K15 promoter-driven enforced expression of NKIRAS exhibits tumor suppressive activity against the development of DMBA/TPA-induced skin tumors.

NKIRAS1 and NKIRAS2 (also called as κB-Ras) were identified as members of the atypical RAS family that suppress the transcription factor NF-κB. However, their function in carcinogenesis is still controversial. To clarify how NKIRAS acts on cellular transformation, we generated transgenic mice in which NKIRAS2 was forcibly expressed using a cytokeratin 15 (K15) promoter, which is mainly activated in follicle bulge cells. The ectopic expression of NKIRAS2 was mainly detected in follicle bulges of transgenic mice with NKIRAS2 but not in wild type mice. K15 promoter-driven expression of NKIRAS2 failed to affect the development of epidermis, which was evaluated using the expression of K10, K14, K15 and filaggrin. However, K15 promoter-driven expression of NKIRAS2 effectively suppressed the development of skin tumors induced by treatment with 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA). This observation suggested that NKIRAS seemed to function as a tumor suppressor in follicle bulges. However, in the case of oncogenic HRAS-driven cellular transformation of murine fibroblasts, knockdown of NKIRAS2 expression drastically suppressed HRAS-mutant-provoked cellular transformation, suggesting that NKIRAS2 was required for the cellular transformation of murine fibroblasts. Furthermore, moderate enforced expression of NKIRAS2 augmented oncogenic HRAS-provoked cellular transformation, whereas an excess NKIRAS2 expression converted its functional role into a tumor suppressive phenotype, suggesting that NKIRAS seemed to exhibit a biphasic bell-shaped enhancing effect on HRAS-mutant-provoked oncogenic activity. Taken together, the functional role of NKIRAS in carcinogenesis is most likely determined by not only cellular context but also its expression level.

Interleukin-33 is expressed in the lesional epidermis in herpes virus infection but not in verruca vulgaris.

Interleukin (IL)-33 is released on cell injury and activates the immune reaction. IL-33 is involved in antiviral reaction in herpes virus infection, but the source that secretes IL-33 has not been identified. We speculate that keratinocytes injured in herpes virus infection secrete IL-33. In order to detect IL-33 in the lesional epidermis of patients with herpes virus infection, we immunostained several cutaneous herpes virus infection samples with an anti-IL-33 antibody, and compared them with cutaneous human papilloma virus (HPV) infection samples. We observed strong nuclear and mild cytoplasmic staining in epidermal keratinocytes of the lesional skin samples with herpes simplex virus and varicella zoster virus infections. However, staining was not observed in the epidermis of verruca vulgaris (VV) samples. We assumed that the strong immune reaction to herpes virus infection may depend on strong IL-33 expression in the epidermis, while very weak immune reaction in samples from patients with VV may be due to low or no expression of IL-33 in the lesional epidermis.

Nuclear expression of IL-33 in epidermal keratinocytes promotes wound healing in mice.

BACKGROUND: Skin is the outermost tissue of the human body, and works as a mechanical, chemical, and biological barrier. The epidermis is the uppermost layer of the skin, and keratinocytes constitute the majority of epidermal cells. Wounds are disruptions of skin integrity, and cause tremendous disadvantages to humans; accordingly, rapid wound healing is very important. Interleukin (IL)-33 is expressed in barrier tissue cells, such as epithelial and endothelial cells. Upon injury, IL-33 is released to stimulate immune cells, functioning as an "alarmin." ST2 is a receptor for IL-33; its soluble form (s)ST2 acts as a decoy receptor and competes for IL-33 binding. OBJECTIVES: We aimed to clarify the role of IL-33 in wound healing. MATERIALS AND METHODS: Wild-type (WT), IL-33 knockout (IL33 KO) mice, and sST2 transgenic (Tg) mice were wounded with a 4-mm punch, and the wound healing process was compared. Immunohistochemical analyses were performed to detect macrophages, neutrophils, and mast cells. Total RNA was extracted from the skin samples and real-time PCR was performed. An in vitro scratch wound assay was performed. RESULTS: Wound healing was delayed in IL33 KO mice compared to WT mice, while wound healing in sST2 Tg mice was comparable to that of WT mice. A histological examination showed delayed elongation of the epidermal tongue in IL-33 KO mice. An immunohistochemical study revealed prolonged neutrophilic infiltration at a later stage in IL-33 KO mice. IL-6, IL-1ß, and CXCL1 transcripts were more abundant in the wounds of IL-33 KO mice than WT mice. Intraperitoneal administration of an NFκB inhibitor to IL-33 KO mice normalized the delayed wound healing and the enhanced expression of IL-6 in IL-33 KO mice. Epidermal keratinocytes from IL-33 KO mice showed delayed wound closure compared to those from WT mice. CONCLUSION: Our results indicate that nuclear IL-33, but not IL-33 as a cytokine, has beneficial effects on wound healing in mice, probably by suppressing NFκB to inhibit excessive inflammation and by maintaining keratinocyte proliferation or migration for epithelialization.

Chemokine receptor CCR8 is required for lipopolysaccharide-triggered cytokine production in mouse peritoneal macrophages.

Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8-/-) and wild-type (WT) mice. We found that CCR8-/- PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca2+ flux and CCL1-driven PMφ aggregation. Similar to CCR8-/- PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. CCR8-/- PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8-/- mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of CCR8-/- mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.

Microbiota-derived lactate accelerates colon epithelial cell turnover in starvation-refed mice.

Oral food intake influences the morphology and function of intestinal epithelial cells and maintains gastrointestinal cell turnover. However, how exactly these processes are regulated, particularly in the large intestine, remains unclear. Here we identify microbiota-derived lactate as a major factor inducing enterocyte hyperproliferation in starvation-refed mice. Using bromodeoxyuridine staining, we show that colonic epithelial cell turnover arrests during a 12- to 36-h period of starvation and increases 12-24 h after refeeding. Enhanced epithelial cell proliferation depends on the increase in live Lactobacillus murinus, lactate production and dietary fibre content. In the model of colon tumorigenesis, mice exposed to a carcinogen during refeeding develop more aberrant crypt foci than mice fed ad libitum. Furthermore, starvation after carcinogen exposure greatly reduced the incidence of aberrant crypt foci. Our results indicate that the content of food used for refeeding as well as the timing of carcinogen exposure influence the incidence of colon tumorigenesis in mice.

Comprehensive analysis of chemokines and cytokines secreted in the peritoneal cavity during laparotomy.

We recently found that chemokine-driven peritoneal cell aggregation is the primary mechanism of postoperative adhesion in a mouse model. To investigate this in humans, paired samples of peritoneal lavage fluid were obtained from seven patients immediately after incision (preoperative) and before closure (postoperative), and were assayed for the presence of 27 cytokines and chemokines using multiplex beads assay. As a result, IL-6 and CCL5 showed the most striking increase during operation. Recombinant CCL5 or lavage fluid induced chemotaxis of human peripheral blood mononuclear cells. We propose that CCL5 is possibly involved in the mechanism of postoperative adhesion in humans.

Interleukin-13 damages intestinal mucosa via TWEAK and Fn14 in mice-a pathway associated with ulcerative colitis.

BACKGROUND & AIMS: TWEAK, a member of the tumor necrosis factor (TNF) superfamily, promotes intestinal epithelial cell injury and signals through the receptor Fn14 following irradiation-induced tissue damage and during development of colitis in mice. Interleukin (IL)-13, an effector of tissue damage in similar models, has been associated with the pathogenesis of ulcerative colitis (UC). We investigated interactions between TWEAK and IL-13 following mucosal damage in mice. METHODS: We compared patterns of gene expression in intestinal tissues from wild-type and TWEAK knockout mice following γ-irradiation. Intestinal explants from these mice were used to detect cell damage induced by IL-13 and TNF-α. Levels of messenger RNA for IL-13, TWEAK, and Fn14 were measured in mucosal samples from patients with UC. RESULTS: Based on gene expression analysis, TWEAK mediates γ-irradiation-induced epithelial cell cycle arrest and apoptosis. However, TWEAK alone did not induce damage or apoptosis of primary intestinal epithelial cells. On the other hand, exogenous IL-13 activated caspase-3 in naïve intestinal explants; this process required TWEAK, Fn14, and secretion of endogenous TNF-α which was mediated by ADAM17. Conversely, activation of caspase by exogenous TNF-α required IL-13, TWEAK, and Fn14. In mucosa from patients with UC, messenger RNA levels of IL-13, TWEAK, and Fn14 increased with level of disease severity. CONCLUSIONS: IL-13-induced damage of intestinal epithelial cells requires TWEAK, its receptor (Fn14), and TNF-α. IL-13, TNF-α, TWEAK, and Fn14 could perpetuate and aggravate intestinal inflammation in patients with UC.

The acute lymphoblastic leukemia-associated JAK2 L611S mutant induces tumorigenesis in nude mice.

JAK2 plays important roles in the regulation of a variety of cellular processes including cell migration, proliferation, and protection from apoptosis. Recently the L611S point mutation in JAK2 has been identified in a child with acute lymphoblastic leukemia. Here we analyzed the mechanism by which JAK2 exhibits its oncogenicity. In BaF3 murine hematopoietic cells, L611S mutant increased the expression of antiapoptotic proteins including X chromosome-linked inhibitor of apoptosis protein, inhibitor of apoptosis protein, and Bcl-XL. We also showed that JAK2 L611S mutant protects BaF3 cells from cytokine withdrawal-induced apoptotic cell death and leads to cytokine-independent cell growth. Furthermore BaF3 cells expressing JAK2 L611S mutant gained the ability to induce tumorigenesis in nude mice. The L611S mutant also exhibited malignancy, including prompt invasion and spreading into various organs, leading to rapid lethality of the mice. Finally we showed that a specific JAK2 inhibitor, AG490, potently inhibited cytokine-independent cell growth induced by JAK2 L611S mutant via the induction of apoptotic cell death. In addition, treatment with AG490 significantly inhibited the JAK2 L611S mutant-induced tumorigenesis in nude mice. Thus, our results both in vitro and in vivo strongly suggest that L611S mutant of JAK2 harbors potent oncogenic activity, and this probably requires the antiapoptotic signaling pathway.

Dermatophagoides farinae extract induces severe atopic dermatitis in NC/Nga mice, which is effectively suppressed by the administration of tacrolimus ointment.

Atopic dermatitis (AD) is a chronic inflammatory skin disease, which is accompanied by marked increases in the levels of inflammatory cells, including mast cells and eosinophils as well as T cells and macrophages. To investigate the expression pattern of chemokines in AD, a house dust mite, Dermatophagoides farinae extracts (DfE)-induced NC/Nga AD model was developed in mice, and this model was used to determine the expression levels of chemokines in atopic lesions using DNA microarrays and RT-PCR. When NC/Nga mice were repeatedly treated with DfE for 4 to 7 weeks on the back skin, the mRNA expression levels of CCL20/LARC, CCL24/eotaxin-2, CCL17/TARC, and CCL11/eotaxin-1 were markedly induced and lesser of CCL2/MCP-1, within the inflammatory lesion of the back skin. Immunohistochemical staining revealed the expression of these chemokines in the epidermis and dermis of DfE-treated NC/Nga mice. Interestingly, repeated application of tacrolimus ointment potently inhibited DfE-induced atopic dermatitis in NC/Nga mice concomitant with the inhibition of these changes in chemokine gene and protein expression levels particularly of CCL20/LARC, CCL17/TARC, and CCL11/eotaxin-1. These data indicate that severe atopic dermatitis induced by DfE accompanies elevated chemokine levels, and it was proposed that tacrolimus ointment is beneficial for the treatment of severe AD.

Mutation of Y925F in focal adhesion kinase (FAK) suppresses melanoma cell proliferation and metastasis.

This study focused on the role of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase important for many cellular processes, in the proliferation, adhesion, and invasion of melanoma cells in vitro and in vivo. We found that the Y925F-mutation of FAK in B16F10 melanoma cells suppressed metastasis in an experimental model, which correlated well with decreased extracellular matrix dependent proliferative capability, adhesive, migrational, and invasive capabilities. Transduction of the mutation Y925F resulted in a down-regulation of the phosphorylation of Erk, the expression of VEGF, and the association of FAK with paxillin. The results provide clear evidence that 925Y of FAK is critical for melanoma metastasis and this phosphorylation site will be an anti-metastatic target.