IQ-Switch is a QF-based innocuous, silencing-free, and inducible gene switch system in zebrafish.

Though various transgene expression switches have been adopted in a wide variety of organisms for basic and biomedical research, intrinsic obstacles of those existing systems, including toxicity and silencing, have been limiting their use in vertebrate transgenesis. Here we demonstrate a novel QF-based binary transgene switch (IQ-Switch) that is relatively free of driver toxicity and transgene silencing, and exhibits potent and highly tunable transgene activation by the chemical inducer tebufenozide, a non-toxic lipophilic molecule to developing zebrafish with negligible background. The interchangeable IQ-Switch makes it possible to elicit ubiquitous and tissue specific transgene expression in a spatiotemporal manner. We generated a RASopathy disease model using IQ-Switch and demonstrated that the RASopathy symptoms were ameliorated by the specific BRAF(V600E) inhibitor vemurafenib, validating the therapeutic use of the gene switch. The orthogonal IQ-Switch provides a state-of-the-art platform for flexible regulation of transgene expression in zebrafish, potentially applicable in cell-based systems and other model organisms.

Promoter methylation changes in ALOX12 and AIRE1: novel epigenetic markers for atherosclerosis.

BACKGROUND: Atherosclerosis is the main cause of cardiovascular diseases such as ischemic stroke and coronary heart disease. Gene-specific promoter methylation changes have been suggested as one of the causes underlying the development of atherosclerosis. We aimed to identify and validate specific genes that are differentially expressed through promoter methylation in atherosclerotic plaques. We performed the present study in four steps: (1) profiling and identification of gene-specific promoter methylation changes in atherosclerotic tissues; (2) validation of the promoter methylation changes of genes in plaques by comparison with non-plaque intima; (3) evaluation of promoter methylation status of the genes in vascular cellular components composing atherosclerotic plaques; and (4) evaluation of promoter methylation differences in genes among monocytes, T cells, and B cells isolated from the blood of ischemic stroke patients. RESULTS: Upon profiling, AIRE1, ALOX12, FANK1, NETO1, and SERHL2 were found to have displayed changes in promoter methylation. Of these, AIRE1 and ALOX12 displayed higher methylation levels in plaques than in non-plaque intima, but lower than those in the buffy coat of blood. Between inflammatory cells, the three genes were significantly less methylated in monocytes than in T and B cells. In the vascular cells, AIRE1 methylation was lower in endothelial and smooth muscle cells. ALOX12 methylation was higher in endothelial, but lower in smooth muscle cells. Immunofluorescence staining showed that co-localization of ALOX12 and AIRE1 was more frequent in CD14(+)-monocytes than in CD4(+)-T cell in plaque than in non-plaque intima. CONCLUSIONS: Promoter methylation changes in AIRE1 and ALOX12 occur in atherosclerosis and can be considered as novel epigenetic markers.

Induction of alopecia areata in C3H/HeJ mice using polyinosinic-polycytidylic acid (poly[I:C]) and interferon-gamma.

Alopecia areata (AA) is a chronic, relapsing hair-loss disorder that is considered to be a T-cell-mediated autoimmune disease. Several animal models for AA have been created to investigate the pathophysiology and screen for effective therapeutic targets. As C3H/HeJ mice develop AA spontaneously in a low frequency, a novel animal model is needed to establish an AA-like condition faster and more conveniently. In this study, we present a novel non-invasive AA rodent model that avoids skin or lymph-node cell transfer. We simply injected C3H/HeJ mice subcutaneously with interferon-gamma (IFNγ) along with polyinosinic:polycytidylic acid (poly[I:C]), a synthetic dsRNA, to initiate innate immunity via inflammasome activation. Approximately 80% of the IFNγ and poly(I:C) co-injected mice showed patchy AA lesions after 8 weeks. None of the mice displayed hair loss in the IFNγ or poly(I:C) solely injection group. Immunohistochemical staining of the AA lesions revealed increased infiltration of CD4+ and CD8+ cells infiltration around the hair follicles. IFNγ and poly(I:C) increased the expression of NLRP3, IL-1ß, CXCL9, CXCL10, and CXCL11 in mouse skin. Taken together, these findings indicate a shorter and more convenient means of AA animal model induction and demonstrate that inflammasome-activated innate immunity is important in AA pathogenesis.

Double-stranded RNA induces inflammation via the NF-κB pathway and inflammasome activation in the outer root sheath cells of hair follicles.

Alopecia areata (AA), a chronic, relapsing, hair-loss disorder, is considered to be a T cell-mediated autoimmune disease. It affects approximately 1.7% of the population, but its precise pathogenesis remains to be elucidated. Despite the recent attention focused on the roles of inflammasomes in the pathogenesis of autoinflammatory diseases, little is known about inflammasome activation in AA. Thus, in this study, we investigated the pattern of NLRP3 inflammasome activation in the outer root sheath (ORS) cells of hair follicles. We found that interleukin (IL)-1ß and caspase-1 expression was increased in hair follicle remnants and inflammatory cells of AA tissue specimens. After stimulation of ORS cells with the double-stranded (ds)RNA mimic polyinosinic:polycytidylic acid (poly[I:C]), the activation of caspase-1 and secretion of IL-1ß were enhanced. Moreover, NLRP3 knockdown decreased this poly(I:C)-induced IL-1ß production. Finally, we found that high-mobility group box 1 (HMGB1) translocated from the nucleus to the cytosol and was secreted into the extracellular space by inflammasome activation. Taken together, these findings suggest that ORS cells are important immunocompetent cells that induce NLRP3 inflammasomes. In addition, dsRNA-induced IL-1ß and HMGB1 secretion from ORS cells may contribute to clarifying the pathogenesis and therapeutic targets of AA.

Targeted deletion of Crif1 in mouse epidermis impairs skin homeostasis and hair morphogenesis.

The epidermis, which consists mainly of keratinocytes, acts as a physical barrier to infections by regulating keratinocyte proliferation and differentiation. Hair follicles undergo continuous cycling to produce new one. Therefore, optimum supply of energy from the mitochondria is essential for maintaining skin homeostasis and hair growth. CRIF1 is a mitochondrial protein that regulates mitoribosome-mediated synthesis and insertion of mitochondrial oxidative phosphorylation polypeptides into the mitochondrial membrane in mammals. Recent studies reveal that conditional knockout (cKO) of Crif1 in specific tissues of mice induced mitochondrial dysfunction. To determine whether the mitochondrial function of keratinocytes affects skin homeostasis and hair morphogenesis, we generated epidermis-specific Crif1 cKO mice. Deletion of Crif1 in epidermis resulted in impaired mitochondrial function and Crif1 cKO mice died within a week. Keratinocyte proliferation and differentiation were markedly inhibited in Crif1 cKO mice. Furthermore, hair follicle morphogenesis of Crif1 cKO mice was disrupted by down-regulation of Wnt/ß-catenin signaling. These results demonstrate that mitochondrial function in keratinocytes is essential for maintaining epidermal homeostasis and hair follicle morphogenesis.

Hyaluronic Acid Decreases Lipid Synthesis in Sebaceous Glands.

Hyaluronic acid (HA) is the major glycosaminoglycan in the extracellular matrix and has been implicated in several functions in skin cells. However, evidence is lacking regarding the HA signaling in sebaceous glands, and its potential role needs to be clarified. We investigated the role of HA in lipid production in sebaceous glands in an experimental study of human sebocytes followed by a clinical study. We first examined the effects of HA on sebaceous glands in hamsters and intradermal injection of HA into hamster auricles decreased both the size of sebaceous glands and the level of lipid production. We demonstrated that human skin sebaceous glands in vivo and sebocytes in vitro express CD44 (HA binding receptor) and that HA downregulates lipid synthesis in a dose-dependent manner. To evaluate the clinical relevance of HA in human skin, 20 oily participants were included in a double-blind, placebo-controlled, split-face study, and the HA-treated side showed a significant decrease in sebum production. The results of this study indicate that HA plays a functional role in human sebaceous gland biology and HA signaling is an effective candidate in the management of disorders in which sebum production is increased.

Retraction: Ampelopsis japonica Makino Extract Inhibits the Inflammatory Reaction Induced by Pathogen-Associated Molecular Patterns in Epidermal Keratinocytes.

[This retracts the article on p. 352 in vol. 28, PMID: 27274634.].

Induction of Interleukin-22 (IL-22) production in CD4+ T Cells by IL-17A Secreted from CpG-Stimulated Keratinocytes.

BACKGROUND: Interleukin-17A (IL-17A) is mainly secreted from Th17 cells that are activated by various stimuli including CpG oligodeoxynucleotide, a Toll-like receptor 9 (TLR9) ligand. Recently, it has been demonstrated that keratinocytes play an important role in the pathogenesis of psoriasis. OBJECTIVE: To investigate the potential role of keratinocytes, we examined whether TLR9 ligand CpG induces IL-17A expression in keratinocytes. METHODS: We used HaCaT keratinocytes as a model system, and determined CpG-induced IL-17A using enzyme-linked immunosorbent assay and Western blot. RESULTS: When HaCaT keratinocytes were treated with CpG, the expression of several cytokines including IL-17A, tumor necrosis factor-α and CCL20 was markedly increased. Treatment with nuclear factor (NF)-κB inhibitor significantly blocked the CpG-induced IL-17A production, indicating that CpG induced IL-17A expression through the NF-κB signaling pathway. In addition, IL-17A secreted from keratinocytes stimulated the CD4+ T cells, resulting in strong induction of IL-22 production. CONCLUSION: Since IL-22 is an important mediator for psoriatic inflammation, our data suggest that keratinocytes can participate in the pathogenesis of psoriasis via the TLR9-dependent IL-17A production.

Ecdysone Receptor-based Singular Gene Switches for Regulated Transgene Expression in Cells and Adult Rodent Tissues.

Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet)-regulated system. Exploiting a Drosophila ecdysone receptor (EcR)-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+) and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site). Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion.

Effect of Botulinum Toxin Type A on TGF-ß/Smad Pathway Signaling: Implications for Silicone-Induced Capsule Formation.

BACKGROUND: One of the most serious complications of breast surgery using implants is capsular contracture. Several preventive treatments have been introduced; however, the mechanism of capsule formation has not been resolved completely. The authors previously identified negative effects of botulinum toxin type A on capsule formation, expression of transforming growth factor (TGF)-ß1, and differentiation of fibroblasts into myofibroblasts. Thus, the authors investigated how to prevent capsule formation by using botulinum toxin type A, particularly by means of TGF-ß1 signaling, in human fibroblasts. METHODS: In vitro, cultured human fibroblasts were treated with TGF-ß1 and/or botulinum toxin type A. Expression of collagen, matrix metalloproteinase, and Smad was examined by Western blotting. The activation of matrix metalloproteinase was observed by gelatin zymography. In vivo, the effect of botulinum toxin type A on the phosphorylation of Smad2 in silicone-induced capsule formation was evaluated by immunocytochemistry. RESULTS: In vitro, the phosphorylation of Smad2 was inhibited by botulinum toxin type A treatment. The expression levels of collagen types 1 and 3 were inhibited by botulinum toxin type A treatment, whereas those of matrix metalloproteinase-2 and matrix metalloproteinase-9 were enhanced. Gelatin zymography experiments confirmed enhanced matrix metalloproteinase-2 activity in collagen degradation. In vivo, botulinum toxin type A treatment reduced capsule thickness and Smad2 phosphorylation in silicone-induced capsules. CONCLUSION: This study suggests that botulinum toxin type A plays an important role in the inhibition of capsule formation through the TGF-ß/Smad signaling pathway. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

The expression pattern and functional role of REIC/Dkk-3 in the development of cutaneous squamous cell carcinoma.

BACKGROUND: The exact physiological function of REIC/Dkk-3 in the development of squamous cell carcinoma(SCC) remains unclear. OBJECTIVE: We aimed to investigate the expression pattern and functional role of REIC/Dkk-3 in the development of SCC. METHODS: We stained normal skin, actinic keratosis (AK) and SCC tissue with REIC/Dkk-3. The proliferation and migration of SCC 12 over-expressed with REIC/Dkk-3 were observed. For in vivo study, SCC12 cells in PBS, SCC12 cells containing LacZ, and REIC/Dkk-3-transduced SCC 12 cells were injected intra-dermally into the left and right backside flanks of SCID mice respectively, and tumor growth was evaluated. RESULTS: REIC/Dkk-3 staining was detected throughout the full epidermis in normal skin, focally positive in AK. Negative or very low stain of REIC/Dkk-3 was observed in SCC in situ, keratoacanthoma, and SCC. REIC/Dkk-3 mRNA level in SCC was very low compared with that in normal skin tissue. REIC/Dkk-3 significantly decreased the proliferation and migration of SCC12 cells comparing with control (p<0.05). Cyclin D1 and CDK4/6 expression was slightly lower and p21 was very higher in REIC/Dkk-3-overexpressed group than in the LacZ group. Fewer ITGA6 cells were found in the REIC/Dkk-3 overexpressed group than in the LacZ control (p<0.01). Mean tumor volume was smallest in the REIC/Dkk-3 overexpressed group (p<0.01) 21days after the intradermal injection of SCC12 cells. CONCLUSION: REIC/Dkk-3 could be involved early in SCC development and have inhibitory effect on the development of SCC.

Ampelopsis japonica Makino Extract Inhibits the Inflammatory Reaction Induced by Pathogen-Associated Molecular Patterns in Epidermal Keratinocytes.

BACKGROUND: Keratinocytes are the major cells in epidermis, providing barrier components such as cornified cells through the sophisticated differentiation process. In addition, keratinocytes exerts their role as the defense cells via activation of innate immunity. It has been known that pathogen-associated molecular patterns (PAMPs) including double-strand RNA and nucleotides can provoke inflammatory reaction in keratinocytes. OBJECTIVE: The aim of this study is to evaluate the effect of Ampelopsis japonica Makino extract (AE) on PAMPs-induced inflammatory reaction of keratinocytes. METHODS: The effects of AE were determined using poly (I:C)-induced inflammation and imiquimod-induced psoriasiform dermatitis models. RESULTS: In cultured keratinocytes, AE significantly inhibited poly(I:C)-induced expression of inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, IL-8 and tumor necrosis factor-α. AE significantly inhibited poly(I:C)-induced release of caspase-1 active form (p20), and down-regulated nuclear factor-κB signaling pathway. In imiquimod-induced psoriasiform dermatitis model, topical application of AE resulted in significant reduction of epidermal hyperplasia. CONCLUSION: These results suggest that AE may be a potential candidate for the treatment of skin inflammation.

The inhibitory effect of A20 on the inflammatory reaction of epidermal keratinocytes.

A20 is a negative regulator of nuclear factor κ-light­chain-enhancer of activated B cells (NF-κB) signaling, and has been implicated in the pathogenesis of psoriasis through genome-wide association study (GWAS). In the present study, we investigated the putative role of A20 in epidermal keratinocytes. Immunohistochemical analysis showed that A20 was expressed in all layers of the epidermis, with an increasing pattern in the upper layers. In our model of calcium-induced keratinocyte differentiation, A20 expression was increased in a time-dependent manner. To investigate whether A20 affected keratinocyte differentiation, we overexpressed A20 in cultured keratinocytes. As a result, we noted that A20 overexpression did not affect keratinocyte differentiation, suggesting that A20 is not a direct modulator of keratinocyte differentiation. Interestingly, we found that A20 levels were decreased in psoriatic lesional skin compared to non-lesional areas. To investigate whether A20 played a role in the innate immune response of keratinocytes, we overexpressed A20 and then examined poly(I:C)-induced cytokine expression. We noted that A20 significantly inhibited poly(I:C)-induced cytokine production, and this effect was related to the inhibition of NF-κB signaling. These results suggest that the downregulation of A20 increased the susceptibility of keratinocytes to external stimuli, thus contributing to the development of psoriasis.

Inhibitory effect of Paeonia lactiflora Pallas extract (PE) on poly (I:C)-induced immune response of epidermal keratinocytes.

Epidermal keratinocytes provide protective role against external stimuli by barrier formation. In addition, kertinocytes exerts their role as the defense cells via activation of innate immunity. Disturbance of keratinocyte functions is related with skin disorders. Psoriasis is a common skin disease related with inflammatory reaction in epidermal cells. We attempted to find therapeutics for psoriasis, and found that Paeonia lactiflora Pallas extract (PE) has an inhibitory potential on poly (I:C)-induced inflammation of keratinocytes. PE significantly inhibited poly (I:C)-induced expression of crucial psoriatic cytokines, such as IL-6, IL-8, CCL20 and TNF-α, via down-regulation of NF-κB signaling pathway in human keratinocytes. In addition, PE significantly inhibited poly (I:C)-induced inflammasome activation, in terms of IL-1ß and caspase-1 secretion. Finally, PE markedly inhibited poly (I:C)-increased NLRP3, an important component of inflammasome. These results indicate that PE has an inhibitory effect on poly (I:C)-induced inflammatory reaction of keratinocytes, suggesting that PE can be developed for the treatment of psoriasis.

Inhibitory effect of cucurbitacin B on imiquimod-induced skin inflammation.

Psoriasis is a common skin disease, of which pathogenesis involves the increase of inflammatory reaction in epidermal cells. In an attempt to find therapeutics for psoriasis, we found that cucurbitacin B has an inhibitory potential on imiquimod-induced inflammation of keratinocytes. Cucurbitacin B significantly inhibited imiquimod-induced expression of crucial psoriatic cytokines, such as IL-8 and CCL20, via down-regulation of NF-κB and STAT3 signaling pathway in human keratinocytes. In addition, keratinocyte proliferation was markedly inhibited by cucurbitacin B. The potential beneficial effect of cucurbitacin B on psoriasis was further validated in imiquimod-induced psoriasiform dermatitis of experimental animal. Topical application of cucurbitacin B resulted in significant reduction of epidermal hyperplasia and inflammatory cytokines production, and ameliorated the psoriatic symptom. Taken together, these results suggest that cucurbitacin B may be a potential candidate for the treatment of psoriasis.

Genkwadaphnin induces reactive oxygen species (ROS)-mediated apoptosis of squamous cell carcinoma (SCC) cells.

Genkwadaphnin is a daphnane diterpene ester molecule isolated from the flower buds of Daphne genkwa. In the present study, we investigated the apoptosis-inducing effect of genkwadaphnin in squamous cell carcinoma (SCC) cells. Apoptosis was triggered in SCC12 cells following genkwadaphnin treatment in a time- and concentration-dependent manner. Genkwadaphnin treatment increased phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Knockdown of JNK and p38 MAPK by recombinant adenovirus expressing microRNA (miR) resulted in significant inhibition of genkwadaphnin-induced apoptosis in SCC12 cells. Finally, pretreatment with the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) markedly reduced SCC12 cell apoptosis, concomitant with significant inhibition of MAPK activation. These results indicate that genkwadaphnin has the potential to induce apoptosis in SCC cells, providing information on which to base further research with the aim of developing a cure for SCC.

Propionibacterium acnes activates the NLRP3 inflammasome in human sebocytes.

Propionibacterium acne and sebaceous glands are considered to have an important role in the development of acne. Although information regarding the activation of innate immunity by P. acnes in the sebaceous gland is limited, different P. acnes phylotypes and a higher prevalence of follicular P. acnes macrocolonies/biofilms in sebaceous follicles of skin biopsies from acne compared with control skin and occasionally single P. acnes clusters in single sebaceous glands have been detected. In this study, we investigated whether P. acnes activates the inflammasome in human sebaceous glands in vivo and in vitro. We found that IL-1ß expression was upregulated in sebaceous glands of acne lesions. After stimulation of human sebocytes with P. acnes, the activation of caspase-1 and secretion of IL-1ß were enhanced significantly. Moreover, knocking down the expression of NLRP3 abolished P. acnes-induced IL-1ß production in sebocytes. The activation of the NLRP3 inflammasome by P. acnes was dependent on protease activity and reactive oxygen species generation. Finally, we found that NALP3-deficient mice display an impaired inflammatory response to P. acnes. These results suggest that human sebocytes are important immunocompetent cells that induce the NLRP3 inflammasome, and that P. acnes-induced IL-1ß activation in sebaceous glands may have a role in combating skin infections and in acne pathogenesis.

Role of nuclear factor E2-related factor 2 (Nrf2) in epidermal differentiation.

Nuclear factor E2-related factor 2 (Nrf2) is one of the most important redox-sensitive transcription factors regulating expression of antioxidative genes and cytoprotective enzymes, which constitute the cellular response to oxidative stress and xenobiotic damage. In this study, we investigated the functional role of Nrf2 during normal epidermal keratinocyte (NHEK) differentiation. Immunohistochemical staining showed that Nrf2 is expressed from basal to granular layer of epidermis. When cultured NHEKs were treated with 1.2 mM calcium, Nrf2 expression was increased gradually in protein levels and Nrf2 translocated into the nucleus in a differentiation-dependent manner. When Nrf2 was overexpressed in NHEK by adenoviral transduction, the expression of the NHEK differentiation marker loricrin and keratin 10 was increased and overexpression of Nrf2 also increased the luciferase activity of loricrin in the absence of calcium. These results suggest that Nrf2 helps to promote the differentiation of epidermal keratinocytes.

Imiquimod induces apoptosis of squamous cell carcinoma (SCC) cells via regulation of A20.

Imiquimod, a nucleoside analogue of the imidazoquinoline family, is being used to treat various cutaneous cancers including squamous cell carcinoma (SCC). Imiquimod activates anti-tumor immunity via Toll-like receptor 7 (TLR7) in macrophage and other immune cells. Imiquimod can also affect tumor cells directly, regardless of its impact on immune system. In this study, we demonstrated that imiquimod induced apoptosis of SCC cells (SCC12) and A20 was involved in this process. When A20 was overexpressed, imiquimod-induced apoptosis was markedly inhibited. Conversely, knockdown of A20 potentiated imiquimod-induced apoptosis. Interestingly, A20 counteracted activation of c-Jun N-terminal kinase (JNK), suggesting that A20-regulated JNK activity was possible mechanism underlying imiquimod-induced apoptosis of SCC12 cells. Finally, imiquimod-induced apoptosis of SCC12 cells was taken place in a TLR7-independent manner. Our data provide new insight into the mechanism underlying imiquimod effect in cutaneous cancer treatment.