Role Of Interferon Regulatory Factor 6 Research Articles

Negative regulation of interferon regulatory factor 6 (IRF6) in interferon and NF-κB signalling pathways of common carp (Cyprinus carpio L.)

BackgroundInterferon (IFN) regulatory factors (IRFs) is a kind of transcription factors, which play an important role in regulating the expression of type I IFN and related genes. In mammals, IRF6 is not relevant with IFN expression, while zebrafish IRF6 was reported to be a positive regulator of IFN expression and could be phosphorylated by both MyD88 and TBK1. However, the role of IRF6 in the immune response and IFN transcription of common carp is unknown.ResultsIn the present study, the cDNA of IRF6 gene (CcIRF6) was cloned from common carp using RACE technique, with a total length of 1905 bp, encoding 471 amino acid residues, which possesses two functional domains of DBD and IAD. Similarity analysis showed that CcIRF6 had more than 50% similarity with IRFs of other vertebrates, and had the highest similarity with grass carp and zebrafish, among which the DBD domain was much more conserved. The phylogenetic analysis showed that CcIRF6 is in the branch of Osteichthyes and has the closest relationship with grass carp. In healthy common carp, the CcIRF6 was expressed in all the examined tissues, with the highest level in the oral epithelium, and the lowest level in the head kidney. After intraperitoneal injection of poly(I:C) or Aeromonas hydrophila, the expression of CcIRF6 increased in spleen, head kidney, foregut and hindgut of common carp. Moreover, poly(I:C), LPS, PGN and flagellin induced the expression of CcIRF6 in peripheral leukocytes and head kidney leukocytes of common carp in vitro. In EPC cells, CcIRF6 inhibited the expression of some IFN-related genes and pro-inflammatory cytokines, and dual luciferase reporter assay showed that CcIRF6 reduced the activity of IFN and NF-κB reporter genes.ConclusionsThe present study suggests that CcIRF6 is involved in the antiviral and antibacterial immune response of common carp, and negatively regulate the expression of IFN and NF-κB signalling pathways, which provides a theoretical basis for the study and prevention of fish disease pathogenesis.

Consistent downregulation of the cleft lip/palate-associated genes IRF6 and GRHL3 in carcinomas.

Interferon Regulatory Factor 6 (IRF6) and Grainyhead Like Transcription Factor 3 (GRHL3) are transcription factors that orchestrate gene regulatory networks required for the balance between keratinocyte differentiation and proliferation. Absence of either protein results in the lack of a normal stratified epidermis with keratinocytes failing to stop proliferating and to terminally differentiate. Numerous pathological variants within IRF6 and GRHL3 have been identified in orofacial cleft-affected individuals and expression of the two transcription factors has been found to be often dysregulated in cancers. However, whether orofacial cleft-associated IRF6 and GRHL3 variants in patients might also affect their cancer risk later in life, is not clear yet. The fact that the role of IRF6 and GRHL3 in cancer remains controversial makes this question even more challenging. Some studies identified IRF6 and GRHL3 as oncogenes, while others could attribute tumor suppressive functions to them. Trying to solve this apparent conundrum, we herein aimed to characterize IRF6 and GRHL3 function in various types of carcinomas. We screened multiple cancer and normal cell lines for their expression, and subsequently proceeded with functional assays in cancer cell lines. Our data uncovered consistent downregulation of IRF6 and GRHL3 in all types of carcinomas analyzed. Reduced levels of IRF6 and GRHL3 were found to be associated with several tumorigenic properties, such as enhanced cell proliferation, epithelial mesenchymal transition, migration and reduced differentiation capacity. Based on our findings, IRF6 and GRHL3 can be considered as tumor suppressor genes in various carcinomas, which makes them potential common etiological factors for cancer and CLP in a fraction of CLP-affected patients.

Investigating the Effects of IRF6 on Focal Adhesions in Keratinocytes

Focal adhesions are one of the two main types of adhesions between cells and the extra‐cellular matrix (ECM). Focal adhesions are dynamic complexes anchored to the actin cytoskeleton through intracellular adaptor proteins, and to the ECM through heterodimers of α and β integrins, forming a transmembrane link between the intra‐ and extra‐cellular space. β‐1 integrin is the primary β subunit found in keratinocytes, and its corresponding heterodimers show affinity for a wide range of ECM proteins including collagen, fibronectin, and laminin. Collagen is the main structural protein in cutaneous ECM (the dermis), while fibronectin is a glycoprotein found in the ECM that binds integrins and plays a crucial role in wound healing. Interferon regulatory factor 6 (IRF6) is a transcription factor expressed by epithelial cells that regulates keratinocyte differentiation, cell‐cell adhesions, and migration. It is well known that focal adhesions are required for cell migration, however the role of IRF6 in regulating focal adhesions is unknown. Therefore, we hypothesize that IRF6 regulates focal adhesion formation. To determine if IRF6 levels affect attachment to the ECM, we seeded wildtype and IRF6 knockdown keratinocytes on collagen IV and plastic and quantified the number of cells attached to the plate after thirty and sixty minutes. No differences were found between wild‐type and IRF6 knockdown keratinocytes on plastic. However, less IRF6 knockdown keratinocytes were attached on collagen IV compared to wildtypes. To explore how IRF6 affects focal adhesions, we plated IRF6 knockdown and wild‐type keratinocytes on different types of ECM that were representative of both normal and wounded conditions of the dermis. Immunofluorescent staining for β‐1 integrin and phalloidin was performed, followed by quantitative analysis of focal adhesion length and intensity. Initial observations indicate that IRF6 knockdown keratinocytes consistently have longer focal adhesions compared to the wildtype. Variations of β‐1 fluorescent intensity was also observed in wild‐type cells compared to IRF6 knockdown keratinocytes. These preliminary results suggest a role for IRF6 in regulating focal adhesions, providing a potential mechanism for the underlying defect observed in the migration of IRF6 deficient keratinocytes.

Disrupted IRF6-NME1/2 Complexes as a Cause of Cleft Lip/Palate

Mutations and common polymorphisms in interferon regulatory factor 6 (IRF6) are associated with both syndromic and nonsyndromic forms of cleft lip/palate (CLP). To date, much of the focus on this transcription factor has been on identifying its direct targets and the gene regulatory network in which it operates. Notably, however, IRF6 is found predominantly in the cytoplasm, with its import into the nucleus tightly regulated like other members of the IRF family. To provide further insight into the role of IRF6 in the pathogenesis of CLP, we sought to identify direct IRF6 protein interactors using a combination of yeast 2-hybrid screens and co-immunoprecipitation assays. Using this approach, we identified NME1 and NME2, well-known regulators of Rho-type GTPases, E-cadherin endocytosis, and epithelial junctional remodeling, as bona fide IRF6 partner proteins. The NME proteins co-localize with IRF6 in the cytoplasm of primary palatal epithelial cells in vivo, and their interaction with IRF6 is significantly enhanced by phosphorylation of key serine residues in the IRF6 C-terminus. Furthermore, CLP associated IRF6 missense mutations disrupt the ability of IRF6 to bind the NME proteins and result in elevated activation of Rac1 and RhoA, compared to wild-type IRF6, when ectopically expressed in 293T epithelial cells. Significantly, we also report the identification of 2 unique missense mutations in the NME proteins in patients with CLP (NME1 R18Q in an IRF6 and GRHL3 mutation-negative patient with van der Woude syndrome and NME2 G71V in a patient with nonsyndromic CLP). Both variants disrupted the ability of the respective proteins to interact with IRF6. The data presented suggest an important role for cytoplasmic IRF6 in regulating the availability or localization of the NME1/2 complex and thus the dynamic behavior of epithelia during lip/palate development.

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.

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

Association of IRF6 (G820A) Gene Variant with Nonsyndromic Cleft Lip and Palate in South Indian Population

Introduction: Cleft lip and palate (CLP) is a major congenital structural anomaly. Cleft lip and palate has significant complex etiology involving both genetic and environmental factors and lifelong morbidity. Mutations in various genes like TGFB3, RARA, IRF6 have been responsible for causing CLP in various populations.IRF6 gene provides instructions for making a protein interferon regulatory factor, which is active in cells that give rise to tissues in the craniofacial region. The mutations in IRF6 gene can lead to NSCLP, tooth agenesis, etc. Aims and objectives: This study was undertaken to evaluate the relationship and determine the degree of association of Interferon Regulatory Factor 6 (IRF6) (G820A) gene variant with nonsyndromic CLP (NSCLP) in South Indian population. IRF6 gene provides instructions for making a protein interferon regulatory factor, which is active in cells that give rise to tissues in the craniofacial region. The mutations in IRF6 gene can lead to NSCLP, tooth agenesis, etc. Materials and methods: The study group consisted of 50 samples who were patients at the department of orthodontics. The samples were divided into Group A with 25 cases having NSCLP and Group B with 25 controls. Two milliliter blood sample was collected from cases and controls each, for DNA isolation. Polymerase chain reaction (PCR) was carried for amplification followed by restriction fragment length polymorphism (RFLP) to assess the genotype of all individuals. z-test was applied for statistical analysis. Results: The study revealed significant differences in the frequency distributions of genotypes of IRF6 (G820A) variant when cases were compared with controls, with high GG genotype frequency in cases whereas GA and AA genotype of IRF6 (G820A) gene variant was found more in control samples. Conclusion: The results of this study further support the role of IRF6 (G820A) gene variants in NSCLP in South Indian population.

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.

Recent insights into the role of IRF6 in craniofacial and skin development

Genetic variation in Interferon Regulatory Factor 6 (IRF6) causes, and contributes risk for, orofacial clefting disorders. In one of these disorders, in addition to orofacial clefts, individuals have skin webbing of the lower limbs. Thus, IRF6 is a key component of palatal and skin development in humans. In mice, Irf6 is expressed in the medial edge epithelium (MEE) that is dependent on Tgfb signaling. And, Irf6 is expressed in the spinous layer of the epidermis. Moreover, mice deficient for Irf6 have abnormal skin, limb and craniofacial development, including cleft palate. In the epidermis, in vivo and in vitro analyses demonstrate that Irf6 is necessary for keratinocyte differentiation. Furthermore, keratinocytes from Irf6–/– epidermis fail to close a scratch wound and display abnormal clonal growth, suggesting abnormal cellular proliferation and migration. In the palate, growth of the shelves is not defective, but they fail to elevate above the tongue, may be due to oral adhesions. The palatal epithelium is highly adherent, but the MEE fails to undergo its normal pattern of dissolution. Mice that are compound heterozygotes for the null and hypomorphic alleles (Irf6–/neo) display less severe abnormalities than the Irf6 deficient mice. In the oral cavity, Irf6–/neo mice have less severe oral adhesions. We are assessing the Irf6–/neo mice for defects in palatal development and epithelial differentiation.

Developmental expression analysis of the mouse and chick orthologues of IRF6: The gene mutated in Van der Woude syndrome

Development of the lip and palate involves a complex series of events that are frequently disturbed resulting in the congenital anomalies cleft lip and cleft palate. Van der Woude syndrome (VWS) is an autosomal dominant disorder that is characterised by cleft lip, cleft palate, lower lip pits, and hypodontia. VWS arises as the result of mutations in the gene encoding interferon regulatory factor 6 (IRF6). To provide insights into the role of IRF6 during embryogenesis, we have analysed the expression of this molecule during mouse and chick facial development. Irf6 was expressed in the ectoderm covering the facial processes during their fusion to form the upper lip and primary palate in both mouse and chick. However, while Irf6 was expressed in the medial edge epithelia of the developing secondary palate of the mouse, which fuses as in man, Irf6 was not expressed in the medial edge epithelia of the naturally cleft chick secondary palate. Similarly, Irf6 was found to be down-regulated in the medial edge epithelia of transforming growth factor beta3-null mice, which also exhibit cleft palate. Together, these results support a role for IRF6 during the fusion events that occur during development of the lip and palate.