Frizzled-1 Research Articles

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.

GPER Agonist G1 Prevents Wnt-Induced JUN Upregulation in HT29 Colorectal Cancer Cells.

Women consistently show lower incidence and mortality rates for colorectal cancer (CRC) compared to men. Epidemiological evidence supports a pivotal role for estrogen in protecting women against CRC. Estrogen protective effects in CRC have been mainly attributed to the estrogen receptor beta (ERβ) however its expression is lost during CRC progression. The role of the G-protein coupled membrane estrogen receptor (GPER/GPER1/GPR30), which remains expressed after ERβ loss in CRC, is currently under debate. We hypothesise that estrogen can protect against CRC progression via GPER by modulating the Wnt/β-catenin proliferative pathway which is commonly hyperactivated in CRC. We sought evidence of sexual dimorphism within the Wnt/β-catenin pathway by conducting Kaplan–Meier analyses based on gene expression of the Wnt receptor FZD1 (Frizzled 1) in multiple public domain CRC patient data sets. High expression of FZD1 was associated with poor relapse-free survival rates in the male but not the female population. In female-derived HT29 CRC cell lines, we show that β-catenin nuclear translocation was not affected by treatment with the GPER agonist G1. However, G1 prevented the Wnt pathway-induced upregulation of the JUN oncogene. These novel findings indicate a mechanistic role for GPER in protecting against CRC progression by selectively reducing the tumorigenic effects of hyperactive Wnt/β-catenin signalling pathways in CRC.

Protein disulfide isomerase family 6 promotes the imatinib-resistance of renal cell carcinoma by regulation of Wnt3a-Frizzled1 axis

ABSTRACT Imatinib is a nontoxic tyrosine kinase inhibitor, used in the treatment of advanced renal cell carcinoma. However, some patients with renal cell carcinoma develop resistance to imatinib. Protein disulfide isomerase family 6 (PDIA6) was involved in the chemo-resistance of lung adenocarcinoma. In this study, the effect of PDIA6 on imatinib-resistance of renal cell carcinoma was investigated. First, PDIA6 was found to be up-regulated in the imatinib-resistant renal cell carcinoma tissues and cells. Functional assays showed that knockdown of PDIA6 sensitized imatinib-resistant renal cell carcinoma cells to imatinib through decreasing the half-maximal inhibitory concentration (IC50) of imatinib-resistant renal cell carcinoma cells. Secondly, cell proliferation of imatinib-resistant renal cell carcinoma cells was suppressed by PDIA6 silencing, and the apoptosis was promoted with reduced Bcl-2, enhanced Bax and cleaved caspase-3. Moreover, the interference of PDIA6 increased phosphorylation of H2A histone family member X (γH2AX), while decreased Rad51 and phosphorylated DNA-dependent protein kinase (DNA-PK) (p-DNA-PK) in imatinib-resistant renal cell carcinoma cells. Lastly, protein expression levels of Wnt3a and Frizzled1 (FZD1) in imatinib-resistant renal cell carcinoma cells were down-regulated by silencing of PDIA6. Over-expression of FZD1 attenuated PDIA6 silencing-induced increase in cell apoptosis and decrease in cell proliferation in imatinib-resistant renal cell carcinoma cells. In conclusion, knockdown of PDIA6 sensitized imatinib-resistant renal cell carcinoma cells into imatinib through inactivation of Wnt3a-FZD1 axis.

LncRNA VIM‑AS1 promotes cell proliferation, metastasis and epithelial‑mesenchymal transition by activating the Wnt/β‑catenin pathway in gastric cancer.

The present study aimed to explore the biological functions and molecular mechanisms of the long non-coding RNA VIM antisense RNA 1 (VIM-AS1) in gastric cancer (GC). The expression of VIM-AS1 was analyzed in tissues from patients with GC and GC cell lines by reverse transcription-quantitative (RT-q)PCR. The relationship between VIM-AS1 expression and overall survival time of patients with GC was also assessed. To determine the biological functions of VIM-AS1, Cell Counting Kit-8 assay, colony formation assay, flow cytometry, wound healing assay and Transwell assay were employed. The targeting relationship among VIM-AS1, microRNA (miR)-8052 and frizzled 1 (FZD1) was verified by the dual luciferase reporter gene assay. The underlying molecular mechanism of VIM-AS1 on GC was determined by RT-qPCR and western blotting. In addition, tumor formation was detected in nude mice. The results of the present study demonstrated that VIM-AS1 was highly expressed in GC tissues and cells. In addition, VIM-AS1 expression was demonstrated to be closely related to the prognosis of patients with GC. Notably, silencing VIM-AS1 inhibited the proliferation, migration and invasion, and enhanced apoptosis of AGS and HGC-27 cells. Silencing VIM-AS1 significantly increased the protein expression levels of cleaved caspase-3, Bax and E-cadherin, but decreased the protein expression levels of Bcl-2, N-cadherin, vimentin, matrix metalloproteinase (MMP)-2, MMP-9, β-catenin, cyclin D1, C-myc and FZD1. Additionally, silencing VIM-AS1 inhibited tumor growth in nude mice. Cumulatively, the present study demonstrated that VIM-AS1 may promote cell proliferation, migration, invasion and epithelial-mesenchymal transition by regulating FDZ1 and activating the Wnt/β-catenin pathway in GC.

Abstract P139: Association of a Potentially Functional Wnt Pathway Mutation With Carotid Artery Wall Traits in African Ancestry Men

The wingless (Wnt) pathway is known to regulate many human systems, including embryonic development, bone mineralization, and cancer. There is emerging basic research evidence that the Wnt pathway is also involved in angiogenesis, atherosclerosis, and vascular damage repair. However, there have been no studies of the Wnt pathway and cardiovascular disease (CVD) in human populations. In a cohort of Afro-Caribbean men (the Tobago Health Study (THS)), we previously identified a potentially functional coding mutation (Ala64Thr) in the Frizzled-1 ( FZD1 ) gene, which acts as a co-receptor with the low-density lipoprotein receptor protein (LRP) to initiate the Wnt signaling cascade. This mutation has a carrier frequency of ~4% in the THS and additional studies reported in the 1000 genomes project have found similar frequencies in other predominantly West African populations. In order to investigate the phenotypic consequences of this Wnt pathway mutation on subclinical CVD, we measured carotid artery wall traits using B-mode ultrasonography in 64 Ala64Thr mutation carriers and 395 controls from the THS. For this analysis, carotid artery measures of interest included carotid plaque prevalence, and common carotid mean intima-media thickness (IMT), inter-adventitial diameter (IAD), and lumen diameter (LD). We assessed differences in carotid traits by Ala64Thr status using multiple linear regression adjusting for CVD risk factors, including age, BMI, smoking, hypertension, diabetes, alcohol intake, physical activity, and sedentary behavior. Men were aged 64 years and were overweight (BMI: 27.5kg/m 2 ), on average. Prevalence of hypertension and type 2 diabetes is high in this sample (66% and 24%, respectively). Men with Ala64Thr tended to be older and had higher prevalence of hypertension than non-carriers (P&lt;0.05 for both). Carrying the Ala64Thr mutation was associated with significantly wider carotid diameters and, after adjustment for traditional CVD risk factors, mutation carriers had &gt;1/3 of a SD greater LD than non-carriers (P=0.01). The effect magnitude of this mutation was similar for IAD, though not statistically significant (P=0.08). There was no significant difference in carotid IMT or plaque prevalence. While this mutation is not expected to be severely deleterious using in silico prediction software, Ala64 is highly conserved and located in a putative signaling peptide sequence. Carriers of a low frequency FZD1 coding mutation show greater remodeling of the carotid artery compared to non-carriers, suggesting that the Wnt pathway may be involved in vascular changes that occur early in the atherosclerotic disease process.

Knockdown of the Wnt receptor Frizzled-1 (FZD1) reduces MDR1/P-glycoprotein expression in multidrug resistant leukemic cells and inhibits leukemic cell proliferation

Multidrug resistance (MDR) is a major obstacle to leukemia treatment. The Frizzled-1 (FZD1) Wnt receptor is involved in MDR in some solid cancers, but has rarely been reported to act in acute myeloid leukemia (AML). We investigated whether the knockdown of FZD1 affects MDR1 expression and P-glycoprotein (P-gp) function in multidrug resistant leukemic cell lines, as well as FZD1 and MDR1/P-gp expression in leukemic cells taken from patients with AML (n = 112). FZD1 knockdown significantly reduced MDR1 expression through the Wnt/β-catenin pathway, disrupted the P-gp efflux function, induced the recovery of sensitivity to chemotherapeutic agents, and hindered cell proliferation in cell lines. FZD1 expression in leukemic cells was significantly higher in patients experiencing relapse (n = 34) than in those with no relapse (n = 44, P = .003). Leukemic cells unable to achieve complete response (CR) showed an increased expression of MDR1 and P-gp, compared to patients who achieved CR. Obtaining CR in patients with higher FZD1 expression at diagnosis is difficult. Moreover, they tend to present instances of relapse, suggesting that AML cells with increased FZD1 expression are resistant to chemotherapy. We conclude that the activated FZD1 observed in leukemic cells likely confers acquired drug resistance, whereas FZD1 silencing may be more effective in reversing MDR.

Recombinant frizzled1 protein attenuated cardiac hypertrophy after myocardial infarction via the canonical Wnt signaling pathway.

Postinfarct cardiac hypertrophy is an independent risk factor for heart failure and sudden death. Regression of cardiac hypertrophy has emerged as a promising strategy in the treatment of myocardial infarction (MI). Here we hypothesized that frizzled1 (FZD1), a receptor of the canonical Wnt signaling pathway, is a novel mediator of ischemia-associated cardiac hypertrophy. MI was induced in mice by left anterior descending (LAD) coronary occlusion. One week after MI, the expression of FZD1 was found to be notably increased in the left ventricles (LVs) of the MI-mice compared to shams. Mouse recombinant FZD1 protein (RFP) was subcutaneously injected in the mice to provoke autoimmunization response. Anti-FZD1 antibody titer was significantly increased in the plasma of RFP-treated mice. RFP significantly mitigated the MI-induced cardiac hypertrophy and improved cardiac function in the MI mouse hearts. Moreover, increased heart and LV weights, myocardial size and the expression of β-myosin heavy chain in the MI-mice were also found to be attenuated by RFP. FZD1 was found to be significantly up-regulated in hypoxia-treated neonatal rat cardiomyocytes (NRCMs). Silencing FZD1 by siRNA transfection notably repressed the hypoxia-induced myocardial hypertrophy in NRCMs. Mechanistically, activation of canonical Wnt signaling induced by MI, e.g., β-catenin and glycogen synthase kinase-3β, was restrained in the LVs of the MI-mice treated by RFP, these inhibition on canonical Wnt signaling was further confirmed in hypoxic NRCMs transfected with FZD1 siRNA. In conclusion, immunization of RFP attenuated cardiac hypertrophy and improved cardiac function in the MI mice via blocking the canonical Wnt signaling pathway.

MiR-135b reverses chemoresistance of non-small cell lung cancer cells by downregulation of FZD1.

Non-small cell lung cancer (NSCLC) chemoresistance usually limits the clinical efficacy of chemotherapeutic approaches. However, few reports have revealed the regulation of miR-135b and Frizzled-1 (FZD1) involved in NSCLC chemoresistance. To identify the mechanism of miR-135b and FZD1 in NSCLC chemoresistance and to observe their biological functions, we detected the expression levels of miR-135b and FZD1 by conducting quantitative real-time polymerase chain reaction (RT-qPCR) and modified the expressions of miR-135b and FZD1 by transiently transfecting cells with miR-135b mimics or FZD1-siRNA. The 3'-untranslated region (3'-UTR) of FZD1 combined with miR-135b was verified through dual-luciferase reporter assay. Compared with that in A549 parental cell lines, the miR-135b expression in drug-resistant lung cancer cell lines (A549/DDP) was decreased and their FZD1 expression was increased. The increased miR-135b expression and silenced FZD1 expression enhanced the sensitivity of resistant cells to cisplatin treatment. The high expression of miR-135b in A549/DDP cells remarkably decreased the mRNA levels of FZD1. FZD1 was further identified as the functional downstream target of miR-135b by directly targeting the 3'-UTR of FZD1. The amplification of miR-135b suppressed NSCLC chemoresistance by directly mediating the FZD1 downregulation.

Frizzled-1 receptor regulates adult hippocampal neurogenesis.

BackgroundIn the adult hippocampus new neurons are continuously generated from neural stem cells (NSCs) present at the subgranular zone of the dentate gyrus. This process is controlled by Wnt signaling, which plays a complex role in regulating multiple steps of neurogenesis including maintenance, proliferation and differentiation of progenitor cells and the development of newborn neurons. Differential effects of Wnt signaling during progression of neurogenesis could be mediated by cell-type specific expression of Wnt receptors. Here we studied the potential role of Frizzled-1 (FZD1) receptor in adult hippocampal neurogenesis.ResultsIn the adult dentate gyrus, we determined that FZD1 is highly expressed in NSCs, neural progenitors and immature neurons. Accordingly, FZD1 is expressed in cultured adult hippocampal progenitors isolated from mouse brain. To evaluate the role of this receptor in vivo we targeted FZD1 in newborn cells using retroviral-mediated RNA interference. FZD1 knockdown resulted in a marked decrease in the differentiation of newborn cells into neurons and increased the generation of astrocytes, suggesting a regulatory role for the receptor in cell fate commitment. In addition, FZD1 knockdown induced an extended migration of adult-born neurons within the granule cell layer. However, no differences were observed in total dendritic length and dendritic arbor complexity between control and FZD1-deficient newborn neurons.ConclusionsOur results show that FZD1 regulates specific stages of adult hippocampal neurogenesis, being required for neuronal differentiation and positioning of newborn neurons into the granule cell layer, but not for morphological development of adult-born granule neurons.

A Role for the Non-Canonical Wnt-β-Catenin and TGF-β Signaling Pathways in the Induction of Tolerance during the Establishment of a Salmonella enterica Serovar Enteritidis Persistent Cecal Infection in Chickens.

Non-typhoidal Salmonella enterica induce an early pro-inflammatory response in chickens. However, the response is short-lived, asymptomatic of disease, resulting in a persistent colonization of the ceca, and fecal shedding of bacteria. The underlying mechanisms that control this persistent infection of chickens by Salmonella are unknown. Recently, we found an expansion of the Treg population and subsequent increased in vitro immunosuppressive functions of the CD4+CD25+ cells isolated from the ceca of the Salmonella-infected chickens by day 4 post-infection that increased steadily throughout the course of the 14 days of infection, whereas the number of CD4+CD25+ cells in the non-infected controls remained steady throughout the study. CD4+CD25+ cells from cecal tonsils of S. enteritidis-infected birds had greater expression of IL-10 mRNA content than the CD4+CD25+ cells from the non-infected controls at all the time points studied. These results suggest the development of a tolerogenic immune response in the cecum of Salmonella-infected chickens may contribute to the persistance of Salmonella cecal colonization. Using a chicken-specific kinome peptide immune array, we have analyzed the signaling pathways altered during the establishment of this tolerogenic state. This analysis has revealed a role for the non-canonical Wnt signaling pathway in the cecum at 4 days post-infection. Infection induced the significant (p < 0.01) phosphorylation of the G-protein-coupled transmembrane protein, Frizzled 1 (FZD1), resulting in an influx of intracellular Ca2+ and the phosphorylation of the Ca2+-dependent effector molecules calcium/calmodulin-dependent kinase II (CamKII), β-catenin, protein kinase C, and the activation of the transcription factor, NFAT. Nuclear translocation of NFAT resulted in a significant increase in the expression of the anti-inflammatory cytokines IL-10 and TGF-β. Increased expression of TGF-β4 mRNA activates the TGF-β signaling pathway that phosphorylates the receptor-activated Smads, Smad2 and Smad3. Combined with the results from our Treg studies, these studies describe kinome-based phenotypic changes in the cecum of chickens during Salmonella Enteritidis infection starting 4 days post-infection that leads to an anti-inflammatory, tolerogenic local environment, and results in the establishment of persistent intestinal colonization.

AP2 suppresses osteoblast differentiation and mineralization through down-regulation of Frizzled-1.

Transcription factor activating protein 2 (AP2) plays an important role in cellular differentiation. Although profound craniofacial and long bone developmental abnormalities have been observed in AP2-knockout mice, the molecular effects of AP2 on osteoblasts are poorly defined. We demonstrated that AP2 regulates the expression of human Frizzled 1 (FZD1), a co-receptor for the Wnt signalling pathway, in human osteoblast cell lines and primary bone marrow stromal cells (BMSCs). We also identified a putative AP2-binding site in the FZD1 proximal promoter in silico and characterized this binding element further in Saos2 invitro by ChIP, electrophoretic mobility shift and promoter reporter assays. The transcriptional repression of the FZD1 promoter by AP2 was confirmed in normal human fetal osteoblasts (hFOB). Furthermore, overexpression of AP2 resulted in a significant reduction in both differentiation and mineralization of Saos2 cells. Knockdown of FZD1 expression before AP2 up-regulation diminished the AP2-dependent inhibition of Saos2 cell differentiation and mineralization. Similarly, overexpressing FZD1 before AP2 treatment in both Saos2 and BMSCs diminished the inhibitory effect of AP2 on osteoblast differentiation and mineralization. Taken together, these results demonstrate that AP2 is a negative regulator of osteoblast differentiation and mineralization, and its inhibitory effect may be mediated in part through down-regulation of FZD1 expression.

Knockdown of a Wnt Receptor FZD1 reduces MDR1/P-Glycoprotein Expression in Human Leukemia Cells through the Wnt/β-Catenin Signaling Pathway

Background: Multidrug resistance (MDR) expressed in leukemia cells is a major obstacle in the use of chemotherapy for leukemia treatment. The Wnt signaling pathway is essential for maintenance, activation, regulated self-renewal and proliferation in both normal hematopoietic and leukemia cells. Frizzled-1 (FZD1), part of the Wnt receptor family, has been reported to be associated with drug resistance in a number of solid cancers, but the involvement of FZD1 in leukemia is yet to be studied. Here, we investigated whether FZD1 is associated with MDR1/P-glycoprotein (P-gp) in multidrug resistance of leukemic cell lines and examined FZD1 and MDR1/P-gp expression levels in AML cells from patients.Methods and Patients: Drug resistant cell lines K562/ADM, K562/Vin and NOMO-1/ADM as well as their parental cell lines were transduced with a GFP-containing FZD1 lentiviral vector (shFZD1) or a scrambled control (shC). The effect of FZD1 knockdown and change in MDR1/P-gp were confirmed by quantitative RT-PCR, western blot analysis, and flow cytometry. Cell growth was evaluated by cell counts over time and colony formation assays. The P-gp-mediated MDR phenotype was verified by assessing the rhodamine123 (Rh123) efflux. Drug sensitivity was examined in both the presence and absence of adriamycin (ADM), daunomycin (DNR) or vincrine (VCR). We also analyzed target genes downstream of the Wnt/β-catenin signaling pathway including β-catenin. Clinical samples were obtained from 112 AML patients with a normal karyotype at initial diagnosis after acquiring written informed consent.Results: The expression level of FZD1 in drug resistant cells was higher than that of their parent cells. Effective knockdown of FZD1 was confirmed and FZD1 expression was reduced by 82% (K562/Vin) and 50%-61% (NOMO-1/ADM and K562/ADM) when compared to shC. Interestingly, knockdown of FZD1 also resulted in a significant reduction of MDR1 by 40%-50% in resistant cells. Western blotting showed a reduction in the P-gp level as well as the FZD1 protein in shFZD1 cells. Mean fluorescence intensities of P-gp were also reduced significantly in K562/Vin (50.6%±16.4%), K562/ADM (68.4%±16.3%) and NOMO-1/ADM (56.9%±0.6%) cells. Rh123 intracellular content in these shFZD1 cells significantly increased 2.5-3.4 folds, indicating a reduction in P-gp function. The cytotoxicity assay revealed restoration of drug sensitivity to a varying extent. In addition, cell growth and colony formation were significantly suppressed. To investigate whether these results were caused by the activation of the Wnt/β-catenin pathway, immunofluorescence of β-catenin was performed, and the results showed that accumulation of nucleic β-catenin was reduced after inhibition of FZD1. We then assessed β-catenin, TCF7L2, LEF1, CyclinD1, p21 and p27 target genes in shFZD1 cells. The expression levels of proteins revealed that β-catenin and TCF7L2 were significantly decreased in shFZD1 cells. Elevated p27 and decreased cyclin D1 protein levels were also observed.A total of 112 untreated AML patients with normal karyotypes who received anthracycline-based chemotherapy were analyzed. Eighty-five patients achieved complete remission (CR), and the remaining 27 patients had no response (NR). Thirty-four of these 85 patients relapsed (CR-Re) after remission, and the remaining 51 patients maintained CR. No significant difference in FZD1 expression was observed between the CR and NR groups. However, a statistical difference was observed between the NR group and the CR group when the CR-Re group was excluded from the analysis (median 5.989 vs. 1.856; p = 0.003). Expression of FZD1 was also significantly higher in the CR-Re group than in the CR group (10.738; p = 0.002). The NR group had significantly higher MDR1/P-gp levels than the CR group. Furthermore, a statistically significant relationship was observed in the NR group between FZD1 and MDR1 (p = 0.005), MDR1 and P-gp expressions (p <0.05).Conclusions: These data indicate that FZD1 promotes the activation of β-catenin/TCF7L2 and increases the proliferative capacity of cells through the regulation of cyclin D1 expression. FZD1 seems to modulate multidrug resistance through the Wnt/β-catenin signaling pathway, suggesting that AML patients who have higher FZD1 expression at diagnosis tend to relapse and show chemotherapy resistance. Therefore, FZD1 likely participates in the development of MDR in leukemic cells. DisclosuresNo relevant conflicts of interest to declare.

Interference of Frizzled 1 (FZD1) reverses multidrug resistance in breast cancer cells through the Wnt/β-catenin pathway

Multidrug resistance (MDR) represents a major obstacle in the successful treatment of breast cancer. The MDR1 gene is a direct target of the Wnt/β-catenin signaling pathway, which controls tumor development. Overexpression of P-glycoprotein, encoded by the MDR1 gene, is one of the most common causes of MDR. We found that the Frizzled 1 (FZD1) protein, which is an essential component of the Wnt/β-catenin pathway, is overexpressed in the multidrug resistant breast cancer cell subline MCF-7/ADM, coincident with MDR1/P-gp. FZD1 silencing induced down-regulation of MDR1/P-gp, restored sensitivity to four chemotherapy drugs, and significantly decreased cytoplasmic and nuclear β-catenin levels. FZD1 appears to mediate multidrug resistance by regulating the Wnt/β-catenin pathway.

Canonical and noncanonical Wnt pathways: a comparison between endometrial cancer type I and atrophic endometrium in Brazil.

The Wnt pathway is involved in tumorigenesis of several tissues. For this reason, we proposed to evaluate Wnt gene expression in endometrial cancer type I. Cross-sectional study on materials gathered from the tissue bank of the Department of Pathology, Universidade Federal de São Paulo. Endometrial specimens were obtained from surgeries performed between 1995 and 2005 at São Paulo Hospital, Universidade Federal de São Paulo. The material was divided into two groups according to tissue type: Group A, atrophic endometrium (n = 15); and Group B, endometrial adenocarcinoma (n = 45). We compared the immunohistochemical expression of Wnt1, Frizzled-1 (FZD1), Wnt5a, Frizzled-5 (FZD5) and beta-catenin between endometrial cancer type I and atrophic endometrium. Regarding Wnt1, FZD1 and Wnt5a expression, no significant association was observed between the groups. A significant association was observed between the groups in relation to FZD5 expression (P = 0.001). The proportion of FZD5-positive samples was significantly higher in group A (80.0%) than in group B (31.1%). Regarding the survival curve for FZD5 in group B, we did not find any significant association between atrophic endometrium and endometrial adenocarcinoma. We also did not find any significant association regarding beta-catenin expression (P = 1.000). FZD5 is downregulated in endometrial adenocarcinoma, in comparison with atrophic endometrium.

Leptin receptor JAK2/STAT3 signaling modulates expression of Frizzled receptors in articular chondrocytes

Differentiated articular chondrocytes express a functional bisoform of the leptin receptor (LRb); however, leptin-LRb signaling in these cells is poorly understood. We hypothesized that leptin-LRb signaling in articular chondrocytes functions to modulate canonical Wnt signaling events by altering the expression of Frizzled (FZD) receptors. Human chondrocyte cell lines and primary articular chondrocytes were grown in serum containing growth media for 24h, followed by a media change to Dulbecco's modified Eagle's medium (DMEM) containing 1% Nutridoma-SP to obtain a serum-deficient environment for 24h before treatment. Treatments included recombinant human leptin (10-100nM), recombinant human IL-6 (0.3-3nM), or recombinant human erythropoietin (Epo) (10mU/ml). Cells were harvested 30min-48h after treatment and whole cell lysates were analyzed using immunoblots or luciferase assays. Treatment of cells with leptin resulted in activation of Janus kinase 2 (JAK2) and subsequent phosphorylation of specific tyrosine residues on LRb, followed by dose- and time-dependent increases in the expression of Frizzled-1 (FZD1) and Frizzled-7 (FZD7). Leptin-mediated increases in the expression of FZD1 were blocked by pre-treatment with the protein synthesis inhibitor cycloheximide or the JAK2 inhibitor AG490. Experiments using a series of hybrid Epo extracellular domain-leptin intracellular domain receptors (ELR) harboring mutations of specific tyrosine residues in the cytoplasmic tail showed that increases in the expression of FZD1 were dependent on LRb-mediated phosphorylation of STAT3, but not ERK1/2 or STAT5. Leptin pre-treatment of chondrocytes prior to Wnt3a stimulation resulted in an increased magnitude of canonical Wnt signaling. These experiments show that leptin-LRb signaling in articular chondrocytes modulates expression of canonical Wnt signaling receptors and suggests that direct cross-talk between these pathways is important in determining chondrocyte homeostasis.

Functional and association analysis of frizzled 1 (FZD1) promoter haplotypes with femoral neck geometry

Frizzleds are receptors for Wnt signaling and are involved in skeletal morphogenesis. Little is known about the transcriptional regulation of frizzleds in bone cells. In the current study, we determined if two common and potentially functional genetic variants (rs2232157, rs2232158) in the frizzled-1 ( FZD1) promoter region and their haplotypes influence FZD1 promoter activity in human osteoblast-like cells. We also determined if these variants are associated with femoral neck bone mineral density (BMD) and geometry in 1319 African ancestry men aged ≥ 40 years. Real-time quantitative PCR and western blot analysis demonstrated FZD1 mRNA and protein expression in the human osteoblast-like cell lines, MG63 and SaOS-2. Promoter activity was next assessed by transient expression of haplotype specific FZD1 promoter reporter plasmids in these cells. In comparison to the common GG haplotype, promoter activity was 3-fold higher for the TC haplotype in both cell lines ( p < 0.05). We previously demonstrated that rs2232158 is associated with differential FZD1 promoter activity and Egr1 binding and thus focused further functional analyses on the rs2232157 G-to-T polymorphism. Electrophoretic mobility shift assay demonstrated that distinct nuclear protein complexes were associated with rs2232157 in an allele specific manner. Bioinformatics analysis predicted that the G to T transversion creates an E2F1 binding site, further supporting the functional significance of rs2232157 in FZD1 promoter regulation. Individual SNPs and haplotypes were not associated with femoral neck BMD. The TC haplotype was associated with larger subperiosteal width and greater CSMI ( p < 0.05). These results suggest that FZD1 expression is regulated in a haplotype-dependent manner in osteoblasts and that these same haplotypes may be associated with biomechanical indices of bone strength.

Functional Characterization of Genetic Variation in the Frizzled 1 (FZD1) Promoter and Association With Bone Phenotypes: More to the LRP5 Story?

WNT signaling is an important determinant of bone formation. The WNT co-receptor, Frizzled homolog 1 (FZD1), initiates WNT signal transduction. To study the influence of FZD1 genetic variation on measures of bone health, we first sequenced a 6.8-kb region surrounding FZD1 in 48 samples of African ancestry. We genotyped all common polymorphisms and performed association analysis with bone phenotypes in a larger sample. Only 3 of 35 SNPs identified were present in >or=5% of the sample and assayed further in 1084 men of African ancestry. Two of these SNPs were in the FZD1 promoter (rs2232157, rs2232158) and were associated with femoral neck areal BMD (p = 0.041 and 0.009, respectively). The minor alleles of these two SNPs were also associated with larger bone size at the radius (p < 0.05 for both), and rs2232158 was associated with greater strength-strain index, an indicator of bone's ability to withstand torsion. Functional experiments were completed to assess the influence of the rs2232158 promoter polymorphism on transcriptional regulation of FZD1. The minor C allele in rs2232158 creates a binding site for the transcription factor Egr1, has higher Egr1 binding affinity, and has greater FZD1 promoter activity in MG63 and SaOS-2 cells, providing a plausible molecular mechanism for the population associations. This study indicates that a cis-regulatory polymorphism in the FZD1 promoter region may have a functional role in determining bone structural geometry.

Frizzled‐1 is down‐regulated in follicular thyroid tumours and modulates growth and invasiveness

The mechanisms of follicular thyroid carcinoma (FTC) transformation and progression are not well understood. Previously, we detected LOH at 7q21 in all FTCs examined, indicating that loss of genetic material in that region is a common trait in these lesions. To analyse the effects of LOH on gene expression, we performed an analysis of the mRNA expression levels of six different genes, located at 7q21.1-7q21.3. A total of 23 lesions, including eight follicular hyperplasias (FHs), eight follicular adenomas (FAs), two FTCs and five papillary thyroid carcinomas (PTCs) were analysed. The Frizzled-1 (FZD-1) gene, located at 7q21.13, showed the lowest levels of mRNA expression. Down-regulation of FZD-1 expression was also confirmed in an independent series of 69 follicular neoplastic lesions compared to 25 PTCs, analysed by quantitative RT-PCR. In vitro studies showed that FZD-1 expression was also markedly reduced at both protein and mRNA levels in three FTC-derived cell lines (FRO, WRO and FTC-133), while it was normal in the three PTC-derived cell lines (Ca300, Ca301 and K1) examined. We demonstrated that over-expression of FZD-1 in 3 FTC-derived cells decreased invasiveness and proliferation rate, indicating a possible pathogenetic role. In addition, FZD-1 RNA interference in the PTC-derived cell line K1 increased invasiveness. Our data indicated that FZD-1 is involved in growth of follicular tumours and may be considered as a novel marker of this type of tumour.

Bone Health in Chronic Kidney Disease–Mineral and Bone Disease

Chronic kidney disease (CKD) is accompanied by disturbances in calcium, phosphate, vitamin D, and parathyroid hormone (PTH) homeostasis that play an important role in the pathophysiology of renal bone disease. The increased cardiovascular morbidity and mortality observed among patients with CKD has recently been recognized to be associated with these disturbances in mineral metabolism. Thus, disturbances in mineral metabolism observed in renal failure results in a multisystem disorder, making the development of a standardized definition of these disorders a top priority. Therefore, the Board of Directors of Kidney Disease: Improving Global Outcomes proposed to define the broader category of mineral disorders associated with CKD as CKD-mineral and bone disorder (CKD-MBD). This newly proposed definition will include the disorders of mineral metabolism, bone histology (renal osteodystrophy), and the extraskeletal manifestations such as vascular calcification. This new definition and stratification of disease should result in improvement not only in the clinical management of patients but also will facilitate the interpretation and translation of clinical research. Renal osteodystrophy is now considered as 1 component of this disorder and will be defined as a morphologic alteration only, based on unification of the histomorphometric definitions that will include parameters of turnover, mineralization, and volume. An internationally accepted classification system will enable the consensus for bone biopsy evaluation as well as for the role of biomarkers. This article will focus on the newly proposed definitions of bone disease as part of CKD-MBD, based on the complex pathophysiologic process underlying bone disease in CKD stages 2 to 5.

Effects of parathyroid hormone on Wnt signaling pathway in bone

The Wnt signaling pathway has recently been demonstrated to play an important role in bone cell function. In previous studies using DNA microarray analyses, we observed a change in some of the molecular components of the canonical Wnt pathway namely, frizzled-1 (FZD-1) and axil, in response to continuous parathyroid hormone (PTH) treatment in rats. In the present study, we further explored other components of the Wnt signaling pathway in rat distal metaphyseal bone in vivo, and rat osteoblastic osteosarcoma cells (UMR 106) in culture. Several Wnt pathway components, including low-density lipoprotein-receptor-related protein 5 (LRP5), LRP6, FZD-1, Dickkopf-1 (Dkk-1), and Kremen-1 (KRM-1), were expressed in bone in vivo and in osteoblasts in vitro. Continuous exposure to PTH (1-38) both in vivo and in vitro upregulated the mRNA expression of LRP6 and FZD-1 and decreased LRP5 and Dkk-1. These effects in UMR 106 cells were associated with an increase in beta-catenin as measured by Western blots and resulted in functional activation (three to six-fold) of a downstream Wnt responsive TBE6-luciferase (TCF/LEF-binding element) reporter gene. Activation of the TBE6-luciferase reporter gene by PTH (1-38) in UMR 106 cells was inhibited by the protein kinase A (PKA) inhibitor, H89. Activation was mimicked by PTH (1-31), PTH-related protein (1-34), and forskolin, but both PTH (3-34) and (7-34) had no effect. These findings suggest that the effect of PTH on the canonical Wnt signaling pathway occurs at least in part via the cAMP-PKA pathway through the differential regulation of the receptor complex proteins (FZD-1/LRP5 or LRP6) and the antagonist (Dkk-1). Taken together, these results reveal a possible role for the Wnt signaling pathway in PTH actions in bone.