Trans-vaccenic acid reprograms CD8+ T cells and anti-tumour immunity.

Diet-derived nutrients are inextricably linked to human physiology by providing energy and biosynthetic building blocks and by functioning as regulatory molecules. However, the mechanisms by which circulating nutrients in the human body influence specific physiological processes remain largely unknown. Here we use a blood nutrient compound library-based screening approach to demonstrate that dietary trans-vaccenic acid (TVA) directly promotes effector CD8+ T cell function and anti-tumour immunity in vivo. TVA is the predominant form of trans-fatty acids enriched in human milk, but the human body cannot produce TVA endogenously1. Circulating TVA in humans is mainly from ruminant-derived foods including beef, lamb and dairy products such as milk and butter2,3, but only around 19% or 12% of dietary TVA is converted to rumenic acid by humans or mice, respectively4,5. Mechanistically, TVA inactivates the cell-surface receptor GPR43, an immunomodulatory G protein-coupled receptor activated by its short-chain fatty acid ligands6-8. TVA thus antagonizes the short-chain fatty acid agonists of GPR43, leading to activation of the cAMP-PKA-CREB axis for enhanced CD8+ T cell function. These findings reveal that diet-derived TVA represents a mechanism for host-extrinsic reprogramming of CD8+ T cells as opposed to the intrahost gut microbiota-derived short-chain fatty acids. TVA thus has translational potential for the treatment of tumours.

Keratin Profiling by Single-Cell RNA-Sequencing Identifies Human Prostate Stem Cell Lineage Hierarchy and Cancer Stem-Like Cells.

Single prostate stem cells can generate stem and progenitor cells to form prostaspheres in 3D culture. Using a prostasphere-based label retention assay, we recently identified keratin 13 (KRT13)-enriched prostate stem cells at single-cell resolution, distinguishing them from daughter progenitors. Herein, we characterized the epithelial cell lineage hierarchy in prostaspheres using single-cell RNA-seq analysis. Keratin profiling revealed three clusters of label-retaining prostate stem cells; cluster I represents quiescent stem cells (PSCA, CD36, SPINK1, and KRT13/23/80/78/4 enriched), while clusters II and III represent active stem and bipotent progenitor cells (KRT16/17/6 enriched). Gene set enrichment analysis revealed enrichment of stem and cancer-related pathways in cluster I. In non-label-retaining daughter progenitor cells, three clusters were identified; cluster IV represents basal progenitors (KRT5/14/6/16 enriched), while clusters V and VI represent early and late-stage luminal progenitors, respectively (KRT8/18/10 enriched). Furthermore, MetaCore analysis showed enrichment of the "cytoskeleton remodeling-keratin filaments" pathway in cancer stem-like cells from human prostate cancer specimens. Along with common keratins (KRT13/23/80/78/4) in normal stem cells, unique keratins (KRT10/19/6C/16) were enriched in cancer stem-like cells. Clarification of these keratin profiles in human prostate stem cell lineage hierarchy and cancer stem-like cells can facilitate the identification and therapeutic targeting of prostate cancer stem-like cells.

Gli activation by the estrogen receptor in breast cancer cells: Regulation of cancer cell growth by Gli3.

BACKGROUND: Gli is an oncogenic transcription factor family thought to be involved in breast cancer (BrCa) cell growth. Gli activity is regulated by a post-translational proteolytic process that is suppressed by Hedgehog signaling. In prostate cancer cells, however, Gli activation is mediated by an interaction of active androgen receptor proteins with Gli3 that stabilizes Gli3 in its un-proteolyzed form. Here we show that the estrogen receptor (ER), ERα, also binds Gli3 and activates Gli in BrCa cells. Moreover, we show that ER + BrCa cells are dependent on Gli3 for cancer cell growth. METHODS: Transfection with Gli-luciferase reporter was used to report Gli activity in 293FT or BrCa cells (MCF7, T47D, MDA-MB-453) with or without steroid ligands. Co-immunoprecipitation and proximity ligation were used to show association of Gli3 with ERα. Gli3 stability was determined by western blots of BrCa cell extracts. ERα knockdown or destabilization (by fulvestrant) was used to assess how loss of ERα affects estradiol-induced Gli reporter activity, formation of intranuclear ERα-Gli3 complexes and Gli3 stability. Expression of Gli1 and/or other endogenous Gli-target genes in BrCa cells were measured by qPCR in the presence or absence of estradiol. Gli3 knockdown was assessed for effects on BrCa cell growth using the Cyquant assay. RESULTS: ERα co-transfection increased Gli reporter activity in 293FT cells that was further increased by estradiol. Gli3 co-precipitated in ERα immunoprecipitates. Acute (2 h) estradiol increased Gli reporter activity and the formation of intranuclear ERα-Gli3 complexes in ER + BrCa cells but more chronic estradiol (48 h) reduced ERα-Gli complexes commensurate with reduced ERα levels. Gli3 stability and endogenous activity was only increased by more chronic estradiol treatment. Fulvestrant or ERα knockdown suppressed E2-induction of Gli activity, intranuclear ERα-Gli3 complexes and stabilization of Gli3. Gli3 knockdown significantly reduced the growth of BrCa cells. CONCLUSIONS: ERα interacts with Gli3 in BrCa cells and estradiol treatment leads to Gli3 stabilization and increased expression of Gli-target genes. Furthermore, we found tthat Gli3 is necessary for BrCa cell growth. These results support the idea that the ERα-Gli interaction and Gli3 may be novel targets for effective control of BrCa growth.

Effects of Inorganic Arsenic on Human Prostate Stem-Progenitor Cell Transformation, Autophagic Flux Blockade, and NRF2 Pathway Activation.

BACKGROUND: Inorganic arsenic (iAs) is an environmental toxicant associated with an increased risk of prostate cancer in chronically exposed populations worldwide. However, the biological mechanisms underlying iAs-induced prostate carcinogenesis remain unclear. OBJECTIVES: We studied how iAs affects normal human prostate stem-progenitor cells (PrSPCs) and drives transformation and interrogated the molecular mechanisms involved. METHODS: PrSPCs were enriched by spheroid culture from normal human primary or immortalized prostate epithelial cells, and their differentiation capability was evaluated by organoid culture. Microarray analysis was conducted to identify iAs-dysregulated genes, and lentiviral infection was used for stable manipulation of identified genes. Soft agar colony growth assays were applied to examine iAs-induced transformation. For in vivo study, PrSPCs mixed with rat urogenital sinus mesenchyme were grafted under the renal capsule of nude mice to generate prostatelike tissues, and mice were exposed to 5 ppm (∼65µM) iAs in drinking water for 3 months. RESULTS: Low-dose iAs (1µM) disturbed PrSPC homeostasis in vitro, leading to increased self-renewal and suppressed differentiation. Transcriptomic analysis indicated that iAs activated oncogenic pathways in PrSPCs, including the KEAP1-NRF2 pathway. Further, iAs-exposed proliferative progenitor cells exhibited NRF2 pathway activation that was sustained in their progeny cells. Knockdown of NRF2 inhibited spheroid formation by driving PrSPC differentiation, whereas its activation enhanced spheroid growth. Importantly, iAs-induced transformation was suppressed by NRF2 knockdown. Mechanistically, iAs suppressed Vacuolar ATPase subunit VMA5 expression, impairing lysosome acidification and inhibiting autophagic protein degradation including p62, which further activated NRF2. In vivo, chronic iAs exposure activated NRF2 in both epithelial and stroma cells of chimeric human prostate grafts and induced premalignant events. CONCLUSIONS: Low-dose iAs increased self-renewal and decreased differentiation of human PrSPCs by activating the p62-NRF2 axis, resulting in epithelial cell transformation. NRF2 is activated by iAs through specific autophagic flux blockade in progenitor cells, which may have potential therapeutic implications. https://doi.org/10.1289/EHP6471.

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution. HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Differential Actions of Estrogen Receptor α and ß via Nongenomic Signaling in Human Prostate Stem and Progenitor Cells.

Human prostate stem and progenitor cells express estrogen receptor (ER)α and ERß and exhibit proliferative responses to estrogens. In this study, membrane-initiated estrogen signaling was interrogated in human prostate stem/progenitor cells enriched from primary epithelial cultures and stem-like cell lines from benign and cancerous prostates. Subcellular fractionation and proximity ligation assays localized ERα and ERß to the cell membrane with caveolin-1 interactions. Exposure to 17ß-estradiol (E2) for 15 to 60 minutes led to sequential phosphorylation of signaling molecules in MAPK and AKT pathways, IGF1 receptor, epidermal growth factor receptor, and ERα, thus documenting an intact membrane signalosome that activates diverse downstream cascades. Treatment with an E2-dendrimer conjugate or ICI 182,870 validated E2-mediated actions through membrane ERs. Overexpression and knockdown of ERα or ERß in stem/progenitor cells identified pathway selectivity; ERα preferentially activated AKT, whereas ERß selectively activated MAPK cascades. Furthermore, prostate cancer stem-like cells expressed only ERß, and brief E2 exposure activated MAPK but not AKT cascades. A gene subset selectively regulated by nongenomic E2 signaling was identified in normal prostate progenitor cells that includes BGN, FOSB, FOXQ1, and MAF. Membrane-initiated E2 signaling rapidly modified histone methyltransferases, with MLL1 cleavage observed downstream of phosphorylated AKT and EZH2 phosphorylation downstream of MAPK signaling, which may jointly modify histones to permit rapid gene transcription. Taken together, the present findings document ERα and ERß membrane-initiated signaling in normal and cancerous human prostate stem/progenitor cells with differential engagement of downstream effectors. These signaling pathways influence normal prostate stem/progenitor cell homeostasis and provide novel therapeutic sites to target the elusive prostate cancer stem cell population.

Evaluation of Bisphenol A (BPA) Exposures on Prostate Stem Cell Homeostasis and Prostate Cancer Risk in the NCTR-Sprague-Dawley Rat: An NIEHS/FDA CLARITY-BPA Consortium Study.

BACKGROUND: Previous work determined that early life exposure to low-dose Bisphenol A (BPA) increased rat prostate cancer risk with aging. Herein, we report on prostate-specific results from CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity), which aims to resolve uncertainties regarding BPA toxicity. OBJECTIVES: We sought to a) reassess whether a range of BPA exposures drives prostate pathology and/or alters prostatic susceptibility to hormonal carcinogenesis, and b) test whether chronic low-dose BPA targets prostate epithelial stem and progenitor cells. METHODS: Sprague-Dawley rats were gavaged daily with vehicle, ethinyl estradiol (EE) or [Formula: see text] BPA/kg-BW during development or chronically, and prostate pathology was assessed at one year. One developmentally exposed cohort was given testosterone plus estradiol ([Formula: see text]) implants at day 90 to promote carcinogenesis with aging. Epithelial stem and progenitor cells were isolated by prostasphere (PS) culture from dorsolateral prostates (DLP) of rats continuously exposed for six months to [Formula: see text] BPA/kg-BW. Gene expression was analyzed by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: Exposure to BPA alone at any dose did not drive prostate pathology. However, rats treated with EE, 2.5, 250, or [Formula: see text] BPA/kg-BW plus [Formula: see text] showed greater severity of lateral prostate intraepithelial neoplasia (PIN), and DLP ductal adenocarcinoma multiplicity was markedly elevated in tumor-bearing rats exposed to [Formula: see text]-BW. DLP stem cells, assessed by PS number, doubled with chronic EE and [Formula: see text] exposures. PS size, reflecting progenitor cell proliferation, was greater at 25 and [Formula: see text] BPA doses, which also shifted lineage commitment toward basal progenitors while reducing luminal progenitor cells. CONCLUSIONS: Together, these results confirm and extend previous evidence using a rat model and human prostate epithelial cells that low-dose BPA augments prostate cancer susceptibility and alters adult prostate stem cell homeostasis. Therefore, we propose that BPA exposures may contribute to the increased carcinogenic risk in humans that occurs with aging. https://doi.org/10.1289/EHP3953.

Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution.

Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland.

Arsenic Induces p62 Expression to Form a Positive Feedback Loop with Nrf2 in Human Epidermal Keratinocytes: Implications for Preventing Arsenic-Induced Skin Cancer.

Exposure to inorganic arsenic in contaminated drinking water poses an environmental public health threat for hundreds of millions of people in the US and around the world. Arsenic is a known carcinogen for skin cancer. However, the mechanism by which arsenic induces skin cancer remains poorly understood. Here, we have shown that arsenic induces p62 expression in an autophagy-independent manner in human HaCaT keratinocytes. In mouse skin, chronic arsenic exposure through drinking water increases p62 protein levels in the epidermis. Nrf2 is required for basal and arsenic-induced p62 up-regulation. p62 knockdown reduces arsenic-induced Nrf2 activity, and induces sustained p21 up-regulation. p62 induction is associated with increased proliferation in mouse epidermis. p62 knockdown had little effect on arsenic-induced apoptosis, while it decreased cell proliferation following arsenic treatment. Our findings indicate that arsenic induces p62 expression to regulate the Nrf2 pathway in human keratinocytes and suggest that targeting p62 may help prevent arsenic-induced skin cancer.

Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta.

Protein Kinase C (PKC) plays a significant role in thrombin-induced loss of endothelial cell (EC) barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue-specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin), dominant negative PKCδ construct and PKCδ silencing (siRNA). In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCµ) and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis.

Serine 1179 Phosphorylation of Endothelial Nitric Oxide Synthase Increases Superoxide Generation and Alters Cofactor Regulation.

Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. In addition to producing NO, eNOS can also generate superoxide (O2-.) in the absence of the cofactor tetrahydrobiopterin (BH4). Previous studies have shown that bovine eNOS serine 1179 (Serine 1177/human) phosphorylation critically modulates NO synthesis. However, the effect of serine 1179 phosphorylation on eNOS superoxide generation is unknown. Here, we used the phosphomimetic form of eNOS (S1179D) to determine the effect of S1179 phosphorylation on superoxide generating activity, and its sensitivity to regulation by BH4, Ca2+, and calmodulin (CAM). S1179D eNOS exhibited significantly increased superoxide generating activity and NADPH consumption compared to wild-type eNOS (WT eNOS). The superoxide generating activities of S1179D eNOS and WT eNOS did not differ significantly in their sensitivity to regulation by either Ca2+ or CaM. The sensitivity of the superoxide generating activity of S1179D eNOS to inhibition by BH4 was significantly reduced compared to WT eNOS. In eNOS-overexpressing 293 cells, BH4 depletion with 10mM DAHP for 48 hours followed by 50ng/ml VEGF for 30 min to phosphorylate eNOS S1179 increased ROS accumulation compared to DAHP-only treated cells. Meanwhile, MTT assay indicated that overexpression of eNOS in HEK293 cells decreased cellular viability compared to control cells at BH4 depletion condition (P<0.01). VEGF-mediated Serine 1179 phosphorylation further decreased the cellular viability in eNOS-overexpressing 293 cells (P<0.01). Our data demonstrate that eNOS serine 1179 phosphorylation, in addition to enhancing NO production, also profoundly affects superoxide generation: S1179 phosphorylation increases superoxide production while decreasing sensitivity to the inhibitory effect of BH4 on this activity.

Sphingosine-1-phosphate lyase is an endogenous suppressor of pulmonary fibrosis: role of S1P signalling and autophagy.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is characterised by accumulation of fibroblasts and myofibroblasts and deposition of extracellular matrix proteins. Sphingosine-1-phosphate (S1P) signalling plays a critical role in pulmonary fibrosis. METHODS: S1P lyase (S1PL) expression in peripheral blood mononuclear cells (PBMCs) was correlated with pulmonary functions and overall survival; used a murine model to check the role of S1PL on the fibrogenesis and a cell culture system to study the effect of S1PL expression on transforming growth factor (TGF)-ß- and S1P-induced fibroblast differentiation. RESULTS: S1PL expression was upregulated in fibrotic lung tissues and primary lung fibroblasts isolated from patients with IPF and bleomycin-challenged mice. TGF-ß increased the expression of S1PL in human lung fibroblasts via activation and binding of Smad3 transcription factor to Sgpl1 promoter. Overexpression of S1PL attenuated TGF-ß-induced and S1P-induced differentiation of human lung fibroblasts through regulation of the expression of LC3 and beclin 1. Knockdown of S1PL (Sgpl1(+/-)) in mice augmented bleomycin-induced pulmonary fibrosis, and patients with IPF reduced Sgpl1 mRNA expression in PBMCs exhibited higher severity of fibrosis and lower survival rate. CONCLUSION: These studies suggest that S1PL is a novel endogenous suppressor of pulmonary fibrosis in human IPF and animal models.

Interaction of integrin ß4 with S1P receptors in S1P- and HGF-induced endothelial barrier enhancement.

We previously reported sphingosine 1-phosphate (S1P) and hepatocyte growth factor (HGF) augment endothelial cell (EC) barrier function and attenuate murine acute lung inury (ALI). While the mechanisms underlying these effects are not fully understood, S1P and HGF both transactivate the S1P receptor, S1PR1 and integrin ß4 (ITGB4) at membrane caveolin-enriched microdomains (CEMs). In the current study, we investigated the roles of S1PR2 and S1PR3 in S1P/HGF-mediated EC signaling and their associations with ITGB4. Our studies confirmed ITGB4 and S1PR2/3 are recruited to CEMs in human lung EC in response to either S1P (1 µM, 5 min) or HGF (25 ng/ml, 5 min). Co-immunoprecipitation experiments identified an S1P/HGF-mediated interaction of ITGB4 with both S1PR2 and S1PR3. We then employed an in situ proximity ligation assay (PLA) to confirm a direct ITGB4-S1PR3 association induced by S1P/HGF although a direct association was not detectable between S1PR2 and ITGB4. S1PR1 knockdown (siRNA), however, abrogated S1P/HGF-induced ITGB4-S1PR2 associations while there was no effect on ITGB4-S1PR3 associations. Moreover, PLA confirmed a direct association between S1PR1 and S1PR2 induced by S1P and HGF. Finally, silencing of S1PR2 significantly attenuated S1P/HGF-induced EC barrier enhancement as measured by transendothelial resistance while silencing of S1PR3 significantly augmented S1P/HGF-induced barrier enhancement. These results confirm an important role for S1PR2 and S1PR3 in S1P/HGF-mediated EC barrier responses that are associated with their complex formation with ITGB4. Our findings elucidate novel mechanisms of EC barrier regulation that may ultimately lead to new therapeutic targets for disorders characterized by increased vascular permeability including ALI.

Role of migratory inhibition factor in age-related susceptibility to radiation lung injury via NF-E2-related factor-2 and antioxidant regulation.

Microvascular injury and increased vascular leakage are prominent features of radiation-induced lung injury (RILI), and often follow cancer-associated thoracic irradiation. Our previous studies demonstrated that polymorphisms in the gene (MIF) encoding macrophage migratory inhibition factor (MIF), a multifunctional pleiotropic cytokine, confer susceptibility to acute inflammatory lung injury and increased vascular permeability, particularly in senescent mice. In this study, we exposed wild-type and genetically engineered mif(-/-) mice to 20 Gy single-fraction thoracic radiation to investigate the age-related role of MIF in murine RILI (mice were aged 8 wk, 8 mo, or 16 mo). Relative to 8-week-old mice, decreased MIF was observed in bronchoalveolar lavage fluid and lung tissue of 8- to 16-month-old wild-type mice. In addition, radiated 8- to 16-month-old mif(-/-) mice exhibited significantly decreased bronchoalveolar lavage fluid total antioxidant concentrations with progressive age-related decreases in the nuclear expression of NF-E2-related factor-2 (Nrf2), a transcription factor involved in antioxidant gene up-regulation in response to reactive oxygen species. This was accompanied by decreases in both protein concentrations (NQO1, GCLC, and heme oxygenase-1) and mRNA concentrations (Gpx1, Prdx1, and Txn1) of Nrf2-influenced antioxidant gene targets. In addition, MIF-silenced (short, interfering RNA) human lung endothelial cells failed to express Nrf2 after oxidative (H2O2) challenge, an effect reversed by recombinant MIF administration. However, treatment with an antioxidant (glutathione reduced ester), but not an Nrf2 substrate (N-acetyl cysteine), protected aged mif(-/-) mice from RILI. These findings implicate an important role for MIF in radiation-induced changes in lung-cell antioxidant concentrations via Nrf2, and suggest that MIF may contribute to age-related susceptibility to thoracic radiation.

Type 2 deiodinase and host responses of sepsis and acute lung injury.

The role of thyroid hormone metabolism in clinical outcomes of the critically ill remains unclear. Using preclinical models of acute lung injury (ALI), we assessed the gene and protein expression of type 2 deiodinase (DIO2), a key driver for synthesis of biologically active triiodothyronine, and addressed potential association of DIO2 genetic variants with ALI in a multiethnic cohort. DIO2 gene and protein expression levels in murine lung were validated by microarrays and immunoblotting. Lung injury was assessed by levels of bronchoalveolar lavage protein and leukocytes. Single-nucleotide polymorphisms were genotyped and ALI susceptibility association assessed. Significant increases in both DIO2 gene and D2 protein expression were observed in lung tissues from murine ALI models (LPS- and ventilator-induced lung injury), with expression directly increasing with the extent of lung injury. Mice with reduced levels of DIO2 expression (by silencing RNA) demonstrated reduced thyroxine levels in plasma and increased lung injury (increased bronchoalveolar lavage protein and leukocytes), suggesting a protective role for DIO2 in ALI. The G (Ala) allele of the Thr92Ala coding single-nucleotide polymorphism (rs225014) was protective in severe sepsis and severe sepsis-associated ALI after adjustments for age, sex, and genetic ancestry in a logistic regression model in European Americans. Our studies indicate that DIO2 is a novel ALI candidate gene, the nonsynonymous Thr92Ala coding variant of which confers ALI protection. Increased DIO2 expression may dampen the ALI inflammatory response, thereby strengthening the premise that thyroid hormone metabolism is intimately linked to the integrated response to inflammatory injury in critically ill patients.

ß-Arrestin1 mediates the endocytosis and functions of macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine, regulating inflammatory and immune responses. MIF binds to cell surface receptor CD74, resulting in both rapid and sustained ERK activation. It was reported that MIF-induced rapid ERK activation requires its co-receptor CD44. But the exact mechanism underlying sustained ERK activation is not well understood. In the current study, we described a detailed mechanism of MIF mediated sustained ERK activation. We found that ß-arrestin1, a scaffold protein involved in the activation of the MAPK cascade, interacts with CD74 upon MIF stimulation, resulting in CD74-mediated MIF endocytosis in a chlorpromazine (CPZ)-sensitive manner. ß-arrestin1 is also involved in endocytotic MIF signaling, leading to sustained ERK activation. Therefore ß-arrestin1 plays a central role in coupling MIF endocytosis to sustained ERK activation.

[Two-dimensional gel electrophoresis analysis of moderately halophilic bacterium Halobacillus dabanensis D-8T under hypoosmotic shock condition].

Halobacillus dabanensis D-8T was isolated from the saline deposits of Daban lake in Xinjiang of China, and is able to grow in complex medium containing 0.5% to 25% salt. To figure out the survival mechanisms of Gram-positive moderately halophilic bacteria under hypoosmotic shock conditions, two-dimensional gel electrophoresis (2-DE) was carried out to investigate differential protein expression profiles of H. dabanensis D-8T in response to low osmotic challenge. The 2-D gels were stored as dry gels and their images were taken by ImageScanner and analyzed by Imagemaster 2D Platinum software. About 650 protein spots were detected in 2-D gel. Most of proteins were distributed in molecular mass of 17.5 - 66kDa and the range of isoelectric point 4.0 - 5.9. A total of 34 protein spots were found to alter their expression after strain D-8T was subjected to hypoosmotic shock from 20% to 0% salinity for 5 min and 50 min. Among them, the expression of 20 protein spots is up-regulated including 6 new protein spots, while that of 14 protein spots is down-regulated in answer to sudden osmotic down-shift. Protein spots of interest were excised from the gels and digested by trypsin. By means of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) and MASCOT search engine, 4 up-regulated protein spots were identified with peptide mass fingerprint, and are similar to heat shock protein DanK, rod shape-determining protein, penicillin-binding protein (PBP-1A) and 5-enolpyruvoylshikimate-3-phosphate synthase, respectively. Noticeably, PBP-1A firstly was up-regulated after shock of 5 min but disappeared after shock of 50 min. This indicated that the strain activate a minor mechanism of peptidoglycan synthesis to compensate the major synthesis mechanism for cells survival through a down-shift challenge. In addition, this paper was the first report that heat shock proteins were up-regulated in response to sudden osmotic down-shift.