ARHGAP29 is required for keratinocyte proliferation and migration.

BACKGROUND: RhoA GTPase plays critical roles in actin cytoskeletal remodeling required for controlling a diverse range of cellular functions including cell proliferation, cell adhesions, migration and changes in cell shape. RhoA cycles between an active GTP-bound and an inactive GDP-bound form, a process that is regulated by guanine nucleotide exchange factors (GEFs), and GTPase-activating proteins (GAPs). ARHGAP29 is a GAP expressed in keratinocytes of the skin and is decreased in the absence of Interferon Regulator Factor 6, a critical regulator of cell proliferation and migration. However, the role for ARHGAP29 in keratinocyte biology is unknown. RESULTS: Novel ARHGAP29 knockdown keratinocyte cell lines were generated using both CRISPR/Cas9 and shRNA technologies. Knockdown cells exhibited significant reduction of ARHGAP29 protein (50-80%) and displayed increased filamentous actin (stress fibers), phospho-myosin light chain (contractility), cell area and population doubling time. Furthermore, we found that ARHGAP29 knockdown keratinocytes displayed significant delays in scratch wound closure in both single cell and collective cell migration conditions. Particularly, our results show a reduction in path lengths, speed, directionality and persistence in keratinocytes with reduced ARHGAP29. The delay in scratch closure was rescued by both adding back ARHGAP29 or adding a ROCK inhibitor to ARHGAP29 knockdown cells. CONCLUSIONS: These data demonstrate that ARHGAP29 is required for keratinocyte morphology, proliferation and migration mediated through the RhoA pathway.

The Mafb cleft-associated variant H131Q is not required for palatogenesis in the mouse.

BACKGROUND: Orofacial clefts (OFCs) are common birth defects with complex etiology. Genome wide association studies for OFC have identified SNPs in and near MAFB. MAFB is a transcription factor critical for structural development of digits, kidneys, skin, and brain. MAFB is also expressed in the craniofacial region. Previous sequencing of MAFB in a Filipino population revealed a novel missense variant significantly associated with an increased risk for OFC. This MAFB variant, leading to the amino acid change H131Q, was knocked into the mouse Mafb, resulting in the MafbH131Q allele. The MafbH131Q construct was engineered to allow for deletion of Mafb ("Mafbdel "). RESULTS: Mafbdel/del animals died shortly after birth. Conversely, MafbH131Q/H131Q mice survived into adulthood at Mendelian ratios. Mafbdel/del and MafbH131Q/H131Q heads exhibited normal macroscopic and histological appearance at all embryonic time points evaluated. The periderm was intact based on expression of keratin 6, p63, and E-cadherin. Despite no effect on craniofacial morphogenesis, H131Q inhibited the Mafb-dependent promoter activation of Arhgap29 in palatal mesenchymal, but not ectodermal-derived epithelial cells in a luciferase assay. CONCLUSIONS: Mafb is dispensable for murine palatogenesis in vivo, and the cleft-associated variant H131Q, despite its lack of morphogenic effect, altered the expression of Arhgap29 in a cell-dependent context.

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis.

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, proliferation and remodeling. Murine wounds have a histologically well-defined and easily recognizable wound bed over which these different phases of the healing process are measurable. Within the field, it is common to use an arbitrarily defined "middle" of the wound for histological analyses. However, wounds are a three-dimensional entity and often not histologically symmetrical, supporting the need for a well-defined and robust method of quantification to detect morphometric defects with a small effect size. In this protocol, we describe the procedure for creating bilateral, full-thickness excisional wounds in mice as well as a detailed instruction on how to measure morphometric parameters using an image processing program on select serial sections. The two-dimension measurements of wound length, epidermal length, epidermal area, and wound area are used in combination with the known distance between sections to extrapolate the three-dimension epidermal area covering the wound, overall wound area, epidermal volume and wound volume. Although this detailed histological analysis is more time and resource consuming than conventional analyses, its rigor increases the likelihood of detecting novel phenotypes in an inherently complex wound healing process.

Interferon regulatory factor 6 is required for proper wound healing in vivo.

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.

Correction: Interferon Regulatory Factor 6 Has a Protective Role in the Host Response to Endotoxic Shock.

[This corrects the article DOI: 10.1371/journal.pone.0152385.].

Interferon Regulatory Factor 6 Controls Proliferation of Keratinocytes From Children With Van der Woude Syndrome.

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 Antigen, IRF6, P63, and Keratin 10 were determined by immunofluorescence. Keratinocytes were isolated and their proliferation potential was assessed by colony-forming efficiency assay. RESULTS: Hip skin from children with VWS showed a thicker epidermis when compared with that from children with NSCLP. Proliferating Cell Nuclear Antigen staining revealed an increase in proliferation in syndromic tissues when compared with controls. However, P63 and Keratin 10 expression were similar between groups. Finally, keratinocytes from VWS showed increased long-term proliferation when compared with NSCLP. CONCLUSIONS: These results support, in vivo and in vitro, a previously described role for IRF6 in epidermal proliferation in humans. They further demonstrate a critical function for IRF6 in cutaneous homeostasis.

Interferon Regulatory Factor 6 Has a Protective Role in the Host Response to Endotoxic Shock.

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.

Sequence variants in oxytocin pathway genes and preterm birth: a candidate gene association study.

BACKGROUND: Preterm birth (PTB) is a complex disorder associated with significant neonatal mortality and morbidity and long-term adverse health consequences. Multiple lines of evidence suggest that genetic factors play an important role in its etiology. This study was designed to identify genetic variation associated with PTB in oxytocin pathway genes whose role in parturition is well known. METHODS: To identify common genetic variants predisposing to PTB, we genotyped 16 single nucleotide polymorphisms (SNPs) in the oxytocin (OXT), oxytocin receptor (OXTR), and leucyl/cystinyl aminopeptidase (LNPEP) genes in 651 case infants from the U.S. and one or both of their parents. In addition, we examined the role of rare genetic variation in susceptibility to PTB by conducting direct sequence analysis of OXTR in 1394 cases and 1112 controls from the U.S., Argentina, Denmark, and Finland. This study was further extended to maternal triads (maternal grandparents-mother of a case infant, N=309). We also performed in vitro analysis of selected rare OXTR missense variants to evaluate their functional importance. RESULTS: Maternal genetic effect analysis of the SNP genotype data revealed four SNPs in LNPEP that show significant association with prematurity. In our case-control sequence analysis, we detected fourteen coding variants in exon 3 of OXTR, all but four of which were found in cases only. Of the fourteen variants, three were previously unreported novel rare variants. When the sequence data from the maternal triads were analyzed using the transmission disequilibrium test, two common missense SNPs (rs4686302 and rs237902) in OXTR showed suggestive association for three gestational age subgroups. In vitro functional assays showed a significant difference in ligand binding between wild-type and two mutant receptors. CONCLUSIONS: Our study suggests an association between maternal common polymorphisms in LNPEP and susceptibility to PTB. Maternal OXTR missense SNPs rs4686302 and rs237902 may have gestational age-dependent effects on prematurity. Most of the OXTR rare variants identified do not appear to significantly contribute to the risk of PTB, but those shown to affect receptor function in our in vitro study warrant further investigation. Future studies with larger sample sizes are needed to confirm the findings of this study.

Transforming growth factor Beta 3 is required for excisional wound repair in vivo.

Wound healing is a complex process that relies on proper levels of cytokines and growth factors to successfully repair the tissue. Of particular interest are the members of the transforming growth factor family. There are three TGF-ß isoforms-TGF- ß 1, 2, and 3, each isoform showing a unique expression pattern, suggesting that they each play a distinct function during development and repair. Previous studies reported an exclusive role for TGF-ß 3 in orofacial development and a potent anti-scarring effect. However, the role of TGF- ß 3 in excisional wound healing and keratinocyte migration remains poorly understood. We tested the effect of TGF-ß 3 levels on excisional cutaneous wounds in the adult mouse by directly injecting recombinant TGF-ß 3 or neutralizing antibody against TGF-ß 3 (NAB) in the wounds. Our results demonstrate that TGF-ß 3 does not promote epithelialization. However, TGF-ß 3 is necessary for wound closure as wounds injected with neutralizing antibody against TGF-ß 3 showed increased epidermal volume and proliferation in conjunction with a delay in keratinocyte migration. Wild type keratinocytes treated with NAB and Tgfb3-deficient keratinocytes closed an in vitro scratch wound with no delay, suggesting that our in vivo observations likely result from a paracrine effect.

Interferon regulatory factor 6 is necessary, but not sufficient, for keratinocyte differentiation.

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.

MCS9.7 enhancer activity is highly, but not completely, associated with expression of Irf6 and p63.

BACKGROUND: DNA variation in Interferon Regulatory Factor 6 (IRF6) contributes risk for orofacial clefting, including a common DNA variant rs642961. This DNA variant is located in a multi-species conserved sequence that is 9.7 kb upstream from the IRF6 transcriptional start site (MCS9.7). The MCS9.7 element was shown to possess enhancer activity that mimicked the expression of endogenous Irf6 at embryonic day 11.5 in transient transgenic embryos, and also contains a p63 binding site that transactivates IRF6 expression. To analyze whether the MCS9.7 enhancer is sufficient to drive IRF6 expression, we generated stable transgenic murine lines that carry a MCS9.7-lacZ transgene. We hypothesized that MCS9.7 was sufficient to recapitulate the endogenous expression of Irf6 at other time-points during embryonic development. RESULTS: We observed that MCS9.7 activity recapitulated endogenous Irf6 expression in most tissues, but not in the medial edge epithelium (MEE) at E14.5, when Irf6 expression was high during secondary palatal fusion. Also, while MCS9.7 activity and Irf6 expression were associated with p63 expression, we observed MCS9.7 activity and Irf6 expression in periderm, although p63 was absent. CONCLUSION: These data suggest that MCS9.7 enhancer activity is not sufficient to recapitulate IRF6 expression, and that p63 expression is not always necessary nor sufficient for transactivation of IRF6.