IRX3 Expression Research Articles

IRX5 promotes colorectal cancer metastasis by negatively regulating the core components of the RHOA pathway.

Colorectal cancer (CRC) is one of the most common malignant tumors worldwide. As tumor metastasis is the leading cause of death in patients with CRC, it is important to elucidate the molecular mechanisms that drive CRC metastasis. Studies have shown a close relationship between Iroquois homeobox (IRX) family genes and multiple cancers, while the mechanism by which IRX5 promotes CRC metastasis is unclear. Therefore, we focused on the involvement of IRX5 in CRC metastasis. In this study, analyses of clinical data indicated that the expression of IRX5 was coincided with metastatic colorectal tumors tissues and was negatively correlated with the overall survival of patients with CRC. Functional analysis showed that IRX5 promoted the migration and invasion of CRC cells, accompanied by a large number of cellular protrusions. IRX5-overexpressing cells were more likely to form metastatic tumors in nude mice. Further analysis demonstrated that the core components of the RHOA/ROCK1/LIMK1 pathway were significantly inhibited in IRX5-overexpressing cells. Overexpression of LIMK1 effectively reversed the enhanced cellular motility caused by IRX5 overexpression. Moreover, we found that high levels of IRX5 in intestinal tissues were correlated with the inflammatory response. IRX5 was significantly increased in azoxymethane/dextran sodium sulfate intestinal tissue of mice and IRX5-overexpressing may also enhance chemokines CXCL1 and CXCL8. In summary, our findings suggested that IRX5 promoted CRC metastasis by inhibiting the RHOA-ROCK1-LIMK1 axis, which correlates with a poor prognosis.

Changes in FTO and IRX3 gene expression in obese and overweight male adolescents undergoing an intensive lifestyle intervention and the role of FTO genotype in this interaction

BackgroundLifestyle intervention may have a critical effect on the association between genetics and obesity. This study aimed to investigate changes in FTO and IRX3 gene expression in obese and overweight male adolescents undergoing a lifestyle intervention and the role of FTO genotype in this interaction.MethodsThis study was a field trial of 62 adolescents from boys’ high schools in Tehran, Iran. Two schools were randomly allocated as the intervention (n = 30) and control (n = 32) schools. The rs9930506 SNP in FTO was genotyped at baseline and the level of FTO and IRX3 expression in peripheral blood mononuclear cells (PBMCs). Anthropometric measurements were assessed at baseline and after 18 weeks of intensive lifestyle intervention.ResultsOur results showed that IRX3 expression in the intervention group was significantly up-regulated compared to baseline (P = 0.007) and compared to the control group (P = 0.011).The intervention group had significantly up-regulated transcripts of IRX3 only in rs9930506 risk allele carriers of the intervention group compared to risk allele carriers of the control group (P = 0.017). Moreover, our data showed that the FTO expression was up-regulated in AA genotype carriers and down-regulated in AG/GG genotype carriers (P = 0.017).ConclusionLifestyle modification may exert its effects on obesity through changes in the expression level of the FTO and IRX3 genes. However, FTO genotype plays a role in the extent of the effect of lifestyle changes on gene expression. Further studies are crucial to have a better understanding of the interaction between lifestyle, genetics and anthropometric measurements.Trial registration This paper reports a comprehensive intervention study (Interactions of Genetics, Lifestyle and Anthropometrics study or IGLA study), which is retrospectively registered in the Iranian Registry of Clinical Trials as IRCT2016020925699N2. Date registered: April 24, 2016. (https://www.irct.ir/searchresult.php?id=25699&number=2)

MON-229 IRX3 and IRX5 Regulate Downstream Targets that Promote Ovarian Follicle Integrity in Mice

Appropriate cell-cell interactions and communication within ovarian follicles are required for their healthy development and maturation and are critical for maintaining female reproductive life quality. Our lab previously showed that two Iroquois transcription factor family genes, Irx3 and Irx5, were important for ovarian follicle integrity. Deletion of Irx3 and Irx5 (Irx3-Irx5EGFP/Irx3-Irx5EGFP, Irx3/5 DKO) resulted in disrupted granulosa cell-oocyte contacts, ectopic gap junction protein expression, and abnormal basement membrane morphology. The mechanisms by which Irx3 and Irx5 promote follicle integrity, however, are unknown. Therefore, we performed RNA-seq experiments on control and Irx3/5 DKO ovarian samples at two critical phases of ovarian development: embryonic day 13.5 when germline nests are established and just after birth when germline nests break down and primordial follicles form. RNA-seq analyses revealed that Inhbb, a TGF-β superfamily gene, was significantly upregulated in Irx3/5 DKO samples at both time points. It was previously reported that Inhbb was also upregulated in Wnt4 and β-catenin mutant ovaries. Notably, similar to the Irx3/5 DKO ovary, cell-cell interaction failure and germ cell death are prominent features of these models and removal of the excessive Inhbb by breeding to Inhbb knockout animals rescued the defects. These data suggest that Inhbb must be blocked to maintain germ cell and follicle survival. Based on the similarities in ovarian phenotype and Inhbb upregulation resulting from both Wnt4/β-catenin and Irx3/5 mutant models, we hypothesized that Irx3 and Irx5 mediate the Wnt4/β-catenin repression of Inhbb while they also regulate the expression of other downstream target genes required for cell contacts and germ cell health within follicles. First, we used gain- and loss-of-function gonad culture paradigms to manipulate β-catenin activity in ovaries and testes, respectively. Results showed that Irx3 and Irx5 expression levels were positively regulated by β-catenin signaling within the canonical Wnt4 pathway. Next, additional analysis of the RNA-seq data using the Database for Annotation, Visualization and Integrated Discovery (DAVID) showed that the differentially expressed genes (DEGs) in Irx3/5 DKO samples included factors critical to extracellular matrix, cell projections, actin-binding and cell adhesion. We are currently validating the DEGs relevant to our Irx3/5 DKO follicle defects and investigating their relationship to Inhbb in ovaries. Results from this study will help us to discover the means by which initial cell contacts are formed between support cells and oocytes in germline nests and new primordial follicles. These interactions provide the foundation for follicle integrity within the ovarian reserve and ultimately ensure that healthy follicles are available for recruitment and maturation.

Interactions between macro-nutrients’ intake, FTO and IRX3 gene expression, and FTO genotype in obese and overweight male adolescents

ABSTRACT This study is the first to identify the effects of FTO genotype on the interactions between the level of macro-nutrients intake and the expression level of fat mass and obesity associated (FTO) and homeobox transcription factor iriquois-3 (IRX3) genes This longitudinal study was carried out on 84 overweight and obese adolescent boys in Tehran, Iran. The rs9930506 SNP in FTO was genotyped at baseline and the level of FTO and IRX3 expression in PBMCs and macro-nutrients’ intake were assessed at baseline and after 18 weeks of the intervention. The results identified that the higher carbohydrates intake significantly up-regulated the FTO gene (P = 0.001) and down-regulated the IRX3 gene (P = 0.01). Protein intake up-regulated the FTO gene (P = 0.001). In carriers of GG genotype of FTO gene, the amount of dietary carbohydrate had a positive association with FTO gene expression (p = 0.001, and p = 0.04, respectively). In AA/AG carriers, dietary protein was positively associated with FTO gene expression (p = 0.001) and dietary carbohydrate was negatively associated with IRX3 gene expression (P = 0.04). Therefore, dietary carbohydrateseems to be associated with FTO and IRX3 genes expression. These associations are influenced by FTO genotype.

The possible mechanisms of the effects of IRX3 gene on body weight: an overview.

IntroductionRecent studies reported that FTO exert its effects on body weight through change the expression IRX3. The aim of this study was investigation of the possible mechanisms of the effects of IRX3 gene on obesity.Material and methodsThe present review was carried out using keywords such as polymorphism and/or obesity and/or BMI and/or IRX3 gene and/or Iroquois homeobox protein 3. Databases including PubMed, Science Direct, web of sciences, Scopus, and Cochran databases were used to collect all related articles published from 2000 to 2019.ResultsBased on this review, there are some evidences on the association between the IRX3 polymorphisms and the IRX3 expression level with body weight. In some studies, the up-regulation of IRX3 expression was related to increased body weight, while in some other studies down-regulation of IRX3 expression was related to obesity.ConclusionsThis review investigated the probable mechanisms of the effects of the IRX3 gene on obesity. Studies in this are limited and reported contradictory results. Further studies are required to evaluate the role of IRX3 gene in the associations between genes, diet, and obesity.

IRX5 regulates adipocyte amyloid precursor protein and mitochondrial respiration in obesity.

ObjectiveA causal obesity risk variant in the FTO locus was recently shown to inhibit adipocyte thermogenesis via increased adipose expression of the homeobox transcription factors IRX3 and IRX5. However, causal effects of IRX5 on fat storage remain to be shown in vivo, and discovery of downstream mediators may open new therapeutic avenues.Methods17 WT and 13 Irx5 knockout (KO) mice were fed low-fat control (Ctr) or high-fat (HF) diet for 10 weeks. Body weight, energy intake and fat mass were measured. Irx5-dependent gene expression was explored by transcriptome analysis of epididymal white adipose tissue (eWAT), confirmatory obesity-dependent expression in human adipocytes in vivo, and in vitro knock-down, overexpression and transcriptional activation assays.ResultsIrx5 knock-out mice weighed less, had diminished fat mass, and were protected from diet-induced fat accumulation. Key adipose mitochondrial genes Pparγ coactivator 1-alpha (Pgc-1α) and uncoupling protein 1 (Ucp1) were upregulated, and a gene network centered on amyloid precursor protein (App) was downregulated in adipose tissue of knock-out mice and in isolated mouse adipocytes with stable Irx5 knock-down. An APP-centered network was also enriched in isolated adipocytes from obese compared to lean humans. IRX5 overexpression increased APP promoter activity and both IRX5 and APP inhibited transactivation of PGC-1α and UCP1. Knock-down of Irx5 or App increased mitochondrial respiration in adipocytes.ConclusionIrx5-KO mice were protected from obesity and this can partially be attributed to reduced adipose App and improved mitochondrial respiration. This novel Irx5-App pathway in adipose tissue is a possible therapeutic entry point against obesity.

The Iroquois homeobox proteins IRX3 and IRX5 have distinct roles in Wilms tumour development and human nephrogenesis.

Wilms tumour is a paediatric malignancy with features of halted kidney development. Here, we demonstrate that the Iroquois homeobox genes IRX3 and IRX5 are essential for mammalian nephrogenesis and govern the differentiation of Wilms tumour. Knock‐out Irx3 − /Irx5 − mice showed a strongly reduced embryonic nephron formation. In human foetal kidney and Wilms tumour, IRX5 expression was already activated in early proliferative blastema, whereas IRX3 protein levels peaked at tubular differentiation. Accordingly, an orthotopic xenograft mouse model of Wilms tumour showed that IRX3 −/− cells formed bulky renal tumours dominated by immature mesenchyme and active canonical WNT/β‐catenin‐signalling. In contrast, IRX5 −/− cells displayed activation of Hippo and non‐canonical WNT‐signalling and generated small tumours with abundant tubulogenesis. Our findings suggest that promotion of IRX3 signalling or inhibition of IRX5 signalling could be a route towards differentiation therapy for Wilms tumour, in which WNT5A is a candidate molecule for enforced tubular maturation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

IRX5 promotes NF-κB signalling to increase proliferation, migration and invasion via OPN in tongue squamous cell carcinoma.

Iroquois homeobox gene 5 (Irx5) is a highly conserved member of the Iroquois homeobox gene family. Members of this family play distinct and overlapping roles in normal embryonic cell patterning and development of malignancies. In this study, we observed that IRX5 was abnormally abundant in tongue squamous cell carcinoma (TSCC) tissues and cell lines. We used gain‐ and loss‐of‐function methods to overexpress and knockdown IRX5 expression in the TSCC cell line CAL27. Our results elucidated that elevated levels of IRX5 promoted proliferation, migration and invasion of TSCC cells, whereas stable or transient knockdown of IRX5 expression suppressed TSCC cell proliferation, migration and invasion. As a transcription factor, IRX5 performed this function by targeting osteopontin (OPN) promoter and activating the NF‐κB pathway. Finally, studies in xenograft tumour model showed that IRX5 significantly enhanced OPN expression and promoted tumour growth. Taken together, our study elucidates a promotive effect of IRX5 in TSCC through the connection with OPN. These findings reveal the new molecular mechanism of TSCC, which may potentiate its use as a novel molecular therapy target for TSCC.

Derepression of the Iroquois Homeodomain Transcription Factor Gene IRX3 Confers Differentiation Block in Acute Leukemia

SummaryThe Iroquois homeodomain transcription factor gene IRX3 is expressed in the developing nervous system, limb buds, and heart, and transcript levels specify obesity risk in humans. We now report a functional role for IRX3 in human acute leukemia. Although transcript levels are very low in normal human bone marrow cells, high IRX3 expression is found in ∼30% of patients with acute myeloid leukemia (AML), ∼50% with T-acute lymphoblastic leukemia, and ∼20% with B-acute lymphoblastic leukemia, frequently in association with high-level HOXA gene expression. Expression of IRX3 alone was sufficient to immortalize hematopoietic stem and progenitor cells (HSPCs) in myeloid culture and induce lymphoid leukemias in vivo. IRX3 knockdown induced terminal differentiation of AML cells. Combined IRX3 and Hoxa9 expression in murine HSPCs impeded normal T-progenitor differentiation in lymphoid culture and substantially enhanced the morphologic and phenotypic differentiation block of AML in myeloid leukemia transplantation experiments through suppression of a terminal myelomonocytic program. Likewise, in cases of primary human AML, high IRX3 expression is strongly associated with reduced myelomonocytic differentiation. Thus, tissue-inappropriate derepression of IRX3 contributes significantly to the block in differentiation, which is the pathognomonic feature of human acute leukemias.

Long-Noncoding RNA Colorectal Neoplasia Differentially Expressed Gene as a Potential Target to Upregulate the Expression of IRX5 by miR-136-5P to Promote Oncogenic Properties in Hepatocellular Carcinoma

Background/Aims: The long-noncoding RNA colorectal neoplasia differentially expressed (CRNDE) gene was first found to be activated in colorectal neoplasia. Now, it also has been found to be upregulated in many other solid tumors. Whether CRNDE affects tumorigenesis remains unknown. Methods: We conducted bioinformatics, real-time polymerase chain reaction (PCR), Western blot analysis, cell proliferation assay, colony formation assay, wound healing assay, cell migration and invasion assays, RNA immunoprecipitation, and reporter vector construction and luciferase assays. Results: CRNDE was upregulated in hepatocellular carcinoma (HCC). The overexpression of CRNDE promoted HCC cellular proliferation, migration, and invasion in intro and in vivo, and acted as an oncogene in HCC progression. Furthermore, CRNDE impaired miR-136-5P expression in a RISC manner, and a reciprocal repression feedback loop was possible between CRNDE and miR-136-5P. We found that the neighboring mRNA of CRNDE was IRX5, and IRX5 increased the tumorigenicity of HCC cells. IRX5 was a potential downstream target gene of miR-136-5P. MiR-136 regulated IRX5 by interacting with its 3’UTR. In addition, miR-136-5P was involved in the CRNDE-regulated expression of IRX5. Conclusion: CRNDE acted as a tumor oncogene by exhibiting oncogenic properties of human HCC and revealed a novel CRNDE-miR-136-5P-IRX5 regulatory network in HCC. CRNDE may be considered to be a potential target for HCC therapies based on its ability to upregulate IRX5, and it deserves further investigation.

Irx3 and Bmp2 regulate mouse mesenchymal cell chondrogenic differentiation in both a Sox9-dependent and -independent manner.

Sox9, a master regulator of cartilage development, controls the cell fate decision to differentiate from mesenchymal to chondrogenic cells. In addition, Sox9 regulates the proliferation and differentiation of chondrocytes, as well as the production of cartilage-specific proteoglycans. The existence of Sox9-independent mechanisms in cartilage development remains to be determined. Here, we attempted to identify genes involved in such putative mechanisms via microarray analysis using a mouse chondrogenic cell line, N1511. We first focused on transcription factors that exhibited upregulated expression following Bmp2 treatment, which was not altered by subsequent treatment with Sox9 siRNA. Among these, we selected positive regulators for chondrogenesis and identified Iroquois-related homeobox 3 (Irx3) as one of the candidate genes. Irx3 expression gradually increased with chondrocyte terminal differentiation in a reciprocal manner to Sox9 expression, and promoted the chondrogenic differentiation of mesenchymal cells upon Bmp2 treatment. Furthermore, Irx3 partially rescued impaired chondrogenesis by upregulating the expression of epiphycan and lumican under reduced Sox9 expression. Finally, Irx3 was shown to act in concert with Bmp2 signaling to activate the p38 MAPK pathway, which in turn stimulated Sox9 expression, as well as the expression of epiphycan and lumican in a Sox9-independent manner. These results indicate that Irx3 represents a novel chondrogenic factor of mesenchymal cells, acts synergistically with Bmp2-mediated signaling, and regulates chondrogenesis independent of the transcriptional machinery associated with Sox9-mediated regulation.

IRX3 is a genetic modifier for birth weight, adolescent obesity and transaminase metabolism.

IRX3 expression has been functionally associated in obesity-associated long-distance susceptibility loci, but the effect of the IRX3 genetic variants on human obesity and obesity-related metabolism remains uncertain. To determine the genetic role of IRX3, we conducted a molecular epidemiological analysis using three haplotype tagging single nucleotide polymorphisms (SNPs; rs8053360, rs3751723 and rs12445085) and one nonsynonymous SNP (rs1126960) at the IRX3 locus in 333 junior and senior high school students from a northeast Chinese population. We identified significant associations between IRX3 SNPs and birth weight, body mass index (BMI), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and AST/ALT ratio. The rs8053360 CC and rs1126960 GG genotypes were associated with increased birth weight and BMI, especially in females. Individuals with the rs12445085 TT genotype had significantly higher levels of AST and ALT, whereas individuals with the rs1126960 GG genotype had a significantly lower AST/ALT ratio than did individuals with other genotypes. However, no significant relationships were found between any of the IRX3 SNPs and metabolic syndrome or diabetes. IRX3 genetic variants associate with birth weight, BMI and AST/ALT-related transaminase metabolism, supporting the role of IRX3 as an obesity-associated susceptibility gene.

Defective Hand1 phosphoregulation uncovers essential roles for Hand1 in limb morphogenesis.

The morphogenesis of the vertebrate limbs is a complex process in which cell signaling and transcriptional regulation coordinate diverse structural adaptations in diverse species. In this study, we examine the consequences of altering Hand1 dimer choice regulation within developing vertebrate limbs. Although Hand1 deletion via the limb-specific Prrx1-Cre reveals a non-essential role for Hand1 in mouse limb morphogenesis, altering Hand1 phosphoregulation, and consequently Hand1 dimerization affinities, results in a severe truncation of proximal-anterior limb elements. Molecular analysis reveals a non-cell-autonomous mechanism that causes widespread cell death within the embryonic limb bud. In addition, we observe changes in proximal-anterior gene regulation, including a reduction in the expression of Irx3, Irx5, Gli3 and Alx4, all of which are upregulated in Hand2 limb conditional knockouts. A reduction of Hand2 and Shh gene dosage improves the integrity of anterior limb structures, validating the importance of the Twist-family bHLH dimer pool in limb morphogenesis.

FTO Obesity Risk Variants Are Linked to Adipocyte IRX3 Expression and BMI of Children - Relevance of FTO Variants to Defend Body Weight in Lean Children?

BackgroundGenome-wide association studies have identified variants within the FTO (fat mass and obesity associated) locus as the strongest predictors of obesity amongst all obesity-associated gene loci. Recent evidence suggests that variants in FTO directly affect human adipocyte function through targeting IRX3 and IRX5 and thermogenesis regulation.AimWe addressed the relevance of this proposed FTO-IRX pathway in adipose tissue (AT) of children.ResultsExpression of IRX3 was higher in adipocytes compared to SVF. We found increased adipocyte-specific expression of IRX3 and IRX5 with the presence of the FTO risk haplotype in lean children, whereas it was unaffected by risk variants in obese peers. We further show that IRX3 expression was elevated in isolated adipocytes and AT of lean compared to obese children, particularly in UCP1-negative adipocytes, and inversely correlated with BMI SDS. Independent of BMI, IRX3 expression in adipocytes was significantly related to adipocyte hypertrophy, and subsequent associations with AT inflammation and HOMA-IR in the children.ConclusionOne interpretation of our observation of FTO risk variants linked to IRX3 expression and adipocyte size restricted to lean children, along with the decreased IRX3 expression in obese compared to lean peers, may reflect a defense mechanism for protecting body-weight, which is pertinent for lean children.

Iroquois homeobox transcription factor (Irx5) promotes G1/S-phase transition in vascular smooth muscle cells by CDK2-dependent activation.

The Iroquois homeobox (Irx5) gene is essential in embryonic development and cardiac electrophysiology. Although recent studies have reported that IRX5 protein is involved in regulation of the cell cycle and apoptosis in prostate cancer cells, little is known about the role of IRX5 in the adult vasculature. Here we report novel observations on the role of IRX5 in adult vascular smooth muscle cells (VSMCs) during proliferation in vitro and in vivo. Comparative studies using primary human endothelial cells, VSMCs, and intact carotid arteries to determine relative expression of Irx5 in the peripheral vasculature demonstrate significantly higher expression in VSMCs. Sprague-Dawley rat carotid arteries were subjected to balloon catherization, and the presence of IRX5 was examined by immunohistochemistry after 2 wk. Results indicate markedly elevated IRX5 signal at 14 days compared with uninjured controls. Total RNA was isolated from injured and uninjured arteries, and Irx5 expression was measured by RT-PCR. Results demonstrate a significant increase in Irx5 expression at 3–14 days postinjury compared with controls. Irx5 genetic gain- and loss-of-function studies using thymidine and 5-bromo-2′-deoxyuridine incorporation assays resulted in modulation of DNA synthesis in primary rat aortic VSMCs. Quantitative RT-PCR results revealed modulation of cyclin-dependent kinase inhibitor 1B (p27kip1), E2F transcription factor 1 (E2f1), and proliferating cell nuclear antigen (Pcna) expression in Irx5-transduced VSMCs compared with controls. Subsequently, apoptosis was observed and confirmed by morphological observation, caspase-3 cleavage, and enzymatic activation compared with control conditions. Taken together, these results indicate that Irx5 plays an important role in VSMC G1/S-phase cell cycle checkpoint control and apoptosis.

Commentary: FTO obesity variant circuitry and adipocyte browning in humans.

Commentary: FTO obesity variant circuitry and adipocyte browning in humans.

Scrutinizing the FTO locus: compelling evidence for a complex, long-range regulatory context.

Single nucleotide polymorphisms (SNPs) within a genetic region including the first two introns of the gene encoding FTO have consistently been shown to be the strongest genetic factors influencing body mass index (BMI). However, this same also contains several regulatory DNA elements that affect the expression of IRX3 and IRX5, which respectively, are located approximately 500 kb and 1.2 Mbp downstream from the BMI-associated FTO locus. Through these affected regulatory elements, genetic variation at the FTO locus influences adipocyte development leading to decreased thermogenesis and increased lipid storage. These findings provide a genomic model for the functional implications of genetic variations at this locus, and also demonstrate the importance of accounting for chromatin-chromatin interactions when constructing hypotheses for the mechanisms of trait and disease-associated common genetic variants. Several consortia have generated genome-wide datasets describing different aspects of chromatin biology which can be utilized to predict functionality and propose biologically relevant descriptions of specific DNA regions. Here, we review some of the publically available data resources on genome function and organization that can be used to gain an overview of genetic regions of interest and to generate testable hypotheses for future studies. We use the BMI- and obesity-associated FTO locus as a subject as it poses an illustrative example on the value of these resources. We find that public databases strongly support long-range interactions between regulatory elements in the FTO locus with the IRXB cluster genes IRX3 and IRX5. Chromatin configuration capture data also support interactions across a large region stretching across from the RPGRIP1L gene, FTO and the IRXB gene cluster.

FTO Obesity Variant Circuitry and Adipocyte Browning in Humans

BackgroundGenomewide association studies can be used to identify disease-relevant genomic regions, but interpretation of the data is challenging. The FTO region harbors the strongest genetic association with obesity, yet the mechanistic basis of this association remains elusive.MethodsWe examined epigenomic data, allelic activity, motif conservation, regulator expression, and gene coexpression patterns, with the aim of dissecting the regulatory circuitry and mechanistic basis of the association between the FTO region and obesity. We validated our predictions with the use of directed perturbations in samples from patients and from mice and with endogenous CRISPR–Cas9 genome editing in samples from patients.ResultsOur data indicate that the FTO allele associated with obesity represses mitochondrial thermogenesis in adipocyte precursor cells in a tissue-autonomous manner. The rs1421085 T-to-C single-nucleotide variant disrupts a conserved motif for the ARID5B repressor, which leads to derepression of a potent preadipocyte enhancer and a doubling of IRX3 and IRX5 expression during early adipocyte differentiation. This results in a cell-autonomous developmental shift from energy-dissipating beige (brite) adipocytes to energy-storing white adipocytes, with a reduction in mitochondrial thermogenesis by a factor of 5, as well as an increase in lipid storage. Inhibition of Irx3 in adipose tissue in mice reduced body weight and increased energy dissipation without a change in physical activity or appetite. Knockdown of IRX3 or IRX5 in primary adipocytes from participants with the risk allele restored thermogenesis, increasing it by a factor of 7, and overexpression of these genes had the opposite effect in adipocytes from nonrisk-allele carriers. Repair of the ARID5B motif by CRISPR–Cas9 editing of rs1421085 in primary adipocytes from a patient with the risk allele restored IRX3 and IRX5 repression, activated browning expression programs, and restored thermogenesis, increasing it by a factor of 7.ConclusionsOur results point to a pathway for adipocyte thermogenesis regulation involving ARID5B, rs1421085, IRX3, and IRX5, which, when manipulated, had pronounced pro-obesity and anti-obesity effects. (Funded by the German Research Center for Environmental Health and others.)

Fat mass- and obesity-associated gene Fto affects the dietary response in mouse white adipose tissue.

Common variants of human fat mass- and obesity-associated gene Fto have been linked with higher body mass index, but the biological explanation for the link has remained obscure. Recent findings suggest that these variants affect the homeobox protein IRX3. Here we report that FTO has a role in white adipose tissue which modifies its response to high-fat feeding. Wild type and Fto-deficient mice were exposed to standard or high-fat diet for 16 weeks after which metabolism, behavior and white adipose tissue morphology were analyzed together with adipokine levels and relative expression of genes regulating white adipose tissue adipogenesis and Irx3. Our results indicate that Fto deficiency increases the expression of genes related to adipogenesis preventing adipocytes from becoming hypertrophic after high-fat diet. In addition, we report a novel finding of increased Irx3 expression in Fto-deficient mice after high-fat feeding indicating a complex link between FTO, IRX3 and fat metabolism.

The Proangiogenic Effect of Iroquois Homeobox Transcription Factor Irx3 in Human Microvascular Endothelial Cells

Angiogenesis is a dynamic process required for embryonic development. However, postnatal vascular growth is characteristic of multiple disease states. Despite insights into the multistep process in which adhesion molecules, extracellular matrix proteins, growth factors, and their receptors work in concert to form new vessels from the preexisting vasculature, there remains a lack of insight of the nuclear transcriptional mechanisms that occur within endothelial cells (ECs) in response to VEGF. Iroquois homeobox gene 3 (Irx3) is a transcription factor of the Iroquois family of homeobox genes. Irx homeodomain transcription factors are involved in the patterning and development of several tissues. Irx3 is known for its role during embryogenesis in multiple organisms. However, the expression and function of Irx3 in human postnatal vasculature remains to be investigated. Here we show that Irx3 is expressed in human microvascular endothelial cells, and expression is elevated by VEGF stimulation. Genetic Irx3 gain and loss of function studies in human microvascular endothelial cells resulted in the modulation of EC migration during wound healing, chemotaxis and invasion, and tubulogenesis. Additionally, we observed increased delta-like ligand 4 (Dll4) expression, which suggests an increase in EC tip cell population. Finally, siRNA screening studies revealed that transient knockdown of Hey1, a downstream Notch signaling mediator, resulted in increased Irx3 expression in response to VEGF treatment. Strategies to pharmacologically regulate Irx3 function in adult endothelial cells may provide new therapies for angiogenesis.