Expression Of Interferon Regulatory Factor 6 Research Articles

IRF6 Is Directly Regulated by ZEB1 and ELF3, and Predicts a Favorable Prognosis in Gastric Cancer.

Interferon regulatory factor 6 (IRF6) acts as a tumor suppressor and controls cell differentiation in ectodermal and craniofacial tissues by regulating expression of target genes. However, its function in gastric cancer (GC) remains unknown to date. In this study, we found that the IRF6 expression was significantly downregulated in GC. And the decreased expression of IRF6 was clinically correlated with poor prognosis of GC. Moreover, loss-of-function and gain-of-function studies showed that IRF6 was negatively regulated by ZEB1 but positively regulated by ELF3. Additionally, transcription factor ZEB1 and ELF3 could directly bind on IRF6 promoter, which suggested that transcription factor IRF6 is transcriptionally regulated by ZEB1 and ELF3. Nevertheless, we found that IRF6 expression was negatively related to its promoter methylation in TCGA stomach cancer cohorts. The downregulation of IRF6 in GC might be due to the overexpression of ZEB1 and the DNA methylation of IRF6 promoter.

A cleft lip and palate gene, Irf6, is involved in osteoblast differentiation of craniofacial bone

Interferon regulatory factor 6 (IRF6) plays a critical role in embryonic tissue development, including differentiation of epithelial cells. Besides orofacial clefting due to haploinsufficiency of IRF6, recent human genetic studies indicated that mutations in IRF6 are linked to small mandible and digit abnormalities. The function of IRF6 has been well studied in oral epithelium; however, its role in craniofacial skeletal formation remains unknown. In this study, we investigated the role of Irf6 in craniofacial bone development using comparative analyses between wild-type (WT) and Irf6-null littermate mice. Immunostaining revealed the expression of IRF6 in hypertrophic chondrocytes, osteocytes, and bone matrix of craniofacial tissues. Histological analysis of Irf6-null mice showed a remarkable reduction in the number of lacunae, embedded osteocytes in matrices, and a reduction in mineralization during bone formation. These abnormalities may explain the decreased craniofacial bone density detected by micro-CT, loss of incisors, and mandibular bone abnormality of Irf6-null mice. To validate the autonomous role of IRF6 in bone, extracted primary osteoblasts from calvarial bone of WT and Irf6-null pups showed no effect on osteoblastic viability and proliferation. However, a reduction in mineralization was detected in Irf6-null cells. Altogether, these findings suggest an autonomous role of Irf6 in regulating bone differentiation and mineralization. Developmental Dynamics 248:221-232, 2019. © 2019 Wiley Periodicals, Inc.

Interferon Regulatory Factor 6 Is Necessary for Salivary Glands and Pancreas Development

Interferon regulatory factor 6 (IRF6) acts as a tumor suppressor and controls cell differentiation in ectodermal and craniofacial tissues by regulating expression of target genes. Haploinsufficiency of IRF6 causes Van der Woude and popliteal pterygium syndrome, 2 syndromic forms of cleft lip and palate. Around 85% of patients with Van der Woude express pits on the lower lip that continuously or intermittently drain saliva, and patients with the common cleft lip and palate have a higher prevalence of dental caries and gingivitis. This study aims to identify the role of IRF6 in development of exocrine glands, specifically the major salivary glands. Our transgenic mouse model that expresses LacZ reporter under the control of the human IRF6 enhancer element showed high expression of IRF6 in major and minor salivary glands and ducts. Immunostaining data also confirmed the endogenous expression of IRF6 in the developing ductal, serous, and mucous acinar cells of salivary glands. As such, we hypothesized that Irf6 is important for proper development of salivary glands and potentially other exocrine glands. Loss of Irf6 in mice causes an increase in the proliferation level of salivary cells, disorganized branching morphogenesis, and a lack of differentiated mucous acinar cells in submandibular and sublingual glands. Expression and localization of the acinar differentiation marker MIST1 were altered in Irf6-null salivary gland and pancreas. The RNA-Seq analysis demonstrated that 168 genes are differentially expressed and confer functions associated with transmembrane transporter activity, spliceosome, and transcriptional regulation. Furthermore, expression of genes involved in the EGF pathway—that is, Ereg, Ltbp4, Matn1, Matn3, and Tpo—was decreased at embryonic day 14.5, while levels of apoptotic proteins were elevated at postnatal day 0. In conclusion, our data report a novel role of Irf6 in exocrine gland development and support a rationale for performing exocrine functional tests for patients with IRF6-damaging mutations.

Down-Regulation of IRF6 Protects Cortical Neurons Against Traumatic Neuronal Injury Through Activating Akt-eNOS Pathway

Interferon regulatory factor 6 (IRF6) is a novel and unique member of the IRF family of transcription factors, and the regulation and function of IRF6 remain unknown. Recently, IRF6 was shown to be upregulated after TBI and could promote neuronal apoptosis under oxidative stress conditions. This study aimed to investigate the role of IRF6 in traumatic neuronal injury (TNI) in primary cultured mouse cortical neurons. We found that the expression of IRF6 was significantly increased within 48 after TNI, and peaked at 24h. Knockdown of IRF6 using specific targeted small interfering RNA (siRNA) attenuated TNI-induced loss of neuronal viability and release of lactate dehydrogenase. The results of TUNEL staining showed that IRF6 knockdown markedly reduced neuronal apoptosis, which was accompanied by decreased activity of caspase-3. Furthermore, downregulation of IRF6 inhibited lipid peroxidation, promoted the activity of endogenous antioxidative enzymes, and differently regulated the expression of inflammatory cytokines after TNI. In addition, IRF6 knockdown significantly increased phosphorylation of Akt and endothelial nitric oxide synthase (eNOS), whereas blocking Akt-eNOS pathway via selective antagonists partly prevented the protective effects of IRF6 knockdown. These data show that downregulation of IRF6 affords protection against TNI through Akt-eNOS pathway-mediated antioxidative and anti-inflammatory activity.

Expression of interferon regulatory factor 6, muscle segment homeobox 1, paired box gene 9, homeo box B3, and related to tyrosine kinases in human cleft-affected tissue

Background and Aim: Recent studies demonstrate direct roles of different genes during formation of secondary palate, but there are no still data about local expression and distribution of gene products in cleft palate affected human tissue. Thus, the aim of our study was to investigate cleft disordered cartilage and bone for detection of local expression of key regulators of palatogenesis and its correlations. Materials and Methods: The study involved 16 patients with unilateral cleft lip and palate. Tissue samples were proceeded for detection of interferon regulatory factor 6 (IRF6), muscle segment homeobox 1 (MSX1), paired box gene 9 (PAX9), homeo box B3 (HOXB3), and related to tyrosine kinases with biotin-streptavidin immunohistochemistry. Distribution of immunoreactive structures was detected semiquantitatively. Statistical analysis included the Mann-Whitney test and Pearson's correlation test. Results: Statistically significant differences were found between expression of IGFR6, MSX1, and HOXB3 in the cartilage and bone. We also detected statistically significant correlation between the expressions of PAX9 and MSX1 in the bone tissue. Conclusions: Cleft lip and palate disordered cartilage is characterized by more pronounced expression of IRF6, MSX1, and PAX9. Expression of HOXB3 is more characteristic for cleft lip and palate affected bone. Considered as a whole, our results suggest that the cleft lip and palate affected cartilage seems more plastic in tissue remodeling what can probably result in qualitative postoperative tissue reconstruction.

Knockdown of IRF6 Attenuates Hydrogen Dioxide-Induced Oxidative Stress via Inhibiting Mitochondrial Dysfunction in HT22 Cells.

Oxidative stress-induced cell damage is involved in many neurological diseases. Interferon regulatory factor 6 (IRF6), a member of the IRF family of transcription factors, is required for the differentiation of skin, breast epithelium, and oral epithelium. However, the regulation and function of IRF6 in central nervous system remain unknown. This study aimed to investigate the role of IRF6 in hydrogen peroxide (H2O2)-induced oxidative neuronal injury in HT22 mouse hippocampal cells. Treatment with H2O2 significantly increased the expression of IRF6 at both mRNA and protein levels, and knockdown of IRF6 using specific small interfering RNA reduced H2O2-induced cytotoxicity, as evidenced by increased cell viability and decreased apoptosis. Knockdown of IRF6 attenuated intracellular reactive oxygen species (ROS) generation and lipid peroxidation, and also preserved endogenous antioxidant enzyme activities. The inhibitory effect of IRF6 knockdown on mitochondrial dysfunction was demonstrated by reduced mitochondrial oxidative level, preserved mitochondrial membrane potential (MMP) and ATP generation, as well as attenuated mitochondrial swelling. In addition, down-regulation of IRF6 inhibited the activation of mitochondrial apoptotic factors, whereas IRF6 knockdown together with caspase inhibitors had no extra effect on cell viability and LDH release. These results suggest that knockdown of IRF6 has protective effects against H2O2-induced oxidative stress by reducing ROS accumulation and apoptosis, and these protective effects are dependent on preservation of mitochondrial function.

IRF6 is the mediator of TGFβ3 during regulation of the epithelial mesenchymal transition and palatal fusion.

Mutation in interferon regulatory factor 6 (IRF6) is known to cause syndromic and non-syndromic cleft lip/palate in human. In this study, we investigated the molecular mechanisms related to IRF6 during palatal fusion using palatal shelves organ culture. The results showed that ablation of Irf6 resulted in a delay in TGFβ3-regulated palatal fusion. Ectopic expression of IRF6 was able to promote palatal fusion and rescue shTgfβ3-induced fusion defect. These findings indicate that IRF6 is involved in TGFβ3-mediated palatal fusion. Molecular analysis revealed that ectopic expression of IRF6 increased the expression of SNAI2, an epithelial mesenchymal transition (EMT) regulator, and diminished the expression of various epithelial markers, such as E-cadherin, Plakophilin and ZO-1. In addition, knockdown of Irf6 expression decreased SNAI2 expression, and restored the expression of ZO-1 and Plakophilin that were diminished by TGFβ3. Blocking of Snai2 expression delayed palatal fusion and abolished the IRF6 rescuing effect associated with shTgfβ3-induced fusion defect. These findings indicate that TGFβ3 increases IRF6 expression and subsequently regulates SNAI2 expression, and IRF6 appears to regulate EMT during palatal fusion via SNAI2. Taken together, this study demonstrates that IRF6 is a mediator of TGFβ3, which regulates EMT and fusion process during the embryonic palate development.

Abstract 3840: A new prognostic marker: interferon regulatory factor 6 in renal cell carcinoma

Abstract According to our previous methylated-CpG island recovery assay (MIRA) and RNA expression array findings, the results indicated methylated status of Interferon regulatory factor 6 (IRF6) could be observed in most of renal cell carcinoma (RCC) cases (40.6%), and presented a negative correlation with gene expression, clear cell type of RCCs especially. As known that IRF6 is one of members of IRF family. It has been described might play an important role in epidermal cells, and correlated with cell proliferation and differentiation. The regulation of feedback loop between IRF6 and ΔNp63, which is known could control the mechanism of keratinocytes differentiation and palate closure, has also been noted. In addition, the tumor suppressor activity of IRF6 had been also demonstrated in squamous cell carcinoma associated with inhibition of tumor cell invasion and migration. The aim of this study is to clarify the clinical significance and role of IRF6 in RCC. We hypothesized low expression of IRF6 via DNA methylation might lead to irregular differentiation of RCC cells, and then further trigger cells proliferated out of control. In total, 105 pairs of clinical RCCs patients and eight RCC cell lines (included chromophobe type RCC98, sacomatoid type RCC52, clear cell type Caki-1, HH332, HOKN-9, RCC100, papillary type HH050 and adenocarcinoma 786-O), as well as Human Embryonic Kidney 293 cell used as control have involved in current study. Firstly, we performed real-time PCR on all cases. Secondly, 5-aza-2′deoxycytidine was treated on all type of RCC cell lines; it is a way of de-methylated DNA. Western blot detections were then to confirm whether the expression level of IRF6 restored in RCC cells. The IRF6 gene expressed levels differ from normal and RCC tissues were applied by the paired- T test, and shown by -ΔCT. Generally, the variant and lower level gene expression of the IRF6 could be observed from all different type of RCC cell lines, except RCC98 and RCC100 cells. After treated with 5-aza-2′deoxycytidine on RCC cell lines, the obvious expression of IRF6 was restored in all clear cell RCC cell lines. The mean - ΔCT of normal tissue was -8.0, and RCC tissue was -11.5, significantly IRF6 expressed levels differ from normal and tumor tissues could be noted (p = 0.013). The IRF6 gene was usually expressed lower level in RCC due to methylation. Our findings demonstrated the expression of IRF6 could be easily resorted by de-methylated in RCC cells, and it might further inhibit cell proliferation in the tumoral progression, and might consider as a prognostic marker to predict patients’ outcomes in RCC. However, the further cell viability and correlation with the clinical information should be further analyzed in the future. Citation Format: Chih-Yang Wei, Ying-Tzu Chen, See-Tong Pang, Wen-Hui Weng. A new prognostic marker: interferon regulatory factor 6 in renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3840. doi:10.1158/1538-7445.AM2015-3840

A novel RIPK4-IRF6 connection is required to prevent epithelial fusions characteristic for popliteal pterygium syndromes.

Receptor-interacting protein kinase 4 (RIPK4)-deficient mice have epidermal defects and fusion of all external orifices. These are similar to Bartsocas-Papas syndrome and popliteal pterygium syndrome (PPS) in humans, for which causative mutations have been documented in the RIPK4 and IRF6 (interferon regulatory factor 6) gene, respectively. Although genetically distinct, these syndromes share the anomalies of marked pterygia, syndactyly, clefting and hypoplastic genitalia. Despite the strong resemblance of these two syndromes, no molecular connection between the transcription factor IRF6 and the kinase RIPK4 was known and the mechanism underlying the phenotype was unclear. Here we describe that RIPK4 deficiency in mice causes epithelial fusions associated with abnormal periderm development and aberrant ectopic localization of E-cadherin on the apical membrane of the outer peridermal cell layers. In Xenopus, RIPK4 depletion causes the absence of ectodermal epiboly and concomitant gastrulation defects that phenocopy ectopic expression of dominant-negative IRF6. We found that IRF6 controls RIPK4 expression and that wild-type, but not kinase-dead, RIPK4 can complement the gastrulation defect in Xenopus caused by IRF6 malfunctioning. In contrast to the mouse, we observed only minor effects on cadherin membrane expression in Xenopus RIPK4 morphants. However, gastrulation defects were associated with a virtual absence of cortical actin in the ectodermal cells that face the blastocoel cavity and this was phenocopied in embryos expressing dominant-negative IRF6. A role for RIPK4 in actin cytoskeleton organization was also revealed in mouse epidermis and in human epithelial HaCaT cells. In conclusion, we showed that in mice RIPK4 is implicated in cortical actin organization and in E-cadherin localization or function, which can explain the characteristic epithelial fusions observed in PPSs. In addition, we provide a novel molecular link between IRF6 and RIPK4 that unifies the different PPSs to a common molecular pathway.

A Novel Heterozygous Mutation (F252Y) in Exon 7 of the IRF6 Gene is Associated with Oral Squamous Cell Carcinomas

Interferon regulatory factor 6 (IRF6) is a transcription factor with distinct and conserved DNA and protein binding domains. Mutations within the protein binding domain have been significantly observed in subjects with orofacial cleft relative to healthy controls. In addition, recent studies have identified loss of expression of IRF6 due to promoter hypermethylation in cutaneous squamous cell carcinomas. Since mutational events occurring within the conserved domains are likely to affect the function of a protein, we investigated whether regions within the IRF6 gene that encodes for the conserved protein binding domain carried mutations in oral squamous cell carcinoma (OSCC). Total chromosomal DNA extracted from 32 post surgical OSCC tissue samples were amplified using intronic primers flanking the exon 7 of IRF6 gene, which encodes for the major region of protein binding domain. The PCR amplicons from all the samples were subsequently resolved in a 1.2% agarose gel, purified and subjected to direct sequencing to screen for mutations. Sequencing analysis resulted in the identification of a mutation within exon 7 of IRF6 that occurred in heterozygous condition in 9% (3/32) of OSCC samples. The wild type codon TTC at position 252 coding for phenylalanine was found to be mutated to TAC that coded for tyrosine (F252Y). The present study identified for the first time a novel mutation within the conserved protein binding domain of IRF6 gene in tissue samples of subjects with OSCC.

IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes.

While the pro-differentiation and tumour suppressive functions of Notch signalling in keratinocytes are well established, the underlying mechanisms remain poorly understood. We report here that interferon regulatory factor 6 (IRF6), an IRF family member with an essential role in epidermal development, is induced in differentiation through a Notch-dependent mechanism and is a primary Notch target in keratinocytes and keratinocyte-derived SCC cells. Increased IRF6 expression contributes to the impact of Notch activation on growth/differentiation-related genes, while it is not required for induction of 'canonical' Notch targets like p21(WAF1/Cip1), Hes1 and Hey1. Down-modulation of IRF6 counteracts differentiation of primary human keratinocytes in vitro and in vivo, promoting ras-induced tumour formation. The clinical relevance of these findings is illustrated by the strikingly opposite pattern of expression of Notch1 and IRF6 versus epidermal growth factor receptor in a cohort of clinical SCCs, as a function of their grade of differentiation. Thus, IRF6 is a primary Notch target in keratinocytes, which contributes to the role of this pathway in differentiation and tumour suppression.

Temporal and spatial expression patterns for the tumor suppressor Maspin and its binding partner interferon regulatory factor 6 during breast development

Interferon regulatory factor 6 (IRF6) is a non-canonical member of the interferon regulatory factor family of transcription factors. We recently identified IRF6 as a novel Maspin-interacting protein in mammary epithelial cells. Maspin is a tumor suppressor in the breast and has also been implicated in mammary gland morphogenesis. To explore a possible role for IRF6 in conjunction with Maspin during mammary gland growth and differentiation, we examined the expression of IRF6 and Maspin during post-utero mammary gland development using a combination of in vitro and in vivo approaches. The data revealed that the expression of IRF6 and Maspin is temporally and spatially regulated throughout mammary gland development, with maximal expression of both proteins occurring in fully differentiated, lactating lobuloalveolar cells. We further show that IRF6 adopts a lumenal localization pattern following complete epithelial cell polarization and present new evidence for the secretion of IRF6 into the milk. These results support the hypothesis that IRF6 and Maspin are important for mammary epithelial cell differentiation, and advance our understanding of the Maspin-IRF6 partnership during normal mammary gland development.