Wnt4 mRNA Research Articles

Effect of total glucosides of paeony on Wnt/β-catenin signal transduction pathway expression in kidney of diabetic rats

The study is to explore the effect of total glucosides of paeony (TGP)on Wnt/β-catenin signal transduction pathway expression in kidney of diabetic rats, and discuss the protection of TGP in diabetic nephropathy and possible mechanism. Ninety male SD rats of 8 weeks age were randomly divided into normal control group (n = 10) and model group (n = 80). Rats of the normal control group were fed with regular diet, while rats of the model group were fed with high-fat high-sugar diet and 4 weeks later were given an intraperitoneal injection of 35 mg x kg(-1) streptozotocin (STZ). The successfully induced type 2 diabetic rat models were then randomly divided into DM group, three TGP (50, 100, 200 mg x kg(-1) x d(-1)) treatment group and tripterygium wilfordii glycosides (8 mg x kg(-1) x d(-1)) control group. Rats of DM group and each treatment group were given high-fat high-sugar diet. At week 14, the levels of blood sugar, 24 hour urine protein, serum creatinine and blood urea nitrogen were tested. The rats were then sacrificed. Renal pathological changes were examined. Renal tissue Wnt-1 and β-catenin expressions were detected by immunohistochemical assay. Wnt-1 mRNA and β-catenin mRNA expression was semi-quantified by RT-PCR. Wnt-1 protein and β-catenin protein expression was semi-quantified by Western blot. The Result show that Wnt-1 and β-catenin expression increased in kidney of high-fat high-sugar induced type 2 diabetic rats. Compared with diabetic group, the level of serum creatinine, blood urea nitrogen, 24 h urine protein, mean glomerular area and mean glomerular volume were decreased, renal histopathology were improved, expression of Wnt-1 and β-catenin mRNA and protein was reduced in TGP group. Tripterygium wilfordii glycosides had the similar effect. In conclusion, these results showed that Wnt/β-catenin abnormal activation in kidney of type 2 diabetic rats, TGP can improve kidney damage in diabetic rats and delay the development of diabetic nephropathy by inhibit the Wnt/β-catenin signaling pathway.

The effects of diet induced obesity on breast cancer associated pathways in mice deficient in SFRP1.

BackgroundSecreted frizzled-related proteins (SFRPs) are a family of proteins that block the Wnt signaling pathway and loss of Sfrp1 expression is observed in breast cancer. The molecular mechanisms by which obesity contributes to breast tumorigenesis are not well defined, but involve increased inflammation. Mice deficient in Sfrp1 show enhanced mammary gland inflammation in response to diet induced obesity (DIO). Furthermore, mammary glands from Sfrp1 −/− mice exhibit increased Wnt signaling, decreased cell death responses, and excessive hyper branching. The work described here was initiated to investigate whether obesity exacerbates the aforementioned pathways, as they each play a key roles in the development of breast cancer.FindingsWnt signaling is significantly affected by DIO and Sfrp1 −/− loss as revealed by analysis of Myc mRNA expression and active β-catenin protein expression. Furthermore, Sfrp1 −/− mice fed a high fat diet (HFD) exhibit an increase in mammary cell proliferation. The death response is also impaired in the mammary gland of Sfrp1 −/− mice fed a normal diet (ND) as well as a HFD. In response to γ-irradiation, mammary glands from Sfrp1−/− mice express significantly less Bax and Bbc3 mRNA, caspase-3 positive cells, and p53 protein. The expression of Wnt4 and Tnfs11 are critical for normal progesterone mediated mammary gland development and in response to obesity, Sfrp1−/− mice express significantly more Wnt4 and Tnfs11 mRNA expression. Evaluation of progesterone receptor (PR) expression showed that DIO increases the number of PR positive cells.ConclusionsOur data indicate that the expression of Sfrp1 is a critical factor required for maintaining appropriate cellular homeostasis in response to the onset of obesity.

Wnt signaling cascade in restenosis: a potential therapeutic target of public health relevance in a North American cohort of Nebraska State

Cardiovascular disease is a leading cause of morbidity and mortality in United States, including Nebraska. Neointimal hyperplasia leading to restenosis is a major public health problem. Identification of key signaling molecules in biochemical pathways is an attractive strategy for development of predictive biomarkers in occlusive vascular diseases (OVD). Our pilot study aimed to identify the role of Wnt-frizzled signaling in restenosis in a North American cohort. North American patients (n = 9) undergoing coronary artery bypass graft surgery at Nebraska Heart Institute, Lincoln, were enrolled. Human saphenous veins (SV) (n = 9) and left internal mammary arteries (LIMA) (n = 9) received post-surgery at Creighton University, Omaha, were harvested using Collagenase-IV digestion method. Isolated primary VSMCs were cultured for 3-4 weeks, and passages P3-P7, were used for molecular biology experiments. The study was approved by the Institutional Review Board. RNA was extracted using trizol method and mRNA transcripts were identified using reverse transcriptase-polymerase chain reaction followed by 2% agarose gel electrophoresis. Mean age of surgery patients (n = 9) was 60.3 years (SD ± 6.5 years). Wnt2 and Wnt5a mRNA transcripts were expressed in human VSMCs; however, Wnt1, Wnt4 and Wnt11 mRNA were not expressed; beta-actin was used as an internal control. Receptor studies demonstrated the expression of Fzd1, Fzd2 and Fzd5 mRNA in hVSMCs. Our preliminary data implicates the public health significance of Wnt signaling in bypass graft patients in Nebraska. Future molecular biology approach-based community health studies targeting Wnt pathway may aid in the development of cost-effective predictive biomarkers for OVD susceptible populations.

A5.5 SMOC2 modulates chondrogenesis by interfering with WNT and BMP signalling

Background and ObjectivesA proteomic analysis of cartilage revealed an increase in SPARC-related modular calcium binding protein-2 (SMOC2) in patients with osteoarthritis (OA). SMOC2 was originally isolated from a chondrogenic extract...

HCV Core Protein-Induced Down-Regulation of microRNA-152 Promoted Aberrant Proliferation by Regulating Wnt1 in HepG2 Cells

BackgroundHepatitis C virus (HCV) has been reported to regulate cellular microRNAs (miRNAs). The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma (HCV-HCC), but HCV core-regulated miRNAs are largely unknown. Our preliminary experiments revealed significant down-regulation of microRNA-152 (miR-152) by HCV core protein in HepG2 cells. Through target gene prediction softwares, Wnt1 was predicted to be a potential target of miR-152. The present study was initiated to investigate whether miR-152 is aberrantly regulated by the HCV core protein, and involved in the regulation of the aberrant proliferation of HCV-HCC cells.MethodsMiR-152 levels were examined by stem-loop real-time RT-PCR (SLqRT-PCR). Cell proliferation was analyzed by MTT and colony formation assay. Cell cycle analysis was performed by flow cytometry. Luciferase reporter assay was conducted to confirm miRNA-target association. Wnt1 expression was determined by real-time qPCR and Western blotting.ResultsHCV core protein significantly suppressed miR-152 expression, and led to significant Wnt1 up-regulation with a concomitant aberrantly promoted proliferation. Moreover, we validated that miR-152 inhibition promoted, while miR-152 mimics inhibited cell proliferation. Using, qRT–PCR and western blot, Wnt1 was demonstrated to be regulated by miR-152. Luciferase activity assay showed that while miR-152 mimics significantly reduced the luciferase activity by 83.76% (P<0.0001), miR-152 inhibitor showed no effect on luciferase reporter. Most notably, salvage expression of miR-152 after Ad-HCV core infection for 24 h almost totally reversed the proliferation-promoting effect of the HCV core protein, and meanwhile, reduced the expression of both Wnt1 mRNA and protein to basal levels.ConclusionThese findings provide important evidence that the reduced miR-152 expression by HCV core protein can indirectly lose an inhibitory effect on Wnt1, which might, at least partially lead to cell proliferation of liver cancer cells. MiR-152 may have a therapeutic potential to suppress liver cancer proliferation.

The Dual PI3K/mTOR Inhibitor NVP-BEZ235 Enhances the Antitumoral Activity of Gemcitabine in Human Pancreatic Cancer Cell Lines

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignant tumors still associated with poor prognosis in advanced stages. Gemcitabine is one of the standard agents for the treatment of PDAC, without having a major impact on the clinical outcome. Combining two compounds acting via different ways of action may result in a better efficacy. Methods: We investigated the effects of gemcitabine in combination with the dual PI3k/mTOR inhibitor BEZ235 in four human pancreatic cancer cell lines (Panc-1, BxPC-3, MiaPaCa-2 and AsPC-1). The cells were analysed with MTT assay for cell viability, FACS-analysis for cell cycle distribution. Real-time RT-PCR and Western blot for survivin/ BIRC5, STAT3, BCl-xL and WNT16 mRNA and protein expression and γH2AX. Results: Application of NVP-BEZ235 or gemcitabine inhibited cell viability of AsPC-1 and BxPC-3 cells while Panc-1 and MiaPaCa-2 remained nearly unaffected. Combined treatment of gemcitabine and BEZ235, however, enhanced the inhibitory effect on cell viability of Panc-1 and MiaPaCa-2 cells of about 80% compared to control cells. This effect was boosted by time-delayed application of the two compounds. The biggest impact on cell growth, viability and downstream gene regulations were achieved by a sequential incubation with gemcitabine followed by BEZ235 24 hours later. Conclusions: Combining gemcitabine with dual PI3K/mTOR inhibitors like NVP-BEZ2235 improved the efficacy on growth inhibition in human pancreatic cell lines especially by sequential application of both agents.

Wnt4 is overexpressed in human pituitary adenomas and is associated with tumor invasion

The Wnt4 molecule is a secretory glycoprotein implicated in proliferation and differentiation of both normal and malignant cells. Despite extensive investigation of Wnt4 expression in various cancers, little is known about its expression pattern in different types of pituitary tumors. In this study, we examined the expression of Wnt4 and its downstream molecule β-catenin in pituitary adenoma specimens. Pituitary adenoma tissues were collected from 43 patients and four normal pituitary tissue samples were obtained at autopsy. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry and western blot were performed to detect the expression of Wnt4 and β-catenin mRNA and protein, respectively. Tumor invasion grade (Knosp grade) was determined on MRI images and was correlated to β-catenin expression. Immunohistochemistry demonstrated elevated Wnt4 expression in follicle-stimulating hormone-producing adenomas, growth hormone-producing adenomas, prolactin-producing adenomas, thyroid-stimulating hormone-producing adenomas and non-functioning adenomas, while adrenocorticotropic hormone-producing adenomas showed a low level of Wnt4 expression that was comparable to normal pituitary tissue. These results were confirmed by real-time RT-PCR and western blot analyses. The expression pattern of β-catenin was similar to that of Wnt4 and was inversely correlated to the Knosp grade of tumor invasion. These data indicate that Wnt4 signaling is deregulated in most pituitary adenomas and its excessive activation may inhibit pituitary tumor invasion.

Chronic Academic Stress Increases a Group of microRNAs in Peripheral Blood

MicroRNAs (miRNAs) play key roles in regulation of cellular processes in response to changes in environment. In this study, we examined alterations in miRNA profiles in peripheral blood from 25 male medical students two months and two days before the National Examination for Medical Practitioners. Blood obtained one month after the examination were used as baseline controls. Levels of seven miRNAs (miR-16, -20b, -26b, -29a, -126, -144 and -144*) were significantly elevated during the pre-examination period in association with significant down-regulation of their target mRNAs (WNT4, CCM2, MAK, and FGFR1 mRNAs) two days before the examination. State anxiety assessed two months before the examination was positively and negatively correlated with miR-16 and its target WNT4 mRNA levels, respectively. Fold changes in miR-16 levels from two days before to one month after the examination were inversely correlated with those in WNT4 mRNA levels over the same time points. We also confirmed the interaction between miR-16 and WNT4 3′UTR in HEK293T cells overexpressing FLAG-tagged WNT4 3′UTR and miR-16. Thus, a distinct group of miRNAs in periheral blood may participate in the integrated response to chronic academic stress in healthy young men.

Spatiotemporal expression of Wnt signaling pathway components during bovine placental development

It is well established that trophoblasts play a crucial role in pregnancy establishment and maintenance through production of various biological substances. In this regard, Wnt signaling is an important regulator of embryo implantation and placentation in various species. However, the role of the Wnt signaling pathway during bovine placental development has remained largely unknown. Employing multiple approaches, we herein found that Wnt2 mRNA was more abundant in cotyledon tissues compared with caruncle tissues, whereas Wnt5b mRNA was more abundant in caruncle tissues compared with cotyledon tissues. Moreover, the Wnt receptor Fzd4 was detected in caruncle epithelial cells and binucleate trophoblasts, but not in uninucleate trophoblasts. In addition, β-catenin, an integral cell-cell adhesion adaptor protein as well as transcriptional co-regulator of Wnt canonical pathway, was spatiotemporally expressed in bovine trophoblasts, with high levels of cellular accumulation and nuclear translocation, particularly in binucleate trophoblasts. Lymphoid enhancer factor-1 mRNA was more abundant in caruncle tissues compared with cotyledon tissues, which was well correlated with the expression profile of Dickkopf-1, a secreted antagonist of the canonical Wnt signaling pathway. These results provided new evidence that precisely regulated canonical Wnt activation may have a very important physiological role during fetal-maternal recognition and pregnancy maintenance in cattle.

Inhibitions of epithelial to mesenchymal transition and cancer stem cells‐like properties are involved in miR‐148a‐mediated anti‐metastasis of hepatocellular carcinoma

The epithelial-mesenchymal transition (EMT) and acquisition of cancer stem cells (CSCs)-like properties are essential steps in the metastasis and postsurgical recurrence of hepatocellular carcinomas (HCCs). The molecular mechanisms involved, however, remain obscure. As determined by an miRNA microarray analysis, there was lower expression of miR-148a in poorly differentiated HCC tissues relative to well-differentiated HCC tissues. MHCC97H and MHCC97L (HCC cells with migratory capacity) and HCC tissues with various differentiation status were selected for further investigation. The results showed that miR-148a levels inversely correlated with the differentiation status of HCC tissues. In MHCC97H and MHCC97L cells, over-expression of miR-148a blocked the EMT process, attenuated the expression of CD90 and CD44 (biomarkers for liver cancer stem cells), and inhibited their migratory capacity. Via TargetScan and microRNA.org algorithms, miR-148a was predicted to bind to the Wnt1 mRNA 3'-UTR. Wnt1 was confirmed as a target gene of miR-148a in HCC cells, and the Wnt signal pathway was determined to be involved in the miR-148a-mediated inhibition of EMT and CSCs-like properties of MHCC97H cells. Moreover, the expression of miR-148a in nonmetastatic HCC tissues was higher than that in metastatic HCC tissues. The results suggest that miR-148a inhibits the metastasis of HCCs by blocking EMT and CSCs-like properties through effects on the Wnt signaling pathway.

Effects of transforming growth factor-β1 on the expression of Wnt signaling pathway related genes in the differentiation of bone marrow stromal cells

Objective To observe the transforming growth factor-β1 (TGF-β1) on the expression of Wnt signaling pathway related genes during the chondrogenesis of bone marrow stromal cells (BMSCs) in vitro.Methods BMSCs cultured in TGF-β1 medium were observed by inverted phase contrast microscope and identified by toluidine blue staining.The expression levels of glycosaminoglycan (GAG) and target genes including Wnt1,Wnt3a,β-catenin,Sox9 and collagen Ⅱ mRNA under different concentrations of TGF-β1 were detected by using enzyme linked immunosorbent assay (ELISA) and real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) respectively.Results At day 21 after TGF-β1 induction,toluidine blue staining showed that more than 95％ cells differentiated into chondrocytes; the amount of GAG in the cell cultured medium in 2 μg/L TGF-β1-induced group was (69.16 ± 1.18) mg/L,significantly higher than other groups (P ＜ 0.05).FQ-PCR results showed that Wnt1,Wnt3a and β-catenin mRNA expression in 2 μg/L TGF-β1-induced group was inhibited [0.0266 ±0.0047,0.0154 ±0.0033,0.0187 ±0.0021,(P ＜0.05)],but in contrast Sox9 and collagen Ⅱ mRNA expression levels were increased [0.0682 ± 0.0024,0.1134 ± 0.0048 (P ＜ 0.05)] as compared with the control group.Conclusion TGF-β1 could promote BMSCs chondrogenic differentiation,and the expression of Wnt/β-catenin signaling pathway related genes Wnt1,Wnt3a and β-catenin mRNA was inhibited and the expression of chondrocyte specific genes Sox9 and collagen Ⅱ mRNA was increased. Key words: Transforming growth factor-beta1; Bone marrow stromal cells; Wnt signaling pathway; Glycosaminoglycan

Sharpening of expression domains induced by transcription and microRNA regulationwithin a spatio-temporal model of mid-hindbrain boundary formation

BackgroundThe establishment of the mid-hindbrain region in vertebrates is mediated by the isthmic organizer, an embryonic secondary organizer characterized by a well-defined pattern of locally restricted gene expression domains with sharply delimited boundaries. While the function of the isthmic organizer at the mid-hindbrain boundary has been subject to extensive experimental studies, it remains unclear how this well-defined spatial gene expression pattern, which is essential for proper isthmic organizer function, is established during vertebrate development. Because the secreted Wnt1 protein plays a prominent role in isthmic organizer function, we focused in particular on the refinement of Wnt1 gene expression in this context.ResultsWe analyzed the dynamics of the corresponding murine gene regulatory network and the related, diffusive signaling proteins using a macroscopic model for the biological two-scale signaling process. Despite the discontinuity arising from the sharp gene expression domain boundaries, we proved the existence of unique, positive solutions for the partial differential equation system. This enabled the numerically and analytically analysis of the formation and stability of the expression pattern. Notably, the calculated expression domain of Wnt1 has no sharp boundary in contrast to experimental evidence. We subsequently propose a post-transcriptional regulatory mechanism for Wnt1 miRNAs which yields the observed sharp expression domain boundaries. We established a list of candidate miRNAs and confirmed their expression pattern by radioactive in situ hybridization. The miRNA miR-709 was identified as a potential regulator of Wnt1 mRNA, which was validated by luciferase sensor assays.ConclusionIn summary, our theoretical analysis of the gene expression pattern induction at the mid-hindbrain boundary revealed the need to extend the model by an additional Wnt1 regulation. The developed macroscopic model of a two-scale process facilitate the stringent analysis of other morphogen-based patterning processes.

Paneth cells expand from newly created and preexisting cells during repair after doxorubicin-induced damage

Paneth cell numbers increase following intestinal damage, but mechanisms driving this process are not understood. We hypothesized that the increase in Paneth cell numbers is due to recruitment of cells from a preexisting pool of secretory progenitors. Mice were given a single injection of doxorubicin (Dox), and intestinal tissue was collected 0-168 h after treatment. Paneth, goblet, and intermediate cells were counted and evaluated for cell morphology. Quantitative RT-PCR was used to measure expression of various genes associated with Paneth cell allocation and maturation. Paneth cells were birth dated using incorporation of thymidine analogs given before or after Dox. Staining revealed "intermediate" cells, which were rarely observed in control crypts but increased significantly in number 96 and 120 h after Dox treatment. Birth dating of intermediate cells 5 days after Dox treatment revealed that 24% of these cells took up thymidine analog given prior to Dox treatment and 36% took up thymidine analog given after Dox treatment. Quantitative RT-PCR demonstrated a significant increase in Spdef, Atoh1, Sox9, EphB3, Mist, Wnt5a, FGF-9, and FGF-18 mRNAs and a significant decrease in Indian hedgehog mRNA. Expansion of the Paneth cell compartment after Dox treatment is due to generation of new cells and recruitment of cells from an existing pool. These cells express Paneth and goblet biomarkers and are found only during repair. Expansion of these cells correlates temporally with reduced Indian hedgehog and increased FGF and Wnt mRNA. These findings are significant, as they provide a first step in understanding mechanisms of Paneth cell expansion during mucosal repair.

Spironolactone inhibits apoptosis in rat mesangial cells under hyperglycaemic conditions via the Wnt signalling pathway

Mesangial cells (MCs) play a crucial role in maintaining structure and function of glomerular tufts, providing structural support for capillary loops and modulating glomerular filtration by their contractility. MCs apoptosis occurs in experimental diabetic nephropathy, and this correlates with worsening albuminuria. Accumulating evidence suggests that mineralocorticoid receptor (MR) blockade effectively reduces proteinuria in diabetic nephropathy; however, it is rarely known whether spironolactone (SPI), a nonspecific MR antagonist, inhibits apoptosis in MCs under hyperglycaemic conditions. The objectives of this study are to determine the relationship between SPI and apoptosis, and investigate the cell signalling pathway by which SPI inhibits apoptosis. Rat MCs were treated with 30 mM D-glucose and 10(-8), 10(-7) or 10(-6) M aldosterone (ALD) for 24 h. In some experiments, MCs were pretreated with 10(-7) M SPI or 10 mM LiCl for 1 h. Apoptosis was evaluated by cell nucleus staining and flow cytometric analyses, and caspase-3 activity was assayed. Gene and protein expression were quantified using quantitative real-time PCR and Western blotting, respectively. SPI directly inhibited high glucose and ALD-induced MCs apoptosis in a caspase-dependent manner. Importantly, SPI inhibited MCs apoptosis via the Wnt signalling pathway. SPI promoted activation of the Wnt signalling pathway in MCs, leading to upregulation of Wnt4 and Wnt5a mRNA expression, decreased GSK-3β protein expression and increased β-catenin protein expression. As a conclusion, this study suggests that SPI may inhibit apoptosis in MCs during hyperglycaemic conditions via the Wnt signalling pathway. Blockade of the ALD system may represent a novel therapeutic strategy to prevent MCs injury under hyperglycaemic conditions.

Inhibition of COG5, a gene from the C7q22 osteoarthritis susceptibility locus, induces glycosylation defects and affects chondrogenesis and osteogenesis by disturbing Wnt signaling

Purpose: Osteoarthritis (OA) is a highly disabling pathology of the bone-cartilage unit, to which both patient-dependent (weight excess, trauma⋯) and genetic factors contribute. Recently, a locus at chromosome 7q22 was linked to knee and hand OA by genome wide association study. In this locus, the Conserved oligomeric Golgi complex subunit (COG) 5 gene encodes a component of Golgi apparatus. Mutations in humans are associated with mild congenital glycosylation disorders, and COG5 expression is higher in 3D than in monolayer cultures of articular chondrocytes. Here we investigated on the role of COG5 during chondrogenesis and osteogenesis. Methods: We used three independent stable clonal colonies of each, controls (non-sliencing vector) and COG5 knocked-down cells (COG5-), established in the chondrogenic ATDC5 and osteogenic MC3T3-E1 cell lines. ATDC5 were cultured as high density micromasses (2x105 cells/10 μl) to mimic the condensation step of developing cartilage. Chondrogenesis was induced by culturing cells for 14 days (D) in DMEM/F12 + 5%FBS, supplemented with ITS (insulin, transferrin, sodium selenite). On D14, cells were switched to mineralization medium (αMEM + 5%FBS, supplemented with ITS, β-glycerophosphate (BGP) and ascorbic acid (AA)) to investigate the mineralization phase of late chondrogenesis. Monolayers of MC3T3-E1 were cultured for 21D in αMEM + 10%FBS, supplemented with BGP and AA. mRNA expression of typical chondrogenesis markers (Aggrecan (Agg), type II (Col2a1) and X (Col10a1)) collagens and osteogenic markers (Osterix (Osx), Osteocalcin (Ocn) and Osteopontin (Opn)) were assessed by quantitative RT-PCR. We also assessed mRNA expression of Wnts (-4, -5a, -5b, -11) and BMPs (-2, -4, -6, -7). Quantification of Alcian Blue, Safranin O, Sirius red and Alizarin red staining were used to evaluate proteoglycans, collagens and mineralized content respectively. Alkaline phosphatase (ALP) activity allowed to monitor osteogenesis. SDS-PAGE was used to assess the global glycosylation status. Western blot allowed to check canonical (β-catenin) and non-canonical (Calmodulin-Kinase II) Wnt signaling, as well as BMP signaling (Smad and MAPK). Results: During early proliferation (D1-7) and late proliferation/pre-hypertrophy phases (D7-14) of chondrogenesis, Col2a1 and Agg mRNA strongly increased, but to a lesser extent in COG5-. Proteoglycan staining intensity was lower in COG5-. BMP-4 levels progressively increased and were significantly higher in COG5-. This was consistent with increased phosphorylation of Smad 1/5/8. Wnt-4, -5a and -5b mRNA were evenly increased in both controls and COG5-. BMP-6 and Col10a1 mRNA were increased at D14, but were significantly lower in COG5-. Wnt-11 mRNA increased in controls, but not in COG5-. In mineralization phase (D14 to D21), Col10a1 mRNA and mineralization were strongly increased, but to a lesser extent in COG5-. During osteogenesis, Opn, Osx and Ocn mRNA were significantly higher in controls, alike Alizarin red staining and ALP activity. Protein analysis suggested differences in global glycosylation status, in particular of Wnt ligands. Conclusions: COG5 defect reduces chondrogenesis and osteogenesis. COG5 particularly affected glycosylation and Wnt signaling, underlining its critical role in the fate of cells from the articular chondrocyte-bone unit in the course of OA.

Role of interleukin-10 in endochondral bone formation

s / Osteoarthritis and Cartilage 21 (2013) S63–S312 S129 (AA)) to investigate the mineralization phase of late chondrogenesis. Monolayers of MC3T3-E1 were cultured for 21D in aMEM + 10%FBS, supplemented with BGP and AA. mRNA expression of typical chondrogenesis markers (Aggrecan (Agg), type II (Col2a1) and X (Col10a1)) collagens and osteogenic markers (Osterix (Osx), Osteocalcin (Ocn) and Osteopontin (Opn)) were assessed by quantitative RT-PCR. We also assessed mRNA expression of Wnts (-4, -5a, -5b, -11) and BMPs (-2, -4, -6, -7). Quantification of Alcian Blue, Safranin O, Sirius red and Alizarin red staining were used to evaluate proteoglycans, collagens and mineralized content respectively. Alkaline phosphatase (ALP) activity allowed to monitor osteogenesis. SDS-PAGE was used to assess the global glycosylation status. Western blot allowed to check canonical (bcatenin) and non-canonical (Calmodulin-Kinase II) Wnt signaling, as well as BMP signaling (Smad and MAPK). Results: During early proliferation (D1-7) and late proliferation/prehypertrophy phases (D7-14) of chondrogenesis, Col2a1 and Agg mRNA strongly increased, but to a lesser extent in COG5-. Proteoglycan staining intensity was lower in COG5-. BMP-4 levels progressively increased and were significantly higher in COG5-. This was consistent with increased phosphorylation of Smad 1/5/8. Wnt-4, -5a and -5b mRNA were evenly increased in both controls and COG5-. BMP-6 and Col10a1 mRNA were increased at D14, but were significantly lower in COG5-. Wnt-11 mRNA increased in controls, but not in COG5-. In mineralization phase (D14 to D21), Col10a1 mRNA and mineralization were strongly increased, but to a lesser extent in COG5-. During osteogenesis, Opn, Osx and Ocn mRNA were significantly higher in controls, alike Alizarin red staining and ALP activity. Protein analysis suggested differences in global glycosylation status, in particular of Wnt ligands. Conclusions: COG5 defect reduces chondrogenesis and osteogenesis. COG5 particularly affected glycosylation andWnt signaling, underlining its critical role in the fate of cells from the articular chondrocyte-bone unit in the course of OA. 232 ROLE OF INTERLEUKIN-10 IN ENDOCHONDRAL BONE FORMATION G. Kim y,z, Y.-K. Jung z, H.-R. Park z, E.-J. Lee z, J.-Y. Choi x, F. Beier {,k, S.-W. Han y,z. yDept. of Rheumatology, Daegu Fatima Hosp., Daegu, Republic of Korea; z Lab. for Arthritis and Bone Biology, Fatima Research Inst., Daegu, Republic of Korea; xDept.s of Biochemistry and Cell Biology, Sch. of Med., WCU program, Kyungpook Natl. Univ., Daegu, Republic of Korea; kDept. of Physiology and Pharmacology, Univ. of Western Ontario, London, ON, Canada; {Children's Hlth.Res. Inst., London, ON,

Xenopus Nkx6.1 and Nkx6.2 are required for mid–hindbrain boundary development

The mid-hindbrain boundary (MHB) organizer is among the best studied local organizers that patterns the midbrain and cerebellum in vertebrates and is established by a regulatory network of several transcription factors and signals, including Wnt signaling. In the present study, we found that Xenopus Nkx6.1 and Nkx6.2, two transcription factors of the Nkx homeobox family, are required for MHB formation. In Xenopus embryos, nkx6.1 and nkx6.2 are expressed in the MHB, and knockdown of either nkx6.1 or nkx6.2 by specific morpholinos disrupts the MHB expression of en2 as well as wnt1, a key regulator for vertebrate MHB formation. In the nkx6.1/nkx6.2 morphants, co-injection of wnt1 or dngsk3β mRNA, which activates Wnt signaling, rescues the expression of en2. Nkx6.1 and Nkx6.2 activate canonical Wnt signaling in reporter assays in both cultured mammalian cells and Xenopus embryos. An Nkx6.2 deletion construct, which inhibits the ability of wild type Nkx6.2 to activate Wnt signaling, also reduces the MHB expression of en2 in Xenopus embryos. These results suggest that Nkx6.1 and Nkx6.2 are involved in MHB formation in Xenopus embryos, likely by modulating wnt1 expression.

Role of aberrant WNT signalling in the airway epithelial response to cigarette smoke in chronic obstructive pulmonary disease

BackgroundWNT signalling is activated during lung tissue damage and inflammation. We investigated whether lung epithelial expression of WNT ligands, receptors (frizzled; FZD) or target genes is dysregulated on cigarette smoking...

Expression of Wnt1 and Wnt8a genes in colons of Hirschsprung disease

Objective To investigate the expression of Wnt1 and Wnt8a genes in tissue samples from the patients with Hirschsprung disease (HD).Methods The expression of Wnt1 and Wnt8a mRNA and proteins were quantitatively analyzed in 60 cases of HD (aganglionic and ganglionic segments).Results In the HD patients,there was statistically significant difference in the differential expression of Wnt1 and Wnt8a mRNA between the stenotic intestine and the normal intestine.The relative expression of Wnt1 mRNA in the stenotic intestine and normal intestine was 17.66 ± 1.28 and 29.61 ± 3.23 respectively.The relative expression of Wnt8a mRNA in the stenotic intestine and normal intestine was 31.77 ± 2.21 and 15.01 ± 1.29 respectively (P ＜ 0.05).The protein expressive levels of Wnt1 and Wnt8a were 19.51 ±1.57 and 33.17 ±2.09 in the stenotic intestine,and 37.05 ± 1.23 and 17.35 ±0.87 in the normal intestine,respectively (P ＜ 0.05).The expression level of Wnt1 protein was lower in the aganglionic segment than in the ganglionic segment.Immunohistochemistry revelaed that Wnt1 was highly expressed in the ganglionic segment,but weakly expressed in the aganglionic segment; Wnt8a was highly expression in the aganglionic segment,but weakly expressed in the ganglionic segment.Conclusion There is the aberrant expression of Wnt1 and Wnt8a mRNA and proteins in HD,suggesting the Wnt1 and Wnt8a are involved in the pathogenesis of HD,which may play a role in the pathogenesis of congenital malformation of the alimentary tract. Key words: Hirschsprung disease ; Wnt1 ; Wnt8a ; Gene expression

Follicle-stimulating hormone regulation of estradiol production: Possible involvement of WNT2 and β-catenin in bovine granulosa cells1,2

Follicle-stimulating hormone regulation of estrogen biosynthesis in the adult rodent ovary requires β-catenin (CTNNB1), but whether CTNNB1 is involved in FSH-induced estrogen production in cattle is unknown. To elucidate the effect of FSH in regulating specific wingless-type mouse mammary tumor virus integration site (WNT)/CTNNB1 pathway components in bovine folliculogenesis and steroidogenesis, granulosa cells and follicular fluid were collected from large antral follicles (8 to 22 mm) from ovaries containing stage-III corpora lutea (d 11 to 17 of an estrous cycle). Follicles were categorized as high estradiol (n = 3; ≥ 25 ng/mL) or low estradiol (n = 3; ≤ 14 ng/mL) based on intra-follicular estradiol concentrations. Protein fractions were collected from granulosa cells and CTNNB1 abundance was analyzed by Western blot. Follicles with increased estradiol concentrations had 6-fold greater (P < 0.001) abundances of CTNNB1 compared with those classified as low-estradiol follicles, indicating that the hormonal milieu responsible for increased estradiol content could result in CTNNB1 accumulation. To ascertain specific contributions of FSH to increases in CTNNB1 protein abundances, granulosa cells were isolated from small ovarian follicles (1 to 5 mm) and cultured in the presence or absence of 100 ng/mL FSH for 24 or 48 h. Real-time PCR quantification of aromatase (CYP19A1) and select WNT family members were evaluated in response to FSH treatment. Successful stimulation of granulosa cells with FSH was confirmed by induction of CYP19A1 mRNA and parallel temporal increases of medium estradiol concentrations. Additionally, protein kinase b (AKT), a known FSH target, increased 1.7-fold (P = 0.07). Of the WNT family members analyzed, only WNT2 mRNA was induced after 24 h of FSH treatment compared with controls (0.12-fold and 3.7-fold for control and FSH-treated, respectively; P < 0.05), and WNT2 expression tended (P = 0.11) to remain increased at 48 h in FSH-treated cells compared with controls (1.0- and 3.14-fold, respectively). Furthermore, FSH-treated granulosa cells had greater abundances of total CTNNB1 (P = 0.04) protein. These data demonstrate for the first time that FSH regulates CTNNB1 protein and WNT2 mRNA expressions in bovine granulosa cells, suggesting a potential role of canonical WNT signaling in ovarian steroidogenesis and follicular growth of cattle. Future studies are necessary to determine if FSH directly regulates CTNNB1 through modulation of AKT or indirectly by up regulating WNT2, which subsequently activates the canonical WNT pathway.