Dickkopf Homolog Research Articles

Effect of 5-azacytidine on growth of thyroid papillary cancer-1 cell line and expression of the dickkopf homolog 3 gene

Objective To study the effects of 5-azacytidine (5-azaC),a methylation inhibitor,on the growth of thyroid papillary cancer-1 (TPC-1) papillary thyroid carcinoma cells and the transcription regulation of DNA guanine cytosine-phosphoric acid-motif (CpG) island demethylation on dickkopf homolog 3 (DKK3) tumor suppressor gene.Methods Methyl thiazol tetrazolium (MTT) method was used to detect the growth of TPC-1 cells after treated with 5-azaC (2,5,10,20,50,100,200 and 400 μmol/L).The expression of DKK3 mRNA and methylation status were examined by using methylation-specific polymerase chain reaction (MSP) and reverse transcription-polymerase chain reaction (RT-PCR) after TPC-1 cells treated with 5-azaC (5,10,20 and 50 μmol/L).Results 5-azaC reduced viability of TPC-1 cells in a dose-and time-dependent manner (P ＜0.05).After TPC-1 cells were treated with 5-azaC (5,10,20 and 50 μ mol/L) for 48 h,the relative expression of DKK3 mRNA was 0.208 ±0.017,0.365 ± 0.013,0.489 ± 0.017 and 0.582 ± 0.011 respectively,which was gradually increased in a dose-dependent manner (F =370.356,P ＜ 0.05).Meanwhile,DKK3 gene was demethylated partly in TPC-1 cells treated with 5-azaC.Conclusion 5-azaC might effectively cause the demethylation of DKK3 gene CpG-rich promoter regions,which may activate the expression of DKK3 gene and inhibit the growth of TPC-1 cells. Key words: Papillary thyroid carcinoma; Dickkopf homolog 3 gene; 5-azacytidine; Demethylation

Novel SNPs in the exon region of bovine DKK4 gene and their association with body measurement traits in Qinchuan cattle

The aim of this study was to determine whether single nucleotide polymorphisms (SNPs) of bovine Dickkopf homolog 4 (DKK4) are associated with body measurement traits in Qinchuan cattle. By using PCR-SSCP technology and DNA sequencing, we discovered 5 DKK4 SNPs in Qingchuan cattle, including -65G>A and -77G>T in the 5'-untranslated region, 1532C>G and 1533T>C in exon 2, and 2088C>T in exon 3. The sequencing map showed that 1532C>G and 1533T>C were in close linkage disequilibrium and were treated as 1532C>G-1533T>C in this study. Allele frequencies were calculated and analyzed by the chi-square test, which showed that -65G>A and 1532C>G-1533T>C were in Hardy-Weinberg equilibrium (P > 0.05), whereas -77G>T and 2088C>T were not in all 633 tested Qinchuan cattle individuals (P < 0.01). Gene heterozygosity (HE), effective allele number (NE), and polymorphism information content (PIC) were 0.407, 1.686, and 0.324 at -65G>A; 0.472, 1.894, and 0.361 at -77G>T; 0.476, 1.908, and 0.363 at 1532C>G-1533T>C; and 0.218, 1.279, and 0.195 at 2088C>T. We also evaluated the potential association of these SNPs with body measurement traits in all 633 individuals; the results suggest that several SNPs in Qinchuan cattle DKK4 were significantly associated with body length, hip height, rump length, hip width, heart girth, and pin bone width (P < 0.05 and P < 0.01). These results suggest that bovine DKK4 could be used as candidate gene for Qinchuan cattle breeding.

Estrogen Receptors β1 and β2 Have Opposing Roles in Regulating Proliferation and Bone Metastasis Genes in the Prostate Cancer Cell Line PC3

The estrogen receptor (ER)β1 is successively lost during cancer progression, whereas its splice variant, ERβ2, is expressed in advanced prostate cancer. The latter form of cancer often metastasizes to bone, and we wanted to investigate whether the loss of ERβ1 and/or the expression of ERβ2 affect such signaling pathways in prostate cancer. Using PC3 and 22Rv1 prostate cancer cell lines that stably express ERβ1 or ERβ2, we found that the ERβ variants differentially regulate genes known to affect tumor behavior. We found that ERβ1 repressed the expression of the bone metastasis regulator Runx2 in PC3 cells. By contrast, RUNX2 expression was up-regulated at the mRNA level by ERβ2 in PC3 cells, whereas Slug was up-regulated by ERβ2 in both PC3 and 22Rv1 cells. In addition, the expression of Twist1, a factor whose expression strongly correlates with high Gleason grade prostate carcinoma, was increased by ERβ2. In agreement with the increased Twist1 expression, we found increased expression of Dickkopf homolog 1; Dickkopf homolog 1 is a factor that has been shown to increase the RANK ligand/osteoprotegerin ratio and enhance osteoclastogenesis, indicating that the expression of ERβ2 can cause osteolytic cancer. Furthermore, we found that only ERβ1 inhibited proliferation, whereas ERβ2 increased proliferation. The expression of the proliferation markers Cyclin E, c-Myc, and p45(Skp2) was differentially affected by ERβ1 and ERβ2 expression. In addition, nuclear β-catenin protein and its mRNA levels were reduced by ERβ1 expression. In conclusion, we found that ERβ1 inhibited proliferation and factors known to be involved in bone metastasis, whereas ERβ2 increased proliferation and up-regulated factors involved in bone metastasis. Thus, in prostate cancer cells, ERβ2 has oncogenic abilities that are in strong contrast to the tumor-suppressing effects of ERβ1.

PAGE4 Positivity Is Associated with Attenuated AR Signaling and Predicts Patient Survival in Hormone-Naive Prostate Cancer

Aberrant activation of the androgen receptor (AR) plays a key role during prostate cancer (PCa) development and progression to castration-resistant prostate cancer (CR-PCa) after androgen deprivation therapy, the mainstay systemic treatment for PCa. New strategies to abrogate AR activity and biomarkers that predict aggressive tumor behavior are essential for improved therapeutic intervention. PCa tissue microarrays herein reveal that prostate-associated gene 4 (PAGE4), an X-linked cancer/testis antigen, is highly up-regulated in the epithelium of preneoplastic lesions compared with benign epithelium, but subsequently decreases with tumor progression. We show that AR signaling is attenuated in PAGE4-expressing cells both in vitro and in vivo, most likely via impaired androgen-induced AR nuclear translocation and subsequently reduced AR protein stabilization and phosphorylation at serines 81 and 213. Consistently, epithelial PAGE4 protein levels inversely correlated with AR activation status in hormone-naive and CR-PCa clinical specimens. Moreover, PAGE4 impaired the development of CR-PCa xenografts, and strong PAGE4 immunoreactivity independently predicted favorable patient survival in hormone-naive PCa. Collectively, these data suggest that dysregulation of epithelial PAGE4 modulates AR signaling, thereby promoting progression to advanced lethal PCa and highlight the potential value of PAGE4 as a prognostic and therapeutic target.

Adalimumab significantly reduces inflammation and serum DKK‐1 level but increases fatty deposition in lumbar spine in active ankylosing spondylitis

To investigate whether adalimumab is effective for active ankylosing spondylitis (AS) patients and whether it has an impact on the formation of fatty deposition lesions (FDL) and serum Dickkopf homolog 1 (Dkk-1) level in AS patients. This was a randomized, double-blind, placebo-controlled study. Active AS patients received 40 mg adalimumab (n = 26) or placebo (n = 20) every other week during an initial 12-week double-blind period, and all switched to adalimumab treatment for another 12 weeks. Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Function Index (BASFI), C-reactive protein (CRP), Ankylosing Spondylitis Disease Activity Scores (ASDAS) and serum DKK-1 levels were measured and magnetic resonance imaging (MRI) of both the lumbar spine and sacroiliac joints were obtained at baseline, week 12 and week 24. Spinal and sacroiliac joint inflammations were evaluated using the Spondyloarthritis Research Consortium of Canada (SPARCC) MRI index, and FDL were assessed in a dichotomous manner. Obvious improvements in clinical assessments (BASDAI, BASFI, CRP and ASDAS reduced, all P < 0.05), as well as MRI inflammation measurements (both lumbar spine and sacroiliac joints SPARCC scores decreased, all P < 0.05) were shown in active AS patients treated by adalimumab for 12 weeks, but FDL in the lumbar spine seen by MRI increased significantly (P < 0.05) accompanied by decrease of serum DKK-1 levels (P < 0.05), while FDL remained stable after the treatment of placebo in AS patients. Our study found that adalimumab was highly effective in reducing inflammation in active AS patients, but it was accompanied by the formation of FDL in the lumbar spine and decrease in serum DKK-1 levels.

Effect of chondroitin sulfate on the factors involved in synovial inflammation

Effect of chondroitin sulfate on the factors involved in synovial inflammation

Beneficial effects of tripterygium glycosides tablet on biomarkers in patients with ankylosing spondylitis.

The aim of the current study was to explore the effects and possible mechanisms of tripterygium glycosides tablet (TGT) in the treatment of active ankylosing spondylitis (AS). Thirty-six patients with active AS were given a 20 mg TGT treatment three times per day for 12 weeks, and 21 unrelated healthy controls were recruited as the control group. Efficacy measures included the Bath AS disease activity index (BASDAI), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) prior and subsequent to TGT treatment. Serum dickkopf homolog 1 (DKK1) and interleukin-17 (IL-17) levels before and after TGT treatment were assessed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and ELISA assay. The levels of several serum biomarkers were determined by ELISA, including receptor activator of nuclear factor κ-B ligand (RANKL), osteoprotegerin (OPG), bone alkaline phosphatase (BAP), bone morphogenetic protein-2 (BMP-2), matrix metalloproteinase-3 (MMP-3), cross-linked telopeptide of type II collagen (CTX-II), vascular endothelial growth factor (VEGF), and prostaglandin E2 (PGE2). After 12 weeks of TGT treatment, the BASDAI score of the patients was significantly reduced (P<0.05), their levels of ESR and CRP were significantly reduced to a normal level (P<0.05, P<0.05), RT-PCR and ELISA showed a significant increase in the level of DKK1 expression (P<0.05) and a significant decreased IL-17 expression (P<0.05), there was a significant increase in the expression of OPG, BAP and BMP-2 (P<0.01, P<0.01, P<0.01) and a significant reduction in the expression levels of RANKL, CTX-II. MMP-3, PGE2, and VEGF (P<0.01, P<0.01, P<0.01, P<0.05, P<0.01) compared with those of the controls. TGT is effective at improving the signs and symptoms of patients with AS through the regulation of serum biomarkers, and the mechanisms may be associated with the anti-inflammatory effect, inhibition of new bone formation and potential bone-protective effects.

A Decrease in DKK1, a WNT Inhibitor, Contributes to Placental Lipid Accumulation in an Obesity-Prone Rat Model1

Placenta, as the sole transport mechanism between mother and fetus, links the maternal physical state and the immediate as well as lifelong outcomes of the offspring. The present study examined the consequences of maternal obesity on placental lipid accumulation and metabolism. Pregnant obesity-prone (OP) and obesity-resistant (OR) rat strains were fed a control diet throughout gestation. Placentas were collected on Gestational Day 21 for mRNA and oxidative stress analysis, and frozen placental sections were analyzed for fat accumulation as well as beta-catenin and Dickkopf homolog 1 (Xenopus laevis) (DKK1) localization. JEG3 trophoblast cells were cultured in vitro to determine the relationship between DKK1 and lipid accumulation. Maternal plasma and placental nonesterified fatty acids and triglycerides (TG) were elevated in OP dams. Placental Dkk1 mRNA content was 4-fold lower in OP placentas, and a significant increase was noted in beta-catenin accumulation as well as in mRNA content of fat transport and TG synthesis genes, including Ppard (peroxisome proliferator-activated receptor delta), Slc27a1 (fatty acid transport protein 1; also known as Fatp1), Cd36 (cluster of differentiation 36; also known as fatty acid translocation [Fat]), Lipin1, and Lipin3. Significant lipid accumulation was found within the decidual zones in OP, but not OR, placentas, and thickness of the decidual and junctional zones was significantly smaller in OP than in OR placentas. Overexpression of DKK1 in JEG3 cells decreased lipid accumulation and mRNA content of PPARD, SLC27A1, CD36, LIPIN1, and LIPIN3. Our results demonstrate that DKK1 is regulating certain aspects of placental lipid metabolism through the WNT signaling pathway.

Effects of administration of hormone therapy or raloxifene on the immune system and on biochemical markers of bone remodeling

Over the last few years, conclusive evidence of the involvement of the immune system in the regulation of bone metabolism has been identified. Consequently, one question that should be formulated concerns the possible effects of antiresorptive therapies on the immune system. Therefore, the purpose of the present work was to evaluate both the functionality of the immune system and bone turnover in women receiving antiresorptive therapies, such as hormone therapy (HT; n = 33) and raloxifene (RLX; n = 66), acting through estrogen receptors. To that end, this study analyzed bone turnover markers in a population of postmenopausal women before and after beginning therapy and compared these with data of women not treated (NT; n = 102). In a subgroup of participants (NT = 33, RLX = 24, and HT = 26), we analyzed the effects of treatments on immune system parameters such as serum levels of interleukin (IL)-6, tumor necrosis factor α, and IL-1β; lymphocyte subpopulations; cell proliferation by peripheral blood mononuclear cells (PBMCs); in vitro production of IL-1β by PBMCs; and the expression of receptor activator of nuclear factor-κB ligand, transforming growth factor β, and IL-4 genes by PBMCs. The results showed that bone resorption was inhibited strongly in women in the RLX and HT groups when compared with women in the NT group. Interestingly, the administration of RLX inhibited the production of the Wnt/β-catenin signaling pathway inhibitor Dickkopf Homolog-1 (P < 0.05) and tended to increase the levels of the osteoclastogenesis inhibitor osteoprotegerin at month 6 (P = 0.059). With regard to the immune system, the different treatments did not markedly perturb the parameters analyzed, with the exception of the increase in serum IL-1β detected in the HT group at month 6 (P < 0.05). The main conclusions of the present work were that HT or RLX do not disturb the immune system and that both treatments have a similar antiresorptive power.

Bone Marrow-Derived Mesenchymal Stromal Cells are Attracted by Multiple Myeloma Cell-Produced Chemokine CCL25 and Favor Myeloma Cell Growth in Vitro and In Vivo

Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells that are predominantly localized in the bone marrow (BM). Mesenchymal stromal cells (MSCs) give rise to most BM stromal cells that interact with MM cells. However, the direct involvement of MSCs in the pathophysiology of MM has not been well addressed. In this study, in vitro and in vivo migration assays revealed that MSCs have tropism toward MM cells, and CCL25 was identified as a major MM cell-produced chemoattractant for MSCs. By coculture experiments, we found that MSCs favor the proliferation of stroma-dependent MM cells through soluble factors and cell to cell contact, which was confirmed by intrafemoral coengraftment experiments. We also demonstrated that MSCs protected MM cells against spontaneous and Bortezomib-induced apoptosis. The tumor-promoting effect of MSCs correlated with their capacity to enhance AKT and ERK activities in MM cells, accompanied with increased expression of CyclinD2, CDK4, and Bcl-XL and decreased cleaved caspase-3 and poly(ADP-ribose) polymerase expression. In turn, MM cells upregulated interleukin-6 (IL-6), IL-10, insulin growth factor-1, vascular endothelial growth factor, and dickkopf homolog 1 expression in MSCs. Finally, infusion of in vitro-expanded murine MSCs in 5T33MM mice resulted in a significantly shorter survival. MSC infusion is a promising way to support hematopoietic recovery and to control graft versus host disease in patients after allogeneic hematopoietic stem cell transplantation. However, our data suggest that MSC-based cytotherapy has a potential risk for MM disease progression or relapse and should be considered with caution in MM patients.

TFAP2E–DKK4 and Chemoresistance in Colorectal Cancer

BackgroundChemotherapy for advanced colorectal cancer leads to improved survival; however, predictors of response to systemic treatment are not available. Genomic and epigenetic alterations of the gene encoding transcription factor AP-2 epsilon (TFAP2E) are common in human cancers. The gene encoding dickkopf homolog 4 protein (DKK4) is a potential downstream target of TFAP2E and has been implicated in chemotherapy resistance. We aimed to further evaluate the role of TFAP2E and DKK4 as predictors of the response of colorectal cancer to chemotherapy.MethodsWe analyzed the expression, methylation, and function of TFAP2E in colorectal-cancer cell lines in vitro and in patients with colorectal cancer. We examined an initial cohort of 74 patients, followed by four cohorts of patients (total, 220) undergoing chemotherapy or chemoradiation.ResultsTFAP2E was hypermethylated in 38 of 74 patients (51%) in the initial cohort. Hypermethylation was associated with decreased expression of TFAP2E in primary and metastatic colorectal-cancer specimens and cell lines. Colorectal-cancer cell lines overexpressing DKK4 showed increased chemoresistance to fluorouracil but not irinotecan or oxaliplatin. In the four other patient cohorts, TFAP2E hypermethylation was significantly associated with nonresponse to chemotherapy (P<0.001). Conversely, the probability of response among patients with hypomethylation was approximately six times that in the entire population (overall estimated risk ratio, 5.74; 95% confidence interval, 3.36 to 9.79). Epigenetic alterations of TFAP2E were independent of mutations in key regulatory cancer genes, microsatellite instability, and other genes that affect fluorouracil metabolism.ConclusionsTFAP2E hypermethylation is associated with clinical nonresponsiveness to chemotherapy in colorectal cancer. Functional assays confirm that TFAP2E-dependent resistance is mediated through DKK4. In patients who have colorectal cancer with TFAP2E hypermethylation, targeting of DKK4 may be an option to overcome TFAP2E-mediated drug resistance. (Funded by Deutsche Forschungsgemeinschaft and others.)

83 EFFECTS OF HUMAN INTERFERON-α ON GENE EXPRESSION IN THE BOVINE ENDOMETRIUM IN COMPARISON TO DAYS 15 AND 18 OF PREGNANCY

Interferon-τ (IFNT), a Type-I interferon (IFN), is the pregnancy recognition signal produced by the ruminant conceptus (Godkin et al. 1984; Hansen et al. 1985; Helmer et al. 1987; Spencer et al. 2007). In addition to these specific functions of IFNT in ruminants, many studies suggest that IFNs play a general role in establishment of pregnancy and conceptus attachment/implantation in most mammalian species (Bazer et al. 2009; Bazer et al. 2010; Johnson et al. 2009; Roberts et al. 2008). To characterise the effects of prototype Type-I IFNs on bovine endometrium, in experiment one, Simmental heifers were treated from Day 14 to Day 16 of the oestrous cycle with a rod-shaped intrauterine device releasing human interferon-α (IFNA) or placebo lipid extrudates or PBS only as controls (n = 4 each). Lipid formulation and concentration of human IFNA were adjusted to release 8–9 × 107 IU of IFNA over a period of 2 days in in vitro release experiments. On Day 16, endometrial biopsy samples were collected after flushing the uterus. In experiment 2, endometrial tissue samples were obtained on Day 12, 15 and 18 post-mating from nonpregnant or pregnant heifers. All samples from both experiments were analysed with an Affymetrix Bovine Genome Array (Santa Clara, CA). In experiment one, IFNA treatment resulted in differential gene expression in the bovine endometrium. Significant differences were found between the IFNA group and both control groups, whereas no differences were observed between the placebo and the PBS control group. In experiment 2, differentially expressed genes were found between pregnant and nonpregnant endometria on Day 15 and 18, but not on Day 12, with many of them known IFN-stimulated genes. The comparison of the data sets from both experiments showed very similar gene expression changes for most of the typical IFN-stimulated genes. In addition, several genes were identified which were differentially expressed after IFNA treatment but not different at Day 15 or 18 of pregnancy compared with nonpregnant animals. Conversely, some genes were found as differentially expressed during pregnancy but not after IFNA treatment. Differential expression of selected genes was verified by quantitative real-time PCR and 4 genes, namely jumonji C domain containing histone demethylase 1 homologue D (JHDM1D), indoleamine 2,3-dioxygenase 1 (IDO1), fatty acid binding protein 3, muscle and heart (mammary-derived growth inhibitor) (FABP3) and dickkopf homologue 1 (DKK1), were selected for localization of mRNA expression in endometrial tissue sections. The findings of this study suggest that there may be differential effects of bovine IFNT compared with human IFNA and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT. This study was supported by the German Ministry for Education and Research (BMBF, FUGATO-plus, COMPENDIUM) and the German Research Foundation (DFG FOR478). The authors are part of the European Union COST action GEMINI.

Case-control study of vitamin D, dickkopf homolog 1 (DKK1) gene methylation, VDR gene polymorphism and the risk of colon adenoma in African Americans.

BackgroundThere are sparse data on genetic, epigenetic and vitamin D exposure in African Americans (AA) with colon polyp. Consequently, we evaluated serum 25(OH) D levels, vitamin D receptor (VDR) polymorphisms and the methylation status of the tumor suppressor gene dickkopf homolog 1 (DKK1) as risk factors for colon polyp in this population.MethodsThe case-control study consisted of 93 patients with colon polyp (cases) and 187 healthy individuals (controls) at Howard University Hospital. Serum levels of 25(OH)D (including D3, D2, and total) were measured by liquid chromatography-mass spectrometry. DNA analysis focused on 49 single nucleotide polymorphisms (SNPs) in the VDR gene. Promoter methylation analysis of DKK1 was also performed. The resulting data were processed in unadjusted and multivariable logistic regression analyses.ResultsCases and controls differed in vitamin D status (D3<50 nmol/L: Median of 35.5 in cases vs. 36.8 in controls nmol/L; P = 0.05). Low levels of 25(OH)D3 (<50 nmol/L) were observed in 86% of cases and 68% of controls and it was associated with higher risks of colon polyp (odds ratio of 2.7, 95% confidence interval 1.3–3.4). The SNP analysis showed no association between 46 VDR polymorphisms and colon polyp. The promoter of the DKK1 gene was unmethylated in 96% of the samples.ConclusionWe found an inverse association between serum 25(OH)D3 and colon polyp in AAs. VDR SNPs and DKK1 methylation were not associated with colon polyp. Vitamin D levels may in part explain the higher incidence of polyp in AAs.

Epigallocatechin gallate inhibits sphere formation of neuroblastoma BE(2)-C cells

A growing number of epidemiological studies have demonstrated that the consumption of green tea inhibits the growth of a variety of cancers. Epigallocatechin gallate (EGCG), the most abundant catechin in green tea, has been shown to have an anti-cancer effect against many cancers. Most cancers are believed to be initiated from and maintained by a small population of tumor-initiating cells (TICs) that are responsible for chemotherapeutic resistance and tumor relapse. In neuroblastoma, an aggressive pediatric tumor that often relapses and has a poor prognosis, TICs were recently identified as spheres grown in a serum-free non-adherent culture used for neural crest stem cell growth. Although EGCG has been reported to induce growth arrest and apoptosis in neuroblastoma cells, its effect on neuroblastoma TICs remains to be defined. Gene expression was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). The effects of EGCG on cell proliferation, apoptosis, and sphere formation were determined by cell counting, propidium iodide staining, and sphere (>100μm in diameter) counting, respectively. Neuroblastoma BE(2)-C cells showed increased expression of stem cell markers (nanog homeobox [NANOG] and octamer-binding transcription factor 4 [OCT4]), as well as decreased expression of neuronal differentiation markers (Cu(2+)-transporting ATPase alpha polypeptide [ATP7A] and dickkopf homolog 2 [DKK2]) in spheres grown in serum-free non-adherent culture, compared to parental cells grown in conventional culture. Although EGCG induced growth arrest and apoptosis in the parental cells in a dose-dependent manner, it was not effective against spheres. However, EGCG potently inhibited sphere formation in the BE(2)-C cells. The present results suggest that EGCG may inhibit the development of TICs in BE(2)-C cells.

Heparan Sulfate Regulates Intraretinal Axon Pathfinding by Retinal Ganglion Cells

PURPOSE. Heparan sulfate (HS) is abundantly expressed in the developing neural retina; however, its role in the intraretinal axon guidance of retinal ganglion cells (RGCs) remains unclear. In this study, the authors examined whether HS was essential for the axon guidance of RGCs toward the optic nerve head. METHODS. The authors conditionally ablated the gene encoding the exostosin-1 (Ext1) enzyme, using the dickkopf homolog 3 (Dkk3)-Cre transgene, which disrupted HS expression in the mouse retina during directed pathfinding by RGC axons toward the optic nerve head. In situ hybridization, immunohistochemistry, DiI tracing, binding assay, and retinal explant assays were performed to evaluate the phenotypes of the mutants and the roles of HS in intraretinal axon guidance. RESULTS. Despite no gross abnormality in RGC distribution, the mutant RGC axons exhibited severe intraretinal guidance errors, including optic nerve hypoplasia, ectopic axon penetration through the full thickness of the neural retina and into the subretinal space, and disturbance of the centrifugal projection of RGC axons toward the optic nerve head. These abnormal phenotypes shared similarities with the RGC axon misguidance caused by mutations of genes encoding Netrin-1 and Slit-1/2. Explant assays revealed that the mutant RGCs exhibited disturbed Netrin-1-dependent axon outgrowth and Slit-2-dependent repulsion. CONCLUSIONS. The present study demonstrated that RGC axon projection toward the optic nerve head requires the expression of HS in the neural retina, suggesting that HS in the retina functions as an essential modulator of Netrin-1 and Slit-mediated intraretinal RGC axon guidance.

Dickkopf homolog 1, a Wnt signaling antagonist, is transcriptionally up-regulated via an ATF4-independent and MAPK/ERK-dependent pathway following amino acid deprivation

Amino acid response (AAR) pathway is activated when cells are deprived of amino acids. In the present study, using the human colon cancer cell line SW480, we observed that DKK1, an antagonist of the Wnt pathway, was significantly induced at the mRNA level after the removal of amino acids from the medium. Addition of the amino alcohol histidinol, which prevents the formation of histidinyl-tRNA His, also increased DKK1 mRNA to a level similar to that observed when cells were deprived of all amino acids. Transcriptional activity and stability of DKK1 mRNA were both increased in the amino acid-deprived condition. The induction of DKK1 gene expression was confirmed by the increased immunofluorescent staining of the DKK1 protein in the amino acid deprived condition. Although chromatin immunoprecipitation assays showed increased RNA Polymerase II binding at the DKK1 promoter in amino acid-limited conditions, ATF4 binding to the promoter is absent. Luciferase reporter assays did not detect any functional AARE within the DKK1 gene structure. Knockdown of ATF4 by siRNA did not affect the increase of DKK1 mRNA during amino acid limitation. Inhibition of ERK phosphorylation abolished the induction of DKK1. Our study revealed that DKK1 is a novel target gene in the response to amino acid deficiency and that the expression of DKK1 is up-regulated through an ATF4-independent and an ERK-dependent pathway.

Expression and Functional Analysis of Dkk1 during Early Gonadal Development

WNT signalling plays a central role in mammalian sex determination by promoting ovarian development and repressing aspects of testis development in the early gonad. Dickkopf homolog 1 (DKK1) is a WNT signalling antagonist that plays critical roles in multiple developmental systems by modulating WNT activity. Here, we examined the role of DKK1 in mouse sex determination and early gonadal development. Dkk1 mRNA was upregulated sex-specifically during testis differentiation, suggesting that DKK1 could repress WNT signalling in the developing testis. However, we observed overtly normal testis development in Dkk1-null XY gonads, and found no significant upregulation of Axin2 or Sp5 that would indicate increased canonical WNT signalling. Nor did we find significant differences in expression of key markers of testis and ovarian development. We propose that DKK1 may play a protective role that is not unmasked by loss-of-function in the absence of other stressors.

Harnessing Expression Data to Identify Novel Candidate Genes in Polycystic Ovary Syndrome

Novel pathways in polycystic ovary syndrome (PCOS) are being identified in gene expression studies in PCOS tissues; such pathways may contain key genes in disease etiology. Previous expression studies identified both dickkopf homolog 1 (DKK1) and DnaJ (Hsp40) homolog, subfamily B, member 1 (DNAJB1) as differentially expressed in PCOS tissue, implicating them as candidates for PCOS susceptibility. To test this, we genotyped a discovery cohort of 335 PCOS cases and 198 healthy controls for three DKK1 single nucleotide polymorphisms (SNPs) and four DNAJB1 SNPs and a replication cohort of 396 PCOS cases and 306 healthy controls for 1 DKK1 SNP and 1 DNAJB1 SNP. SNPs and haplotypes were determined and tested for association with PCOS and component phenotypes. We found that no single nucleotide polymorphisms were associated with PCOS risk; however, the major allele of rs1569198 from DKK1 was associated with increased total testosterone (discovery cohort P = 0.0035) and dehydroepiandrosterone sulfate (replication cohort P = 0.05). Minor allele carriers at rs3962158 from DNAJB1 had increased fasting insulin (discovery cohort P = 0.003), increased HOMA-IR (discovery cohort P = 0.006; replication cohort P = 0.036), and increased HOMA-%B (discovery cohort P = 0.004). Carriers of haplotype 2 at DNAJB1 also had increased fasting insulin, HOMA-IR, and HOMA-%B. These findings suggest that genetic variation in DKK1 and DNAJB1 may have a role in the hyperandrogenic and metabolic dysfunction of PCOS, respectively. Our results also demonstrate the utility of gene expression data as a source of novel candidate genes in PCOS, a complex and still incompletely defined disease, for which alternative methods of gene identification are needed.

Effect of Imids on Gene Expression Profiles of Fresh Human Myeloma Cells

AbstractAbstract 5013Immunomodulatory drugs represent a major therapeutic advance in the treatment of patients with multiple myeloma. While these agents appear to exert various effects on the microenvironment, including effect on immune cells and angiogenesis, a direct effect on the tumor cells themselves is also likely. To describe and compare the effect of the three clinically available agents (thalidomide, lenalidomide, pomalidomide) we analyzed the gene expression profiles of fresh human myeloma cells exposed to thalidomide, lenalidomide or pomalidomide, using high density DNA arrays. Fresh human myeloma samples were obtained from bone marrow aspirates of patients with myeloma, and myeloma cells were immunopurified using anti CD138 magnetic beads. Purified myeloma cells (1.106 cells/ml) were incubated for 24 hours in RPMI 1640 medium supplemented with 10% fetal calf serum under each of the four following conditions: 1) DMSO; 2) thalidomide 40 microM; 3) lenalidomide 1 microM; 4) pomalidomide 100 nM. These levels are achievable in the plasma of MM pts. Pangenomic array experiments were performed usingWhole Human Genome 4 × 44K Agilent one-color microarrays. Data were normalized using the quantile normalization method. Samples were analysed for differentially expressed genes, taking into account both the level of significance and the fold-change. Ten evaluable samples were processed. Exposure to thalidomide, lenalidomide and pomalidomide induced differential expression of 36, 50 and 75 genes, respectively, in comparison to DMSO-exposed controls, the total list including 101 genes. Twelve of these were found to be differentially expressed after exposure to all of the three agents, including trophoblast glycoprotein, WAS protein family member 1, dickkopf homolog 1, pentraxin-related gene, CD28, interleukin 12B, tissue factor pathway inhibitor 2, phospholipase A2, dehydrogenase/reductase (SDR family) member 9, hypothetical LOC145788 and betacellulin. These commonly altered genes could be mechanistically involved in themultiple activities of these agents in multiple myeloma or may represent epiphenoma mechanistically unrelated to drug-induced cell death. Genes differentially expressed between the treatment with each of these agents could be indicative of the different and non-overlapping actions these agents have in multiple myeloma. An example of this is the recent demonstration that pomalidomide is clinically active in lenalidomide refractory patients. These results suggest that exposure to IMIDs induce various intracellular signalization pathways in myeloma cells which might be involved in the cytotoxic activity of these compounds. Disclosures:Dumontet:Celgene: Research Funding.

DICKKOPF‐4 activates the noncanonical c‐Jun‐NH2 kinase signaling pathway while inhibiting the Wnt‐canonical pathway in human renal cell carcinoma

To the authors' knowledge, the functional significance of the Wnt antagonist dickkopf homolog 4 (DKK4) has not been investigated previously in renal cancer. The authors initially observed that the expression of DKK4 was significantly higher in renal cancer tissues compared with adjacent normal kidney tissues. To assess the function of DKK4, stable DKK4-transfected cells were established, and functional analyses were performed, including a T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter assay and tests for cell viability, colony formation, apoptosis, cell cycle, invasive capability, wound-healing capability, and in vivo tumor growth. The relative TCF/LEF activity was significantly lower in DKK4-transfected cells compared with empty vector, and nuclear β-catenin expression was decreased in DKK4 transfectants. In addition, expression levels of the β-catenin downstream effector proteins cyclin D1 and c-Myc were decreased in DKK4 transfectants. However, greater invasiveness and migration were observed in stably transfected DKK4 cells. Increased growth of DKK4-transfected tumors also was observed in nude mice. Members of the Wnt noncanonical/c-Jun-NH2 kinase (JNK) signaling pathway also were effected, such as c-Jun, which had significantly increased expression and phosphorylation in DKK4-stable transfectants, and matrix metalloproteinase-2, which had significantly increased expression in DKK4-stable transfectants. This is the first study to indicate that DKK4 expression is increased in renal cancer tissues and that DKK4 activates the noncanonical JNK signaling pathway while inhibiting the Wnt-canonical pathway.