BC-1215 inhibits ATP-induced IL-1ß secretion via the FBXL2-mediated ubiquitination and degradation of not only NLRP3, but also pro-IL-1ß in LPS-primed THP-1 cells.

BC-1215, bis-pyridinyl benzyl ethanediamine, is an inhibitor of F-box only protein 3 (FBXO3) and exerts anti-inflammatory effects. BC-1215 inhibits interactions between FBXO3-F-box and the leucine rich repeat protein 2 (FBXL2), leading to the upregulation of FBXL2 expression, FBXL2-mediated ubiquitination and the degradation of tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) or NOD-, LRR- and the pyrin domain-containing protein 3 (NLRP3), which subsequently results in the down-regulation of inflammatory cytokine production. In the current study, we investigated the issue of whether or how BC-1215 suppresses the ATP-induced secretion of IL-1ß in LPS-primed human macrophage-like cells, THP-1 cells. Our result show that pre-treatment with BC-1215 attenuated the ATP-induced secretion of IL-1ß in LPS-primed THP-1 cells. Treatment of the LPS-primed THP-1 cells with BC-1215 resulted in a decrease in the level of NLRP3 and pro-IL-1ß at the protein level, but not at the mRNA level. In addition, treatment with MG-132, but not leupeptin, inhibited the BC-1215-induced degradation of NLRP3 and pro-IL-1ß proteins, and restored their levels, suggesting that BC-1215 decreases the stability of NLRP3 and pro-IL-1ß at the protein level via proteasome-dependent degradation. Our results also show that FBXL2, which is increased by BC-1215, bound to and ubiquitinated NLRP3 and pro-IL-1ß, but not pro-caspase-1. These collective results indicate that treatment with BC-1215, an inhibitor of FBXO3, inhibits ATP-induced IL-1ß secretion via the FBXL2-mediated ubiquitination and degradation of pro-IL-1ß as well as NLRP3 in LPS-primed THP-1 cells, suggesting that FBXO3 is a potential therapeutic target for developing agents against inflammatory diseases.

Tear-Derived Exosome Proteins Are Increased in Patients with Thyroid Eye Disease.

Exosomes contain proteins, lipids, RNA, and DNA that mediate intercellular signaling. Exosomes can contribute to the pathological processes of various diseases, although their roles in ocular diseases are unclear. We aimed to isolate exosomes from tear fluids (TF) of patients with Thyroid eye disease (TED) and analyze the exosomal proteins. TFs were collected from eight patients with TED and eight control subjects. The number of TF exosomes were measured using nanoparticle-tracking analysis. The expression of specific proteins in the purified exosome pellets were analyzed using a Proteome Profiler Array Kit. Cultured normal orbital fibroblasts were incubated with TF exosomes from patients with TED and control subjects, and changes in inflammatory cytokine levels were compared. TF exosomes from TED patients showed more exosomes than the control subjects. The expression levels of exosomal proteins vitamin D-binding (VDB) protein, C-reactive protein (CRP), chitinase 3-like 1 (CHI3L1), matrix metalloproteinase-9 (MMP-9), and vascular adhesion molecule-1 (VCAM-1) were significantly increased in patients with TED, compared to those of controls. Orbital fibroblasts exposed to TF exosomes from patients with TED showed significantly higher levels of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) production than those treated with control TF exosomes. Specific proteins showed higher expression in exosomes from TED patients, implying that they may play keys roles in TED pathogenesis.

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.

Hypoxia-induced cancer stemness acquisition is associated with CXCR4 activation by its aberrant promoter demethylation.

BACKGROUND: A hypoxic microenvironment leads to an increase in the invasiveness and the metastatic potential of cancer cells within tumors via the epithelial-mesenchymal transition (EMT) and cancer stemness acquisition. However, hypoxia-induced changes in the expression and function of candidate stem cell markers and their possible molecular mechanism is still not understood. METHODS: Lung cell lines were analyzed in normoxic or hypoxic conditions. For screening among the stem cell markers, a transcriptome analysis using next-generation sequencing was performed. For validation, the EMT and stem cell characteristics were analyzed. To determine whether an epigenetic mechanism was involved, the cell lines were treated with a DNA methyltransferase inhibitor (AZA), and methylation-specific PCR and bisulfite sequencing were performed. RESULTS: Next-generation sequencing revealed that the CXCR4 expression was significantly higher after the hypoxic condition, which functionally resulted in the EMT and cancer stemness acquisition. The acquisition of the EMT and stemness properties was inhibited by treatment with CXCR4 siRNA. The CXCR4 was activated by either the hypoxic condition or treatment with AZA. The methylation-specific PCR and bisulfite sequencing displayed a decreased CXCR4 promoter methylation in the hypoxic condition. CONCLUSIONS: These results suggest that hypoxia-induced acquisition of cancer stem cell characteristics was associated with CXCR4 activation by its aberrant promoter demethylation.

Modeling the Cooperative Adsorption of Solid-Binding Proteins on Silica: Molecular Insights from Surface Plasmon Resonance Measurements.

Combinatorially selected solid-binding peptides (SBPs) provide a versatile route for synthesizing advanced materials and devices, especially when they are installed within structurally or functionally useful protein scaffolds. However, their promise has not been fully realized because we lack a predictive understanding of SBP-material interactions. Thermodynamic and kinetic binding parameters obtained by fitting quartz crystal microbalance and surface plasmon resonance (SPR) data with the Langmuir model whose assumptions are rarely satisfied provide limited information on underpinning molecular interactions. Using SPR, we show here that a technologically useful SBP called Car9 confers proteins to which is fused a sigmoidal adsorption behavior modulated by partner identity, quaternary structure, and ionic strength. We develop a two-step cooperative model that accurately captures the kinetics of silica binding and provides insights into how SBP-SBP interactions, fused scaffold, and solution conditions modulate adsorption. Because cooperative binding can be converted to Langmuir adhesion by mutagenesis, our approach offers a path to identify and to better understand and design practically useful SBPs.

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.

CD11b+Gr-1low cells that accumulate in M.leprae-induced granulomas of the footpad skin of nude mice have the characteristics of monocytic-myeloid-derived suppressor cells.

CD11b+Gr-1low cells that are increased in the lungs of a Mycobacterium (M) tuberculosis-infection mouse model have the characteristics of monocytic (M)-myeloid-derived suppressor cells (MDSCs) and harbor M.tuberculosis. Interestingly, a high number of M-MDSCs have also been observed in skin lesions of patients with lepromatous leprosy. We hypothesized that CD11b+Gr-1low cells might be involved in the pathogenesis of leprosy, as they are in tuberculosis. In the current study, we investigated the issue of whether CD11b+Gr-1low cells accumulate in Mycobacterium (M) leprae-induced granulomas of the footpad skin of nude mice. Our results show that CD11b+Gr-1low cells began to accumulate in the 7-month-old M.leprae-induced granulomas and were replaced by other leukocytes, including CD11b+Gr-1high over time during M.leprae infections. CD11b + Gr-1low cells expressed the surface markers of M-MDSC, Ly6Chigh and Ly6Glow. In addition, CD11b+Gr-1low cells have the nuclei of a mononuclear cell type and expressed higher levels of arginase 1 (Arg1) and inducible NO synthetase (iNOS). Furthermore, they showed a higher infection rate by M.leprae. Taken together, our results indicate that the inoculation with M.leprae induced an accumulation of CD11b + Gr-1low at a relatively early stage, 7-month-old M.leprae-induced granulomas, and that CD11b+Gr-1low have the characteristics of M-MDSC and may act as a reservoir for M.leprae.

Leprosy-specific subsets of macrophages and Schwann cells identified by single-cell RNA-sequencing.

In Mycobacterium leprae (M. leprae)-infection, inflammatory cells' subsets and dynamics as well as the interactions with Schwann cells have remained elusive. We investigated individual cells in M. leprae-inoculated nude mice by single-cell RNA-sequencing (scRNA-seq). For macrophages, we dissected two M1-like subsets and five M2-like subsets, where lipid-associated signatures were pervasive in both M1-like and M2-like subsets. There were four macrophage trajectories showing: (i) pro-inflammatory (M1), (ii) lipid metabolism-related (M2), (iii) anti-inflammatory (M2), and (iv) interferon-stimulated gene-related (M2) fates. They displayed early divergence without ever rejoining along the paths, suggesting simultaneous or continuous stimuli for macrophage activation in leprosy. The scRNA-seq predicted Schwann cell-macrophage interactions (Notch1-Jag1, Plxnb1-Sema4d interactions). An immature Schwann cell subset showing Tfap2a expression was identified, indicating Schwann cell dedifferentiation in leprosy tissues. Expressions of Notch1, Jag1, Plxnb1, Sema4d, and Tfap2a were validated in mouse or human leprosy tissues by immunohistochemistry. We identified both pro-inflammatory and inflammation-resolution signatures, where lipid-associated signatures were pervasive to the macrophages, representing leprosy-specific macrophage states for prolonged and repeated episodes of inflammation and resolution. Our study identified refined molecular states and interactions of macrophages and Schwann cells, suggesting novel insights into the pathogenesis of unhealed inflammation with neuropathy and potential therapeutic targets for leprosy.

Volunteer Plants' Occurrence and the Environmental Adaptability of Genetically Modified Fodder Corn upon Unintentional Release into the Environment.

The number of corn cultivars that have been improved using genetically modified technology continues to increase. However, concerns about the unintentional release of living-modified organisms (LMOs) into the environment still exist. Specifically, there are cases where LMO crops grown as fodder are released into the environment and form a volunteer plant community, which raises concerns about their safety. In this study, we analyzed the possibility of weediness and volunteer plants' occurrence when GMO fodder corn grains distributed in Korea are unintentionally released into the environment. Volunteer plants' occurrence was investigated by directly sowing grains in an untreated field. The results showed that the germination rate was extremely low, and even if a corn seed germinated, it could not grow into an adult plant and would die due to weed competition. In addition, the germination rate of edible and fodder grains was affected by temperature (it was high at 20 °C and 30 °C but low at 40 °C and extremely low at 10 °C), and it was higher in the former than in the latter. And the germination rate was higher in Daehakchal (edible corn grains) than in Gwangpyeongok (fodder corn grains). The environmental risk assessment data obtained in this study can be used for future evaluations of the weediness potential of crops and the development of volunteer plant suppression technology in response to unintentional GMO release.

Generation of double knockout cattle via CRISPR-Cas9 ribonucleoprotein (RNP) electroporation.

BACKGROUND: Genome editing has been considered as powerful tool in agricultural fields. However, genome editing progress in cattle has not been fast as in other mammal species, for some disadvantages including long gestational periods, single pregnancy, and high raising cost. Furthermore, technically demanding methods such as microinjection and somatic cell nuclear transfer (SCNT) are needed for gene editing in cattle. In this point of view, electroporation in embryos has been risen as an alternative. RESULTS: First, editing efficiency of our electroporation methods were tested for embryos. Presence of mutation on embryo was confirmed by T7E1 assay. With first combination, mutation rates for MSTN and PRNP were 57.6% ± 13.7% and 54.6% ± 13.5%, respectively. In case of MSTN/BLG, mutation rates were 83.9% ± 23.6% for MSTN, 84.5% ± 18.0% for BLG. Afterwards, the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep sequencing. Thirteen recipients were transferred for MSTN/PRNP, 4 calves were delivered, and one calf underwent an induction for double KO. Ten surrogates were given double-KO embryos for MSTN/BLG, and four of the six calves that were born had mutations in both genes. CONCLUSIONS: These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively applied. Finally, MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.

Palmitate induced secretion of IL-6 and MCP-1 in orbital fibroblasts derived from patients with thyroid-associated ophthalmopathy.

PURPOSE: Orbital fibroblasts are now recognized as the key effectors in the development of thyroid associated ophthalmopathy (TAO). TAO is clinically apparent in approximately 50% of patients with Graves' hyperthyroidism. High levels of plasma free fatty acids (FFAs) are frequently seen in patients with hyperthyroidism. Palmitate is one of the most abundant FFAs in plasma and aggravates inflammation by promoting secretion of pro-inflammatory cytokines in various cells. In the present study, we characterized orbital fibroblasts from patients with TAO and then examined the effect of palmitate on the production of pro-inflammatory cytokines and hyaluronic acid (HA) in orbital fibroblasts. METHODS: Orbital fat explants were obtained from patients with TAO undergoing orbital decompression surgery (n=5). The fibroblasts were characterized by antibodies specific for fibroblast markers and Thy-1 (cluster differentiation 90, CD90) by immunostaining and flow cytometry. We then investigated the capability of orbital fibroblasts to secrete cytokines and HA in response to interleukin (IL)-1ß using an enzyme-linked immunosorbent assay (ELISA). The effect of palmitate on cytokine and HA production in orbital fibroblasts was examined at the protein level by ELISA and at the mRNA level by quantitative real time RT-PCR. The level of phosphorylation of mitogen-activated protein kinase (MAPK)s, including p38 MAPK (p38), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), was measured by immunoblot analysis. We then examined the role of MAPKs on palmitate-induced cytokine production using specific inhibitors to p38, ERK, and JNK, respectively. RESULTS: The orbital fibroblasts from patients with TAO were Thy-1- positive fibroblasts (>90%) with the ability to secrete IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), and HA in response to IL-1ß. Treatment with palmitate induced significant production of IL-6 and MCP-1, but not IL-8 and HA, in orbital fibroblasts. IL-6 and MCP-1 expression by palmitate were differentially regulated by MAPKs. IL-6 expression was mediated by the p38, ERK, JNK pathways, whereas MCP-1 expression was mediated by ERK and JNK, but not by p38, in palmitate-treated orbital fibroblasts. CONCLUSIONS: We show the possible involvement of palmitate in the promotion of inflammation within orbital tissues. This finding may be helpful for understanding the development of TAO in patients with hyperthyroidism.

Vacuolar H+ -ATPase c protects glial cell death induced by sodium nitroprusside under glutathione-depleted condition.

We examined the role of the c subunit (ATP6L) of vacuolar H(+) -ATPase and its molecular mechanisms in glial cell death induced by sodium nitroprusside (SNP). ATP6L siRNA-transfected cells treated with SNP showed a significant increase in cytotoxicity under glutathione (GSH)-depleted conditions after pretreatment with buthionine sulfoximine, but reduction of ATP6L did not affect the regulation of lysosomal pH in analyses with lysosomal pH-dependent fluorescence probes. Photodegraded SNP and ferrous sulfate induced cytotoxicity with the same pattern as that of SNP, but SNAP and potassium cyanide did not show activity. Pretreatment of the transfected cells with deferoxamine (DFO) reduced ROS production and significantly inhibited the cytotoxicity, which indicates that primarily iron rather than nitric oxide or cyanide from SNP contributes to cell death. Involvement of apoptotic processes in the cells was not shown. Pretreatment with JNK or p38 chemical inhibitor significantly inhibited the cytotoxicity, and we also confirmed that the MAPKs were activated in the cells by immunoblot analysis. Significant increase of LC3-II conversion was observed in the cells, and the conversions were inhibited by cotransfection of the MAPK siRNAs and pretreatment with DFO. Introduction of Atg5 siRNA inhibited the cytotoxicity and inhibited the activation of MAPKs and the conversion of LC3. We finally confirmed autophagic cell death and involvement of MAPKs by observation of autophagic vacuoles via electron microscopy. These data suggest that ATP6L has a protective role against SNP-induced autophagic cell death via inhibition of JNK and p38 in GSH-depleted glial cells.

Deletion of the bis gene results in a marked increase in the production of corticosterone that is associated with thymic atrophy in mice.

Bis (Bag3) is known to be involved in cell survival, migration, the regulating of chaperones, and protein quality control. We reported recently on the production of bis gene-deleted mice, which show early lethality within 3 wk after birth with a phenotype showing severe malnutrition and shrinkage of the thymus. In this report, we provide evidence to show that an intrinsic problem of adrenal gland is the the primary cause for the severe atrophy of the thymus in bis(-/-) mice. The bis(-/-) mice show significantly higher levels of corticosterone, but CRH and ACTH levels were considerably lower than those of wild littermates. The transcription of steroidogenic enzymes was increased in the adrenal glands of bis(-/-) mice, accompanied by an increase in the thickness of the zona reticularis. An analysis of thymus tissue from bis(-/-) mice revealed that the severe atrophy of the thymus is due to the specific loss of immature double-positive (CD4(+)CD8(+)) cortical thymocytes by apoptosis, as evidenced by immunohistochemical examination and flow cytometric analysis, which were restored by injection of an inhibitor of glucocorticoid synthesis. In vitro cultures of thymocytes with increasing doses of dexamethasone exhibited a similar degree of apoptosis between wild and bis(-/-) thymocytes. The corticosterone levels from fasted wild littermates were one-half those of bis(-/-) mice, although serum glucose levels were similar. Thus, the deletion of the bis gene resulted in the intrinsic defect in the adrenal gland, leading to a marked increase in glucocorticoid levels, probably upon starvation stress, which accounts for the massive apoptosis of the thymus.

Octreotide inhibits secretion of IGF-1 from orbital fibroblasts in patients with thyroid-associated ophthalmopathy via inhibition of the NF-κB pathway.

PURPOSE: We investigated the effect of octreotide, a long-acting somatostatin (SST) analogue, on IGF-1 secretion and its possible mechanism of action in orbital fibroblasts (OFs) from patients with thyroid-associated ophthalmopathy (TAO). MATERIALS AND METHODS: OFs were isolated from the orbital fat of patients with TAO or healthy individuals. The expression level of insulin-like growth factor (IGF)-1, at the protein and mRNA level, was determined with ELISA and quantitative RT-PCR, respectively. The expression pattern of somatostatin receptor (SSTR) 2, which has the highest affinity for octreotide, was examined by flow cytometry. The activity of NF-κB pathway was determined by examining the levels of phosphorylation of IKKα/ß and p65, and degradation of IκB via western blot analysis, and by measuring the activity of NF-kB-dependent luciferase via transfection with plasmids containing luciferase and NF-κB binding site. RESULTS: OFs from patients with TAO showed significantly higher levels of IGF-1 secretion and NF-κB activity even in the absence of stimulation, compared to those from controls. Treatment with octreotide reduced the level of IGF-1 secretion in OFs from patients with TAO, but not in OFs from controls. OFs from patients with TAO expressed higher levels of SSTR2 on the cell surface, compared to controls. In addition, the expression of IGF-1 at the protein and mRNA level was dependent on the activity of NF-κB pathway in OFs from patients with TAO. Furthermore, treatment with octreotide reduced on the activity of NF-κB pathway in OFs from patients with TAO. CONCLUSION: OFs from patients with TAO showed significantly higher levels of IGF-1 secretion via up-regulation of NF-κB activity. Treatment with octreotide inhibited the secretion of IGF-1 by reducing the NF-κB pathway in OFs, which expressed higher levels of SSRT2 on the cell surface, from patients with TAO.

Sodium nitroprusside induces autophagic cell death in glutathione-depleted osteoblasts.

Previous studies reported that high levels of nitric oxide (NO) induce apoptotic cell death in osteoblasts. We examined molecular mechanisms of cytotoxic injury induced by sodium nitroprusside (SNP), a NO donor, in both glutathione (GSH)-depleted and control U2-OS osteoblasts. Cell viability was reduced by much lower effective concentrations of SNP in GSH-depleted cells compared to normal cells. The data suggest that the level of intracellular GSH is critical in SNP-induced cell death processes of osteoblasts. The level of oxidative stress due to SNP treatments doubled in GSH-depleted cells when measured with fluorochrome H2DCFDA. Pretreatment with the NO scavenger PTIO preserved the viability of cells treated with SNP. Viability of cells treated with SNP was recovered by pretreatment with Wortmannin, an autophagy inhibitor, but not by pretreatment with zVAD-fmk, a pan-specific caspase inhibitor. Large increases of LC3-II were shown by immunoblot analysis of the SNP-treated cells, and the increase was blocked by pretreatment with PTIO or Wortmannin; this implies that under GSH-depleted conditions SNP induces different molecular signaling that lead to autophagic cell death. The ultrastructural morphology of SNP-treated cells in transmission electron microscopy showed numerous autophagic vacuoles. These data suggest NO produces oxidative stress and cellular damage that culminate in autophagic cell death of GSH-depleted osteoblasts.

Insulin-like growth factor-1 enhances the expression of functional TSH receptor in orbital fibroblasts from thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO), an autoimmune disease, occurs in approximately 50 % of patients with Graves' hyperthyroidism. Thyroid-stimulating hormone receptor (TSHR) that is expressed in orbital fibroblasts is the autoimmune target in the development of TAO. In addition to thyroid-stimulating immunoglobulin (TSI), insulin-like growth factor (IGF)-1 is also involved in the development of TAO. IGF-1 has been reported to potentiate the effects of thyroid-stimulating hormone (TSH) and TSI on TSHR signaling. In the current study, we investigated the effects of IGF-1 on the cell surface expression of the functional TSHR and its possible mechanism of action in human orbital fibroblasts. Our results show that orbital fibroblasts from the TAO patients expressed higher levels of IGF-1 receptor (IGF-R), compared to control subjects. Treatment with IGF-1 enhanced the expression of surface TSHR in orbital fibroblasts from TAO patients, but not from control subjects. In addition, treatment with IGF-1 increased the level of TSHR at both the protein and mRNA levels. Furthermore, pre-treatment with IGF-1 potentiated TSH-induced cAMP production, compared to cells that were treated with only TSH. Our results also show that pre-treatment with cycloheximide, an inhibitor of mRNA translation, partially, but not completely, inhibited the expression of TSHR on the cell surfaces of orbital fibroblasts from TAO patients. These collective results show that IGF-1enhances the cell surface expression of functional TSHR, not only by increasing TSHR expression, but also by inducing TSHR translocation to the plasma membrane in orbital fibroblasts from TAO.