Expression Of Lymphoid Enhancer Factor 1 Research Articles

The WNT/Ca2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria.

WNT signaling plays an important role in cardiac development, but abnormal activity is often associated with cardiac hypertrophy, myocardial infarction, remodeling, and heart failure. The effect of WNT signaling on regulation of atrial natriuretic peptide (ANP) secretion is unclear. Therefore, the purpose of this study was to investigate the effect of Wnt agonist 1 (Wnta1) on ANP secretion and mechanical dynamics in beating rat atria. Wnta1 treatment significantly increased atrial ANP secretion and pulse pressure; these effects were blocked by U73122, an antagonist of phospholipase C. U73122 also abolished the effects of Wnta1-mediated upregulation of protein kinase C (PKC) β and γ expression, and the PKC antagonist Go 6983 eliminated Wnta1-induced secretion of ANP. In addition, Wnta1 upregulated levels of phospho-transforming growth factor-β activated kinase 1 (p-TAK1), TAK1 banding 1 (TAB1) and phospho-activating transcription factor 2 (p-ATF2); these effects were blocked by both U73122 and Go 6983. Wnta1-induced ATF2 was abrogated by inhibition of TAK1. Furthermore, Wnta1 upregulated the expression of T cell factor (TCF) 3, TCF4, and lymphoid enhancer factor 1 (LEF1), and these effects were blocked by U73122 and Go 6983. Tak1 inhibition abolished the Wnta1-induced expression of TCF3, TCF4, and LEF1 and Wnta1-mediated ANP secretion and changes in mechanical dynamics. These results suggest that Wnta1 increased the secretion of ANP and mechanical dynamics in beating rat atria by activation of PKC-TAK1-ATF2-TCF3/LEF1 and TCF4/LEF1 signaling mainly via the WNT/Ca2+ pathway. It is also suggested that WNT-ANP signaling is implicated in cardiac physiology and pathophysiology.

Abstract 6387: Inhibition of Lymphoid Enhancer Factor1(LEF1) in acute myeloid leukemia overcomes acquired cytarabine resistance

Abstract Canonical Wnt-β-catenin signalling is frequently dysregulated in myeloid leukemia. LEF1 (Lymphoid Enhancer Factor 1), a key mediator of Wnt signalling, functions as an interacting partner with β-catenin. Previous studies have shown that LEF1 is associated with leukemic transformation and is an independent prognostic factor in normal karyotype AML. Through high throughput transcriptome profiling between the parental and acquired cytarabine resistant cell lines, we identified that LEF1 to be a potential target mediating this resistance. Here we evaluated the role, of molecular and pharmacological inhibition of LEF1 in overcoming acquired cytarabine resistance. The differential expression of LEF1 in THP1, MV4-11 and U937 parental and Ara-C resistant AML cell lines was checked by quantitative RT-PCR, immunoblot and immunofluorescence. LEF1 knock-out (KO) cells were generated using the CRISPR-Cas9 in the THP1 cytarabine resistant cell line. Lentiviral based over-expression of LEF1 was carried out in THP1 parental cell line to ensure that LEF1 promotes Ara-C resistance. For nuclear translocation of LEF1, the small molecule CHIR99021 was used in over-expressed cells.The knock-out and over-expressed cells were characterized by analysing proliferation, cell cycle and cytotoxicity assay. The effect of pharmacological inhibition of LEF1 using niclosamide was examined by in-vitro-cytotoxicity assay.Although LEF1 transcript expression was high in all the three cytarabine resistant cell lines compared to the parental cell lines, THP1-araC-R cell line showed around ≈ 500-fold change in expression and hence was used for further experiments. THP1-araC-R was marginally cross resistant to both DNR (IC50 Parent -0.19µM, AraC 0.26µM) and ATO (IC50 Parent -2.1µM, AraC >4µM). LEF1 knock-out showed complete absence of LEF1 protein, decreased rate of proliferation and improved sensitivity to Ara-C (Ara-C IC50- 1430 µM in the untransduced vs. 387.4 µM after LEF1 KO). Over-expression of LEF1 with a fold change of ≈1000 followed by enforced nuclear translocation using the small molecule CHIR99021 in parental cell line resulted in increased resistance to Ara-C (IC50 Parental- 50.7µM vs OE CHIR treated 240.05µM). Further, niclosamide, the pharmacological inhibitor of LEF1 was able to re-sensitize the THP1 AraC -resistant cells (IC50: 1430 µM for UT vs 516.8 µM treated with niclosamide). Our results suggest that inhibition of LEF1 by molecular and pharmacological means showed considerable increase in cytarabine sensitivity in-vitro while we are testing its effect in-vivo using a transplantable AML mouse model. Citation Format: Saswati Das, Raveen Stephen Illangeswaran, Daniel Zechariah Paul Jebanesan, Rakhi Thalayatta Vidhyadharan, Nayanthara Karpillymoola Bijukumar, Bharathi Murugan Rajamani, Martin Michaelis, Jindrich Cinatl Jr., Vikram Mathews, Shaji Ramchandran Velayudhan, Poonkuzhali Balasubramanian. Inhibition of Lymphoid Enhancer Factor1(LEF1) in acute myeloid leukemia overcomes acquired cytarabine resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6387.

Screen and Function Study of the Key Regulative Odontogenic Genes in Mice

To screen the key odontogenic genes in mice and verify the odontogenic inducing effect on amniotic epithelial cells (WISH). The spatially and temporally different expression of bone morphogenetic proteins 4 (BMP4), fibroblast growth factor 8 (FGF8), sonic hedgehog (SHH), lymphoid enhancer factor 1 (LEF1) proteins and their genes expression in the early odontogenesis stage (embryo day 10.5 (E10.5)、E11.5、E14.5) in fetal mice were detected by immunohistochemistry staining and quantitative real-time PCR (RT-qPCR). According to the results, we screened the probable key odontogenic genes. Then adding osteogenic inducing solution to induce non-odontogenic epithelium cells, WISH. After 3 weeks culture of non-odontogenic epithelial WISH for osteogenic induction, the epithelial-mesenchymal transformation cap ability was evaluated by using Alizarin (ALZ) red staining and RT-qPCR on the alkaline phosphatase ( ALP) mRNA expression level. Using germ layer recombination experiment to observe and verify whether the screened genes can induce non-odontogenic epithelium cells acquire odontogenesis ability. The recombined tissue grafts containing key genes were transplanted beneath the renal capsule of mice. The results of immunohistochemistry staining and RT-qPCR showed that on E10.5 BMP4 protein and gene were differently expressed in the first and second branchial arch epithelium, which synchronized the odontogenic capability transferring from epithelium to mesenchyme from E10.5-E14.5. Though the expression of FGF8 protein and gene existed such difference in the first and second branchial arch epithelium, there was no synchronization in transfer. The expression of LEF1 and SHH proteins and genes had neither difference nor synchronization. So far, we considered the BMP4 was the probable key odontogenic gene. Through 3 weeks' osteogenic induction, ALZ red stained positively and calcium nodules were observed in WISH, and the expression level of ALP mRNA increased. In the germ layer recombination experiment, exogenous BMP4 protein enabled the second branchial arch mesenchyme forming tooth-like structures after recombined with the second branchial arch epithelium or WISH. The proteins and genes of BMP4, FGF8, SHH and LEF1 are spatially and temporally differently expressed in the early tooth development stage in mice. The protein and gene of BMP4 are differently expressed between the first and second branchial arch epithelium and enables the non-odontogenic epithelium acquiring odontogenic ability. BMP4 is the possible key odontogenic gene.

Prognostic Impact of Lymphoid Enhancer Factor 1 Expression and Serum Galectin.3 in Egyptian AML Patients

Background Deregulation of the Wnt signaling pathway had a role in haematological malignancies. Previous studies reported that lymphoid enhancer factor 1 (LEF1) expression and serum Galectin-3 level could affect clinical parameters and outcome in acute myeloid leukemia patients, but as far as we know, no study has addressed their combined effect on AML patients. Aim We studied the expression of LEF1 by real-time qPCR and measured serum level of Gal.3 by ELISA technique in peripheral blood of 69 AML patients and correlated it with different clinicopathological criteria of patients, response, PFS and OS. Results We found high expression (LEF1high) was associated with better OS (p = 0.02) and EFS (p = 0.019) compared to LEF1low, low serum Gal.3 level had better OS (p = 0.014) and EFS (p = 0.02) compared to high serum Gal.3 level. LEF1high less likely to carry a FLT3-ITD (p = 0.047) compared to LEF1low patient, also LEF1high characterized by favorable risk (p = 0.02) than LEF1low patients. While patients with higher Gal-3 levels characterized by poor risk (p = 0.02) than lower Gal.3 lels, also more likely to carry a FLT3-ITD with borderline significance (p = 0.054). Combined LEF1high/Gal.3 low patients had lower baseline blast percentages (p = 0.02), favorable risk (p = 0.01), less likely to carry FLT3-ITD (p = 0.02), higher CR rate (p = 0.055), shorter time to CR (0.001) than other groups. Among high Gal.3 level group, LEF1highexpression improved OS and EFS (20 and 15 months respectively) vs LEF1low expression (13 and 8 months respectively). Conclusion We conclude that high LEF1 expression was a favorable prognostic marker which can define AML patient risk and outcome independent from assessing the serum galectin.3 level.

Epstein-Barr virus stably confers an invasive phenotype to epithelial cells through reprogramming of the WNT pathway.

Epstein-Barr virus (EBV)-associated carcinomas, such as nasopharyngeal carcinoma (NPC), exhibit an undifferentiated and metastatic phenotype. To determine viral contributions involved in the invasive phenotype of EBV-associated carcinomas, EBV-infected human telomerase-immortalized normal oral keratinocytes (NOK) were investigated. EBV-infected NOK were previously shown to undergo epigenetic reprogramming involving CpG island hypermethylation and delayed responsiveness to differentiation. Here, we show that EBV-infected NOK acquired an invasive phenotype that was epigenetically retained after viral loss. The transcription factor lymphoid enhancer factor 1 (LEF1) and the secreted ligand WNT5A, expressed in NPC, were increased in EBV-infected NOK with sustained expression for more than 20 passages after viral loss. Increased LEF1 levels involved four LEF1 variants, and EBV-infected NOK showed a lack of responsiveness to β-catenin activation. Although forced expression of WNT5A and LEF1 enhanced the invasiveness of parental NOK, LEF1 knockdown reversed the invasive phenotype of EBV-infected NOK in the presence of WNT5A. Viral reprogramming of LEF1 and WNT5A was observed several passages after EBV infection, suggesting that LEF1 and WNT5A may provide a selective advantage to virally-infected cells. Our findings suggest that EBV epigenetically reprogrammed epithelial cells with features of basal, wound healing keratinocytes, with LEF1 contributing to the metastatic phenotype of EBV-associated carcinomas.

Strong Correlation Between CTLA-4 and LEF1 Gene Expression Levels in CLL: Targeting of the Wnt/β-Catenin Pathway May Adversely Affect CTLA-4 Expression and Function

Recently published clinical trials have confirmed the effectiveness of anti-CD38 monoclonal antibody therapy in myeloma. Furthermore, in vitro studies of chronic lymphocytic leukaemia (CLL) cells suggest that CD38 expression can be enhanced by treatment with retinoid derivatives and thus may enhance the cytotoxic effects of anti-CD38 therapy. However, retinoids have been shown to have diverse effects on cellular function and we have previously shown that the retinoid drug acitretin upregulates CD38 expression while also reducing cell homing to the chemokine CXCL12 in primary CLL cells.To investigate possible key mechanisms for these effects, we purified CD20+ B cells from the peripheral blood of 20 CLL patients (9 previously treated, 11 untreated) and, using flow cytometry, measured percentage cell surface expression of CD38 and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4, CD152). We also measured gene expression levels of the key retinoid receptor, stimulated by retinoic acid 6 (STRA6) and it's agonist, retinol-binding protein 4 (RBP4), as well as CTLA-4, cyclin D1 (CCND1) and the transcription factors, lymphoid enhancer factor 1 (LEF1) and signal transducer and activator of transcription 3 (STAT3) using RT-PCR. GAPDH was used as a reference gene.Mean percentage surface expression of CD38 and CTLA-4 was 21.96% and 45.25% respectively. Mean ΔCT gene expression levels of CCND1, CTLA-4, LEF1 and STAT3 were 12.03, 5.57 , 5.99 and 8.98 respectively. RBP4 and STRA6 gene expression levels were undetectable in all 20 patients. Gene expression of LEF1 showed significant correlations with CTLA-4 (rs=0.572, p=0.008), CCND1 (rs=0.61, p=0.004) and STAT3 (rs=0.587, p=0.006). There was also a significant correlation between gene expression of CCND1 and of STAT3 (r =0.499, p=0.025). No significant correlations were found between percentage surface expression of CTLA-4 and gene expression levels of either CTLA-4 or of LEF1. A weak negative correlation between percentage surface expression of CTLA-4 and of CD38 was not statistically significant. Comparing untreated and previously treated patients, there was no significant difference in gene expression levels of CTLA-4 and of LEF1 or in surface expression of CTLA-4.The failure to detect RBP4 and STRA6 gene expression in unstimulated peripheral blood CLL cells is evidence against an autocrine retinoid effect in CLL, although upregulation of STRA6 gene expression following stimulation by retinoids might be anticipated. The Wnt signalling pathway has been shown to be active in CLL, including aggressive disease subtypes, highlighting the potential benefits in targeting this pathway. Intriguingly, CTLA-4 expression, although found to be the most highly induced gene following treatment with recombinant Wnt-3a in melanoma cell lines, is associated with a favourable outcome in CLL, possibly by inhibiting cell proliferation and survival. In contrast, expression of LEF1, which is a direct target of the Wnt signalling pathway, is associated with disease progression in CLL. Our finding that CTLA-4 and LEF1 gene expression levels are strongly correlated suggests that further investigation of the relationship between CTLA-4 and the Wnt/β-Catenin pathway in CLL is required and that targeting of the Wnt/β-catenin pathway may have unwanted consequences on CTLA-4 expression and function. DisclosuresQuinn:Celgene: Honoraria; Janssen Cilag: Honoraria.

Simvastatin inhibits neural cell apoptosis and promotes locomotor recovery via activation of Wnt/β-catenin signaling pathway after spinal cord injury.

Statins exhibit neuroprotective effects after spinal cord injury (SCI). However, the molecular mechanism underlying these effects remains unknown. This study demonstrates that the hydroxymethylglutaryl coenzyme A reductase inhibitor simvastatin (Simv) exhibits neuroprotective effects on neuronal apoptosis and supports functional recovery in a rat SCI model by activating the Wnt/β‐catenin signaling pathway. In specific, Simv administration after SCI significantly up‐regulated the expression of low density lipoprotein receptor‐related protein 6 phosphorylation and β‐catenin protein, increased the mRNA expression of lymphoid enhancer factor‐1 and T‐cell factor‐1, and suppressed the expression of β‐catenin phosphorylation in the spinal cord neurons. Simv enhanced motor neuronal survival in the spinal cord anterior horn and decreased the lesion of spinal cord tissues after SCI. Simv administration after SCI also evidently reduced the expression levels of Bax, active caspase‐3, and active caspase‐9 in the spinal cord neurons and the proportion of transferase UTP nick end labeling (TUNEL)‐positive neuron cells, but increased the expression level of Bcl‐2 in the spinal cord neurons. However, the anti‐apoptotic effects of Simv were reduced in cultured spinal cord nerve cells when the Wnt/β‐catenin signaling pathway was suppressed in the lipopolysaccharide‐induced model. Furthermore, the Basso, Beattie, and Bresnahan scores indicated that Simv treatment significantly improved the locomotor functions of rats after SCI. This study is the first to report that Simv exerts neuroprotective effects by reducing neuronal apoptosis, and promoting functional and pathological recovery after SCI by activating the Wnt/β‐catenin signaling pathway. We verified the neuroprotective properties associated with simvastatin following spinal cord injury (SCI). Simvastatin reduced neuronal apoptosis, improved the functional and pathological recovery via activating Wnt/β‐catenin signal pathway, however, the anti‐apoptosis effects of simvastatin were reversed following suppressing Wnt/β‐catenin signaling pathway in primary spinal cord neurons. The significant findings may provide clinical therapeutic value of simvastatin for treating SCI.

Resveratrol attenuates hyperoxia-induced oxidative stress, inflammation and fibrosis and suppresses Wnt/β-catenin signalling in lungs of neonatal rats.

Although survival rate of infants born prematurely has been raised by supplemental oxygen treatment, it is followed by high morbidity of hyperoxia-induced bronchopulmonary dysplasia. In this study, the effect of resveratrol on the lung injury was evaluated in hyperoxia-exposed rats of preterm birth. The results demonstrated that hyperoxia led to thickened alveolar wall, simplified alveolar architecture and fibrosis. In addition, elevated methane dicarboxylic aldehyde level, decreased glutathione level and superoxide dismutase activity were also found in hyperoxic lungs, as well as the increased tumor necrosis factor-α, interleukin-1β and interleukin-6 in the bronchoalveolar lavage fluid. Fibrotic-associated proteins transforming growth factor-β1, α-smooth muscle actin, collagen I and fibronectin deposition were also found in interstitial substance of lungs. Furthermore, Wnt/β-catenin signalling was found to be active in hyperoxia-induced lungs. In addition, expression of SP-C was increased and T1α was decreased in hyperoxia-exposed lungs. Resveratrol intraperitoneal administration alleviated hyperoxia-induced histological injury of lungs, regulated redox balance, decreased pro-inflammatory cytokine release, and down-regulated expression of fibrotic-associated proteins. Furthermore, Wnt/β-catenin signalling was also suppressed by resveratrol, as represented by diminished expression of lymphoid enhancer factor-1, Wnt induced signalling protein-1 and cyclin D1. In addition, the increase of SP-C and decrease of T1α expression was prevented as well. The present study showed that resveratrol could protect lungs from hyperoxia-induced injury through its antioxidant, anti-inflammatory and anti-fibrotic effects. The transdifferentiation of alveolar epithelial type II cells to alveolar epithelial type I cells promotion and Wnt/β-catenin signalling suppression are also involved in the protective effect.

Mutation and expression of LEF1 in adult acute lymphocytic leukemia and their clinical significance

Lymphoid enhancer factor 1 (LEF1) is a key transcription factor in Wingless-type (Wnt) pathway. The present study was aimed to explore the genetic mutation and expression of LEF1, and their clinical significance in adult patients with acute lymphocytic leukemia (ALL). Genomic DNA was amplified and sequenced to detect the mutation of LEF1 in 131 newly diagnosed adult patients with ALL. Quantitative PCR (qPCR) was performed to detect the expression of LEF1. Moreover, the correlations between mutations and expression of LEF1 with clinical characteristics were analyzed. The results showed that the frequency of LEF1 mutation in adult ALL was 3.1% (4/131) and all of them were point mutations located in exon 2 and 3; the median white blood cell count and median percentage of blasts at diagnosis were significantly higher in LEF1 high expression group than in low expression group (70.6 × 10⁹/L vs 26.2 × 10⁹/L)(P = 0.010); (81.0% vs 57.0%) (P = 0.014); in addition, the percentage of patients with Philadelphia chromosome positive and patients in high-risk group significantly increased in LEF1 high expression group compared with that in low expression group (66.7% vs 36.5%) (P = 0.038); (79.2% vs 56.2%) (P = 0.044). It is concluded that high expression of LEF1 may play an important role on development of adult ALL.

Increased LEF1 Expression and Decreased Notch2 Expression Are Strong Predictors of Poor Outcomes in Colorectal Cancer Patients

Background/Objective. We aimed to examine the expression of lymphoid enhancer factor 1 (LEF1) and Notch2 in colorectal cancer (CRC) and their association with clinicopathologic variables and CRC patients' prognosis. Methods. Immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot analysis were performed to assess the expression of LEF1 and Notch2 in 184 patients with CRC. Results. We observed a strong negative correlation between LEF1 expression and Notch2 expression (P < 0.001). Both LEF1 mRNA and protein expression increased while the Notch2 mRNA and protein expression decreased in tumor specimens compared with the matched paratumorous normal tissue (P < 0.001). An increase in LEF1 protein expression was significantly associated with lymph node metastases, distant metastasis, advanced TNM (tumor-node-metastasis) stage, and shorter overall survival. A decrease in Notch2 protein expression was associated with poorly differentiated tumors, lymph node metastases, distant metastasis, advanced TNM stage, and shorter overall survival of patients. In the multivariate Cox regression analysis, the LEF1 protein expression (P < 0.001), Notch2 protein expression (P < 0.001), TNM stage (P < 0.001), and the combination of increased LEF1 protein coexpression and decreased Notch2 protein coexpression (P < 0.001) were found to be independent prognostic indicators in CRC. Conclusion. Our results suggest that increased LEF1 coexpression and decreased Notch2 coexpression represent a risk factor for poor overall survival of CRC patients.

New G-CSF-Dependent Signaling Pathway and Its Role In Patients with Severe Congenital Neutropenia and Acute Myeloid Leukemia

AbstractAbstract 385We demonstrated that stimulation of hematopoietic CD34+ cells with granulocyte-colony stimulating factor (G-CSF) leads to phosphorylation of hematopoietic cell-specific Lyn substrate 1 (HCLS1) protein and HCLS1 interaction with G-CSF receptor-associated tyrosine kinases Lyn and Syk. Activated HCLS1 binds to lymphoid-enhancer factor 1 (LEF-1) protein, inducing LEF-1-dependent autoregulation of the LEF-1 gene promoter. Transcription factor LEF-1 is a downstream effector of Wnt signaling and a master regulator of myeloid differentiation. In patients with severe congenital neutropenia (CN), inherited mutations in the HCLS1-associated protein × 1 (HAX1) lead to profound defects in the G-CSF-triggered expression and phosphorylation of HCLS1 protein and subsequently, to reduced expression of LEF-1 and abrogated granulopoiesis. Using immunoprecipitation assay, we identified endogenous HAX1 protein in the complex with LEF-1 and HCLS1 proteins. HAX1 is known as a mitochondrial protein, however using cell fractionation experiments of different myeloid cell lines (NB4, HL60, THP-1) and of primary CD34+ cells we identified endogenous HAX1 protein in different intracellular compartments. Thus, HAX1 protein was expressed in mitochondria, cytoplasm, nucleus, cytosol and in membranous fraction. Expression levels of HAX1 protein varied between different cell lines and between maturation stage of the cells. In vitro inhibition of HCLS1 or HAX1 by shRNA severely disrupted granulocytic differentiation of CD34+ cells, due to a lack of LEF-1 and C/EBPa protein expression and subsequently abolished activation of LEF-1 and C/EBPa gene promoters. Intriguingly, LEF-1 in turn induced HCLS1 mRNA and protein expression by direct binding to the HCLS1 gene promoter and inhibition of LEF-1 in CD34+ cells leads to severe downregulation of HCLS1 protein levels. These findings demonstrated a reciprocal feed-back regulation between LEF-1 and HCLS1 proteins. Moreover, HCLS1-/- mice are neutropenic due to a lack of LEF-1 protein expression and defective in vitro G-CSF-triggered F-actin rearrangement in bone marrow myeloid progenitor cells, as compared to WT mice. At the same time, in bone marrow biopsies of 88.46 % of tested patients (46 out of 52 patients) with acute myeloid leukemia (AML), we found significantly elevated levels of HCLS1 protein. Inhibition of HCLS1 in the AML cell lines and primary blasts of AML patients resulted in reduced proliferation and increased apoptosis. Taken together, we described a novel G-CSFR downstream signaling pathway, with dose-dependent effects on myelopoiesis: diminished expression of HCLS1 and LEF-1 resulted in a “maturation arrest” of granulopoiesis and severe neutropenia, but hyperactivation of one of these proteins led to a hyperproliferation of myeloid progenitors and AML. Disclosures:No relevant conflicts of interest to declare.

Wnt signaling inhibits cementoblast differentiation and promotes proliferation

Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions; however the role of Wnt signaling on cementogenesis has not been examined. In this study, we have identified a consistent expression profile of Wnt signaling molecules in cementoblasts, in vitro by RT-PCR. Exposure of cells to LiCl, which promotes canonical Wnt signaling by inhibiting GSK-3β, increased β-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive promoters, suggesting that an endogenous canonical Wnt pathway functions in cementoblasts. Activation of endogenous canonical Wnt signaling with LiCl suppressed alkaline phosphatase (ALP) activity and expression of genes associated with cementum function; ALP, bone sialoprotein (BSP), and osteocalcin (OCN). Exposure to Wnt3a, as a representative canonical Wnt member, also inhibited the expression of ALP, BSP, and OCN gene. This effect was accompanied by decreased gene expression of Runx2 and Osterix and by increased gene expression of lymphoid enhancer factor-1. Pretreatment with Dickkopf (Dkk)-1, a potent canonical Wnt antagonist, which binds to a low-density lipoprotein-receptor-related protein (LRP)-5/6 co-receptor, attenuated the suppressive effects of Wnt3a on mRNA expression of Runx2 and OCN on cementoblasts. These findings suggest that canonical Wnt signaling inhibits cementoblast differentiation via regulation of expression of selective transcription factors. Wnt3a also increased the expression of cyclin D1, known as a cell cycle regulator, as well as cell proliferation. In conclusion, these observations suggest that Wnt signaling inhibits cementoblast differentiation and promotes cell proliferation. Elucidating the role of Wnt in controlling cementoblast function will provide new tools needed to improve on existing periodontal regeneration therapies.

Wnt signaling in osteoblast differentiation and bone formation

Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions; however the role of Wnt signaling on cementogenesis has not been examined. In this study, we have identified a consistent expression profile of Wnt signaling molecules in cementoblasts, in vitro by RT-PCR. Exposure of cells to LiCl, which promotes canonical Wnt signaling by inhibiting GSK-3β, increased β-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive promoters, suggesting that an endogenous canonical Wnt pathway functions in cementoblasts. Activation of endogenous canonical Wnt signaling with LiCl suppressed alkaline phosphatase (ALP) activity and expression of genes associated with cementum function; ALP, bone sialoprotein (BSP), and osteocalcin (OCN). Exposure to Wnt3a, as a representative canonical Wnt member, also inhibited the expression of ALP, BSP, and OCN gene. This effect was accompanied by decreased gene expression of Runx2 and Osterix and by increased gene expression of lymphoid enhancer factor-1. Pretreatment with Dickkopf (Dkk)-1, a potent canonical Wnt antagonist, which binds to a low-density lipoprotein-receptor-related protein (LRP)-5/6 co-receptor, attenuated the suppressive effects of Wnt3a on mRNA expression of Runx2 and OCN on cementoblasts. These findings suggest that canonical Wnt signaling inhibits cementoblast differentiation via regulation of expression of selective transcription factors. Wnt3a also increased the expression of cyclin D1, known as a cell cycle regulator, as well as cell proliferation. In conclusion, these observations suggest that Wnt signaling inhibits cementoblast differentiation and promotes cell proliferation. Elucidating the role of Wnt in controlling cementoblast function will provide new tools needed to improve on existing periodontal regeneration therapies.

Alterations of Lymphoid Enhancer Factor-1 Isoform Expression in Solid Tumors and Acute Leukemias

Two major transcripts of lymphoid enhancer factor-1 (LEF-1) have been described. The long isoform with b-catenin binding domain functions as a transcriptional enhancer factor. The short isoform derives from an intronic promoter and exhibits dominant negative activity. Recently, alterations of LEF-1 isoforms distribution have been described in colon cancer. In the current study we employed a quantitative real-time reverse transcription PCR method (TaqMan) to analyze expression of LEF-1 isoforms in a large cohort of human tumor (n = 304) and tumor-free control samples (n = 56). The highest expression level of LEF-1 was found in carcinoma samples whereas brain cancer samples expressed little. Expression of LEF-1 was different in distinct cancer types. For example, the mRNA level of LEF-1 was lower in testicular tumor samples compared with tumor-free control samples. Besides epithelial cancers, significant LEF-1 expression was also found in hematopoietic cells. In hematological malignancies, overall LEF-1 level was higher in lymphocytic leukemias compared with myeloid leukemias and normal hematopoiesis. However, acute myeloid leukemia and acute lymphocytic leukemia showed a significantly increased fraction of the oncogenic LEF-1 compared with chronic lymphocytic leukemia and chronic myeloid leukemia. Taken together, these data suggest that LEF-1 is abundantly expressed in human tumors and the ratio of the oncogenic and the dominant negative short isoform altered not only in carcinomas but also in leukemia.

Alterations of Lymphoid Enhancer Factor-1 Isoform Expression in Solid Tumors and Acute Leukemias

<title>Abstract</title> Two major transcripts of lymphoid enhancer factor-1 (LEF-1) have been described. The long isoform with β-catenin binding domain functions as a transcriptional enhancer factor. The short isoform derives from an intronic promoter and exhibits dominant negative activity. Recently, alterations of LEF-1 isoforms distribution have been described in colon cancer. In the current study we employed a quantitative real-time reverse transcription PCR method (TaqMan) to analyze expression of LEF-1 isoforms in a large cohort of human tumor (<italic>n</italic>=304) and tumor-free control samples (<italic>n</italic>=56). The highest expression level of LEF-1 was found in carcinoma samples whereas brain cancer samples expressed little. Expression of LEF-1 was different in distinct cancer types. For example, the mRNA level of LEF-1 was lower in testicular tumor samples compared with tumor-free control samples. Besides epithelial cancers, significant LEF-1 expression was also found in hematopoietic cells. In ematological malignancies, overall LEF-1 level was higher in lymphocytic leukemias compared with myeloid leukemias and normal hematopoiesis. However, acute myeloid leukemia and acute lymphocytic leukemia showed a significantly increased fraction of the oncogenic LEF-1 compared with chronic lymphocytic leukemia and chronic myeloid leukemia. Taken together, these data suggest that LEF-1 is abundantly expressed in human tumors and the ratio of the oncogenic and the dominant negative short isoform altered not only in carcinomas but also in leukemia. <b>FullText for HTML: </b><a target="_blank" class="col_4A90E2" href="https://doi.org/10.1093/abbs/37.3.173">https://doi.org/10.1093/abbs/37.3.173</a>

Expression of enamel proteins and LEF1 in adamantinomatous craniopharyngioma: evidence for its odontogenic epithelial differentiation

Adamantinomatous craniopharyngioma (ACP) resembles histologically some odontogenic tumours, such as ameloblastoma and calcifying odontogenic cyst. However, there has been no evidence that ACP differentiates also functionally as odontogenic epithelium. The aim of this study was to gain evidence of odontogenic epithelial differentiation in ACP by means of immunohistochemistry. Among normal human tissues, enamel proteins are expressed exclusively in teeth, and lymphoid enhancer factor 1 (LEF1), in co-operation with beta-catenin, play an important role in tooth development. The expression of these proteins is therefore indicative of odontogenic epithelial differentiation. The expression of enamel proteins and LEF1 was examined in 10 adamantinomatous and six papillary craniopharyngiomas. All the ACPs showed a variable degree of enamel protein expression, including amelogenin, enamelin and enamelysin, mainly in ghost cells. LEF1 was also heterogeneously expressed in ACPs; remarkably, its expression pattern was identical to that of nuclear beta-catenin accumulation. In contrast, none of the papillary craniopharyngiomas expressed enamel proteins or LEF1. These results suggest that ACP consistently shows odontogenic epithelial differentiation. Since ACPs harbour beta-catenin mutation, the inappropriate activation of beta-catenin/LEF1 complex-dependent transcription may play a critical role in ACP tumorigenesis.

Lymphoid Enhancer Factor-1 Blocks Adenomatous Polyposis Coli-mediated Nuclear Export and Degradation of β-Catenin: REGULATION BY HISTONE DEACETYLASE 1

The oncogenic protein beta-catenin is overexpressed in many cancers, frequently accumulating in nuclei where it forms active complexes with lymphoid enhancer factor-1 (LEF-1)/T-cell transcription factors, inducing genes such as c-myc and cyclin D1. In normal cells, nuclear beta-catenin levels are controlled by the adenomatous polyposis coli (APC) protein through nuclear export and cytoplasmic degradation. Transient expression of LEF-1 is known to increase nuclear beta-catenin levels by an unknown mechanism. Here, we show that APC and LEF-1 compete for nuclear beta-catenin with opposing consequences. APC can export nuclear beta-catenin to the cytoplasm for degradation. In contrast, LEF-1 anchors beta-catenin in the nucleus by blocking APC-mediated nuclear export. LEF-1 also prevented the APC/CRM1-independent nuclear export of beta-catenin as revealed by in vitro assays. Importantly, LEF-1-bound beta-catenin was protected from degradation by APC and axin in SW480 colon cancer cells. The ability of LEF-1 to trap beta-catenin in the nucleus was down-regulated by histone deacetylase 1, and this correlated with a decrease in LEF1 transcription activity. Our findings identify LEF-1 as key regulator of beta-catenin nuclear localization and stability and suggest that overexpression of LEF-1 in colon cancer and melanoma cells may contribute to the accumulation of oncogenic beta-catenin in the nucleus.

Alx4 Binding to LEF-1 Regulates N-CAM Promoter Activity

During murine embryogenesis, expression of the paired-like homeodomain protein Alx4 is restricted to tissues whose development depends on the expression of lymphoid enhancer factor-1 (LEF-1). Given the defects seen in hair follicle development in both LEF-1 and Alx4 knockout and mutant animals and the overlapping expression patterns, we predicted that LEF-1 and Alx4 might form physical complexes. We demonstrate here the interaction between LEF-1 and Alx4. This interaction is mediated through a specific proline-rich domain in the N-terminal region of Alx4 and requires the DNA-binding domain (HMG-box) of LEF-1. We also demonstrate that LEF-1 and Alx4 can bind simultaneously to adjacent sites on the neural cell adhesion molecule (N-CAM) promoter and that this binding alters N-CAM promoter activity. Furthermore, when expressed in primary mammary stromal cells, Alx4 decreases the expression of endogenous N-CAM protein. These results reveal a potential mechanism that gives rise to mesenchymal-specific activities of LEF-1.

Nuclear localization of β-catenin by interaction with transcription factor LEF-1

Vertebrate β-catenin and Drosophila Armadillo share structural similarities suggesting that β-catenin, like Armadillo, has a developmental signaling function. Both proteins are present as components of cell adherens junctions, but accumulate in the cytoplasm upon Wingless/Wnt signaling. β-Catenin has axis-inducing properties like Wnt when injected into Xenopus blastomeres, providing evidence for participation of β-catenin in the Wnt-pathway, but until now no downstream targets for β-catenin have been identified. Here we demonstrate that β-catenin binds to the HMG-type transcription factor lymphoid enhancer factor-1 (LEF-1), resulting in a nuclear translocation of β-catenin both in cultured mouse cells and after ectopic expression of LEF-1 in two-cell mouse embryos. LEF-1/β-catenin complexes bind to the promoter region of the E-cadherin gene in vitro, suggesting that this interaction could regulate E-cadherin transcription. As shown for β-catenin, ectopic expression of LEF-1 in Xenopus embryos caused duplication of the body axis, indicating a regulatory role for a LEF-1-like molecule in dorsal mesoderm formation.

Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate.

T cell-specific transcription factor (TCF-1) and lymphoid enhancer factor 1 (LEF-1) have been implicated exclusively in the regulation of T cell-specific genes. The only adult tissue other than thymus known to express these factors is spleen and lymph node, which contain low levels of LEF-1 and no TCF-1. We noticed that genes involved in hair-specific gene expression possess LEF-1/TCF-1 consensus motifs located in similar positions relative to their TATA box. We show that of the two factors only LEF-1 is expressed in hair follicles; it can be cloned in both splice forms from human skin keratinocytes and it can bind to these sites in the hair promoters. We show that LEF-1 mRNA is present in pluripotent ectoderm, and it is up-regulated in a highly restricted pattern just before the formation of underlying mesenchymal condensates and commitment of overlying ectodermal cells to invaginate and become hair follicles. New waves of ectodermal LEF-1 spots appear concomitant with new waves of follicle morphogenesis. To test whether LEF-1 patterning might be functionally important for hair patterning and morphogenesis, we used transgenic technology to alter the patterning and timing of LEF-1 over the surface ectoderm. Striking abnormalities arose in the positioning and orientation of hair follicles, leaving a marked disruption of this normally uniform patterning. This provides the first direct evidence that ectodermal cues are critical in establishing these developmental processes, which at later stages are known to be influenced by underlying mesenchyme. Remarkably, elevated LEF-1 in the lip furrow epithelium of developing transgenic animals triggered these cells to invaginate, sometimes leading to the inappropriate adoption of hair follicle and tooth cell fates. Collectively, our findings demonstrate that ectodermal expression of LEF-1 plays a central role in gene expression, pattern formation, and other developmental processes involving epithelial-mesenchymal associations.