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Martine Dunnwald - One of the best experts on this subject based on the ideXlab platform.
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Interferon Regulatory factor 6 is required for proper wound healing in vivo
Developmental dynamics : an official publication of the American Association of Anatomists, 2019Co-Authors: Lindsey Rhea, Leah C. Biggs, Franklin J. Canady, Tanner Reeb, John W. Canady, Deborah S. F. Kacmarynski, Rishika Avvari, Martine DunnwaldAbstract:Background Van der Woude syndrome (VWS) is the most common form of syndromic orofacial cleft caused predominantly by mutations in Interferon Regulatory Factor 6 (IRF6). We previously reported that individuals with VWS have increased risk of wound healing complications following cleft repair compared with individuals with nonsyndromic orofacial clefts (nonsyndromic cleft lip and palate-NSCLP). In vitro, absence of IRF6 leads to impaired keratinocyte migration and embryonic wound healing. However, there is currently no data on tissue repair in adult animals and cells with reduced levels of IRF6 like in VWS. Results Excisional wounds of Irf6+/- and wild-type animals were analyzed 4 and 7 days post-wounding. Although all wounds were reepithelialized after 7 days, the epidermal and wound volume of repaired wounds was larger in Irf6+/- . These data were supported by increased keratinocyte proliferation in the neoformed epidermis and a less mature granulation tissue with increased cytokine levels. This effect was not cell autonomous, as Irf6+/- neonatal keratinocytes in vitro did not exhibit defects in scratch wound closure or proliferation. Keratinocytes from individuals with VWS also migrated similarly to keratinocytes from NSCLP individuals. Conclusions These data support a role for IRF6 in wound healing by regulating keratinocyte proliferation, granulation tissue maturation, and cytokine levels.
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Correction: Interferon Regulatory Factor 6 Has a Protective Role in the Host Response to Endotoxic Shock.
PloS one, 2017Co-Authors: Sophie Joly, Lindsey Rhea, Paige Volk, Jessica G Moreland, Martine DunnwaldAbstract:[This corrects the article DOI: 10.1371/journal.pone.0152385.].
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Interferon Regulatory Factor 6 Controls Proliferation of Keratinocytes From Children With Van der Woude Syndrome.
The Cleft palate-craniofacial journal : official publication of the American Cleft Palate-Craniofacial Association, 2016Co-Authors: Katherine R. Hixon, Lindsey Rhea, Franklin J. Canady, John W. Canady, Jennifer Standley, Martine DunnwaldAbstract:Objective: Interferon Regulatory Factor 6 (IRF6) is critical for craniofacial development, epidermal differentiation, and tissue repair. IRF6 mutations cause Van der Woude Syndrome (VWS) and Popliteal Pterygium Syndrome. Individuals with VWS exhibit craniofacial anomalies, including cleft lip and palate and lip pits. Furthermore, they have an increased risk for wound-healing complications following surgical repair when compared with patients with nonsyndromic cleft lip and palate (NSCLP). However, nothing is known about the skin of these patients. The objective was to characterize the skin of patients with VWS. We hypothesize that IRF6 is required for proper skin homeostasis in humans. Design: Discarded tissue from a hip was collected during surgical alveolar bone graft. Samples from children with VWS harboring IRF6 mutations (n = 2) were compared with samples from children with NSCLP (n = 7). Histology was assessed following hematoxylin and eosin staining. The expressions of Proliferating Cell Nuclear ...
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Interferon Regulatory factor 6 has a protective role in the host response to endotoxic shock
PLOS ONE, 2016Co-Authors: Sophie Joly, Lindsey Rhea, Paige Volk, Jessica G Moreland, Martine DunnwaldAbstract:Interferon Regulatory Factor (IRF) 6, a member of the IRF family, is essential for epidermal and orofacial embryonic development. Irf6 is strongly expressed in keratinocytes, in which it regulates epidermal proliferation, differentiation, and migration. A recent role for Irf6 in Toll-like receptor 2-dependent chemokine gene expression was also reported in an epithelial cell line. However, a function for Irf6 in innate immune cells was not previously reported. In the present study, we investigated the expression and function of Irf6 in bone marrow-derived neutrophils and macrophages. We show here, using a conditional knockout of Irf6 in lysosymeM expressing cells, that Irf6 is required for resistance to LPS-induced endotoxic shock. In addition, Irf6-deficient bone marrow-derived neutrophils exhibited increased chemotactic index and velocity compared with wild-type cells in vitro. TLR4-specific KC and IL6 secretions were upregulated in Irf6-deficient bone marrow-derived macrophages in vitro. These cells also exhibited an increased level of phosphorylated IkBa. Collectively, our findings suggest a role for Irf6 in the resistance to endotoxic shock due to NFk-B-mediated alteration of cytokine production.
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Interferon Regulatory factor 6 regulates keratinocyte migration.
Journal of Cell Science, 2014Co-Authors: Leah C. Biggs, Rachelle Naridze, Kris A. Demali, Daniel F. Lusche, Spencer Kuhl, David R. Soll, Brian C. Schutte, Martine DunnwaldAbstract:Interferon Regulatory factor 6 (Irf6) regulates keratinocyte proliferation and differentiation. In this study, we tested the hypothesis that Irf6 regulates cellular migration and adhesion. Irf6-deficient embryos at 10.5 days post-conception failed to close their wound compared with wild-type embryos. In vitro, Irf6-deficient murine embryonic keratinocytes were delayed in closing a scratch wound. Live imaging of the scratch showed deficient directional migration and reduced speed in cells lacking Irf6. To understand the underlying molecular mechanisms, cell–cell and cell–matrix adhesions were investigated. We show that wild-type and Irf6-deficient keratinocytes adhere similarly to all matrices after 60 min. However, Irf6-deficient keratinocytes were consistently larger and more spread, a phenotype that persisted during the scratch-healing process. Interestingly, Irf6-deficient keratinocytes exhibited an increased network of stress fibers and active RhoA compared with that observed in wild-type keratinocytes. Blocking ROCK, a downstream effector of RhoA, rescued the delay in closing scratch wounds. The expression of Arhgap29, a Rho GTPase-activating protein, was reduced in Irf6-deficient keratinocytes. Taken together, these data suggest that Irf6 functions through the RhoA pathway to regulate cellular migration.
Brian C. Schutte - One of the best experts on this subject based on the ideXlab platform.
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Intercellular Genetic Interaction Between Irf6 and Twist1 during Craniofacial Development
Scientific Reports, 2017Co-Authors: Walid D. Fakhouri, Youssef A. Kousa, Kareem Metwalli, Ali Naji, Sarah Bakhiet, Angela Quispe-salcedo, Larissa Nitschke, Brian C. SchutteAbstract:Interferon Regulatory Factor 6 ( IRF6 ) and TWIST1 are transcription factors necessary for craniofacial development. Human genetic studies showed that mutations in IRF6 lead to cleft lip and palate and mandibular abnormalities. In the mouse, we found that loss of Irf6 causes craniosynostosis and mandibular hypoplasia. Similarly, mutations in TWIST1 cause craniosynostosis, mandibular hypoplasia and cleft palate. Based on this phenotypic overlap, we asked if Irf6 and Twist1 interact genetically during craniofacial formation. While single heterozygous mice are normal, double heterozygous embryos ( Irf6 ^+ /− ; Twist1 ^+ /− ) can have severe mandibular hypoplasia that leads to agnathia and cleft palate at birth. Analysis of spatiotemporal expression showed that Irf6 and Twist1 are found in different cell types. Consistent with the intercellular interaction, we found reduced expression of Endothelin1 (EDN1) in mandible and transcription factors that are critical for mandibular patterning including DLX5, DLX6 and HAND2, were also reduced in mesenchymal cells. Treatment of mandibular explants with exogenous EDN1 peptides partially rescued abnormalities in Meckel’s cartilage. In addition, partial rescue was observed when double heterozygous embryos also carried a null allele of p53 . Considering that variants in IRF6 and TWIST1 contribute to human craniofacial defects, this gene-gene interaction may have implications on craniofacial disorders.
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Generation and characterization of a conditional allele of Interferon Regulatory Factor 6.
Genesis (New York N.Y. : 2000), 2017Co-Authors: Arianna L. Smith, Akira Kinoshita, Baoli Yang, Youssef A. Kousa, Kate Fodor, Brian C. SchutteAbstract:Interferon Regulatory Factor 6 (IRF6) is a critical regulator of differentiation, proliferation and migration of keratinocytes. Mutations in IRF6 cause two autosomal dominant disorders characterized by cleft lip with or without cleft palate. In addition, DNA variation in IRF6 confers significant risk for non-syndromic cleft lip and palate. IRF6 is also implicated in adult onset development and disease processes, including mammary gland development and squamous cell carcinoma. Mice homozygous for a null allele of Irf6 die shortly after birth due to severe skin, limb, and craniofacial defects, thus impeding the study of gene function after birth. To circumvent this, a conditional allele of Irf6 was generated. To validate the functionality of the conditional allele, we used three “deleter” Cre strains: Gdf9-Cre, CAG-Cre, and Ella-Cre. When Cre expression was driven by the Gdf9-Cre or CAG-Cre transgenes, 100% recombination was observed as indicated by DNA genotyping and phenotyping. In contrast, use of the Ella-Cre transgenic line resulted in incomplete recombination, despite expression at the one-cell stage. In sum, we generated a novel tool to delete Irf6 in a tissue specific fashion, allowing for study of gene function past perinatal stages. However, recombination efficiency of this allele was dictated by the Cre-driver used. This article is protected by copyright. All rights reserved.
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Interferon Regulatory factor 6 regulates keratinocyte migration.
Journal of Cell Science, 2014Co-Authors: Leah C. Biggs, Rachelle Naridze, Kris A. Demali, Daniel F. Lusche, Spencer Kuhl, David R. Soll, Brian C. Schutte, Martine DunnwaldAbstract:Interferon Regulatory factor 6 (Irf6) regulates keratinocyte proliferation and differentiation. In this study, we tested the hypothesis that Irf6 regulates cellular migration and adhesion. Irf6-deficient embryos at 10.5 days post-conception failed to close their wound compared with wild-type embryos. In vitro, Irf6-deficient murine embryonic keratinocytes were delayed in closing a scratch wound. Live imaging of the scratch showed deficient directional migration and reduced speed in cells lacking Irf6. To understand the underlying molecular mechanisms, cell–cell and cell–matrix adhesions were investigated. We show that wild-type and Irf6-deficient keratinocytes adhere similarly to all matrices after 60 min. However, Irf6-deficient keratinocytes were consistently larger and more spread, a phenotype that persisted during the scratch-healing process. Interestingly, Irf6-deficient keratinocytes exhibited an increased network of stress fibers and active RhoA compared with that observed in wild-type keratinocytes. Blocking ROCK, a downstream effector of RhoA, rescued the delay in closing scratch wounds. The expression of Arhgap29, a Rho GTPase-activating protein, was reduced in Irf6-deficient keratinocytes. Taken together, these data suggest that Irf6 functions through the RhoA pathway to regulate cellular migration.
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Haploinsufficiency of Interferon Regulatory factor 6 alters brain morphology in the mouse.
American Journal of Medical Genetics Part A, 2013Co-Authors: Andrea M. Aerts, Martine Dunnwald, Brian C. Schutte, Ian Devolder, Seth M. Weinberg, Dan Thedens, Peg NopoulosAbstract:Orofacial clefts are among the commonest birth defects. Among many genetic contributors to orofacial clefting, Interferon Regulatory Factor 6 (IRF6) is unique since mutations in this gene cause Van der Woude (VWS), the most common clefting syndrome. Furthermore, variants in IRF6 contribute to increased risk for non-syndromic cleft lip and/or palate (NSCL/P). Our previous work shows that individuals with either VWS or NSCL/P may have cerebral anomalies (larger anterior, smaller posterior regions), and a smaller cerebellum. The objective of this study was to test the hypothesis that disrupting Irf6 in the mouse will result in quantitative brain changes similar to those reported for humans with VWS and NSCL/P. Male mice heterozygous for Irf6 (Irf6gt1/+; n = 9) and wild type (Irf6+/+; n = 6) mice at comparable age underwent a 4.7T MRI scan to obtain quantitative measures of cortical and subcortical brain structures. There was no difference in total brain volume between groups. However, the frontal cortex was enlarged in the Irf6gt1/+ mice compared to that of wild types (p = 0.028) while the posterior cortex did not differ. In addition, the volume of the cerebellum of Irf6gt1/+ mice was decreased (p = 0.004). Mice that were heterozygous for Irf6 showed a similar pattern of brain anomalies previously reported in humans with VWS and NSCL/P. These structural differences were present in the absence of overt oral clefts. These results support a role for IRF6 in brain morphometry and provide evidence for a potential genetic link to abnormal brain development in orofacial clefting.
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Interferon Regulatory Factor 6 Is Necessary, but Not Sufficient, for Keratinocyte Differentiation
The Journal of investigative dermatology, 2011Co-Authors: Leah C. Biggs, Brian C. Schutte, Lindsey Rhea, Martine DunnwaldAbstract:Regulation of epidermal proliferation and differentiation is critical for maintenance of cutaneous homeostasis. Interferon Regulatory Factor 6 (Irf6)-deficient mice die perinatally and exhibit ectopic proliferation and defective epidermal differentiation. We sought to determine whether these disruptions of epidermal function were cell autonomous, and used embryonic Irf6(-/-) keratinocytes to understand the specific role of Irf6 in keratinocyte proliferation and differentiation. In the absence of Irf6, keratinocytes exhibited a heterogeneous phenotype with the presence of large cells. Irf6(-/-) keratinocytes displayed increased colony-forming efficiency compared with wild-type cells, suggesting that Irf6 represses long-term proliferation. Irf6 was present at low levels in wild-type keratinocytes in culture, and upregulated after induction of differentiation in vitro, along with upregulation of markers of early differentiation. However, Irf6(-/-) keratinocytes did not express markers of terminal differentiation. Overexpression of Irf6 in wild-type keratinocytes was insufficient to induce expression of markers of differentiation under growing conditions. Together, these results indicated that Irf6 is necessary, but not sufficient, for keratinocyte differentiation. Finally, using a transgenic mouse expressing Lac-Z under the regulation of an enhancer element 9.7 kb upstream of the Irf6 start site, we demonstrated that this element contributes to the regulation of Irf6 in the epidermis and keratinocytes in culture.
Shinji Kondo - One of the best experts on this subject based on the ideXlab platform.
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Two missense mutations of the IRF6 gene in two Japanese families with popliteal pterygium syndrome.
American journal of medical genetics. Part A, 2010Co-Authors: Noriko Matsuzawa, Shinji Kondo, Norio Niikawa, Kazuo Shimozato, Toru Nagao, Motoi Nakano, Masayoshi Tsuda, Akiyoshi Hirano, Koh-ichiro YoshiuraAbstract:Mutations in the Interferon Regulatory factor 6 gene (IRF6) cause either popliteal pterygium syndrome (PPS) or Van der Woude syndrome (VWS), allelic autosomal dominant orofacial clefting conditions. To further investigate the IRF6 mutation profile in PPS, we performed mutation analysis of patients from two unrelated Japanese families with PPS and identified mutations in IRF6: c.251G>T (R84L) and c.1271C>T (S424L). We also found R84L, which together with previous reports on R84 mutations, provided another line of evidence that both syndromes could result from the same mutation probably under an influence of a modifier gene(s). This supports the idea that the R84 residue in the DNA binding domain of IRF6 is a mutational hot spot for PPS. A luciferase assay of the S424L protein in the other family demonstrated that the mutation decreased the IRF6 transcriptional activity significantly to 6% of that of the wild-type. This finding suggests that the C-terminus region of IRF6 could have an important function in phosphorylation or protein interaction. To our knowledge, this is the first report of mutations observed in Japanese PPS patients.
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Prevalence and nonrandom distribution of exonic mutations in Interferon Regulatory factor 6 in 307 families with Van der Woude syndrome and 37 families with popliteal pterygium syndrome
Genetics in Medicine, 2009Co-Authors: Renata L L Ferreira De Lima, Shinji Kondo, Sarah A Hoper, Michella Ghassibe, Margaret E Cooper, Nicholas K Rorick, Lori Katz, Mary L Marazita, John Compton, Sherri BaleAbstract:Purpose: Interferon Regulatory factor 6 encodes a member of the IRF family of transcription factors. Mutations in Interferon Regulatory factor 6 cause Van der Woude and popliteal pterygium syndrome, two related orofacial clefting disorders. Here, we compared and contrasted the frequency and distribution of exonic mutations in Interferon Regulatory factor 6 between two large geographically distinct collections of families with Van der Woude and between one collection of families with popliteal pterygium syndrome. Methods: We performed direct sequence analysis of Interferon Regulatory factor 6 exons on samples from three collections, two with Van der Woude and one with popliteal pterygium syndrome. Results: We identified mutations in Interferon Regulatory factor 6 exons in 68% of families in both Van der Woude collections and in 97% of families with popliteal pterygium syndrome. In sum, 106 novel disease-causing variants were found. The distribution of mutations in the Interferon Regulatory factor 6 exons in each collection was not random; exons 3, 4, 7, and 9 accounted for 80%. In the Van der Woude collections, the mutations were evenly divided between protein truncation and missense, whereas most mutations identified in the popliteal pterygium syndrome collection were missense. Further, the missense mutations associated with popliteal pterygium syndrome were localized significantly to exon 4, at residues that are predicted to bind directly to DNA. Conclusion: The nonrandom distribution of mutations in the Interferon Regulatory factor 6 exons suggests a two-tier approach for efficient mutation screens for Interferon Regulatory factor 6. The type and distribution of mutations are consistent with the hypothesis that Van der Woude is caused by haploinsufficiency of Interferon Regulatory factor 6. On the other hand, the distribution of popliteal pterygium syndrome-associated mutations suggests a different, though not mutually exclusive, effect on Interferon Regulatory factor 6 function.
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Abnormal skin, limb and craniofacial morphogenesis in mice deficient for Interferon Regulatory factor 6 (Irf6)
Nature Genetics, 2006Co-Authors: Christopher R. Ingraham, Akira Kinoshita, Shinji Kondo, Baoli Yang, Samin A. Sajan, Kurt J. Trout, Martine Dunnwald, Stephen L. Goudy, Margaret I Malik, Michael LovettAbstract:Transcription factor paralogs may share a common role in staged or overlapping expression in specific tissues, as in the Hox family. In other cases, family members have distinct roles in a range of embryologic, differentiation or response pathways (as in the Tbx and Pax families). For the Interferon Regulatory factor (IRF) family of transcription factors, mice deficient in Irf1 , Irf2 , Irf3 , Irf4 , Irf5 , Irf7 , Irf8 or Irf9 have defects in the immune response but show no embryologic abnormalities^ 1 , 2 , 3 , 4 , 5 , 6 , 7 . Mice deficient for Irf6 have not been reported, but in humans, mutations in IRF6 cause two mendelian orofacial clefting syndromes^ 8 , 9 , 10 , and genetic variation in IRF6 confers risk for isolated cleft lip and palate^ 11 , 12 , 13 , 14 , 15 . Here we report that mice deficient for Irf6 have abnormal skin, limb and craniofacial development. Histological and gene expression analyses indicate that the primary defect is in keratinocyte differentiation and proliferation. This study describes a new role for an IRF family member in epidermal development.
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Abnormal skin, limb and craniofacial morphogenesis in mice deficient for Interferon Regulatory factor 6 ( Irf6 )
Nature genetics, 2006Co-Authors: Christopher R. Ingraham, Akira Kinoshita, Shinji Kondo, Baoli Yang, Samin A. Sajan, Kurt J. Trout, Margaret Malik, Martine Dunnwald, Stephen L. Goudy, Michael LovettAbstract:Abnormal skin, limb and craniofacial morphogenesis in mice deficient for Interferon Regulatory factor 6 ( Irf6 )
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Mammary serine protease inhibitor (Maspin) binds directly to Interferon Regulatory factor 6: identification of a novel serpin partnership.
The Journal of biological chemistry, 2005Co-Authors: Caleb M. Bailey, Shinji Kondo, Brian C. Schutte, Naira V. Margaryan, Zhila Khalkhali-ellis, Richard E.b. Seftor, William W. Wheaton, Sumaira Amir, Michael R. Pins, Mary J. C. HendrixAbstract:Since its reported discovery in 1994, maspin (mammary serine protease inhibitor) has been characterized as a class II tumor suppressor by its ability to promote apoptosis and inhibit cell invasion. Maspin is highly expressed in normal mammary epithelial cells but reduced or absent in aggressive breast carcinomas. However, despite efforts to characterize the mechanism(s) by which maspin functions as a tumor suppressor, its molecular characterization has remained somewhat elusive. Therefore, in an attempt to identify maspin-interacting proteins and thereby gain insight into the functional pathways of maspin, we employed a maspin-baited yeast two-hybrid system and subsequently identified Interferon Regulatory Factor 6 (IRF6) as a maspin-binding protein. IRF6 belongs to the IRF family of transcription factors, which is best known for its regulation of Interferon and Interferon-inducible genes following a pathogenic stimulus. Although many of the IRF family members have been well characterized, IRF6 remains poorly understood. We report that IRF6 is expressed in normal mammary epithelial cells and that it directly associates with maspin in a yeast two-hybrid system and in vitro. The interaction occurs via the conserved IRF protein association domain and is regulated by phosphorylation of IRF6. We have shown that, similar to maspin, IRF6 expression is inversely correlated with breast cancer invasiveness. We further demonstrated that the transient re-expression of IRF6 in breast cancer cells results in an increase of N-cadherin and a redistribution of vimentin commensurate with changes in cell morphology, suggestive of an epithelial-to-mesenchymal transition event. Concomitantly, we showed that maspin acts as a negative regulator of this process. These findings help to elucidate the molecular mechanisms of maspin and suggest an interactive role between maspin and IRF6 in regulating cellular phenotype, the loss of which can lead to neoplastic transformation.
Linda P Jakobsen - One of the best experts on this subject based on the ideXlab platform.
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A novel mutation in IRF6 resulting in VWS-PPS spectrum disorder with renal aplasia.
American journal of medical genetics. Part A, 2008Co-Authors: Filipe De Medeiros, Lars Hansen, Evete Mawlad, Hans Eiberg, Camilla Asklund, Niels Tommerup, Linda P JakobsenAbstract:Popliteal pterygium syndrome (PPS) and Van der Woude syndrome (VWS) are caused by mutations in the gene Interferon Regulatory factor 6 (IRF6). Skeletal, genital malformations and involvement of the skin occur in PPS and orofacial clefting and lip pits occur in both. We report on a patient with unilateral cleft lip and palate, ankyloblepharon, paramedian lip pits, unilateral renal aplasia, and a coronal hypospadias. By sequencing IRF6, we detected a novel missense mutation (Arg339Ile). The other family members were unaffected and had no IRF6 mutations, including the patient's brother who was also born with hypospadias. The patient and his brother were both conceived by in vitro fertilization (IVF). It is discussed whether the renal malformation in the patient is related to the IVF procedure or to the IRF6 mutation.
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A novel mutation in IRF6 resulting in VWS-PPS spectrum disorder with renal aplasia.
American Journal of Medical Genetics Part A, 2008Co-Authors: Filipe De Medeiros, Lars Hansen, Evete Mawlad, Hans Eiberg, Camilla Asklund, Niels Tommerup, Linda P JakobsenAbstract:Popliteal pterygium syndrome (PPS) and Van der Woude syndrome (VWS) are caused by mutations in the gene Interferon Regulatory factor 6 (IRF6). Skeletal, genital malformations and involvement of the skin occur in PPS and orofacial clefting and lip pits occur in both. We report on a patient with unilateral cleft lip and palate, ankyloblepharon, paramedian lip pits, unilateral renal aplasia, and a coronal hypospadias. By sequencing IRF6, we detected a novel missense mutation (Arg339Ile). The other family members were unaffected and had no IRF6 mutations, including the patient's brother who was also born with hypospadias. The patient and his brother were both conceived by in vitro fertilization (IVF). It is discussed whether the renal malformation in the patient is related to the IVF procedure or to the IRF6 mutation. © 2008 Wiley-Liss, Inc.
Jennifer Huynh - One of the best experts on this subject based on the ideXlab platform.
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Interferon Regulatory Factor 6 Promotes Keratinocyte Differentiation in Response to Porphyromonas gingivalis
Infection and immunity, 2017Co-Authors: Jennifer Huynh, Glen M. Scholz, Eric C. ReynoldsAbstract:We recently demonstrated that the expression of the Interferon Regulatory factor 6 (IRF6) transcription factor in oral keratinocytes was stimulated by the periodontal pathogen Porphyromonas gingivalis . Here, we have established that IRF6 promotes the differentiation of oral keratinocytes in response to P. gingivalis . This was evidenced by the IRF6-dependent upregulation of specific markers of keratinocyte terminal differentiation (e.g. Involucrin, IVL; Keratin 13, KRT13), together with additional transcriptional regulators of keratinocyte differentiation, including Grainyhead-like 3 (GRHL3) and Ovo-like zinc finger 1 (OVOL1). We have previously established that the transactivator function of IRF6 is activated by Receptor-interacting protein kinase 4 (RIPK4). Consistently, the silencing of RIPK4 inhibited the stimulation of IVL, KRT13, GRHL3, and OVOL1 gene expression. IRF6 was shown to also regulate the stimulation of Transglutaminase-1 (TGM1) gene expression by P. gingivalis , as well as that of small proline-rich proteins (e.g. SPRR1), which are covalently cross-linked by TGM1 to other proteins, including IVL, during cornification. The expression of the tight junction protein Occludin (OCLN) was found to also be upregulated in an IRF6-dependent manner in response to P. gingivalis . IRF6 was demonstrated to be important for the barrier function of oral keratinocytes; specifically, silencing of IRF6 increased P. gingivalis -induced intercellular permeability and cell invasion. Taken together, our findings potentially position IRF6 as an important mediator of barrier defense against P. gingivalis .
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receptor interacting protein kinase 4 and Interferon Regulatory factor 6 function as a signaling axis to regulate keratinocyte differentiation
Journal of Biological Chemistry, 2014Co-Authors: Jennifer Huynh, Eric C. Reynolds, Mei Qi Kwa, Lianyi Zhang, Thao P Nguyen, Matthew J Sweet, John A Hamilton, Glen M. ScholzAbstract:Receptor-interacting protein kinase 4 (RIPK4) and Interferon Regulatory factor 6 (IRF6) are critical regulators of keratinocyte differentiation, and their mutation causes the related developmental epidermal disorders Bartsocas-Papas syndrome and popliteal pterygium syndrome, respectively. However, the signaling pathways in which RIPK4 and IRF6 operate to regulate keratinocyte differentiation are poorly defined. Here we identify and mechanistically define a direct functional relationship between RIPK4 and IRF6. Gene promoter reporter and in vitro kinase assays, coimmunoprecipitation experiments, and confocal microscopy demonstrated that RIPK4 directly regulates IRF6 trans-activator activity and nuclear translocation. Gene knockdown and overexpression studies indicated that the RIPK4-IRF6 signaling axis controls the expression of key transcriptional regulators of keratinocyte differentiation, including Grainyhead-like 3 and OVO-like 1. Additionally, we demonstrate that the p.Ile121Asn missense mutation in RIPK4, which has been identified recently in Bartsocas-Papas syndrome, inhibits its kinase activity, thereby preventing RIPK4-mediated IRF6 activation and nuclear translocation. We show, through mutagenesis-based experiments, that Ser-413 and Ser-424 in IRF6 are important for its activation by RIPK4. RIPK4 is also important for the regulation of IRF6 expression by the protein kinase C pathway. Therefore, our findings not only provide important mechanistic insights into the regulation of keratinocyte differentiation by RIPK4 and IRF6, but they also suggest one mechanism by which mutations in RIPK4 may cause epidermal disorders (e.g. Bartsocas-Papas syndrome), namely by the impaired activation of IRF6 by RIPK4.
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Interferon Regulatory factor 6 differentially regulates toll like receptor 2 dependent chemokine gene expression in epithelial cells
Journal of Biological Chemistry, 2014Co-Authors: Jennifer Huynh, Eric C. Reynolds, Mei Qi Kwa, Thao P Nguyen, Divya Ramnath, Dominic De Nardo, Pui Yeng Lam, John A HamiltonAbstract:Epidermal and mucosal epithelial cells are integral to host defense. They not only act as a physical barrier but also utilize pattern recognition receptors, such as the Toll-like receptors (TLRs), to detect and respond to pathogens. Members of the Interferon Regulatory factor (IRF) family of transcription factors are key components of TLR signaling as they impart specificity to downstream responses. Although IRF6 is a critical regulator of epithelial cell proliferation and differentiation, its role in TLR signaling has not previously been addressed. We show here that IRF6 is activated by IRAK1 as well as by MyD88 but not by TRIF or TBK1. Co-immunoprecipitation experiments further demonstrated that IRF6 can interact with IRAK1. Gene silencing in epithelial cells along with gene promoter reporter assays showed that IRAK1 mediates TLR2-inducible CCL5 gene expression at least in part by promoting IRF6 activation. Conversely, IRAK1 regulated CXCL8 gene expression independently of IRF6, thus identifying a molecular mechanism by which TLR2 signaling differentially regulates the expression of specific chemokines in epithelial cells. Bioinformatics analysis and mutagenesis-based experiments identified Ser-413 and Ser-424 as key Regulatory sites in IRF6. Phosphomimetic mutation of these residues resulted in greatly enhanced IRF6 dimerization and trans-activator function. Collectively, our findings suggest that, in addition to its importance for epithelial barrier function, IRF6 also contributes to host defense by providing specificity to the regulation of inflammatory chemokine expression by TLR2 in epithelial cells.