Induction Of Sonic Hedgehog Research Articles

Polysaccharides from Basella alba Protect Post-Mitotic Neurons against Cell Cycle Re-Entry and Apoptosis Induced by the Amyloid-Beta Peptide by Blocking Sonic Hedgehog Expression.

The amyloid-beta peptide (Aβ) is the neurotoxic component in senile plaques of Alzheimer's disease (AD) brains. Previously we have reported that Aβ toxicity is mediated by the induction of sonic hedgehog (SHH) to trigger cell cycle re-entry (CCR) and apoptosis in post-mitotic neurons. Basella alba is a vegetable whose polysaccharides carry immunomodulatory and anti-cancer actions, but their protective effects against neurodegeneration have never been reported. Herein, we tested whether polysaccharides derived from Basella alba (PPV-6) may inhibit Aβ toxicity and explored its underlying mechanisms. In differentiated rat cortical neurons, Aβ25-35 reduced cell viability, damaged neuronal structure, and compromised mitochondrial bioenergetic functions, all of which were recovered by PPV-6. Immunocytochemistry and western blotting revealed that Aβ25-35-mediated induction of cell cycle markers including cyclin D1, proliferating cell nuclear antigen (PCNA), and histone H3 phosphorylated at Ser-10 (p-Histone H3) in differentiated neurons was all suppressed by PPV-6, along with mitigation of caspase-3 cleavage. Further studies revealed that PPV-6 inhibited Aβ25-35 induction of SHH; indeed, PPV-6 was capable of suppressing neuronal CCR and apoptosis triggered by the exogenous N-terminal fragment of sonic hedgehog (SHH-N). Our findings demonstrated that, in the fully differentiated neurons, PPV-6 exerts protective actions against Aβ neurotoxicity via the downregulation of SHH to suppress neuronal CCR and apoptosis.

Impaired Sonic Hedgehog Responsiveness of Induced Pluripotent Stem Cell-Derived Floor Plate Cells Carrying the LRRK2-I1371V Mutation Contributes to the Ontogenic Origin of Lower Dopaminergic Neuron Yield.

Lower population of dopaminergic (DA) neurons is known to increase susceptibility to Parkinson's disease (PD), and our earlier study showed a lower yield of DA neurons in Leucine-Rich Repeat Kinase Isoleucine 1371 Valine (LRRK2-I1371V) mutation-carrying PD patient-derived induced Pluripotent Stem Cells (iPSCs). Although the role of Sonic Hedgehog (SHH) in DA neurogenesis of floor plate cells (FPCs) is known, the effect of LRRK2 mutations on SHH responsiveness of FPCs impacting DA neuronal yield has not been studied. We investigated SHH responsiveness of FPCs derived from LRRK2-I1371V PD patient iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 (glioma-associated oncogene 1) expression, intracellular Ca2+ rise, and cytosolic cyclic adenosine monophosphate (cAMP) levels upon SHH induction. In addition, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane fluidity and Rab8A and Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC) FPCs overexpressing LRRK2-I1371V as well as FPCs. Although total expression of Ptch1 and Smo was comparable, receptor expression on cell surface was significantly lower in LRRK2-I1371V FPCs than in HC FPCs, with distinctly lower nuclear expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2-I1371V exhibited a similarly reduced cell surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared with HC FPCs upon SHH stimulation. The LRRK2-I1371V mutant FPCs and LRRK2-I1371V-transfected SH-SY5Y and HC FPCs too exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A and Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker caveolin 1 were also observed in them. These findings suggest that impaired SHH responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA neurons during ontogeny. Reduced cell surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation.

Tubule-derived exosomes play a central role in fibroblast activation and kidney fibrosis

Extracellular vesicles such as exosomes are involved in mediating cell-cell communication by shuttling an assortment of proteins and genetic information. Here, we tested whether renal tubule-derived exosomes play a central role in mediating kidney fibrosis. The production of exosomes was found to be increased in the early stage of unilateral ureteral obstruction, ischemia reperfusion injury or 5/6 nephrectomy models of kidney disease. Exosome production occurred primarily in renal proximal tubular epithelium and was accompanied by induction of sonic hedgehog (Shh). Invitro, upon stimulation with transforming growth factor-β1, kidney proximal tubular cells (HKC-8) increased exosome production. Purified exosomes from these cells were able to induce renal interstitial fibroblast (NRK-49F) activation. Conversely, pharmacologic inhibition of exosome secretion with dimethyl amiloride, depletion of exosome from the conditioned media or knockdown of Shh expression abolished the ability of transforming growth factor-β1-treated HKC-8 cells to induce NRK-49F activation. Invivo, injections of tubular cell-derived exosomes aggravated kidney injury and fibrosis, which was negated by an Shh signaling inhibitor. Blockade of exosome secretion invivo ameliorated renal fibrosis after either ischemic or obstructive injury. Furthermore, knockdown of Rab27a, a protein that is essential for exosome formation, also preserved kidney function and attenuated renal fibrotic lesions in mice. Thus, our results suggest that tubule-derived exosomes play an essential role in renal fibrogenesis through shuttling Shh ligand. Hence, strategies targeting exosomes could be a new avenue in developing therapeutics against renal fibrosis.

Abstract 777: Down-regulation of miR-506 contributes to EGFR-TKI resistance through inducing sonic hedgehog signaling in non-small cell lung cancer

Abstract Background: Lung cancer is the leading cause of cancer related death in the United States. Epidermal Growth Factor Receptor (EGFR) mutation predicts response to a tyrosine kinase inhibitor (TKI) of EGFR in approximately 25% of patients. Non-small cell lung cancer (NSCLC) patients with EGFR mutation eventually develop resistance to EGFR-TKI. The mechanism of resistance is not fully elucidated but majority of the cases is related to emergence of clones with T790M mutation in EGFR, amplification of cMET and Epidermal Mesenchymal Transition (EMT). Sonic Hedgehog (SHh) signaling activation is involved in EMT through FGF, notch, TGFβ signaling and microRNA (miRs) networks. Several miRs have been shown to correlate with TKI resistance indicating that miRs may serve as novel targets and/or biomarkers for anti-EGFR therapy. MiR-506 is involved in Hedgehog signaling and it is abnormally expressed in several cancers. However, the role of miR-506 in modulating TKI resistance in NSCLC remains unexplored. In this study, we investigated the role of miR-506 in the regulation of SHh in EGFR-TKI-resistant lung cancer cell lines. Methods: To generate resistant cell lines, two EGFR-TKI sensitive NSCLC cells (HCC4006 and HCC827) were exposed to increasing concentrations of erlotinib up to 20 µM over 6 months. The resultant clones were verified for sensitivity to erlotinib using a crystal violet staining assay. The expression of miR-506 in parental and resistant clones was measured using Taqman microRNA assay. Invasive/migratory ability of parental and resistant clones was measured by Boyden chamber assays. EMT and stemness markers were evaluated by Western blotting. Results: The studies demonstrated that miR-506 levels were ~90% and 75% lower in most resistant clones of HCC4006 (HCC4006ER4) and HCC827 (HCC827ER3), respectively. These clones showed overexpression of SHh (5-fold in HCC4006ER4 and 2.5-fold in HCC827ER3 as compared to their respective parental cells). Ectopic expression of miR-506 in these resistant cells inhibits the expression of SHh by ~80% as well as its downstream mediator, GLI1 by 55%. Our results showed that the IC50 of erlotinib was 22.4 μM for ER cells transfected with ctrl-miR and 4.7 μM for cells with miR-506 mimics. Moreover, an epithelial marker, E-cadherin is upregulated (~2.7-fold) and mesenchymal/cancer stem cell markers (e.g., N-cadherin, vimentin, Sox-2, Notch1, CD44 and ALDH1A1) were significantly downregulated (75-90%, p-value<0.001) in miR-506 overexpressing cells. Conclusions: Our data showed that miR-506 downregulation and induction of SHh are associated with EGFR-TKI resistance in EGFR mutated NSCLC cells. MiR-506 interference and inhibition of SHh pathway may be potential therapeutic strategies to reverse resistance to EGFR-TKI in NSCLC with EGFR mutation. (This study was funded by the support from IRG-16-194-07(IH) and Godwin's lab). Citation Format: Inamul Haque, Mukut Sharma, Andrew K. Godwin, Chao H. Huang. Down-regulation of miR-506 contributes to EGFR-TKI resistance through inducing sonic hedgehog signaling in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 777.

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

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.

Reemergence of hedgehog mediates epithelial-mesenchymal crosstalk in pulmonary fibrosis.

Hedgehog signaling plays important roles in cell development and differentiation. In this study, the ability of Sonic Hedgehog (SHH) to induce myofibroblast differentiation was analyzed in isolated human lung fibroblasts, and its in vivo significance was evaluated in rodent bleomycin-induced pulmonary fibrosis. The results showed that SHH could induce myofibroblast differentiation in human lung fibroblasts in a Smo- and Gli1-dependent manner. Gel shift analysis, chromatin immunoprecipitation assay, and site-directed mutagenesis revealed that a Gli1 binding consensus in the α-SMA gene promoter was important for mediating SHH-induced myofibroblast differentiation. Analysis of Hedgehog reemergence in vivo revealed that of all three Hedgehog isoforms, only SHH was significantly induced in bleomycin-injured lung along with Gli1. The induction of SHH was only noted in epithelial cells, and its expression was undetectable in lung fibroblasts or macrophages. transforming growth factor (TGF)-β induced SHH significantly in cultured alveolar epithelial cells, whereas SHH induced TGF-β in lung fibroblasts. Pulmonary fibrosis and α-smooth muscle actin (α-SMA) expression were significantly reduced in mice that were Smo deficient only in type I collagen-expressing cells. Thus, the reemergence of SHH in epithelial cells could result in induction of myofibroblast differentiation in a Smo-dependent manner and subsequent Gli1 activation of the α-SMA promoter.

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

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.

Abstract 1966: A unique cross-talk between HER2 and sonic hedgehog signaling promotes anoikis resistance in breast cancer cells

Abstract Metastatic cancer cells possess a unique capacity to survive without appropriate signaling from the extracellular matrix (ECM). The epithelial cells surrounded by proper ECM within tissues are designed to grow and differentiate whereas epithelial cells lacking suitable EMC are destined to anoikis, a form of apoptosis. The metastatic cancer cells resist anoikis to survive under the absence of ECM. Current study identifies a novel interplay between HER2 and sonic hedgehog signaling in promoting anoikis resistance. MDA-MB-231 cells exhibited an up-regulation of HER2 expression in cells cultured under anchorage- independent conditions as compared to adherent cells. Similar phenomenon was also observed in 4T-1 cells. The survival of MDA-MB-231 cells with stable HER2 overexpression (HH) was significantly enhanced when cultured under anchorage-independent conditions. However, the enhanced survival of MDA-MB-231 (HH) was reduced when HER2 was silenced using shRNA. These observations indicate the obligatory role of HER2 in anoikis resistance. Furthermore cells grown under anchorage-independent conditions induced the expression of sonic hedgehog (SHH). Increase in SHH was accompanied with enhanced expressions of PTCH2 and GLI2, the receptor and subsequent transcription factors of the sonic hedgehog signaling. Our results further showed that the induction of SHH was significantly higher in MDA-MB-231 (HH) cells relative to parent cells when cultured under anchorage-independent conditions, suggesting a correlation between HER2 and SHH signaling. Treatment of MDA-MB-231 and 4T-1 cells with cyclopamine (SHH signaling inhibitor) reduced the survival of cells under anchorage-independent conditions. Interestingly, cyclopamine treatment also inhibited the induction of HER2 during anchorage-independent culture conditions. Taken together, our study suggests a SHH-mediated induction of HER2 to promote anoikis resistance in breast cancer cells. Further mechanistic and in vivo studies are under progress. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: Parul Gupta, Sanjay K. Srivastava. A unique cross-talk between HER2 and sonic hedgehog signaling promotes anoikis resistance in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1966. doi:10.1158/1538-7445.AM2014-1966

Direct functional consequences of ZRS enhancer mutation combine with secondary long range SHH signalling effects to cause preaxial polydactyly

Sonic hedgehog (SHH) plays a central role in patterning numerous embryonic tissues including, classically, the developing limb bud where it controls digit number and identity. This study utilises the polydactylous Silkie (Slk) chicken breed, which carries a mutation in the long range limb-specific regulatory element of SHH, the ZRS. Using allele specific SHH expression analysis combined with quantitative protein analysis, we measure allele specific changes in SHH mRNA and concentration of SHH protein over time. This confirms that the Slk ZRS enhancer mutation causes increased SHH expression in the posterior leg mesenchyme. Secondary consequences of this increased SHH signalling include increased FGF pathway signalling and growth as predicted by the SHH/GREM1/FGF feedback loop and the Growth/Morphogen models. Manipulation of Hedgehog, FGF signalling and growth demonstrate that anterior-ectopic expression of SHH and induction of preaxial polydactyly is induced secondary to increased SHH signalling and Hedgehog-dependent growth directed from the posterior limb. We predict that increased long range SHH signalling acts in combination with changes in activation of SHH transcription from the Slk ZRS allele. Through analysis of the temporal dynamics of anterior SHH induction we predict a gene regulatory network which may contribute to activation of anterior SHH expression from the Slk ZRS.

Abstract 043: Regulation of cardiac excitability by non-canonical Sonic Hedgehog signaling.

Introduction: Induction of Sonic Hedgehog (Shh) in the myocardium post myocardial infarction promotes neovascularization and reduces infarct size. Shh signals through the 7-TM protein Smoothened (SMO), which we have found to couple to Gi proteins. Our goal is to investigate the effect of Shh on myocardial excitability and electrical remodeling and the role of SMO/Gi coupling. Methods and Results: We performed whole-cell patch clamp action potential and K+ currents measurements, biochemistry and volume-conducted ECG measurements in Langerdoff preparations using wild type and transgenic GiCT mice with inducible cardiomyocyte-specific impaired Gi protein signaling. In normal hearts and isolated cardiomyocytes, Shh reduced by 80% the isoproterenol-induced cAMP increase and protein kinase A (PKA) activation (p<0.01). Shh also induced a 2-2.5 fold prolongation of the action potential duration (APD) (p<0.001) and a reduction of the IK, slow (9.3±0.8 vs. 1.9±0.5 pA/pF; p<0.05). In Langerdorff-perfused hearts, Shh agonists induced a ~30% increase of the QT interval (p<0.01) and premature ventricular contractions (PVC) (0 out of 6 controls vs. 8 out of 8 Shh) and ventricular tachycardia (VT) (0 out of 6 controls vs. 7 out of 8 Shh), while pre-perfusion with KAAD-cyclopamine, a SMO inhibitor, reduced the arrhythmic episodes (PVC in 3 out of 8 and VT in 0 out 8). These effects we mediated by Gi proteins, as cAMP reduction, prolongation of the QT segment, and development of arrhythmias were abolished in hearts of GiCT mice. Conclusion: Our study demonstrates that acute Shh signaling in the heart leads to reshaping of the action potential (AP) and results in a dramatic increase of ventricular arrhythmias. Our data constitute the first evidence that acute, noncanonical Shh signaling may be implicated in the pathological electrical remodeling that sets the substrate for life-threatening arrhythmias in HF patients.

Upregulation of sonic hedgehog by fibroblast growth factor: Is TFAP2C a downstream intermediate?

Fibroblast growth factors (FGFs) from the apical ectodermal ridge (AER) and sonic hedgehog (SHH) from the zone of polarizing activity (ZPA) in the distal posterior limb bud mesoderm are critical for limb outgrowth and patterning. Maintenance of SHH expression is dependent on FGF, but the mechanism by which FGF regulates SHH has not been determined. Previous gene arrays have identified 300+ genes up‐regulated in response to FGF application. Among the genes up‐regulated by FGF was Transcription Factor Activating Protein‐2 gamma (TFAP2C). TFAP2C was up‐regulated within 3 hours of FGF application, which led us to hypothesize that TFAP2C was a downstream intermediate in the FGF to SHH regulation pathway.We analyzed the effects of ectopic TFAP2C expression on SHH induction in a region of the posterior limb bud known to be inducible for SHH by FGF. A TFAP2C expression plasmid was injected into Hamburger/Hamilton stage 22–23 chick embryos and harvested at 24 hrs. In situ hybridizations for TFAP2C and SHH were performed.We confirmed ectopic TFAP2C expression following injection of a beta‐actin promoter‐driven construct. However, no SHH expression was seen in 18 out of 18 embryos.We conclude that TFAP2C expression alone in the SHH inducible domain is not sufficient to up‐regulate SHH expression.Grant Funding Source: Departmental

Lymphoid Enhancer-binding Factor-1 (LEF1) Interacts with the DNA-binding Domain of the Vitamin D Receptor

Ligand-independent actions of the vitamin D receptor (VDR) are required for normal post-morphogenic hair cycles; however, the molecular mechanisms by which the VDR exerts these actions are not clear. Previous studies demonstrated impaired regulation of the canonical Wnt signaling pathway in primary keratinocytes lacking the VDR. To identify the key effector of canonical Wnt signaling that interacts with the VDR, GST pulldown studies were performed. A novel interaction between the VDR and LEF1 (lymphoid enhancer-binding factor-1) that is independent of β-catenin was identified. This interaction is dependent upon sequences within the N-terminal region of the VDR, a domain required for VDR-DNA interactions and normal hair cycling in mice. Mutation of specific residues within the N-terminal region of the VDR not only abrogated interactions between the VDR and LEF1 but also impaired the ability of the VDR to enhance Wnt signaling in vdr(-/-) primary keratinocytes. Thus, this study demonstrates a novel interaction between the VDR and LEF1 that is mediated by the DNA-binding domain of the VDR and that is required for normal canonical Wnt signaling in keratinocytes.

Erythropoietin and sonic hedgehog mediate the neuroprotective effects of brain-derived neurotrophic factor against mitochondrial inhibition

Brain-derived neurotrophic factor (BDNF) deficiency and mitochondrial dysfunction have been implicated in the pathogenesis of Huntington's disease (HD). 3-Nitropropionic acid (3-NP) is a mitochondrial inhibitor commonly used as a pharmacological model mimicking HD. We have recently reported that preconditioning of primary rat cortical cultures with BDNF induces sonic hedgehog (SHH), which contributes to the protective effects of BDNF against 3-NP neurotoxicity. Because carbamylated erythropoietin (EPO) may induce SHH, we investigated whether BDNF-dependent SHH expression and 3-NP resistance require prior induction of EPO. We found that BDNF induced EPO expression at both mRNA and protein levels. BDNF-mediated SHH induction and 3-NP resistance were abolished by the soluble EPO receptor (sEPO-R), an EPO inhibitor. Recombinant rat EPO (rEPO) induced SHH and attenuated 3-NP neurotoxicity. The rEPO-dependent neuroprotection was suppressed by the SHH inhibitor cyclopamine (CPM); however, sEPO-R failed to affect SHH neuroprotection. Furthermore, the rEPO-dependent neuroprotection was not suppressed by the BDNF neutralizing antibody, which completely abolished BDNF-mediated 3-NP resistance at the same dosage. Overall, our results demonstrate that BDNF-dependent SHH expression and 3-NP resistance require prior induction of EPO, thus establishing a signaling cascade of “BDNF → EPO → SHH → 3-NP resistance” in rat cortical neurons.

Abstract 995: Integrin-Linked Kinase localizes in the primary cilia to mediate Hedgehog signaling

Abstract The Hedgehog (Hh) signaling pathway is a highly conserved system for regulating metazoan development and cell fate. Integrin linked kinase (ILK) is required for proliferation of Hh-dependent neurons in the mammalian cerebellum, which also give rise to medulloblastoma, the most common childhood brain tumour. Sonic Hedgehog (SHh) binds to the Ptch transmembrane receptor, derepressing the Smo receptor to activate Hh signaling and key downstream targets such as Gli transcription factors. We find that SHh-induced osteogenic differentiation of C3H10T1/2 cells is blocked (>90% inhibition) by a small molecule ILK inhibitor, or by ILK-specific RNAi. SHh induces alkaline phosphatase (AP) activity in control 10T1/2 cells, but not cells transfected with ILK siRNA (>75% knockdown). Moreover we find that the small molecule ILK inhibitor suppresses SHh-induced differentiation in a dose-dependent manner, as effectively and to the same extent as the well-characterized selective Hh inhibitor, cyclopamine. Activation of Smo with the agonist, SAG, induces ILK-dependent AP induction in these cells, suggesting ILK acts downstream of Smo in the Hh pathway. SHh induction of the Gli1 transcription factor is also blocked by inhibiting ILK, suggesting ILK acts upstream of direct Hh targets. ILK inhibitor or siRNA suppresses constitutively activated Gli1 in medulloblastoma cells, derived from Ptch+/-:p53 compound heterozygote mice. In support of ILK involvment in Hh signaling, we have discovered a novel localization of ILK that indicates spatial proximity to Hh signaling molecules. The primary cilium is a microtubule-based organelle that is required for Hedgehog signaling, and we now find that ILK localizes in this structure. Using a selective ciliary marker, acetylated α-tubulin, we find that ILK localizes in the primary cilium along with components of the Hh pathway. Inhibition of ILK does not inhibit cilia formation, indicating that ILK is not involved in ciliogenesis. IFT proteins (eg. kinesin II motor subunit Kif3a), regulate both the assembly of, and the entry of Hh signaling proteins to, the cilium. Knockdown of Kif3a inhibits ciliogenesis and efficiently blocks SHh signaling, strongly suggesting the ciliary pool of ILK is critical for Hh signaling. Our work points to ILK as a novel therapeutic target in Hh-activated cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 995.

Sonic hedgehog mediates BDNF-induced neuroprotection against mitochondrial inhibitor 3-nitropropionic acid

Sonic hedgehog (SHH), a morphogen critical for embryogenesis, has also been shown to be neuroprotective. We have recently reported that pretreatment of rat cortical neurons for 8 h with brain-derived neurotrophic factor (BDNF; 100 ng/ml) affords protection against neurotoxicity of 3-nitropropionic acid (3-NP; 2.5 mM for 24 h), a mitochondrial complex II inhibitor. However, whether SHH is involved in BDNF-mediated neuroprotection remains unknown. Herein we tested whether BDNF induces SHH expression and if so, whether BDNF induction of SHH contributes to the observed neuroprotective effects. We found BDNF (100 ng/ml) increased SHH expression at both mRNA and protein levels. BDNF protection against 3-NP was abolished by cyclopamine (CPM; 5 μM), the SHH pathway inhibitor. Preconditioning of cortical neurons with N-terminal fragment of SHH (SHH-N; 0.1–1 ng/ml) was sufficient to confer resistance. These results indicate that BDNF induces SHH expression, which contributes to neuroprotection against 3-NP toxicity in rat cortical neurons.

Nuclear Factor-κB Contributes to Hedgehog Signaling Pathway Activation through Sonic Hedgehog Induction in Pancreatic Cancer

The hedgehog (Hh) signaling pathway, which functions as an organizer in embryonic development, is implicated in the development of various tumors. In pancreatic cancer, pathway activation is reported to result from aberrant expression of the ligand, sonic Hh (Shh). However, the details of the mechanisms regulating Shh expression are not yet known. We hypothesized that nuclear factor-kappaB (NF-kappaB), a hallmark transcription factor in inflammatory responses, contributes to the overexpression of Shh in pancreatic cancer. In the present study, we found a close positive correlation between NF-kappaB p65 and Shh expression in surgically resected pancreas specimens, including specimens of chronic pancreatitis and pancreatic adenocarcinoma. We showed that blockade of NF-kappaB suppressed constitutive expression of Shh mRNA in pancreatic cancer cells. Further activation of NF-kappaB by inflammatory stimuli, including interleukin-1beta, tumor necrosis factor-alpha, and lipopolysaccharide, induced overexpression of Shh, resulting in activation of the Hh pathway. Overexpression of Shh induced by these stimuli was also suppressed by blockade of NF-kappaB. NF-kappaB-induced Shh expression actually activated the Hh pathway in a ligand-dependent manner and enhanced cell proliferation in pancreatic cancer cells. In addition, inhibition of the Hh pathway as well as NF-kappaB suppressed the enhanced cell proliferation. Our data suggest that NF-kappaB activation is one of the mechanisms underlying Shh overexpression in pancreatic cancer and that proliferation of pancreatic cancer cells is accelerated by NF-kappaB activation in part through Shh overexpression.

Interferon-γ Induced Medulloblastoma in the Developing Cerebellum

We have generated a mouse model system with a high incidence of medulloblastoma, a malignant neoplasm believed to arise from immature precursors of cerebellar granule neurons. These animals ectopically express interferon-gamma (IFN-gamma) in astrocytes in the CNS in a controlled manner, exploiting the tetracycline-controllable system. More than 80% of these mice display severe ataxia and develop cerebellar tumors that express synaptophysin, the mouse atonal homolog MATH1, sonic hedgehog (SHH), and Gli1. IFN-gamma-induced tumorigenesis in these mice is associated with increased expression of SHH, and SHH induction and tumorigenesis are dependent on signal transducer and activator of transcription 1 (STAT1). When IFN-gamma expression is shut down with doxycycline at postnatal day 12 (P12), the clinical symptoms dissipate and the mice do not develop tumors, whereas if transgene expression is shut down at P16, the clinical symptoms and tumors progress to lethality, indicating that IFN-gamma is required for tumor induction but not progression. The tumors that occur in the continued presence of IFN-gamma display extensive necrosis and apoptosis as well as macrophage and lymphocytic infiltration, whereas the tumors that develop in mice in which IFN-gamma expression is shut down at P16 do not. Thus, IFN-gamma expression in the perinatal period can induce SHH expression and medulloblastoma in the cerebellum by a STAT1-dependent mechanism, and its continued presence appears to promote a host response to the tumor.

Shh-dependent differentiation of intestinal tissue from embryonic pancreas by activin A.

The pancreas develops from the endoderm to give rise to ducts, acini and islets of Langerhans. This process involves extracellular signals of the Transforming Growth Factor beta (TGFbeta) family. The aim of this work was to study the effects of activin A, a member of this family, whose potential role in pancreas differentiation is controversial. To this end, we used pancreatic explants from E12.5 mouse embryos. In culture these explants exhibited spontaneous growth, epithelial morphogenesis and endocrine and exocrine differentiation. Exposure to activin A did not affect exocrine or endocrine differentiation. Surprisingly, activin A induced in the explants the appearance of a large contractile structure surrounded by a cylindrical epithelium, a thick basal lamina and a smooth muscle layer. This structure, the formation of which was prevented by follistatin, was typical of an intestinal wall. Consistent with this interpretation, activin A rapidly induced in the explants the mRNAs for fatty acid binding proteins (FABPs), which are markers of the intestine, but not of the pancreas. We also found that induction of the FABPs was preceded by induction of Sonic hedgehog (Shh), a known inducer of intestinal differentiation in the endoderm. Activin B induced neither Shh nor intestinal differentiation. The activin A-mediated intestinal differentiation was blocked by cyclopamine, an inhibitor of Hedgehog signaling, and it was mimicked by Shh. We conclude that activin A does not appear to affect the exocrine or endocrine components of the pancreas, but that it can promote differentiation of pancreatic tissue into intestine via a Shh-dependent mechanism. These findings illustrate the plasticity of differentiation programs in response to extracellular signals in the pancreas and they shed new light on the regulation of pancreas and intestinal development.

Induction of sonic hedgehog and patched by retinoic acid in the developing rat lung in vitro. • 292

Induction of sonic hedgehog and patched by retinoic acid in the developing rat lung in vitro . • 292