Wnt/ß-catenin signaling activation promotes lipogenesis in the steatotic liver via physical mTOR interaction.

Background and aims: Wnt/ß-catenin signaling plays an important role in regulating hepatic metabolism. This study is to explore the molecular mechanisms underlying the potential crosstalk between Wnt/ß-catenin and mTOR signaling in hepatic steatosis. Methods: Transgenic mice (overexpress Wnt1 in hepatocytes, Wnt+) mice and wild-type littermates were given high fat diet (HFD) for 12 weeks to induce hepatic steatosis. Mouse hepatocytes cells (AML12) and those transfected to cause constitutive ß-catenin stabilization (S33Y) were treated with oleic acid for lipid accumulation. Results: Wnt+ mice developed more hepatic steatosis in response to HFD. Immunoblot shows a significant increase in the expression of fatty acid synthesis-related genes (SREBP-1 and its downstream targets ACC, AceCS1, and FASN) and a decrease in fatty acid oxidation gene (MCAD) in Wnt+ mice livers under HFD. Wnt+ mice also revealed increased Akt signaling and its downstream target gene mTOR in response to HFD. In vitro, increased lipid accumulation was detected in S33Y cells in response to oleic acid compared to AML12 cells reinforcing the in vivo findings. mTOR inhibition by rapamycin led to a down-regulation of fatty acid synthesis in S33Y cells. In addition, ß-catenin has a physical interaction with mTOR as verified by co-immunoprecipitation in hepatocytes. Conclusions: Taken together, our results demonstrate that ß-catenin stabilization through Wnt signaling serves a central role in lipid metabolism in the steatotic liver through up-regulation of fatty acid synthesis via Akt/mTOR signaling. These findings suggest hepatic Wnt signaling may represent a therapeutic strategy in hepatic steatosis.

ß-catenin and Its Relation to Alveolar Bone Mechanical Deformation - A Study Conducted in Rats With Tooth Extraction.

The aim of this study was to analyze the relationship between alveolar bone deformation and ß-catenin expression levels in response to the mechanical load changed by dental extraction in adult rats. Twenty-four male rats (Rattus norvegicus albinus), Wistar linage, at 2 months of age, were used. The right upper incisor tooth was extracted, and euthanasia occurred in periods 5 (n = 6), 7 (n = 6), and 14 (n = 6) days after Day 0. In the control group (n = 6), the dentition was maintained. The euthanasia occurred within 14 days after day 0. After euthanasia, the rats of all groups had their left jaw with tooth removed and separated in the middle. The pieces were undergone routine histological processing and then the immunohistochemical marking were performed to label expression of the primary ß-catenin antibody, which was evaluated by qualitative and quantitative analysis. One head by each group (control and experimental) was submitted to computerized microtomography. After the three-dimensional reconstruction of the skull of the rat in each group, the computational simulation for finite elements analysis were performed to simulate a bite in the incisors. In finite element analysis, the strain patterns were evaluated after the application of bite force. The results were analyzed considering the areas in which changes in the amount of deformations were detected. The action of the bite force in the experimental condition, resulted in a uniform distribution of the amount of deformations, in addition to lower amount of deformation areas, differentiating from the control group. Comparing with the control group, the levels of ß-catenin signaled in the lingual bone of the middle third of the alveolar bone were raised in the periods of 5 and 14 days. The increased ß-catenin positive staining intensity was concentrated on osteocytes and gaps of osteocytes. The findings of the present study were in accordance with our hypothesis that the condition of dental extraction can cause the expression of ß-catenin and alter the regimes of bone deformation.

Aberrant activation of hepatocyte growth factor/MET signaling promotes ß-catenin-mediated prostatic tumorigenesis.

Co-occurrence of aberrant hepatocyte growth factor (HGF)/MET proto-oncogene receptor tyrosine kinase (MET) and Wnt/ß-catenin signaling pathways has been observed in advanced and metastatic prostate cancers. This co-occurrence positively correlates with prostate cancer progression and castration-resistant prostate cancer development. However, the biological consequences of these abnormalities in these disease processes remain largely unknown. Here, we investigated the aberrant activation of HGF/MET and Wnt/ß-catenin cascades in prostate tumorigenesis by using a newly generated mouse model in which both murine Met transgene and stabilized ß-catenin are conditionally co-expressed in prostatic epithelial cells. These compound mice displayed accelerated prostate tumor formation and invasion compared with their littermates that expressed only stabilized ß-catenin. RNA-Seq and quantitative RT-PCR analyses revealed increased expression of genes associated with tumor cell proliferation, progression, and metastasis. Moreover, Wnt signaling pathways were robustly enriched in prostate tumor samples from the compound mice. ChIP-qPCR experiments revealed increased ß-catenin recruitment within the regulatory regions of the Myc gene in tumor cells of the compound mice. Interestingly, the occupancy of MET on the Myc promoter also appeared in the compound mouse tumor samples, implicating a novel role of MET in ß-catenin-mediated transcription. Results from implanting prostate graft tissues derived from the compound mice and controls into HGF-transgenic mice further uncovered that HGF induces prostatic oncogenic transformation and cell growth. These results indicate a role of HGF/MET in ß-catenin-mediated prostate cancer cell growth and progression and implicate a molecular mechanism whereby nuclear MET promotes aberrant Wnt/ß-catenin signaling-mediated prostate tumorigenesis.

Hepatocyte nuclear factor 1ß suppresses canonical Wnt signaling through transcriptional repression of lymphoid enhancer-binding factor 1.

Hepatocyte nuclear factor-1ß (HNF-1ß) is a tissue-specific transcription factor that is required for normal kidney development and renal epithelial differentiation. Mutations of HNF-1ß produce congenital kidney abnormalities and inherited renal tubulopathies. Here, we show that ablation of HNF-1ß in mIMCD3 renal epithelial cells results in activation of ß-catenin and increased expression of lymphoid enhancer-binding factor 1 (LEF1), a downstream effector in the canonical Wnt signaling pathway. Increased expression and nuclear localization of LEF1 are also observed in cystic kidneys from Hnf1b mutant mice. Expression of dominant-negative mutant HNF-1ß in mIMCD3 cells produces hyperresponsiveness to exogenous Wnt ligands, which is inhibited by siRNA-mediated knockdown of Lef1. WT HNF-1ß binds to two evolutionarily conserved sites located 94 and 30 kb from the mouse Lef1 promoter. Ablation of HNF-1ß decreases H3K27 trimethylation repressive marks and increases ß-catenin occupancy at a site 4 kb upstream to Lef1. Mechanistically, WT HNF-1ß recruits the polycomb-repressive complex 2 that catalyzes H3K27 trimethylation. Deletion of the ß-catenin-binding domain of LEF1 in HNF-1ß-deficient cells abolishes the increase in Lef1 transcription and decreases the expression of downstream Wnt target genes. The canonical Wnt target gene, Axin2, is also a direct transcriptional target of HNF-1ß through binding to negative regulatory elements in the gene promoter. These findings demonstrate that HNF-1ß regulates canonical Wnt target genes through long-range effects on histone methylation at Wnt enhancers and reveal a new mode of active transcriptional repression by HNF-1ß.

ß-Catenin mutations as determinants of hepatoblastoma phenotypes in mice.

Hepatoblastoma (HB) is the most common pediatric liver cancer. Although long-term survival of HB is generally favorable, it depends on clinical stage, tumor histology, and a variety of biochemical and molecular features. HB appears almost exclusively before the age of 3 years, is represented by seven histological subtypes, and is usually associated with highly heterogeneous somatic mutations in the catenin ß1 (CTNNB1) gene, which encodes ß-catenin, a Wnt ligand-responsive transcriptional co-factor. Numerous recurring ß-catenin mutations, not previously documented in HB, have also been identified in various other pediatric and adult cancer types. Little is known about the underlying factors that determine the above HB features and behaviors or whether non-HB-associated ß-catenin mutations are tumorigenic when expressed in hepatocytes. Here, we investigated the oncogenic properties of 14 different HB- and non-HB-associated ß-catenin mutants encoded by Sleeping Beauty vectors following their delivery into the mouse liver by hydrodynamic tail-vein injection. We show that all ß-catenin mutations, as well as WT ß-catenin, are tumorigenic when co-expressed with a mutant form of yes-associated protein (YAP). However, tumor growth rates, histologies, nuclear-to-cytoplasmic partitioning, and metabolic and transcriptional landscapes were strongly influenced by the identities of the ß-catenin mutations. These findings provide a context for understanding at the molecular level the notable biological diversity of HB.

Histone demethylase KDM5A is transactivated by the transcription factor C/EBPß and promotes preadipocyte differentiation by inhibiting Wnt/ß-catenin signaling.

ß-Catenin signaling is triggered by WNT proteins and is an important pathway that negatively regulates adipogenesis. However, the mechanisms controlling the expression of WNT proteins during adipogenesis remain incompletely understood. Lysine demethylase 5A (KDM5A) is a histone demethylase that removes trimethyl (me3) marks from lysine 4 of histone 3 (H3K4) and serves as a general transcriptional corepressor. Here, using the murine 3T3-L1 preadipocyte differentiation model and an array of biochemical approaches, including ChIP, immunoprecipitation, RT-qPCR, and immunoblotting assays, we show that Kdm5a is a target gene of CCAAT/enhancer-binding protein ß (C/EBPß), an important early transcription factor required for adipogenesis. We found that C/EBPß binds to the Kdm5a gene promoter and transactivates its expression. We also found that siRNA-mediated KDM5A down-regulation inhibits 3T3-L1 preadipocyte differentiation. The KDM5A knockdown significantly up-regulates the negative regulator of adipogenesis Wnt6, having increased levels of the H3K4me3 mark on its promoter. We further observed that WNT6 knockdown significantly rescues adipogenesis inhibited by the KDM5A knockdown. Moreover, we noted that C/EBPß negatively regulates Wnt6 expression by binding to the Wnt6 gene promoter and repressing Wnt6 transcription. Further experiments indicated that KDM5A interacts with C/EBPß and that their interaction cooperatively inhibits Wnt6 transcription. Of note, C/EBPß knockdown impaired the recruitment of KDM5A to the Wnt6 promoter, which had higher H3K4me3 levels. Our results suggest a mechanism involving C/EBPß and KDM5A activities that down-regulates the Wnt/ß-catenin pathway during 3T3-L1 preadipocyte differentiation.

Neuron activity-induced Wnt signaling up-regulates expression of brain-derived neurotrophic factor in the pain neural circuit.

Brain-derived neurotrophic factor (BDNF) is a master regulator of synaptic plasticity in various neural circuits of the mammalian central nervous system. Neuron activity-induced BDNF gene expression is regulated through the Ca2+/CREB pathway, but other regulatory factors may also be involved in controlling BDNF levels. We report here that Wnt/ß-catenin signaling plays a key role in controlling neuron activity-regulated BDNF expression. Using primary cortical cultures, we show that blockade of Wnt/ß-catenin signaling inhibits the BDNF up-regulation that is induced by activation of the N-methyl-d-aspartic acid (NMDA) receptor and that activation of the Wnt/ß-catenin signaling pathway stimulates BDNF expression. In vivo, Wnt/ß-catenin signaling activated BDNF expression and was required for peripheral pain-induced up-regulation of BDNF in the mouse spine. We also found that conditional deletion of one copy of either Wntless (Wls) or ß-catenin by Nestin-Cre-mediated recombination is sufficient to inhibit the pain-induced up-regulation of BDNF. We further show that the Wnt/ß-catenin/BDNF axis in the spinal neural circuit plays an important role in regulating capsaicin-induced pain. These results indicate that neuron activity-induced Wnt signaling stimulates BDNF expression in the pain neural circuits. We propose that pain-induced Wnt secretion may provide an additional mechanism for intercellular coordination of BDNF expression in the neural circuit.

ß-Catenin directs the transformation of testis Sertoli cells to ovarian granulosa-like cells by inducing Foxl2 expression.

Sertoli and granulosa cells are two major types of somatic cells in male and female gonads, respectively. Previous studies have shown that Sertoli and granulosa cells are derived from common progenitor cells and that differentiation of these two cell types is regulated by sex differentiation genes. The signaling pathway including the adhesion and transcription factor Ctnnb1 (cadherin-associated protein, ß1, also known as ß-catenin) regulates differentiation of granulosa cells in the absence of the transcription factor Sry, and overactivation of ß-catenin in the presence of Sry leads to granulosa prior to sex determination. Surprisingly, our previous study found that ß-catenin overactivation in Sertoli cells after sex determination can also cause disruption of the testicular cord and aberrant testis development. However, the underlying molecular mechanism was unclear. In this study, we found that constitutive activation of Ctnnb1 in Sertoli cells led to ectopic expression of the granulosa cell-specific marker FOXL2 in testes. Co-staining experiments revealed that FOXL2-positive cells were derived from Sertoli cells, and Sertoli cells were transformed into granulosa-like cells after Ctnnb1 overactivation. Further studies demonstrated that CTNNB1 induced Foxl2 expression by directly binding to transcription factor Tcf/Lef-binding sites in the FOXL2 promoter region. We also found that direct overexpression of Foxl2 decreased the expression of Sertoli cell-specific genes in primary Sertoli cells. Taken together, these results demonstrate that repression of ß-catenin (CTNNB1) signaling is required for lineage maintenance of Sertoli cells. Our study provides a new mechanism for Sertoli cell lineage maintenance during gonad development.

Knockdown of sodium-calcium exchanger 1 induces epithelial-to-mesenchymal transition in kidney epithelial cells.

Mesenchymal-to-epithelial transition (MET) and epithelial-to-mesenchymal transition (EMT) are important processes in kidney development. Failure to undergo MET during development leads to the initiation of Wilms tumor, whereas EMT contributes to the development of renal cell carcinomas (RCC). The role of calcium regulators in governing these processes is becoming evident. We demonstrated earlier that Na+/Ca2+ exchanger 1 (NCX1), a major calcium exporter in renal epithelial cells, regulates epithelial cell motility. Here, we show for the first time that NCX1 mRNA and protein expression was down-regulated in Wilms tumor and RCC. Knockdown of NCX1 in Madin-Darby canine kidney cells induced fibroblastic morphology, increased intercellular junctional distance, and induced paracellular permeability, loss of apico-basal polarity in 3D cultures, and anchorage-independent growth, accompanied by expression of mesenchymal markers. We also provide evidence that NCX1 interacts with and anchors E-cadherin to the cell surface independent of NCX1 ion transport activity. Consistent with destabilization of E-cadherin, NCX1 knockdown cells showed an increase in ß-catenin nuclear localization, enhanced transcriptional activity, and up-regulation of downstream targets of the ß-catenin signaling pathway. Taken together, knockdown of NCX1 in Madin-Darby canine kidney cells alters epithelial morphology and characteristics by destabilization of E-cadherin and induction of ß-catenin signaling.

Coordinated Activities of Multiple Myc-dependent and Myc-independent Biosynthetic Pathways in Hepatoblastoma.

Hepatoblastoma (HB) is associated with aberrant activation of the ß-catenin and Hippo/YAP signaling pathways. Overexpression of mutant ß-catenin and YAP in mice induces HBs that express high levels of c-Myc (Myc). In light of recent observations that Myc is unnecessary for long-term hepatocyte proliferation, we have now examined its role in HB pathogenesis using the above model. Although Myc was found to be dispensable for in vivo HB initiation, it was necessary to sustain rapid tumor growth. Gene expression profiling identified key molecular differences between myc+/+ (WT) and myc-/- (KO) hepatocytes and HBs that explain these behaviors. In HBs, these included both Myc-dependent and Myc-independent increases in families of transcripts encoding ribosomal proteins, non-structural factors affecting ribosome assembly and function, and enzymes catalyzing glycolysis and lipid bio-synthesis. In contrast, transcripts encoding enzymes involved in fatty acid ß-oxidation were mostly down-regulated. Myc-independent metabolic changes associated with HBs included dramatic reductions in mitochondrial mass and oxidative function, increases in ATP content and pyruvate dehydrogenase activity, and marked inhibition of fatty acid ß-oxidation (FAO). Myc-dependent metabolic changes included higher levels of neutral lipid and acetyl-CoA in WT tumors. The latter correlated with higher histone H3 acetylation. Collectively, our results indicate that the role of Myc in HB pathogenesis is to impose mutually dependent changes in gene expression and metabolic reprogramming that are unattainable in non-transformed cells and that cooperate to maximize tumor growth.

A Critical Role for ß-Catenin in Modulating Levels of Insulin Secretion from ß-Cells by Regulating Actin Cytoskeleton and Insulin Vesicle Localization.

The processes regulating glucose-stimulated insulin secretion (GSIS) and its modulation by incretins in pancreatic ß-cells are only partly understood. Here we investigate the involvement of ß-catenin in these processes. Reducing ß-catenin levels using siRNA knockdown attenuated GSIS in a range of ß-cell models and blocked the ability of GLP-1 agonists and the depolarizing agent KCl to potentiate this. This could be mimicked in both ß-cell models and isolated islets by short-term exposure to the ß-catenin inhibitory drug pyrvinium. In addition, short-term treatment with a drug that increases ß-catenin levels results in an increase in insulin secretion. The timing of these effects suggests that ß-catenin is required for the processes regulating trafficking and/or release of pre-existing insulin granules rather than for those regulated by gene expression. This was supported by the finding that the overexpression of the transcriptional co-activator of ß-catenin, transcription factor 7-like 2 (TCF7L2), attenuated insulin secretion, consistent with the extra TCF7L2 translocating ß-catenin from the plasma membrane pool to the nucleus. We show that ß-catenin depletion disrupts the intracellular actin cytoskeleton, and by using total internal reflectance fluorescence (TIRF) microscopy, we found that ß-catenin is required for the glucose- and incretin-induced depletion of insulin vesicles from near the plasma membrane. In conclusion, we find that ß-catenin levels modulate Ca2+-dependent insulin exocytosis under conditions of glucose, GLP-1, or KCl stimulation through a role in modulating insulin secretory vesicle localization and/or fusion via actin remodeling. These findings also provide insights as to how the overexpression of TCF7L2 may attenuate insulin secretion.

Charged Amino Acid-rich Leucine Zipper-1 (Crlz-1) as a Target of Wnt Signaling Pathway Controls Pre-B Cell Proliferation by Affecting Runx/CBFß-targeted VpreB and λ5 Genes.

The proliferation of pre-B cells is known to further increase the clonal diversity of B cells at the stage of pre-B cells by allowing the same rearranged heavy chains to combine with differently rearranged light chains in a subsequent developmental stage. Crlz-1 (charged amino acid-rich leucine zipper-1) was found to control this proliferation of pre-B cells by working as a Wnt (wingless-related mouse mammary tumor virus integration site) target gene in these cells. Mechanistically, Crlz-1 protein functioned by mobilizing cytoplasmic CBFß (core binding factor ß) into the nucleus to allow Runx (runt-related transcription factor)/CBFß heterodimerization. Runx/CBFß then turned on its target genes such as EBF (early B cell factor), VpreB, and λ5 and thereby pre-B cell receptor signaling, leading to the expression of cyclins D2 and D3 Actually, the proliferative function of Crlz-1 was demonstrated by not only Crlz-1 or ß-catenin knockdown but also Crlz-1 overexpression. Furthermore, the mechanistic view that the proliferative function of Crlz-1 is caused by relaying Wnt/ß-catenin to pre-B cell receptor signaling pathways through the regulation of Runx/CBFß heterodimerization was also verified by employing niclosamide, XAV939, and LiCl as Wnt inhibitors and activator, respectively.

A Cytosolic Multiprotein Complex Containing p85α Is Required for ß-Catenin Activation in Colitis and Colitis-associated Cancer.

Wnt/ß-catenin signaling is required for crypt structure maintenance. We previously observed nuclear accumulation of Ser-552 phosphorylated ß-catenin (pß-Cat(Ser-552)) in intestinal epithelial cells (IEC) during colitis and colitis-associated cancer. Data here delineate a novel multiprotein cytosolic complex (MCC) involved in ß-catenin signaling in the intestine. The MCC contains p85α, the class IA subunit of PI3K, along with ß-catenin, 14-3-3ζ, Akt, and p110α. MCC levels in IEC increase in colitis and colitis-associated cancer patients. IEC-specific p85α-deficient (p85(ΔIEC)) mice develop more severe dextran sodium sulfate colitis due to delayed ulcer healing and reduced epithelial ß-catenin activation. In colonic IEC, p85α deficiency did not alter PI3K signaling. In vitro shRNA depletion of individual complex members disrupts the MCC and reduces ß-catenin signaling. Despite worse colitis, p85(ΔIEC) mice have reduced tumor burden after azoxymethane/dextran sodium sulfate treatment. Together the data indicate that the ß-catenin MCC is needed for mucosal repair and carcinogenesis. This novel MCC may be an attractive therapeutic target in preventing cancer in colitis patients.

Pertussis Toxin Exploits Specific Host Cell Signaling Pathways for Promoting Invasion and Translocation of Escherichia coli K1 RS218 in Human Brain-derived Microvascular Endothelial Cells.

Pertussis toxin (PTx), an AB5 toxin and major virulence factor of the whooping cough-causing pathogen Bordetella pertussis, has been shown to affect the blood-brain barrier. Dysfunction of the blood-brain barrier may facilitate penetration of bacterial pathogens into the brain, such as Escherichia coli K1 (RS218). In this study, we investigated the influence of PTx on blood-brain barrier permissiveness to E. coli infection using human brain-derived endothelial HBMEC and TY10 cells as in vitro models. Our results indicate that PTx acts at several key points of host cell intracellular signaling pathways, which are also affected by E. coli K1 RS218 infection. Application of PTx increased the expression of the pathogen binding receptor gp96. Further, we found an activation of STAT3 and of the small GTPase Rac1, which have been described as being essential for bacterial invasion involving host cell actin cytoskeleton rearrangements at the bacterial entry site. In addition, we showed that PTx induces a remarkable relocation of VE-cadherin and ß-catenin from intercellular junctions. The observed changes in host cell signaling molecules were accompanied by differences in intracellular calcium levels, which might act as a second messenger system for PTx. In summary, PTx not only facilitates invasion of E. coli K1 RS218 by activating essential signaling cascades; it also affects intercellular barriers to increase paracellular translocation.

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-Troglitazone-induced Apoptosis in Prostate Cancer Cells Involve AMP-activated Protein Kinase.

Prostate cancer (PCa) is one of the most frequently diagnosed cancers in men with limited treatment options for the hormone-resistant forms. Development of novel therapeutic options is critically needed to target advanced forms. Here we demonstrate that combinatorial treatment with the thiazolidinedione troglitazone (TZD) and TNF-related apoptosis-inducing ligand (TRAIL) can induce significant apoptosis in various PCa cells independent of androgen receptor status. Because TZD is known to activate AMP-activated protein kinase (AMPK), we determined whether AMPK is a molecular target mediating this apoptotic cascade by utilizing PCa cell lines stably overexpressing AMPKα1 dominant negative (C4-2-DN) or empty vector (C4-2-EV). Our results indicated a significantly higher degree of apoptosis with TRAIL-TZD combination in C4-2-EV cells compared with C4-2-DN cells. Similarly, results from a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed a larger reduction of viability of C4-2-EV cells compared with C4-2-DN cells when treated with TRAIL-TZD, thus suggesting that C4-2-DN cells were more apoptosis-resistant. Additionally, siRNA-mediated knockdown of endogenous AMPKα1 expression showed a reduction of TRAIL-TZD-induced apoptosis, further confirming the participation of AMPK in mediating this apoptosis. Apoptosis induction by this combinatorial treatment was also associated with a cleavage of ß-catenin that was inhibited in both C4-2-DN cells and those cells in which AMPKα1 was knocked down. In addition, time course studies showed an increase in pACC(S79) (AMPK target) levels coinciding with the time of apoptosis. These studies indicate the involvement of AMPK in TRAIL-TZD-mediated apoptosis and ß-catenin cleavage and suggest the possibility of utilizing AMPK as a therapeutic target in apoptosis-resistant prostate cancer.

Inhibition of transient receptor potential channel 5 reverses 5-Fluorouracil resistance in human colorectal cancer cells.

5-Fluorouracil (5-Fu) is commonly used in the chemotherapy of colorectal cancer (CRC), but resistance to 5-Fu occurs in most cases, allowing cancer progression. Suppressing ABCB1 (ATP-binding cassette, subfamily B, member 1), which is a pump overproduced in cancer cells to export cytotoxic drugs, is an attractive strategy to overcome drug resistance. In the present study, transient receptor potential channel TrpC5 was found to be overproduced at the mRNA and protein levels together with ABCB1 in 5-Fu-resistant human CRC HCT-8 (HCT-8/5-Fu) and LoVo (LoVo/5-Fu) cells. More nuclear-stabilized ß-catenin accumulation was found in HCT-8/5-Fu and LoVo/5-Fu cells than in HCT-8 and LoVo cells. Suppressing TrpC5 expression with TrpC5-specific siRNA inhibited the canonical Wnt/ß-catenin signal pathway, reduced the induction of ABCB1, weakened the ABCB1 efflux pump, and caused a remarkable reversal of 5-Fu resistance in HCT-8/5-Fu and LoVo/5-Fu cells. On the contrary, enforcing TrpC5 expression resulted in an activated Wnt/ß-catenin signal pathway and up-regulation of ABCB1. Taken together, we demonstrated an essential role of TrpC5 in ABCB1 induction and drug resistance in human CRC cells via promoting nuclear ß-catenin accumulation.

WITHDRAWN: The hydrophobic rich N- and C-terminal tails of beta-catenin facilitate nuclear import of beta-catenin.

This manuscript has been withdrawn by the author.

Secreted Frizzled-related protein 1 (Sfrp1) regulates the progression of renal fibrosis in a mouse model of obstructive nephropathy.

Renal fibrosis is responsible for progressive renal diseases that cause chronic renal failure. Sfrp1 (secreted Frizzled-related protein 1) is highly expressed in kidney, although little is known about connection between the protein and renal diseases. Here, we focused on Sfrp1 to investigate its roles in renal fibrosis using a mouse model of unilateral ureteral obstruction (UUO). In wild-type mice, the expression of Sfrp1 protein was markedly increased after UUO. The kidneys from Sfrp1 knock-out mice showed significant increase in expression of myofibrobast markers, α-smooth muscle actin (αSMA). Sfrp1 deficiency also increased protein levels of the fibroblast genes, vimentin, and decreased those of the epithelial genes, E-cadherin, indicated that enhanced epithelial-to-mesenchymal transition. There was no difference in the levels of canonical Wnt signaling; rather, the levels of phosphorylated c-Jun and JNK were more increased in the Sfrp1(-/-) obstructed kidney. Moreover, the apoptotic cell population was significantly elevated in the obstructed kidneys from Sfrp1(-/-) mice following UUO but was slightly increased in those from wild-type mice. These results indicate that Sfrp1 is required for inhibition of renal damage through the non-canonical Wnt/PCP pathway.

A pituitary homeobox 2 (Pitx2):microRNA-200a-3p:ß-catenin pathway converts mesenchymal cells to amelogenin-expressing dental epithelial cells.

Pitx2, Wnt/ß-catenin signaling, and microRNAs (miRs) play a critical role in the regulation of dental stem cells during embryonic development. In this report, we have identified a Pitx2:ß-catenin regulatory pathway involved in epithelial cell differentiation and conversion of mesenchymal cells to amelogenin expressing epithelial cells via miR-200a. Pitx2 and ß-catenin are expressed in the labial incisor cervical loop or epithelial stem cell niche, with decreased expression in the differentiating ameloblast cells of the mouse lower incisor. Bioinformatics analyses reveal that miR-200a-3p expression is activated in the pre-ameloblast cells to enhance epithelial cell differentiation. We demonstrate that Pitx2 activates miR-200a-3p expression and miR-200a-3p reciprocally represses Pitx2 and ß-catenin expression. Pitx2 and ß-catenin interact to synergistically activate gene expression during odontogenesis and miR-200a-3p attenuates their expression and directs differentiation. To understand how this mechanism controls cell differentiation and cell fate, oral epithelial and odontoblast mesenchymal cells were reprogrammed by a two-step induction method using Pitx2 and miR-200a-3p. Conversion to amelogenin expressing dental epithelial cells involved an up-regulation of the stem cell marker Sox2 and proliferation genes and decreased expression of mesenchymal markers. E-cadherin expression was increased as well as ameloblast specific factors. The combination of Pitx2, a regulator of dental stem cells and miR-200a converts mesenchymal cells to a fully differentiated dental epithelial cell type. This pathway and reprogramming can be used to reprogram mesenchymal or oral epithelial cells to dental epithelial (ameloblast) cells, which can be used in tissue repair and regeneration studies.