Analysis of apoptosis-associated molecules Erythroid differentiation regulator 1, bcl-2 and p53 in actinic keratosis after treatment with ingenol mebutate.

Actinic keratosis (AK) is the most common cutaneous premalignant neoplasm precursor of malignant skin tumors. The aberrant apoptotic pathway is thought to be associated with pathogenesis of AK. Ingenol mebutate has been shown to be effective and safe for treatment of AK. However, the effect of ingenol mebutate on apoptosis-related molecules using human skin samples has not been studied well. Erythroid differentiation regulator 1 (Erdr1) was recently reported to play a crucial role in malignant skin cancers like malignant melanoma. The role of Erdr1 in premalignant actinic keratosis (AK) has not been explored. The purpose of this study was to investigate whether the expression of apoptosis-associated molecules such as Erdr1, p53 and bcl-2 was affected by the treatment of ingenol mebutate in AK. Nine patients with AK underwent skin biopsy at baseline and 8 weeks after treatment with ingenol mebutate for immunohistochemical evaluation with Erdr1, p53 and bcl-2. In addition, skin samples from five control subjects were retrieved. Upregulation of Erdr1 and a significant decrease in expression of p53 and bcl-2 were observed after treatment with ingenol mebutate. Ingenol mebutate treatment for AK resulted in the modulation of apoptosis-associated molecules with an increase in the expression of Erdr1 and a decrease in the expression of p53 and bcl-2.

Molecular Mechanisms and Management of a Cutaneous Inflammatory Disorder: Psoriasis.

Psoriasis is a complex chronic inflammatory cutaneous disorder. To date, robust molecular mechanisms of psoriasis have been reported. Among diverse aberrant immunopathogenetic mechanisms, the current model emphasizes the role of Th1 and the IL-23/Th17 axis, skin-resident immune cells and major signal transduction pathways involved in psoriasis. The multiple genetic risk loci for psoriasis have been rapidly revealed with the advent of a novel technology. Moreover, identifying epigenetic modifications could bridge the gap between genetic and environmental risk factors in psoriasis. This review will provide a better understanding of the pathogenesis of psoriasis by unraveling the complicated interplay among immunological abnormalities, genetic risk foci, epigenetic modification and environmental factors of psoriasis. With advances in molecular biology, diverse new targets are under investigation to manage psoriasis. The recent advances in treatment modalities for psoriasis based on targeted molecules are also discussed.

Erythroid Differentiation Regulator 1 as a Novel Biomarker for Hair Loss Disorders.

Erythroid differentiation regulator 1 (Erdr1) is known to be involved in the inflammatory process via regulating the immune system in many cutaneous disorders, such as psoriasis and rosacea. However, the role of Erdr1 in various hair loss disorders remains unclear. The aim of this study was to investigate the putative role of Erdr1 in alopecias. Skin samples from 21 patients with hair loss disorders and five control subjects were retrieved, in order to assess their expression levels of Erdr1. Results revealed that expression of Erdr1 was significantly downregulated in the epidermis and hair follicles of patients with hair loss disorders, when compared to that in the control group. In particular, the expression of Erdr1 was significantly decreased in patients with alopecia areata. We propose that Erdr1 downregulation might be involved in the pathogenesis of hair loss, and could be considered as a novel biomarker for hair loss disorders.

Molecular Mechanisms of Cutaneous Inflammatory Disorder: Atopic Dermatitis.

Atopic dermatitis (AD) is a multifactorial inflammatory skin disease resulting from interactions between genetic susceptibility and environmental factors. The pathogenesis of AD is poorly understood, and the treatment of recalcitrant AD is still challenging. There is accumulating evidence for new gene polymorphisms related to the epidermal barrier function and innate and adaptive immunity in patients with AD. Newly-found T cells and dendritic cell subsets, cytokines, chemokines and signaling pathways have extended our understanding of the molecular pathomechanism underlying AD. Genetic changes caused by environmental factors have been shown to contribute to the pathogenesis of AD. We herein present a review of the genetics, epigenetics, barrier dysfunction and immunological abnormalities in AD with a focus on updated molecular biology.

Rosacea: Molecular Mechanisms and Management of a Chronic Cutaneous Inflammatory Condition.

Rosacea is a chronic cutaneous inflammatory disease that affects the facial skin. Clinically, rosacea can be categorized into papulopustular, erythematotelangiectatic, ocular, and phymatous rosacea. However, the phenotypic presentations of rosacea are more heterogeneous. Although the pathophysiology of rosacea remains to be elucidated, immunologic alterations and neurovascular dysregulation are thought to have important roles in initiating and strengthening the clinical manifestations of rosacea. In this article, we present the possible molecular mechanisms of rosacea based on recent laboratory and clinical studies. We describe the genetic predisposition for rosacea along with its associated diseases, triggering factors, and suggested management options in detail based on the underlying molecular biology. Understanding the molecular pathomechanisms of rosacea will likely aid toward better comprehending its complex pathogenesis.

Melphalan-induced apoptosis of EBV-transformed B cells through upregulation of TAp73 and XAF1 and nuclear import of XPA.

Melphalan (Mel) is widely used to treat patients with hematologic cancer, including multiple myeloma, but its mechanism of action in EBV-transformed B cells is poorly described. In this study, we demonstrate a novel mechanism by which transcriptionally active p73 (TAp73) induces translocation of X-linked inhibitor of apoptosis protein-associated factor 1 (XAF1) and xeroderma pigmentosum group A (XPA) during apoptosis caused by Mel treatment. We observed that Mel induced significant generation of reactive oxygen species (ROS) and subsequent apoptosis, as well as an early phosphorylation of p38 MAPK that preceded expression of the mitochondria membrane potential disruption-related molecules and the cleavage of caspases. In particular, Mel led to upregulation of TAp73, XAF1, and Puma and induced XPA nuclear import and translocation of Bax into mitochondria. Mel-induced apoptosis was inhibited by pretreatment with the ROS scavenger 4-amino-2,4-pyrrolidine-dicarboxylic acid (APDC) and the p38 MAPK inhibitor SB203580. We supposed that ROS generation might be the first event in Mel-induced apoptosis, because APDC blocked the increase in ROS, p38 MAPK, and TAp73, but SB203580 did not block ROS generation. Moreover, Mel elicited activation of ATR, and APDC inhibited phosphorylation of ATR but not SB203580. APDC and SB203580 completely blocked XPA and Bax translocation. We conclude that Mel promotes TAp73-mediated XAF1 and Puma expression via ROS generation and ATR/p38 MAPK pathway activation, thereby triggering apoptosis. Our results provide evidence of a novel alternate regulatory mechanism of TAp73 and reveal that Mel may be a therapeutic drug for curing EBV-related malignancies.

IL-18 and Cutaneous Inflammatory Diseases.

Interleukin (IL)-18, an IL-1 family cytokine, is a pleiotropic immune regulator. IL-18 plays a strong proinflammatory role by inducing interferon (IFN)-γ. Previous studies have implicated IL-18 in the pathogenesis of various diseases. However, it is not well understood biologic activities of IL-18 in the diverse skin diseases. Here, we have reviewed the expression and function of IL-18 in skin diseases including inflammatory diseases. This article provides an evidence-based understanding of the role of IL-18 in skin diseases and its relationship with disease activities.

The combination of DHEA, histamine, and insulin increases adipogenic differentiation and enhances tissue transplantation outcome in mice.

Adipose stem cells (ASCs) are pluripotent cells that can generate pure fat tissue for regeneration. Differentiated adipose cells have been generated by a common inducer cocktail composed of dexamethasone, insulin, and isobutylmethylxanthine (DIM). The major drawbacks of adipose cells are their tendency to float on the culture media and their cost. To overcome some of these disadvantages, a new inducer cocktail that includes insulin, dehydroepiandrosterone, and histamine (DH IH) was tested. As a result, lipid accumulation was elevated more than twofold with DH IH than with DIM. Cell adhesion and viability, which are important factors for stable differentiation, were increased with DH IH and were proven through measurement of mRNA expression levels of adhesion marker genes, N-cadherin and vascular cell adhesion molecule, as well as through an alamar blue assay. The expression of adipogenesis-related genes, adiponectin, and glucose transporter type 4 lasted for a long time. To improve the efficiency of grafting, cell adhesion and neovascularization need to be increased. Neovascularization was observed around the transplanted adipose cells, which showed a higher number of vessel formation in DH IH than in DIM. The above results suggest that DH IH can produce pure differentiated adipose cells effectively and enhance their adhesion onto the target location when these differentiated adipose cells were applied as a clinical resource.

Expression of hypothalamic-pituitary-adrenal axis in common skin diseases: evidence of its association with stress-related disease activity.

Hypothalamic-pituitary-adrenal (HPA) axis hormones and their receptors expressed in the skin are known to function locally, but how these hormones affect the maintenance of skin homeostasis or the pathogenesis of skin diseases is not fully understood. We comprehensively reviewed the distribution and function of the central and peripheral HPA axis in various stress-related skin diseases. Previous studies have shown altered expression of central and peripheral HPA axis hormones in chronic inflammatory skin diseases and skin tumours, and that hyper-active lesional HPA axis hormones may negatively feedback to the central HPA axis and interact with some cytokines and neuropeptides, leading to symptom deterioration. This provides an evidence-based understanding of the expression of the central and peripheral HPA axis in common skin diseases and its association with disease activity.

Endoplasmic reticulum stress-mediated apoptosis of EBV-transformed B cells by cross-linking of CD70 is dependent upon generation of reactive oxygen species and activation of p38 MAPK and JNK pathway.

CD70 is expressed in normal activated immune cells as well as in several types of tumors. It has been established that anti-CD70 mAb induces complement-dependent death of CD70(+) tumor cells, but how anti-CD70 mAb affects the intrinsic signaling is poorly defined. In this report, we show that ligation of CD70 expressed on EBV-transformed B cells using anti-CD70 mAb induced production of reactive oxygen species (ROS) and subsequent apoptosis. We observed an early expression of endoplasmic reticulum (ER) stress response genes that preceded the release of apoptotic molecules from the mitochondria and the cleavage of caspases. CD70-induced apoptosis was inhibited by pretreatment with the ER stress inhibitor salubrinal, ROS quencher N-acetylcysteine, and Ca(2+) chelator BAPTA. We supposed that ROS generation might be the first event of CD70-induced apoptosis because N-acetylcysteine blocked increases of ROS and Ca(2+), but BAPTA did not block ROS generation. We also found that CD70 stimulation activated JNK and p38 MAPK. JNK inhibitor SP600125 and p38 inhibitor SB203580 effectively blocked upregulation of ER stress-related genes and cleavage of caspases. Inhibition of ROS generation completely blocked phosphorylation of JNK and p38 MAPK and induction of ER stress-related genes. Taken together, we concluded that cross-linking of CD70 on EBV-transformed B cells triggered ER stress-mediated apoptosis via ROS generation and JNK and p38 MAPK pathway activation. Our report reveals alternate mechanisms of direct apoptosis through CD70 signaling and provides data supporting CD70 as a viable target for an Ab-based therapy against EBV-related tumors.

LL-37 suppresses sodium nitroprusside-induced apoptosis of systemic sclerosis dermal fibroblasts.

The human cathelicidin antimicrobial peptide LL-37 regulates apoptosis of several cell types. Defective apoptosis of skin fibroblasts may contribute to systemic sclerosis (SSc). Here, we show that LL-37 inhibited apoptosis of SSc fibroblasts and identified the signalling pathways by which LL-37 mediates apoptosis. Immunohistochemistry showed that cathelicidin expression was enhanced in SSc patients compared with healthy controls. In addition, LL-37 decreased sodium nitroprusside (SNP)-induced apoptosis of SSc fibroblasts. LL-37 significantly increased expression of Bcl-2 and decreased levels of BAX protein. Pretreatment with LL-37 decreased activation of caspase-3 following SNP-treatment. Moreover, exposure of SSc fibroblasts to LL-37 resulted in increased expression of COX-2 and stimulation of prostaglandin E(2) (PGE(2)). Furthermore, LL-37 induced phosphorylation of ERK and the ERK inhibitor PD98059 blocked the inhibitory effect of LL-37 on apoptosis. Our data indicate that LL-37 may be associated with skin sclerosis by inhibiting apoptosis of dermal fibroblasts.

Erythroid differentiation regulator 1 (Erdr1) is a proapototic factor in human keratinocytes.

Skin is constantly exposed to physical and chemical stressors. The exposure of keratinocytes to ultraviolet B (UVB) irradiation causes epidermal damage via induction of apoptosis. Erythroid differentiation regulator 1 (Erdr1) modulates growth and survival of cells under various stressful conditions, but the function of Erdr1 in human keratinocyte apoptosis has not been investigated so far. Here, we investigated the effect of Erdr1 on UVB-induced apoptosis in human keratinocytes and also examined the underlying regulatory mechanism. First, Erdr1 expression was detected in human primary keratinocytes and normal human skin tissues. Expression of Erdr1 was enhanced in human keratinocytes following UVB irradiation. Knock-down of Erdr1 led to resistance to UVB-induced apoptosis. Also, Erdr1 overexpression increased UVB-induced apoptosis and induced caspase-3 activation. Furthermore, the extracellular signal-regulated kinase (ERK) inhibitor PD98059 and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 significantly reduced Erdr1 expression following UVB irradiation. These results indicate that UVB induces Erdr1 via a MAPK-dependent mechanism. Taken together, these findings suggest that Erdr1 has a role as a proapoptotic factor in human keratinocytes and acts via ERK and p38 MAPK pathways. Therefore, Erdr1 may be a potential therapeutic target to reduce apoptosis in keratinocytes in conditions such as psoriasis and skin cancer.

Expression of ADAM33 is a novel regulatory mechanism in IL-18-secreted process in gastric cancer.

IL-18 has recently been reported to play a critical role in tumor migration, invasion, and metastasis. Because IL-18 has various biological activities after its secretion as an 18 kDa mature form, the regulation of the IL-18 secretion process is an important step in tumor progression. This study investigated the implication of IL-18 in vascular endothelial growth factor (VEGF)-D-regulated migration, along with the role of the IL-18 secretion process. VEGF-D enhanced cell migration, which was then blocked by inhibiting IL-18. VEGF-D increased IL-18 expression and secretion, suggesting that IL-18 is a critical mediator for VEGF-D-enhanced migration. VEGF-D induced a disintegrin and metalloprotease 33 (ADAM33) expression, which has a metalloproteinase domain. VEGF-D-enhanced IL-18 secretion and cell migration were inhibited by ADAM33 knock-down. Moreover, cell proliferation was considerably reduced in ADAM33 small interfering RNA transfectants. In conclusion, ADAM33 has a key role in gastric cancer pathogenesis by up-regulating IL-18 secretion process, resulting in increased cell migration and proliferation.

Black tea extract prevents lipopolysaccharide-induced NF-κB signaling and attenuates dextran sulfate sodium-induced experimental colitis.

BACKGROUND: Black tea has been shown to elicit anti-oxidant, anti-carcinogenic, anti-inflammatory and anti-mutagenic properties. In this study, we investigated the impact of black tea extract (BTE) on lipopolysaccharide (LPS)-induced NF-κB signaling in bone marrow derived-macrophages (BMM) and determined the therapeutic efficacy of this extract on colon inflammation. METHODS: The effect of BTE on LPS-induced NF-κB signaling and pro-inflammatory gene expression was evaluated by RT-PCR, Western blotting, immunofluorescence and electrophoretic mobility shift assay (EMSA). The in vivo efficacy of BTE was assessed in mice with 3% dextran sulfate sodium (DSS)-induced colitis. The severity of colitis was measured by weight loss, colon length and histologic scores. RESULTS: LPS-induced IL-12p40, IL-23p19, IL-6 and IL-1ß mRNA expressions were inhibited by BTE. LPS-induced IκBα phosphorylation/degradation and nuclear translocation of NF-κB/p65 were blocked by BTE. BTE treatment blocked LPS-induced DNA-binding activity of NF-κB. BTE-fed, DSS-exposed mice showed the less weight loss, longer colon length and lower histologic score compared to control diet-fed, DSS-exposed mice. DSS-induced IκBα phosphorylation/degradation and phosphorylation of NF-κB/p65 were blocked by BTE. An increase of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) in DSS-exposed mice was blocked by BTE. CONCLUSIONS: These results indicate that BTE attenuates colon inflammation through the blockage of NF-κB signaling and apoptosis in DSS-induced experimental colitis model.

Vitamin C attenuates ERK signalling to inhibit the regulation of collagen production by LL-37 in human dermal fibroblasts.

Vitamin C is used as an anti-ageing agent because of its collagen enhancing effects. The precise cellular signalling mechanism of vitamin C is not well known. Here, we investigate the profibrotic mechanism of vitamin C against LL-37. Antimicrobial peptide LL-37 decreases collagen expression at mRNA and protein levels in human dermal fibroblasts (HDFs). The ability of LL-37 to inhibit collagen expression is dependent on phosphorylation of extracellular signal-regulated kinase (ERK). HDFs and human keloid fibroblasts were treated with vitamin C followed by 2 h of LL-37 treatment. Collagen mRNA expression and total soluble collagen production inhibited by LL-37 was enhanced by treatment with 0.5 mm vitamin C. Vitamin C also decreased intracellular reactive oxygen intermediates (ROI) levels that were increased by LL-37. Furthermore, the phosphorylation of ERK was analysed by Western blot following treatment with vitamin C and LL-37. Vitamin C turned off phosphorylation of ERK that was induced by LL-37. Ets-1 transcriptional factor, which is involved in the regulation of collagen expression by LL-37, was also inhibited by vitamin C. This study shows that vitamin C enhances collagen production by inhibiting the ERK pathway induced by LL-37.

Cell cycle arrest induced by engagement of B7-H4 on Epstein-Barr virus-positive B-cell lymphoma cell lines.

B7-H4 is a recently discovered B7 family member that has inhibitory effects on T-cell immunity. However, the reverse signalling mechanism of the B7-H4-expressing cells remains unclear. Previous work has shown that B7-H4 expression was enhanced on B cells following Epstein-Barr virus (EBV) infection, and engagement of cell-surface-expressed B7-H4 induces cell death of EBV-transformed B cells. Here we found that B7-H4 was constitutively expressed on EBV-positive lymphoma cells, Raji and IM-9 cells, but was not expressed on EBV-negative lymphoma cells (Ramos). Engagement of B7-H4 significantly reduced cell growth of Raji and IM-9 cells and resulted in cell cycle arrest at G0-G1 phase in a dose- and time-dependent manner. To clarify the mechanism of cell cycle arrest via activation of B7-H4, cell cycle regulatory factors were examined by reverse transcription-polymerase chain reaction and immunoblotting. We found that B7-H4 triggered down-regulation of CDK4/6 and up-regulation of p21 expression at both protein and RNA levels. Furthermore, CDK2 and cyclin E/D expression was down-regulated by B7-H4 triggering. Additionally, the down-regulation of phospho-AKT and phospho-cyclin E were clearly detected in B7-H4-activated Raji cells, but the phosphorylation of p53 was constitutively maintained. These results indicate that B7-H4-mediated signalling on EBV-positive B-cell lymphoma cells modulates the cell cycle through down-regulation of the AKT pathway. Consequently, B7-H4 may be a new potential target for use in EBV-positive lymphoma therapy.

Expression of human NDRG2 by myeloid dendritic cells inhibits down-regulation of activated leukocyte cell adhesion molecule (ALCAM) and contributes to maintenance of T cell stimulatory activity.

We reported previously that N-myc downstream-regulated gene 2 (NDRG2), a member of a new family of differentiation-related genes, is expressed specifically in dendritic cells (DC) differentiated from monocytes, CD34(+) progenitor cells, and the myelomonocytic leukemic cell line. In this study, we demonstrate that NDRG2 protein expression is detected, not only in in vitro-differentiated DC but also in primary DC from lymph nodes, thymus, and skin when anti-NDRG2 antibodies are used. As predicted from previous studies investigating the mRNA expression pattern of several types of cell lines, progenitor cells, and DC, NDRG2 protein was expressed strongly in DC. Its expression was detected at significant levels after differentiation from progenitor cells. RNA interference of NDRG2 demonstrated that activated leukocyte cell adhesion molecule (ALCAM) expression is down-regulated specifically in DC differentiated from NDRG2 small interfering RNA (siRNA)-transfected monocytes. This was consistent with our observation that U937 cells transfected with NDRG2 became resistant to the GM-CSF/IL-4-induced ALCAM reduction. Furthermore, DC, which had differentiated from NDRG2 siRNA-transfected monocytes, showed a reduced ability to induce T cell proliferation. Taken together, our results indicate that NDRG2 is able to preserve ALCAM expression during DC differentiation from monocytes under cytokine culture conditions and that its expression helps DC maintain costimulatory signals necessary for T cell stimulation.

B7-H4 reverse signaling induces the apoptosis of EBV-transformed B cells through Fas ligand up-regulation.

B7-H4 has an inhibitory effect on immune responses via the down-regulation of T cell-mediated immunity, but how the engagement of B7-H4 molecules by counter molecules affects the signaling mechanism of the B7-H4-expressing cells is poorly defined. In this study, we found that B7-H4 expression was enhanced on B cells infected with Epstein-Barr virus (EBV) and that triggering of these molecules induced apoptosis of EBV-transformed B cells. Engagement of B7-H4 initially increased intracellular level of ROS, which then induced the expression of FasL. Engagement of B7-H4 subsequently provoked Fas-mediated and caspase-dependent apoptosis in association with cytochrome c and AIF, and EndoG was released from the mitochondria on EBV-transformed B cells. These results suggest that B7-H4 may be a potential therapeutic target for EBV involved malignancy diseases.

Gene expression profile related to prognosis of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous group of diseases with respect to biology and clinical course. Through genome-wide scanning, we can have an improvement of the diagnosis and assay system of AML. Microarray was performed for the identification of acute myeloid leukemia prognosis. We divided patients into two groups (good prognosis group, GPG and poor prognosis group, PPG) based on differences in the individual reactions to treatment. Gene expression profiles were analyzed using microarray. Among genes up-regulated at least two-fold and down-regulated at least 0.5-fold in HL-60, we chose three up-regulated genes (PPP2CA, ME3, and CCDN2) and three down-regulated genes (GLO1, ANXA2, and BMI1) and confirmed the expression of these six genes by RT-PCR. We created a leukemia-specific subclass microarray, based on the gene expression profiles. Clinical samples from the bone marrow of four patients were hybridized on this microarray. Among the genes selected by the microarray technology, NB4, silenced TRIB3 and overexpressed XRN2 were not differentiated in spite of treatment with ATRA. This indicates that XRN2 and TRIB3 play an important role in cell differentiation. These data provided an expression profile for the diagnosis and prognosis of AML patients and identified candidate genes that might allow the prognosis of AML through the relative comparison of the expression level of genes between GPG and PPG.

Tumor necrosis factor-alpha and interleukin-1beta increases CTRP1 expression in adipose tissue.

CTRP1, a member of the CTRP superfamily, consists of an N-terminal signal peptide sequence followed by a variable region, a collagen repeat domain, and a C-terminal globular domain. CTRP1 is expressed at high levels in adipose tissues of LPS-stimulated Sprague-Dawley rats. The LPS-induced increase in CTRP1 gene expression was found to be mediated by TNF-alpha and IL-1beta. Also, a high level of expression of CTRP1 mRNA was observed in adipose tissues of Zucker diabetic fatty (fa/fa) rats, compared to Sprague-Dawley rats in the absence of LPS stimulation. These findings indicate that CTRP1 expression may be associated with a low-grade chronic inflammation status in adipose tissues.