Dickkopf Homolog Research Articles

Effects of miR-335-5p on the osteoblast function in high glucose condition

Objective To investigate the effects of miR-335-5p on the proliferation and apoptosis of osteoblasts which were exposed to high glucose condition, and explore its possible molecular mechanisms. Methods MC3T3-E1 osteoblasts were divided into four groups: control group(5.5 mmol/L glucose), high glucose group(HG group, 22.0 mmol/L glucose), agomir-335-5p group(transfected with agomir-335-5p and exposed to 22.0 mmol/L glucose), and agomir negative control group(agomir NC group, transfected with agomir negative control and exposed to 22.0 mmol/L glucose), cultured for 7 days. Cell proliferaton, cell apoptosis, expressions of miR-335-5p and dickkopf homolog 1(DKK1) mRNA, protein levels of DKK1 and cysteinyl aspartate-specific proteinase-3(caspase-3) were detected using MTT, flow cytometry, quantitative realtime PCR and western blot, respectively. Results Compared with control group, the expression of miR-335-5p mRNA and cell proliferation in HG group were significantly decreased(P 0.05). The miR-335-5p mRNA expression and cell proliferation in agomir-335-5p group were higher than those in HG group and agomir NC group(P<0.05). However, Cell apoptosis and the protein levels of DKK1 and caspase-3 in agomir-335-5p group were lower than those in HG group(P<0.05). Conclusion High glucose inhibits the proliferation and induce the apoptosis of MC3T3-E1 osteoblast through decreasing the expression of miR-335-5p and subsequently increasing the DKK1 expression. (Chin J Endocrinol Metab, 2015, 31: 712-716) Key words: miR-335-5p; Osteoblast; Proliferation; Apoptosis

FoxM1 transactivates PTTG1 and promotes colorectal cancer cell migration and invasion.

BackgroundMetastasis is the major cause of cancer-related death. Forkhead Box M1 (FoxM1) is a master regulator of tumor metastasis. This study aims to identify new FoxM1 targets in regulating tumor metastasis using bioinformatics tools as well as biological experiments.MethodsIllumina microarray was used to profile WT and PTTG1 knockout HCT116 cells. R2 Genomics Analysis was used to identify PTTG1 as a potential FoxM1 targeted gene. Luciferase reporter array, EMSA and Chromatin Immunoprecipitation (ChIP) were used to determine the binding of FoxM1 to PTTG1 promoter. Boyden chamber assay was used to evaluate the effects of FoxM1-PTTG1 on cell migration and invasion. Splenic-injection induced liver metastasis model was used to evaluate the effects of FoxM1-PTTG1 on liver metastasis of colorectal cancer.ResultsAnalyses of multiple microarray datasets derived from human colorectal cancer indicated that correlation levels of FoxM1 and pituitary tumor transforming gene (PTTG1) are highly concordant (R = 0.68 ~ 0.89, p = 2.1E-226 ~ 9.6E-86). FoxM1 over-expression increased and knock-down decreased PTTG1 expression. Luciferase reporter assay identified that the −600 to −300 bp region of PTTG1 promoter is important for FoxM1 to enhance PTTG1 promoter activity. EMSA and ChIP assays confirmed that FoxM1 directly binds to PTTG1 promoter at the −391 to −385 bp region in colorectal cancer cells. Boyden chamber assay indicated that both FoxM1 and PTTG1 regulate migration and invasion of HCT116 and SW620 colorectal cancer cells. Further in vivo assays indicated that PTTG1 knock out decreased the liver metastasis of FoxM1 over-expressing HCT116 cells. Microarray analyses identified 662 genes (FDR < 0.05) differentially expressed between WT and PTTG1−/− HCT116 cells. Among them, dickkopf homolog 1 (DKK1), a known WNT pathway inhibitor, was suppressed by PTTG1 and FoxM1.ConclusionsPTTG1 is a FoxM1 targeted gene. FoxM1 binds to PTTG1 promoter to enhance PTTG1 transcription, and FoxM1-PTTG1 pathway promotes colorectal cancer migration and invasion.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0126-9) contains supplementary material, which is available to authorized users.

Dickkopf Homolog 3 Induces Stem Cell Differentiation into Smooth Muscle Lineage via ATF6 Signalling

Smooth muscle cells (SMCs) are a key component of healthy and tissue engineered vessels and play a crucial role in vascular development and the pathogenic events of vascular remodeling i.e. restenosis. However, the cell source from which they can be isolated is limited. Embryonic stem (ES) cells that have the remarkable capability to differentiate into vascular SMCs in response to specific stimuli provide a useful model for studying SMC differentiation. Previous studies suggested that dickkopf homolog 3 (DKK3) has a role in human partially induced pluripotent stem cell to SMC differentiation. Here, we demonstrate that the expression of DKK3 is essential for the expression of SMC markers and myocardin at both the mRNA and protein levels during mouse ES cell differentiation into SMCs (ESC-SMC differentiation). Overexpression of DKK3 leads to further up-regulation of the aforementioned markers. Further investigation indicates that DKK3 added as a cytokine activates activating transcription factor 6 (ATF6), leading to the increased binding of ATF6 on the myocardin promoter and increased its expression. In addition, inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) promotes the expression of ATF6 and leads to further increase of myocardin transcription. Our findings offer a novel mechanism by which DKK3 regulates ESC-SMC differentiation by activating ATF6 and promoting myocardin expression.

High Bone Mass-Causing Mutant LRP5 Receptors Are Resistant to Endogenous Inhibitors In Vivo.

Certain missense mutations affecting LRP5 cause high bone mass (HBM) in humans. Based on in vitro evidence, HBM LRP5 receptors are thought to exert their effects by providing resistance to binding/inhibition of secreted LRP5 inhibitors such as sclerostin (SOST) and Dickkopf homolog-1 (DKK1). We previously reported the creation of two Lrp5 HBM knock-in mouse models, in which the human p.A214V or p.G171V missense mutations were knocked into the endogenous Lrp5 locus. To determine whether HBM knock-in mice are resistant to SOST- or DKK1-induced osteopenia, we bred Lrp5 HBM mice with transgenic mice that overexpress human SOST in osteocytes ((8kb) Dmp1-SOST) or mouse DKK1 in osteoblasts and osteocytes ((2.3kb) Col1a1-Dkk1). We observed that the (8kb) Dmp1-SOST transgene significantly lowered whole-body bone mineral density (BMD), bone mineral content (BMC), femoral and vertebral trabecular bone volume fraction (BV/TV), and periosteal bone-formation rate (BFR) in wild-type mice but not in mice with Lrp5 p.G171V and p.A214V alleles. The (2.3kb) Col1a1-Dkk1 transgene significantly lowered whole-body BMD, BMC, and vertebral BV/TV in wild-type mice and affected p.A214V mice more than p.G171V mice. These in vivo data support in vitro studies regarding the mechanism of HBM-causing mutations, and imply that HBM LRP5 receptors differ in their relative sensitivity to inhibition by SOST and DKK1.

Heterogeneous Mixed-Lineage Differentiation of Mouse Embryonic Stem Cells Induced by Conditioned Media from A549 Cells

Conditioned media (CM) of transformed cells, such as the human lung-derived A549 cells, is a useful tool for directing differentiation of embryonic stem cells (ESCs). Previous work indicates that A549-CM induced pulmonary differentiation of mouse ESCs (mESCs). In this study, we compared the effects of A549-CM treatment on the differentiation of mESCs organized in monolayer or embryoid bodies. We analyzed the cultures treated with A549-CM using specific lineage markers by quantitative polymerase chain reaction (qPCR) and lineage-focused PCR arrays and demonstrated heterogeneous CM-induced differentiation. We then constructed bioinformatics-based gene networks to establish correlations between the upregulated lineage-specific genes and proteins in the A549-CM identified by proteomic analysis. Network analysis supported the phenotypic and genotypic heterogeneic differentiation of mESCs into multiple cell lineages via enriched stemness, cardiovascular, neuronal, and lung development gene ontologies (GOs). The significant enrichment toward lung ontologies was specific for treatment with A549-CM, but not CM of liver (HepG2) and pancreas (Capan-1) cells. Based on network analysis, we identified laminin alpha5, prosaposin, lamin A/C, dickkopf homolog 1, clusterin, and calreticulin as the most relevant proteins related to the enrichment of lung GOs. We validated the effects of laminin isoforms on mESC differentiation in vitro and found enriched differential induction of surfactant protein gene expression. Our data suggest that A549-CM can be used for identifying secreted proteins for the heterogeneous mixed-lineage differentiation of mESCs toward a variety of lung-relevant cells. Such a heterogeneous cell population will be required for the in vitro generation of complex lung tissue and mixed cell populations for regenerative pulmonary therapy.

Parathyroid hormone-related protein (107-111) improves the bone regeneration potential of gelatin–glutaraldehyde biopolymer-coated hydroxyapatite

Biopolymer-coated nanocrystalline hydroxyapatite (HA) made as macroporous foams which are degradable and flexible are promising candidates as orthopaedic implants. The C-terminal (107-111) epitope of parathyroid hormone-related protein (PTHrP) exhibits osteogenic properties. The main aim of this study was to evaluate whether PTHrP (107-111) loading into gelatin–glutaraldehyde biopolymer-coated HA (HAGlu) scaffolds would produce an optimal biomaterial for tissue engineering applications. HAGlu scaffolds with and without PTHrP (107-111) were implanted into a cavitary defect performed in both distal tibial metaphysis of adult rats. Animals were sacrificed after 4weeks for histological, microcomputerized tomography and gene expression analysis of the callus. At this time, bone healing occurred only in the presence of PTHrP (107-111)-containing HAGlu implant, related to an increase in bone volume/tissue volume and trabecular thickness, cortical thickness and gene expression of osteocalcin and vascular cell adhesion molecule 1, but a decreased gene expression of Wnt inhibitors, SOST and dickkopf homolog 1. The autonomous osteogenic effect of the PTHrP (107-111)-loaded HAGlu scaffolds was confirmed in mouse and human osteoblastic cell cultures. Our findings demonstrate the advantage of loading PTHrP (107-111) into degradable HAGlu scaffolds for achieving an optimal biomaterial that is promising for low load bearing clinical applications.

Elevated miR-29a expression is not correlated with disease activity index in PBMCs of patients with ankylosing spondylitis

Objectives. Ankylosing spondylitis (AS) is a chronic inflammatory disease characterized by new bone formation. Recent evidence suggests that new bone formation in AS may be due to upregulation of Wnt signaling in the osteoblastic pathway secondary to low serum Dickkopf homolog 1 (Dkk-1) levels. And miR-29a orchestrates osteoblast differentiation through direct targeting and negative regulation of Dkk-1.Methods. We initially validated the expression levels of miR-29a in the peripheral blood mononuclear cells (PBMCs) of AS patients (n = 30), rheumatoid arthritis (RA) patients (n = 30) and healthy controls (n = 30) using real-time quantitative reverse transcription PCR (qRT-PCR). Correlation analysis was assessed between miR-29a level in PBMCs of AS patients and disease activity indexes, including erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), Bath ankylosing spondylitis disease activity index (BASDAI), Bath ankylosing spondylitis function index (BASFI) and modified Stoke ankylosing spondylitis spinal score (mSASSS).Results. Significantly higher expression of miR-29a was observed in PBMCs of AS patients (Ct 9.18 ± 1.96) compared with that in RA patients (10.97 ± 0.70, p < 0.001) and healthy controls (Ct 11.45 ± 1.23, p < 0.001). There was no significant difference between RA patients and healthy controls in miR-29a expression (p > 0.05). Elevated miR-29a expression is not correlated with disease activity index (p > 0.05). A weak correlation was found between elevated miR-29a expression and mSASSS (r = −0.393, p = 0.032).Conclusions. We report for the first time elevated miR-29a expression in PBMCs of patients with ankylosing spondylitis, and miR-29a might be used as a useful diagnostic marker in new bone formation but cannot reflect disease activity.

High expression of the secreted protein dickkopf homolog 4: roles in invasion and metastasis of renal cell carcinoma and its association with Von Hippel-Lindau gene.

The aim of this study was to investigate the effects of the dickkopf homolog 4 (DKK4)/Wnt/β-catenin signaling pathway on tumorigenesis and metastasis in clear cell renal cell carcinoma (ccRCC), as well as to elucidate the underlying mechanisms. We examined the expression of DKK4 in 30 cases of ccRCC and matched adjacent normal tissues, and investigated its correlation with clinicopathological characteristics. Stable DKK4-transfected cells were established, and DKK4 functional analyses were performed, including a T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter assay, and experiments on cell viability, apoptosis, invasive capability and tumor growth in vivo. Finally, western blot analysis was performed to detect Von Hippel-Lindau (VHL) expression in 50 clinical specimens. The expression levels of the DKK4, β-catenin and β-catenin downstream target genes, cyclin D1 and c-myc, were determined in the these specimens, as well as in RCC4(-), T3-14(+) cell lines by qRT-PCR and western blot analysis. The same tests were also performed in human embryonic kidney (HEK)293 cells which were transfected with the pCDH-DKK4 plasmid. After 6 weeks the tumor weight significantly increased in the mice transfected with the tumor cells. DKK4 mRNA and protein expression levels were significantly upregulated (p<0.001). DKK4 was distinctly overexpressed (68.0%) in all patient tissues. VHL(-) samples accounted for 60.0% of all samples, while DKK4 expression was significantly upregulated in 50% of these samples, indicating a correlation with VHL(-) expression (r=0.403, p<0.05). We also observed reduced expression levels of cyclin D1, c-myc and β-catenin (to a greater extent) in the VHL(-), RCC4(-) and T3-14(+) cells, as well as in the stably transfected HEK293 cells. DKK4 may be an oncogene, and its upregulated expression may be involved in the pathogenesis of ccRCC as a downstream gene of VHL. By activating other pathways apart from the Wnt/β-catenin pathway, DKK4 may play an important role in ccRCC tumorigenesis and metastasis.

Effects of Er-Zhi-Wan on microarchitecture and regulation of Wnt/β-catenin signaling pathway in alveolar bone of ovariectomized rats

Recent studies have shown that Er-Zhi-Wan (EZW), a traditional Chinese medicine consisting of Herba Ecliptae (HE) and Fructus Ligustri Lucidi (FLL), had a definite antiosteoporotic effect on osteoporotic femur, but its effect on osteoporosis of alveolar bone remains unknown. In the present study, we investigated the effects of Er-Zhi-Wan (EZW) on the microarchitecture and the regulation of Wnt/β-catenin signaling pathway in the alveolar bone of ovariectomized rats. Thirty Sprague-Dawley rats were randomly divided into three groups: sham operation group (sham, n=10), ovariectomy (OVX) group (n=10), and OVX with EZW treatment group (EZW group, n=10). From one week after ovariectomy, EZW (100 mg/mL) or vehicle (distilled water) was fed (1 mL/100 g) once per day for 12 weeks until the sacrifice of the rats. The body weights were measured weekly. After sacrifice, the sera and mandible were collected and routinely prepared for the measurement of alveolar trabecular microarchitecture, serum levels of E2, bone-specific alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase 5b (TRAP5b), as well as mandibular mRNA expression of Wnt/β-catenin signaling pathway molecules wnt3a, low-density lipoprotein receptor-related protein 5 (LRP5), β-catenin and dickkopf homolog 1 (DKK1). The results showed that EZW treatment significantly prevented the body weight gain, degradation of alveolar trabecular microarchitecture and alveolar bone loss in the OVX rats. Furthermore, we observed that EZW could increase the serum levels of E2 and BALP, and decrease levels of serum TRAP5b in EZW group compared with vehicle group. In addition, RT-PCR results revealed that EZW upregulated the expression levels of wnt3a, LRP5 and β-catenin, and reduced the expression of DKK1 in OVX rats. Taken together, our results suggested that EZW may have potential anti-osteoporotic effects on osteoporotic alveolar bone by stimulating Wnt/LRP5/β-catenin signaling pathway.

Tyrosine Kinase Inhibitor Tyrphostin AG490 Retards Chronic Joint Inflammation in Mice

Tyrphostin AG490 is a Janus kinase (JAK) 2 inhibitor that is clinically used as an anticancer agent and is also effective in various models of inflammatory and autoimmune diseases. In this study, we examined the effects of tyrphostin AG490 on the development of collagenase-induced osteoarthritis (CIOA). Our results showed that tyrphostin-ameliorated cartilage and bone destructions. This effect was associated with decreased expression of signal transducers and activators of transcription 3 (STAT3), phosphorylated JAK2, Dickkopf homolog 1, and receptor activator of nuclear factor κB ligand (RANKL) in the joints of arthritic mice. Tyrphostin AG490 suppressed STAT3 phosphorylation and the expression of tumor necrosis factor-related apoptosis-inducing ligand and RANKL by synovial fluid cells. The drug inhibited RANKL-induced osteoclast differentiation in vitro. Molecules, such as tyrphostin AG490 that limit bone erosion and influence osteoclast generation, might have therapeutic utility in joint degenerative disorders.

Correction: Dickkopf Homolog 3 (DKK3) Plays a Crucial Role Upstream of WNT/β-CATENIN Signaling for Sertoli Cell Mediated Regulation of Spermatogenesis

Correction: Dickkopf Homolog 3 (DKK3) Plays a Crucial Role Upstream of WNT/β-CATENIN Signaling for Sertoli Cell Mediated Regulation of Spermatogenesis

The Role for Dickkopf-Homolog-1 in the Pathogenesis of Crohn’s Disease-Associated Fistulae

BackgroundOne of the most challenging conditions in Crohn’s disease (CD) patients is the treatment of perianal fistulae. We have recently shown that epithelial-to-mesenchymal transition (EMT) plays a crucial role during CD-fistulae development. Dickkopf-homolog 1 (DKK-1) is known to play a key role during EMT. Here, we investigated a role for DKK-1 in the pathogenesis of CD-associated fistulae.MethodsDkk-1 protein expression in CD-fistula specimens were investigated by immunohistochemistry. Colonic lamina propria fibroblasts (CLPF) were obtained from either non-IBD control patients or patients with fistulizing CD. HT-29 intestinal epithelial cells (IEC) were either grown as monolayers or spheroids. Cells were treated with either TNF-α, TGF-β or IL-13. Knock-down of DKK-1 or β-Catenin was induced in HT-29-IEC by siRNA technique. mRNA expression was determined by real-time-PCR.ResultsDkk-1 protein was specifically expressed in transitional cells lining the fistula tracts. TGF-β induced DKK-1 mRNA expression in HT-29-IEC, but decreased it in fistula CLPF. On a functional level, DKK-1 knock-down prevented TGF-β-induced IL-13 mRNA expression in HT-29-IEC. Further, loss of β-Catenin was accompanied by reduced levels of DKK-1 and, again, IL-13 in IEC in response to TGF-β. In turn, treatment of HT-29-IEC as well as fistula CLPF with IL-13 resulted in decreased levels of DKK-1 mRNA. Treatment with TNF-α or the bacterial wall component, muramyl-dipeptide, decreased DKK-1 mRNA levels in HT-29-IEC, but enhanced it in fistula CLPF.DiscussionWe demonstrate that DKK-1 is strongly expressed in cells lining the CD-fistula tracts and regulates factors involved in EMT initiation. These data provide evidence for a role of DKK-1 in the pathogenesis of CD-associated perianal fistulae.

Breast Cancer Cells Induce Osteolytic Bone Lesions In vivo through a Reduction in Osteoblast Activity in Mice

Bone metastases are severely debilitating and have a significant impact on the quality of life of women with metastatic breast cancer. Treatment options are limited and in order to develop more targeted therapies, improved understanding of the complex mechanisms that lead to bone lesion development are warranted. Interestingly, whilst prostate-derived bone metastases are characterised by mixed or osteoblastic lesions, breast-derived bone metastases are characterised by osteolytic lesions, suggesting unique regulatory patterns. This study aimed to measure the changes in bone formation and bone resorption activity at two time-points (18 and 36 days) during development of the bone lesion following intratibial injection of MDA-MB-231 human breast cancer cells into the left tibiae of Severely Combined Immuno-Deficient (SCID) mice. The contralateral tibia was used as a control. Tibiae were extracted and processed for undecalcified histomorphometric analysis. We provide evidence that the early bone loss observed following exposure to MDA-MB-231 cells was due to a significant reduction in mineral apposition rate, rather than increased levels of bone resorption. This suggests that osteoblast activity was impaired in the presence of breast cancer cells, contrary to previous reports of osteoclast-dependent bone loss. Furthermore mRNA expression of Dickkopf Homolog 1 (DKK-1) and Noggin were confirmed in the MDA-MB-231 cell line, both of which antagonise osteoblast regulatory pathways. The observed bone loss following injection of cancer cells was due to an overall thinning of the trabecular bone struts rather than perforation of the bone tissue matrix (as measured by trabecular width and trabecular separation, respectively), suggesting an opportunity to reverse the cancer-induced bone changes. These novel insights into the mechanisms through which osteolytic bone lesions develop may be important in the development of new treatment strategies for metastatic breast cancer patients.

AB0061 Effects of chondroitin sulfate on the gene expression profile in il-1b stimulated synovial fibroblast cells cultures

BackgroundChondroitin sulfate (CS) is one the most used molecules in the management of OA. Its mechanism of action remains to be detailed.ObjectivesTo perform a microarray analysis to identify a differential...

FRI0058 Expression of specific pathways in the inflamed synovial membrane of osteoarthritis patient: Identification of new potential key intermediates

BackgroundSynovitis is a key factor in osteoarthritis (OA) pathophysiology.ObjectivesWe investigated the gene expression profiles of synovial cells from two different areas and identified differentially regulated pathways, using an original methodology...

AB0063 Effects of chondroitin sulfate on the gene expression profile in the inflamed synovial membrane

BackgroundChondroitin sulfate (CS) is one the most used molecules in the management of OA. Its mechanism of action remains to be detailed.ObjectivesTo identify the differentially expressed genes between the inflammatory...

DKK3 is a potential tumor suppressor gene in papillary thyroid carcinoma

The expression of the Dickkopf homolog 3 (DKK3) gene is downregulated in some human cancers, suggesting a possible tumor suppressor role of this gene. The role and regulation of DKK3 in thyroid cancer have not been examined. In this study, we explored the relationship of promoter methylation with the inactivation of DKK3 and tumor behaviors in papillary thyroid carcinoma (PTC). We used methylation-specific PCR and RT-PCR to examine the promoter methylation and expression of DKK3 and tumor characteristics. We found mRNA expression of DKK3 in 44.9% of the PTC tissue samples vs 100% of the matched normal thyroid tissue samples (P<0.01). In contrast, an opposite distribution pattern of DKK3 gene methylation was observed; specifically, 38.8% of the PTC tissue samples vs 0% of the matched normal thyroid tissue samples harbored DKK3 methylation. An inverse correlation between the promoter methylation and mRNA expression of DKK3 in PTC tissue samples was also observed. Moreover, we also found an inverse correlation between DKK3 expression and some aggressive pathological characteristics of PTC, including high TNM stages and lymph node metastasis, but a positive correlation between DKK3 promoter hypermethylation and pathological aggressiveness of the tumor. Treatment of the PTC cell line TPC-1 with the demethylating agent 5-azaC reduced DKK3 promoter methylation and enhanced its expression, establishing functionally the impact of DKK3 methylation on its expression. Our data thus for the first time demonstrate that the DKK3 gene is a potential tumor suppressor gene in thyroid cancer and that aberrant promoter methylation is an important mechanism for its downregulation, which may play a role in the tumorigenesis and aggressiveness of PTC.

Dickkopf Homolog 3 (DKK3) Plays a Crucial Role Upstream of WNT/β-CATENIN Signaling for Sertoli Cell Mediated Regulation of Spermatogenesis

Testicular Sertoli cells (Sc) are main somatic component of seminiferous tubules that govern the differentiation of germ cells (Gc) and provide them physical support. Sc are the target of follicle stimulating hormone (FSH) and testosterone (T) which are known to regulate spermatogenesis. FSH and T levels in human and sub-human male primates remain high during infancy (4–6 months post birth), similar to those during puberty. Subsequently, juvenile phase is marked with low levels of these hormones. In spite of prolonged hormonal exposure, spermatogenesis is not discerned during infancy unlike that during puberty. Situation during infancy is similar to certain idiopathic male infertility, where prolonged hormone supplementation fails to initiate spermatogenesis. In our quest to determine non hormonal causes of idiopathic infertility which may reside within the Sc, we investigated the association between spermatogenesis and Sc specific gene(s) expressed differentially during puberty and infancy. Although products of several genes may be necessary for quantitatively normal spermatogenesis, one needs to investigate their roles one by one. Differential display and real time PCR analysis revealed higher expression of a known tumor suppressor, Dickkopf homolog 3 (DKK3), by pubertal monkey Sc as compared to infant Sc. To evaluate role of DKK3 in spermatogenesis, we generated DKK3 knock down mice (DKDM) using shRNA construct targeted to DKK3. In testis of adult DKDM, expression of DKK3 mRNA and protein were significantly (p<0.05) low and was associated with elevated WNT-4/β-CATENIN activity. Elevated β-CATENIN activity is known to restrict Sc maturation. Abundant expression of infant Sc marker, Mullerian inhibiting substance (MIS), in the testes of adult DKDM confirmed lack of Sc maturation in DKDM. Gc differentiation and fertility was severely compromised in DKDM. This is the first report of role of DKK3 in the testis and DKK3 mediated regulation of spermatogenesis via WNT-4/β-CATENIN modulation.

Elevated miR-29a expression is not correlated with disease activity index in PBMCs of patients with ankylosing spondylitis.

OBJECTIVES: Ankylosing spondylitis (AS) is a chronic inflammatory disease characterized by new bone formation. Recent evidence suggests that new bone formation in AS may be due to upregulation of Wnt signaling in the osteoblastic pathway secondary to low serum Dickkopf homolog 1 (Dkk-1) levels. And miR-29a orchestrates osteoblast differentiation through direct targeting and negative regulation of Dkk-1. METHODS: We initially validated the expression levels of miR-29a in the peripheral blood mononuclear cells (PBMCs) of AS patients (n=30), rheumatoid arthritis (RA) patients (n=30) and healthy controls (n=30) using real-time quantitative reverse transcription PCR (qRT-PCR). Correlation analysis was assessed between miR-29a level in PBMCs of AS patients and disease activity indexes, including erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), Bath ankylosing spondylitis disease activity index (BASDAI), Bath ankylosing spondylitis function index (BASFI) and modified Stoke ankylosing spondylitis spinal score (mSASSS). RESULTS: Significantly higher expression of miR-29a was observed in PBMCs of AS patients (Ct 9.18±1.96) compared with that in RA patients (10.97±0.70, p<0.001) and healthy controls (Ct 11.45±1.23, p<0.001). There was no significant difference between RA patients and healthy controls in miR-29a expression (p>0.05). Elevated miR-29a expression is not correlated with disease activity index (p>0.05). A weak correlation was found between elevated miR-29a expression and mSASSS (r=-0.393, p=0.032). CONCLUSIONS: We report for the first time elevated miR-29a expression in PBMCs of patients with ankylosing spondylitis, and miR-29a might be used as a useful diagnostic marker in new bone formation but cannot reflect disease activity.

Investigation of potential new targets for the diagnosis and/or the treatment of osteoarthritis

appear differentially expressed. The interferon alpha-inductible protein 6 (IFI6) was the most up-regulated. We also identified the hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1), the cathepsin K (CTSK), the chemokine (C-X-C motif) ligand 1 (CXCL1) and the EBV-induced Gprotein coupled receptor 2 (EBI2). The differential expression of intermediates involved in angiogenesis pathway was also revealed between N/R and I areas. Among them, R-spondin-3 (RSPO3), the secreted phopshoprotein 1 (SPP1) and aquaporin 9 (AQP9) were up-regulated whereas ADAMTS1 was down-regulated. Finally, in the Wnt signaling, RSPO3 was up-regulated unlike dickkopf homolog 3 (DKK3) which was in turn down-regulated. We next performed a class comparison test between N/R and N/R-CS in one hand and between I and I-CS the other hand. 489 genes were identified as differentially expressed genes between N/R and N/R-CS conditions while 219 genes were identified between I and I-CS conditions. In this latter, our attention was focused on the down-regulated genes. Among them, we identified a number implicated in angiogenesis and cell migration pathways. Thus, the endothelial cell-specific molecule-1 (ESM1), the Transmembrane-4-Lsix-family-1 (TM4SF1), the 50-Ectonucleotidase (NT5E) and the growth arrest-specific gene 6 (GAS6) were down-regulated by CS. Conclusions: Our work demonstrates the differential gene expression profile between paired inflammatory and normal/reactive areas of synovial membrane as well as the modulatory effects of CS on gene expression in the inflammatory areas, especially regarding genes involved in both angiogenesis and cell migration.