Chemotherapy-Induced Long Non-coding RNA 1 Promotes Metastasis and Chemo-Resistance of TSCC via the Wnt/ß-Catenin Signaling Pathway.

Increasing evidence has shown that chemo-resistance is related to the process of epithelial-mesenchymal transition (EMT) and increased invasiveness by tongue squamous cell carcinoma (TSCC) cells. Long non-coding RNAs (lncRNAs) play pivotal roles in tumor metastasis and progression. However, the roles and mechanisms of lncRNAs in cisplatin-resistance-induced EMT and metastasis are not well understood. In this study, a chemotherapy-induced lncRNA 1 (CILA1) was discovered by using microarrays and was functionally identified as a regulator of chemo-sensitivity in TSCC cells. Upregulation of CILA1 promotes EMT, invasiveness, and chemo-resistance in TSCC cells, whereas the inhibition of CILA1 expression induces mesenchymal-epithelial transition (MET) and chemo-sensitivity, and inhibits the invasiveness of cisplatin-resistant cells both in vitro and in vivo. We also found that CILA1 exerts its functions via the activation of the Wnt/ß-catenin signaling pathway. High CILA1 expression levels and low levels of phosphorylated ß-catenin were closely associated with cisplatin resistance and advanced disease stage, and were predictors of poor prognosis in TSCC patients. These findings provided a new biomarker for the chemo-sensitivity of TSCC tumors and a therapeutic target for TSCC treatment.

Determination of Rate-Limiting Factor for Formation of Beta-Catenin Destruction Complexes Using Absolute Protein Quantification.

Wnt/ß-catenin signaling plays important roles in both ontogenesis and development. In the absence of a Wnt stimulus, ß-catenin is degraded by a multiprotein "destruction complex" that includes Axin, APC, GSK3B, and FBXW11. Although the key molecules required for transducing Wnt signals have been identified, a quantitative understanding of this pathway has been lacking. Here, we calculated the absolute number of ß-catenin destruction complexes by absolute protein quantification using LC-MS/MS. Similar amounts of destruction complex-constituting proteins and ß-catenin interacted, and the number of destruction complexes was calculated to be about 1468 molecules/cell. We demonstrated that the calculated number of destruction complexes was valid for control of the ß-catenin destruction rate under steady-state conditions. Interestingly, APC had the minimum expression level among the destruction complex components at about 2233 molecules/cell, and this number approximately corresponded to the calculated number of destruction complexes. Decreased APC expression by siRNA transfection decreased the number of destruction complexes, resulting in ß-catenin accumulation and stimulation of the transcriptional activity of T-cell factor. Taken together, our results suggest that the amount of APC expression is the rate-limiting factor for the constitution of ß-catenin destruction complexes.

A Simple Method to Assess Abundance of the ß-Catenin Signaling Pool in Cells.

ß-catenin (CTNNB1) is a dual-function cell-cell adhesion/transcriptional co-activator protein and an essential transducer of canonical Wnt signals. Although a number of established techniques and reagents are available to quantify the nuclear signaling activity of ß-catenin (e.g., TCF-dependent reporter assays, nuclear accumulation of ß-catenin, and generation of N-terminally hypophosphorylated ß-catenin), there are cell-type and context-dependent limitations of these methods. Since the posttranscriptional stabilization of ß-catenin outside of the cadherin complex appears universally required for ß-catenin signaling, the following method allows for simple assessment of the cadherin-free fraction of ß-catenin in cells, using a GST-tagged form of ICAT (Inhibitor of ß-Catenin and Tcf) as an affinity matrix. This method is more sensitive and quantitative than immunofluorescence and may be useful in studies that implicate TCF-independent signaling events.

Isolation and nucleotide sequence analysis of the of Rhinella arenarum ß-catenin: an mRNA and protein expression study during the larval stages of the digestive tract development.

ß-catenin interacts with several proteins mediating key biological processes, such as cadherin-mediated cell-cell adhesion as well as signal transduction. This work was done to establish the molecular basis and regulation of the formation pattern of cadherin/ß-catenin-mediated adherens junctions, using an animal model of unknown gene sequence, the toad Rhinella arenarum. A Rhinella arenarum ß-catenin homolog was isolated from larval tissue, their sequence compared and analyzed with those of eight other vertebrates using bioinformatics tools. The mRNA and protein expression levels of ß-catenin were determined during the development of Rhinella arenarum digestive tract both by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and immunohistochemistry-morphometry respectively. Using Xenopus laevis frog specific primers, a fragment 539 bp of Rhinella arenarum toad ß-catenin cDNA was obtained and sequenced. The resulting putative sequence of 177 amino acids showed high similarity at the amino acid level (97%) when compared to other six vertebrates (Xenopus laevis, Xenopus tropicalis, Mus musculus, Rattus norvegicus, Bos taurus and Homo sapiens), with sequences and structural domains characteristic of catenins. Subsequently, using primers specifically designed for Rhinella arenarum nucleotide sequence, ß-catenin-mRNA increasing levels were found during the Rhinella arenarum metamorphosis. Finally, increasing ß-catenin protein expression during development has confirmed the specificity the detection of Rhinella arenarum ß-catenin. Summarizing, we have isolated and sequenced a ß-catenin-homologue sequence from the Rhinella arenarum toad, which is highly conserved between species, and following we have detected ß-catenin mRNA and protein levels during their digestive tract development.

Functional phosphosite screening for targeted protein-protein interactions by combining phosphoproteomics strategies and mammalian two-hybrid assays.

An effective new platform for phosphosite mapping and subsequent functional screening was developed to analyze the targeted protein-protein interactions of p300 and CBP with ß-catenin. Two novel functional phosphosites, Ser12 of p300 and Ser92 of CBP, were revealed to modulate p300/ß-catenin and CBP/ß-catenin interactions, respectively.

Activated beta-catenin induces myogenesis and inhibits adipogenesis in BM-derived mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells (MSC) have been thought to be attractive candidates for the treatment of degenerative muscle diseases. However, little is known about the molecular mechanisms governing the myogenic differentiation in MSC. As the Wnt signaling pathway has been associated with myogenesis in embryogenesis and post-natal muscle regeneration, we hypothesized that the Wnt signaling pathway may be involved in governing the myogenic differentiation in MSC. METHODS: Primary MSC were isolated from Sprague-Dawley rats and expanded in proliferation medium. The rMSC were transfected with a constitutively active hbeta-catenin (S37A) plasmid or control vector by Lipofectamine followed by G418 selection. The transfected rMSC were grown to 80% confluence and then cultured in myogenic or adipogenic differentiation medium. Cells were characterized by light microscopy, immunofluorescence and RT-PCR at different time points after myogenic or adipogenic introduction. RESULTS: Ectopic expression of activated beta-catenin located primarily in the nucleus and activated transcription in rMSC. Overexpression of stabilized beta-catenin induced 27.1 +/- 3.91% rMSC forming long multinucleated cells expressing MyoD, myogenin, desmin and myosin heavy chain (MHC) via evoking the expression of skeletal muscle-specific transcription factors. In addition, overexpression of activated beta-catenin inhibited the adipogenic differentiation in rMSC through down-regulated expressions of C/EBPalpha and PPARgamma. DISCUSSION: To our knowledge, this is the first evidence that activated beta-catenin can induce myogenic differentiation in rMSC. The ability of stabilized beta-catenin to induce myogenic differentiation in rMSC may allow for its therapeutic application.

PKCdelta and cofilin activation affects peripheral actin reorganization and cell-cell contact in cells expressing integrin alpha5 but not its tailless mutant.

Integrin-mediated cell adhesion transduces signaling activities for actin reorganization, which is crucially involved in cellular function and architectural integrity. In this study, we explored the possibility of whether cell-cell contacts might be regulated via integrin-alpha5beta1-mediated actin reorganization. Ectopic expression of integrin alpha5 in integrin-alpha5-null intestinal epithelial cells resulted in facilitated retraction, cell-cell contact loss, and wound healing depending on Src and PI3K (phosphoinositide 3-kinase) activities by a reagent that affects actin organization. However, cytoplasmic tailless integrin alpha5 (hereafter referred to as alpha5/1) expression caused no such effects but rather sustained peripheral actin fibers, regardless of Src and PI3K signaling activities. Furthermore, integrin alpha5 engagement with fibronectin phosphorylated Ser643 of PKCdelta, upstream of FAK and Src and at a transmodulatory loop with PI3K/Akt. Pharmacological PKCdelta inactivation, dominant-negative PKCdelta adenovirus or inactive cofilin phosphatase (SSH1L mutant) retrovirus infection of alpha5-expressing cells sustained peripheral actin organization and blocked the actin reorganizing-mediated loss of cell-cell contacts. Meanwhile, wild-type PKCdelta expression sensitized alpha5/1-expressing cells to the actin disruptor to induce cell scattering. Altogether, these observations indicate that integrin alpha5, but not alpha5/1, mediates PKCdelta phosphorylation and cofilin dephosphorylation, which in turn modulate peripheral actin organization presumably leading to an efficient regulation of cell-cell contact and migration.

Cooperative roles of Par-3 and afadin in the formation of adherens and tight junctions.

Par-3 is a cell-polarity protein that regulates the formation of tight junctions (TJs) in epithelial cells, where claudin is a major cell-cell adhesion molecule (CAM). TJs are formed at the apical side of adherens junctions (AJs), where E-cadherin and nectin are major CAMs. We have revealed that nectin first forms cell-cell adhesions, and then recruits cadherin to nectin-based cell-cell adhesion sites to form AJs and subsequently recruits claudin to the apical side of AJs to form TJs. The cytoplasmic tail of nectin binds afadin and Par-3. Afadin regulates the formation of AJs and TJs cooperatively with nectin. Here, we studied the role of Par-3 in the formation of these junctions by using Par-3-knockdown MDCK cells. Par-3 was necessary for the formation of AJs and TJs but was not necessary for nectin-based cell-cell adhesion. Par-3 promoted the association of afadin with nectin, whereas afadin was not necessary for the association of Par-3 with nectin. However, the association of afadin with nectin alone was not sufficient for the formation of AJs or TJs, and Par-3 and afadin cooperatively regulated it. We describe here these novel roles of Par-3 in the formation of junctional complexes.

Switch of cadherin expression from E- to N-type during the activation of rat hepatic stellate cells.

The activation of hepatic stellate cell (HSC) is a common pathway leading to hepatic fibrosis. However, the molecular mechanisms underlying HSC activation remain obscure. To elucidate the nature of the HSC activation, we investigated the expression of E-cadherin and its switch to N-cadherin during rat HSC activation, in vivo and in vitro. Immunohistochemical and immunocytochemical staining were performed to identify the expressions of E-cadherin, N-cadherin, and beta-catenin in rat HSCs, in vivo and in vitro. Serial changes in the expressions of these adhesion molecules during the spontaneous activation of cultured rat HSCs were also demonstrated by RT-PCR and by immunoblotting. E-cadherin and beta-catenin were expressed on opposing cell membranes of GFAP-positive rat HSCs and adjacent hepatocytes in vivo, and between desmin-positive rat HSCs in vitro. With the progression of rat HSC activation in tissue and in culture, E-cadherin disappeared gradually, whereas N-cadherin appeared at the cell periphery. The results of RT-PCR and immunoblotting were concordant with immunocytochemistry findings. In conclusion, resting rat HSCs express E-cadherin and beta-catenin both in vivo and in vitro, and E-cadherin switches to N-cadherin during HSC activation. These results suggest that HSC activation represents transdifferentiation from an epithelial to a mesenchymal phenotype.