Wnt4 activates the canonical β-catenin pathway and regulates negatively myostatin: functional implication in myogenesis

Expression of Wnt proteins is known to be important for developmental processes such as embryonic pattern formation and determination of cell fate. Previous studies have shown that Wn4 was involved in the myogenic fate of somites, in the myogenic proliferation, and differentiation of skeletal muscle. However, the function of this factor in adult muscle homeostasis remains not well understood. Here, we focus on the roles of Wnt4 during C2C12 myoblasts and satellite cells differentiation. We analyzed its myogenic activity, its mechanism of action, and its interaction with the anti-myogenic factor myostatin during differentiation. Established expression profiles indicate clearly that both types of cells express a few Wnts, and among these, only Wnt4 was not or barely detected during proliferation and was strongly induced during differentiation. As attested by myogenic factors expression pattern analysis and fusion index determination, overexpression of Wnt4 protein caused a strong increase in satellite cells and C2C12 myoblast differentiation leading to hypertrophic myotubes. By contrast, exposure of satellite and C2C12 cells to small interfering RNA against Wnt4 strongly diminished this process, confirming the myogenic activity of Wnt4. Moreover, we reported that Wnt4, which is usually described as a noncanonical Wnt, activates the canonical β-catenin pathway during myogenic differentiation in both cell types and that this factor regulates negatively the expression of myostatin and the regulating pathways associated with myostatin. Interestingly, we found that recombinant myostatin was sufficient to antagonize the differentiation-promoting activities of Wnt4. Reciprocally, we also found that the genetic deletion of myostatin renders the satellite cells refractory to the hypertrophic effect of Wnt4. These results suggest that the Wnt4-induced decrease of myostatin plays a functional role during hypertrophy. We propose that Wnt4 protein may be a key factor that regulates the extent of differentiation in satellite and C2C12 cells.     

SNX25 regulates TGF-β signaling by enhancing the receptor degradation

SNXs (sorting nexin), a family of proteins playing roles in cargo sorting and signaling from compartments within the endocytic network, regulate traffic of membrane proteins including TGF-β receptors. Here we report that the full length human and mouse SNX25, a SNX member with PX, PXA and RGS domains, co-localizes with TGF-β receptors, and forms internalized cytosolic punctae upon treatment with TGF-β. While overexpression of SNX25 inhibits TGF-β induced luciferase reporter activity, knocking down endogenous SNX25 by siRNA in NIH3T3 cells elevates the TGF-β receptor levels and facilitates TGF-β signaling. Immunoprecipitation experiments demonstrate that SNX25 interacts with TβRI. Western blot analyses indicate that SNX25 enhances the degradation of TGF-β receptors. SNX25 induced TGF-β receptor degradation is shown via the clathrin dependent endocytosis pathway into lysosome. We have characterized that PXA domain of SNX25 is required for the degradation of TβRI. Our findings demonstrate that SNX25 negatively regulates TGF-β signaling by enhancing the receptor degradation through lysosome pathway.     

An F-box protein, FBXW5, negatively regulates TAK1 MAP3K in the IL-1β signaling pathway

TAK1, a member of the MAP3K family, plays an essential role in activation of JNK/p38 MAPKs and IKK in the IL-1β and TNFα signaling pathway. Upon stimulation, TAK1 is rapidly and transiently activated. While the activation mechanism of TAK1 in these signaling pathways is well characterized, how its activity is terminated still remains unclear. To identify the molecule(s) involved in TAK1 regulation, we performed tandem affinity purification (TAP) in HeLa cells stably expressing TAP-tagged TAK1. FBXW5, an F-box family protein, was identified as a previously unknown component of the IL-1β-induced TAK1 complex. FBXW5 associated with endogenous TAK1 in an IL-1β-dependent manner. Overexpression of FBXW5 inhibited IL-1β-induced activation of JNK/p38 MAPKs and NF-κB as well as phosphorylation of TAK1 on Thr187. Conversely, knockdown of FBXW5 resulted in the prolonged activation of TAK1 upon IL-1β stimulation. These results suggest that FBXW5 negatively regulates TAK1 in the IL-1β signaling pathway.     

Niclosamide suppresses cancer cell growth by inducing Wnt co-receptor LRP6 degradation and inhibiting the Wnt/β-catenin pathway

The Wnt/β-catenin signaling pathway is important for tumor initiation and progression. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling and represents a promising anticancer target. Recently, the antihelminthic drug, niclosamide was found to inhibit Wnt/β-catenin signaling, although the mechanism was not well defined. We found that niclosamide was able to suppress LRP6 expression and phosphorylation, block Wnt3A-induced β-catenin accumulation, and inhibit Wnt/β-catenin signaling in HEK293 cells. Furthermore, the inhibitory effects of niclosamide on LRP6 expression/phosphorylation and Wnt/β-catenin signaling were conformed in human prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. Moreover, we showed that the mechanism by which niclosamide suppressed LRP6 resulted from increased degradation as evident by a shorter half-life. Finally, we demonstrated that niclosamide was able to induce cancer cell apoptosis, and displayed excellent anticancer activity with IC(50) values less than 1 μM for prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. The IC(50) values are comparable to those shown to suppress the activities of Wnt/β-catenin signaling in prostate and breast cancer cells. Our data indicate that niclosamide is a unique small molecule Wnt/β-catenin signaling inhibitor targeting the Wnt co-receptor LRP6 on the cell surface, and that niclosamide has a potential to be developed a novel chemopreventive or therapeutic agent for human prostate and breast cancer.     

Cholest-4-en-3-one attenuates TGF-β responsiveness by inducing TGF-β receptors degradation in Mv1Lu cells and colorectal adenocarcinoma cells

Purpose: The transforming growth factor-beta (TGF-β) pathway is an important in the initiation and progression of cancer. Due to a strong association between an elevated colorectal cancer risk and increase fecal excretion of cholest-4-en-3-one, we aim to determine the effects of cholest-4-en-3-one on TGF-β signaling in the mink lung epithelial cells (Mv1Lu) and colorectal cancer cells (HT29) in vitro.

Methods: The inhibitory effects of cholest-4-en-3-one on TGF-β-induced Smad signaling, cell growth inhibition, and the subcellular localization of TGF-β receptors were investigated in epithelial cells using a Western blot analysis, luciferase reporter assays, DNA synthesis assay, confocal microscopy, and subcellular fractionation.

Results: Cholest-4-en-3-one attenuated TGF-β signaling in Mv1Lu cells and HT29 cells, as judged by a TGF-β-specific reporter gene assay of plasminogen activator inhibitor-1 (PAI-1), Smad2/3 phosphorylation and nuclear translocation. We also discovered that cholest-4-en-3-one suppresses TGF-β responsiveness by increasing lipid raft and/or caveolae accumulation of TGF-β receptors and facilitating rapid degradation of TGF-β and thus suppressing TGF-β-induced signaling.

Conclusions: Our results suggest that cholest-4-en-3-one inhibits TGF-β signaling may be due, in part to the translocation of TGF-β receptor from non-lipid raft to lipid raft microdomain in plasma membranes. Our findings also implicate that cholest-4-en-3-one may be further explored for its potential role in colorectal cancer correlate to TGF-β deficiency.     

RIOK3 interacts with caspase-10 and negatively regulates the NF-κB signaling pathway

RIOK3 was initially characterized as a homolog of Aspergillus nidulans sudD and showed down-regulation at the invasive front of malignant melanomas, but the molecular mechanism remains elusive. Here, we report that overexpression of RIOK3 inhibits TNFα-induced NF-κB activation, but down-regulation of endogenous RIOK3 expression by siRNA potentiates it. A yeast two-hybrid experiment revealed that RIOK3 interacted with caspase-10, and further, a GST pull-down assay and endogenous coimmunoprecipitation validated the interaction. We subsequently showed that the interaction was mediated by the RIO domain of RIOK3 and each death effector domain of caspase-10. Interestingly, our data demonstrated that RIOK3 suppressed caspase-10-mediated NF-κB activation by competing RIP1 and NIK to bind to caspase-10. Importantly, the kinase activity of RIOK3 was confirmed to be relevant to NF-κB signaling. Taken together, our findings strongly suggest that RIOK3 negatively regulates NF-κB signaling pathway activated by TNFα dependent on its kinase activity and NF-κB signaling pathway activated by caspase-10 independent of its kinase activity.     

TET1 suppresses colon cancer proliferation by impairing β-catenin signal pathway

The function of ten-eleven translocation methylcytosine dioxygenase 1 (TET1) in cancer is background dependent and may be involved in the initial step of active DNA demethylation, while there is little research to decipher the role of TET1 in DNA methylation-sensitive colon cancer. Downregulated TET1 expression assayed by quantitative real-time PCR (qRT-PCR) was observed in both colon cancer samples and cancer cell lines of HT29, HCT116, and SW48. Such downregulation could promote colon cancer cells proliferation as indicated by the fact that shTET1 could increase the viability of HT29 and HCT116 cells determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and cell count assay accompanied with upregulation of β-catenin (CTNNB1) and WNT luciferase activity, which was further confirmed as shTET1 could increase the tumor volume and tumor weight, and decrease the body weight in HT29 cells inoculated BALB/C nude mice. The CTNNB1 transfection could rescue the cell growth diminished by normal expression of TET1. shTET1 could promote axis inhibition protein1 (AXIN1) expression and the cell proliferation effect induced by TET1 short hairpin RNA was attenuated by co-inhibition of AXIN1. All of these indicate that TET1 can suppress colon cancer proliferation and the inhibition of the β-catenin pathway is AXIN1 dependent.     

Neopterin negatively regulates expression of ABCA1 and ABCG1 by the LXRα signaling pathway in THP-1 macrophage-derived foam cells

To investigate the effects of neopterin on ABCA1 expression and cholesterol efflux in human THP-1 macrophage-derived foam cells, and to explore the role of the liver X receptor alpha (LXRα) involved. In the present study, THP-1 cells were pre-incubated with ox-LDL to become foam cells. The protein and mRNA expression were examined by Western blot assays and real-time quantitative PCR, respectively. Liquid scintillation counting and high performance liquid chromatography assays were used to test cellular cholesterol efflux and cholesterol content. Neopterin decreased ABCA1 expression and cholesterol efflux in a time- and concentration-dependent manner in THP-1 macrophage-derived foam cells, and the LXRα siRNA can reverse the inhibitory effects induced by neopterin. Neoterin has a negative regulation on ABCA1 expression via the LXRα signaling pathway, which suggests the aggravated effects of neopterin on atherosclerosis.     

Plk1 regulates mitotic Aurora A function through βTrCP-dependent degradation of hBora

Polo-like kinase 1 (Plk1) and Aurora A play key roles in centrosome maturation, spindle assembly, and chromosome segregation during cell division. Here we show that the functions of these kinases during early mitosis are coordinated through Bora, a partner of Aurora A first identified in Drosophila. Depletion of human Bora (hBora) results in spindle defects, accompanied by increased spindle recruitment of Aurora A and its partner TPX2. Conversely, hBora overexpression induces mislocalization of Aurora A and monopolar spindle formation, reminiscent of the phenotype seen in Plk1-depleted cells. Indeed, Plk1 regulates hBora. Following Cdk1-dependent recruitment, Plk1 triggers hBora destruction by phosphorylating a recognition site for [Formula: see text]. Plk1 depletion or inhibition results in a massive accumulation of hBora, concomitant with displacement of Aurora A from spindle poles and impaired centrosome maturation, but remarkably, co-depletion of hBora partially restores Aurora A localization and bipolar spindle formation. This suggests that Plk1 controls Aurora A localization and function by regulating cellular levels of hBora.     

Tripartite motif-containing protein 38 negatively regulates TLR3/4- and RIG-I-mediated IFN-β production and antiviral response by targeting NAP1

Recognition of RNA virus through TLR and RIG-I-like receptor results in rapid expression of type I IFNs, which play an essential role in host antiviral responses. However, the mechanisms to terminate the production of type I IFNs are not well defined. In the current study, we identified a member of the tripartite motif (TRIM) family, TRIM38, as a negative regulator in TLR3/4- and RIG-I-mediated IFN-β signaling. Knockdown of TRIM38 expression by small interfering RNA resulted in augmented activation of IFN regulatory factor 3 and enhanced expression of IFN-β, whereas overexpression of TRIM38 had opposite effects. Coimmunoprecipitation and colocalization experiments demonstrated that TRIM38 interacted with NF-κB-activating kinase-associated protein 1 (NAP1), which is required for TLR-induced IFN regulatory factor 3 activation and IFN-β production. As an E3 ligase, TRIM38 promoted K48-linked polyubiquitination and proteasomal degradation of NAP1. Thus, knockdown of TRIM38 expression resulted in higher protein level of NAP1 in primary macrophages. Consistent with the inhibitory roles in TLR3/4- and RIG-I-mediated IFN-β signaling, knockdown of TRIM38 significantly inhibited the replication of vesicular stomatitis virus. Overexpression of TRIM38 resulted in enhanced replication of vesicular stomatitis virus. Therefore, our results demonstrate that TRIM38 is a negative regulator for TLR and RIG-I-mediated IFN-β production by targeting NAP1 for ubiquitination and subsequent proteasome-mediated degradation.