C10orf99/GPR15L Regulates Proinflammatory Response of Keratinocytes and Barrier Formation of the Skin.

The epidermis, outermost layer of the skin, forms a barrier and is involved in innate and adaptive immunity in an organism. Keratinocytes participate in all these three protective processes. However, a regulator of keratinocyte protective responses against external dangers and stresses remains elusive. We found that upregulation of the orphan gene 2610528A11Rik was a common factor in the skin of mice with several types of inflammation. In the human epidermis, peptide expression of G protein-coupled receptor 15 ligand (GPR15L), encoded by the human ortholog C10orf99, was highly induced in the lesional skin of patients with atopic dermatitis or psoriasis. C10orf99 gene transfection into normal human epidermal keratinocytes (NHEKs) induced the expression of inflammatory mediators and reduced the expression of barrier-related genes. Gene ontology analyses showed its association with translation, mitogen-activated protein kinase (MAPK), mitochondria, and lipid metabolism. Treatment with GPR15L reduced the expression levels of filaggrin and loricrin in human keratinocyte 3D cultures. Instead, their expression levels in mouse primary cultured keratinocytes did not show significant differences between the wild-type and 2610528A11Rik deficient keratinocytes. Lipopolysaccharide-induced expression of Il1b and Il6 was less in 2610528A11Rik deficient mouse keratinocytes than in wild-type, and imiquimod-induced psoriatic dermatitis was blunted in 2610528A11Rik deficient mice. Furthermore, repetitive subcutaneous injection of GPR15L in mouse ears induced skin inflammation in a dose-dependent manner. These results suggest that C10orf99/GPR15L is a primary inducible regulator that reduces the barrier formation and induces the inflammatory response of keratinocytes.

Cutaneous p38 mitogen-activated protein kinase activation triggers psoriatic dermatitis.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease characterized by IL-17-mediated immune responses. p38 is known to be highly activated in the psoriatic epidermis; however, whether p38 is involved in the development of psoriasis is unclear. OBJECTIVE: We sought to demonstrate that activation of p38 mitogen-activated protein kinase is sufficient to induce psoriatic inflammation in mice and that cutaneous p38 activities are the topical therapeutic targets for psoriasis. METHODS: A p38 activator, anisomycin, was applied daily to murine skin. Transcriptomic analyses were performed to evaluate the similarities of the skin responses to those in human psoriasis and the existing animal model. BIRB796, a small-molecule inhibitor targeting p38 activities, was applied to the murine psoriatic models topically or to human psoriatic skin specimens ex vivo. RESULTS: Topical treatment with anisomycin induced key signatures in psoriasis, such as epidermal thickening, neutrophil infiltration, and gene expression of Il1a, Il1b, Il6, Il24, Cxcl1, Il23a, and Il17a, in treated murine skin. These responses were fully abrogated by topical treatment with BIRB796, and were reduced in IL-17A-deficient mice. Transcriptomic analyses demonstrated the similarities of anisomycin-induced dermatitis to human psoriasis and imiquimod-induced murine psoriatic dermatitis. Furthermore, BIRB796 targeting of p38 activities reduced expression of psoriasis-related genes in both human keratinocytes stimulated with recombinant IL-17A in vitro and psoriatic skin specimens ex vivo. CONCLUSION: Therefore our findings suggest that cutaneous p38 activation can be a key event in patients with psoriasis and a potential topical therapeutic target of a small molecule.

Immune Control by TRAF6-Mediated Pathways of Epithelial Cells in the EIME (Epithelial Immune Microenvironment).

In the protective responses of epithelial tissues, not only immune cells but also non-immune cells directly respond to external agents. Epithelial cells can be involved in the organization of immune responses through two phases. First, the exogenous harmful agents trigger the primary responses of the epithelial cells leading to various types of immune cell activation. Second, cytokines produced by the immune cells that are activated directly by the external agents and indirectly by the epithelial cell products elicit the secondary responses giving rise to further propagation of immune responses. TRAF6 is a ubiquitin E3 ligase, which intermediates between various types of receptors for exogenous agents or endogenous mediators and activation of subsequent transcriptional responses via NF-kappaB and MAPK pathways. TRAF6 ubiquitously participates in many protective responses in immune and non-immune cells. Particularly, epithelial TRAF6 has an essential role in the primary and secondary responses via driving type 17 response in psoriatic inflammation of the skin. Consistently, many psoriasis susceptibility genes encode the TRAF6 signaling players, such as ACT1 (TRAF3IP2), A20 (TNFAIP3), ABIN1 (TNIP1), IL-36Ra (IL36RN), IkappaBzeta (NFKBIZ), and CARD14. Herein, we describe the principal functions of TRAF6, especially in terms of positive and regulatory immune controls by interaction between immune cells and epithelial cells. In addition, we discuss how TRAF6 in the epithelial cells can organize the differentiation of immune responses and drive inflammatory loops in the epithelial immune microenvironment, which is termed EIME.

Epithelial TRAF6 drives IL-17-mediated psoriatic inflammation.

Epithelial cells are the first line of defense against external dangers, and contribute to induction of adaptive immunity including Th17 responses. However, it is unclear whether specific epithelial signaling pathways are essential for the development of robust IL-17-mediated immune responses. In mice, the development of psoriatic inflammation induced by imiquimod required keratinocyte TRAF6. Conditional deletion of TRAF6 in keratinocytes abrogated dendritic cell activation, IL-23 production, and IL-17 production by γδ T cells at the imiquimod-treated sites. In contrast, hapten-induced contact hypersensitivity and papain-induced IgE production were not affected by loss of TRAF6. Loss of psoriatic inflammation was not solely due to defective imiquimod sensing, as subcutaneous administration of IL-23 restored IL-17 production but did not reconstitute psoriatic pathology in the mutant animals. Thus, TRAF6 was required for the full development of IL-17-mediated inflammation. Therefore, epithelial TRAF6 signaling plays an essential role in both triggering and propagating IL-17-mediated psoriatic inflammation.

Possible roles of tumor necrosis factor-α and angiotensin II type 1 receptor on high glucose-induced damage in renal proximal tubular cells.

Recent studies have identified that high glucose-induced renal tubular cell damage. We previously demonstrated that high glucose treatment induced oxidative stress in human renal proximal tubular epithelial cells (RPTECs), and angiotensin II type 1 (AT1) receptor blockers reduce high glucose-induced oxidative stress in RPTEC possibly via blockade of intracellular as well as extracellular AT1 receptor. However, exact roles of tumor necrosis factor (TNF)-α and AT1 receptor on high glucose-induced renal tubular function remain unclear. N-acetyl-beta-glucosaminidase (NAG), concentrations of TNF-α/angiotensin II and p22(phox) protein levels after high glucose treatment with or without AT1 receptor blocker or thalidomide, an inhibitor of TNF-α protein synthesis, were measured in immortalized human renal proximal tubular epithelial cells (HK2 cells). AT1 receptor knockdown was performed with AT1 receptor small interfering RNA (siRNA). High glucose treatment (30 mM) significantly increased NAG release, TNF-α/angiotensin II concentrations in cell media and p22(phox) protein levels compared with those in regular glucose medium (5.6 mM). Candesartan, an AT1R blocker, showed a significant reduction on high glucose-induced NAG release, TNF-α concentrations and p22(phox) protein levels in HK2 cells. In addition, significant decreases of NAG release, TNF-α concentrations and p22(phox) protein levels in HK2 cells were observed in high glucose-treated group with thalidomide. AT1R knockdown with siRNA markedly reversed high glucose, angiotensin II or TNF-α-induced p22(phox) protein levels in HK2 cells. TNF-α may be involved in high glucose-induced renal tubular damage in HK2 cells possibly via AT1 receptor signaling.

Polychlorinated biphenyl (PCB) concentrations and congener composition in masu salmon from Japan: a study of all 209 PCB congeners by high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS).

We collected two subspecies of masu salmon: Oncorhynchus masou masou from four localities (southern Sea of Japan northward to Hokkaido) and O. masou ishikawae from upstream from Ise Bay close to a heavy industrial area. All 209 PCB congeners were analyzed using HRGC/HRMS. PCA ordination of congener concentrations divided data into three groups: (i) ssp. masou from Hokkaido, (ii) ssp. masou from the other regions and (iii) ssp. ishikawae. The highest ∑ PCB concentration (40.39 ng/wet wt) was in ssp. ishikawae followed by ssp. masou from southern waters; however the TEQdioxin-like PCBs was highest in ssp. masou from southern water (1.96 pg-TEQdioxin-like PCBs/g wet wt.) due to the high proportion of congener #126 in its complement (#126 has the highest toxic equivalency factor among congeners). There is likely a contamination source offshore in the southern Sea of Japan and/or along the migratory route of ssp. masou.

Initial performance evaluation of iterative model reconstruction in abdominal computed tomography.

OBJECTIVE: The aim of this study was to present initial evaluation of the performance of the iterative model reconstruction algorithm (IMR) in abdominal computed tomography (CT). METHODS: Computed tomographic examinations were performed for clinical study of 36 patients and for phantom study. We reconstructed the raw data with 1.0- and 5.0-mm slice thicknesses using filtered back projection (FBP), iDose4, and IMR and evaluated image quality objectively and subjectively. RESULTS: For almost all subjective characteristics, the image quality was better using IMR than iDose4. Objective image noise was significantly less using IMR than iDose4 (P < 0.0001). The contrast-noise ratio of both slice thicknesses increased in order from FBP to iDose4 to IMR. The spatial resolution of reconstructed images was almost identical using IMR, FBP, and iDose4. CONCLUSIONS: The IMR can significantly improve image noise and low-contrast resolution and maintain edge sharpness in abdominal CT images compared with iDose4 or FBP.

MR findings of ruptured endometrial cyst: comparison with tubo-ovarian abscess.

OBJECTIVE: To evaluate the MR findings of ruptured endometrial cyst, focusing on the differentiation from tubo-ovarian abscess (TOA). PATIENTS AND METHODS: We reviewed the records of 21 patients who underwent preoperative MR examinations for TOAs (n=15) or ruptured endometrial cysts (n=6). We evaluated the presence of hyper-intense ascites and hyper-intense peritoneum in T1-weighted sequences, strong enhancement of the peritoneum, hyper-intense content and hyper-intense rim of the ovarian lesion in T1-weighted sequences, and strong wall enhancement of the ovarian lesion. χ2 test was used to assess the relationship between TOA cases versus cases with ruptured endometrial cysts, and the three MR peritoneal findings. We evaluated the relationship between TOA versus non-infected endometrial cysts, and the ovarian MR peritoneal findings, too. RESULTS: Hyper-intense ascites was found in all of the patients with ruptured endometrial cyst and none with TOA (p<0.0001). Hyper-intense peritoneum was observed in only TOAs cases (4 of 8). Strong peritoneal enhancement was seen in 3 of the 3 patients with ruptured endometrial cyst and 7 of the 13 patients with TOA (p=0.1366). Hyper-intense content of the ovarian lesion was seen more often in the non-infected endometrial cysts than in the TOAs (p=0.001607), while hyper-intense rim was more frequent in TOAs (p=0.000402). Strong wall enhancement was observed only in TOAs (11 of 15) (p=0.001355). CONCLUSIONS: MR images are useful to differentiate ruptured endometrial cyst from TOA.

Possible involvement of intracellular angiotensin II receptor in high-glucose-induced damage in renal proximal tubular cells.

BACKGROUND: Recent studies have identified high glucose as a potent stimulus for the intracellular synthesis of angiotensin II. However, the exact roles of angiotensin II and angiotensin II type 1 receptor blockers (ARB) in high-glucose-induced renal tubular function remain unclear. METHODS: N-Acetyl-beta-glucosaminidase (NAG), angiotensin II and 8-hydroxy-2'-deoxyguanosine (8-OHdG) concentrations in renal proximal tubular epithelial cells (RPTECs) with or without high glucose/ARB were determined using a modified commercial procedure. The changes of p22phox and cytoplasmic inhibitory kappa B (IkB) protein levels in RPTECs were measured using Western blotting. RESULTS: High-glucose treatment (4x10-2 mol/L) significantly increased NAG release, angiotensin II concentrations in cell lysates and 8-OHdG and p22phox protein levels compared with those in regular glucose medium (1.75x10(-2) mol/L). ARBs (candesartan, olmesartan or valsartan; 1x10(-9)-10(-7) mol/L) showed a significant reduction in high-glucose-induced NAG, 8-OHdG and p22phox protein levels in RPTECs. Significant decreases of cytoplasmic IkB protein levels were observed in the high-glucose-treated group in RPTECs. ARBs markedly reversed high-glucose-induced reduction of IkB protein levels in RPTECs. CONCLUSIONS: ARBs reduce high-glucose-induced oxidative stress in RPTECs possibly via blockade of intracellular as well as extracellular AT1 receptor signaling, which possibly protects renal tubular cell function during diabetic nephropathy.

Soft-tissue perineurioma of the retroperitoneum in a 63-year-old man, computed tomography and magnetic resonance imaging findings: a case report.

INTRODUCTION: Soft-tissue perineuriomas are rare benign peripheral nerve sheath tumors in the subcutis of the extremities and the trunks of young patients. To our knowledge, this the first presentation of the computed tomography and magnetic resonance imaging of a soft-tissue perineurioma in the retroperitoneum with pathologic correlation. CASE PRESENTATION: A 63-year-old Japanese man was referred for assessment of high blood pressure. Abdominal computed tomography and magnetic resonance imaging showed a well-defined, gradually enhancing tumor without focal degeneration or hemorrhage adjacent to the pancreatic body. Tumor excision with distal pancreatectomy and splenectomy was performed, as a malignant tumor of pancreatic origin could not be ruled out. No recurrence has been noted in the 16 months since the operation. Pathologic examination of the tumor revealed a soft-tissue perineurioma of the retroperitoneum. CONCLUSION: Although the definitive diagnosis of soft-tissue perineurioma requires biopsy and immunohistochemical reactivity evaluation, the computed tomography and magnetic resonance imaging findings described in this report suggest inclusion of this rare tumor in the differential diagnosis when such findings occur in the retroperitoneum.

Regulation of glucocorticoid receptor transcription and nuclear translocation during single and repeated immobilization stress.

We have previously reported reduced glucocorticoid receptor (GR) mRNA levels in the hippocampus and hypothalamic paraventricular nucleus (PVN) during repeated immobilization, which is potentially associated with persistent activation of the hypothalamic-pituitary-adrenocortical axis. We used in situ hybridization and Western blot to examine the transcriptional regulation of the GR gene, GR nuclear translocation, and expression of cytosolic heat shock protein 90 (hsp90), a chaperone protein essential for GR nuclear translocation, in the hippocampus, PVN, and anterior pituitary (AP) during single immobilization (sIMO) and the final immobilization on d 7 after daily IMO for 6 days (rIMO). As with GR mRNA, GR heteronuclear RNA levels decreased in the hippocampus and PVN and increased in the AP during sIMO and rIMO, indicating that the GR mRNA levels in these regions were regulated at the transcriptional level. In both sIMO and rIMO, nuclear GR levels were significantly increased in the hippocampus, medial basal hypothalamus (MBH), and AP. However, GR nuclear translocation was reduced in the hippocampus, unchanged in the MBH, and enhanced in the AP during rIMO, as compared with sIMO. Cytosolic hsp90 expression was unchanged in the hippocampus and MBH, whereas it significantly increased in the AP at 30 min during rIMO but not during sIMO. These results suggest that the site-specific changes in GR nuclear translocation during sIMO vs. rIMO are partially linked to hsp90 responses to immobilization. The reduced nuclear translocation of GR in the hippocampus during rIMO may reflect decreased glucocorticoid-mediated negative feedback on the hypothalamic-pituitary-adrenocortical axis.

Characterization of GPIT-1 and GPIT-2, two auxiliary components of the Neurospora crassa GPI transamidase complex.

The glycosylphosphatidylinositol (GPI) transamidase contains five known subunits and functions in the lumen of the ER to produce GPI-anchored proteins. The transamidase cleaves proteins containing a GPI anchor attachment signal at their C terminus and generates an amide bond between the newly generated carboxyl terminus of the protein and a GPI anchor. We have identified and characterized GPIT-1 and GPIT-2, two of the transamidase subunits from Neurospora crassa. GPIT-1 and GPIT-2 are homologs of the human PIG-T and PIG-U transamidase subunits respectively. We demonstrated that GPIT-2 is required for the addition of GPI anchors onto GPI-anchored proteins. We employed the Neurospora RIP (repeat-induced point mutation) phenomenon to generate 106 "noncritical" amino acid changes in GPIT-1 and 84 "noncritical" amino acid changes in GPIT-2. We used the data to evaluate three-dimensional models for the structures of GPIT-1 and GPIT-2. The mutational data for GPIT-1 is consistent with a multiple-blade propeller structure containing a central channel. The mutational analysis for GPIT-2 supports a structural model based on the karyopherin alpha subunit.

Effects of angiotensin II type 1 receptor blocker on albumin-induced cell damage in human renal proximal tubular epithelial cells.

BACKGROUND/AIMS: Proteinuria is not merely a marker of chronic nephropathies, but may also be involved in the progression to end-stage renal failure. We investigated the effect of angiotensin II type 1 receptor blockers (ARBs) on albumin-induced cell damage in human renal proximal tubular epithelial cells (RPTEC). METHODS: The N-acetyl-beta-D-glucosaminidase (NAG) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in the medium after albumin treatment with ARBs were determined by commercially available kits. The levels of p22(phox) protein in RPTEC were measured using Western blotting after albumin treatment with ARBs. Angiotensin II concentrations in cell media and cell lysates were assayed with a commercially available kit. RESULTS: Human albumin (0.1-10 mg/ml) dose-dependently increased NAG release and olmesartan or valsartan (10(-9)-10(-7) mol/l) showed a significant reduction on albumin (1 mg/ml)-induced NAG release in RPTEC. Albumin treatment (1 mg/ml) showed significant increases in p22(phox) protein levels in RPTEC and ARBs significantly decreased albumin-induced p22(phox) protein levels. Significant increases in 8-OHdG levels were observed in the albumin (1 mg/ml)-treated group and ARBs markedly reduced albumin-induced 8-OHdG levels in RPTEC. Human albumin dose-dependently increased angiotensin II concentrations in both cell media and lysates. CONCLUSION: These observations suggest renal tubular cell-protective properties of ARBs related to decreased oxidative stress during proteinuria.

Angiotensin II receptor blocker inhibits tumour necrosis factor-alpha-induced cell damage in human renal proximal tubular epithelial cells.

AIM: We investigated the effect of angiotensin II (AII) type 1 (AT1) and angiotensin II type 2 (AT2) receptor blockers on tumour necrosis factor alpha (TNF-alpha)-induced cell damage in human renal proximal tubular epithelial cells (RPTEC). METHODS: The lactate dehydrogenase (LDH) and N-acetyl-beta-glucosaminidase (NAG) release into the medium after TNF-alpha treatment in RPTEC were determined using modified commercial procedures. In addition, the levels of caspase 3/7 activity in RPTEC were measured after TNF-alpha treatment with AlphaTau1 or AT2 receptor blockers. Finally we investigated the change of p22phox protein levels after TNF-alpha with AlphaTau1 or AT2 receptor blockers in RPTEC. RESULTS: Tumour necrosis factor alpha (10(-8) mol/L) significantly increased LDH and NAG release into the medium from RPTEC. AlphaTau1 receptor blockers, olmesartan and valsartan (10(-9)-10(-6) mol/L) showed a significant reduction on TNF-alpha-induced LDH and NAG release in RPTEC. AT2 receptor blocker, PD123319 (10(-7)-10(-5) mol/L) also decreased TNF-alpha-induced LDH and NAG release in RPTEC. Blockade of both AlphaTau1 and AT2 receptor indicated additional reduction on TNF-alpha-induced LDH and NAG release. TNF-alpha (10(-8) mol/L) treatment showed small but significant increases of caspase 3/7 activity in RPTEC, and AT1 and AT2 receptor blockers (10(-8) mol/L) comparably decreased TNF-alpha-induced caspase 3/7 activity. Significant increases of p22phox protein levels were observed in TNF-alpha-treated group in RPTEC. However, only AlphaTau1 (10(-8) mol/L) but not AT2 (10(-5) mol/L) receptor blocker significantly decreased TNF-alpha-induced p22phox protein levels. CONCLUSION: The present study demonstrates that TNF-alpha induces renal tubular cell damage in RPTEC and AT1/AT2 receptor blockers showed cytoprotective effects probably via at least partly different mechanism.

Dermal matrix proteins initiate re-epithelialization but are not sufficient for coordinated epidermal outgrowth in a new fish skin culture model.

We have established a new culture system to study re-epithelialization during fish epidermal wound healing. In this culture system, fetal bovine serum (FBS) stimulates the epidermal outgrowth of multi-cellular layers from scale skin mounted on a coverslip, even when cell proliferation is blocked. The rate of outgrowth is about 0.4 mm/h, and at 3 h after incubation, the area occupied by the epidermal sheet is nine times larger than the area of the original scale skin. Cells at the bottom of the outgrowth show a migratory phenotype with lamellipodia, and "purse string"-like actin bundles have been found over the leading-edge cells with polarized lamellipodia. In the superficial cells, re-development of adherens junctions and microridges has been detected, together with the appearance and translocation of phosphorylated p38 MAPK into nuclear areas. Thus, this culture system provides an excellent model to study the mechanisms of epidermal outgrowth accompanied by migration and re-differentiation. We have also examined the role of extracellular matrix proteins in the outgrowth. Type I collagen or fibronectin stimulates moderate outgrowth in the absence of FBS, but development of microridges and the distribution of phosphorylated p38 MAPK are attenuated in the superficial cells. In addition, the leading-edge cells do not have apparent "purse string"-like actin bundles. The outgrowth stimulated by FBS is inhibited by laminin. These results suggest that dermal substrates such as type I collagen and fibronectin are able to initiate epidermal outgrowth but require other factors to enhance such outgrowth, together with coordinated alterations in cellular phenotype.

The CARMA1-Bcl10 signaling complex selectively regulates JNK2 kinase in the T cell receptor-signaling pathway.

Members of the c-Jun NH(2)-terminal kinase (JNK) family play crucial roles in cell activation, differentiation, and apoptosis. Although many studies have indicated that JNK1 and JNK2 have functional differences and redundancy, the upstream signaling pathway that selectively activates JNK1 or JNK2 remains unknown. In this study, we have revealed a selective mechanism of JNK activation, in which JNK2, but not JNK1, was regulated by CARMA1, a scaffold molecule, after stimulation of the T cell receptor (TCR). This CARMA1-dependent regulation of JNK2 worked through the scaffold molecule Bcl10, which was inducibly associated with JNK2 and served as a JNK-interacting protein (JIP)-like scaffold to assemble the kinases JNK2, MKK7, and TAK1. Finally, we showed that CARMA1- and Bcl10-mediated JNK2 activation had a critical role in regulating the amount of c-Jun protein. Together, our studies provide genetic evidence that JNK1 and JNK2 are differentially regulated in the TCR-signaling pathway and play different functions.