Soluble Heparin and Heparan Sulfate Glycosaminoglycans Interfere with Sonic Hedgehog Solubilization and Receptor Binding

Sonic hedgehog (Shh) signaling plays a tumor-promoting role in many epithelial cancers. Cancer cells produce soluble a Shh that signals to distant stromal cells that express the receptor Patched (Ptc). These receiving cells respond by producing other soluble factors that promote cancer cell growth, generating a positive feedback loop. To interfere with reinforced Shh signaling, we examined the potential of defined heparin and heparan sulfate (HS) polysaccharides to block Shh solubilization and Ptc receptor binding. We confirm in vitro and in vivo that proteolytic cleavage of the N-terminal Cardin–Weintraub (CW) amino acid motif is a prerequisite for Shh solubilization and function. Consistent with the established binding of soluble heparin or HS to the Shh CW target motif, both polysaccharides impaired proteolytic Shh processing and release from source cells. We also show that HS and heparin bind to, and block, another set of basic amino acids required for unimpaired Shh binding to Ptc receptors on receiving cells. Both modes of Shh activity downregulation depend more on HS size and overall charge than on specific HS sulfation modifications. We conclude that heparin oligosaccharide interference in the physiological roles of HS in Shh release and reception may be used to expand the field of investigation to pharmaceutical intervention of tumor-promoting Shh functions.

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FoxF1 is Required for Ciliogenesis and Distribution of Sonic Hedgehog Signaling Components in Cilium

Current Molecular Medicine ◽

10.2174/1566524019666190405115420 ◽

2019 ◽

Vol 19 (5) ◽

pp. 326-334

Author(s):

Lu Huang ◽

Marco Tjakra ◽

Desha Luo ◽

Lin Wen ◽

Daoxi Lei ◽

...

Keyword(s):

Sonic Hedgehog ◽

Hedgehog Signaling ◽

3T3 Cells ◽

Shh Signaling ◽

Nih 3T3 ◽

Signaling Components ◽

Nih 3T3 Cells

Background: In vertebrates, cilium is crucial for Hedgehog signaling transduction. Forkhead box transcriptional factor FoxF1 is reported to be associated with Sonic Hedgehog (Shh) signaling in many cases. However, the role of FoxF1 in cilium remains unknown. Here, we showed an essential role of FoxF1 in the regulation of ciliogenesis and in the distribution of Shh signaling components in cilium. Methods: NIH/3T3 cells were serum starved for 24h to induce cilium. Meanwhile, shRNA was used to knockdown the FoxF1 expression in the cells and CRISPR/Cas9 was used to generate the FoxF1 zebrafish mutant. The mRNA and protein expression of indicated genes were detected by the qRT-PCR and western blot, respectively. Immunofluorescence staining was performed to detect the cilium and Shh components distribution. Results: FoxF1 knockdown decreased the cilium length in NIH/3T3 cells. Meanwhile, the disruption of FoxF1 function inhibited the expression of cilium-related genes and caused an abnormal distribution of Shh components in the cilium. Furthermore, homozygous FoxF1 mutants exhibited defective development of pronephric cilium in early zebrafish embryos. Conclusion: Together, our data illustrated that FoxF1 is required for ciliogenesis in vitro and in vivo and for the proper localization of Shh signaling components in cilium.

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Mediator Modulates Gli3-Dependent Sonic Hedgehog Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.00443-06 ◽

2006 ◽

Vol 26 (23) ◽

pp. 8667-8682 ◽

Cited By ~ 78

Author(s):

Haiying Zhou ◽

Seokjoong Kim ◽

Shunsuke Ishii ◽

Thomas G. Boyer

Keyword(s):

Gene Transcription ◽

Sonic Hedgehog ◽

Target Gene ◽

Dominant Negative ◽

Transactivation Domain ◽

Functional Interaction ◽

Shh Signaling ◽

Target Gene Transcription

ABSTRACT The physiological and pathological manifestations of Sonic hedgehog (Shh) signaling arise from the specification of unique transcriptional programs dependent upon key nuclear effectors of the Ci/Gli family of transcription factors. However, the underlying mechanism by which Gli proteins regulate target gene transcription in the nucleus remains poorly understood. Here, we identify and characterize a physical and functional interaction between Gli3 and the MED12 subunit within the RNA polymerase II transcriptional Mediator. We show that Gli3 binds to MED12 and intact Mediator both in vitro and in vivo through a Gli3 transactivation domain (MBD; MED12/Mediator-binding domain) whose activity derives from concerted functional interactions with both Mediator and the histone acetyltransferase CBP. Analysis of MBD truncation mutants revealed an excellent correlation between the in vivo activation strength of an MBD derivative and its ability to bind MED12 and intact Mediator in vitro, indicative of a critical functional interaction between the Gli3 MBD and the MED12 interface in Mediator. Disruption of the Gli3-MED12 interaction through dominant-negative interference inhibited, while RNA interference-mediated MED12 depletion enhanced, both MBD transactivation function and Gli3 target gene induction in response to Shh signaling. We propose that activated Gli3 physically targets the MED12 interface within Mediator in order to functionally reverse Mediator-dependent suppression of Shh target gene transcription. These findings thus link MED12 to the modulation of Gli3-dependent Shh signaling and further implicate Mediator in a broad range of developmental and pathological processes driven by Shh signal transduction.

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Investigation of Structure-Activity Relationships of Oxyntomodulin (Oxm) Using Oxm Analogs

Endocrinology ◽

10.1210/en.2008-0828 ◽

2008 ◽

Vol 150 (4) ◽

pp. 1712-1721 ◽

Cited By ~ 66

Author(s):

Maralyn R. Druce ◽

James S. Minnion ◽

Benjamin C. T. Field ◽

Sejal R. Patel ◽

Joyceline C. Shillito ◽

...

Keyword(s):

Food Intake ◽

Receptor Binding ◽

Neutral Endopeptidase ◽

Duration Of Action ◽

Taste Avoidance ◽

Peptide Analogs ◽

C Terminus ◽

And Function

Oxyntomodulin (Oxm) is an intestinal peptide that inhibits food intake and body weight in rodents and humans. These studies used peptide analogs to study aspects of structure and function of Oxm, and the sensitivity of parts of the Oxm sequence to degradation. Analogs of Oxm were synthesized and studied using receptor binding and degradation studies in vitro. Their effects on food intake and conditioned taste avoidance were measured in vivo in rodents. Oxm breakdown by the enzyme dipeptidyl peptidase IV (DPPIV) was demonstrated in vitro and in vivo. In vitro degradation was reduced and in vivo bioactivity increased by inhibitors of DPPIV. Modifications to the N terminus of Oxm modulated binding to the glucagon-like peptide (GLP)-1 receptor and degradation by DPPIV. Modifications to the midsection of Oxm modulated binding to the GLP-1 receptor and degradation by neutral endopeptidase. These modifications also altered bioactivity in vivo. The C-terminal octapeptide of Oxm was shown to contribute to the properties of Oxm in vitro and in vivo but was not alone sufficient for the effects of the peptide. Elongation and acylation of the C terminus of Oxm altered GLP-1 receptor binding and duration of action in vivo, which may be due to changes in peptide clearance. An Oxm analog was developed with enhanced pharmaceutical characteristics, with greater potency and longevity with respect to effects on food intake. These studies suggest that Oxm is a potential target for antiobesity drug design.

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Sonic Hedgehog Signaling Drives Proliferation of Synoviocytes in Rheumatoid Arthritis: A Possible Novel Therapeutic Target

Journal of Immunology Research ◽

10.1155/2014/401903 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 10

Author(s):

Mingxia Wang ◽

Shangling Zhu ◽

Weixiang Peng ◽

Qiuxia Li ◽

Zhaoxia Li ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Sonic Hedgehog ◽

Therapeutic Target ◽

Hedgehog Signaling ◽

Specific Inhibitor ◽

Sonic Hedgehog Signaling ◽

Shh Signaling ◽

Novel Therapeutic

Sonic hedgehog (Shh) signaling controls many aspects of human development, regulates cell growth and differentiation in adult tissues, and is activated in a number of malignancies. Rheumatoid arthritis (RA) is characterized by chronic synovitis and pannus formation associated with activation of fibroblast-like synoviocytes (FLS). We investigated whether Shh signaling plays a role in the proliferation of FLS in RA. Expression of Shh signaling related components (Shh, Ptch1, Smo, and Gli1) in RA synovial tissues was examined by immunohistochemistry (IHC) and in FLS by IHC, immunofluorescence (IF), quantitative RT-PCR, and western blotting. Expression of Shh, Smo, and Gli1 in RA synovial tissue was higher than that in control tissue (P<0.05). Cyclopamine (a specific inhibitor of Shh signaling) decreased mRNA expression of Shh, Ptch1, Smo, and Gli1 in cultured RA FLS, Shh, and Smo protein expression, and significantly decreased FLS proliferation. Flow cytometry analysis suggested that cyclopamine treatment resulted in cell cycle arrest of FLS in G1phase. Our data show that Shh signaling is activated in synovium of RA patientsin vivoand in cultured FLS form RA patientsin vitro, suggesting a role in the proliferation of FLS in RA. It may therefore be a novel therapeutic target in RA.

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Astrocytic Sonic Hedgehog Alleviates Intracerebral Hemorrhagic Brain Injury via Modulation of Blood-Brain Barrier Integrity

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2020.575690 ◽

2020 ◽

Vol 14 ◽

Author(s):

Gebeili Xing ◽

Tianman Zhao ◽

Xiyue Zhang ◽

He Li ◽

Xiuping Li ◽

...

Keyword(s):

Brain Injury ◽

Signaling Pathway ◽

Blood Brain Barrier ◽

Sonic Hedgehog ◽

Critical Role ◽

Brain Barrier ◽

Shh Signaling ◽

Blood Brain

Background: Intracerebral hemorrhage (ICH) is a fatal subtype of stroke that lacks effective therapy. Blood-brain barrier (BBB) damage is a hallmark of ICH-induced brain injury that leads to edema formation, leukocytes infiltration, influx of blood components into the perihematomal (PHE) region, and eventually brain injury. Astrocytes are essential for the formation and maintenance of the BBB by providing secreted molecules that contribute to the association between these cells. Sonic hedgehog (SHH) derived from astrocytes promotes the maturity and integrity of the BBB by upregulating tight junctions (TJs) in brain capillary endothelial cells (ECs). However, the effect of SHH on BBB in ICH has not been investigated.Methods: Cyclopamine (CYC) is a potent, selective inhibitor that specifically blocks the SHH signaling pathway. Here, we used pharmacological inhibitions (CYC and its derivatives) to determine a critical role of the SHH signaling pathway in promoting BBB integrity after ICH by mechanisms of regulating the TJ proteins in vivo and in vitro.Results: The expression of astrocytic SHH was upregulated in mouse brains after ICH. Compared with the vehicle-treated group, inhibition of the SHH signaling pathway with CYC and its derivatives treatments aggravated neurological function deficits, brain edema, hematoma volume, and BBB impairment by downregulating TJs in ECs through the SHH-Gli-1 axis in vivo and in vitro.Conclusions: SHH signaling pathway at the level of the BBB provides a barrier-promoting effect, suggesting that the SHH signaling pathway may function as a potential therapeutic target for restoring BBB function in ICH.

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Disruption of the ciliary GTPase Arl13b suppresses Sonic hedgehog overactivation and inhibits medulloblastoma formation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1706977115 ◽

2018 ◽

Vol 115 (7) ◽

pp. 1570-1575 ◽

Cited By ~ 20

Author(s):

Sarah N. Bay ◽

Alyssa B. Long ◽

Tamara Caspary

Keyword(s):

Sonic Hedgehog ◽

Pediatric Brain Tumor ◽

Resistance Mechanisms ◽

Tumor Formation ◽

Tumor Resistance ◽

Shh Signaling ◽

Genetic Ablation ◽

Primary Mouse

Medulloblastoma (MB) is the most common malignant pediatric brain tumor, and overactivation of the Sonic Hedgehog (Shh) signaling pathway, which requires the primary cilium, causes 30% of MBs. Current treatments have known negative side effects or resistance mechanisms, so new treatments are necessary. Shh signaling mutations, like those that remove Patched1 (Ptch1) or activate Smoothened (Smo), cause tumors dependent on the presence of cilia. Genetic ablation of cilia prevents these tumors by removing Gli activator, but cilia are a poor therapeutic target since they support many biological processes. A more appropriate strategy would be to identify a protein that functionally disentangles Gli activation and ciliogenesis. Our mechanistic understanding of the ciliary GTPase Arl13b predicts that it could be such a target. Arl13b mutants retain short cilia, and loss of Arl13b results in ligand-independent, constitutive, low-level pathway activation but prevents maximal signaling without disrupting Gli repressor. Here, we show that deletion of Arl13b reduced Shh signaling levels in the presence of oncogenic SmoA1, suggesting Arl13b acts downstream of known tumor resistance mechanisms. Knockdown of ARL13B in human MB cell lines and in primary mouse MB cell culture decreased proliferation. Importantly, loss of Arl13b in a Ptch1-deleted mouse model of MB inhibited tumor formation. Postnatal depletion of Arl13b does not lead to any overt phenotypes in the epidermis, liver, or cerebellum. Thus, our in vivo and in vitro studies demonstrate that disruption of Arl13b inhibits cilia-dependent oncogenic Shh overactivation.

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Sonic hedgehog signaling is required during the appearance of spinal cord oligodendrocyte precursors

Development ◽

10.1242/dev.126.11.2419 ◽

1999 ◽

Vol 126 (11) ◽

pp. 2419-2429 ◽

Cited By ~ 8

Author(s):

D.M. Orentas ◽

J.E. Hayes ◽

K.L. Dyer ◽

R.H. Miller

Keyword(s):

Spinal Cord ◽

Sonic Hedgehog ◽

Ventricular Zone ◽

Developmental Period ◽

Dorsal Spinal Cord ◽

Shh Signaling ◽

Oligodendrocyte Precursors ◽

Dorsal Spinal

Spinal cord oligodendrocyte precursors arise in the ventral ventricular zone as a result of local signals. Ectopic oligodendrocyte precursors can be induced by sonic hedgehog (Shh) in explants of chick dorsal spinal cord over an extended developmental period. The role of Shh during normal oligodendrocyte development is, however, unclear. Here we demonstrate that Shh is localized to the ventral spinal cord immediately prior to, and during the appearance of oligodendrocyte precursors. Continued expression of Shh is required for the appearance of spinal cord oligodendrocyte precursors as neutralization of Shh signaling both in vivo and in vitro during a defined developmental period blocked their emergence. The inhibition of oligodendrocyte precursor emergence in the absence of Shh signaling was not the result of inhibiting precursor cell proliferation, and the neutralization of Shh signaling after the emergence of oligodendrocyte precursors had no effect on the appearance of additional cells or their subsequent differentiation. Similar concentrations of Shh induce motor neurons and oligodendrocytes in dorsal spinal cord explants. However, in explants from early embryos the motor neuron lineage is preferentially expanded while in explants from older embryos the oligodendrocyte lineage is preferentially expanded.

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Multifaceted functions of Rab23 on primary cilium and Hedgehog signaling-mediated granule cell proliferation

10.1101/2020.08.01.231985 ◽

2020 ◽

Author(s):

CHH Hor ◽

WY Leong ◽

ELK Goh

Keyword(s):

Sonic Hedgehog ◽

Primary Cilium ◽

Granule Cell ◽

Hedgehog Signaling ◽

Cell Precursor ◽

Shh Signaling ◽

Shh Pathway ◽

Granule Cell Precursor

AbstractSonic Hedgehog (Shh) signaling from the primary cilium drives cerebellar granule cell precursor (GCP) proliferation. Mutations of hedgehog (Hh) pathway repressors could cause medulloblastoma, the most prevalent and malignant childhood brain tumor that arises from aberrant GCP proliferation. We demonstrate that brain-specific knockout of a Shh pathway repressor Rab23 in mice caused mis-patterning of cerebellar folia and elevated GCP proliferation during early development, but with no prevalent occurrence of medulloblastoma at adult stage. Strikingly, Rab23-depleted GCPs exhibited up-regulated basal level of Shh pathway activities despite reduced ciliation, and were desensitized against stimulations by Shh and Smoothened (Smo) agonist in primary GCP culture. These results illustrate dual functions of Rab23 in repressing the basal level of Shh signaling, while facilitating Shh signal transduction via Shh/Smo on primary cilium. Collectively, our findings unravel instrumental roles of Rab23 in GCP proliferation and ciliogenesis. Rab23’s potentiation of Shh signaling pathway through the primary cilium and Smo, suggests a potential new therapeutic for Smo/primary cilium-driven medulloblastoma.Author SummaryC.H.H conceived, designed, lead, and performed all in vitro and in vivo experiments, analyzed data and wrote the manuscript. W.Y performed QPCR experiments and primary GCP cultures and analyzed data. E.L.G conceived and directed the study.

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The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis

Development ◽

10.1242/dev.128.24.5201 ◽

2001 ◽

Vol 128 (24) ◽

pp. 5201-5212 ◽

Cited By ~ 1

Author(s):

Nadia Dahmane ◽

Pilar Sánchez ◽

Yorick Gitton ◽

Verónica Palma ◽

Tao Sun ◽

...

Keyword(s):

Brain Tumors ◽

Sonic Hedgehog ◽

Brain Structures ◽

Brain Growth ◽

Shh Signaling ◽

In Vivo Assays ◽

Plastic Process ◽

The Brain

The mechanisms that regulate the growth of the brain remain unclear. We show that Sonic hedgehog (Shh) is expressed in a layer-specific manner in the perinatal mouse neocortex and tectum, whereas the Gli genes, which are targets and mediators of SHH signaling, are expressed in proliferative zones. In vitro and in vivo assays show that SHH is a mitogen for neocortical and tectal precursors and that it modulates cell proliferation in the dorsal brain. Together with its role in the cerebellum, our findings indicate that SHH signaling unexpectedly controls the development of the three major dorsal brain structures. We also show that a variety of primary human brain tumors and tumor lines consistently express the GLI genes and that cyclopamine, a SHH signaling inhibitor, inhibits the proliferation of tumor cells. Using the in vivo tadpole assay system, we further show that misexpression of GLI1 induces CNS hyperproliferation that depends on the activation of endogenous Gli1 function. SHH-GLI signaling thus modulates normal dorsal brain growth by controlling precursor proliferation, an evolutionarily important and plastic process that is deregulated in brain tumors.

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