FOXE1-Dependent Regulation of Macrophage Chemotaxis by Thyroid Cells In Vitro and In Vivo.
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ABSTRACT:
SUBMITTER: Credendino SC
PROVIDER: S-EPMC8307198 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundFoxE1 is a thyroid-specific forkhead transcription factor essential for thyroid gland development, as well as for the maintenance of the thyroid differentiated state in adults. FoxE1 recognizes and binds to a short DNA sequence present in thyroglobulin (Tg) and thyroperoxidase (Tpo) promoters, but FoxE1 binding to regulatory regions other than Tg and Tpo promoters remains almost unexplored. Improving knowledge of the regulatory functions of FoxE1 is necessary to clarify its role in endocrine syndromes and cancer susceptibility.Methodology/principal findingIn order to further investigate downstream FoxE1 targets, we performed a genome-wide expression screening after knocking-down FoxE1 and obtained new insights into FoxE1 transcriptional networks in thyroid follicular cells. After validation, we confirmed Adamts9, Cdh1, Duox2 and S100a4 as upregulated genes and Casp4, Creld2, Dusp5, Etv5, Hsp5a, Nr4a2 and Tm4sf1 as downregulated genes when FoxE1 was silenced. In promoter regions of putative FoxE1-regulated genes and also in the promoters of the classical thyroid genes Nis, Pax8 and Titf1, we performed an in silico search of the FoxE1 binding motif that was in close proximity to the NF1/CTF binding sequence, as previously described for other forkhead factors. Using chromatin immunoprecipitation we detected specific in vivo FoxE1 binding to novel regulatory regions in two relevant thyroid genes, Nis and Duox2. Moreover, we demonstrated simultaneous binding of FoxE1 and NF1/CTF to the Nis upstream enhancer region, as well as a clear functional activation of the Nis promoter by both transcription factors.Conclusions/significanceIn search for potential downstream mediators of FoxE1 function in thyroid cells, we identified two novel direct FoxE1 target genes. To our knowledge, this is the first evidence regarding the implication of Nis and Duox2 in executing the transcriptional program triggered by FoxE1. Furthermore, this study points out the important role of FoxE1 in the regulation of a large number of genes in thyroid cells.
| S-EPMC3652843 | biostudies-literature
Project description:The transcription factor Forkhead box E1 (FOXE1) is a key player in thyroid development and function and has been identified by genome-wide association studies as a susceptibility gene for papillary thyroid cancer. Several cancer-associated polymorphisms fall into gene regulatory regions and are likely to affect FOXE1 expression levels. However, the possibility that changes in FOXE1 expression modulate thyroid cancer development has not been investigated. Here, we describe the effects of FOXE1 gene dosage reduction on cancer phenotype in vivo. Mice heterozygous for FOXE1 null allele (FOXE1+/-) were crossed with a BRAFV600E-inducible cancer model to develop thyroid cancer in either a FOXE1+/+ or FOXE1+/- genetic background. In FOXE1+/+ mice, cancer histological features are quite similar to that of human high-grade papillary thyroid carcinomas, while cancers developed with reduced FOXE1 gene dosage maintain morphological features resembling less malignant thyroid cancers, showing reduced proliferation index and increased apoptosis as well. Such cancers, however, appear severely undifferentiated, indicating that FOXE1 levels affect thyroid differentiation during neoplastic transformation. These results show that FOXE1 dosage exerts pleiotropic effects on thyroid cancer phenotype by affecting histology and regulating key markers of tumor differentiation and progression, thus suggesting the possibility that FOXE1 could behave as lineage-specific oncogene in follicular cell-derived thyroid cancer.
| S-EPMC7792778 | biostudies-literature
Project description:FOXE1 is a thyroid-specific transcription factor essential for thyroid gland development and maintenance of the differentiated state. Interestingly, a strong association has been recently described between FOXE1 expression and susceptibility to thyroid cancer, but little is known about the mechanisms underlying FOXE1-induced thyroid tumorigenesis. Here, we used a panel of human thyroid cancer-derived cell lines covering the spectrum of thyroid cancer phenotypes to examine FOXE1 expression and to test for correlations between FOXE1 expression, the allele frequency of two SNPs and a length polymorphism in or near the FOXE1 locus associated with cancer susceptibility, and the migration ability of thyroid cancer cell lines. Results showed that FOXE1 expression correlated with differentiation status according to histological sub-type, but not with SNP genotype or cell migration ability. However, loss-and-gain-of-function experiments revealed that FOXE1 modulates cell migration, suggesting a role in epithelial-to-mesenchymal transition (EMT). Our previous genome-wide expression analysis identified Zeb1, a major EMT inducer, as a putative Foxe1 target gene. Indeed, gene silencing of FOXE1 decreased ZEB1 expression, whereas its overexpression increased ZEB1 transcriptional activity. FOXE1 was found to directly interact with the ZEB1 promoter. Lastly, ZEB1 silencing decreased the ability of thyroid tumoral cells to migrate and invade, pointing to its importance in thyroid tumor mestastases. In conclusion, we have identified ZEB1 as a bona fide target of FOXE1 in thyroid cancer cells, which provides new insights into the role of FOXE1 in regulating cell migration and invasion in thyroid cancer.
| S-EPMC6993207 | biostudies-literature
Project description:CXCL16 is a multifunctional chemokine that is highly expressed by macrophages and other immune cells in response to bacterial and viral pathogens; however, little is known regarding the role of CXCL16 during parasitic infections. The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis. Even though chemokine production is a host defense mechanism during infection, subversion of the host chemokine system constitutes a survival strategy adopted by the parasite. Here, we report that L. donovani promastigotes upregulate CXCL16 synthesis and secretion by bone marrow-derived macrophages (BMDM). In contrast to wild-type parasites, a strain deficient in the virulence factor lipophosphoglycan (LPG) failed to induce CXCL16 production. Consistent with this, cell treatment with purified L. donovani LPG augmented CXCL16 expression and secretion. Notably, the ability of BMDM to promote migration of cells expressing CXCR6, the cognate receptor of CXCL16, was augmented upon L. donovani infection in a CXCL16- and LPG-dependent manner. Mechanistically, CXCL16 induction by L. donovani required the activity of AKT and the mechanistic target of rapamycin (mTOR) but was independent of Toll-like receptor signaling. Collectively, these data provide evidence that CXCL16 is part of the inflammatory response elicited by L. donovani LPG in vitro Further investigation using CXCL16 knockout mice is required to determine whether this chemokine contributes to the pathogenesis of visceral leishmaniasis and to elucidate the underlying molecular mechanisms.
| S-EPMC6479025 | biostudies-literature
Project description:BackgroundDiscordance of monozygotic twins for thyroid dysgenesis suggests that epigenetic mechanisms may underlie defects in thyroid gland development. This prompted us to evaluate whether differentially methylated regions (DMRs) can be found between human thyroids (either eutopic or ectopic) and matched leukocytes.MethodsTo compare the genome-wide methylation profile of thyroids and leukocytes, immunoprecipitated methylated DNA was interrogated on human promoter plus CpG island tiling arrays. In addition, the methylation profile of the human FOXE1, PAX8, and NKX2.1 promoter was examined using bisulfite sequencing. Finally, the functional impact of CpG methylation of the promoter on FOXE1 expression was assessed with luciferase assays.ResultsGenome-wide methylation profiling and bisulfite sequencing of CpG islands of PAX8 and NKX2.1 promoters revealed no DMR between thyroid and leukocytes. However, bisulfite sequencing revealed that the methylation level of two consecutive CpG dinucleotides (CpG14 and CpG15, which were not covered by the genome-wide array) in one CpG island of the FOXE1 promoter (-1600 to -1140 from the transcription start site) is significantly higher in leukocytes than in eutopic or ectopic thyroid tissues, suggesting that methylation of this region may decrease FOXE1 gene expression. Indeed, luciferase activities were decreased when FOXE1 promoter constructs were methylated in vitro. Moreover, derepression of luciferase activity was observed when the methylation of CpG14 and CpG15 was prevented by mutations.ConclusionWe report a tissue-dependent DMR in the FOXE1 promoter. This DMR contains two consecutive CpG dinucleotides, which are epigenetic modifiers of FOXE1 expression in nontumoral tissues.
| S-EPMC5050036 | biostudies-literature
Project description:UnlabelledChemokines (CK) provide directional cues that mediate the recruitment of leukocytes to sites of inflammation. Broad-spectrum blockade of the CC-CK family, using the vaccinia virus 35K protein, has been shown to cause a potent reduction of systemic inflammation in models of atherosclerosis, vein graft disease and arthritis. We have used a cell membrane-targeted form of 35K, Mem35K, to probe whether cell-associated blockade of chemokine response is sufficient to reduce cell recruitment in inflammation. In Tie2cre mice, activation of a flox-stop Mem35K transgene directed conditional expression of Mem35K in leukocytes and endothelial cells, confirmed by Western blotting, flow cytometry and immunofluorescence microscopy. This conditional Mem35K expression was sufficient to increase cell surface CCL5 binding and reduce chemotaxis in vitro to CCL5, CCL2 and CCL3 but not to non-CC-CK chemoattractants, LTB4, C5a or chemerin. However, in vivo monocyte recruitment into the peritoneum driven by zymosan or CC-chemokine injection, which was demonstrated to be CC-CK dependent using CCR2-/- mice, was not reduced by Mem35K expression, despite the expression of functional Mem35K protein. These findings highlight differing requirements for cell-associated anti-inflammatory activity in in vitro and in vivo models.Key messageMem35K is a cell-associated CC-chemokine binding protein. Conditional Mem35K transgenic mice show expression Mem35K in leukocytes. Mem35K blocks in vitro primary macrophage chemotaxis specifically towards CC-chemokines. Mem35K expression is not sufficient to reduce inflammation in vivo. The requirements for anti-inflammatory activity in vitro and in vivo are different.
| S-EPMC4206339 | biostudies-literature
Project description:BackgroundThe forkhead transcription factor gene E1 (FOXE1) plays an important role in regulation of thyroid development, palate formation and hair morphogenesis in mammals. However, avian FOXE1 genes have not been characterized and as such, codon evolution of FOXE1 orthologs in a broader evolutionary context of mammals and birds is not known.ResultsIn this study we identified the avian FOXE1 gene in chicken, turkey and zebra finch, all of which consist of a single exon. Chicken and zebra finch FOXE1 are uniquely located on the sex-determining Z chromosome. In situ hybridization shows that chicken FOXE1 is specifically expressed in the developing thyroid. Its expression is initiated at the placode stage and is maintained during the stages of vesicle formation and follicle primordia. Based on this expression pattern, we propose that avian FOXE1 may be involved in regulating the evagination and morphogenesis of thyroid. Chicken FOXE1 is also expressed in growing feathers. Sequence analysis identified two microdeletions in the avian FOXE1 genes, corresponding to the loss of a transferable repression domain and an engrailed homology motif 1 (Eh1) C-terminal to the forkhead domain. The avian FOXE1 proteins exhibit a significant sequence divergence of the C-terminus compared to those of amphibian and mammalian FOXE1. The codon evolution analysis (dN/dS) of FOXE1 shows a significantly increased dN/dS ratio in the avian lineages, consistent with either a relaxed purifying selection or positive selection on a few residues in avian FOXE1 evolution. Further site specific analysis indicates that while relaxed purifying selection is likely to be a predominant cause of accelerated evolution at the 3'-region of avian FOXE1, a few residues might have evolved under positive selection.ConclusionsWe have identified three avian FOXE1 genes based on synteny and sequence similarity as well as characterized the expression pattern of the chicken FOXE1 gene during development. Our evolutionary analyses suggest that while a relaxed purifying selection is likely to be the dominant force driving accelerated evolution of avian FOXE1 genes, a few residues may have evolved adaptively. This study provides a basis for future genetic and comparative biochemical studies of FOXE1.
| S-EPMC3207924 | biostudies-literature
Project description:Macrophages migrate and invade into damaged muscle rapidly and are important for muscle repair and subsequent regeneration. The exact cellular and biological events that cause macrophage migration toward injured muscle are not completely understood. In this study, the effect of macrophage differentiation on the chemotactic capability to invade local damaged muscle was investigated using an in vitro model of muscle injury. We used C2C12 cell myoblasts and J774 cell macrophages, and the "killed-C2C12" cells were combined with live C2C12 cells as a partially damaged muscle model. The cultured J774 cells, with or without lipopolysaccharide (LPS), were treated with Ly294002 (Ly), which is an inhibitor of phosphoinositide 3-kinase (PI3K). In order to evaluate the polarization effect of LPS stimulation on J774 cells, expression of cell surface Toll-like receptor 4 (TLR4), CD11c and CCR2, and expression of F-actin intensity, were analyzed by flow cytometry. The real-time horizontal chemotaxis assay of J774 cells was tested using the TAXIScan device. The expressions of TLR4, CD11c, and F-actin intensity in LPS-treated cells were significantly higher than those in Ctrl cells. In LPS-treated cells, the chemotactic activity toward damaged muscle cells completely disappeared. Moreover, the reduced chemotaxis depended far more on directionality than velocity. However, Ly treatment reversed the reduced chemotactic activity of the LPS-treated cells. In addition, cell-adhesion and F-actin intensity, but not CCR2 expression, in LPS-treated cells, was significantly reduced by Ly treatment. Taken together, our results suggest that the PI3K/Akt activation state drives migration behavior towards damaged muscle cells.
| S-EPMC6210562 | biostudies-literature
Project description:BACKGROUND:Recent reports indicated that incidence of thyroid carcinoma is increasing throughout the worldwide. The aim of our study was to determine a possible relationship between Forkhead box E1 (FOXE1) gene variants and histopathological features of papillary thyroid carcinoma. METHODS:FOXE1 gene variations; rs894673, rs1867277 and rs3758249 were analyzed in 57 Papillary thyroid carcinoma patients and 51 age matched healthy control subjects. Restriction fragment length polymorphism (RFLP) technique was used to specifically detect the variations. RESULTS:There was a significant difference in the distribution of rs894673 genotypes in Papillary thyroid carcinoma cases (P=0.01). AA genotype presence of rs1867277 was more significantly associated with several histopathological parameters such as focal and diffuse capsular invasion, lymphatic invasion, P3 with P4 tumor grade and surgical margins. AA genotype presence in rs1867277 variation was significantly associated with the classical variant which is subtype of papillary thyroid carcinoma. Furthermore, the presence of the allel A was found to be related with lymph node invasion risk by 2.46 fold, capsular invasion risk by 2.97 fold, and pT3 with pT4 pathological stage risk by 4.13 fold and the presence of allele A in rs1867277 was significantly associated with classic variants. The presence of allele A in rs1867277 was more significantly associated with several histopathological parameters in classic variant in papillary thyroid carcinoma cases such as, the presence of the A allele was found relationship with lymph node invasion risk by 2.0 fold, capsular invasion risk by 2.39 fold , and pT3 with pT4 pathological stage risk by 3.57 fold. In addition, AATT, AAAA and GATT haplotypes (rs1867277 and rs894673) were evaluated for association with papillary thyroid carcinoma cases. Our results indicate that the significant difference according to two-allele haplotype distribution between papillary thyroid carcinoma cases and control groups. CONCLUSION:Our findings suggest that FOXE1 variations generate a higher risk for poor histopathological features of papillary thyroid carcinoma.
| S-EPMC4680502 | biostudies-literature
Project description:BACKGROUND: Single nucleotide polymorphisms (SNPs) near thyroid transcription factor genes (FOXE1 rs965513/NKX2-1 rs944289) have been shown to be associated with differentiated thyroid cancer (DTC) in Caucasoid populations. We investigated the role of those SNPs in German patients with DTC and also extended our analysis to tumor stages and lymphocytic infiltration of the tumors (ITL). METHODS: Patients with DTC (n=243; papillary, PTC; follicular, FTC) and healthy controls (HC; n=270) were analyzed for the rs965513 and rs944289 SNPs. RESULTS: The case-control analysis for rs965513 SNP showed that the genotypes "AA," "AG," and minor allele "A" were more frequent in patients with DTC than in HC (pronounced in PTC p(genotype)=0.000084, p(allele)=0.006 than FTC p(genotype)=0.29 and p(allele)=0.06). Furthermore, subgroup analysis of the DTC patients stratified for primary tumor stage (T1-T2, T3-T4), the absence or presence of regional lymph node metastases (N0, N1), for distant metastases (M0, M1), as well as for ITL, showed an association of rs965513 with stages T1-T2, T1-T3, N1, and absence of ITL. The NKX2-1 SNP rs944289, however, was not associated with DTC. CONCLUSION: Our results confirm that the FOXE1 rs965513 SNP confers an increased risk for DTC in the German population, particularly allele "A" and the genotypes "AA" and "AG" for PTC. This increased risk was also observed in advanced tumor stages and absence of ITL, which may reflect the course of a more aggressive disease. The NKX2-1 rs944289 SNP, however, appears to play a secondary role in the development of DTC in the German population.
| S-EPMC4026307 | biostudies-literature