Dickkopf Homolog Research Articles

WTAP and METTL14 regulate the m6A modification of DKK3 in renal tubular epithelial cells of diabetic nephropathy

Diabetic nephropathy (DN) is an important cause of death in diabetes patients, which is mainly due to its complex pathogenesis. Here, we explored the role of N6-methyladenosine (m6A) RNA methylation in DN development. Renal tubular epithelial cells from DN patients and experimental DN mice treated with streptozotocin (STZ) exhibited a considerable increase in METTL14 and WTAP expression as well as overall m6A methylation. Knocking down the expression of METTL14 and WTAP inhibited the migration and proliferation of tubular epithelial cells. MeRIP-seq analysis of the renal tissues of DN patients revealed that the genes with elevated m6A methylation were concentrated in the Wnt/β-Catenin signaling pathway. Dickkopf homolog 3 (DKK3) was screened out as the gene with the most significant increase in m6A methylation. In addition, the expression change pattern of DKK3 under DN circumstances is in line with those of METTL14 and WTAP. DKK3's m6A methylation sites were confirmed to be located in the 3′UTR region, which is how METTL14 and WTAP improved DKK3's mRNA stability. Finally, YTHDF1, a m6A reader, was demonstrated to recognize m6A-methylated DKK3 and promote DKK3 expression.

Genome-wide DNA methylation and transcriptome expression profiles of peripheral blood mononuclear cells in patients with systemic sclerosis with interstitial lung disease.

This study aims to investigate the genome-wide DNA methylation and transcriptome expression profiles of peripheral blood mononuclear cells (PBMCs) in patients with systemic sclerosis (SSc) with interstitial lung disease (ILD), and to analyze the effects of DNA methylation on Wnt/β-catenin and chemokine signaling pathways. PBMCs were collected from 19 patients with SSc (SSc group) and 18 healthy persons (control group). Among SSc patients, there were 10 patients with ILD (SSc with ILD subgroup) and 9 patients without ILD (SSc without ILD subgroup). The genome-wide DNA methylation and gene expression level were analyzed by using Illumina 450K methylation chip and Illumina HT-12 v4.0 gene expression profiling chip. The effect of DNA methylation on Wnt/β-catenin and chemokine signal pathways was investigated. Genome-wide DNA methylation analysis identified 71 hypermethylated CpG sites and 98 hypomethylated CpG sites in the SSc with ILD subgroup compared with the SSc without ILD subgroup. Transcriptome analysis distinguished 164 upregulated genes and 191 downregulated genes in the SSc with ILD subgroup as compared with the SSc without ILD subgroup. In PBMCs of the SSc group, 35 genes in Wnt/β-catenin signaling pathway were hypomethylated, while frizzled-1 (FZD1), mitogen-activated protein kinase 9 (MAPK9), mothers against DPP homolog 2 (SMAD2), transcription factor 7-like 2 (TCF7L2), and wingless-type MMTV integration site family, member 5B (WNT5B) mRNA expressions were upregulated as compared with the control group (all P<0.05). Compared with the SSc without ILD subgroup, the mRNA expressions of dickkopf homolog 2 (DKK2), FZD1, MAPK9 were upregulated in the SSc with ILD subgroup, but the differences were not statistically significant (all P>0.05). In PBMCs of the SSc group, 38 genes in chemokine signaling pathway were hypomethylated, while β-arrestin 1 (ARRB1), C-X-C motif chemokine ligand 10 (CXCL10), C-X-C motif chemokine ligand 16 (CXCL16), FGR, and neutrophil cytosolic factor 1C (NCF1C) mRNA expressions were upregulated as compared with the control group (all P<0.05). Compared with the SSc without ILD subgroup, the mRNA expressions of ARRB1, CXCL10, CXCL16 were upregulated in the SSc with ILD subgroup, but the differences were not statistically significant (all P>0.05). There are differences in DNA methylation and transcriptome profiles between SSc with ILD and SSc without ILD. The expression levels of multiple genes in Wnt/β- catenin and chemokine signaling pathways are upregulated, which might be associatea with the pathogenesis of SSc.

Vitamin D receptor polymorphism and prostate cancer prognosis.

Prostatic epithelial cells synthesize the active form of vitamin D (1,25-dihydroxyvitamin D3), which participates in regulating prostate growth. Calcitriol, a synthetic form of vitamin D3, exhibits antiproliferative and prodifferentiation activities in prostate cancer. The function of 1,25-dihydroxyvitamin D3 is mediated by its binding to vitamin D receptor (VDR). VDR forms a heterodimer, typically with retinoid X receptor, to regulate vitamin D target genes. We evaluated the relationship between VDR polymorphism and clinical characteristics associated with prostate cancer risk and prognosis among Egyptian men. This case-control study included 2 groups of patients: group A, a control group of 50 subjects with benign prostate hyperplasia, and group B, 50 subjects newly diagnosed with prostate cancer. All participants performed complete blood count, liver and kidney function tests, prostate specific antigen measurement, histopathological analysis and immunohistochemistry for Dickkopf Homolog 3. Restriction fragment length polymorphism-polymerase chain reaction as performed to detect VDR polymorphism. Patients with prostate cancer and controls showed a significantly different CA genotype frequency (p = 0.007). Furthermore, prostate-specific antigen levels were significantly different in different genotypes in patients with prostate cancer (p < 0.001). Finally, T stage and the VDR ApaI C/A polymorphism were significantly associated (p < 0.041). The VDR ApaI C/A polymorphism may be a diagnostic and prognostic marker for prostate cancer in Egyptian men.

Dickkopf Homolog 3 (DKK3) as a Prognostic Marker in Lupus Nephritis: A Prospective Monocentric Experience.

Background: The gold standard for diagnosis of lupus nephritis (LN) is still represented by renal biopsy, and serological prognostic biomarkers are still lacking. Dickkopf homolog-3 (DKK3) has been suggested as a marker of tissue fibrosis in different conditions; however, its role in autoimmune diseases needs to be elucidated. Here, we investigated the prognostic role of DKK3 in systemic lupus erythematosus (SLE) patients with and without LN, assessing its changes in relation to kidney function, flares, and interstitial fibrosis. Methods: Overall, 132 SLE patients (57 with LN) were included and prospectively followed up for at least 36 months. DKK3 was measured in serum at baseline. Biopsies were evaluated for glomerular involvement, interstitial fibrosis, and tubular atrophy. Results: Patients with biopsy-proven LN had significantly higher levels of DKK3 than those without (median [min–max]: 215 ng/mL [81–341] vs. 21.1 ng/mL [1–69], p < 0.01). DKK3 levels were associated with prevalent chronic kidney diseases (OR: 4.31 [C.I. 2.01–6.61] per DKK3 doubling, p < 0.01), higher chronicity index at biopsy (1.75 [1.51–2.77] per DKK3 doubling, p < 0.01), and flares rate (OR: 1.45 [C.I. 1.1–5.71] per DKK3 doubling, p < 0.044). Conclusions: While kidney biopsy still represents the gold standard for diagnostic and prognostic assessment in LN, DKK3 could represent an additional prognostic tool to monitor SLE patients and guide therapeutic choices.

Integration of Long Non-Coding RNA and mRNA Profiling Reveals the Mechanisms of Different Dietary NFC/NDF Ratios Induced Rumen Development in Calves.

Simple SummaryAltering carbohydrate source and form affects the development of the rumen epithelium. Calves fed diets with one of three different ratios of non-fibrous carbohydrate to neutral detergent fiber (NFC/NDF) was conducted to reveal putative mechanisms and pathways affected by and responsible for rumen development. Calves in the high NFC/NDF ratio (1.10) group had higher average daily gain, and ruminal papillae developed flatter than calves in the low NFC/NDF ratio (0.60) group. Transcriptomics identified that a large number of differentially expressed genes were significantly enriched in pathways related to rumen epithelia development including focal adhesion, Wnt signaling pathway, thyroid hormone signaling pathway, regulation of actin cytoskeleton and cGMP-PKG signaling pathway. The lncRNA-mRNA network analysis revealed that some target genes including XLOC_068691 and monoamine oxidase B (MOAB), XLOC_023657 and dickkopf homolog 2 (DKK2), XLOC_064331 and protein phosphatase 1 regulatory subunit 12A (PPP1R12A) were affected by the NCF/NDF ratios fed. The pathways and affected genes may serve as markers for the further investigation into the mechanisms regulating dietary and luminal factors impacting rumen development in growing ruminants.The aim of the present study was to explore the effects of dietary non-fibrous carbohydrate to neutral detergent fiber (NFC/NDF) ratios on rumen development of calves, and to investigate the mechanisms by integrating of lncRNA and mRNA profiling. Forty-five weaned Charolais hybrid calves [body weight = 94.38 ± 2.50 kg; age = 70 ± 2.69 d] were randomly assigned to 1 of 3 treatment groups with different dietary NFC/NDF ratios: 1.10 (H group), 0.94 (M group) and 0.60 (L group), respectively. The ventral sac of the rumen was sampled for morphological observation and transcriptional sequencing. The average daily gain of calves in the high NFC/NDF ratio group was significantly higher than that in other groups (p < 0.05). Papillae width was largest in high NFC/NDF ratio group calves (p < 0.05). Identified differentially expressed genes that were significantly enriched in pathways closely related to rumen epithelial development included focal adhesion, Wingless-int signaling pathway, thyroid hormone signaling pathway, regulation of actin cytoskeleton and cGMP-PKG signaling pathway. The lncRNA-mRNA network included XLOC_068691 and MOAB, XLOC_023657 and DKK2, XLOC_064331 and PPP1R12A which we interpret to mean they have important regulatory roles in calve rumen development. These findings will serve as a theoretical basis for further analysis of the molecular genetic mechanism of dietary factors affecting rumen development in calves.

A Review of Specific Biomarkers of Chronic Renal Injury and Their Potential Application in Nonclinical Safety Assessment Studies.

A host of novel renal biomarkers have been developed over the past few decades which have enhanced monitoring of renal disease and drug-induced kidney injury in both preclinical studies and in humans. Since chronic kidney disease (CKD) and acute kidney injury (AKI) share similar underlying mechanisms and the tubulointerstitial compartment has a functional role in the progression of CKD, urinary biomarkers of AKI may provide predictive information in chronic renal disease. Numerous studies have explored whether the recent AKI biomarkers could improve upon the standard clinical biomarkers, estimated glomerular filtration rate (eGFR), and urinary albumin to creatinine ratio, for predicting outcomes in CKD patients. This review is an introduction to alternative assays that can be utilized in chronic (>3 months duration) nonclinical safety studies to provide information on renal dysfunction and to demonstrate specific situations where these assays could be utilized in nonclinical drug development. Novel biomarkers such as symmetrical dimethyl arginine, dickkopf homolog 3, and cystatin C predict chronic renal injury in animals, act as surrogates for GFR, and may predict changes in GFR in patients over time, ultimately providing a bridge from preclinical to clinical renal monitoring.

Prospective phase II trial of levonorgestrel intrauterine device: nonsurgical approach for complex atypical hyperplasia and early-stage endometrial cancer

The incidence of complex atypical hyperplasia and early-stage endometrioid endometrial cancer is increasing, in part owing to the epidemic of obesity, which is a risk factor tightly linked to the development of endometrial hyperplasia and cancer. The standard upfront treatment for complex atypical hyperplasia and early-stage endometrial cancer is hysterectomy. However, nonsurgical treatment of early-stage endometrial neoplasia may be necessary owing to medical comorbidities precluding surgery or desired future fertility. This study aimed to evaluate the efficacy of the levonorgestrel intrauterine device to treat complex atypical hyperplasia and grade 1 endometrioid endometrial carcinoma. A single-institution, single-arm, phase II study of the levonorgestrel intrauterine device (52 mg levonorgestrel, Mirena) was conducted in patients with complex atypical hyperplasia or grade 1 endometrioid endometrial cancer. The primary endpoint was pathologic response rate at 12 months, including complete or partial response. Quality of life and toxicity were assessed. Molecular analyses for proliferation markers, hormone-regulated genes, and wingless-related integration site pathway activation were performed at baseline and 3 months. A total of 57 patients were treated (21 endometrial cancer, 36 complex atypical hyperplasia). The median age was 48.0 years, and the median body mass index was 45.5 kg/m2. Of the 47 evaluable patients, 12-month response rate was 83% (90% credible interval, 72.7-90.3)-37 were complete responders (8 endometrial cancer; 29 complex atypical hyperplasia), 2 were partial responders (2 endometrial cancer), 3 had stable disease (2 endometrial cancer; 1 complex atypical hyperplasia), and 5 had progressive disease (3 endometrial cancer; 2 complex atypical hyperplasia). After stratification for histology, the response rate was 90.6% for complex atypical hyperplasia and 66.7% for grade 1 endometrioid endometrial cancer. Notably, 4 patients (9.5%) experienced relapse after the initial response. Adverse events were mild, primarily irregular bleeding and cramping. Quality of life was not negatively affected. At 3 months, exogenous progesterone effect was present in 96.9% of responders (31 of 32) vs 25% of nonresponders (2 of 8) (P=.001). Nonresponders had higher baseline proliferation (Ki67) and lower dickkopf homolog 3 gene expression than responders (P=.023 and P=.030). Nonresponders had significantly different changes in secreted frizzled-related protein 1, frizzled class receptor 8, and retinaldehyde dehydrogenase 2 compared with responders. The levonorgestrel intrauterine device has a substantial activity in complex atypical hyperplasia and grade 1 endometrioid endometrial cancer, with a modest proportion demonstrating upfront progesterone resistance. Potential biomarkers were identified that may correlate with resistance to therapy; further exploration is warranted.

Dickkopf Homolog 3 (DKK3) Acts as a Potential Tumor Suppressor in Gallbladder Cancer.

Gallbladder cancer (GBC) is a common malignancy of biliary tract cancers and its incidence has been rising rapidly worldwide. The prognosis for this disease is dismal as most of the symptoms are non-specific leading to a definitive diagnosis only at a late stage. Loss of DKK3 gene is associated with a possible tumor suppressor role in human cancers. The role and regulation of DKK3 in GBC have not been studied. We found that DKK3 expression levels were low in GBC patients and cell lines. Treatment of GBC cell lines with demethylating agent 5-Aza- 2'-deoxycytidine enhances its expression, establishing impact of methylation on DKK3 expression. We observed low expression of DKK3 in gallbladder adenocarcinoma tumors and highly invasive GBC cell lines. We showed that overexpression of DKK3 can decrease cell invasion, proliferation, and colony forming ability of GBC cells. Our data thus demonstrated the DKK3 gene is a potential tumor suppressor gene in GBC and aberrant promoter methylation could be involved in its downregulation, which may play a role in the tumorigenesis and aggressiveness of GBC.

The effect of lithium chloride on the attenuation of cognitive impairment in experimental hypoglycemic rats

BackgroundHypoglycemia is the most common complication in the treatment of diabetes mellitus. Accumulating evidence indicated that severe hypoglycemia could induce cognitive impairment. However, the molecular mechanism of regulating this progress is largely unknown. MethodsWe established a model of insulin-induced recurrent hypoglycemia in adult male Wistar rats (n = 40). Lithium chloride was injected after hypoglycemia once a day for consecutive 30 days. The loss of cognition function was evaluated by water maze test in these hypoglycemic rats. Glial cells activation and Wnt and inflammatory cytokines IL-1β, IL-6, IL-4, IL-10, TGFβ and TNFα expression were further examined to determine the mechanism of cognitive function impairment. ResultsHypoglycemia could induce impairment of cognitive function in rats and administration of lithium chloride could partly attenuate cognitive impairment compared to the control (p < 0.05). Lithium chloride could significantly up-regulate Wnt signaling and reduce hypoglycemia-induced neuronal death, glial cells activation and inflammatory response in the hippocampus of rats compared to the control (p < 0.05). The efficacy of lithium chloride could be reversed by injecting canonical Wnt signaling antagonist the dickkopf homolog 1. ConclusionLithium chloride attenuated hypoglycemia-induced cognitive function impairment in rats; and it was associated with Wnt signaling up-regulation and reduction of inflammatory response. Our results suggested that activating Wnt signaling pathways and inhibiting inflammatory response were the therapeutic potential to prevent hypoglycemia-induced neurological damage.

Induction of Lrp5 HBM-causing mutations in Cathepsin-K expressing cells alters bone metabolism.

High-bone-mass (HBM)-causing missense mutations in the low density lipoprotein receptor-related protein-5 (Lrp5) are associated with increased osteoanabolic action and protection from disuse- and ovariectomy-induced osteopenia. These mutations (e.g., A214V and G171V) confer resistance to endogenous secreted Lrp5/6 inhibitors, such as sclerostin (SOST) and Dickkopf homolog-1 (DKK1). Cells in the osteoblast lineage are responsive to canonical Wnt stimulation, but recent work has indicated that osteoclasts exhibit both indirect and direct responsiveness to canonical Wnt. Whether Lrp5-HBM receptors, expressed in osteoclasts, might alter osteoclast differentiation, activity, and consequent net bone balance in the skeleton, is not known. To address this, we bred mice harboring heterozygous Lrp5 HBM-causing conditional knock-in alleles to Ctsk-Cre transgenic mice and studied the phenotype using DXA, μCT, histomorphometry, serum assays, and primary cell culture. Mice with HBM alleles induced in Ctsk-expressing cells (TG) exhibited higher bone mass and architectural properties compared to non-transgenic (NTG) counterparts. In vivo and in vitro measurements of osteoclast activity, population density, and differentiation yielded significant reductions in osteoclast-related parameters in female but not male TG mice. Droplet digital PCR performed on osteocyte enriched cortical bone tubes from TG and NTG mice revealed that ~8-17% of the osteocyte population (depending on sex) underwent recombination of the conditional Lrp5 allele in the presence of Ctsk-Cre. Further, bone formation parameters in the midshaft femur cortex show a small but significant increase in anabolic action on the endocortical but not periosteal surface. These findings suggest that Wnt/Lrp5 signaling in osteoclasts affects osteoclastogenesis and activity in female mice, but also that some of the changes in bone mass in TG mice might be due to Cre expression in the osteocyte population.

DKK1 and Kremen Expression Predicts the Osteoblastic Response to Bone Metastasis

Bone metastasis is a complication of advanced breast and prostate cancer. Tumor-secreted Dickkopf homolog 1 (DKK1), an inhibitor of canonical Wnt signaling and osteoblast differentiation, was proposed to regulate the osteoblastic response to metastatic cancer in bone. The objectives of this study were to compare DKK1 expression with the in vivo osteoblastic response in a panel of breast and prostate cancer cell lines, and to discover mechanisms that regulate cancer DKK1 expression. DKK1 expression was highest in MDA-MB-231 and PC3 cells that produce osteolytic lesions, and hence a suppressed osteoblastic response, in animal models of bone metastasis. LnCaP, C4-2B, LuCaP23.1, T47D, ZR-75-1, MCF-7, ARCaP and ARCaPM cancer cells that generate osteoblastic, mixed or no bone lesions had the lowest DKK1 expression. The cell lines with negligible expression, LnCaP, C4-2B and T47D, exhibited methylation of the DKK1 promoter. Canonical Wnt signaling activity was then determined and found in all cell lines tested, even in the MDA-MB-231 and PC3 cell lines despite sizeable amounts of DKK1 protein expression expected to block canonical Wnt signaling. A mechanism of DKK1 resistance in the osteolytic cell lines was investigated and determined to be at least partially due to down-regulation of the DKK1 receptors Kremen1 and Kremen2 in the MDA-MB-231 and PC3 cell lines. Combined DKK1 and Kremen expression in cancer cells may serve as predictive markers of the osteoblastic response of breast and prostate cancer bone metastasis.

SATB1 downregulation induced by oxidative stress participates in trophoblast invasion by regulating β-catenin.

Preeclampsia (PE) is characterized by abnormal placentation in the early stages of pregnancy. Adequate migration and invasion of trophoblasts into the uterine wall and spiral arteries to form a functional maternal-fetal interface are pivotal for normal placentation, but the exact mechanism remains unclear. Growing evidence has revealed that special AT-rich sequence binding protein 1 (binds to nuclear matrix/scaffold-associating DNA) (SATB1) is a tumor promoter that participates in cancer cell migration and invasion. However, the expression and function of SATB1 in trophoblasts is unknown. Here, we characterize the stimulatory effect of SATB1 on the migration and invasion of trophoblasts and identify the regulatory events and downstream signaling components. Downregulated SATB1 was detected in PE placentae and villous explants cultured under hypoxia/reoxygenation (H/R) conditions. H/R-treated trophoblasts with lower SATB1 levels exhibited weaker invasive and growth capacities, whereas upregulation of the SATB1 level with recombinant SATB1 restored these impairments. This restoration was especially apparent with the sumoylation-deficient SATB1 variant, which contained a mutated site that blocked sumoylation. Moreover, the elevated concentration of SATB1 also increased the expression of β-catenin, which is involved in human placental trophoblast invasion and differentiation is downregulated in PE. However, a specific activator, namely, lithium chloride (LiCl), increased β-catenin expression but had no evident influence on SATB1 expression. Furthermore, upregulated SATB1 failed to restore trophoblast function when Wnt/β-catenin was suppressed by dickkopf (Xenopus laevis) homolog 1, dickkopf 1 homolog (Xenopus laevis) (DKK1). Together, these data show that SATB1expression in the human placenta is affected by oxidative stress and might regulate the migration and invasion of trophoblasts via β-catenin signaling.

The therapeutic effects of Yougui pill on knee osteoarthritis and the expression of Wnt signal pathway related factors in rats

To observe the effects of Yougui pill (Traditional Chinese Medicine) on the related factors of Wnt signal pathway of rats with knee osteoarthritis (KOA), and explore its protective mechanism. Sixty SPF SD rats were randomly divided into the sham-operative group, model group, glucosamine sulfate group, high-dose, middle-dose, low-dose of Yougui pill treated group (n=10). KOA model was established by modified Hulth method for six weeks. The rats in the high, middle and low-dose of Yougui pill group were treated with Yougui pills at the doses of 20,10 and 5 g/kg respectively by gastrogavage once a day for 8 weeks, while equal volume of normal saline was given to those in the sham and model control group and an equal volume of glucosamine sulfate (1.7 g/kg·d) was given to those in glucosamine sulfate group for 8 weeks. The knee joint was removed after the last dose of drug. The pathological changes of cartilaginous tissues were observed under a microscope. The mRNA levels of Dickkopf homolog 1(DKK1), Wnt induced secreted protein 1(WISP1), Wnt1, low density lipoprotein receptor related protein 5(LRP5) and beta -catenin in rats cartilaginous tissues were analyzed by using RT-PCR method, and the protein contents of DKK1, WISP1, Wnt1, LRP5 and beta-catenin in cartilaginous tissues were detected by Western blot. Compared with the sham group, the articular cartilage was severely damaged, the Mankin score was increased significantly (P<0. 05), the mRNA and protein expression levels of DKK1 in cartilaginous tissue were markedly decreased(P<0.05), while those of WISP, Wnt1, LRP5 and beta-catenin were increased significantly in model group(P<0.05). Compared with model group, the articular cartilage lesions was light (P<0.05), the Mankin Score was decreased significantly(P<0.05), and the mRNA and protein levels of DKK1 in cartilaginous tissue were increased(P<0.05), while those of WISP, Wnt1, LRP5 and beta-catenin were decreased in Yougui pill high-dose group and glucosamine sulfate group (P<0.05). Yougui pill has protective effects on the KOA by inhibiting the expressions of WISP, Wnt1, LRP5, beta-catenin and increasing the expression of DKK1 cytokine in the Wnt signaling pathway.

Dickkopf homolog 3 (DKK3): A candidate for detection and treatment of cancers?

Wnt signaling is an evolutionary highly conserved pathway that is modulated by several inhibitors and activators, and plays a key role in numerous physiological processes. One of the extracellular Wnt inhibitors is the DKK (Dickkopf Homolog) family which has four members (Dkk1-4) and a unique Dkk3-related gene, Dkkl1 (soggy). DKK3 is a divergent member of the DKK protein family. Evidence suggests that DKK3 may serve as a potential therapeutic target in several types of human cancers. We review here the biological role of DKK3 as a tumor suppressor gene (TSG) or oncogene, and its correlation with various miRNAs. In addition, we discuss the role of polymorphisms and promoter methylation of the DKK3 gene, and of its expression in regulating cancer cell proliferation. Finally, we propose that DKK3 may be considered as both a biomarker and a therapeutic target in different cancers.

Combined activity of temozolomide and the mTOR inhibitor temsirolimus in metastatic melanoma involves DKK1.

The BRAFV600E inhibitor vemurafenib achieves remarkable clinical responses in patients with BRAF-mutant melanoma, but its effects are limited by the onset of drug resistance. In the case of resistance, chemotherapy can still be applied as second line therapy. However, it yields low response rates and strategies are urgently needed to potentiate its effects. In a previous study, we showed that the inhibition of the PI3K-AKT-mTOR pathway significantly increases sensitivity of melanoma cells to chemotherapeutic drugs (J. Invest. Dermatol. 2009, 129, 1500). In this study, the combination of the mTOR inhibitor temsirolimus with the chemotherapeutic agent temozolomide significantly increases growth inhibition and apoptosis in melanoma cells compared to temsirolimus or temozolomide alone. The combination of temozolomide with temsirolimus is not only effective in established but also in newly isolated and vemurafenib-resistant metastatic melanoma cell lines. These effects are associated with the downregulation of the anti-apoptotic protein Mcl-1 and the upregulation of the Wnt antagonist Dickkopf homologue 1 (DKK1). Knock-down of DKK1 suppresses apoptosis induction by the combination of temsirolimus and temozolomide. These data suggest that the inhibition of the mTOR pathway increases sensitivity of melanoma cells towards temozolomide. Chemosensitisation is associated with enhanced expression of the Wnt antagonist DKK1.

Bone morphogenetic proteins and Dickkopf-1 in ankylosing spondylitis

Objectives: To determine bone morphogenetic proteins (BMPs) and Dickkopf homologue-1 (Dkk-1) levels in ankylosing spondylitis (AS).Method: Serum BMPs and Dkk-1 were measured in 72 AS patients and 30 healthy controls. For AS patients, we recorded the demographic data, disease activity, functional index, and global assessment with questionnaires, and image changes with roentgenography. We also measured human leucocyte antigen-B27 and systemic inflammatory reactants.Results: BMPs were higher but Dkk-1 was significantly lower in AS patients than in controls. Dkk-1 was higher in AS patients who received non-steroidal anti-inflammatory drugs (NSAIDs) regularly in the past year (p = 0.001). Serum BMP-7 level and the BMP-7/Dkk-1 ratio correlated significantly with sacroiliitis severity, Bath Ankylosing Spondylitis Radiology Index (BASRI)-total, modified Stoke Ankylosing Spondylitis Spinal Score, and disease duration. There were also significant positive correlations among serum levels of BMP-2, -4, and -6, BASRI-total, and disease duration (p < 0.05). However, BMP-2/Dkk-1 was only significantly correlated with disease duration. The calculated area under the standard receiver operating characteristics curve suggested that BMP-2/Dkk-1 and serum BMP-2 are good indicators to predict disease activity, functional index, and patient global assessment in AS patients.Conclusion: BMPs and BMPs/Dkk-1 were significantly correlated with disease activity, and radiological and functional indices in AS patients. Dkk-1 was lower in AS patients than in controls. Among AS patients, Dkk-1 was higher in those taking NSAIDs regularly. BMP or Dkk-1 may be taken as a biomarker for disease severity or a treatment outcome predictor in AS, but this needs further study.

The Involvement ofβ-Catenin/COX-2/VEGF Axis in NMDA-Caused Retinopathy

NMDA, a molecule that is capable of producing the loss of retinal ganglia cells (RGCs), has been widely studied; however, the detailed mechanism is not yet clarified. Previously, Wnt/β-catenin signaling has been suggested to be involved in the NMDA-induced retinopathy. In addition, previous investigations in our group demonstrated the presence of a Wnt/β-catenin/COX-2 axis in dorsal root ganglions (DRGs). Therefore, here in this paper, we tested whether there is an association of such axis with NMDA-induced RGC loss. Rat retinal damage models generated by intravitreal injection of NMDA were used to measure the expression levels of β-catenin, COX-2, and VEGF in retinas, and the neuron numbers of the retinal GCL of rats were counted. Then, pharmacological tools (MK801, a NMDA receptor inhibitor; Dickkopf homolog 1, a specific inhibitor of the Wnt pathway; NS-398, a COX-2 inhibitor; and bevacizumab, IVB, a VEGF inhibitor) were introduced to evaluate the detailed roles of Wnt/β-catenin, COX-2, and VEGF in retinopathy of rats. Results demonstrated that all three factors in sequence are positively regulated neuronal loss induced by NMDA. These observations indicated that the Wnt pathway/COX-2/VEGF axis plays a pathogenic role in retinopathy and represented novel therapeutic targets.

R-Spondin 1 Regulates WNT Signaling Pathway by Antagonizing the Function of Dickkopf Homolog 1

The WNT family of secreted proteins play important roles in diverse biological processes, including development, proliferation, and differentiation, which regulate various diseases, such as prostate cancer, breast cancer, glioblastoma, type II diabetes and others. The WNT ligands could activate two major intracellular pathways: known as the canonical pathway which is β-catenin-dependent, and non-canonical pathway which is β-catenin-independent.

Interferon-Alpha Promotes Th1 Response and Epithelial Apoptosis via Inflammasome Activation in Human Intestinal Mucosa.

Backgound & AimsSeveral lines of investigation suggest that interferon (IFN) alpha can alter human intestinal mucosa homeostasis. These include the endogenous production of IFN alpha in celiac disease or inflammatory bowel diseases, as well as the occurrence of intestinal side effects of exogenous IFN alpha used as a therapeutic tool. Here, we present an ex vivo translational approach to investigate the effects of IFN alpha on the human normal intestinal mucosa, as well as its underlying mechanisms.MethodsHuman normal colonic mucosa explants were cultured in the presence or absence of IFN alpha 2a. Epithelial homeostasis was assessed using the immunohistochemical marker of apoptosis M30. The Wnt inhibitor Dickkopf-Homolog-1 (DKK1) was assayed in the supernatants by enzyme-linked immunosorbent assay. Activation of the inflammasome (caspase-1/interleukin [IL]18) and of a Th1 response was determined by in situ detection of active caspase-1, as well as by measurement of mature IL18 production and the prototype Th1 cytokine IFN gamma by enzyme-linked immunosorbent assay. In addition, mechanistic studies were performed using the specific caspase-1 inhibitor Tyr-Val-Ala-Asp(OMe)-fluoromethylketone (YVAD-FMK), IL18-binding protein, neutralizing anti–IFN gamma, and anti-DKK1 antibodies.ResultsIFN alpha 2a elicited a rapid (24 hours) disruption of surface and crypt colonic epithelial cells via apoptosis that was variable in intensity among the 20 individuals studied. This apoptotic effect was dependent on the initiation of an IFN gamma response elicited by resident T box expressed in T cells–positive lamina propria cells. Both apoptosis and Th1 response were subordinated to active caspase-1 and IL18 production. Finally, neutralization of IFN gamma–induced DKK1 partially protected against IFN alpha–induced epithelial apoptosis.ConclusionsBy using an ex vivo model, we show an interindividual heterogeneity of IFN alpha effects. We show that IFN alpha is able to disrupt both epithelial and immune homeostasis in the human intestine, by activation of an innate immunity platform, the inflammasome, which drives a Th1 response and leads to epithelial barrier disruption.

Pathogenesis of bone metastasis

Abstract Bone metastases are more frequently seen as a complication of cancer than primary bone tumors. For example, it can be seen in as many as 70% of advanced stage breast and prostate cancer cases. Metastatic bone disease is generally categorized as osteoblastic, and osteolytic disease. However most of the cancer types demonstrate a wide spectrum between these two extremes. Paracrine interaction between parathyroid hormone–related protein (PTHrP) which increases the rate of bone osteolysis, and transforming growth factor-β (TGF-β) plays a role in osteolytic metastasis. Increased local bone PTHrP concentration increases expression of receptor activator of nuclear factor kappa-B ligand (RANKL) with resultant activation of osteoclastogenesis. Endothelin – 1 (ET-1), and dickkopf homolog -1 (DKK-1) produced by tumor involve in osteoblastic metastasis. DKK-1 is the central regulator of osteoblastic activity, and osteoblastic bone metastasis. For the elaboration of treatment strategies against frequently seen complication, that is, bone metastases, targets involving in pathogenesis of these complications should be taken into consideration.