Pharmacologic Inhibition of Porcupine, Disheveled, and ß-Catenin in Wnt Signaling Pathway Ameliorates Diabetic Peripheral Neuropathy in Rats.

Wnt signaling pathway has been investigated extensively for its diverse metabolic and pain-modulating mechanisms; recently its involvement has been postulated in the development of neuropathic pain. However, there are no reports as yet on the involvement of Wnt signaling pathway in one of the most debilitating neurovascular complication of diabetes, namely, diabetic peripheral neuropathy (DPN). Thus, in the present study, involvement of Wnt signaling was investigated in DPN using Wnt signaling inhibitors namely LGK974 (porcupine inhibitor), NSC668036 (disheveled inhibitor), and PNU74654 (ß-catenin inhibitor). Diabetes was induced by a single intraperitoneal injection of streptozotocin (50 mg/kg) to male Sprague-Dawley rats. Diabetic rats after 6 weeks of diabetes induction showed increased expression of Wnt signaling proteins in the spinal cord (L4-L6 lumbar segment), dorsal root ganglions and sciatic nerves. Subsequent increase in inflammation, endoplasmic reticulum stress and loss of intraepidermal nerve fiber density was also observed, leading to neurobehavioral and nerve functional deficits in diabetic rats. Intrathecal administration of Wnt signaling inhibitors (each at doses of 10 and 30 µmol/L) in diabetic rats showed improvement in pain-associated behaviors (heat, cold, and mechanical hyperalgesia) and nerve functions (motor, sensory nerve conduction velocities, and nerve blood flow) by decreasing the expression of Wnt pathway proteins, inflammatory marker, matrix metalloproteinase 2, endoplasmic reticulum stress marker, glucose-regulated protein 78, and improving intraepidermal nerve fiber density. All these results signify the neuroprotective potential of Wnt signaling inhibitors in DPN. PERSPECTIVE: This study emphasizes the involvement of Wnt signaling pathway in DPN. Blockade of this pathway using Wnt inhibitors provided neuroprotection in experimental DPN in rats. This study may provide a basis for exploring the therapeutic potential of Wnt inhibitors in DPN patients.

Discovery of a small-molecule inhibitor of Dvl-CXXC5 interaction by computational approaches.

The Wnt/ß-catenin signaling pathway plays a significant role in the control of osteoblastogenesis and bone formation. CXXC finger protein 5 (CXXC5) has been recently identified as a negative feedback regulator of osteoblast differentiation through a specific interaction with Dishevelled (Dvl) protein. It was reported that targeting the Dvl-CXXC5 interaction could be a novel anabolic therapeutic target for osteoporosis. In this study, complex structure of Dvl PDZ domain and CXXC5 peptide was simulated with molecular dynamics (MD). Based on the structural analysis of binding modes of MD-simulated Dvl PDZ domain with CXXC5 peptide and crystal Dvl PDZ domain with synthetic peptide-ligands, we generated two different pharmacophore models and applied pharmacophore-based virtual screening to discover potent inhibitors of the Dvl-CXXC5 interaction for the anabolic therapy of osteoporosis. Analysis of 16 compounds selected by means of a virtual screening protocol yielded four compounds that effectively disrupted the Dvl-CXXC5 interaction in the fluorescence polarization assay. Potential compounds were validated by fluorescence spectroscopy and nuclear magnetic resonance. We successfully identified a highly potent inhibitor, BMD4722, which directly binds to the Dvl PDZ domain and disrupts the Dvl-CXXC5 interaction. Overall, CXXC5-Dvl PDZ domain complex based pharmacophore combined with various traditional and simple computational methods is a promising approach for the development of modulators targeting the Dvl-CXXC5 interaction, and the potent inhibitor BMD4722 could serve as a starting point to discover or design more potent and specific the Dvl-CXXC5 interaction disruptors.

Non-canonical Wnt induces chondrocyte de-differentiation through Frizzled 6 and DVL-2/B-raf/CaMKIIα/syndecan 4 axis.

Dysregulation of Wnt signaling has been implicated in developmental defects and in the pathogenesis of many diseases such as osteoarthritis; however, the underlying mechanisms are poorly understood. Here, we report that non-canonical Wnt signaling induced loss of chondrocyte phenotype through activation of Fz-6/DVL-2/SYND4/CaMKIIα/B-raf/ERK1/2 cascade. We show that in response to Wnt-3a, Frizzled 6 (Fz-6) triggers the docking of CaMKIIα to syndecan 4 (SYND4) and that of B-raf to DVL-2, leading to the phosphorylation of B-raf by CaMKIIα and activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling, which leads to chondrocyte de-differentiation. We demonstrate that CaMKIIα associates and phosphorylates B-raf in vitro and in vivo. Our study reveals the mechanism by which non-canonical Wnt activates ERK1/2 signaling that induces loss of chondrocyte phenotype, and demonstrates a direct functional relationship between CaMKIIα and B-raf during chondrocyte de-differentiation. The identification of Fz-6, SYND4, and B-raf as novel physiological regulators of chondrocyte phenotype may provide new potential anti-osteoarthritic targets.

The preventive effects of taurine on neural tube defects through the Wnt/PCP-Jnk-dependent pathway.

The aim of this study was to clarify the protective role of taurine in neuronal apoptosis and the role of the Wnt/PCP-Jnk pathway in mediating the preventive effects of taurine on neural tube defects (NTDs). HT-22 cells (a hippocampal neuron cell line) were divided into a control group, a glutamate-induced apoptosis group, and glutamate (4.0 mmol/L) plus low-dose taurine (L; 0.5 mmol/L) and high-dose taurine (H; 2.0 mmol/L) groups. The MTT assay was used to monitor cell proliferation and cell survival. Immunofluorescence and Western blot analyses were used to determine caspase 9 expression. Retinoic acid (RA) induced embryonic NTDs in Kunming mice, thus establishing an NTD model. Pregnant mice were divided into a control group, an RA (30 mg/kg body weight) group, and an RA (30 mg/kg body weight) plus taurine (free drinking of 2 g/L solution) group. Immunohistochemistry and Western blot analyses were used to detect the expression of Dvl, RhoA and phosphorylated (p)-Jnk/Jnk in the embryonic neural tubes. In HT-22 cells, the apoptosis rate was significantly higher and caspase 9 activation was also significantly increased in the glutamate-induced apoptosis group compared to the L and H taurine groups. In the NTD model, the expression levels of Dvl, RhoA, and p-Jnk were significantly higher in the RA group than in the control group, whereas they were significantly reduced in the RA + taurine group. This study suggests that taurine has positive effects on neuronal protection and NTD prevention. Moreover, the Wnt/PCP-Jnk-dependent pathway plays an important role in taurine-mediated prevention of NTDs.

The Fungal Metabolite Brefeldin A Inhibits Dvl2-Plk1-Dependent Primary Cilium Disassembly.

The primary cilium is a non-motile microtubule-based organelle that protrudes from the surface of most human cells and works as a cellular antenna to accept extracellular signals. Primary cilia assemble from the basal body during the resting stage (G0 phase) and simultaneously disassemble with cell cycle re-entry. Defective control of assembly or disassembly causes diverse human diseases including ciliopathy and cancer. To identify the effective compounds for studying primary cilium disassembly, we have screened 297 natural compounds and identified 18 and 17 primary cilium assembly and disassembly inhibitors, respectively. Among them, the application of KY-0120, identified as Brefeldin A, disturbed Dvl2-Plk1-mediated cilium disassembly via repression of the interaction of CK1ɛ-Dvl2 and the expression of Plk1 mRNA. Therefore, our study may suggest useful compounds for studying the cellular mechanism of primary cilium disassembly to prevent ciliopathy and cancer.

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/ß-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/ß-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/ß-catenin signaling are potential candidates for the development of bone anabolic anti-osteoporosis drugs.

Cyclin G2 inhibits epithelial-to-mesenchymal transition by disrupting Wnt/ß-catenin signaling.

Epithelial ovarian cancer (EOC) has the highest mortality rate among gynecological malignancies owing to poor screening methods, non-specific symptoms and limited knowledge of the cellular targets that contribute to the disease. Cyclin G2 is an unconventional cyclin that acts to oppose cell cycle progression. Dysregulation of the cyclin G2 gene (CCNG2) in a variety of human cancers has been reported; however, the role of cyclin G2 in tumorigenesis remains unclear. In this study, we investigated the function of cyclin G2 in EOC. In vitro and in vivo studies using several EOC-derived tumor cell lines revealed that cyclin G2 inhibited cell proliferation, migration, invasion and spheroid formation, as well as tumor formation and invasion. By interrogating cDNA microarray data sets, we found that CCGN2 mRNA is reduced in several large cohorts of human ovarian carcinoma when compared with normal ovarian surface epithelium or borderline tumors of the ovary. Mechanistically, cyclin G2 was found to suppress epithelial-to-mesenchymal transition (EMT), as demonstrated by the differential regulation of various EMT genes, such as Snail, Slug, vimentin and E-cadherin. Moreover, cyclin G2 potently suppressed the Wnt/ß-catenin signaling pathway by downregulating key Wnt components, namely LRP6, DVL2 and ß-catenin, which could be linked to inhibition of EMT. Taken together, our novel findings demonstrate that cyclin G2 has potent tumor-suppressive effects in EOCs by inhibiting EMT through attenuating Wnt/ß-catenin signaling.