Hyun Yi Kim

Persicaria hydropiper (L.) spach and its flavonoid components, isoquercitrin and isorhamnetin, activate the Wnt/β-catenin pathway and inhibit adipocyte differentiation of 3T3-L1 cells.

Obesity, which is related to metabolic syndrome and is associated with liver disease, represents an epidemic problem demanding effective therapeutic strategies. Evidence shows that the Wnt/β‐catenin pathway is closely associated with obesity and that small molecules regulating the Wnt/β‐catenin pathway can potentially control adipogenesis related to obesity. Eleven plant extracts activating the Wnt/β‐catenin pathway were screened by using HEK 293‐TOP cells retaining the Wnt/β‐catenin signaling reporter gene. An extract of Persicaria hydropiper (L.) Spach was found to activate Wnt/β‐catenin signaling. P. hydropiper is grown worldwide in temperate climates and is found widely in Southeast Asia. The P. hydropiper extract inhibited the differentiation of adipocyte 3T3‐L1 cells. Isoquercitrin and isorhamnetin, constituents of P. hydropiper, also activated Wnt/β‐catenin signaling and suppressed the differentiation of 3T3‐L1 cells. These results indicate that isoquercitrin in P. hydropiper suppresses the adipogenesis of 3T3‐L1 cells via the inhibition of Wnt/β‐catenin signaling. P. hydropiper and isoquercitrin may therefore be potential therapeutic agents for obesity and its associated disorders. Copyright

CXXC5 is a negative-feedback regulator of the Wnt/ β -catenin pathway involved in osteoblast differentiation

The positive roles of the Wnt/β-catenin pathway in osteoblast differentiation and bone mineral density (BMD) maintenance have been clearly demonstrated in both animal experiments and clinical investigations. CXXC finger protein 5 (CXXC5), a recently identified negative regulator of the Wnt/β-catenin pathway, showed altered cellular localization and function, which were dependent on the cell type in previous studies. However, the in vivo function of CXXC5 has not been clearly investigated yet. Here, we characterized CXXC5 as a negative regulator of osteoblast differentiation and bone formation. Deficiency of CXXC5 resulted in elevated BMD in mice without any severe gross developmental abnormalities. CXXC5 exerted a negative-feedback effect on the Wnt/β-catenin pathway via Wnt-dependent binding to Dishevelled (Dvl) during osteoblast differentiation. Suppression of the Dvl–CXXC5 interaction using a competitor peptide resulted in the activation of the Wnt/β-catenin pathway and osteoblast differentiation, and accelerated thickness growth of ex vivo-cultured calvariae. Overall, CXXC5 is a negative-feedback regulator induced by Wnt/β-catenin signaling that inhibits osteoblast differentiation and bone formation via interaction with Dvl.

The Dishevelled-binding protein CXXC5 negatively regulates cutaneous wound healing

In human melanoma biopsies and a murine cutaneous wound model, Lee et al. identify the Dishevelled-binding protein CXXC5 as a negative modulator of skin wound healing. CXXC5-deficient mice present accelerated wound healing as well as keratin and collagen synthesis. CXXC5, interacting with Dvl, operates as a negative feedback regulator of Wnt/β-catenin signaling and may represent a potential target for wound treatment.

CXXC5 is a transcriptional activator of Flk-1 and mediates bone morphogenic protein-induced endothelial cell differentiation and vessel formation

CXXC5 is a member of a small subset of proteins containing CXXC‐type zinc‐finger domain. Here, we show that CXXC5 is a transcription factor activating Flk‐1, a receptor for vascular endothelial growth factor. CXXC5 and Flk‐1 were accmulated in nucli and membrane of mouse embryonic stem cells (mESCs), respectively, during their endothelial differentiation. CXXC5 overexpression induced Flk‐1 transcription in both endothelium‐differentiated mESCs and human umbilical vein endothelial cells (HUVECs). In vitro DNA binding assay showed direct interaction of CXXC5 on the Flk‐1 promoter region, and mutation on its DNA‐binding motif abolished transcriptional activity. We showed that bone morphorgeneic protein 4 (BMP4) induced CXXC5 transcription in the cells, and inhibitors of BMP signaling suppressed the CXXC5 induction and the consequent Flk‐1 induction by BMP4 treatment. CXXC5 knockdown resulted in suppression of BMP4‐induced stress fiber formation (56.8±1.3% decrease, P<0.05) and migration (54.6±1.9% decrease, P<0.05) in HUVECs. The in vivo roles of CXXC5 in BMP‐signaling‐specific vascular development and angiogenesis were shown by specific defect of caudal vein plex vessel formation (57.9±11.8% decrease, P<0.05) in cxxc5 morpholino‐injected zebrafish embryos and by supression of BMP4‐induced angigogensis in subcutaneously injected Matrigel plugs in CXXC5‐/‐ mice. Overall, CXXC5 is a transcriptional activator for Flk‐1, mediating BMP signaling for differentiation and migration of endothelial cell and vessel formation.—Kim, H.‐Y., Yang, D.‐H., Shin, S.‐W., Kim, M.‐Y., Yoon, J.‐H., Kim, S., Park, H.‐C., Kang, D. W., Min, D., Hur, M.‐W., Choi, K‐Y. CXXC5 is a transcriptional activator of Flk‐1 and mediates bone morphogenic protein‐induced endothelial cell differentiation and vessel formation. FASEB J. 28, 615–626 (2014). www.fasebj.org

Hovenia dulcis Thunb Extract and Its Ingredient Methyl Vanillate Activate Wnt/β-Catenin Pathway and Increase Bone Mass in Growing or Ovariectomized Mice

The Wnt/β-catenin pathway is a potential target for development of anabolic agents to treat osteoporosis because of its role in osteoblast differentiation and bone formation. However, there is no clinically available anti-osteoporosis drug that targets this Wnt/β-catenin pathway. In this study, we screened a library of aqueous extracts of 350 plants and identified Hovenia dulcis Thunb (HDT) extract as a Wnt/β-catenin pathway activator. HDT extract induced osteogenic differentiation of calvarial osteoblasts without cytotoxicity. In addition, HDT extract increased femoral bone mass without inducing significant weight changes in normal mice. In addition, thickness and area of femoral cortical bone were also significantly increased by the HDT extract. Methyl vanillate (MV), one of the ingredients in HDT, also activated the Wnt/β-catenin pathway and induced osteoblast differentiation in vitro. MV rescued trabecular or cortical femoral bone loss in the ovariectomized mice without inducing any significant weight changes or abnormality in liver tissue when administrated orally. Thus, natural HDT extract and its ingredient MV are potential anabolic agents for treating osteoporosis.

The small molecule indirubin-3′-oxime activates Wnt/β-catenin signaling and inhibits adipocyte differentiation and obesity

Objectives:Activation of the Wnt/β-catenin signaling pathway inhibits adipogenesis by maintaining preadipocytes in an undifferentiated state. We investigated the effect of indirubin-3′-oxime (I3O), which was screened as an activator of the Wnt/β-catenin signaling, on inhibiting the preadipocyte differentiation in vitro and in vivo.Methods:3T3L1 preadipocytes were differentiated with 0, 4 or 20u2009μM of I3O. The I3O effect on adipocyte differentiation was observed by Oil-red-O staining. Activation of Wnt/β-catenin signaling in I3O-treated 3T3L1 cells was shown using immunocytochemical and immunoblotting analyses for β-catenin. The regulation of adipogenic markers was analyzed via real-time reverse transcription-PCR (RT-PCR) and immunoblotting analyses. For the in vivo study, mice were divided into five different dietary groups: chow diet, high-fat diet (HFD), HFD supplemented with I3O at 5, 25 and 100u2009mgu2009kg−1. After 8 weeks, adipose and liver tissues were excised from the mice and subject to morphometry, real-time RT-PCR, immunoblotting and histological or immunohistochemical analyses. In addition, adipokine and insulin concentrations in serum of the mice were accessed by enzyme-linked immunosorbent assay.Results:Using a cell-based approach to screen a library of pharmacologically active small molecules, we identified I3O as a Wnt/β-catenin pathway activator. I3O inhibited the differentiation of 3T3-L1 cells into mature adipocytes and decreased the expression of adipocyte markers, CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ, at both mRNA and protein levels. In vivo, I3O inhibited the development of obesity in HFD-fed mice by attenuating HFD-induced body weight gain and visceral fat accumulation without showing any significant toxicity. Factors associated with metabolic disorders such as hyperlipidemia and hyperglycemia were also improved by treatment of I3O.Conclusion:Activation of the Wnt/β-catenin signaling pathway can be used as a therapeutic strategy for the treatment of obesity and metabolic syndrome and implicates I3O as a candidate anti-obesity agent.

House dust mite allergen der f 2 induces interleukin-13 expression by activating the PI3K/Akt pathway

House dust mites (HDMs) are a common cause of allergic asthma. The group 2 allergen from Dermatophagoides farinae, Der f 2, is one of the major HDM allergens. Elevated Der f 2 immunoglobulin E (IgE) levels are observed in most of the allergic patients. Interleukin-13 (IL-13), a gene associated with asthma pathology, was induced by Der f 2 in BEAS-2B human airway epithelial cells; however, the signaling pathways associated with Der f 2 are not fully understood. In this study, we identified a role of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway, a well-known potential target for anti-asthma drugs, in the IL-13 induction by Der f 2. First, Der f 2 activated the PI3K/Akt pathway, which subsequently activated the nuclear factor-kappa B (NF-κB) pathway and induced IL-13 expression in BEAS-2B cells. Treatment with the PI3K inhibitor LY294002 abolished Der f 2-induced activation of Akt and NF-κB and the expression of IL-13. Furthermore, Der f 2-induced activation of the PI3K/Akt and NF-κB pathways, expression of IL-13, and the blockade of these effects with a PI3K inhibitor were confirmed in the lungs of mice that were intranasally exposed to Der f 2. Taken together, these results indicate that the PI3K/Akt pathway regulates Der f 2-induced IL-13 expression via activation of the NF-κB pathway.

Small-molecule binding of the axin RGS domain promotes β-catenin and Ras degradation.

Both the Wnt/β-catenin and Ras pathways are aberrantly activated in most human colorectal cancers (CRCs) and interact cooperatively in tumor promotion. Inhibition of these signaling may therefore be an ideal strategy for treating CRC. We identified KY1220, a compound that destabilizes both β-catenin and Ras, via targeting the Wnt/β-catenin pathway, and synthesized its derivative KYA1797K. KYA1797K bound directly to the regulators of G-protein signaling domain of axin, initiating β-catenin and Ras degradation through enhancement of the β-catenin destruction complex activating GSK3β. KYA1797K effectively suppressed the growth of CRCs harboring APC and KRAS mutations, as shown by various in vitro studies and by in vivo studies using xenograft and transgenic mouse models of tumors induced by APC and KRAS mutations. Destabilization of both β-catenin and Ras via targeting axin is a potential therapeutic strategy for treatment of CRC and other type cancers activated Wnt/β-catenin and Ras pathways.

Small molecule inhibitors of the Dishevelled‐CXXC5 interaction are new drug candidates for bone anabolic osteoporosis therapy

Bone anabolic agents promoting bone formation and rebuilding damaged bones would ideally overcome the limitations of anti‐resorptive therapy, the current standard prescription for osteoporosis. However, the currently prescribed parathyroid hormone (PTH)‐based anabolic drugs present limitations and adverse effects including osteosarcoma during long‐term use. Also, the antibody‐based anabolic drugs that are currently being developed present the potential limits in clinical application typical of macromolecule drugs. We previously identified that CXXC5 is a negative feedback regulator of the Wnt/β‐catenin pathway via its interaction with Dishevelled (Dvl) and suggested the Dvl–CXXC5 interaction as a potential target for anabolic therapy of osteoporosis. Here, we screened small‐molecule inhibitors of the Dvl–CXXC5 interaction via a newly established in vitro assay system. The screened compounds were found to activate the Wnt/β‐catenin pathway and enhance osteoblast differentiation in primary osteoblasts. The bone anabolic effects of the compounds were shown using ex vivo‐cultured calvaria. Nuclear magnetic resonance (NMR) titration analysis confirmed interaction between Dvl PDZ domain and KY‐02061, a representative of the screened compounds. Oral administration of KY‐02327, one of 55 newly synthesized KY‐02061 analogs, successfully rescued bone loss in the ovariectomized (OVX) mouse model. In conclusion, small‐molecule inhibitors of the Dvl–CXXC5 interaction that block negative feedback regulation of Wnt/β‐catenin signaling are potential candidates for the development of bone anabolic anti‐osteoporosis drugs.

CXXC5 plays a role as a transcription activator for myelin genes on oligodendrocyte differentiation

Myelination in corpus callosum plays important role for normal brain functions by transferring neurological information between various brain regions. However, the factors controlling expression of myelin genes in myelination are poorly understood. Here, CXXC5, a recently identified protein with CXXC‐type zinc finger DNA binding motif, was characterized as a transcriptional activator of major myelin genes. We identified expression of CXXC5 expression was increased by Wnt/β‐catenin signaling. CXXC5 specifically expressed in the white matter induced expression of myelin genes through the direct binding of CXXC DNA‐binding motif of CXXC5 on the MBP promoter. During the differentiation of neural stem cells (NSCs) of CXXC5−/− mice, the expressions of myelin genes were simultaneously reduced. The CXXC5−/− mice exhibited severely reduction of myelin genes expression in corpus callosum as well as abnormalities in myelin structure. The disrupted structural integrity of myelin in the CXXC5−/− mice resulted in reduced electrical conduction amplitudes at corpus callosum. These findings indicate that the regulation of myelin genes expression by CXXC5 is important for forming myelin structure involved with axonal electrical signal transfer in the corpus callosum. GLIA 2016;64:350–362