LEF1 Research Articles

Bmp7 and Lef1 Are the Downstream Effectors of Androgen Signaling in Androgen-Induced Sex Characteristics Development in Medaka

Androgens play key roles in the morphological specification of male type sex attractive and reproductive organs, whereas little is known about the developmental mechanisms of such secondary sex characters. Medaka offers a clue about sexual differentiation. They show a prominent masculine sexual character for appendage development, the formation of papillary processes in the anal fin, which has been induced in females by exogenous androgen exposure. This current study shows that the development of papillary processes is promoted by androgen-dependent augmentation of bone morphogenic protein 7 (Bmp7) and lymphoid enhancer-binding factor-1 (Lef1). Androgen receptor (AR) subtypes, ARα and ARβ, are expressed in the distal region of outgrowing bone nodules of developing papillary processes. Development of papillary processes concomitant with the induction of Bmp7 and Lef1 in the distal bone nodules by exposure to methyltestosterone was significantly suppressed by an antiandrogen, flutamide, in female medaka. When Bmp signaling was inhibited in methyltestosterone-exposed females by its inhibitor, dorsomorphin, Lef1 expression was suppressed accompanied by reduced proliferation in the distal bone nodules and retarded bone deposition. These observations indicate that androgen-dependent expressions of Bmp7 and Lef1 are required for the bone nodule outgrowth leading to the formation of these secondary sex characteristics in medaka. The formation of androgen-induced papillary processes may provide insights into the mechanisms regulating the specification of sexual features in vertebrates.

Aberrant G-CSFR Signaling in Congenital Neutropenia

Clinical observations revealed that congenital neutropenia (CN) patients harboring either ELANE or HAX mutations have similar bone marrow morphology, responses to G-CSF therapy, requirements of G-CSF dosages, and the risk of developing leukemia. Therefore, we suggested a common pathomechanism of defective G-CSFR-triggered granulopoiesis downstream of both mutated genes in these patients. We identified severely diminished expression and functions of the transcription factors lymphoid enhancer binding factor-1 (LEF-1) and C/EBPa in myeloid cells of CN patients, in comparison to healthy individuals and patients with cyclic neutropenia (CyN). LEF-1 expression was abrogated in patients harboring either ELANE or HAX1 mutations, which suggested LEF-1 as a possible common candidate factor for defective G-CSFR signaling. We further identified a mechanism of the diminished LEF-1 expression downstream of HAX1 or ELANE mutations. HAX1 is HCLS1-Associated protein X1. HCLS1 is Hematopoietic Cell-Specific Lyn Substrate 1. We found that HCLS1 protein is expressed at high levels in human myeloid cells and is phosphorylated upon stimulation with G-CSF. HCLS1 interacted with LEF-1 protein, inducing nuclear translocation of LEF-1, LEF-1 autoregulation, C/EBPa activation, and granulocytic differentiation. In CN patients with HAX1 mutations, we found profound defects in the G-CSF-triggered phosphorylation of HCLS1, subsequently leading to abrogated nuclear transport and autoregulation of LEF-1. In CN patients with ELANE mutations we detected severely reduced levels of the natural inhibitor of neutrophil elastase (NE), and secretory leukocyte protease inhibitor (SLPI). We demonstrated the important role of SLPI in myeloid differentiation by activation of Erk1/2 phosphorylation and subsequent phErk1/2-triggered tyrosine phosphorylation and activation of the LEF-1 protein. Therefore, the direct link between ELANE mutations and diminished LEF-1 expression was established: in these patients LEF-1 protein expression is diminished due to the reduced levels of SLPI. We further evaluated how G-CSF treatment overcomes maturation arrest of granulopoiesis in CN patients despite the absence of LEF-1 and C/EBPa in myeloid cells. We identified nicotinamide phosphoribosyltransferase (NAMPT) as an essential enzyme mediating G-CSF-triggered granulopoiesis in healthy individuals and in CN patients. Treatment of healthy individuals with G-CSF resulted in upregulation of NAMPT levels in myeloid cells and in plasma. NAMPT and NAD+ amounts were even more dramatically elevated by G-CSF treatment of CN individuals. The molecular events triggered by NAMPT included elevation of NAD+, NAD+-dependent activation of protein deacetylase sirtuin-1 (SIRT1), binding of SIRT1 to the myeloid specific transcription factors C/EBPα and C/EBPβ, and activation of these transcription factors. In CN patients, C/EBPα expression is severely diminished; therefore “steady-state” granulopoiesis could not be activated. G-CSF treatment induces expression of C/EBPβ in these patients via NAMPT and SIRT1 and operated via the “emergency” pathway. We also investigated the patterns of acquisition of leukemia-associated-mutations in 31 CN patients developing leukemia using next-generation DNA deep sequencing. Intriguingly, 20 of the 31 patients (64.5%) demonstrated mutations within RUNX1. The majority of patients with RUNX1 mutations (85%) had acquired CSF3R mutations. Other leukemia-associated mutations in the patients with RUNX1 mutations were found infrequently. Cytogenetics of the leukemic cells revealed that 10 patients with RUNX1 mutations developed monosomy 7, and six patients had trisomy 21. Single cell analysis in two patients revealed that RUNX1 and CSF3R mutations were segregated in the same malignant clone. Functional studies demonstrated proliferative advantage of CD34+ cells transduced with mutated RUNX1 and CSF3R. By analysis of the leukemogenic role of the defective G-CSFR signaling in CN patients we identified a significant and sustained elevation in the levels of phospho-STAT5 in hematopoietic CD34+ cells of CN patients which were even higher in CN/ acute myeloid leukemia patients. The other possible reason for the leukemogenic transformation could be elevated NAMPT/SIRT-triggered deacetylation of tumor supressor protein p53, proto-oncogene FOXO3a and Akt proteins. Disclosures:No relevant conflicts of interest to declare.

Investigating The Role Of MLL2 (Mll4) In B Cell Development

BackgroundThe myeloid/lymphoid or mixed-lineage leukaemia 2 (MLL2) histone methyltransferase (referred to as Mll4 in mice) forms part of a large multiprotein complex, which catalyses the methylation of lysine 4 on histone H3 (H3K4). High levels of this histone modification are detected at promoter regions of actively transcribed genes. Loss-of-function mutations in MLL2 have been identified in 80-90% of follicular lymphoma (FL) cases. These mutations are distributed throughout the coding region and lead to the loss of the C-terminal catalytic SET domain and reduction in H3K4 methylation. We generated a Mll4 knockout (Mll4-/-) mouse model to elucidate the effects of Mll4 loss on B cell development and understand how mutations in this gene contribute to FL pathogenesis. Results and DiscussionCre:ERT2-mediated recombination was used to induce the deletion of exons 2–4 of Mll4 in adult mice. CD19+ B lymphocytes were purified from the spleens of wild-type (Mll4WT) and Mll4-/- mice and confirmed the loss of Mll4 mRNA and protein by quantitative RT-PCR (qRT-PCR) and immunoblotting, respectively. As expected, B cells lacking Mll4 (Mll4ΔB/ΔB) exhibited a global reduction in H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3), compared with normal B cells. Gene expression profiling (GEP) using the GeneChip® Mouse Genome 430 2.0 Array (n=5 Mll4WT + 5 Mll4-/- mice) was carried out to determine the transcriptional changes upon loss of Mll4. We identified >200 genes differentially expressed (>2-fold) between Mll4WT and Mll4ΔB/ΔB CD19+ B cells. The top 40 candidate genes (p<0.05) were verified in an independent series of experiments using qRT-PCR. We noted a significant decrease in the expression of the transcription factor lymphoid enhancer-binding factor 1 (Lef1) and the histone acetyltransferase nuclear receptor coactivator 3 (Ncoa3). The downregulation of these genes is consistent with published data, as the promoters of both are marked by H3K4me3 in normal mouse CD19+ B cells. Lef1 is a key regulator of lymphoid differentiation, while Ncoa3 depletion has been shown to induce B-cell lymphoma in mice as a result of constitutive NF-κB activation. A significant number of genes (p<0.001) involved in cell cycle and immune response were upregulated in Mll4ΔB/ΔB CD19+ B cells. Furthermore, loss of Mll4 led to an elevated expression (2.4-fold) of activation-induced cytidine deaminase (Aicda), an enzyme required for germinal centre-derived lymphomagenesis. Immunophenotyping was used to examine the role of Mll4 at different stages of B cell development. We detected a significant reduction in the number of pre-B cells (B220+CD43–IgM–) in the bone marrow of Mll4-/- mice (n=12), compared with their littermate controls (n=10; p<0.01), although this did not affect the number of peripheral mature splenic B cells (B220+IgM+). Mll4 loss had an adverse effect on immune response, with CD19+ B cells failing to induce expression of the MHC-II, CD86, CD40 and CD69 activation markers upon in vitro stimulation with lipopolysaccharide and interleukin 4. ConclusionOur findings provide the first insight into the potential mechanistic link between MLL2 loss and the onset of FL using a mouse model. Mll4-/- mice do not develop any lymphoproliferative disorders, but show defects in B cell development and an impaired immune response. Furthermore, loss of Mll4 leads to the global depletion of H3K4 methylation in mouse B lymphocytes, thus affecting the expression of Lef1, Ncoa3 and Aicda. Although these MLL2/Mll4 target genes have defined roles in B cell biology, their contribution to the pathogenesis of FL will depend on when MLL2 mutations arise during FL development. Disclosures:No relevant conflicts of interest to declare.

Altered canonical Wingless-Int signaling in the ovine fetal lung after exposure to intra-amniotic lipopolysaccharide and antenatal betamethasone

Antenatal inflammation and maternal corticosteroids induce fetal lung maturation but interfere with late lung development. Canonical Wingless-Int (Wnt) signaling directs lung development and repair. We showed that intra-amniotic (IA) lipopolysaccharide (LPS) exposure disrupted developmental signaling pathways in the preterm lamb lungs. Therefore, we hypothesized that pulmonary Wnt signaling was altered by exposure to IA LPS and/or antenatal corticosteroids. Ovine fetuses were exposed to IA LPS, maternal intramuscular betamethasone, a control saline injection, or a combination thereof at 107 and/or 114 d gestational age (term = 150 d gestational age) before delivery at 121 d gestational age. IA LPS exposure decreased the lung expression of lymphoid enhancer-binding factor 1 (LEF1), a major Wnt pathway effector. WNT1, WNT4, and downstream messenger β-catenin decreased after LPS exposure. WNT7b mRNA increased fourfold 14 d post-LPS exposure. Betamethasone treatment 7 d before LPS exposure prevented the reduction in LEF1 expression, whereas betamethasone administration after LPS normalized the LPS-induced increase in Wnt7b mRNA. IA LPS exposure decreased canonical Wnt signaling in the developing lung. Antenatal corticosteroids before or after IA inflammation had different effects on pulmonary Wnt signaling. This study provides new insights into possible mechanisms by which prenatal inflammation affects lung development and how corticosteroid can be beneficial in this setting.

Innate immune control of EBV-infected B cells by invariant natural killer T cells

AbstractIndividuals with X-linked lymphoproliferative disease lack invariant natural killer T (iNKT) cells and are exquisitely susceptible to Epstein-Barr virus (EBV) infection. To determine whether iNKT cells recognize or regulate EBV, resting B cells were infected with EBV in the presence or absence of iNKT cells. The depletion of iNKT cells increased both viral titers and the frequency of EBV-infected B cells. However, EBV-infected B cells rapidly lost expression of the iNKT cell receptor ligand CD1d, abrogating iNKT cell recognition. To determine whether induced CD1d expression could restore iNKT recognition in EBV-infected cells, lymphoblastoid cell lines (LCL) were treated with AM580, a synthetic retinoic acid receptor-α agonist that upregulates CD1d expression via the nuclear protein, lymphoid enhancer-binding factor 1 (LEF-1). AM580 significantly reduced LEF-1 association at the CD1d promoter region, induced CD1d expression on LCL, and restored iNKT recognition of LCL. CD1d-expressing LCL elicited interferon γ secretion and cytotoxicity by iNKT cells even in the absence of exogenous antigen, suggesting an endogenous iNKT antigen is expressed during EBV infection. These data indicate that iNKT cells may be important for early, innate control of B cell infection by EBV and that downregulation of CD1d may allow EBV to circumvent iNKT cell-mediated immune recognition.

Clinical significance of lymphoid enhancer-binding factor 1 expression in acute myeloid leukemia

Lymphoid enhancer-binding factor 1 (LEF1) is a downstream effector of the Wnt/β-catenin signaling pathway and its dysregulation is associated with a number of malignant diseases such as leukemia. We explored the expression profile of LEF1 in acute myeloid leukemia (AML) and determined its specific prognostic significance in this disease. The LEF1 mRNA level in patients with previously untreated AML was significantly higher than in normal controls. Patients with AML with relatively higher LEF1 expression were more likely to achieve a complete remission (CR) following induction therapy in comparison to those with a lower LEF1 level. Moreover, we provide the first evidence that primary AML samples with AML1–ETO or PML–RARα have a higher LEF1 level compared with those without each fusion gene. High LEF1 expression predicts a significantly better overall survival for patients with intermediate-risk cytogenetics. High LEF1 level was associated with a favorable relapse-free survival in patients with FLT3-ITD wild-type. Finally, a scoring system based on LEF1 level and mutation status of FLT3-ITD or NPM1 is reliable to predict the outcome for AML with intermediate-risk cytogenetics. Our results indicate that LEF1 contributes to the pathophysiology of AML and could serve as a novel predictor of better treatment response. LEF1 level may be incorporated into an improved risk classification system for certain specific subtypes of AML.

Identification of HDAC4 as a target of γ-catenin that regulates the oncogenic K-Ras-mediated malignant phenotype of Rat2 cells

The mechanisms by which activated Ras accelerates malignant transformation of normal cells are not fully understood. Here, we characterized the role and molecular mechanism of γ-catenin in regulating the malignant phenotype of Rat2 cells induced by codon 12-mutant K-Ras (K-Ras12V). Suppression of γ-catenin signaling by K-Ras12V was an early event and played a crucial role in promoting the acquisition of a highly metastatic phenotype of Rat2 cells. Notably, the gene encoding histone deacetylase 4 (HDAC4) was identified as a target of γ-catenin during this process. The transcription factor, lymphoid enhancer-binding factor-1 (Lef1), was involved in the modulation of HDAC4 transcription, and disruption of this pathway was a key event in promoting the invasion and migration of K-Ras12V-transduced Rat2 cells. Thus, our findings extend the range of targets for the development of new drugs for the therapy of oncogenic K-Ras-driven cancer.

PAD4 regulates proliferation of multipotent haematopoietic cells by controlling c-myc expression

Peptidylarginine deiminase 4 (PAD4) functions as a transcriptional coregulator by catalyzing the conversion of histone H3 arginine residues to citrulline residues. Although the high level of PAD4 expression in bone marrow cells suggests its involvement in haematopoiesis, its precise contribution remains unclear. Here we show that PAD4, which is highly expressed in lineage− Sca-1+ c-Kit+ (LSK) cells of mouse bone marrow compared with other progenitor cells, controls c-myc expression by catalyzing the citrullination of histone H3 on its promoter. Furthermore, PAD4 is associated with lymphoid enhancer-binding factor 1 and histone deacetylase 1 at the upstream region of the c-myc gene. Supporting these findings, LSK cells, especially multipotent progenitors, in PAD4-deficient mice show increased proliferation in a cell-autonomous fashion compared with those in wild-type mice. Together, our results strongly suggest that PAD4 regulates the proliferation of multipotent progenitors in the bone marrow by controlling c-myc expression.

Invariant natural killer T cells recognize and restrict the transformation of B cells by Epstein-Barr virus (P4363)

Abstract Individuals with X-linked lymphoproliferative disease (XLP) lack invariant natural killer T (iNKT) cells and are uniquely susceptible to Epstein-Barr virus (EBV). To determine whether iNKT cells recognize or regulate EBV replication, human B cells were infected with GFP+ EBV 95.8 in the presence or absence of iNKT cells. We observed that iNKT cells significantly reduced early EBV titres and GFP+ B cell expansion but that within 72 hours of infection, B cells transformed into lymphoblastoid cell lines (LCL) by EBV had downregulated the iNKT antigen receptor CD1d, completely abrogating iNKT recognition of the lipid agonist α-galactosylceramide (αGalCer). To determine whether ectopic CD1d expression could restore iNKT recognition, LCL were treated with a synthetic retinoic acid receptor-α agonist AM580 known to modulate CD1d expression by regulating the nuclear protein lymphoid enhancer-binding factor-1 (LEF-1). AM580 treatment significantly reduced LEF-1 at the CD1d promoter, induced surface CD1d expression and enabled iNKT recognition of LCL loaded with αGalCer. Unexpectedly, AM580-treated LCL also activated iNKT function even in the absence of αGalCer suggesting that EBV infection and transformation triggers endogenous iNKT antigen expression. These observations indicate that iNKT cells may be important for the early recognition and control of EBV-infected B cells prior to CD1d downregulation and the loss of CD1d may allow EBV to circumvent iNKT-mediated immunosurveillance.

Wnt3a stimulates maturation of impaired neutrophils developed from severe congenital neutropenia patient-derived pluripotent stem cells

The derivation of induced pluripotent stem (iPS) cells from individuals of genetic disorders offers new opportunities for basic research into these diseases and the development of therapeutic compounds. Severe congenital neutropenia (SCN) is a serious disorder characterized by severe neutropenia at birth. SCN is associated with heterozygous mutations in the neutrophil elastase [elastase, neutrophil-expressed (ELANE)] gene, but the mechanisms that disrupt neutrophil development have not yet been clarified because of the current lack of an appropriate disease model. Here, we generated iPS cells from an individual with SCN (SCN-iPS cells). Granulopoiesis from SCN-iPS cells revealed neutrophil maturation arrest and little sensitivity to granulocyte-colony stimulating factor, reflecting a disease status of SCN. Molecular analysis of the granulopoiesis from the SCN-iPS cells vs. control iPS cells showed reduced expression of genes related to the wingless-type mmtv integration site family, member 3a (Wnt3a)/β-catenin pathway [e.g., lymphoid enhancer-binding factor 1], whereas Wnt3a administration induced elevation lymphoid enhancer-binding factor 1-expression and the maturation of SCN-iPS cell-derived neutrophils. These results indicate that SCN-iPS cells provide a useful disease model for SCN, and the activation of the Wnt3a/β-catenin pathway may offer a novel therapy for SCN with ELANE mutation.

Lymphoid Enhancer-binding Factor 1 Expression Precedes Dentin Sialophosphoprotein Expression during Rat Odontoblast Differentiation and Regeneration

IntroductionThe molecular mechanisms behind odontoblast differentiation remain obscure. Lymphoid enhancer-binding factor 1 (Lef1) is a transcription factor that mediates Wnt signaling and has been suggested to regulate dentin sialophosphoprotein (Dspp) expression in vitro. This study aimed to clarify their precise relationship in the process of odontoblast differentiation in vivo. MethodsThe detailed spatiotemporal expression patterns of Lef1 and Dspp together with other known and putative odontoblast differentiation markers such as P21 and heat-shock protein 25 (Hsp25) were examined by in situ hybridization and immunohistochemistry on paraffin sections of rat incisors and developing molars at postnatal days 1–100. To observe odontoblast regeneration following tooth injury, a cavity was prepared on the upper first molar of 10-week-old rats and the expressions of Lef1 and Dspp were investigated. ResultsFollowing undifferentiated state expressing none of these examined markers, preodontoblasts begun to express P21, Lef1 and Hsp25 according to their progress of differentiation, although Dspp was undetectable. Immature odontoblasts commenced transcribing Dspp simultaneously with dentin calcification. Lef1, Dspp and Hsp25 were co-expressed in mature odontoblasts. In contrast to continuously growing incisors, Lef1, Dspp and P21 were down-regulated in the resting odontoblasts in molars when primary dentin formation was completed. Remarkably, Lef1 expression also preceded Dspp expression in newly differentiated odontoblast-like cells during the pulpal healing process after tooth injury. ConclusionsLef1 expression precedes Dspp expression without exception in both primary and reparative dentinogeneses. Our results suggest that Lef1 might play a key role in odontoblast differentiation through regulating Dspp expression.

Lef1 Contributes to the Differentiation of Bulge Stem Cells by Nuclear Translocation and Cross-Talk with the Notch Signaling Pathway

Lymphoid enhancer binding factor-1 (Lef1) is an essential regulatory protein in the Wnt signal pathway, which controls cell growth and differentiation. Investigators in the field of skin biology have confirmed that multipotent bulge stem cells (BSCs) are responsible for hair follicle development and regeneration. However, the role of Lef1 remains poorly understood. In this study, we investigated the pattern of Lef1 expression at different stages of the hair growth cycle. Lef1 was strongly expressed during anagen but attenuated in both catagen- and telogen-phase hair follicles in vivo. When stem cells were induced to differentiate toward a hair fate in a co-culture system, Lef1 was notably up-regulated and accumulated in the nucleus, appearing to activate the target protein c-myc and jagged1. Simultaneously, the Wnt and Notch signaling pathways were co-activated, as confirmed by the increased expression of β-catenin and notch1. Plasmids expressing Lef1 and ΔNLef1, a construct in which the β-catenin-binding domain of Lef1 was deleted, were used to evaluate the effects of Lef1 on stem cell differentiation. Lef1 overexpression promoted bulge stem cell differentiation toward a hair fate, which was accompanied by the subsequent migration of β-catenin into the nucleus, whereas no changes were observed in the control group. Taken together, our results demonstrate that Lef1 plays an important role in bulge stem cell differentiation, promoting β-catenin translocation into the nucleus, activating downstream signaling molecules, eventually causing hair follicle bulge stem cells to adopt the hair fate.

Transcriptome Profiling Reveals Differentially Expressed Transcripts Between the Human Adrenal Zona Fasciculata and Zona Reticularis

The human adrenal zona fasciculata (ZF) and zona reticularis (ZR) are responsible for the production of cortisol and 19-carbon steroids (often called adrenal androgens), respectively. However, the gene profiles and exact molecular mechanisms leading to the functional phenotype of the ZF and ZR are still not clearly defined. In the present study, we identified the transcripts that are differentially expressed in the ZF and ZR. The objective of the study was to compare the transcriptome profiles of ZF and ZR. ZF and ZR were microdissected from 10 human adrenals. Total RNA was extracted from 10 ZF/ZR pairs and hybridized to Illumina microarray chips. The 10 most differentially expressed transcripts were studied with quantitative RT-PCR (qPCR). Immunohistochemistry was also performed on four zone-specific genes. Microarray results demonstrated that only 347 transcripts of the 47 231 were significantly different by 2-fold or greater in the ZF and ZR. ZF had 195 transcripts with 2-fold or greater increase compared with its paired ZR, whereas ZR was found to have 152 transcripts with 2-fold or greater higher expression than in ZF. Microarray and qPCR analysis of transcripts encoding steroidogenic enzymes (n = 10) demonstrated that only 3β-hydroxysteroid dehydrogenase, steroid sulfotransferase, type 5 17β-hydroxysteroid dehydrogenase, and cytochrome b5 were significantly different. Immunohistochemistry and qPCR studies confirmed that the ZF had an increased expression of lymphoid enhancer-binding factor 1 and nephroblastoma overexpressed, whereas ZR showed an increased expression of solute carrier family 27 (fatty acid transporter) (SLC27A2), member 2 and TSPAN12 (tetraspanin 12) CONCLUSION: Microarray revealed several novel candidate genes for elucidating the molecular mechanisms governing the ZF and ZR, thereby increasing our understanding of the functional zonation of these two adrenocortical zones.

High Expression of Lymphoid Enhancer-Binding Factor 1 (LEF1) Is Related to Ummutated Status of Igvh and Is an Unfavorable Prognostic Marker for Aggressive CLL Patients

Abstract 4573Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal CD5+ B lymphocytes. CLL is highly variable and heterogeneous in clinical courses, prognosis and outcomes, so efficient prognostic markers are essential to better identify different clinical courses of CLL. The prognostic influence of mutations in IGVH, NOTCH1, and other genes, as well as high expression of CD38 and ZAP70 has been identified in recent years. However, these prognostic markers are still not enough to accurately evaluate clinical courses and outcomes of CLL patients. The canonical Wnt pathway is widely involved in multiple physiological and pathological processions. As a member of the TCF/LEF family, lymphoid enhancer-binding factor-1 (LEF1) plays a critical role in Wnt pathway by directly interacting with β-catenin. LEF1 has a regulatory effect in cell proliferation, development and apoptosis of hematopoietic tissues, especially lymphoid lineages. Recent reports identified the potential participation of LEF1 in the pathogenesis of both B-cell and T-cell acute lymphoblastic leukemia. In CLL, it has been demonstrated that LEF1 is significantly overexpressed, and serves as a prosurvival factor for CLL cells.In the present study, we analyzed the relevance of LEF1 expression with clinical characteristics and outcomes in a cohort of 100 CLL patients. All the patients were previously untreated, or did not receive any treatment in the latest 6 months. The median age was 69 years. LEF1 expression was assessed using quantitative real-time PCR and β-actin mRNA served as an inner control gene. We found that LEF1 expression was significantly relevant with the somatic mutation status of IGVH (P<0.05). In patients with unmutated IGVH, LEF1 expression level was far higher than patients with mutated IGVH. We also found a correlation between LEF1 expression and ZAP70 protein expression. LEF1 mRNA expression level is higher among ZAP70-positive patients. However, this correlation is not statistically significant. No obvious association between LEF1 expression and CD38 was found. Patients were classified into high and low LEF1 subgroups according to a LEF1 media split. Then we analyzed LEF1 effect on overall survival (OS) in CLL patients in different clinical stages classified according to Binet or Rai staging system. Among patients in Binet C stage, high LEF1 expression predicted a shorter OS compared with low LEF1 patients (median, 120 months vs. undefined, P<0.05). For patients staging in Rai III-IV, patients with high LEF1 expression had a much shorter OS than low LEF1 patients (median, 108 months vs. undefined, P<0.05). As CLL patients generally share a relative longer term of survival, we concentrated mainly on the patients with a follow-up for more than 24 months for survival analysis. Low LEF1 patients, compared with high LEF1 patients, had a prolonged overall survival (undefined vs. 144 months; P=0.068). Although the correlation between LEF1 expression and overall survival did not get statistically significance yet, the correlation is quite obvious, indicating the prognostic potential of LEF1 for CLL patients.In summary, our data showed that LEF1 expression level is significantly correlated with IGVH mutation status. High LEF1 level is an unfavorable prognostic marker for CLL patients in aggressive stages and patients with a long clinical course. The results suggest that LEF1 may be involved in the pathological process of CLL and has the potential for serving as a prognostic marker of CLL. Disclosures:No relevant conflicts of interest to declare.

Postnatal isoform switch and protein localization of LEF1 and TCF7L2 transcription factors in cortical, thalamic, and mesencephalic regions of the adult mouse brain.

β-Catenin signaling, leading to the activation of lymphoid enhancer-binding factor 1/T cell factor (LEF1/TCF) transcription factors, plays a well-established role in transcription regulation during development and tissue homeostasis. In the adult organism, the activity of this pathway has been found in stem cell niches and postmitotic thalamic neurons. Recently, studies show that mutations in components of β-catenin signaling networks have been associated with several psychiatric disorders, indicating the involvement of β-catenin and LEF1/TCF proteins in the proper functioning of the brain. Here, we report a comprehensive analysis of LEF1/TCF protein localization and the expression profile of their isoforms in cortical, thalamic, and midbrain regions in mice. We detected LEF1 and TCF7L2 proteins in neurons of the thalamus and dorsal midbrain, i.e., subcortical regions specialized in the integration of diverse sources of sensory information. These neurons also exhibited nuclear localization of β-catenin, suggesting the involvement of β-catenin/LEF1/TCF7L2 in the regulation of gene expression in these regions. Analysis of alternative splicing and promoter usage identified brain-specific TCF7L2 isoforms and revealed a developmentally coordinated transition in the composition of LEF1 and TCF7L2 isoforms. In the case of TCF7L2, the typical brain isoforms lack the so-called C clamp; in addition, the dominant-negative isoforms are predominant in the embryonic thalamus but disappear postnatally. The present study provides a necessary framework to understand the role of LEF1/TCF factors in thalamic and midbrain development until adulthood and predicts that the regulatory role of these proteins in the adult brain is significantly different from their role in the embryonic brain or other non-neural tissues.

Wnt Inhibitory Factor 1 (Wif1) Is Regulated by Androgens and Enhances Androgen-Dependent Prostate Development

Fetal prostate development from urogenital sinus (UGS) epithelium requires androgen receptor (AR) activation in UGS mesenchyme (UGM). Despite growing awareness of sexually dimorphic gene expression in the UGS, we are still limited in our knowledge of androgen-responsive genes in UGM that initiate prostate ductal development. We found that WNT inhibitory factor 1 (Wif1) mRNA is more abundant in male vs. female mouse UGM in which its expression temporally and spatially overlaps androgen-responsive steroid 5α-reductase 2 (Srd5a2). Wif1 mRNA is also present in prostatic buds during their elongation and branching morphogenesis. Androgens are necessary and sufficient for Wif1 expression in mouse UGS explant mesenchyme, and testicular androgens remain necessary for normal Wif1 expression in adult mouse prostate stroma. WIF1 contributes functionally to prostatic bud formation. In the presence of androgens, exogenous WIF1 protein increases prostatic bud number and UGS basal epithelial cell proliferation without noticeably altering the pattern of WNT/β-catenin-responsive Axin2 or lymphoid enhancer binding factor 1 (Lef1) mRNA. Wif1 mutant male UGSs exhibit increased (Sfrp)2 and (Sfrp)3 expression and form the same number of prostatic buds as the wild-type control males. Collectively our results reveal Wif1 as one of the few known androgen-responsive genes in the fetal mouse UGM and support the hypothesis that androgen-dependent Wif1 expression is linked to the mechanism of androgen-induced prostatic bud formation.

HTLV-1 bZIP factor dysregulates the Wnt pathways to support proliferation and migration of adult T-cell leukemia cells

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL). HTLV-1 bZIP factor (HBZ), the viral gene transcribed from the antisense strand, is consistently expressed in ATL cells and promotes their proliferation. In this study, we found that a Wnt pathway-related protein, disheveled-associating protein with a high frequency of leucine residues (DAPLE), interacts with both HTLV-1 Tax and HBZ. In the presence of DAPLE, Tax activated canonical Wnt signaling. Conversely, HBZ markedly suppressed canonical Wnt activation induced by either Tax/DAPLE or β-catenin. As a mechanism of HBZ-mediated Wnt suppression, we found that HBZ targets lymphoid enhancer-binding factor 1, one of the key transcription factors of the pathway, and impairs its DNA-binding ability. We also observed that the canonical Wnt pathway was not activated in HTLV-1-infected cells, whereas the representative of noncanonical Wnt ligand, Wnt5a, which antagonizes canonical Wnt signaling, was overexpressed. HBZ was able to induce Wnt5a transcription by enhancing its promoter activity through the TGF-β pathway. Importantly, knocking down of Wnt5a in ATL cells repressed cellular proliferation and migration. Our results implicate novel roles of HBZ in ATL leukemogenesis through dysregulation of both the canonical and noncanonical Wnt pathways.

Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models

Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targetable transformation characteristics, we established stable populations of BPH-1, PNT1B and RWPE-1 immortalized human PrEC lines that constitutively express flag-tagged ERG3 (fERG). All fERG-expressing populations exhibited characteristics of in vitro and in vivo transformation. Microarray analysis revealed >2000 commonly dysregulated genes in the fERG-PrEC lines. Functional analysis revealed evidence that fERG cells underwent EMT and acquired invasive characteristics. The fERG-induced EMT transcript signature was exemplified by suppressed expression of E-cadherin and keratins 5, 8, 14 and 18; elevated expression of N-cadherin, N-cadherin 2 and vimentin, and of the EMT transcriptional regulators Snail, Zeb1 and Zeb2, and lymphoid enhancer-binding factor-1 (LEF-1). In BPH-1 and RWPE-1-fERG cells, fERG expression is correlated with increased expression of integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1. Interfering RNA suppression of ERG decreased expression of ILK, Snail and LEF-1, whereas small interfering RNA suppression of ILK did not alter fERG expression. Interfering RNA suppression of ERG or ILK impaired fERG-PrEC Matrigel invasion. Treating fERG-BPH-1 cells with the small molecule ILK inhibitor, QLT-0267, resulted in dose-dependent suppression of Snail and LEF-1 expression, Matrigel invasion and reversion of anchorage-independent growth. These results suggest that ILK is a therapeutically targetable mediator of ERG-induced EMT and transformation in PCa.

Interactions among HCLS1, HAX1 and LEF-1 proteins are essential for G-CSF–triggered granulopoiesis

We found that hematopoietic cell-specific Lyn substrate 1 (HCLS1 or HS1) is highly expressed in human myeloid cells and that stimulation with granulocyte colony-stimulating factor (G-CSF) leads to HCLS1 phosphorylation. HCLS1 binds the transcription factor lymphoid-enhancer binding factor 1 (LEF-1), transporting LEF-1 into the nucleus upon G-CSF stimulation and inducing LEF-1 autoregulation. In patients with severe congenital neutropenia, inherited mutations in the gene encoding HCLS1-associated protein X-1 (HAX1) lead to profound defects in G-CSF-triggered phosphorylation of HCLS1 and subsequently to reduced autoregulation and expression of LEF-1. Consistent with these results, HCLS1-deficient mice are neutropenic. In bone marrow biopsies of the majority of tested patients with acute myeloid leukemia, HCLS1 protein expression is substantially elevated, associated with high levels of G-CSF synthesis and, in some individuals, a four-residue insertion in a proline-rich region of HCLS1 protein known to accelerate intracellular signaling. These data demonstrate the importance of HCLS1 in myelopoiesis in vitro and in vivo.

Cutting edge: generation of memory precursors and functional memory CD8+ T cells depends on T cell factor-1 and lymphoid enhancer-binding factor-1.

T cell factor (TCF)-1 and lymphoid enhancer-binding factor (LEF)-1 transcription factors have redundant roles in promoting thymocyte maturation. TCF-1 has been recently shown to critically regulate memory CD8+ T cell differentiation and persistence. The complete spectra of regulatory roles for TCF-1 and LEF-1 in CD8+ T cell responses are yet unknown. We conditionally targeted LEF-1, and by combination with germline deletion of TCF-1, we found that loss of both factors completely abrogated the generation of KLR G1(lo)IL-7Rα+ memory precursors in effector CD8+ T cell populations in response to Listeria monocytogenes infection. Whereas CD8+ effectors deficient for TCF-1 and LEF-1 retained the capacity to express IFN-γ, granzyme B, and perforin, they were defective in TNF-α production. In the memory phase, the Ag-specific CD8+ T cells lacking TCF-1 and LEF-1 exhibited an effector phenotype and were severely impaired in secondary expansion upon rechallenge. Thus, TCF-1 and LEF-1 cooperatively regulate generation of memory precursors and protective memory CD8+ T cells.