Expression Of Ptc1 Research Articles

Procalcitonin and Pancreatic Stone Protein Function as Biomarkers in Early Diagnosis of Pediatric Acute Osteomyelitis.

BackgroundHigh plasma levels of procalcitonin (PCT) are typically seen in children with severe bacterial infection, particularly in cases of septic shock or bacteremia. Similarly, pancreatic stone protein (PSP) is associated with inflammation, infection, and other disease-related stimuli. However, the prognostic value of PSP in critically ill pediatric patients is unknown. This study investigated the early diagnostic value of PCT and PSP in pediatric acute osteomyelitis.Material/MethodsA total of 187 patients with suspected acute osteomyelitis and 80 healthy control children were enrolled. The serum expression of PTC and PSP was measured. Pearson correlation analysis was conducted to correlate PTC with PSP. ROC analysis was used to test the value of PTC and PSP in early diagnosis of pediatric acute osteomyelitis.ResultsAcute osteomyelitis was diagnosed in 49.2% of the patients (n=92) based on the layered bone puncture. The serum levels of PTC and PSP in pediatric acute osteomyelitis were higher than in the non-acute osteomyelitis group (P<0.01). Serum PTC concentrations showed a significantly positive correlation with PSP levels (P<0.001). ROC analysis showed that the AUC values of PTC and PSP were 0.767 (95% CI, 0.700–0.826), and 0.796 (95% CI, 0.731–0.855), respectively. The AUC value of PTC & PSP was 0.903 (95% CI: 0.851–0.941), which was markedly increased compared with PTC or PSP (P<0.01).ConclusionsSerum levels of PCT and PSP are promising biomarkers for early diagnosis of pediatric acute osteomyelitis.

Primary cilium alterations and expression changes of Patched1 proteins in niemann-pick type C disease.

Niemann-Pick type C disease (NPC) is a disorder characterized by abnormal intracellular accumulation of unesterified cholesterol and glycolipids. Two distinct disease-causing genes have been isolated, NPC1 and NPC2. The NPC1 protein is involved in the sorting and recycling of cholesterol and glycosphingolipids in the late endosomal/lysosomal system. It has extensive homology with the Patched1 (Ptc1) receptor, a transmembrane protein localized in the primary cilium, and involved in the Hedgehog signaling (Shh) pathway. We assessed the presence of NPC1 and Ptc1 proteins and evaluated the relative distribution and morphology of primary cilia in fibroblasts from five NPC1 patients and controls, and in normal fibroblasts treated with 3-ß-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A), a cholesterol transport-inhibiting drug that is widely used to mimic NPC. Immunofluorescence and western blot analyses showed a significant decrease in expression of NPC1 and Ptc1 in NPC1 fibroblasts, while they were normally expressed in U18666A-treated fibroblasts. Moreover, fibroblasts from NPC1 patients and U18666A-treated cells showed a lower percentage distribution of primary cilia and a significant reduction in median cilia length with respect to controls. These are the first results demonstrating altered cytoplasmic expression of Ptc1 and reduced number and length of primary cilia, where Ptc1 is located, in fibroblasts from NPC1 patients. We suggest that the alterations in Ptc1 expression in cells from NPC1 patients are closely related to NPC1 expression deficit, while the primary cilia alterations observed in NPC1 and U18666A-treated fibroblasts may represent a secondary event derived from a defective metabolic pathway.

Phase 2 Trial of Smoothened (SMO) Inhibitor PF-04449913 (PF-04) in Refractory Myelodysplastic Syndromes (MDS)

▪Introduction: MDS encompasses a heterogeneous group of clonal neoplastic hematopoietic stem cell diseases characterized by ineffective hematopoiesis and frequent progression to acute myeloid leukemia (AML). While the advent of hypomethylating agent (HMA) therapy has modestly improved survival in higher-risk MDS, outcomes following HMA failure are dismal. The hedgehog (HH) signaling pathway is an important regulator of hematopoietic cell survival and differentiation, with upregulation of HH target genes and signaling proteins a frequent occurrence in MDS and AML. PF-04449913 (PF-04) is an orally bioavailable inhibitor of Smoothened (SMO), which demonstrated antileukemic activity in a prior phase 1 study. We performed a phase 2 study of PF-004449913 in patients with MDS following HMA failure.Objectives: 1) primary: to estimate overall IWG-2006 response rate (CR + PR + HI + marrow CR) to PF-04449913 in patients with relapsed/refractory MDS and CMML; 2) secondary: to assess safety , overall survival, time to AML transformation, and effect of PF-04 on HH target gene expression.Methods: This was a single center, open-label phase 2 study. Key eligibility criteria included: 1) patients age ≥ 18 with MDS, CMML, or AML (20-29% blasts) following failure of HMA; 2) ECOG 0-2; 3) Adequate kidney and hepatic function. Treatment consisted of PF-04 at 100 mg/day in 4-week cycles for 4 cycles, with continuation allowed for achievement of stable disease or better. Dose increase to 200 mg/day was permitted after cycle 2 for patients with stable disease. Bone marrow biopsy for response assessment occurred after cycle 2, cycle 4, and every 4th cycle thereafter. For correlative studies, total cellular RNA was isolated from ficolled mononuclear cells in baseline marrow samples, and qPCR performed for expression of GLI1, PTCH1, and SMO).Results: Thirty five patients were enrolled, 24M and 11F. Median age was 75 years (range 55-88). A majority of patients had higher-risk (54%) vs lower-risk (43%) MDS by IPSS at time of enrollment. A total of thirty four patients (97%) had received prior azacitidine for a median of 9 cycles (range 1-88), while 6 patients had received decitabine for a median of 4 cycles (5 of whom had also received azacitidine). The median number of cycles of PF-04 received was 3 (range 1-11). All 35 patients were evaluable for response. Two of 35 patients (6%) achieved response per MDS IWG 2006 criteria, including one patient with HI-N/HI-P and another with marrow CR and HI-N. Nineteen additional patients (54%) had stable disease following cycle 2 (8 weeks). With a median follow-up of 10.1 months, the median survival for all treated patients was 10.2 months (95% CI 6.8-13.6 mo)(Figure 1). Median survival for patients with low/INT-1 risk MDS was longer than for INT-2/high risk patients (22.4 mo vs 7.5 mo, p=0.013). Six patients (17%) died while on-treatment, but none of the deaths were considered related to PF-04. The most common treatment-emergent adverse events included myalgia/muscle cramps (65%), dysgeusia (60%), nausea (31%), anorexia (31%), oral mucositis (25%), and AST elevation (22%). The vast majority of these events were grade 1/2. Except for infection (11%), there were no treatment-emergent grade 3/4 events in > 10% of patients. Only 5 patients (14%) required dose reduction to 50 mg/day due to intolerance. In the pharmacodynamics analysis, responding patients had higher baseline expression of SMO, PTC ,and GLI1 than non-responders, while non-responders had higher post-treatment increases in these transcripts compared with responders.Conclusions: PF-04 was well tolerated in patients with advanced, HMA refractory MDS, but had limited efficacy in this unselected, heavily pre-treated patient population. The pharmacodynamic analysis suggests predictive potential of HH pathway gene expression for response warranting further investigation. [Display omitted] DisclosuresKomrokji:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy. Sweet:Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Pfizer: Speakers Bureau; Incyte Corporation: Research Funding; Karyopharm: Honoraria, Research Funding.

Associations of miR-146a and miR-146b expression and clinical characteristics in papillary thyroid carcinoma.

Human miRNAs have emerged as potentially useful diagnostic and prognostic markers in cancer within the past decade. Abnormal expression of microRNA-146 (miR-146) has been found in several classes of cancers. To explore expression and clinical associations of miR-146a and miR-146b in papillary thyroid carcinoma. We investigated expressions of miR-146a and miR-146b in tissues and peripheral blood among 128 patients with primary PTC, 120 patients with nodular goiter and 120 healthy controls by RT-PCR. Furthermore, the associations between the miR-146a and miR-146b expression and clinical parameters of PTC were analyzed. Levels of miR-146a and miR-146b expression in PTC tissues were higher compared to the nodular goiter tissues (P=0.014 and 0.001) and perineoplastic thyroid tissues (P=0.023 and 0.002). Positive associations were found between levels of miR-146a expression in PTC tissues and gender (female), cervical lymph node metastasis, multifocality, extra tyroidal invasion and advanced clinical TNM stages (III and IV). For miR-146b, there were positive association between levels of miR-146b expression and female, extra tyroidal invasion and advanced clinical TNM stages. Up-regulation of miR-146a and miR-146b expression in tissues was related to carcinogenesis and deterioration of PTC. MicroRNA-146a and miR-146b expressed in thyroid tissue may act as potential biomarkers for PTC patients.

Sonic Hedgehog Improves Redifferentiation of Dedifferentiated Chondrocytes for Articular Cartilage Repair

Sonic hedgehog (Shh) is involved in the induction of early cartilaginous differentiation of mesenchymal cells in the limb. We investigated whether Shh could promote redifferentiation of dedifferentiated chondrocytes and have a favorable effect on the regeneration of cartilage. Articular chondrocytes of rats were separated and cultured. The redifferentiation of dedifferentiated chondrocytes transfected with Shh was evaluated using monolayer and pellet culture system. The signaling molecules (Ptc 1, Gli 1 and Sox9) of the hedgehog pathway were investigated. A rat model of articular cartilage defect was used to evaluate cartilage repair after transplantation with dedifferentiated chondrocytes. After Shh gene transfer, the hedgehog pathway was upregulated in dedifferentiated chondrocytes. Real time-PCR and western blot analysis verified the stronger expression of Ptc1, Gli1 and Sox9 in Shh transfected cells. Shh upregulates the Shh signaling pathway and multiple cytokines (bone morphogenetic protein 2 and insulin-like growth factor 1) in dedifferentiated chondrocytes. After transplantation in the joint, histologic analysis of the regenerative tissues revealed that significantly better cartilage repair in rats transplanted with Shh transfected cells. These data suggest that Shh could induce redifferentiation of dedifferentiated chondrocytes through up-regulating Shh signaling pathway, and have considerable therapeutic potential in cartilage repair.

The Hedgehog Receptor Patched Functions in Multidrug Transport and Chemotherapy Resistance

Most anticancer drugs fail to eradicate tumors, leading to the development of drug resistance and disease recurrence. The Hedgehog signaling plays a crucial role during embryonic development, but is also involved in cancer development, progression, and metastasis. The Hedgehog receptor Patched (Ptc) is a Hedgehog signaling target gene that is overexpressed in many cancer cells. Here, we show a link between Ptc and resistance to chemotherapy, and provide new insight into Ptc function. Ptc is cleared from the plasma membrane upon interaction with its ligand Hedgehog, or upon treatment of cells with the Hedgehog signaling antagonist cyclopamine. In both cases, after incubation of cells with doxorubicin, a chemotherapeutic agent that is used for the clinical management of recurrent cancers, we observed an inhibition of the efflux of doxorubicin from Hedgehog-responding fibroblasts, and an increase of doxorubicin accumulation in two different cancer cell lines that are known to express aberrant levels of Hedgehog signaling components. Using heterologous expression system, we stringently showed that the expression of human Ptc conferred resistance to growth inhibition by several drugs from which chemotherapeutic agents such as doxorubicin, methotrexate, temozolomide, and 5-fluorouracil. Resistance to doxorubicin correlated with Ptc function, as shown using mutations from Gorlin's syndrome patients in which the Ptc-mediated effect on Hedgehog signaling is lost. Our results show that Ptc is involved in drug efflux and multidrug resistance, and suggest that Ptc contributes to chemotherapy resistance of cancer cells.

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.

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.

Abstract 713: Active hedgehog pathway in human primary gastric tumor models

Abstract The hedgehog (Hh) signaling pathway is known to be essential for multiple aspects of embryonic development, and activation of the Hh pathway leads to the development of several human malignancies such as basal cell carcinoma and medulloblastoma. Recent data have implicated a much broader role of the Hh pathway in other human cancer as well as the maintenance of cancer stem cells. Loss-function mutation of Ptc and gain of gunction mutation of Smo in human malignancies make the components in Hh pathway attractive therapeutic targets. However, this pathway is readily deactivated once human tumor cells are propagated in cell culture, a prerequisite of most preclinical human cancer models. In order to identify useful models for the evaluation of novel therapeutics targeting the Hh pathway, we profiled the expression of Ptc, Smo, Gli in a cohort of twenty established human primary gastric tumor models by TaqMan real-time quantitative PCR reaction and immunohistochemistry (IHC). Seven of the twenty models displayed high expression of either Gli-1 or Gli-2. Eight models were found to have high expression level of Shh. And three models were found to have high level of Ptc. The Gli protein expression level is also examined with IHC. High levels of Gli and Shh in the selected models will be very useful to evaluate anticancer drugs against Hh signaling pathway for their drug activity and pharmacodynamic responses. These models, together with the associated clinical and pathological information, provide a unique platform to evaluate the in vivo activities of pharmaceutical agents targeting the Hh pathway. 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 713.

The effect of PTC1 transfection on the growth and apoptosis of human pulmonary adenocarcinoma cell A549

To investigate the effects of the tumor suppressor gene PTC on the growth inhibition and down-regulation of epidermal growth factor receptors (EGFR) in pulmonary adenocarcinoma cell A549. Pulmonary adenocarcinoma cell A549 were divided into wild type group, mutant type group, blank group and control group. They were transfected with wild-type PTC1 plasmids, mutant-PTC1 plasmids and blank plasmids, respectively. After transfection, the cell growth curve was drown every day for a week. The expression of PTC1 and EGFR were detected by western blot. Flow cytometry was used to analyze apoptosis and cell cycle of the transfected cells. After transfected with wild-type PTC1, the growth rates of A549 cells were slow down, but the other groups of cells had no change. Compared with the control group, the expression of EGFR were down-regulated. The apoptosis rates in wild type group was 24.5%, and the mutant type group was 8.3% (P < 0.01). But the apoptosis rate of blank group has no change. Wild-type PTC1 could induce apoptosis and inhibitory effect on A549 cells.

Cellular plasticity of trans- and dedifferentiation markers in human hepatoma cells in vitro and in vivo

Tumor cells have the capability to trans- and to dedifferentiate, for example by reactivating embryonic development genes and stem cell characteristics. The aim of our study was to show the differential expression of stem- and progenitor cell markers in human hepatocellular carcinoma cell lines (HCC). Different human HCC cell lines (HUH7, HUH7 5-15, HUH7 pcDNA3.1, Hep3B and HepG2) were cultured under standard conditions in vitro or implanted subcutaneously (5x10(6) cells) in male NMRI mice. Specimens were characterized by quantitative real-time PCR, Western blotting, methylation-specific PCR and immunohistochemistry for markers of differentiation (cytokeratins, vimentin), embryonic development or stem cells (PTC, PDX-1, SHH, Thy1, c-kit, CD34, beta-catenin, Ki-67). The investigated HCC cell lines showed different patterns of marker expression allowing to distinguish four distinct groups: the classical cholangiocellular type (Huh-7, Huh-7 pcDNA3.1, Hep3B) with expression of CK7/19, beta-catenin and CD34; a dedifferentiated mesenchymal-proliferative type (Huh-7 5-15) characterized by CK19, Vimentin and Ki-67; a dedifferentiated embryonic-development type (Hep3B implanted in matrigel) with expression of CK19, beta-catenin and PTC and a classical HCC type (HepG2) showing CK18/19 and beta-catenin expression. HCC cell lines showed significantly different expression patterns of differentiation markers in a xenograft model. Furthermore, direct association of some markers was observed. The groups differ from each other in expression patterns, but also show that environmental factors play an important role in the behaviour of cells.

Expression of patched‐1 and smoothened in testicular meiotic and post‐meiotic cells

Desert hedgehog (Dhh) signaling plays an essential role in the normal development of the testis and in the process of spermatogenesis. Little is known about the involvement in spermatogenesis of the prototypic member of the family, Ptc1, which acts to suppress hedgehog signaling through Smoothened (Smo). Here, we have examined the expression of Ptc1, Smo, and Dhh in mouse and rat seminiferous epithelium. Our findings demonstrate that Ptc1 and Smo are expressed by primary spermatocytes and by round and condensing spermatids whereas Dhh is expressed by Sertoli cells. The findings suggest that Sertoli cells coordinate Dhh-dependent spermatogenesis events via Ptc1 and Smo prior to the first meiotic division and in postmeiotic (haploid) cells, particularly during the first half of spermiogenesis.

The Expression Pattern of PDX-1, SHH, Patched and Gli-1 Is Associated with Pathological and Clinical Features in Human Pancreatic Cancer

Background and Aims: Pancreatic cancer cells have been shown to possess stem-cell-like properties, especially by reactivating embryonic transcription factors involved in tissue differentiation. We therefore investigated if and to what extent developmental genes of the human pancreas are expressed in pancreatic ductal adenocarcinomas and precursor lesions, pancreatic intraepithelial neoplasia (PanIN), and if this correlates or predicts response to treatment and overall survival. Material and Methods: Invasive ductal adenocarcinomas of the pancreas [UICC pT3pN0 (n = 13) vs. pT3pN1 (n = 25)] and tumors after neoadjuvant chemotherapy [5-fluorouracil (FU)/folic-acid and gemcitabine; UICC ypNO (n = 7) vs. ypN1 (n = 6)] resected between 1997 and 2003 were characterized histochemically and immunohistochemically [pancreas duodenum homeobox 1 (PDX-1), Sonic hedgehog protein (SHH), Patched (Ptc) and Gli-1]. Gene distribution was compared with morphological patterns of the pancreatic carcinoma and PanIN as well as with peritumorous reactions of normal pancreas. Results: The overall expression of PDX-1, SHH, Ptc and Gli-1 was low, but showed a distinctive and topographic linkage inside pancreatic carcinomas as well as inside PanlNs. Additionally, a topographic and significant association of these markers with nodal status (PDX-1, Ptc, Gli-1), tumor size (PDX-1, Gli-1) and R status (PDX-1) was found. After stratification with the strongest outcome predictor, grading, survival analysis revealed that Ptc expression in grade 2 and PDX-1 expression in grade 3 carcinomas are independent survival factors. Conclusions: Markers of pancreas development are reexpressed in invasive ductal adenocarcinomas and their expression is essentially associated with general clinical and pathological features such as survival Or nodal Status.

The effects of Hedgehog-Gli 1 signaling pathway on proliferation and apoptosis of hepatic stellate cells

To investigate the effect of Hedgehog-Gli1 signaling pathway on proliferation, apoptosis and activation of hepatic stellate cells (HSCs) in vitro. The expression of Shh, Smo, Ptc and Gli-1 in HSC-T6 cells was analyzed by RT-PCR. HSC-T6 cells were incubated with various concentration of cyclopamine (0, 50, 100, 150, 200, 250 mumol/L) for 24 hours, cell viability was checked by MTT colorimetric assay, cell cycle was analyzed by flow cytometry, apoptosis was assayed by agarose electrophoresis of DNA and PI-Annexin V fluorescent staining, and the mRNA levels of Gli-1, TGF beta 1, PDGF and Bcl-2 were quantified by real-time RT-PCR. RT-PCR indicated that the components of the Hedgehog-Gli1 signaling pathway were expressed in HSC-T6 cells. MTT assay indicated that cyclopamine inhibited cell viability in a concentration dependant manner (F = 636.81, P less than 0.01). Flow cytometry indicated that cells were piled up at G0/G1 phase in cyclopamine treated cells (65.08%+/-1.50%) as compared to control cells (55.41%+/-2.54%, t = -8.05, P less than 0.01). Cyclopamine treatment resulted in apoptosis as indicated by DNA fragmentation and PI-Annexin V staining. The mRNA levels of Gli-1, TGF beta 1, PDGF and Bcl-2 in cyclopamine treated cells were significantly lower than that in control cells (P less than 0.01). Cyclopamine may inhibit the Hedgehog-Gli1 signaling, and hence repress proliferation and promote apoptosis in hepatic stellate cells.

Abstract 3656: Estradiol Improves Neural Functional Recovery After Nerve Injury by Reducing Inhibition of the Hedgehog-signaling Pathway

Background: Prior data indicate that estradiol (E2) favorably affects neovascularization in injured tissue, and that Sonic Hedgehog (Shh)-induced neovascularization is critical for nerve regeneration; however, little is known about the relationship between E2 and Shh signaling. We tested the hypothesis that E2 potentiates nerve regeneration via Shh-activated neoangiogen-esis. Methods and Results: Ovariectomized mice were subjected to unilateral sciatic nerve crush injury and treated with either E2 (via locally administered biodegradable polymer) or placebo (polymer alone). E2 treatment resulted in greater functional recovery of the nerve as evaluated by both exercise duration (P&lt;0.05) and motor nerve conduction velocity (P&lt;0.05), and intraneural vascularity was significantly higher in E2-treated animals than in the placebo-treatment group (P&lt;0.05). Analyses of mRNA expression in the injured nerves of placebo-treated mice showed that the Shh pathway was activated immediately after injury, but Shh expression declined substantially within 7 days. Shh activation persisted in the E2-treatment group; Shh expression on day 7 was greater in E2-treated animals than in the placebo-treatment group (P&lt;0.05). The expression of Hedgehog-interacting protein (HIP), an endogenous Shh inactivator, was significantly downregulated 24 hours after E2 treatment, and HIP downregulation was attenuated by the E2-receptor blocker ICI, suggesting that E2 augments Shh signaling both my enhancing Shh expression and by inhibiting HIP. Inhibition of HIP expression by E2 was also observed in vitro in Schwann cells and endothelial cells. The expression of Ptc1 (Shh receptor) and Gli1 (a downstream target of Shh) was evaluated in Ptc1-LacZ and Gli1-LacZ mice after nerve-crush injury. X-gal staining demonstrated that E2 treatment upregulated both Ptc1 and Gli1 expression within 3 days of surgery. Conclusions: E2 accelerates the functional recovery of injured nerves by enhancing angiogenesis. We propose that these effects occur in part through the inhibition of the Shh inactivator HIP, thereby preserving Shh activity after nerve injury. These findings may have identified a novel regulatory mechanism for nerve repair via E2-enhaced, Shh-mediated angiogenesis.

Expression of hedgehog signal molecules in pancreatic cancer and their correlation with clinical and pathologic characteristics

Objective To investigate the expression of hedgehog signal molecules (Ihh,Ptc and Stop) in human pancreatic cancer and their correlation with clinical and pathologic characteristics.Methods Fifty-four pancreatic cancer samples and 5 normal pancreatic tissues were examined by im-munohistochemistry for the expression and localization of hedgehog signal molecules (Ihh, Ptc andStop). Meanwhile,correlation of hedgehog signal molecules to clinical and pathologic characteristics ofpancreatic cancer was analyzed. Western blot was carried out to analyze the expression of Ihh,Ptc and Smo in 21 freshly removed pancreatic cancer tissues and paraneoplastic pancreatic tissues. Results Of the 54 pancreatic cancer samples, immunoreactivity of Ihh, Ptc and Smo was observed in 70. 4%,64.8% and 88.9 % of the cancer tissues, respectively. No immunoreaetivity was observed in the ductal cells of all the 5 normal pancreas tissues. Expression of Ptc was strongly correlated with tumor size,differentiation grade of tumor cells,lymph node metastasis and TNM stage of tumor,and Stop expres-sion strongly correlated with the differentiated grade of tumor cells (P~0. 05,respectively). Western blot analysis revealed that the expression of Ihh, Ptc and Stop was significantly higher in pancreatic cancer compared to paraneoplastic pancreatic tissues (P < 0. 05). Conclusion The expression of Hedgehog signal molecules is significantly higher in pancreatic cancer tissues. These molecules are correlated to clinical and pathologic characteristics of pancreatic cancer. Key words: Pancreatic neoplasm; Hedgehog; Immunohistochemistry; Western blot

Sonic Hedgehog Signaling Pathway in Pancreatic Cystic Neoplasms and Ductal Adenocarcinoma

Hedgehog (Hh) signaling is an important mediator of tumorigenesis of pancreatic ductal adenocarcinoma (PA). It is intriguing to explore whether Hh signaling is also involved in pancreatic cystic neoplasms, which are phenotypically different from PA. Patients with solid and pseudopapillary tumor (SPT; n = 12), mucinous cystic neoplasm (MCN; n = 18), intraductal papillary mucinous neoplasm (IPMN; n = 18), and PA (n = 20) were studied. Expression of Hh signaling molecules including sonic Hh (sHh), smoothened (Smo), patched 1 (Ptc1), and Gli were determined using immunohistochemistry and/or Western blotting. Cell cycle-regulator genes, including cyclin A, B, C, and D1 messenger RNA, were determined using ribonuclease protection assay. Six of 12 SPT, 8 of 18 MCN, 17 of 18 IPMN, and 20 of 20 PA displayed Hh signaling using immunohistochemistry. Sonic Hh was predominantly expressed in stromal cells neighboring to the neoplastic cells of SPT and IPMN; in contrast, sHh was expressed in both stromal cells and neoplastic epithelial cells of MCN and PA. The quantitative expression of sHh signaling detected by Western blotting showed that expression of Ptc1 and Gli, but not Smo, corresponded to the magnitude of sonic hedgehog ligand. The expression of cyclin D1 messenger RNA was highest in PA, followed by MCN, IPMN, and SPT, which matches with Ptc1 and Gli. Hedgehog signaling pathway might play a role during tumorigenesis of SPT, MCN, IPMN, and PA. Mucinous cystic neoplasm and PA exhibit an autocrine regulation of sHh, whereas SPT and IPMN do not. Overexpression of Ptc1 and Gli, reflected by cyclin D1, might represent proliferative potential of various pancreatic neoplasms.

Androgen regulation of prostate morphoregulatory gene expression: Fgf10-dependent and -independent pathways.

Androgens are essential and sufficient for prostate gland morphogenesis; however, the downstream gene targets that mediate this action are unclear. To identify androgen-regulated genes involved in prostate development, we used short-term organ culture and examined the effect of testosterone on the expression of several critical prostate morphoregulatory genes. Rat ventral prostates (VP) and lateral prostates (LP) were collected at birth, and contralateral lobes were cultured for 18 h in the presence or absence of 10 nM testosterone with or without OH-flutamide to block residual androgens. Gene expression was quantitated using real-time RT-PCR. Although expression of Fgf10, Nkx3.1, and Ptc was increased in both prostate lobes, other genes were regulated by testosterone in a lobe-specific manner. This included up-regulation of epithelial genes FgfR2iiib, Shh, Hoxb13, and Bmp7 in the VP specifically and down-regulation of mesenchymal genes Wnt5a (VP) and Bmp4 (LP). Thus, in addition to stimulation of homeobox genes and paracrine-acting growth factors, androgens may positively regulate prostatic development through suppression of growth inhibitory genes. Because previous studies revealed a similar gene regulation pattern in response to exogenous Fgf10, experiments were performed to identify androgen-regulated genes mediated through Fgf10 signaling. Short-term VP and LP cultures with FgfR antagonist PD173074 and Mek inhibitor U0126 identified epithelial Shh and Hoxb13 up-regulation by androgens to be Fgf10-dependent. We propose that androgen regulation of prostate development is mediated through positive and negative regulation of multiple morphoregulatory genes acting in combination through complex gene networks. Lobe-specific responses may provide a developmental basis for prostate gland heterogeneity.

Age‐related expression of molecular regulators of hypertrophy and maturation in articular cartilage

The purpose of this study was to determine changes in the expression of regulatory molecules in normal equine articular cartilage throughout development up to 18 months of age. The hypothesis was that expression of these regulatory molecules would decrease from birth to postpubescence. Cartilage was harvested from normal femoropatellar or scapulohumeral joints from 34 fresh horse cadavers. Horses were placed in four age groups [prenatal (n = 5); prepubertal, 0-6 months (n = 11); pubertal, 7-14 months (n = 13); and postpubertal, 15-18 months (n = 5)]. Indian hedgehog (Ihh), Gli1, Gli3, Patched1 (Ptc1), Smoothened (Smo), Noggin, bone morphogenetic protein-6 (BMP-6), BMP-2, parathyroid hormone-related peptide (PTHrP), and PTH/PTHrP receptor mRNA expression levels were evaluated by real-time quantitative PCR. Spatial tissue mRNA and protein expression was determined by in situ hybridization and immunohistochemistry. The expression of PTHrP decreased (p = 0.002) in the pubertal group, while PTH/PTHrP receptor expression significantly increased (p = 0.001). No significant difference was found between groups for Ihh (p = 0.6) or Smo (p = 0.3) expression. In contrast, there was significantly increased expression of Ptc1 (p = 0.006), Gli1 (p = 0.04), and Gli3 (p = 0.007) in the pubertal group, and Gli3 (p = 0.007) remained elevated in the postpubertal group. The expression of BMP-6 significantly increased from prenatal to postnatal groups (p = 0.03) while BMP-2 expression increased during puberty and postpuberty (p = 0.03). The changes in expression of hedgehog and BMP signaling molecules in articular cartilage during postnatal development have not been shown previously. The increased expression of hedgehog receptor and transcription factors during puberty may indicate maturation of the deep articular layer during this time period.

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

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