Hedgehog Signal Transduction Research Articles

Rab23 is required for cardiac progenitor cell differentiation and positively-regulates Wnt11/AP-1 signalling in zebrafish

There has been considerable recent interest in the roles of vesicle transport proteins in ciliary biology and regulation of signal transduction. We have previously discovered RAB23 mutations in Carpenter syndrome, in which patients present with craniofacial and cardiac malformations, postnatal obesity and tumour formation, and Rab23 regulates trafficking of hedgehog signalling components between cilia and the cytoplasm. In the current study we investigated whether Rab23 may also regulate other signalling pathways. Wnt signalling pathways regulate a variety of developmental processes, yet the mechanisms of Wnt signal transduction remain unclear. We show that zebrafish deficient for Rab23 or its GTPase-activating protein, Evi5l, exhibit abnormal heart formation, which we attribute to a requirement for rab23 in the differentiation of cardiac progenitor cells. We delineate a Wnt11 signalling pathway that is implicated in cardiomyocyte differentiation, which acts via phospholipase C, inositol triphosphate receptor, and protein kinase C, to regulate activator protein 1 (AP-1) transcriptional activity downstream of c-Jun N-terminal kinase. We also show that Rab23 interacts functionally with several components of this pathway to regulate transcription by AP-1. Collectively, these findings identify Rab23 as a novel positive-regulator of non-canonical Wnt11/AP-1 signalling, which may be relevant to cardiovascular disease and stem cell biology. We also present evidence that Rab23 is dispensible for hedgehog signalling in zebrafish, and we propose that this reflects previously reported evolutionary differences in the requirement for cilia in hedgehog signal transduction. Collectively, our findings suggest that Rab23 may integrate multiple signalling pathways, possibly playing both cilia-dependent and –independent roles.

Primary cilia and forebrain development

With a microtubule-based axoneme supporting its plasma membrane-ensheathed projection from the basal body of almost all cell types in the human body, and present in only one copy per cell, the primary cilium can be considered an organelle sui generis. Although it was first observed and recorded in histological studies from the late 19th century, the tiny structure was essentially forgotten for many decades. In the past ten years, however, scientists have turned their eyes once again upon primary cilia and realized that they are very important for the development of almost all organs in the mammalian body, especially those dependent upon the signaling from members Hedgehog family, such as Indian and Sonic hedgehog. In this review, we outline the roles that primary cilia play in forebrain development, not just in the crucial transduction of Sonic hedgehog signaling, but also new results showing that cilia are important for cell cycle progression in proliferating neural precursors. We will focus upon cerebral cortex development but will also discuss the importance of cilia for the embryonic hippocampus, olfactory bulb, and diencephalon.

Targeting the hedgehog signal transduction pathway at the level of GLI inhibits neuroblastoma cell growth in vitro and in vivo

Hedgehog (HH) signaling is an important regulator of embryogenesis that has been associated with the development of several types of cancer. HH signaling is characterized by Smoothened (SMO)-dependent activation of the GLI transcription factors, which regulate the expression of critical developmental genes. Neuroblastoma, an embryonal tumor of the sympathetic nervous system, was recently shown to express high levels of key molecules in this signaling cascade. Using compounds blocking SMO (cyclopamine and SANT1) or GLI1/GLI2 (GANT61) activity revealed that inhibition of HH signaling at the level of GLI was most effective in reducing neuroblastoma growth. GANT61 sensitivity positively correlated to GLI1 and negatively to MYCN expression in the neuroblastoma cell lines tested. GANT61 downregulated GLI1, c-MYC, MYCN and Cyclin D1 expression and induced apoptosis of neuroblastoma cells. The effects produced by GANT61 were mimicked by GLI knockdown but not by SMO knockdown. Furthermore, GANT61 enhanced the effects of chemotherapeutic drugs used in the treatment of neuroblastoma in an additive or synergistic manner and reduced the growth of established neuroblastoma xenografts in nude mice. Taken together this study suggests that inhibition of HH signaling is a highly relevant therapeutic target for high-risk neuroblastoma lacking MYCN amplification and should be considered for clinical testing.

Identification of a novel Smoothened antagonist that potently suppresses Hedgehog signaling

The Hedgehog signaling pathway plays an essential role in embryo development and adult tissue homeostasis, in regulating stem cells and is abnormally activated in many cancers. Given the importance of this signaling pathway, we developed a novel and versatile high-throughput, cell-based screening platform using confocal imaging, based on the role of β-arrestin in Hedgehog signal transduction, that can identify agonists or antagonist of the pathway by a simple change to the screening protocol. Here we report the use of this assay in the antagonist mode to identify novel antagonists of Smoothened, including a compound (A8) with low nanomolar activity against wild-type Smo also capable of binding the Smo point mutant D473H associated with clinical resistance in medulloblastoma. Our data validate this novel screening approach in the further development of A8 and related congeners to treat hedgehog related diseases, including the treatment of basal cell carcinoma and medulloblastoma.

Identification of Vascular Changes Required for Ovulation Using Multiphoton Microscopy.

Mice in which the hedgehog signaling pathway is conditionally activated in the ovary were used as a model to determine vascular events required for ovulation. In Amhr2cre/+SmoM2 mutant mice, CRE-mediated recombination induces expression of a dominant active allele of the hedgehog signal transducer smoothened, beginning in the neonatal ovary. Preovulatory follicles develop in these mice and undergo many of the expected changes during the periovulatory period, but they fail to rupture. Vessels in the theca of growing follicles do not become appropriately associated with vascular smooth muscle. This defect appears to be due to changes elicited by over-activation of hedgehog signaling during neonatal development, leading to persistent failure throughout life of vessels within the theca to undergo maturation. These previous findings are consistent with the known importance of hedgehog signaling in vascular development. In the current study, multiphoton microscopy was used to image vascular changes in preovulatory follicles in vivo. Prepubertal mutant and genotype-matched control mice were primed with eCG followed 48h later by treatment with hCG. In this model, ovulation normally occurs 12-13h post hCG. An ovary with intact bursa was exteriorized and immobilized, and the entire preparation, including the abdomen of the mouse, was bathed with warm Ringer's solution. Rhodamine-labeled 2x106 MW dextran was injected retro-orbitally, and individual vessels were imaged over a four hour time period using a submersion objective. Vessel diameter and red blood cell velocity were measured repeatedly in individual capillary-scale vessels, and blood flow was calculated. At each time point, multiple vessels in the apical and lateral areas of preovulatory follicles from 3 control and 3 mutant mice were studied. At 48h after eCG, apical and lateral vessels did not differ in diameter or blood flow between control and mutant mice. The apex of mutant and control follicles had equivalent density of blood vessels. Following hCG treatment in control mice, blood flow in apical and lateral vessels decreased at 12h and 13h and was <5% of that observed at 11h (P<0.05). By 13h, 73% of the apical vessels and 20% of the lateral vessels were no longer detectable, and flow rates in the vessels that remained visible were low. Vessels in which blood flow slowed dramatically or stopped at 12h were undetectable one hour later, suggesting a process of vasoconstriction or remodeling. By 14h, the remodeling associated with ovulation prevented identification of previously measured vessels. In mutant mice, the flow rate in apical vessels did not change between 11h and 12h after hCG, but it decreased moderately at 13h to levels 44% of that at 11h (P<0.05). The apparent disappearance of vessels from the apex that was observed in control mice did not occur in mutants. In lateral vessels of mutant mice, flow was constant between 11h and 13h. In summary, the diameter and flow rate of thecal vessels in preovulatory follicles decreased during the periovulatory period in control mice. Thecal vessels in mutant mice failed to undergo these changes, likely due to lack of maturation of the vessels by association with smooth muscle. These findings suggest that vascular changes such as vasoconstriction are required for follicle rupture. Supported by NICHD R03HD057648, NIH/NIBIB P41 EB001976-20, Cornell Center for Vertebrate Genomics, and NSF ADVANCE 0547373

Cholesterol and its derivatives in Sonic Hedgehog signaling and cancer

The connection between the Hedgehog (HH) 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.

Overactivation of Hedgehog Signaling Alters Development of the Ovarian Vasculature in Mice1

The hedgehog (HH) signaling pathway is critical for ovarian function in Drosophila, but its role in the mammalian ovary has not been defined. Previously, expression of a dominant active allele of the HH signal transducer protein smoothened (SMO) in Amhr2(cre/+)SmoM2 mice caused anovulation in association with a lack of smooth muscle in the theca of developing follicles. The current study examined events during the first 2 wk of life in Amhr2(cre/+)SmoM2 mice to gain insight into the cause of anovulation. Expression of transcriptional targets of HH signaling, Gli1, Ptch1, and Hhip, which are used as measures of pathway activity, were elevated during the first several days of life in Amhr2(cre/+)SmoM2 mice compared to controls but were similar to controls in older mice. Microarray analysis showed that genes with increased expression in 2-day-old mutants compared to controls were enriched for the processes of vascular and tube development and steroidogenesis. The density of platelet endothelial cell adhesion molecule (PECAM)-labeled endothelial tubes was increased in the cortex of newborn ovaries of mutant mice. Costaining of preovulatory follicles for PECAM and smooth muscle actin showed that muscle-type vascular support cells are deficient in theca of mutant mice. Expression of genes for steroidogenic enzymes that are normally expressed in the fetal adrenal gland were elevated in newborn ovaries of mutant mice. In summary, overactivation of HH signaling during early life alters gene expression and vascular development and this is associated with the lifelong development of anovulatory follicles in which the thecal vasculature fails to mature appropriately.

Novel Mechanism of Action on Hedgehog Signaling by a Suppressor of Fused Carboxy Terminal Variant

The Suppressor of Fused (SUFU) protein plays an essential role in the Hedgehog (HH) signaling pathway, by regulation of the GLI transcription factors. Two major isoforms of human SUFU are known, a full-length (SUFU-FL) and a carboxy-terminal truncated (SUFU- ΔC) variant. Even though SUFU- ΔC is expressed at an equivalent level as SUFU-FL in certain tissues, the function of SUFU-ΔC and its impact on HH signal transduction is still unclear. In two cell lines from rhabdomyosarcoma, a tumor type associated with deregulated HH signaling, SUFU-ΔC mRNA was expressed at comparable levels as SUFU-FL mRNA, but at the protein level only low amounts of SUFU-ΔC were detectable. Heterologous expression provided support to the notion that the SUFU-ΔC protein is less stable compared to SUFU-FL. Despite this, biochemical analysis revealed that SUFU-ΔC could repress GLI2 and GLI1ΔN, but not GLI1FL, transcriptional activity to the same extent as SUFU-FL. Moreover, under conditions of activated HH signaling SUFU-ΔC was more effective than SUFU-FL in inhibiting GLI1ΔN. Importantly, co-expression with GLI1FL indicated that SUFU-ΔC but not SUFU-FL reduced the protein levels of GLI1FL. Additionally, confocal microscopy revealed a co-localization of GLI1FL with SUFU-ΔC but not SUFU-FL in aggregate structures. Moreover, specific siRNA mediated knock-down of SUFU-ΔC resulted in up-regulation of the protein levels of GLI1FL and the HH signaling target genes PTCH1 and HHIP. Our results are therefore suggesting the presence of novel regulatory controls in the HH signaling pathway, which are elicited by the distinct mechanism of action of the two alternative spliced SUFU proteins.

Scube activity is necessary for Hedgehog signal transduction in vivo

The Hedgehog (HH) signaling pathway is a central regulator of embryonic development, controlling the pattern and proliferation of a wide variety of organs. Previous studies have implicated the secreted protein, Scube2, in HH signal transduction in the zebrafish embryo (Hollway et al., 2006; Kawakami et al., 2005; Woods and Talbot, 2005) although the nature of the molecular function of Scube2 in this process has remained undefined. This analysis has been compounded by the fact that removal of Scube2 activity in the zebrafish embryo leads to only subtle defects in HH signal transduction in vivo (Barresi et al., 2000; Hollway et al., 2006; Ochi and Westerfield, 2007; van Eeden et al., 1996; Wolff et al., 2003). Here we present the discovery of two additional scube genes in zebrafish, scube1 and scube3, and demonstrate their roles in facilitating HH signal transduction. Knocking down the function of all three scube genes simultaneously phenocopies a complete loss of HH signal transduction in the embryo, revealing that Scube signaling is essential for HH signal transduction in vivo. We further define the molecular role of scube2 in HH signaling.

Abstract 149: Antitumor effect of itraconazole on hematological malignant cells by suppression of Hedgehog signal transduction via inhibition of lipid raft formation

Abstract [Introduction and aim] Itraconazole (ITCZ) is one of the antifungal agents that inhibits fungal cell membrane ergosterol synthesis. ITCZ has been widely used for the treatment of fungal infections with high efficiency. Recently, anti-tumor effect of ITCZ was reported for solid tumors (Kim J et al. Cancer Cell 2010;17:388-399). During the treatment of hematological malignancies, ITCZ has been quite often used for invasive fungal infections; however, there has been no report for the effect of ITCZ on hematological malignancies. Therefore, we aimed to investigate the anti-tumor effect of ITCZ on hematological malignancies in the present study. [Methods] DAUDI, Jurkat, K562, Karpas299 cell lines were used as a model of hematological malignancies in the present study. At first, MTT assays were performed by treating the cells to determine if ITCZ possessed anti-tumor effect on the cell growth. Then, comprehensive analysis of intracellular signal transductions was performed utilizing reporter assay system. To evaluate the involvement of lipid raft formation by ITCZ, Gli1 expression concerning the Hedgehog signal transduction was determined by real-time PCR and western blotting, and fluorescent staining of the cells using lipid raft marker was also performed. [Results] ITCZ inhibited the cell growth by 40 to 60% determined by MTT assays in all four cell lines tested in the present study. Comprehensive analysis of intracellular signal transductions utilizing reporter assay system in Karpas299 cells showed the strong decrease of Gli1 transcription, implying the inhibition of Hedgehog signal transduction. Concerning Hedgehog signal pathway, Gli1 protein has been reported to be overexpressed in tumor cells and considered to be involved in the tumor growth, and Gli1 has also been reported to localize in lipid raft. Therefore, we hypothesized that ITCZ might influenced on the lipid raft of cell membrane and then Gli1 expression was suppressed, leading to the weakened Hedgehog signal transduction and cell growth inhibition. Fluorescent staining of Jurkat cells using lipid raft marker showed that the presence of ITCZ apparently inhibited the formation of lipid raft on cell membrane. This finding was quite similar compared to that observed when lipid raft inhibitor was added to cell culture medium. [Conclusions] ITCZ has an inhibitory effect on the cell growth in the cultured cell lines derived from hematological malignancies as same as the previous finding observed in solid tumors. Our results further indicated that ITCZ inhibited the formation of lipid raft on cell membrane, and then Hedgehog signal transduction should be weakened, finally leading to the cell growth inhibition. Taken together, the tumor inhibition by ITCZ should be favorable effect and may enhance the efficacy of the treatment of various hematological malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 149. doi:1538-7445.AM2012-149

Abstract 5106: A HHIP derived gene expression signature for the prediction of patient survival after adjuvant chemotherapy of pancreatic cancer

Abstract Prognosis for patients with pancreatic carcinoma (PDAC) remains poor. Despite of increasing knowledge about the molecular basis of PDAC neither a specific marker for early diagnosis nor target proteins for a new therapeutic approach have been identified so far. However, reliable prediction of survival might help to improve the therapeutic outcome for patients with PDAC. In recent years it has become evident, that the Hedgehog (Hh) signal transduction pathway is a key regulator of pancreatic cancer development. Hedgehog interacting protein (HHIP) is a key modulator of Hh action and is overexpressed in the tumor stroma of PDACs. Reasoning that HHIP could serve as an indicator of Hh signaling activation we identified HHIP associated genes (HAG) using guilt by association (GBA) analysis in a set of PDAC gene expression profiles from microdissected tissue. Using the Affymetrix GeneChip U133 2.0 array and TNASAS we analyzed the gene expression profile of 59 PDAC samples and identified prognostic marker from HAG for survival of PDAC patients with adjuvant therapy. GBA analysis of gene expression profiles from microdissected PDAC tissue yielded 276 HAGs. TNASAS analysis using these genes revealed a prognostic gene expression signature of 50 genes, which was able to identify patients receiving adjuvant therapy with gemcitabine surviving more than 24 month with a total error of 0.087. TNASAS analysis of samples from patients treated without adjuvant therapy failed to identify a gene set with prognostic significance. Gene expression analysis is able to identify genes with prognostic significance in patients with adjuvant chemotherapy. These genes seem to be correlated with the efficacy of adjuvant gemcitabine therapy. Further analysis of those genes can reveal more insights in the signal transduction mechanisms associated with those genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5106. doi:1538-7445.AM2012-5106

Abstract 3285: Hedgehog pathway inhibition delays regrowth of ovarian cancer following paclitaxel and carboplatinum only if initiated immediately after completion of chemotherapy

Abstract The high mortality associated with ovarian cancer is attributed to the lack of any reliable early detection method, unknown pathogenesis of the disease, and the development of recurrent and chemoresistant tumors. Current efforts have focused on the identification of therapeutics that may be used independently or in combination with current chemotherapeutic regimens to reduce tumor volume. To date, limited research has focused on preventing or delaying disease recurrence. The Hedgehog (Hh) signal transduction pathway is inactive in most adult cells. Malignant activation of the Hh pathway through the signaling protein Smoothened (Smo) occurs in a broad range of cancers, including ovarian. IPI-926 is a potent orally delivered small molecule that targets the Hh pathway by inhibiting Smo. Recent studies from our laboratory provide evidence that IPI-926 slows serous ovarian cancer growth in a primary human tumor xenograft model. More importantly, IPI-926 delays the resurgence of tumor growth typically observed after cytoreduction with paclitaxel and carboplatinum (T/C) treatment. Our current objective was to assess whether this effect of IPI-926 required that the Smo inhibitor be administered during a critical window following T/C treatment to prevent the resurgence of tumor growth. To test our hypothesis, mice bearing human ovarian cancer xenografts were treated with vehicle or T/C. T/C treatment was withdrawn following significant reduction (30-50%) in tumor volume. The original vehicle treated cohort was divided into 2 arms which then either received IPI-926 or continued on vehicle for the duration of the experiment. Mice in the T/C cohort were divided into 3 groups. Group 1 received vehicle alone (T/C-Vehicle), group 2 received IPI-926 immediately following the last T/C dose (T/C-IPI-926) and group 3 received no vehicle or IPI treatment for 14 days following the last T/C dose (Window). Group 3 was then maintained on IPI-926 treatment starting at day 15 post-T/C withdrawal. The withdrawal of T/C led to a dramatic increase in tumor volume in the T/C-Vehicle group. As previously observed, tumor growth inhibition was maintained in mice receiving IPI-926 immediately following the cessation of T/C treatment. In contrast, delaying the administration of IPI-926 following T/C prevented the suppression of tumor growth as evidenced by the increased tumor volume. Our data suggest that blocking Hh pathway activity immediately following chemotherapy maintains and prolongs the inhibitory effect of chemotherapy on ovarian tumor growth. The absence of an IPI-926-mediated inhibition of tumor resurgence following a 14-day delay in treatment supports the concept that there is a critical period for tumor re-establishment. Our data, along with those of others, suggest that the early stages of ovarian tumor re-growth may be dependent on Hh pathway signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3285. doi:1538-7445.AM2012-3285

Abstract SY37-01: Cilia and wounding cooperate with Hedgehog signaling to initiate basal cell carcinoma

Abstract Primary cilia are microtubule-based projections found on many human cells. These cilia do not move, but function as cellular antennae to receive and interpret signals. For example, olfactory cilia sense odorants in the environment. Other cilia sense cues from other cells, such as Hedgehog proteins, to coordinate tissue patterning and growth. We have identified mechanisms of ciliary signaling, cilium formation, and the regulation of ciliary composition. Defects in ciliary signaling cause some forms of birth defects, cancer, polycystic kidney disease, and other disorders collectively called ciliopathies. To examine whether cilia can participate in disorders beyond recognized ciliopathies, we assessed the prevalence of cilia in human tumors and the role of cilia in cancer. Human basal cell carcinomas (BCCs) are associated with misactivation of Hedgehog signaling, commonly through acquisition of mutations in Smoothened. We found that human BCCs are frequently ciliated. We tested the role of cilia in BCC by conditionally deleting genes required for ciliogenesis in a Hedgehog pathway-dependent mouse model of BCC. Ciliary ablation strongly inhibited BCC-like tumors induced by misactivation of the Hedgehog pathway at the level of Smoothened, revealing that BCC can be addicted to cilia. Skin tumors, like many other cancers, are associated with injury. Although stem cells participate in tissue regeneration after wounding, it is unclear whether these cells also contribute to epithelial tumors. Surprisingly, expression of an activated form of Smoothened by stem cells of the hair-follicle bulge and secondary hair germ does not induce robust Hedgehog signaling or produce BCCs. However, wounding recruits these cells from the follicle to the wound site, where downstream Hedgehog signal transduction is derepressed, giving rise to superficial BCC-like tumors. Thus, BCCs can originate from follicular stem cells and wounding can promote tumorigenesis by moving oncogene-expressing stem cells out of a suppressive niche. As we found that oncogenic Smoothened requires the cilium to promote tumorigenesis, we investigated how Smoothened moves to the cilium. We found that proteins of the Tectonic family form part of the transition zone, a region at the base of the cilium. Tectonic1 is an extracytosolic glycoprotein that interacts with transmembrane components of the transition zone to connect to a cytosolic transition zone complex comprised of the products of genes associated with two ciliopathies, Joubert and Meckel syndromes. Loss of components of the Tectonic transition zone complex in mice compromise ciliogenesis in some tissues, but in others, it is required for Smoothened and select other membrane proteins to localize to cilia. A complementary small molecule screen is identifying new chemicals that also prevent Smoothened localization to cilia and block oncogenic Hedgehog signaling. Interfering with the cellular machinery that moves Smoothened to cilia may be a complementary means of treating Hedgehog pathway-associated cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY37-01. doi:1538-7445.AM2012-SY37-01

Combination of Hedgehog Signaling Blockage and Chemotherapy Leads to Tumor Reduction in Pancreatic Adenocarcinomas

Activation of the hedgehog signal transduction pathway, triggered by hedgehog binding to the transmembrane receptor patched 1 (PTCH1) or by mutations in the PTCH1 gene, plays an important role in the development of various tumors. To investigate whether the Hedgehog signaling pathway is also active in human pancreatic adenocarcinomas, we determined the expression levels of the known Hedgehog target genes PTCH1 and GLI-1 in pancreatic tumors. To determine whether alterations in the PTCH1 gene are responsible for this pathway activation, we screened pancreatic carcinomas for mutations in PTCH. To investigate the contribution of hedgehog signaling to the tumorigenicity of pancreatic tumor cells, we blocked the Hedgehog pathway in cultured tumor cells and xenografts using the steroidal alkaloid cyclopamine and the small-molecule Hedgehog inhibitor Hh-Antag. We identified single nucleotide polymorphisms (SNPs) within the PTCH1 gene but no somatic PTCH1 mutations. Pathway-blockage resulted in a significant dose-dependent reduction of tumor cell growth in vitro and in vivo. Moreover, combined treatment with cyclopamine and the conventional antimetabolite gemcitabine revealed a synergistic effect on the reduction of tumor growth in pancreatic adenocarcinoma xenografts. Inhibition of Hedgehog signaling could be a promising approach for the treatment of pancreatic adenocarcinomas.

Gastric Sonic Hedgehog Acts as a Macrophage Chemoattractant During the Immune Response to Helicobacter pylori

Macrophages mediate the epithelial response to Helicobacter pylori and are involved in the development of gastritis. Sonic Hedgehog (Shh) regulates gastric epithelial differentiation and function, but little is known about its immunoregulatory role in the stomach. We investigated whether gastric Shh acts as a macrophage chemoattractant during the innate immune response to H pylori infection. Mice with parietal cell-specific deletion of Shh (PC-Shh(KO)) and control mice were infected with H pylori. Levels of gastric Shh, cytokines, and chemokines were assayed by quantitative reverse-transcriptase polymerase chain reaction or by a Luminex-based multiplex assay 2, 7, or 180 days after infection. Circulating concentrations of Shh were measured by enzyme-linked immunosorbent assay. Bone marrow chimera experiments were performed with mice that have myeloid cell-specific deletion of the Hedgehog signal transduction protein Smoothened (LysMCre/Smo(KO)). Macrophage recruitment was measured in gastric tissue and peripheral blood by fluorescence-activated cell sorting analysis. Control mice infected with H pylori for 6 months developed an inflammatory response characterized by infiltration of CD4(+) T cells and increased levels of interferon gamma and interleukin 1β in the stomach. PC-Shh(KO) mice did not develop gastritis, even after 6 months of infection with H pylori. Control mice had increased concentrations of Shh, accompanied by the recruitment of CD11b(+)F4/80(+)Ly6C(high) macrophages 2 days after infection. Control mice that received bone marrow transplants from control mice had an influx of macrophages to the gastric mucosa in response to H pylori infection; this was not observed in H pylori-infected control mice that received bone marrow transplants from LysMCre/Smo(KO) mice. H pylori induces release of Shh from the stomach; Shh acts as a macrophage chemoattractant during initiation of gastritis.

Abstract 18219: Endothelial Canonical Sonic Hedgehog Signaling is Not Required for Sonic Hedgehog Mediated Angiogenesis or Ischemic Tissue Repair

Introduction: Hedgehog signaling is a critical mediator of development, ischemic tissue repair, and carcinogenesis. Sonic hedgehog (Shh) signaling has been shown to mediate ischemic tissue repair in experimental models. However, there is controversy on whether Shh signaling improves tissue perfusion by the canonical hedgehog pathway in endothelial cells or via indirect effects on stromal cells. We tested the hypothesis that direct canonical endothelial Shh signaling via the Hedgehog signal transducer Smoothened (Smo) is required for Shh mediated angiogenesis and ischemic tissue repair. Methods: We utilized Cre-Lox mouse models to test this hypothesis. Two endothelial Cre mouse lines, the Tie2-Cre and Vascular Endothelial Cadherin-Cre lines were crossed with mice in which Smo was homozygously floxed to generate endothelial specific Cre+ Smo flox/flox (eSmoNull) mice. Baseline cardiac phenotype was assessed with echocardiography. eSmoNull and SmoWT mice were compared using the corneal angiogenesis and hind limb ischemia (HLI) mouse models. Results: eSmoNull mice were viable and appeared normal. Baseline echo demonstrated normal LV function (fractional shortening: 34.39±1.9 vs. 34.07±1.1, P=0.93). Collagenase digestion of eSmoNull and SmoWT hearts followed by CD31+ cell selection and RT-PCR confirmed effective deletion of Smo. Corneal angiogenesis was induced with Shh pellets revealing similar degrees of Shh induced angiogenesis in eSmoNull and WT mice. HLI was induced to determine the role of endothelial, Smo-mediated hedgehog signaling after ischemic injury. In striking contrast to the impairment in recovery following antibody-mediated global blockade of Shh (Pola, Nature Med, 2001), laser Doppler perfusion demonstrated no significant difference in perfusion at days 7, 14, 21, and 28 (Day 28 perfusion ratio ischemic/nonischemic: 0.56 vs. 0.74, P=0.07). Limb preservation and functional scores were not significantly different. The ratio of lectin+ capillaries per muscle fiber in the ischemic limb was similar (1.58±0.15 vs. 1.65±0.11, P=0.36). Conclusion: Endothelial canonical Shh signaling is dispensable for vascular development, resting cardiac function, Shh induced angiogenesis, and recovery after ischemic injury.

Neuropilins are positive regulators of Hedgehog signal transduction

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.

A novel protein LZTFL1 regulates ciliary trafficking of the BBSome and Smoothened.

Many signaling proteins including G protein-coupled receptors localize to primary cilia, regulating cellular processes including differentiation, proliferation, organogenesis, and tumorigenesis. Bardet-Biedl Syndrome (BBS) proteins are involved in maintaining ciliary function by mediating protein trafficking to the cilia. However, the mechanisms governing ciliary trafficking by BBS proteins are not well understood. Here, we show that a novel protein, Leucine-zipper transcription factor-like 1 (LZTFL1), interacts with a BBS protein complex known as the BBSome and regulates ciliary trafficking of this complex. We also show that all BBSome subunits and BBS3 (also known as ARL6) are required for BBSome ciliary entry and that reduction of LZTFL1 restores BBSome trafficking to cilia in BBS3 and BBS5 depleted cells. Finally, we found that BBS proteins and LZTFL1 regulate ciliary trafficking of hedgehog signal transducer, Smoothened. Our findings suggest that LZTFL1 is an important regulator of BBSome ciliary trafficking and hedgehog signaling.

Protein kinase A acts at the basal body of the primary cilium to prevent Gli2 activation and ventralization of the mouse neural tube

Protein kinase A (PKA) is an evolutionarily conserved negative regulator of the hedgehog (Hh) signal transduction pathway. PKA is known to be required for the proteolytic processing event that generates the repressor forms of the Ci and Gli transcription factors that keep target genes off in the absence of Hh. Here, we show that complete loss of PKA activity in the mouse leads to midgestation lethality and a completely ventralized neural tube, demonstrating that PKA is as strong a negative regulator of the sonic hedgehog (Shh) pathway as patched 1 (Ptch1) or suppressor of fused (Sufu). Genetic analysis shows that although PKA is important for production of the repressor form of Gli3, the principal function of PKA in the Shh pathway in neural development is to restrain activation of Gli2. Activation of the Hh pathway in PKA mutants depends on cilia, and the catalytic and regulatory subunits of PKA are localized to a compartment at the base of the primary cilia, just proximal to the basal body. The data show that PKA does not affect cilia length or trafficking of smoothened (Smo) in the cilium. Instead, we find that there is a significant increase in the level of Gli2 at the tips of cilia of PKA-null cells. The data suggest a model in which PKA acts at the base of the cilium after Gli proteins have transited the primary cilium; in this model the sequential movement of Gli proteins between compartments in the cilium and at its base controls accessibility of Gli proteins to PKA, which determines the fates of Gli proteins and the activity of the Shh pathway.

HSulf-1 suppresses cell growth and down-regulates Hedgehog signaling in human gastric cancer cells

Gastric cancer is the second most lethal cancer worldwide. Despite the current surgical and adjuvant therapies, 5-year survival remains less than 20-25% in the US, Europe and China. Therefore, there is an urgent need to identify new therapeutic targets for treating this malignant disease. Accumulating evidence has supported that aberrant activation of the Hedgehog signaling pathway plays a crucial role in tumorigenesis and progression of gastric cancer. Human sulfatase-1 (HSulf-1) is a recently identified enzyme that desulfates cell surface heparan sulfate proteoglycans (HSPGs), which is critical for Hedgehog signal transduction under a highly sulfated state. HSulf-1 has recently emerged as a tumor suppressor gene in certain types of cancer, including ovarian, breast, myeloma and hepatocellular carcinoma; however, its role in gastric cancer remains to be elucidated. Therefore, we established HSulf-1-expressing monoclonal MKN28 gastric cancer cells to investigate its function in gastric cancer. Expression of HSulf-1 significantly suppressed cellular proliferation and growth in MKN28 gastric cancer cells. Notably, HSulf-1 inhibits Gli-mediated transcription and down-regulates the expression of Hedgehog target genes, including GLI1, PTCH1/2, HHIP, CCND1, C-MYC and BCL-2. Collectively, the study provides evidence that HSulf-1 may function as a tumor suppressor in gastric cancer. It suppresses gastric cancer cell proliferation, possibly through abrogating the Hedgehog signaling pathway. The study provides new mechanistic insight into HSulf-1- mediated tumor suppression, and supports the use of HSulf-1 as a potential new therapeutic target in treating gastric cancer.