Shh Overexpression Research Articles

Empowering sonic hedgehog to rescue brain cells after ischemic stroke.

Ischemic stroke occurs when blood supply to the brain is interrupted. This can cause irreversible injury to the central nervous system (CNS) tissue. Each year in the United States almost 800,000 people experience a new or recurrent stroke. 15% of stroke patients die shortly after insult and only 10% recover completely, leaving the majority of surviving stroke patients with disabilities. Tissue-type plasminogen activator (tPA) is the only available therapy for stroke but its clinical use is limited because of associated danger of intracranial hemorrhage. Therefore, there is an emergent need for stroke therapeutics that are safe and effective when administered at a later time point after insult.

The significance of sonic hedgehog immunohistochemical expression in colorectal carcinoma

Colorectal carcinoma is a significant source of major morbidity and mortality. Sonic hedgehog (Shh) is expressed in normal gastrointestinal tract mucosa and in many malignancies. The purpose of the present study is to investigate the relationship between Shh immunoexpression in CRC and clinicopathological characteristics. Paraffin blocks of 155 primary CRCs and 37 nodal metastases were retrieved and tissue microarrays were constructed. Immunohistochemistry was performed using anti-Shh antibody. Immunostaining was scored and results were analysed in relation to the clinicopathological parameters. Shh was overexpressed in primary CRC (p=0.02) and in nodal metastasis (p=0.004). There was no difference between Shh immunoexpression in primary CRC and in nodal metastasis (p=0.941). High Shh immunoexpression was associated with well differentiated tumours (p=0.004). However, there was no association with other clinicopathological parameters. Shh overexpression was not associated disease free survival (log-rank=0.079, p=0.778). Shh is overexpressed in well differentiated CRC. However, Shh is not associated with other clinicopathological and prognostic factors. Loss of Shh may be associated with proliferation and loss of differentiation in CRC. Further molecular studies are required to address the potential importance of Shh signalling in CRC and to test Shh inhibitors and activators as potential therapeutic targets in CRC.

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

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

Expressions of sonic hedgehog, smoothened and Glioblastoma transcriptional factor in human pancreatic cancer and their correlation with clinicopathologic characteristics

Objective To investigate the differential expression of sonic hedgehog (Shh),Smoothened (Smo) and Glioblastoma transcriptional factor (Gli1) in the pancreatic cancers and their adjacent tissues,and their correlation with pathogenesis of pancreatic cancer.Methods Reverse transcriptionpolymerase chain reaction (RT-PCR) and Western blotting were used to determine the mRNA and protein expression of Shh,Smo and Gli1 in 50 cases of pancreatic cancers and their adjacent tissues.The relationship between the expression of Shh,Smo and Gli1 and the clinicopathological features of pancreatic cancer was analyzed.Results The expression of Shh,Smo and Gli1 mRNA in pancreatic cancer was 0.309 ± 0.162,0.327 ±0.264 and 0.341 ±0.132,and that in the para-cancerous adjacent tissue was 0.184 ± 0.227,0.148 ±0.276 and 0.105 ±0.351 respectively (P ＜0.05).The expression of Shh,Smo and Gli1 protein in pancreatic cancer was 0.624 ±0.139,0.547 ±0.418 and 0.563 ±0.271,and that in paracancerous adjacent tissue was 0.388 ± 0.294,0.294 ± 0.152 and 0.328 ± 0.129 respectively (P ＜ 0.05).The expression levels of Shh,Smo and Gli1 mRNA and protein in pancreatic cancer tissue were higher than in paracancerous adjacent tissue.The overexpression of Shh,Smo and Gli1 was correlated with differentitation of pancreatic cancer cells (P ＜ 0.05),but not with age,tumor size,TNM grade,invasion to nerves and vascellum,lymph node metastasis and distant metastasis (P ＞ 0.05).Conclusion The expression level of Shh,Smo and Gli1 is increased in human pancreatic cancer cells.The overexpression of hedgehog signaling pathway might play an important role in carcinogenesis and development of pancreatic cancer. Key words: Pancreatic cancer; Sonic hedgehog; Smoothened ; Glioblastoma transcriptional factor ; Hedgehog pathway

Sonic hedgehog is a novel tubule-derived growth factor for interstitial fibroblasts after kidney injury.

Tubular epithelium constitutes the majority of the renal parenchyma and is the primary target of various kidney injuries. However, how the injured tubules drive interstitial fibroblast activation and proliferation remains poorly understood. Here, we investigated the role of sonic hedgehog (Shh), a secreted extracellular signaling protein, in fibroblast proliferation. Shh was induced in renal tubular epithelia in animal models of CKD induced by ischemia/reperfusion injury (IRI), adriamycin, or renal mass ablation, and in renal tubules of kidney biopsy specimens from CKD patients with different etiologies. Using Gli1-CreER(T2) reporter mice, we identified interstitial fibroblasts as the principal targets of renal Shh signaling in vivo. In vitro, incubation with Shh promoted normal rat kidney fibroblast proliferation, which was assessed by cell counting, MTT assay, and BrdU incorporation assay, and stimulated the induction of numerous proliferation-related genes. However, Shh had no effect on the proliferation of renal tubular epithelial cells. In vivo, overexpression of Shh promoted fibroblast expansion and aggravated kidney fibrotic lesions after IRI. Correspondingly, blockade of Shh signaling by cyclopamine, a small molecule inhibitor of Smoothened, inhibited fibroblast proliferation, reduced myofibroblast accumulation, and attenuated renal fibrosis. These studies identify Shh as a novel, specific, and potent tubule-derived growth factor that promotes interstitial fibroblast proliferation and activation. Our data also suggest that blockade of Shh signaling is a plausible strategy for therapeutic intervention of renal fibrosis.

Hedgehog acyltransferase as a target in pancreatic ductal adenocarcinoma.

Sonic Hedgehog (Shh) is abnormally expressed in pancreatic cancer and is associated with disease onset and progression. Inhibition of Shh signaling is thus an attractive clinical target for therapeutic intervention. Most efforts to block Shh signaling have focused on inhibitors of Smoothened, which target the canonical Shh signaling pathway. These approaches have met with limited success, in part due to development of resistance-conferring mutations and contributions from non-canonical signaling pathways. Here, we show that Hedgehog acyltransferase (Hhat), the enzyme responsible for the attachment of palmitate onto Shh, is a novel target for inhibition of Shh signaling in pancreatic cancer cells. Depletion of Hhat with lentivirally delivered small hairpin RNA decreased both anchorage-dependent and independent proliferation of human pancreatic cancer cells. In vivo, Hhat knockdown led to reduction of tumor growth in a mouse xenograft model of pancreatic cancer. RU-SKI 43, a small molecule inhibitor of Hhat recently developed by our group, reduced pancreatic cancer cell proliferation and Gli-1 activation through Smoothened-independent non-canonical signaling. In addition, RU-SKI 43 treatment inhibited two key proliferative pathways regulated by Akt and mTOR. This work demonstrates that Hhat has a critical role in pancreatic cancer and that a small molecule inhibitor of Hhat can successfully block pancreatic cancer cell proliferation. It also highlights the importance of developing optimized Hhat inhibitors to be used as therapeutics in pancreatic cancer, as well as in other malignancies characterized by Shh overexpression.

Transient Activation of Hedgehog Pathway Rescued Irradiation-Induced Hyposalivation by Preserving Salivary Stem/Progenitor Cells and Parasympathetic Innervation

To examine the effects and mechanisms of transient activation of the Hedgehog pathway on rescuing radiotherapy-induced hyposalivation in survivors of head and neck cancer. Mouse salivary glands and cultured human salivary epithelial cells were irradiated by a single 15-Gy dose. The Hedgehog pathway was transiently activated in mouse salivary glands, by briefly overexpressing the Sonic hedgehog (Shh) transgene or administrating smoothened agonist, and in human salivary epithelial cells, by infecting with adenovirus encoding Gli1. The activity of Hedgehog signaling was examined by the expression of the Ptch1-lacZ reporter and endogenous Hedgehog target genes. The salivary flow rate was measured following pilocarpine stimulation. Salivary stem/progenitor cells (SSPC), parasympathetic innervation, and expression of related genes were examined by flow cytometry, salisphere assay, immunohistochemistry, quantitative reverse transcription PCR, Western blotting, and ELISA. Irradiation does not activate Hedgehog signaling in mouse salivary glands. Transient Shh overexpression activated the Hedgehog pathway in ductal epithelia and, after irradiation, rescued salivary function in male mice, which is related with preservation of functional SSPCs and parasympathetic innervation. The preservation of SSPCs was likely mediated by the rescue of signaling activities of the Bmi1 and Chrm1-HB-EGF pathways. The preservation of parasympathetic innervation was associated with the rescue of the expression of neurotrophic factors such as Bdnf and Nrtn. The expression of genes related with maintenance of SSPCs and parasympathetic innervation in female salivary glands and cultured human salivary epithelial cells was similarly affected by irradiation and transient Hedgehog activation. These findings suggest that transient activation of the Hedgehog pathway has the potential to restore salivary gland function after irradiation-induced dysfunction.

Hedgehog Signaling in Neonatal and Adult Lung

Sonic Hedgehog (Shh) signaling is essential during embryonic lung development, but its role in postnatal lung development and adult lung are not known. Using Gli1(nlacZ) reporter mice to identify cells with active Hh signaling, we found that Gli1(nlacZ)-positive mesenchymal cells are densely and diffusely present up to 2 weeks after birth and decline in number thereafter. In adult mice, Gli1(nlacZ)-positive cells are present around large airways and vessels and are sparse in alveolar septa. Hh-stimulated cells are mostly fibroblasts; only 10% of Gli1(nlacZ)-positive cells are smooth muscle cells, and most smooth muscle cells do not have activation of Hh signaling. To assess its functional relevance, we influenced Hh signaling in the developing postnatal lung and adult injured lung. Inhibition of Hh signaling during early postnatal lung development causes airspace enlargement without diminished alveolar septation. After bleomycin injury in the adult lung, there are abundant Gli1(nlacZ)-positive mesenchymal cells in fibrotic lesions and increased numbers of Gli1(nlacZ)-positive cells in preserved alveolar septa. Inhibition of Hh signaling with an antibody against all Hedgehog isoforms does not reduce bleomycin-induced fibrosis, but adenovirus-mediated overexpression of Shh increases collagen production in this model. Our data provide strong evidence that Hh signaling can regulate lung stromal cell function in two critical scenarios: normal development in postnatal lung and lung fibrosis in adult lung.

Enhanced Dentin-Like Mineralized Tissue Formation by AdShh-Transfected Human Dental Pulp Cells and Porous Calcium Phosphate Cement

The aim of the present study was to investigate the effect of Sonic hedgehog (Shh) on human dental pulp cells (hDPCs) and the potential of complexes with Shh gene modified hDPCs and porous calcium phosphate cement (CPC) for mineralized tissue formation. hDPCs were cultured and transfected with adenoviral mediated human Shh gene (AdShh). Overexpression of Shh and cell proliferation was tested by real-time PCR analysis, western blotting analysis, and MTT analysis, respectively. The odontoblastic differentiation was assessed by alkaline phosphatase (ALP) activity and real-time PCR analysis on markers of Patched-1 (Ptc-1), Smoothened (Smo), Gli 1, Gli 2, Gli 3, osteocalcin (OCN), dentin matrix protein-1 (DMP-1), and dentin sialophosphoprotein (DSPP). Finally, AdShh-transfected hDPCs were combined with porous CPC and placed subcutaneously in nude mice for 8 and 12 weeks, while AdEGFP-transfected and untransfected hDPCs were treated as control groups. Results indicated that Shh could promote proliferation and odontoblastic differentiation of hDPCs, while Shh/Gli 1 signaling pathway played a key role in this process. Importantly, more mineralized tissue formation was observed in combination with AdShh transfected hDPCs and porous CPC, moreover, the mineralized tissue exhibited dentin-like features such as structures similar to dentin-pulp complex and the positive staining for DSPP protein similar to the tooth tissue. These results suggested that the constructs with AdShh-transfected hDPCs and porous CPC might be a better alternative for dental tissue regeneration.

Effects of electromagnetic pulse on polydactyly of mouse fetuses

There is an increasing public concern regarding potential health impacts from electromagnetic radiation exposure. Embryonic development is sensitive to the external environment, and limb development is vital for life quality. To determine the effects of electromagnetic pulse (EMP) on polydactyly of mouse fetuses, pregnant mice were sham-exposed or exposed to EMP (400 kV/m with 400 pulses) from Days 7 to 10 of pregnancy (Day 0 = day of detection of vaginal plug). As a positive control, mice were treated with 5-bromodeoxyuridine on Days 9 and 10. On Days 11 or 18, the fetuses were isolated. Compared with the sham-exposed group, the group exposed to EMP had increased rates of polydactyly fetuses (5.1% vs. 0.6%, P < 0.05) and abnormal gene expression (22.2% vs. 2.8%, P < 0.05). Ectopic expression of Fgf4 was detected in the apical ectodermal ridge, whereas overexpression and ectopic expression of Shh were detected in the zone of polarizing activity of limbs in the EMP-exposed group and in the positive control group. However, expression of Gli3 decreased in mesenchyme cells in those two groups. The percentages of programmed cell death of limbs in EMP-exposed and positive control group were decreased (3.57% and 2.94%, respectively, P < 0.05, compared with 7.76% in sham-exposed group). In conclusion, polydactyly induced by EMP was accompanied by abnormal expression of the above-mentioned genes and decreased percentage of programmed cell death during limb development.

ABCB2 (TAP1) as the downstream target of SHH signaling enhances pancreatic ductal adenocarcinoma drug resistance

Hedgehog signaling plays critical roles in drug resistance of PDAC. We demonstrate that SHH is highly expressed in PDAC patients and cell lines. SHH signaling protects PDAC cells against gemcitabine induced apoptosis, because either over-expression or knockdown of SHH in PDAC cells affects the sensitivity to gemcitabine. Mechanistic studies show that ABCB2 serves as the downstream target of SHH signaling, leading to the drug resistance of PDAC cells. Combinational treatments with gemcitabine and cyclopamine yield synergistic antitumor effects in vitro and in vivo. Our study suggests that inhibiting SHH signaling or targeting ABCB2 gene improves the efficacy of chemotherapy in patients with PDAC.

Hedgehog signaling displays a biphasic expression pattern during intestinal injury and repair

Background/PurposeGastrointestinal injury is common clinically. The exact mechanism by which gastrointestinal repair occurs has yet to be well defined. Hedgehog (Hh) signaling is known to be involved in gastrointestinal development and repair of tissues such as skin and heart. The present study aimed to investigate the role of Hh in the repair of the small intestine. Methodsi) To study acute intestinal injury, we optimized a mouse model of 5-flurouracil (5-FU) induced injury of the small intestine. Ileal tissues were evaluated for injury and repair markers at day 0, 2, 5, and 9. ii) Immunohistochemistry (Sonic hedgehog, Shh), in situ hybridization (Shh), and Ptch/LacZ transgenic mice were carried out to localize hedgehog expression. A33CrPr × ShhTg knock-in mice were bred to study the effect of Shh over-expression. qPCR of Shh, Ihh, Ptch, Bmp4 was carried out to quantify hedgehog signaling. iii) 5FU treated mice were then treated with a hedgehog inhibitor or saline (control) and the effects of Shh inhibition including apoptosis, proliferation, and mitosis were then compared. Resultsi) Immunohistochemistry and in situ hybridization of Shh, qPCR of hedgehog signaling pathway genes, and Ptch/LacZ staining results consistently showed down-regulation during the injury phase (P<0.05) followed by up-regulation during the repair phase (P<0.005). ii) Hh signaling inhibition following 5-FU induced injury augmented apoptotic activity (P<0.05), suppressed mitotic activity (P<0.005) in intestinal crypts, and reduced Paneth cell hyperplasia (P<0.005). iii) Shh over-expression in conditionally knock-mice led to increased mitotic, Paneth, and goblet cells. ConclusionHedgehog signaling pathway displays a biphasic expression pattern during the injury/repair of small intestine. It may play an important regulatory role in intestinal repair.

Evolution of an adaptive behavior and its sensory receptors promotes eye regression in blind cavefish.

BackgroundHow and why animals lose eyesight during adaptation to the dark and food-limited cave environment has puzzled biologists since the time of Darwin. More recently, several different adaptive hypotheses have been proposed to explain eye degeneration based on studies in the teleost Astyanax mexicanus, which consists of blind cave-dwelling (cavefish) and sighted surface-dwelling (surface fish) forms. One of these hypotheses is that eye regression is the result of indirect selection for constructive characters that are negatively linked to eye development through the pleiotropic effects of Sonic Hedgehog (SHH) signaling. However, subsequent genetic analyses suggested that other mechanisms also contribute to eye regression in Astyanax cavefish. Here, we introduce a new approach to this problem by investigating the phenotypic and genetic relationships between a suite of non-visual constructive traits and eye regression.ResultsUsing quantitative genetic analysis of crosses between surface fish, the Pachón cavefish population and their hybrid progeny, we show that the adaptive vibration attraction behavior (VAB) and its sensory receptors, superficial neuromasts (SN) specifically found within the cavefish eye orbit (EO), are genetically correlated with reduced eye size. The quantitative trait loci (QTL) for these three traits form two clusters of congruent or overlapping QTL on Astyanax linkage groups (LG) 2 and 17, but not at the shh locus on LG 13. Ablation of EO SN in cavefish demonstrated a major role for these sensory receptors in VAB expression. Furthermore, experimental induction of eye regression in surface fish via shh overexpression showed that the absence of eyes was insufficient to promote the appearance of VAB or EO SN.ConclusionsWe conclude that natural selection for the enhancement of VAB and EO SN indirectly promotes eye regression in the Pachón cavefish population through an antagonistic relationship involving genetic linkage or pleiotropy among the genetic factors underlying these traits. This study demonstrates a trade-off between the evolution of a non-visual sensory system and eye regression during the adaptive evolution of Astyanax to the cave environment.

Sonic hedgehog promotes autophagy of vascular smooth muscle cells

Sonic hedgehog (Shh) is a morphogen critically involved in development that is reexpressed in atherosclerotic lesions. It also stimulates proliferation of vascular smooth muscle cells (SMCs). Autophagy in vascular SMCs is known to promote SMC survival and increase plaque stability. The aim of this study was to investigate whether Shh induces autophagy of vascular SMCs. Our study showed that both Shh protein and microtubule-associated protein 1 light chain 3 (LC3)-II were increased in SMCs within neointimal lesions of mouse common carotid arteries. In cultured mouse aortic SMCs, recombinant mouse Shh stimulated LC3-II levels. Overexpression of wild-type mouse Shh through the tetracycline-regulated expression-inducible system in human aortic SMCs time-dependently increased the levels of LC3-II and also stimulated protein kinase B (AKT) phosphorylation. Pretreatment with AKT inhibitor IV (AKTI IV) inhibited AKT phosphorylation and the increase in LC3-II. Shh-induced autophagy was further confirmed by the formation of autophagosomes as detected by immunostaining and transmission electron microscopy, which was inhibited by AKTI IV. Shh further increased SMC LC3-II in the presence of bafilomycin A1, (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, and pepstatin A or siRNA for the autophagy-related gene 7 (ATG7). In addition, Shh induced SMC proliferation, which was inhibited not only by AKTI IV but also by cyclopamine, an inhibitor of Shh receptor. Inhibition of autophagy with 3-methyladenine (3-MA), bafilomycin A1, or ATG7 siRNA resulted in inhibition of cell proliferation. Treatment with 3-MA, AKTI IV, or cyclopamine inhibited neointima formation in mouse common carotid arteries. Taken together, our results have shown that Shh induces autophagy of vascular SMCs involving AKT activation, suggesting a role of autophagy in Shh-induced cellular responses.

Role of Microparticles as Messengers Enhancing Stem Cell Activity After Genetic Engineering

Despite significant therapeutic advances over several decades, myocardial infarction remains the most common cause of death worldwide. In this context, cell-based therapy was considered a promising novel strategy for regeneration of healthy, functionally integrated, myocardial tissue. Unfortunately, the clinical trials using cell-based therapy did not always confirm the preclinical experimental results.1,2 Therefore, in the last decade, the efforts were focused on studying the complex molecular and cellular mechanisms contributing to improved myocardial remodeling and function after stem cell transplantation.3 Article, see p 312 Because cell-based therapy has been proposed, various cell types were tested in transplantation experiments.4–10 Among all, autologous bone marrow-derived CD34+ stem cells demonstrate a major benefit, showing favorable impact on angiogenic growth, without the toxicity and inflammatory response observed in nonautologous cell-based therapy.11,12 However, insufficient number of donor stem cells, low cell viability, and inefficient expansion techniques limit the effects of this therapy. In this regard, overexpressing angiogenic growth factors in transplanted cells has been reported to be beneficial by attenuating myocardial ischemia and preserving the heart function.13–17 In this issue of Circulation Research , Mackie et al investigate the mechanisms of preserving the heart function after transplantation of CD34+ cells overexpressing sonic hedgehog (Shh) protein, a well-established angiogenic factor.18 After …

Hedgehog pathway activation in human transitional cell carcinoma of the bladder.

Background:The Hedgehog (Hh) signalling pathway functions as an organiser in embryonic development. Recent studies have shown constitutive activation of this pathway in various malignancies, but its role in bladder cancer remains poorly studied.Methods:Expression levels of 31 genes and 9 microRNAs (miRNAs) involved in the Hh pathway were determined by quantitative real-time RT–PCR in 71 bladder tumour samples (21 muscle-invasive (MIBC) and 50 non-muscle-invasive (NMIBC) bladder cancers), as well as in 6 bladder cancer cell lines.Results:The SHH ligand gene and Gli-inducible target genes (FOXM1, IGF2, OSF2, H19, and SPP1) were overexpressed in tumour samples as compared with normal bladder tissue. SHH overexpression was found in 96% of NMIBC and 52% of MIBC samples, as well as in two bladder cancer cell lines. Altered expression of miRNAs supported their oncogene or tumour-suppressor gene status. In univariate analysis, high expression levels of PTCH2, miRNA-92A, miRNA-19A, and miRNA-20A were associated with poorer overall survival in MIBC (P=0.02, P=0.012, P=0.047, and P=0.036, respectively).Conclusion:We observed constitutive activation of the Hh pathway in most NMIBC and about 50% of MIBC. We also found that some protein-coding genes and miRNAs involved in the Hh pathway may have prognostic value at the individual level.

Thyroid hormone-induced sonic hedgehog signal up-regulates its own pathway in a paracrine manner in the Xenopus laevis intestine during metamorphosis.

During Xenopus laevis metamorphosis, Sonic hedgehog (Shh) is directly induced by thyroid hormone (TH) at the transcription level as one of the earliest events in intestinal remodeling. However, the regulation of other components of this signaling pathway remains to be analyzed. Here, we analyzed the spatiotemporal expression of Patched (Ptc)-1, Smoothened (Smo), Gli1, Gli2, and Gli3 during natural and TH-induced intestinal remodeling. We show that all of the genes examined are transiently up-regulated in the mesenchymal tissues during intestinal metamorphosis. Interestingly, in the presence of protein synthesis inhibitors, Gli2 but not the others was induced by TH, suggesting that Gli2 is a direct TH response gene, while the others are likely indirect ones. Furthermore, we demonstrate by the organ culture experiment that overexpression of Shh enhances the expression of Ptc-1, Smo, and Glis even in the absence of TH, indicating that Shh regulates its own pathway components during intestinal remodeling.

Aberrant Hedgehog Ligands Induce Progressive Pancreatic Fibrosis by Paracrine Activation of Myofibroblasts and Ductular Cells in Transgenic Zebrafish

Hedgehog (Hh) signaling is frequently up-regulated in fibrogenic pancreatic diseases including chronic pancreatitis and pancreatic cancer. Although recent series suggest exclusive paracrine activation of stromal cells by Hh ligands from epithelial components, debates still exist on how Hh signaling works in pathologic conditions. To explore how Hh signaling affects the pancreas, we investigated transgenic phenotypes in zebrafish that over-express either Indian Hh or Sonic Hh along with green fluorescence protein (GFP) to enable real-time observation, or GFP alone as control, at the ptf1a domain. Transgenic embryos and zebrafish were serially followed for transgenic phenotypes, and investigated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), in situ hybridization, and immunohistochemistry. Over-expression of Ihh or Shh reveals virtually identical phenotypes. Hh induces morphologic changes in a developing pancreas without derangement in acinar differentiation. In older zebrafish, Hh induces progressive pancreatic fibrosis intermingled with proliferating ductular structures, which is accompanied by the destruction of the acinar structures. Both myofibroblasts and ductular are activated and proliferated by paracrine Hh signaling, showing restricted expression of Hh downstream components including Patched1 (Ptc1), Smoothened (Smo), and Gli1/2 in those Hh-responsive cells. Hh ligands induce matrix metalloproteinases (MMPs), especially MMP9 in all Hh-responsive cells, and transform growth factor-ß1 (TGFß1) only in ductular cells. Aberrant Hh over-expression, however, does not induce pancreatic tumors. On treatment with inhibitors, embryonic phenotypes are reversed by either cyclopamine or Hedgehog Primary Inhibitor-4 (HPI-4). Pancreatic fibrosis is only prevented by HPI-4. Our study provides strong evidence of Hh signaling which induces pancreatic fibrosis through paracrine activation of Hh-responsive cells in vivo. Induction of MMPs and TGFß1 by Hh signaling expands on the current understanding of how Hh signaling affects fibrosis and tumorigenesis. These transgenic models will be a valuable platform in exploring the mechanism of fibrogenic pancreatic diseases which are induced by Hh signaling activation.

The Antagonistic Action of B56-containing Protein Phosphatase 2As and Casein Kinase 2 Controls the Phosphorylation and Gli Turnover Function of Daz Interacting Protein 1

The Hedgehog (Hh) pathway is evolutionarily conserved and plays critical roles during embryonic development and adult tissue homeostasis. Defective Hh signaling has been linked to a wide range of birth defects and cancers. Hh family proteins regulate the expression of their downstream target genes through the control of proteolytic processing and the transcriptional activation function of Gli transcription factors. Although Hh-dependent regulation of Gli has been studied extensively, other Gli regulatory mechanisms remain relatively unappreciated. Here we report our identification of a novel signaling cascade that controls the stability of Gli proteins. This cascade consists of Daz interacting protein 1 (Dzip1), casein kinase 2 (CK2), and B56 containing protein phosphatase 2As (PP2As). We provide evidence that Dzip1 is involved in a novel Gli turnover pathway. We show that CK2 directly phosphorylates Dzip1 at four serine residues, Ser-664/665/706/714. B56-containing PP2As, through binding to a domain located between amino acid residue 474 and 550 of Dzip1, dephosphorylate Dzip1 on these CK2 sites. Our mutagenesis analysis further demonstrates that the unphosphorylatable form of Dzip1 is more potent in promoting Gli turnover. Consistently, we found that the stability of Gli proteins was decreased upon CK2 inhibition and increased by inhibition of B56-containing PP2As. Thus, reversible phosphorylation of Dzip1, which is controlled by the antagonistic action of CK2 and B56-containing PP2As, has an important impact on the stability of Gli transcription factors and Hh signaling.

Expression of sonic hedgehog and the downstream transcriptional factor Gli1 in sonic hedgehog pathway in gastric cancer

Objective To study the expression and prognostic value of Sonic hedgehog (Shh) and the downstream transcriptional factor Cli1 in Shh signal pathway in gastric cancer. Methods 100 primary gastric cancer samples and 10 normal gastric mucosa samples were selected in histopathological tissue bank of 1st affiliated hospital of Sun Yat-sen University. The expression of Shh and Gli1 was detected by using immunochemistry staining. Results Shh and Cli1 staining was positive in all gastric cancer samples,while only three samples were positive in normal gastric mucosa ( 3/10, 30% ) , and the staining intensity was lower in normal tissues than in cancer samples (P＜0. 01). Shh and Gli1 were associated with worse differentiation, metastasis, late stage and shorter survival. Shh and Gli1 showed linear positive correlation in gastric cancer with a correlation coefficient equal to 0. 747 ( P ＜ 0. 01 ). The survival was shortened along with the increases in Shh and Gli1 staining intensity (P＜0. 05). Conclusion Shh signal pathway played an important role in gastric carcinogenesis, and Shh and Gli1 overexpression was associated with worse prognosis. Key words: Gastric carcinoma; Sonic hedgehog; Prognosis