T-cell factor 7L2 is a novel regulator of osteoblast functions that acts in part by modulation of hypoxia signaling.

T-cell-like factor (TCF)7l2, a key effector of canonical Wnt signaling, is highly expressed in bone but nothing is known about its role in regulating osteoblast function. To test this, we generated mice with conditional disruption of Tcf7l2 gene in osteoblast lineages using Tcf7l2 floxed and Col1α2-Cre mice. Skeletal parameters were evaluated using heterozygous conditional knockdown (HCKD) mice since homozygous conditional knockout died during pregnancy or immediately after birth. At 5 wk of age, trabecular bone mass of long bones was reduced by 35% as measured by microcomputed tomography (µCT). Histology data showed a 42% reduction in femur trabecular bone mass caused by reduced bone formation. Knockdown of Tcf7l2 expression in osteoblasts decreased proliferation and differentiation by 20%-40%. Expression levels of genes (Hif1α, Vegf, and ß-catenin) targeted by TCF7L2 were decreased by 50% in Tcf7l2-deficient osteoblasts and bones of HCKD mice. We found that the Hif1α gene promoter contained multiple putative TCF7L2 motifs and stabilization of HIF1α protein levels rescued expression of TCF7L2 target genes and alkaline phosphatase (ALP) activity in Tcf7l2-deficient osteoblasts. Furthermore, Tcf7l2 overexpression increased proliferation in the presence of canonical Wnt3a that was not affected by ß-catenin inhibitor providing evidence for a noncanonical signaling in mediating TCF7L2 effects. Tcf7l2 expression was increased in response to mechanical strain (MS) in vitro and in vivo, and disruption of Tcf7l2 expression in osteoblasts reduced MS-induced ALP activity by 35%. We conclude that Tcf7l2, a mechanoresponsive gene, is an important regulator of osteoblast function acting, in part, via hypoxia signaling.NEW & NOTEWORTHY TCF7L2 is expressed by bone but it was not known whether TCF7L2 expression influenced bone development. By using a mouse model with conditional disruption of Tcf7l2 in osteoblast lineage cells, we have demonstrated for the first time, that TCF7L2 plays an important role in regulating osteoblasts via a noncanonical pathway.

Diverse LEF/TCF Expression in Human Colorectal Cancer Correlates with Altered Wnt-Regulated Transcriptome in a Meta-Analysis of Patient Biopsies.

Aberrantly activated Wnt signaling causes cellular transformation that can lead to human colorectal cancer. Wnt signaling is mediated by Lymphoid Enhancer Factor/T-Cell Factor (LEF/TCF) DNA-binding factors. Here we investigate whether altered LEF/TCF expression is conserved in human colorectal tumor sample and may potentially be correlated with indicators of cancer progression. We carried out a meta-analysis of carefully selected publicly available gene expression data sets with paired tumor biopsy and adjacent matched normal tissues from colorectal cancer patients. Our meta-analysis confirms that among the four human LEF/TCF genes, LEF1 and TCF7 are preferentially expressed in tumor biopsies, while TCF7L2 and TCF7L1 in normal control tissue. We also confirm positive correlation of LEF1 and TCF7 expression with hallmarks of active Wnt signaling (i.e., AXIN2 and LGR5). We are able to correlate differential LEF/TCF gene expression with distinct transcriptomes associated with cell adhesion, extracellular matrix organization, and Wnt receptor feedback regulation. We demonstrate here in human colorectal tumor sample correlation of altered LEF/TCF gene expression with quantitatively and qualitatively different transcriptomes, suggesting LEF/TCF-specific transcriptional regulation of Wnt target genes relevant for cancer progression and survival. This bioinformatics analysis provides a foundation for future more detailed, functional, and molecular analyses aimed at dissecting such functional differences.

Developmentally regulated Tcf7l2 splice variants mediate transcriptional repressor functions during eye formation.

Tcf7l2 mediates Wnt/ß-Catenin signalling during development and is implicated in cancer and type-2 diabetes. The mechanisms by which Tcf7l2 and Wnt/ß-Catenin signalling elicit such a diversity of biological outcomes are poorly understood. Here, we study the function of zebrafish tcf7l2alternative splice variants and show that only variants that include exon five or an analogous human tcf7l2 variant can effectively provide compensatory repressor function to restore eye formation in embryos lacking tcf7l1a/tcf7l1b function. Knockdown of exon five specific tcf7l2 variants in tcf7l1a mutants also compromises eye formation, and these variants can effectively repress Wnt pathway activity in reporter assays using Wnt target gene promoters. We show that the repressive activities of exon5-coded variants are likely explained by their interaction with Tle co-repressors. Furthermore, phosphorylated residues in Tcf7l2 coded exon5 facilitate repressor activity. Our studies suggest that developmentally regulated splicing of tcf7l2 can influence the transcriptional output of the Wnt pathway.

Celecoxib inhibits osteoblast maturation by suppressing the expression of Wnt target genes.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to impair bone healing. We previously reported that in colon cancer cells, celecoxib, a COX-2-selective NSAID, inhibited the canonical Wnt/ß-catenin signaling pathway. Since this pathway also plays an important role in osteoblast growth and differentiation, we examined the effect of celecoxib on maturation of osteoblast-like cell line MC3T3-E1. Celecoxib induced degradation of transcription factor 7-like 2, a key transcription factor of the canonical Wnt pathway. Subsequently, we analyzed the effect of celecoxib on two osteoblast differentiation markers; runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP), both of which are the products of the canonical Wnt pathway target genes. Celecoxib inhibited the expression of both RUNX2 and ALP by suppressing their promoter activity. Consistent with these observations, celecoxib also strongly inhibited osteoblast-mediated mineralization. These results suggest that celecoxib inhibits osteoblast maturation by suppressing Wnt target genes, and this could be the mechanism that NSAIDs inhibit bone formation and fracture healing.

Geniposide promotes beta-cell regeneration and survival through regulating ß-catenin/TCF7L2 pathway.

T-cell factor 7-like 2 (TCF7L2) is an important transcription factor of Wnt/ß-catenin signaling, which has critical roles in ß-cell survival and regeneration. In preliminary screening assay, we found geniposide, a naturally occurring compound, was able to increase TCF7L2 mRNA level in Min6 cells. Here we aimed to investigate the role of geniposide in ß-cell and underlying mechanism involved. Geniposide was found to promote ß-cell survival by increasing ß-cell proliferation and decreasing ß-cell apoptosis in cultured mouse islets after challenge with diabetic stimuli. Geniposide protected ß-cell through activating Wnt signaling, enhanced expressions of TCF7L2 and GLP-1R, activated AKT, inhibited GSK3ß activity, and promoted ß-catenin nuclear translocation. The protective effect of geniposide was remarkably suppressed by siRNAs against ß-catenin, or by ICG001 (ß-catenin/TCF-mediated transcription inhibitor). Moreover, geniposide promoted ß-cell regeneration in vivo to normalize blood glucose in high-fat diet and db/db mice. Increased ß-cell proliferation was observed in pancreatic sections of geniposide-treated diabetic mice. Most importantly, geniposide triggered small islet-like cell clusters formation as a result of ß-cell neogenesis from ductal epithelium, which was well correlated with the increase in TCF7L2 expression. In exocrine cells isolated from mouse pancreas, geniposide could induce duct cell differentiation through upregulating TCF7L2 expression and activating JAK2/STAT3 pathway. Taken together, we identified a novel role of geniposide in promoting ß-cell survival and regeneration by mechanisms involving the activation of ß-catenin/TCF7L2 signaling. Our finding highlights the potential value of geniposide as a possible treatment for type 2 diabetes.

Genome-wide analysis of DNA methylation variations caused by chronic glucolipotoxicity in beta-cells.

There is a growing body of literature suggesting the role of interactions between genes and the environment in development of type 2 diabetes mellitus (T2DM). However, the interplay between environment and genetic in developing and progressing T2MD is not fully understood. To determine the effects of high-glucose-lipid on the status of DNA methylation in beta cells, and clarify the mechanism of glucolipotoxicity on beta-cell deterioration, the DNA methylation profile was detected in beta-cells cultured with high-glucose-lipid medium.We utilized a high throughput NimbleGen RN34 CpG Island & Promoter Microarray to investigate the DNA methylation profile in beta-cells cultured with high-glucose-lipid medium. To validate the results of microarray, the immunoprecipitation (MeDIP) PCR was used to test the methylation status of some selected genes. The mRNA and protein expression of insulin and Tcf7l2 in these cells were quantified by RT-PCR and western blot, respectively.We have identified a lot of loci which experienced aberrant DNA methylation in beta-cells cultured with high-glucose-lipid medium. The results of MeDIP PCR were consistency to the microarray. An opposite regulation in transcription and translation of Tcf7l2 gene was found. Furthermore, the insulin mRNA and protein expression in beta-cells also decreased after cultured with high-glucose-lipid medium compared with the control cells.We conclude that chronic glucolipotoxicity could induce aberrant DNA methylation of some genes and may affect these genes expression in beta-cells, which might contribute to beta-cell function failure in T2DM and be helpful to explain, at least partially, the mechanism of glucolipotoxicity on beta-cells deterioration.

Tcf7l2 is tightly controlled during myelin formation.

Recent, studies have shown that Tcf7l2, an important transcription factor in Wnt pathway, plays critical roles in oligodendrocyte development. In this article we report a study showing that Tcf7l2 is under tight regulation during myelin formation. We have found that during early development, Tcf7l2 mRNA appears much earlier than the protein, suggesting a regulation at the translational level. We induced demyelination in a mouse model by a dietary toxin, where remyelination followed after a few weeks, and found that Tcf7l2 protein was expressed specifically during the active remyelination phase. Similarly, in human patients with demyelination diseases, Tcf7l2 protein expression was specifically promoted in regions undergoing active remyelination. During remyelination, Tcf7l2 was only expressed in non-dividing oligodendrocyte precursors and was associated with modest levels of nuclear beta-catenin. We also documented that Tcf7l2 could form protein complex with Olig2, but not with Olig1. Our data showed that during myelin formation, Tcf7l2/beta-catenin is regulated temporally, spatially, and also at levels of expression. These data suggest a key role for Tcf7l2 in myelination/remyelination processes via a tightly controlled activation of Wnt/beta-catenin pathway and the interaction with Olig2.

DIF-1 inhibits the Wnt/ß-catenin signaling pathway by inhibiting TCF7L2 expression in colon cancer cell lines.

We previously reported that differentiation-inducing factor-1 (DIF-1), a morphogen in Dictyostelium discoideum, inhibits the proliferation of human cancer cell lines by inducing ß-catenin degradation and suppressing the Wnt/ß-catenin signaling pathway. To determine whether ß-catenin degradation is essential for the effect of DIF-1, we examined the effect of DIF-1 on human colon cancer cell lines (HCT-116, SW-620 and DLD-1), in which the Wnt/ß-catenin signaling pathway is constitutively active. DIF-1 strongly inhibited cell proliferation and arrested the cell cycle in the G(0)/G(1) phase via the suppression of cyclin D1 expression at mRNA and protein levels without reducing ß-catenin protein. TCF-dependent transcriptional activity and cyclin D1 promoter activity were revealed to be inhibited via suppression of transcription factor 7-like 2 (TCF7L2) expression. Luciferase reporter assays and EMSAs using the TCF7L2 promoter fragments indicated that the binding site for the transcription factor early growth response-1 (Egr-1), which is located in the -609 to -601 bp region relative to the start codon in the TCF7L2 promoter, was involved in DIF-1 activity. Moreover, RNAi-mediated depletion of endogenous TCF7L2 resulted in reduced cyclin D1 promoter activity and protein expression, and the overexpression of TCF7L2 overrode the inhibition of the TCF-dependent transcriptional activity and cyclin D1 promoter activity induced by DIF-1. Therefore, DIF-1 seemed to inhibit the Wnt/ß-catenin signaling pathway by suppressing TCF7L2 expression via reduced Egr-1-dependent transcriptional activity in these colon cancer cell lines. Our results provide a novel insight into the mechanisms by which DIF-1 inhibits the Wnt/ß-catenin signaling pathway.