HH pathway Gli3 binds to a conserved genomic loci near Osr1 promoter region. Molecular analysis of mouse germline Osr1 mutants reveals multiple functions of Osr1 during foregut development. Osr1 mutants exhibit fewer lung progenitors in the ventral foregut. Osr is then required for the proper branching of the primary lung buds, with mutants exhibiting miss-located lung lobes. Finally, Osr1 is essential for proper mesenchymal differentiation including pulmonary arteries, esophageal and tracheal smooth muscle as well as tracheal cartilage rings. Tissue specific conditional knockouts in combination with lineage tracing indicate that Osr1 is required cell autonomously in the foregut mesenchyme. We conclude that Osr1 is a novel downstream target of HH pathway, required for lung specification, branching morphogenesis and foregut mesenchymal differentiation. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. 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.

  20. Hedgehog-mediated paracrine interaction between hepatic stellate cells and marrow-derived mesenchymal stem cells

    SciTech Connect

    Lin Nan Tang Zhaofeng; Deng Meihai; Zhong Yuesi; Lin Jizong; Yang Xuhui; Xiang Peng; Xu Ruiyun

    2008-07-18

    During liver injury, bone marrow-derived mesenchymal stem cells (MSCs) can migrate and differentiate into hepatocytes. Hepatic stellate cell (SC) activation is a pivotal event in the development of liver fibrosis. Therefore, we hypothesized that SCs may play an important role in regulating MSC proliferation and differentiation through the paracrine signaling pathway. We demonstrate that MSCs and SCs both express hedgehog (Hh) pathway components, including its ligands, receptors, and target genes. Transwell co-cultures of SCs and MSCs showed that the SCs produced sonic hedgehog (Shh), which enhanced the proliferation and differentiation of MSCs. These findings demonstrate that SCs indirectly modulate the activity of MSCs in vitro via the Hh pathway, and provide a plausible explanation for the mechanisms of transplanted MSCs in the treatment of liver fibrosis.

  1. 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

  2. Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling

    PubMed Central

    Lin, Chuwen; Yao, Erica; Wang, Kevin; Nozawa, Yoko; Shimizu, Hirohito; Johnson, Jeffrey R.; Chen, Jau-Nian; Krogan, Nevan J.

    2014-01-01

    Control of Gli function by Suppressor of Fused (Sufu), a major negative regulator, is a key step in mammalian Hedgehog (Hh) signaling, but how this is achieved in the nucleus is unknown. We found that Hh signaling results in reduced Sufu protein levels and Sufu dissociation from Gli proteins in the nucleus, highlighting critical functions of Sufu in the nucleus. Through a proteomic approach, we identified several Sufu-interacting proteins, including p66β (a member of the NuRD [nucleosome remodeling and histone deacetylase] repressor complex) and Mycbp (a Myc-binding protein). p66β negatively and Mycbp positively regulate Hh signaling in cell-based assays and zebrafish. They function downstream from the membrane receptors, Patched and Smoothened, and the primary cilium. Sufu, p66β, Mycbp, and Gli are also detected on the promoters of Hh targets in a dynamic manner. Our results support a new model of Hh signaling in the nucleus. Sufu recruits p66β to block Gli-mediated Hh target gene expression. Meanwhile, Mycbp forms a complex with Gli and Sufu without Hh stimulation but remains inactive. Hh pathway activation leads to dissociation of Sufu/p66β from Gli, enabling Mycbp to promote Gli protein activity and Hh target gene expression. These studies provide novel insight into how Sufu controls Hh signaling in the nucleus. PMID:25403183

  3. 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.

  4. 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.

  5. Two Lamprey Hedgehog Genes Share Non-Coding Regulatory Sequences and Expression Patterns with Gnathostome Hedgehogs

    PubMed Central

    Ekker, Marc; Hadzhiev, Yavor; Müller, Ferenc; Casane, Didier; Magdelenat, Ghislaine; Rétaux, Sylvie

    2010-01-01

    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. PMID:20967201

  6. 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.

  7. 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.

  8. 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.

  9. Indian Hedgehog, a critical modulator in osteoarthritis, could be a potential therapeutic target for attenuating cartilage degeneration disease.

    PubMed

    Zhou, Jingming; Wei, Xiaochun; Wei, Lei

    2014-08-01

    The Hedgehog (Hh) family of proteins consists of Indian hedgehog (Ihh), sonic hedgehog (Shh), and desert hedgehog (Dhh). These proteins serve as essential regulators in a variety of developmental events. Ihh is mainly produced and secreted by prehypertrophic chondrocytes and regulates chondrocyte hypertrophy and endochondral bone formation during growth plate development. Tissue-specific deletion of the Ihh gene (targeted by Col2a1-Cre) causes early lethality in mice. Transgenic mice with induced Ihh expression exhibit increased chondrocyte hypertrophy and cartilage damage resembling human osteoarthritis (OA). During OA development, chondrocytes recapitulate the differentiation process that happens during the fetal status and which does not occur to an appreciable degree in adult articular cartilage. Ihh expression is up-regulated in human OA cartilage, and this upregulation correlates with OA progression and changes in chondrocyte morphology. A genetic study in mice further showed that conditional deletion of Ihh in chondrocytes attenuates OA progression, suggesting the possibility that blocking Ihh signaling can be used as a therapeutic approach to prevent or delay cartilage degeneration. However, Ihh gene deletion is currently not a therapeutic option as it is lethal in animals. RNA interference (RNAi) provides a means to knockdown Ihh without the severe side effects caused by chemical inhibitors. The currently available delivery methods for RNAi are nanoparticles and liposomes. Both have problems that need to be addressed. In the future, it will be necessary to develop a safe and effective RNAi delivery system to target Ihh signaling for preventing and treating OA.

  10. A switch from low to high Shh activity regulates establishment of limb progenitors and signaling centers

    USDA-ARS?s Scientific Manuscript database

    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...

  11. 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.

  12. 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.

  13. Cytoneme-Mediated Delivery of Hedgehog Regulates the Expression of Bone Morphogenetic Proteins to Maintain Germline Stem Cells in Drosophila

    PubMed Central

    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. PMID:22509132

  14. Hedgehog compactification

    SciTech Connect

    Guendelman, E.I.; Rabinowitz, A.I. )

    1993-04-15

    We show that the presence of a hedgehog field configuration of a Higgs field, which causes spontaneous symmetry breaking of the internal symmetry, can also cause spontaneous compactification of two of the dimensions into a two-sphere. In contrast with other mechanisms of spontaneous compactification, when the higher-dimensional cosmological constant is zero, we still get a zero effective four-dimensional cosmological constant, and not a large negative curvature of four-dimensional space. In the models in which this mechanism works, the scale of symmetry breaking is fixed self-consistently to be the Planck scale.

  15. 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.

  16. 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.

  17. 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.

  18. Nkx factors specifically regulate expression of Hedgehog receptor isoforms in early embryonic development

    USDA-ARS?s Scientific Manuscript database

    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...

  19. 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

  20. 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.

  1. 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.

  2. Secretion and Signaling Activities of Lipoprotein-Associated Hedgehog and Non-Sterol-Modified Hedgehog in Flies and Mammals

    PubMed Central

    Kumari, Veena; Ehrhart-Bornstein, Monika; Bornstein, Stefan R.; Eaton, Suzanne

    2013-01-01

    Hedgehog (Hh) proteins control animal development and tissue homeostasis. They activate gene expression by regulating processing, stability, and activation of Gli/Cubitus interruptus (Ci) transcription factors. Hh proteins are secreted and spread through tissue, despite becoming covalently linked to sterol during processing. Multiple mechanisms have been proposed to release Hh proteins in distinct forms; in Drosophila, lipoproteins facilitate long-range Hh mobilization but also contain lipids that repress the pathway. Here, we show that mammalian lipoproteins have conserved roles in Sonic Hedgehog (Shh) release and pathway repression. We demonstrate that lipoprotein-associated forms of Hh and Shh specifically block lipoprotein-mediated pathway inhibition. We also identify a second conserved release form that is not sterol-modified and can be released independently of lipoproteins (Hh-N*/Shh-N*). Lipoprotein-associated Hh/Shh and Hh-N*/Shh-N* have complementary and synergistic functions. In Drosophila wing imaginal discs, lipoprotein-associated Hh increases the amount of full-length Ci, but is insufficient for target gene activation. However, small amounts of non-sterol-modified Hh synergize with lipoprotein-associated Hh to fully activate the pathway and allow target gene expression. The existence of Hh secretion forms with distinct signaling activities suggests a novel mechanism for generating a diversity of Hh responses. PMID:23554573

  3. 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

  4. Human germline hedgehog pathway mutations predispose to fatty liver.

    PubMed

    Guillen-Sacoto, Maria J; Martinez, Ariel F; Abe, Yu; Kruszka, Paul; Weiss, Karin; Everson, Joshua L; Bataller, Ramon; Kleiner, David E; Ward, Jerrold M; Sulik, Kathleen K; Lipinski, Robert J; Solomon, Benjamin D; Muenke, Maximilian

    2017-10-01

    Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease. Activation of hedgehog (Hh) signaling has been implicated in the progression of NAFLD and proposed as a therapeutic target; however, the effects of Hh signaling inhibition have not been studied in humans with germline mutations that affect this pathway. Patients with holoprosencephaly (HPE), a disorder associated with germline mutations disrupting Sonic hedgehog (SHH) signaling, were clinically evaluated for NAFLD. A combined mouse model of Hh signaling attenuation (Gli2 heterozygous null: Gli2(+/-)) and diet-induced NAFLD was used to examine aspects of NAFLD and hepatic gene expression profiles, including molecular markers of hepatic fibrosis and inflammation. Patients with HPE had a higher prevalence of liver steatosis compared to the general population, independent of obesity. Exposure of Gli2(+/-) mice to fatty liver-inducing diets resulted in increased liver steatosis compared to wild-type mice. Similar to humans, this effect was independent of obesity in the mutant mice and was associated with decreased expression of pro-fibrotic and pro-inflammatory genes, and increased expression of PPARγ, a potent anti-fibrogenic and anti-inflammatory regulator. Interestingly, tumor suppressors p53 and p16INK4 were found to be downregulated in the Gli2(+/-) mice exposed to a high-fat diet. Our results indicate that germline mutations disrupting Hh signaling promotes liver steatosis, independent of obesity, with reduced fibrosis. While Hh signaling inhibition has been associated with a better NAFLD prognosis, further studies are required to evaluate the long-term effects of mutations affecting this pathway. Lay summary: Non-alcoholic fatty liver disease (NAFLD) is characterized by excess fat deposition in the liver predominantly due to high calorie intake and a sedentary lifestyle. NAFLD progression is usually accompanied by activation of the Sonic hedgehog (SHH) pathway leading to fibrous

  5. EFCAB7 and IQCE regulate Hedgehog signaling by tethering the EVC-EVC2 complex to the base of primary cilia

    PubMed Central

    Pusapati, Ganesh V.; Hughes, Casey E; Dorn, Karolin V.; Zhang, Dapeng; Sugianto, Priscilla; Aravind, L.; Rohatgi, Rajat

    2014-01-01

    The Hedgehog (Hh) pathway depends on primary cilia in vertebrates, but the signaling machinery within cilia remains incompletely defined. We report the identification of a complex between two ciliary proteins, EFCAB7 and IQCE, which positively regulates the Hh pathway. The EFCAB7-IQCE module anchors the EVC-EVC2 complex in a signaling microdomain at the base of cilia. EVC and EVC2 genes are mutated in Ellis van Creveld and Weyers syndromes, characterized by impaired Hh signaling in skeletal, cardiac and orofacial tissues. EFCAB7 binds to a C-terminal disordered region in EVC2 that is deleted in Weyers patients. EFCAB7 depletion mimics the Weyers cellular phenotype— the mis-localization of EVC-EVC2 within cilia and impaired activation of the transcription factor GLI2. Evolutionary analysis suggests that emergence of these complexes might have been important for adaptation of an ancient organelle, the cilium, for an animal-specific signaling network. PMID:24582806

  6. Treatment of Medulloblastoma with Hedgehog Pathway Inhibitor GDC-0449

    PubMed Central

    Rudin, Charles M.; Hann, Christine L.; Laterra, John; Yauch, Robert L.; Callahan, Christopher A.; Fu, Ling; Holcomb, Thomas; Stinson, Jeremy; Gould, Stephen E.; Coleman, Barbara; LoRusso, Patricia M.; Von Hoff, Daniel D.; de Sauvage, Frederic J.; Low, Jennifer A.

    2017-01-01

    SUMMARY Medulloblastoma is the most common malignant brain tumor in children. Aberrant activation of the hedgehog signaling pathway is strongly implicated in the development of some cases of medulloblastoma. A 26-year-old man with metastatic medulloblastoma that was refractory to multiple therapies was treated with a novel hedgehog pathway inhibitor, GDC-0449; treatment resulted in rapid (although transient) regression of the tumor and reduction of symptoms. Molecular analyses of tumor specimens obtained before treatment suggested that there was activation of the hedgehog pathway, with loss of heterozygosity and somatic mutation of the gene encoding patched homologue 1 (PTCH1), a key negative regulator of hedgehog signaling. PMID:19726761

  7. 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

  8. Hedgehog signaling pathway in small bovine ovarian follicles

    USDA-ARS?s Scientific Manuscript database

    The hedgehog signaling pathway is involved in the regulation of cell proliferation, differentiation, and turnover in a variety of mammalian embryonic and a