β-Catenin regulation during matrigel-induced rat hepatocyte differentiation

Hepatocytes in primary cultures de-differentiate and re-differentiate following addition of Engelbreth-Holm-Swarm mouse sarcoma (matrigel) to the cultures. The Wnt/β-catenin pathway has been shown to be important in liver growth and development. Here, we investigate changes in β-catenin and its mechanism, during matrigel-induced hepatocyte differentiation. Primary rat hepatocytes were cultured for 8 days, and matrigel was added to half of the cultures. Total and nuclear protein and total RNA were extracted at different days of culture and examined for β-catenin and other Wnt pathway components. A significant increase in total β-catenin protein was observed upon matrigel addition, during hepatocyte differentiation, despite a decrease in β-catenin and frizzled-1 (Wnt receptor) expression. A concurrent decrease in the glycogen synthase kinase-3β (GSK3β), axin, and ser45/thr41-phosphorylated β-catenin proteins was observed in matrigel-treated cultures, implying decreased degradation of β-catenin. Interestingly, a decrease in nuclear β-catenin and total active β-catenin was observed in the presence of matrigel. Matrigel also induced an increased association of β-catenin with Met (hepatocyte growth factor receptor), whereas association with E-cadherin remained unchanged. This coexisted with decreased β-catenin tyrosine phosphorylation. Thus, β-catenin undergoes multifactorial regulation during matrigel-induced hepatocyte differentiation and maturation; this induces its stabilization and membrane translocation, possibly contributing to hepatocyte differentiation. A. Micsenyi and M. Germinaro contributed equally to this work. Grant Support: Rango's fund for Enhancement of Pathology Research, American Cancer Society Grant-RSG-03-141-01 and National Institute of Health Grant 1RO1DK62277, to SPSM.     

Wnt/β-catenin signaling is required for development of the exocrine pancreas

Background  

β-catenin is an essential mediator of canonical Wnt signaling and a central component of the cadherin-catenin epithelial adhesion complex. Dysregulation of β-catenin expression has been described in pancreatic neoplasia. Newly published studies have suggested that β-catenin is critical for normal pancreatic development although these reports reached somewhat different conclusions. In addition, the molecular mechanisms by which loss of β-catenin affects pancreas development are not well understood. The goals of this study then were; 1] to further investigate the role of β-catenin in pancreatic development using a conditional knockout approach and 2] to identify possible mechanisms by which loss of β-catenin disrupts pancreatic development. A Pdx1-cre mouse line was used to delete a floxed β-catenin allele specifically in the developing pancreas, and embryonic pancreata were studied by immunohistochemistry and microarray analysis.     

Accumulation of β-catenin by lithium chloride in porcine myoblast cultures accelerates cell differentiation

The Wnt/β-catenin signaling pathway regulates cell proliferation and differentiation to determine cell fate during embryogenesis. Lithium chloride (LiCl) is known to activate canonical Wnt signaling by inhibiting glycogen synthetase kinase-3β and consequently stabilizing free cytosolic β-catenin. To understand the role of the Wnt/β-catenin pathway in the regulation of porcine myoblast differentiation, we studied the effects of LiCl on cultured porcine myoblasts and β-catenin expression. A supplementation of 25 mM LiCl induced myoblast differentiation into myotubes over 3 days of culture. By semi-quantitative RT-PCR analyses, levels of mRNA encoding MyoD, Myogenin, Myf5 and several Wnt-responsive genes in the cultured myoblast cells were significantly increased after LiCl treatment. Using Western blotting and immunofluorescence analysis, we found that the protein levels of β-catenin were consistently increased by LiCl. Meanwhile, phosphorylated GSK-3β at Ser9 levels were also increased as an indicator of GSK-3β inactivation. Additionally, the nuclear staining of endogenous β-catenin was also significantly increased in porcine myoblasts 48 h after LiCl treatment. These results provided additional evidence that Wnt/β-catenin is a significant pathway that regulates myogenic differentiation. An enhanced level of β-catenin plays a positive role in porcine myoblast differentiation.     

Retracted: PRC1 gene silencing inhibits proliferation,invasion, and angiogenesis of retinoblastoma cells through the inhibition of the Wnt/β-catenin signaling pathway

Retinoblastoma is an ocular malignancy occurring in childhood. The current study evaluates the ability of silenced PRC1 on retinoblastoma cell proliferation, and angiogenesis via the Wnt/β-catenin signaling pathway. A total of 36 cases of retinoblastoma tissues (n = 36) and normal retinal tissues (n = 10) were selected in the current study. Retinoblastoma cells presenting with the high PRC1 messenger RNA (mRNA) expression were selected among the WERI-Rb-1, HXO-RB44, Y79, SO-Rb50, and SO-Rb70 cells lines, and were transfected with siRNA-PRC1 and LiCl (the activator of the Wnt/β-catenin pathway). The expressions of PRC1, VEGF, Wnt1, β-catenin, CyclinD1, extent of β-catenin, and GSK-3β phosphorylation were evaluated. Cell proliferation, cell-cycle distribution, and cell invasion of retinoblastoma cells were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and Transwell assay. The angiogenesis of retinoblastoma cells was detected by tube formation assay. HXO-RB44 and WERI-Rb-1 cells were selected owing to the highest PRC1 mRNA expression. Meanwhile, PRC2 gene silencing presented lower expression levels of PRC1, VEGF, Wnt1, β-catenin, CyclinD1, extent of β-catenin and GSK-3β phosphorylation, decreased proliferation and invasion abilities, extended G0/G1 phase, and shortened S and G2/M phases of HXO-RB44 and WERI-Rb-1 cells, suggesting the silenced PRC2 inactivated Wnt/β-catenin pathway, so as to further restrain the retinoblastoma cell proliferation, invasion, and angiogenesis. These results support the view that PRC1 gene silencing could suppress the proliferation, and angiogenesis of retinoblastoma cells by repressing the Wnt/β-catenin pathway.     

Activation of extracellular signal-regulated kinase by TGF-β1 via TβRII and Smad7 dependent mechanisms in human bronchial epithelial BEP2D cells

Transforming growth factor-β1 (TGF-β1) can activate mitogen-activated protein kinases (MAPKs) in many types of cells. The mechanism of this activation is not well elucidated. Here, we explore the role of TGF-β/Smads signaling compounds in TGF-β1-mediated activation of extracellular signal-regulated kinase (ERK) MAPK in human papillomavirus (HPV)-18 immortalized human bronchial epithelial cell line BEP2D and the role of TGF-β1-induced phosphorylation of ERK in proliferation and apoptosis of BEP2D. The cell models of siRNA-mediated silencing of TGF-β receptor type II (TβRII), Smad2, Smad3, Smad4, and Smad7 were employed in this study. Our results demonstrate that TGF-β1 activates ERK in a time-dependent manner with a maximum effect at 60 min; overexpression of Smad7 increased this TGF-β1-mediated phosphorylation of the ERK; and siRNA-mediated silencing of TβRII, Smad3, Smad4, and Smad7 abrogated this effect. Moreover, we observed that overexpression of Smad7 restored TGF-β1-mediated ERK phosphorylation in Smad4 knockdown cells but not in TβRII knockdown cells. In BEP2D cells, TGF-β1 treatment effectively inhibited cells’ proliferation and induced their apoptosis. Pretreatment with U0126, an inhibitor of ERK1/2, significantly enhanced the TGF-β1-mediated antiproliferative and apoptosis induction effects in BEP2D cells. These data revealed that TβRII and Smad7 play the critical roles in TGF-β1-mediated activation of ERK; Smad3 and Smad4 can play an indirect role through up-regulating Smad7 expression; and TGF-β1-induced phosphorylation of ERK may participate in BEP2D cell proliferation and apoptosis regulation.     

Overexpression of miR-623 suppresses progression of hepatocellular carcinoma via regulating the PI3K/Akt signaling pathway by targeting XRCC5

It has been reported that miR-623 is deregulated in lung adenocarcinoma and inhibits tumor growth and invasion. However, it is unclear whether miR-623 has a role in the progression of hepatocellular carcinoma (HCC). Herein, we found that miR-623 was significantly downregulated in HCC, and that its expression was related to poor clinical outcomes of patients with HCC. Upregulation of miR-623 decreased cell proliferation, viability, migration, and invasion and further promoted apoptosis in 7721, Huh7, and Bel-7402 cells. Moreover, we also observed that miR-623 regulated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), Wnt/β-catenin, and extracellular regulated protein kinases/c-Jun N-terminal kinase (ERK/JNK) signaling pathways as well as the expression level of related proteins. Further, X-ray repair cross complementing 5 (XRCC5) was a direct target for miR-623, and the suppression of PI3K/Akt, Wnt/β-catenin, and ERK/JNK signaling pathways and cell proliferation and invasion abilities caused by miR-623 in HCC cells was significantly reversed by the upregulation of XRCC5. Collectively, our data suggested that miR-623 suppressed the progression of HCC by regulating the PI3K/Akt, Wnt/β-catenin, and ERK/JNK pathways by targeting XRCC5 in HCC in vitro, indicating that miR-623 may be a target for the therapy of HCC.     

Phosphatidylcholine regulates NF-κB activation in attenuation of LPS-induced inflammation: evidence from in vitro study

Phosphatidylcholine (PC) has been demonstrated as anti-inflammatory and antioxidant/pro-oxidant molecules. In this study, we investigated the protective effects of PC on inflammatory bowel disease (IBD) caused by lipopolysaccharide (LPS)-induced injury in intestinal epithelia cells. The IEC-6 cells (intestinal epithelia cells) were stimulated with LPS (1 μg/mL) for 24 h with or without PC pretreatment, in the next steps: (1) the level of the inflammatory cytokine tumor necrosis factor (TNF)-α was measured with ELISA; (2) the nuclear translocation and phosphorylation of NF-κB was investigated with Western blot, EMSA, immunofluorence assay; (3) the protein phosporylation levels in MAPK signaling pathway were detected with Western blot method. The results showed: (1) compared with the normal group, 10 and 20?μg/mL of PC significantly inhibited the production and activation of TNF-α, (P < 0.01); (2) pretreatment with PC inhibited LPS-induced nuclear translocation and phosphorylation of p65 in IEC-6 cells; (3) pretreatment with PC inhibited the protein phosphorylation levels in MAPK signaling pathway. Our findings indicated that PC had the effect to protect IEC-6 cells from LPS-induced injury and this effect was exerted possibly through inhibiting the TNF-ɑ secretion, down-regulating nuclear translocation and phosphorylation of p65 and inhibiting MAPK signaling pathways.     

TGF-β acts as a dual regulator of COX-2/PGE2 tumor promotion depending of its cross-interaction with H-Ras and Wnt/β-catenin pathways in colorectal cancer cells

Transforming growth factor-β (TGF-β) plays a dual role acting as tumor promoter or suppressor. Along with cyclooxygenase-2 (COX-2) and oncogenic Ras, this multifunctional cytokine is deregulated in colorectal cancer. Despite their individual abilities to promote tumor growth and invasion, the mechanisms of cross regulation between these pathways is still unclear. Here, we investigate the effects of TGF-β, Ras oncogene and COX-2 in the colorectal cancer context. We used colon adenocarcinoma cell line HT-29 and Ras-transformed IEC-6 cells, both treated with prostaglandin E₂ (PGE₂), TGF-β or a combined treatment with these agents. We demonstrated that PGE₂ alters the subcellular localization of E-cadherin and β-catenin and enhanced the tumorigenic potential in HT-29 cells. This effect was inhibited by TGF-β, indicating a tumor suppressor role. Conversely, in Ras-transformed IEC-6 cells, TGF-β induced COX-2 expression and increased invasiveness, acting as a tumor promoter. In IEC-6 Ras-transformed cells, TGF-β increased nuclear β-catenin and Wnt/β-catenin activation, opposite to what was seen in the PGE₂ and TGF-β joint treatment in HT-29 cells. Together, our findings show that TGF-β increases COX-2 levels and induces invasiveness cooperating with Ras in a Wnt/β-catenin activation-dependent manner. This shows TGF-β dual regulation over COX-2/PGE₂ tumor promotion depending on the H-Ras and Wnt/β-catenin pathways activation status in intestinal cancer cells.     

Plant stanols induce intestinal tumor formation by up-regulating Wnt and EGFR signaling in ApcMin mice

The rate of APC mutations in the intestine increases in middle-age. At the same period of life, plant sterol and stanol enriched functional foods are introduced to diet to lower blood cholesterol. This study examined the effect of plant stanol enriched diet on intestinal adenoma formation in the Apc^Min mouse. Apc^Min mice were fed 0.8% plant stanol diet or control diet for nine weeks. Cholesterol, plant sterols and plant stanols were analyzed from the caecum content and the intestinal mucosa. Levels of β-catenin, cyclin D1, epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase 1/2 (ERK1/2) were measured from the intestinal mucosa by Western blotting. Gene expression was determined from the intestinal mucosa using Affymetrix and the data were analyzed for enriched categories and pathways. Plant stanols induced adenoma formation in the small intestine, however, the adenoma size was not affected. We saw increased levels of nuclear β-catenin, phosphorylated β-catenin (Ser675 and Ser552), nuclear cyclin D1, total and phosphorylated EGFR and phosphorylated ERK1/2 in the intestinal mucosa after plant stanol feeding. The Affymetrix data demonstrate that several enzymes of cholesterol synthesis pathway were up-regulated, although the cholesterol level in the intestinal mucosa was not altered. We show that plant stanols induce adenoma formation by activating Wnt and EGFR signaling. EGFR signaling seems to have promoted β-catenin phosphorylation and its translocation into the nucleus, where the expression of cyclin D1 was increased. Up-regulated cholesterol synthesis may partly explain the increased EGFR signaling in the plant stanol-fed mice.     

Wnt/β-catenin/Tcf Signaling Pathway Activation in Malignant Progression of Rat Gliomas Induced by Transplacental <Emphasis Type="Italic">N-</Emphasis>Ethyl-<Emphasis Type="Italic">N-</Emphasis>Nitrosourea Exposure

Although Wnt/β-catenin/Tcf signaling pathway has been shown to be a crucial factor in the development of many cancers, little is known about its role in glioma malignancy. In the present study, we report the first evidence that Wnt/β-catenin/Tcf signaling pathway is constitutively activated in experimental gliomas induced by single transplacental dose of N-ethyl-N-nitrosourea (ENU). In the present study we analyzed ENU induced rat gliomas of different stages (P90, P135 and P180) for the expression of β-catenin, Lef1, Tcf4 and their targets c-Myc, N-Myc and cyclin D1. Western blot analysis revealed upregulation of β-catenin, Lef1, Tcf4, c-Myc, N-Myc and cyclin D1 in gliomas compared to controls and their levels were progressively increased from initial stage (P90) to progression stage (P180). In consistent with this, immunohistochemistry revealed the cytoplasmic and nuclear accumulation of β-catenin, and nuclear positivity was evident for Lef1, Tcf4, c-Myc, N-Myc and cyclin D1. Based on these results, we conclude that Wnt/β-catenin pathway may play a major role in the tumorigenesis and tumor progression in ENU induced rat gliomas.