Formation of SUMO3-conjugated chains of MAVS induced by poly(dA:dT), a ligand of RIG-I, enhances the aggregation of MAVS that drives the secretion of interferon-ß in human keratinocytes.

The retinoic-acid inducible gene (RIG)-I is a cytoplasmic pattern recognition receptor that senses single-stranded (ss) or double-stranded (ds) RNA. RIG-I also senses AT-rich dsDNA, poly(dA:dT), through the action of an RNA polymerase III-transcribed RNA intermediate. Upon the binding of an RNA ligand, RIG-I binds to the mitochondrial antiviral-signaling protein (MAVS) and induces the formation of filamentous aggregates of MAVS, leading to the formation of a signaling complex that drives Type I interferon (IFN) responses. In the current study, we investigated the issue of whether the SUMOylation of MAVS induced by poly(dA:dT) affects the aggregation of MAVS in the RIG-I/MAVS pathway in human keratinocytes. Our results show that the poly(dA:dT)-induced secretion of IFN-ß was dependent on RIG-I and MAVS. The inhibition of SUMOylation by Ginkgolic acid or Ubc9 siRNA was found to inhibit the poly(dA:dT)-induced secretion of IFN-ß, suggesting that the SUMOylation is required for the poly(dA:dT)-activated RIG-I/MAVS pathway, which drives the secretion of IFN-ß. In addition, treatment with poly(dA:dT) enhanced the formation of polymeric chains of small-ubiquitin like modifiers (SUMO)3, but not SUMO1 and SUMO2, on MAVS. Our results also show that the conjugation of SUMO3 to MAVS induced by poly (dA:dT) enhanced the aggregation of MAVS. These collective results show that the formation of SUMO3-conjugated chains of MAVS induced by poly (dA:dT), a ligand of RIG-I, enhances the aggregation of MAVS which, in turn, drives the secretion of IFN-ß in human keratinocytes.

Quercetin inhibits the poly(dA:dT)-induced secretion of IL-18 via down-regulation of the expressions of AIM2 and pro-caspase-1 by inhibiting the JAK2/STAT1 pathway in IFN-γ-primed human keratinocytes.

Quercetin, a polyphenol, belongs to a class of flavonoids that exerts anti-inflammatory effects. Interleukin (IL)-18 is a member of the IL-1 family cytokine that regulates immune responses and is implicated in various inflammatory skin diseases. Absent in melanoma 2 (AIM2) is a cytosolic double-stranded (ds) DNA sensor that recognizes the dsDNA of a microbial or host origin. Binding of dsDNA to AIM2 simulates caspase-1-dependent inflammasome activity, which leads to the production of IL-1ß and IL-18. Increased levels of AIM2 have been observed in patients with inflammatory skin diseases. In the current study, we investigated the issue of whether or how Quercetin attenuates poly (dA:dT), a synthetic analog of microbial dsDNA, -induced IL-18 secretion in IFN-γ-primed human keratinocytes. Treatment with 5 and 10 µM of Quercetin inhibited the poly (dA:dT)-induced secretion of IL-18 after IFN-γ priming and before poly (dA:dT)-induced AIM2 activation. In addition, treatment with Quercetin at 10 µM, significantly inhibited the phosphorylation of JAK2 and STAT1, and the nuclear translocation of phosphorylated STAT1 in poly (dA:dT)-treated and IFN-γ-primed keratinocytes. These results suggest that treatment with Quercetin inhibits the poly (dA:dT)-induced secretion of IL-18 via down-regulation of the expressions of AIM2 and pro-caspase-1 by inhibiting the JAK2/STAT1 pathway in IFN-γ-primed keratinocytes.

Palmitate induces RIP1/RIP3-dependent necrosis via MLKL-mediated pore formation in the plasma membrane of RAW 264.7 cells.

We previously reported that palmitate induces receptor-interacting protein (RIP)1-dependent necrosis in RAW 264.7 macrophage cells. In response to death receptor stimuli, RIP1 is reported to activate RIP3, which causes the phosphorylation and translocation of mixed-lineage kinase domain-like (MLKL) protein to the plasma membrane, subsequent pore formation in the plasma membrane, and necrotic cell death. In the current study, we investigated the role of MLKL in palmitate-induced, RIP1/RIP3-dependent necrotic cell death in RAW 264.7 cells. The down-regulation of RIP1 or RIP3 by siRNA transfection protected the cells from palmitate-induced cell death. In addition, MLKL was phosphorylated at the serine residue and translocated to the plasma membrane in palmitate-treated cells. In these cells, MLKL was observed as aggregate dots on the plasma membrane. The findings also show that palmitate induced the formation of pores with varied shapes and sizes, and an increase in propidium iodide (PI) uptake and lactate dehydrogenase (LDH) release. Furthermore, the down-regulation of MLKL by siRNA transfection significantly decreased palmitate-induced PI uptake and LDH release, resulting in protection against palmitate-induced necrotic cell death. The findings reported here indicate that palmitate induces RIP1/RIP3-dependent necrosis via MLKL-mediated pore formation of RAW 264.7 cells in the plasma membrane, which could provide a new mechanism to explain the link between elevated levels of free fatty acids (FFAs), palmitate in particular, and macrophage death.

Epigallocatechin-3-gallate attenuates the AIM2-induced secretion of IL-1ß in human epidermal keratinocytes.

The pro-inflammatory cytokine interleukin-1ß (IL-1ß) plays a central role in the pathogenesis of psoriasis. Keratinocytes are a major source of IL-1ß and express absent in melanoma 2 (AIM2). AIM2 recognizes a double-stranded DNA and initiates the IL-1ß-processing of inflammasome. The AIM2 inflammasome is a cytosolic multiprotein complex composed of AIM2, an apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1. Epigallocatechin-3-Gallate (EGCG), a major polyphenolic component of green tea, has anti-inflammatory properties. In the current study, we investigated the issue of whether or how EGCG suppresses AIM2 inflammasome in human epidermal keratinocytes, neonatal (HEKn). Treatment with EGCG, before or after IFN-γ priming, attenuated poly(dA:dT)-induced IL-1ß secretion in HEKn cells. Pre-treatment with EGCG reduced the level of IFN-γ-induced priming signal via the down-regulation of pro-IL-1ß and pro-capspase-1 in HEKn cells. Furthermore, treatment with EGCG attenuated poly(dA:dT)-induced ASC oligomerization and caspase-1 activation in IFN-γ-primed HEKn cells. These results suggest that EGCG attenuates AIM2-induced IL-1ß secretion by suppressing both IFN-γ-mediated priming and poly(dA:dT)-induced ASC oligomerization of inflammasomes in human epidermal keratinocytes.

Palmitate induces cisternal ER expansion via the activation of XBP-1/CCTα-mediated phospholipid accumulation in RAW 264.7 cells.

BACKGROUND: Endoplasmic reticulum (ER) stress induces ER expansion. The expansion of the intracisternal space of the ER was found in macrophages associated with human atherosclerotic lesions. We also previously reported that palmitate induces cisternal ER expansion and necrosis in RAW 264.7 cells. In this study, we report on an investigation of the likely mechanism responsible for this palmitate-induced cisternal ER expansion in a mouse macrophage cell line, RAW 264.7 cells. METHODS: RAW 264.7 cells were pre-treated with the designated inhibitor or siRNA, followed by treatment with palmitate. Changes in the ER structure were examined by transmission electron microscopy. The induction of ER stress was confirmed by an increase in the extent of phosphorylation of PERK, the expression of BiP and CHOP, and the splicing of XBP-1 mRNA. Phospholipid staining was performed with the LipidTOX Red phospholipidosis detection reagent. Related gene expressions were detected by quantitative real time-RT-PCR or RT-PCR. RESULTS: Palmitate was found to induce ER stress and cisternal ER expansion. In addition, palmitate-induced cisternal ER expansion was attenuated by ER stress inhibitors, such as 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid (TUDCA). The findings also show that palmitate induced-mRNA expression of CCTα, which increases phospholipid synthesis, was attenuated by the down-regulation of XBP-1, a part of ER stress. Furthermore, palmitate-induced phospholipid accumulation and cisternal ER expansion were attenuated by the down-regulation of XBP-1 or CCTα. CONCLUSIONS: The findings reported herein indicate that palmitate-induced cisternal ER expansion is dependent on the activation of XBP-1/CCTα-mediated phospholipid accumulation in RAW 264.7 cells.

The Effect of (-)-Epigallocatechin-3-Gallate on IL-1ß Induced IL-8 Expression in Orbital Fibroblast from Patients with Thyroid-Associated Ophthalmopathy.

Orbital fibroblasts have been reported to be an important effector cells for the development of thyroid-associated ophthalmopathy (TAO). Orbital fibroblasts secrete various inflammatory cytokines in response to an inflammatory stimulation, leading to TAO-related tissue swelling. It has also been reported that (-)-epigallocatechin-3-gallate (EGCG), a major polyphenolic constituent of green tea, has antioxidant and anti-inflammatory properties. In the current study, we investigated the issue of whether or how EGCG affects the interleukin (IL)-1ß-induced secretion of IL-8 in human orbital fibroblasts from TAO patients. Treatment with EGCG significantly reduced the level of IL-1ß-induced secretion of IL-8 and the expression of IL-8 mRNA. IL-1ß-induced the degradation of IκBα, and the phosphorylation of p38 and ERK, and the IL-1ß-induced expression of IL-8 mRNA was inhibited by specific inhibitors, such as BAY-117085 for NF-kB, SB203580 for p38, and PD98059 for ERK. In addition, treatment with EGCG inhibited the IL-1ß-induced degradation of IκBα, and the phosphorylation of p38 and ERK. However, pre-treatment with antioxidants, NVN and NAC, which suppressed ROS generation, did not reduce IL-8 expression in IL-1ß-treated orbital fibroblasts, suggesting that the IL-1ß-induced IL-8 expression is not mediated by the generation of ROS. These results show that EGCG suppresses the IL-1ß-induced expression of IL-8 through inhibition of the NF-κB, p38, and ERK pathways. These findings could contribute to the development of new types of EGCG-containing pharmacological agents for use in the treatment of TAO.

PDGF-BB Enhances the Proliferation of Cells in Human Orbital Fibroblasts by Suppressing PDCD4 Expression Via Up-Regulation of microRNA-21.

PURPOSE: The aim of this study was to investigate the effect of platelet-derived growth factor (PDGF)-BB on the proliferation of cells and its possible mechanism in human orbital fibroblasts. METHODS: Human orbital fibroblasts were obtained from orbital fat from decompression surgery in patients with thyroid-associated ophthalmopathy (TAO). The cells were treated with PDGF-BB, and the number of cells was counted using an Advanced Detection and Accurate Measurement (ADAM) automatic cell counter. The expression of programmed cell death 4 (PDCD4) was determined by Western blotting. The effect of PDCD4 on cell proliferation was evaluated using PDCD4 small interfering RNA (siRNA)-transfected cells. The level of microRNA-21 (miRNA-21) was measured by quantitative real-time RT-PCR. In addition, the role of miRNA-21 in the proliferation of PDGF-BB-treated cells was assessed by means of anti-miRNA-21 siRNA and resveratrol (trans-3,4',5-trihydroxys-tilbene), an inhibitor of miRNA-21. RESULTS: PDGF-BB was found to enhance cell proliferation, whereas it inhibited PDCD4 expression in human orbital fibroblasts. Down-regulation of PDCD4 by PDCD4 siRNA transfection significantly increased the number of human orbital fibroblasts. In addition, PDGF-BB increased the level of miRNA-21 in human orbital fibroblasts. Transfection with anti-miRNA-21 and treatment with resveratrol partially restored the expression of PDCD4 and led to a reduction in cell number in PDGF-BB-treated orbital fibroblasts. CONCLUSIONS: PDGF-BB enhances proliferation by suppressing PDCD4 expression by up-regulation of miRNA-21 in human orbital fibroblasts. These results suggest that PDGF-BB stimulates cell proliferation through microRNA-21-mediated PDCD4 down-regulation, leading to the development of TAO.

Palmitate induces COX-2 expression via the sphingolipid pathway-mediated activation of NF-κB, p38, and ERK in human dermal fibroblasts.

It has been suggested that free fatty acids (FFA) such as palmitate, which are secreted from enlarged adipocytes in the subcutaneous fat of obese subjects, serve as a link between obesity and altered skin functions. Cyclooxygenease-2 (COX-2) and prostanoids participate in the induction of impaired dermal function. In the current study, we investigated the issue of whether palmitate induces COX-2 expression via the sphingolipid pathway-mediated activation of NF-κB or mitogen-activated protein kinase (MAPK) pathways in human dermal fibroblasts. Palmitate treatment significantly induced COX-2 expression and prostaglandin E2 (PGE2) release in human dermal fibroblasts. In addition, pre-treatment with triacsin C, an inhibitor of acyl-CoA synthetase in de novo ceramide synthesis, was found to reduce palmitate-induced COX-2 expression and PGE2 release in human dermal fibroblast. The findings also show that palmitate-induced COX-2 expression and PGE2 release are mediated by the NF-κB, p38, and extracellular signal-regulated kinase (ERK) MAPK pathways. These findings point to a new mechanism for explaining the link between increased FFAs in obesity and impaired dermal function.