Loureirin B inhibits fibroblast proliferation and extracellular matrix deposition in hypertrophic scar via TGF‐β/Smad pathway

The ethanolic extract of Resina Draconis (RDEE) has been reported beneficial to normal wound healing yielding more regularly arranged collagen fibres. Loureirin B, a major component in RDEE, has been supposed to be effective on the prevention and treatment of pathological scars. To investigate the therapeutic effects of loureirin B on hypertrophic scar (HS), fibroblasts from human HS and normal skin (NS) were isolated. Results showed that loureirin B dose‐dependently downregulated both mRNA and protein levels of type I collagen (ColI), type III collagen (ColIII) and α ‐ smooth muscle actin (α ‐ SMA) in HS fibroblasts. Loureirin B also suppressed fibroblast proliferative activity and redistributed cell cycle, but did not affect cell apoptosis. In vivo rabbit ear scar model, loureirin B significantly improved the arrangement and deposition of collagen fibres, decreased protein levels of ColI, ColIII and α ‐ SMA and suppressed myofibroblast differentiation and scar proliferative activity. In NS fibroblasts, loureirin B effectively inhibited TGF‐β1‐induced upregulation of ColI, ColIII and α‐SMA levels, myofibroblast differentiation and the activation of Smad2 and Smad3. Loureirin B also affected mRNA levels of major MMPs and TIMPs in TGF‐β1‐stimulated fibroblasts. Taken together, this study demonstrates that loureirin B could downregulate the expression of fibrosis‐related molecules by regulating MMPs and TIMPs levels, inhibit scar fibroblast proliferation and suppress TGF‐β1‐induced fibrosis, during which TGF‐β1/Smad2/3 pathway is likely involved. These findings suggest that loureirin B is a potential therapeutic compound for HS treatment.     

Transforming growth factor‐β enhances matrix metalloproteinase‐2 expression and activity through AKT in fibroblasts derived from angiofibromas in patients with tuberous sclerosis complex

Background Patients with tuberous sclerosis complex (TSC) develop fibrous tumours in the brain, skin, kidney, heart and lungs due to TSC1/2 mutations. In the skin, patients develop angiofibromas that have vascular and fibrotic components in which transforming growth factor (TGF)‐β and matrix metalloproteinase (MMP)‐2 are important. Objectives To investigate if the TGF‐β axis and MMP‐2 play an important role in the pathogenesis of TSC angiofibromas. Methods Samples from TSC angiofibromas and normal skin were measured for expression of TGF‐β and MMP‐2 by immunohistochemistry and real‐time polymerase chain reaction. Fibroblasts grown from TSC angiofibromas (TSC fibroblasts) were incubated with TGF‐β. Expression of ERK, AKT and S6K was measured by Western blotting, and MMP‐2 expression and activity were determined by enzyme‐linked immunosorbent assay and gelatin zymography, respectively. Results There was an increase in the expression of TGF‐β and MMP‐2 in TSC tumours compared with those in normal skin. The baseline expression of MMP‐2 was increased in conditioned medium from TSC fibroblasts. In addition, TGF‐β enhanced MMP‐2 production and activity, which could be abrogated by pretreatment with an AKT inhibitor (LY294002) but not with rapamycin. Finally, there was a significant colocalization of TGF‐β and MMP‐2 in the TSC tumours. Conclusions There is an increase of MMP‐2 as a result of TGF‐β acting through AKT in TSC tumour cells. This regulation of the TGF‐β–AKT–MMP‐2 axis is independent of mammalian target of rapamycin (mTOR) signalling. In addition to targeting the mTOR pathway, targeting TGF‐β simultaneously could block dysregulated tissue remodelling in TSC tumours.     

REGγ accelerates melanoma formation by regulating Wnt/β‐catenin signalling pathway

It has been reported that the proteasome activator REGγ is associated with multiple oncogenic pathways in human cancers. However, the role of REGγ in the development of melanoma and the underlying mechanisms remain unclear. In this study, we attempted to investigate the effects of REGγ on human melanoma cell proliferation in vitro and in vivo. We demonstrated that knockdown of REGγ inhibited melanoma cell growth and arrested melanoma cell at G1 phase. Furthermore, depletion of REGγ also inhibited the xenograft growth of human melanoma. Mechanistically, REGγ activates Wnt/β‐catenin signal pathway by degrading GSK‐3β in melanoma cell lines and mouse models. Transient knockdown of β‐catenin effectively blocked cell proliferation in REGγ wild‐type melanoma cells. In human melanoma samples, REGγ was overexpressed and positively correlated with β‐catenin levels. This study demonstrates that REGγ is a central molecule in the development of melanoma by regulating Wnt/β‐catenin pathway. This suggests that targeting REGγ could be an alternative therapeutic approach for melanoma.     

The role of transforming growth factor‐β1 and oxidative stress in podoconiosis pathogenesis

Background Podoconiosis (endemic nonfilarial elephantiasis) occurs in susceptible individuals who go barefoot in regions of irritant volcanic soil. Silicate particles absorbed via the skin are thought to induce an inflammatory process and a consequent endolymphangitis of the lower leg lymphatics. Objectives To establish which oxidative stress biomarkers play a part in the inflammatory process, and to test whether transforming growth factor (TGF)‐β1 also has a pathogenetic role. Patients and methods We enrolled 50 patients with early clinical stage disease, 43 patients with advanced stage disease and 35 local healthy controls. Oxidative stress biomarkers included serum total peroxides (TP), total antioxidant capacity (TAC), total nitrate plus nitrite (TN), malondialdehyde (MDA) and total superoxide dismutase (SOD) activity. The oxidative stress index (OSI) was also determined. Serum total TGF‐β1 was assayed using sandwich enzyme‐linked immunosorbent assay. Results Compared with healthy controls, patients with early stage disease showed significantly higher mean levels of TP (P < 0·001), MDA (P < 0·05) and OSI (P < 0·01); and significantly lower mean concentrations of SOD (P < 0·001) and TGF‐β1 (P < 0·001). Mean levels of TGF‐β1 were even lower among patients with advanced stage disease (P < 0·001). Mean TAC levels were significantly lower among patients with advanced disease than either other group (P < 0·001). Conclusions This is the first study, to our knowledge, to attempt to elucidate the molecular pathogenetic events in podoconiosis. We conclude that TGF‐β1 may have a pathogenetic role, with oxidative stress playing a minor role in the early stages of disease.     

Azelaic acid reduced senescence‐like phenotype in photo‐irradiated human dermal fibroblasts: possible implication of PPARγ

Azelaic acid (AzA) has been used for the treatment for inflammatory skin diseases, such as acne and rosacea. Interestingly, an improvement in skin texture has been observed after long‐time treatment with AzA. We previously unrevealed that anti‐inflammatory activity of AzA involves a specific activation of PPARγ, a nuclear receptor that plays a relevant role in inflammation and even in ageing processes. As rosacea has been considered as a photo‐aggravated disease, we investigated the ability of AzA to counteract stress‐induced premature cell senescence (SIPS). We employed a SIPS model based on single exposure of human dermal fibroblasts (HDFs) to UVA and 8‐methoxypsoralen (PUVA), previously reported to activate a senescence‐like phenotype, including long‐term growth arrest, flattened morphology and increased synthesis of matrix metalloproteinases (MMPs) and senescence‐associated β‐galactosidase (SA‐β‐gal). We found that PUVA‐treated HDFs grown in the presence of AzA maintained their morphology and reduced MMP‐1 release and SA‐β‐galactosidase‐positive cells. Moreover, AzA induced a reduction in ROS generation, an up‐modulation of antioxidant enzymes and a decrease in cell membrane lipid damages in PUVA‐treated HDFs. Further evidences of AzA anti‐senescence effect were repression of p53 and p21, increase in type I pro‐collagen and abrogation of the enhanced expression of growth factors, such as HGF and SCF. Interestingly, PUVA‐SIPS showed a decreased activation of PPARγ and AzA counteracted this effect, suggesting that AzA effect involves PPARγ modulation. All together these data showed that AzA interferes with PUVA‐induced senescence‐like phenotype and its ability to activate PPAR‐γ provides relevant insights into the anti‐senescence mechanism.     

Overexpression of miR‐200b inhibits the cell proliferation and promotes apoptosis of human hypertrophic scar fibroblasts in vitro

Hypertrophic scarring leads to a deformed appearance and contracted neogenetic tissue, resulting in physiological and psychological problems for patients. Millions of people suffer these discomforts each year. Emerging evidence has reported that miRNA contributed to hypertrophic scarring or keloid formation. In this study, nine hypertrophic scar samples and the matched normal skin tissues were used to perform a miRNA microarray. The results of miRNA array showed that miR‐200b was downregulated by more than 2‐fold, validated by qPCR in hypertrophic scar tissues and human hypertrophic scar fibroblasts, suggesting that there was an important correlation between miR‐200b and hypertrophic scarring. We also found that miR‐200b affected hypertrophic scarring through regulating the cell proliferation and apoptosis of human hypertrophic scar fibroblasts by affecting the collagen I and III synthesis, fibronectin expression and TGF‐β1/α‐SMA signaling. Thus, our study provides evidence to support that miR‐200b may be a useful target for hypertrophic scarring management.     

Melanocyte‐protective effect of afzelin is mediated by the Nrf2‐ARE signalling pathway via GSK‐3β inactivation

Vitiligo is an acquired condition characterized by depigmented, cutaneous lesions that result from the death of pigment‐producing cells, melanocytes. The occurrence of oxidative stress has been proposed as a pathogenetic mechanism for melanocyte degeneration in vitiligo. Therefore, in this study, we investigated the cytoprotective effects of afzelin against oxidative stress and its mechanism of action in human epidermal melanocytes. We found that afzelin significantly inhibited hydrogen peroxide‐induced cell death, cellular reactive oxygen species production and lipid peroxidation in melanocytes. In an antioxidant response element (ARE)‐luciferase reporter assay, afzelin increased ARE‐luciferase reporter activity in a concentration‐dependent manner. Consistently, the expression of antioxidant genes, including NF‐E2‐related factor 2 (Nrf2), haem oxygenase‐1 (HO‐1) and catalase, was enhanced by afzelin treatment. Nuclear translocation of Nrf2 also increased in response to afzelin treatment. In addition, the phosphorylation of glycogen synthase kinase‐3β (GSK‐3β) was induced by afzelin treatment. The enhancement of HO‐1 gene expression by afzelin treatment was reduced by Nrf2‐siRNA expression. Furthermore, we found that the expression of Nrf2‐siRNA significantly attenuated the cytoprotective effect of afzelin against hydrogen peroxide. These data suggest that the cytoprotective effects of afzelin against hydrogen peroxide may be mediated by Nrf2‐ARE signalling via GSK‐3β inactivation. Our data suggest the novel use of afzelin for the prevention of oxidative stress‐induced damage in melanocytes and its potential as a therapeutic agent for vitiligo.     

Blue light inhibits transforming growth factor‐β1‐induced myofibroblast differentiation of human dermal fibroblasts

Transforming growth factor‐β1 (TGF‐β1) is the major promoter of phenotypic shift between fibroblasts and myofibroblasts accompanied by the expression and incorporation of α‐smooth muscle actin (α‐SMA). This differentiation is crucial during normal wound healing and wound closure; however, myofibroblasts are considered as the main effecter cell type in fibrosis, for example in scleroderma and hypertrophic scarring. As blue light has exerted antiprolific and toxic effects in several cell types, we investigated whether blue light irradiations with a light‐emitting diode array (420 nm) were able to affect proliferation and differentiation of human dermal fibroblasts (HDF). We found that repeated irradiation with non‐toxic doses significantly inhibits TGF‐β1‐induced differentiation of HDF into myofibroblasts shown by α‐SMA immunocytochemistry and Western blotting. Additionally, used doses reduced proliferation and myofibroblast contractibility measured by resazurin and collagen gel contraction assays. It could be demonstrated that blue light mediates cell toxicity by oxidative stress due to the generation of singlet oxygen. We postulate that irradiations at non‐toxic doses induce low‐level oxidative stress and energy‐consuming cellular responses, which both may effect proliferation stop and interfere with myofibroblast differentiation. Thus, targeting differentiation, proliferation and activity of myofibroblasts by blue light may represent a useful strategy to prevent or reduce pathological fibrotic conditions.     

Effective induction of melanoma‐antigen‐specific CD8+ T cells via Vγ9γδT cell expansion by CD56high+ Interferon‐α‐induced dendritic cells

Dendritic cells (DCs) can be differentiated from CD14⁺ monocytes in the presence of interferon‐α (IFNα) and granulocyte/macrophage‐colony stimulating factor (GM‐CSF) in vitro and are known as IFN‐DCs. Circulating blood CD56⁺ cells expressing high levels of CD14, HLA‐DR and CD86 have been shown to spontaneously differentiate into DC‐like cells in vitro after their isolation from blood. We show here that IFN‐DCs expressing high levels of CD56 (hereafter, CD56^high+ IFN‐DCs) can be differentiated in vitro from monocytes obtained as adherent cells from healthy donors and patients with metastatic melanoma. These cells expressed high levels of CD14, HLA‐DR and CD86 and possessed many pseudopodia. These CD56^high+ IFN‐DCs may be an in vitro counterpart of the circulating CD56⁺ CD14⁺ CD86⁺ HLA‐DR⁺ cells in blood. Conventional mature DCs differentiated from monocytes as adherent cells in the presence of GM‐CSF, IL‐4 and TNF‐α (hereafter, mIL‐4DCs) did not express CD56 or CD14. In contrast to mIL‐4DCs, the CD56^high+ IFN‐DCs exhibited a stronger capacity to stimulate autologous CD56⁺ Vγ9γδT cells highly producing IFNγ in the presence of zoledronate and IL‐2. The CD56^high+ IFN‐DCs possessing HLA‐A*0201 effectively induced Mart‐1‐modified melanoma peptide (A27L)‐specific CD8⁺ T cells through preferential expansion of CD56⁺ Vγ9γδT cells in the presence of A27L, zoledronate and IL‐2. Vaccination with CD56^high+ IFN‐DCs copulsed with tumor antigens and zoledronate may orchestrate the induction of various CD56⁺ immune cells possessing high effector functions, resulting in strong immunological responses against tumor cells. This study may be relevant to the design of future clinical trials of CD56^high+ IFN‐DCs‐based immunotherapies for patients with melanoma.     

Epidermal keratinocytes sense dsRNA via the NLRP3 inflammasome,mediating interleukin (IL)‐1β and IL‐18 release

Skin epidermis, in addition to its barrier function, is able to actively sense harmful pathogens using pattern recognition receptors. In immune cells, the nucleotide‐binding oligomerization domain, leucine‐rich repeat and pyrin domain containing 3 (NLRP3) inflammasome can mediate innate immunity against viral infection via a mechanism involving viral dsRNA recognition. Epidermal keratinocytes express NLRP3 inflammasome, which can sense contact sensitizers and mite allergens, leading to pro‐interleukin (IL)‐1β and pro‐IL‐18 cleavage into their active forms. Skin often faces viral infection. However, it is unknown whether viral dsRNA can be detected by the keratinocyte NLRP3 inflammasome. We transfected polyinosinic:polycytidylic acid (poly I:C), a synthetic viral dsRNA analogue, into cultured primary human keratinocytes at the aid of Lipofectamine 2000, and found that transfected poly I:C activated caspase‐1 and induced caspase‐1‐dependent release of IL‐1β and IL‐18, which were suppressed on transfection with NLRP3 siRNA. The activation of keratinocyte NLRP3 inflammasome by transfected poly I:C was dependent on dsRNA‐induced protein kinase (PKR) activation, and priming with type I interferons upregulated NLRP3 inflammasome activation through promoting PKR activation in poly I:C‐transfected keratinocytes. In conclusion, the NLRP3 inflammasome can act as a sensor of dsRNA in epidermal keratinocytes, which may be important in both skin innate immune defense against viral infection and skin inflammation.     

NADPH oxidase‐2 is a key regulator of human dermal fibroblasts: a potential therapeutic strategy for the treatment of skin fibrosis

The proliferation of human skin dermal fibroblasts (HDFs) is a critical step in skin fibrosis, and transforming growth factor‐beta1 (TGF‐β1) exerts pro‐oxidant and fibrogenic effects on HDFs. In addition, the oxidative stress system has been implicated in the pathogenesis of skin disease. However, the role of NADPH oxidase as a mediator of TGF‐β1‐induced effects in HDFs remains unknown. Thus, our aim was to investigate the role of NADPH in human skin dermal fibroblasts. Primary fibroblasts were cultured and pretreated with various stimulants. Real‐time Q‐PCR and Western blotting analyses were used for mRNA and protein detection. In addition, siRNA technology was applied for gene knock‐down analysis. Hydrogen peroxide production and 2′,7′‐dichlorofluorescein diacetate (DCFDA) measurement assay were performed. Here, our findings demonstrated that HDFs express key components of non‐phagocytic NADPH oxidase mRNA. TGF‐β1 induced NOX2 and reactive oxygen species formation via NADPH oxidase activity. In contrast, NOX3 was barely detectable, and other NOXs did not display significant changes. In addition, TGF‐β1 phosphorylated MAPKs and increased activator protein‐1 (AP‐1) in a redox‐sensitive manner, and NOX2 suppression inhibited baseline and TGF‐β1‐mediated stimulation of Smad2 phosphorylation. Moreover, TGF‐β1 stimulated cell proliferation, migration, collagen I and fibronectin expression, and bFGF and PAI‐1 secretion: these effects were attenuated by diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and NOX2 siRNA. Importantly, NOX2 siRNA suppresses collagen production in primary keloid dermal fibroblasts. These findings provide the proof of concept for NADPH oxidase as a potential target for the treatment of skin fibrosis.     

Sorafenib induces pigmentation via the regulation of β-catenin signalling pathway in melanoma cells

We conducted large-scale screening test on drugs that were already approved for other diseases to find pigmentation-modulating agents. Among drugs with potential for pigmentation control, we selected sorafenib and further investigated the effect on pigmentation using HM3KO melanoma cells. As a result of treating melanoma cells with sorafenib, pigmentation was promoted in terms of melanin content and tyrosinase activity. Sorafenib increased mRNA and protein levels of pigmentation-related genes such as MITF, tyrosinase and TRP1. To uncover the action mechanism, we investigated the effect of sorafenib on the intracellular signalling pathways. Sorafenib reduced phosphorylation of AKT and ERK, suggesting that sorafenib induces pigmentation through inhibition of the AKT and ERK pathways. In addition, sorafenib significantly increased the level of active β-catenin, together with activation of β-catenin signalling. Mechanistic study revealed that sorafenib decreased phosphorylation of serine 9 (S9) of GSK3β, while it increased phosphorylation of tyrosine 216 (Y216) of GSK3β. These results suggest that sorafenib activates the β-catenin signalling through the regulation of GSK3β phosphorylation, thereby affecting the pigmentation process.