Frizzled-8 Research Articles

In Silico Optimization of Frizzled-8 Receptor Inhibition Activity of Carbamazepine: Designing New Anti-Cancer Agent.

Frizzled-8 (FZD8) receptor is a therapeutic target for cancer treatment and recent research has shown that carbamazepine (CBZ) can inhibit this receptor. In this work, it has been tried to optimize CBZ to enhance its binding capacity to the N6W binding site of FZD8 by using structure-based drug design methods. CBZ and its 83 derivatives were docked to the N6W binding site of FZD8. Docking results show that two compounds 79 and 82 have the smallest binding energies and are fitted to the N6W binding site. Compounds C79 and C82 have been synthesized by replacing a hydrogen atom of the seven-membered ring in CBZ with benzoate and nicotinate groups, respectively. In addition, docking results show that a trifluoromethyl on one of the phenyl rings is favorable for improving the FZD8 inhibition activity of the molecule. Both molecules C79 and C82 were subjected to molecular dynamics (MD) simulation. MD results show that FZD8-C82 complex is stable and this compound binds to the N6W binding site more strongly than compounds C79 and CBZ.

MiR-99a alleviates apoptosis and extracellular matrix degradation in experimentally induced spine osteoarthritis by targeting FZD8

BackgroundOur previous study identified miR-99a as a negative regulator of early chondrogenic differentiation. However, the functional role of miR-99a in the pathogenesis of osteoarthritis (OA) remains unclear.MethodsWe examined the levels of miR-99a and Frizzled 8 (FZD8) expression in tissue specimens. Human SW1353 chondrosarcoma cells were stimulated with IL-6 and TNF-α to construct an in vitro OA environment. A luciferase reporter assay was performed to analyze the relationship between miR-99a and FZD8. CCK-8 assays, flow cytometry, and ELISA assays were used to assess cell viability, apoptosis, and inflammatory molecule expression, respectively. Percutaneous intra-spinal injections of papain mixed solution were performed to create an OA Sprague–Dawley rat model. Alcian Blue staining, Safranin O Fast Green staining, and Toluidine Blue O staining were performed to detect the degrees of cartilage injury.ResultsMiR-99a expression was downregulated in the severe spine OA patients when compared with the mild spine OA patients, and was also decreased in the experimentally induced in vitro OA environment when compared with the control environment. Functionally, overexpression of miR-99a significantly suppressed cell apoptosis and extracellular matrix degradation stimulated by IL-6 and TNF-α. FZD8 was identified as a target gene of miR-99a. Furthermore, the suppressive effects of miR-99a on cell injury induced by IL-6 and TNF-α were reversed by FZD8 overexpression. Moreover, the levels of miR-99a expression were also reduced in the induced OA model rats, and miR-99a agomir injection relieved the cartilage damage. At the molecular level, miR-99a overexpression downregulated the levels of MMP13, β-catenin, Bax, and caspase-3 protein expression and upregulated the levels of COL2A1 and Bcl-2 protein expression in the in vitro OA-like chondrocyte model and also in the experimental OA model rats.ConclusionsOur data showed that miR-99a alleviated apoptosis and extracellular matrix degradation by targeting FZD8, and thereby suppressed the development and progression of experimentally induced spine osteoarthritis.

The role of exosomal miR-375-3p: A potential suppressor in bladder cancer via the Wnt/β-catenin pathway.

miR-375-3p is a significantly downregulated miRNA in bladder cancer (BC). However, its role in BC regulation is still unclear. In this study, we reported that miR-375-3p overexpression inhibited proliferation and migration and promoted apoptosis in BC cells. Frizzled-8 (FZD8) gene is identified as the direct miR-375-3p targeting gene. miR-375-3p blocks the Wnt/β-catenin pathway and downstream molecules Cyclin D1 and c-Myc by inhibiting the expression of FZD8 directly, it could increase caspase 1 and caspase 3 expression and promote T24 cell apoptosis as well. miR-375-3p also showed a significant inhibitory effect in vivo in bladder tumor-bearing nude mice, as demonstrated by the reduced tumor volume and Ki67 proliferation index in tumor tissue. Collectively, miR-375-3p is a suppressor of BC that inhibits proliferation and metastasis, and promotes apoptosis in BC cells as well as suppresses tumor growth in a T24 xenograft mouse model, which could be used as a potential therapeutic approach for BC in future.

Wnt receptor Frizzled 8 is a target of ERG in prostate cancer.

Prostate cancer (PCa) is one of the most frequently diagnosed cancers among men. Many molecular changes have been detailed during PCa progression. The gene encoding the transcription factor ERG shows recurrent rearrangement, resulting in the overexpression of ERG in the majority of prostate cancers. Overexpression of ERG plays a critical role in prostate oncogenesis and development of metastatic disease. Among the downstream effectors of ERG, Frizzled family member FZD4 has been shown to be a target of ERG. Frizzled-8 (FZD8) has been shown to be involved in PCa bone metastasis. In the present study, we show that the expression of FZD8 is directly correlated with ERG expression in PCa. Furthermore, we show that ERG directly targets and activates FZD8 by binding to its promoter. This activation is specific to ETS transcription factor ERG and not ETV1. We propose that ERG overexpression in PCa leads to induction of Frizzled family member FZD8, which is known to activate the Wnt pathway. Taken together, these findings uncover a novel mechanism for PCa metastasis, and indicate that FZD8 may represent a potential therapeutic target for PCa.

MicroRNA-520b Suppresses Proliferation, Migration, and Invasion of Spinal Osteosarcoma Cells via Downregulation of Frizzled-8.

Spinal osteosarcoma (OS) is a malignant tumor that has a poor outcome. MicroRNA-520b (miR-520b) acts as a cancer suppressor in various types of cancer. Because of the limited amount of literature on OS, we aimed to identify the role of miR-520b in OS. The miR-520b level in clinical spinal OS tissues and adjacent nontumor tissues as well as in cell lines was assessed. The effect of miR-520b on cell proliferation, migration, invasion, and frizzled-8 (FZD8) degradation were all evaluated. Alterations of key proteins involved in the Wnt/β-catenin pathway were assessed by Western blot analysis. In the present study, miR-520b was downregulated in human spinal OS tissues and OS cell lines (p < 0.01 or p < 0.001). Overexpression of miR-520b inhibited cell proliferation (p < 0.01 or p < 0.001), migration (p < 0.01), and invasion (p < 0.01). FZD8 expression was negatively regulated by infection with a lentivirus vector carrying an miR-520b precursor in dose- and time-dependent manners. In OS tissues, miR-520b was inversely correlated with FZD8 expression. FZD8 was upregulated in human spinal OS tissues and cell lines. Finally, miR-520b inactivated the Wnt/β-catenin pathway through downregulation of FZD8. miR-520b inhibited cell proliferation, migration, and invasion through inactivating the Wnt/β-catenin pathway by downregulation of FZD8, providing a novel therapeutic target for spinal OS.

Frizzled 8 promotes the cell proliferation and metastasis of renal cell carcinoma.

Recent reports have shown a rapid rise in the incidence of renal cell carcinoma (RCC), and Wnt (Wingless-related integration site) signaling pathway is important in RCC. Frizzled 8 (FZD8) is a member of Frizzled (FZD) receptor family which could activate canonical or non-canonical Wnt/β-catenin pathways. Nevertheless, the role of FZD8 in RCC is poorly investigated. The immunohistochemical analysis showed high expression of FZD8 in RCC tissues compared with peri-tumor tissues. FZD8 knockdown decreased the ability of proliferation and metastasis of RCC cells. Research revealed that the FZD8 regulated the transcription of Cyclin D1, c-Myc, and could promote the epithelial to mesenchymal transition (EMT) by mediating Vimentin and Snail through the Wnt/β-catenin signaling pathway. In addition, the results of our experiment revealed that FZD8 is involved in the regulation of non-canonical Wnt signaling pathway. These data suggested that the expression of FZD8 may play an important role in the proliferation and metastasis of RCC, and serve as a putative promising drug target for human RCC therapy.

Feedback Activation of Basic Fibroblast Growth Factor Signaling via the Wnt/β-Catenin Pathway in Skin Fibroblasts.

Skin wound healing is a complex process requiring the coordinated behavior of many cell types, especially in the proliferation and migration of fibroblasts. Basic fibroblast growth factor (bFGF) is a member of the FGF family that promotes fibroblast migration, but the underlying molecular mechanism remains elusive. The present RNA sequencing study showed that the expression levels of several canonical Wnt pathway genes, including Wnt2b, Wnt3, Wnt11, T-cell factor 7 (TCF7), and Frizzled 8 (FZD8) were modified by bFGF stimulation in fibroblasts. Enzyme-linked immunosorbent assay (ELISA) analysis also showed that Wnt pathway was activated under bFGF treatment. Furthermore, treatment of fibroblasts with lithium chloride or IWR-1, an inducer and inhibitor of the Wnt signaling pathway, respectively, promoted and inhibited cell migration. Also, levels of cytosolic glycogen synthase kinase 3 beta phosphorylated at serine9 (pGSK3β Ser9) and nuclear β-catenin were increased upon exposure to bFGF. Molecular and biochemical assays indicated that phosphoinositide 3-kinase (PI3K) signaling activated the GSK3β/β-catenin/Wnt signaling pathway via activation of c-Jun N-terminal kinase (JNK), suggesting that PI3K and JNK act at the upstream of β-catenin. In contrast, knock-down of β-catenin delayed fibroblast cell migration even under bFGF stimulation. RNA sequencing analysis of β-catenin knock-down fibroblasts demonstrated that β-catenin positively regulated the transcription of bFGF and FGF21. Moreover, FGF21 treatment activated AKT and JNK, and accelerated fibroblast migration to a similar extent as bFGF does. In addition, ELISA analysis demonstrated that both of bFGF and FGF21 were auto secretion factor and be regulated by Wnt pathway stimulators. Taken together, our analyses define a feedback regulatory loop between bFGF (FGF21) and Wnt signaling acting through β-catenin in skin fibroblasts.

Dickkopf-1 Is a Biomarker for Systemic Lupus Erythematosus and Active Lupus Nephritis.

An early diagnosis of lupus nephritis (LN) has an important clinical implication in guiding treatments of systemic lupus erythematosus (SLE) in clinical settings. In this study, the concentrations of Wnt-3A, Frizzled-8 (FZD-8), and Dickkopf-1 (DKK-1) of Wnt signaling, as well as their diagnostic values for accessing LN, were evaluated by ELISA in sera and urine of 111 SLE patients (31 with LN and 80 without LN) and 70 healthy cohorts. Significantly more abundances of DKK-1 protein were determined in both of sera and urine of SLE patients compared to healthy cohorts (p < 0.0001); in particular the serum DKK-1 concentration was even higher in LN-SLE patients relative to non-LN SLE subjects (p < 0.0001). Intriguingly, concentrations of above examined proteins in SLE patients showed no correlation between serum and urine. Moreover, a combination of DKK-1 with anti-dsDNA and/or levels of complement C3 and C4 could not increase the specificity and/or sensitivity for identification of patients with LN diseases, but both ROC curve and multiple-factor nonconditional logistic regression analysis showed that serum DKK-1 was considered better positive biomarker for identification of LN in SLE patients. These results imply that serum and/or urine DKK-1 may be a valuable and independent biomarker for identification of SLE patients with LN.

MicroRNA-375 suppresses human colorectal cancer metastasis by targeting Frizzled 8.

microRNAs are aberrantly expressed during the development and progression of a variety of human cancers, including colorectal cancer (CRC). Of these microRNAs, microRNA-375 (miR-375) was previously observed to be downregulated in human colorectal cancer(CRC) plasma and tissues, but its functions are largely unknown. Here, we investigated the impact of miR-375 on CRC metastasis. Specifically, miR-375 expression was significantly decreased in human CRC tissues compared with their matched noncancerous tissues (NCTs), and low levels of miR-375 predicted tumor metastatic potential. The up-regulation of miR-375 suppressed colorectal cancer cell migration and invasion in vitro and reduced tumor metastases in murine models established by both orthotopic implantation and spleen injection. Furthermore, we identified Frizzled 8 (FZD8) as a direct target of miR-375 in CRC, and miR-375 negatively regulated Wnt/β-catenin signaling by suppressing FZD8. More importantly, FZD8 expression inversely correlated with overall survival in human CRC patients and is a likely independent predictor of survival. Therefore, we concluded that miR-375 functions as a tumor-suppressive microRNA by directly acting upon FZD8, which may serve as a new therapeutic target to inhibit tumor metastasis in CRC.

Soluble (pro)renin receptor via β-catenin enhances urine concentration capability as a target of liver X receptor

The extracellular domain of the (pro)renin receptor (PRR) is cleaved to produce a soluble (pro)renin receptor (sPRR) that is detected in biological fluid and elevated under certain pathological conditions. The present study was performed to define the antidiuretic action of sPRR and its potential interaction with liver X receptors (LXRs), which are known regulators of urine-concentrating capability. Water deprivation consistently elevated urinary sPRR excretion in mice and humans. A template-based algorithm for protein-protein interaction predicted the interaction between sPRR and frizzled-8 (FZD8), which subsequently was confirmed by coimmunoprecipitation. A recombinant histidine-tagged sPRR (sPRR-His) in the nanomolar range induced a remarkable increase in the abundance of renal aquaporin 2 (AQP2) protein in primary rat inner medullary collecting duct cells. The AQP2 up-regulation relied on sequential activation of FZD8-dependent β-catenin signaling and cAMP-PKA pathways. Inhibition of FZD8 or tankyrase in rats induced polyuria, polydipsia, and hyperosmotic urine. Administration of sPRR-His alleviated the symptoms of diabetes insipidus induced in mice by vasopressin 2 receptor antagonism. Administration of the LXR agonist TO901317 to C57/BL6 mice induced polyuria and suppressed renal AQP2 expression associated with reduced renal PRR expression and urinary sPRR excretion. Administration of sPRR-His reversed most of the effects of TO901317. In cultured collecting duct cells, TO901317 suppressed PRR protein expression, sPRR release, and PRR transcriptional activity. Overall we demonstrate, for the first time to our knowledge, that sPRR exerts antidiuretic action via FZD8-dependent stimulation of AQP2 expression and that inhibition of this pathway contributes to the pathogenesis of diabetes insipidus induced by LXR agonism.

Can a few non-coding mutations make a human brain?

The recent finding that the human version of a neurodevelopmental enhancer of the Wnt receptor Frizzled 8 (FZD8) gene alters neural progenitor cell cycle timing and brain size is a step forward to understanding human brain evolution. The human brain is distinctive in terms of its cognitive abilities as well as its susceptibility to neurological disease. Identifying which of the millions of genomic changes that occurred during human evolution led to these and other uniquely human traits is extremely challenging. Recent studies have demonstrated that many of the fastest evolving regions of the human genome function as gene regulatory enhancers during embryonic development and that the human‐specific mutations in them might alter expression patterns. However, elucidating molecular and cellular effects of sequence or expression pattern changes is a major obstacle to discovering the genetic bases of the evolution of our species. There is much work to do before human‐specific genetic and genomic changes are linked to complex human traits.Also watch the Video .

MiR-375 regulates the canonical Wnt pathway through FZD8 silencing in arthritis synovial fibroblasts

Whether the rheumatoid arthritis (RA) pathogenesis is regulated by microRNA (miRNA) is not entirely clear. In this study, we found that miR-375 was down-regulated significantly in fibroblast-like synoviocytes (FLS) in adjuvant-induced arthritis (AIA) rat model compared with control. Because the web-based software TargetScan and PicTar predict Frizzled 8 (FZD8) as the target of miR-375, we investigated whether up-regulated miR-375 plays a role in the activation of the canonical Wnt signaling by targeting the FZD8. Furthermore, the purpose of the present experiments was also to determine the role of miR-375 in the pathogenesis of AIA rat model and to ascertain the effects of FZD8 in this process. Real time qPCR, Western blotting, ELISA and ChIP assay were used to assess the inhibited role of miR-375 in the pathogenesis of AIA rat model and the canonical Wnt signaling. RNA interference was also used to detect the role of knockdown of dephosphorylated β-catenin. Luciferase reporter gene and related methods were performed to determine the FZD8 as the target of miR-375. The increased miR-375 inhibited the pathogenesis of AIA rat model as indicated by decreases in the several disease markers, such as MMP3 and fibronectin. Interestingly, miR-375 also inhibited the canonical Wnt signaling, and the stabilized form of β-catenin blocked the miR-375 effects. FZD8 was identified as the target of miR-375 in AIA rat model by the firefly luciferase reporter gene. In summary, our results demonstrate that miR-375 regulates the pathogenesis of AIA rat model through the canonical Wnt signaling pathway. This discovery may provide new targets for therapeutic intervention to benefit RA patients.

Noncanonical WNT‐5A signaling regulates TGF‐β‐induced extracellular matrix production by airway smooth muscle cells

Transforming growth factor β (TGF-β), a key mediator of fibrotic responses, is increased in asthma and drives airway remodeling by inducing expression of extracellular matrix (ECM) proteins. We investigated the molecular mechanisms underlying TGF-β-induced ECM expression by airway smooth muscle cells and demonstrate a novel link between TGF-β and Wingless/integrase 1 (WNT) signaling in ECM deposition. Airway smooth muscle expresses abundant WNT ligands, with the noncanonical WNT-5A being the most profoundly expressed. Interestingly, WNT-5A shows ∼2-fold higher abundance in airway smooth muscle cells isolated from individuals with asthma than individuals without asthma. WNT-5A is markedly induced in response to TGF-β (4-16-fold; EC₅₀ 0.3 ng/ml) and is required for collagen and fibronectin expression by airway smooth muscle. WNT-5A engages noncanonical WNT signaling pathways, as inhibition of Ca(2+) and c-Jun N-terminal kinase (JNK) signaling attenuated this TGF-β response, whereas the canonical WNT antagonist Dickkopf 1 (DKK-1) did not. Accordingly, WNT-5A induced JNK phosphorylation and nuclear translocation of nuclear factor of activated T cells c1 (NFATc1). Furthermore, silencing of the WNT-5A receptors Frizzled 8 (FZD8) and RYK attenuated TGF-β-induced ECM expression. Collectively, these findings demonstrate that noncanonical WNT-5A signaling is activated by and necessary for TGF-β-induced ECM production by airway smooth muscle cells, which could have significance in asthma pathogenesis.

Molecular cloning and characterization of human Frizzled-8 gene on chromosome 10p11.2.

Mouse Frizzled-8, encoding a WNT receptor, is a potent cancer-associated gene to activate the beta-catenin-TCF pathway. However, these is a possibility that mouse Frizzled-8 might be a pseudogene, because structure and expression profile of mouse Frizzled-8 mRNA are still unclear. We have cloned and characterized the human Frizzled-8 (FZD8) gene, a human homologue of mouse Frizzled-8. Comparison between FZD8 genome clones and FZD8 cDNA clones isolated in this study revealed no intron within the FZD8 gene. FZD8 was found to encode a 694 amino-acid polypeptide with the frizzled-like cysteine-rich domain, seven transmembrane domains, and the C-terminal Ser/Thr-X-Val motif. Among human FZD family, FZD8 was most homologous to FZD5 (total amino-acid identity 69.1%). The 4.0-kb FZD8 mRNA was detected in fetal kidney and brain, and also in adult kidney, heart, pancreas, and skeletal muscle. These results indicate that human FZD8 is not a pseudogene. The FZD8 gene was mapped to human chromosome 10p11.2 by fluorescence in situ hybridization. Among human cancer cell lines, FZD8 was relatively highly expressed in HeLa S3 (cervical uterus cancer) and A549 (lung cancer). Up-regulation of FZD8 might play key roles in several types of human cancer through activation of the beta-catenin-TCF pathway.