Explore the vast range of titles available.

A subset of environmental chemicals acts as \"obesogens\" as they increase adipose mass and lipid content in livers of treated rodents. One of the most studied class of obesogens are the tin-containing chemicals that have as a central moiety tributyltin (TBT), which bind and activate two nuclear hormone receptors, Peroxisome Proliferator Activated Receptor Gamma (PPARG) and Retinoid X Receptor Alpha (RXRA), at nanomolar concentrations. Here, we have tested whether TBT chloride at such concentrations may affect the neutral lipid level in two cell line models of human liver. Indeed, using high content image analysis (HCA), TBT significantly increased neutral lipid content in a time- and concentration-dependent manner. Consistent with the observed increased lipid accumulation, RNA fluorescence in situ hybridization (RNA FISH) and RT-qPCR experiments revealed that TBT enhanced the steady-state mRNA levels of two key genes for de novo lipogenesis, the transcription factor SREBF1 and its downstream enzymatic target, FASN. Importantly, pre-treatment of cells with 2-deoxy-D-glucose reduced TBT-mediated lipid accumulation, thereby suggesting a role for active glycolysis during the process of lipid accumulation. As other RXRA binding ligands can promote RXRA protein turnover via the 26S proteasome, TBT was tested for such an effect in the two liver cell lines. We found that TBT, in a time- and dose-dependent manner, significantly reduced steady-state RXRA levels in a proteasome-dependent manner. While TBT promotes both RXRA protein turnover and lipid accumulation, we found no correlation between these two events at the single cell level, thereby suggesting an additional mechanism may be involved in TBT promotion of lipid accumulation, such as glycolysis.

Here we investigate the role of Phosphatidylinositol (4,5) bisphosphate (PIP(2)) in the physiological activation of primary murine T cells by antigen presenting cells (APC) by addressing two principal challenges in PIP(2) biology. First, PIP(2) is a regulator of cytoskeletal dynamics and a substrate for second messenger generation. The relative importance of these two processes needs to be determined. Second, PIP(2) is turned over by multiple biosynthetic and metabolizing enzymes. The joint effect of these enzymes on PIP(2) distributions needs to be determined with resolution in time and space. We found that T cells express four isoforms of the principal PIP(2)-generating enzyme phosphatidylinositol 4-phosphate 5-kinase (PIP5K) with distinct spatial and temporal characteristics. In the context of a larger systems analysis of T cell signaling, these data identify the T cell/APC interface and the T cell distal pole as sites of differential PIP(2) turnover. Overexpression of different PIP5K isoforms, as corroborated by knock down and PIP(2) blockade, yielded an increase in PIP(2) levels combined with isoform-specific changes in the spatiotemporal distributions of accessible PIP(2). It rigidified the T cell, likely by impairing the inactivation of Ezrin Moesin Radixin, delayed and diminished the clustering of the T cell receptor at the cellular interface, reduced the efficiency of T cell proximal signaling and IL-2 secretion. These effects were consistently more severe for distal PIP5K isoforms. Thus spatially constrained cytoskeletal roles of PIP(2) in the control of T cell rigidity and spatiotemporal organization dominate the effects of PIP(2) on T cell activation.

Diverse toxicants and mixtures that affect hormone responsive cells [endocrine disrupting chemicals (EDCs)] are highly pervasive in the environment and are directly linked to human disease. They often target the nuclear receptor family of transcription factors modulating their levels and activity. Many high-throughput assays have been developed to query such toxicants; however, single-cell analysis of EDC effects on endogenous receptors has been missing, in part due to the lack of quality control metrics to reproducibly measure cell-to-cell variability in responses. We began by developing single-cell imaging and informatic workflows to query whether the single cell distribution of the estrogen (ER), used as a model system, can be used to measure effects of EDCs in a sensitive and reproducible manner. We used high-throughput microscopy, coupled with image analytics to measure changes in single cell ER nuclear levels on treatment with toxicants, over a large number of biological and technical replicates. We developed a two-tiered quality control pipeline for single cell analysis and tested it against a large set of biological replicates, and toxicants from the EPA and Agency for Toxic Substances and Disease Registry lists. We also identified a subset of potentially novel EDCs that were active only on the endogenous ER level and activity as measured by single molecule RNA fluorescence hybridization (RNA FISH). We demonstrated that the distribution of ER levels per cell, and the changes upon chemical challenges were remarkably stable features; and importantly, these features could be used for quality control and identification of endocrine disruptor toxicants with high sensitivity. When coupled with orthogonal assays, ER single cell distribution is a valuable resource for high-throughput screening of environmental toxicants. https://doi.org/10.1289/EHP9297.

A subset of environmental chemicals acts as \"obesogens\" as they increase adipose mass and lipid content in livers of treated rodents. One of the most studied class of obesogens are the tin-containing chemicals that have as a central moiety tributyltin (TBT), which bind and activate two nuclear hormone receptors, Peroxisome Proliferator Activated Receptor Gamma (PPARG) and Retinoid X Receptor Alpha (RXRA), at nanomolar concentrations. Here, we have tested whether TBT chloride at such concentrations may affect the neutral lipid level in two cell line models of human liver. Indeed, using high content image analysis (HCA), TBT significantly increased neutral lipid content in a time- and concentration-dependent manner. Consistent with the observed increased lipid accumulation, RNA fluorescence in situ hybridization (RNA FISH) and RT-qPCR experiments revealed that TBT enhanced the steady-state mRNA levels of two key genes for de novo lipogenesis, the transcription factor SREBF1 and its downstream enzymatic target, FASN. Importantly, pre-treatment of cells with 2-deoxy-D-glucose reduced TBT-mediated lipid accumulation, thereby suggesting a role for active glycolysis during the process of lipid accumulation. As other RXRA binding ligands can promote RXRA protein turnover via the 26S proteasome, TBT was tested for such an effect in the two liver cell lines. We found that TBT, in a time- and dose-dependent manner, significantly reduced steady-state RXRA levels in a proteasome-dependent manner. While TBT promotes both RXRA protein turnover and lipid accumulation, we found no correlation between these two events at the single cell level, thereby suggesting an additional mechanism may be involved in TBT promotion of lipid accumulation, such as glycolysis.

Here we investigate the role of Phosphatidylinositol (4,5) bisphosphate (PIP.sub.2) in the physiological activation of primary murine T cells by antigen presenting cells (APC) by addressing two principal challenges in PIP.sub.2 biology. First, PIP.sub.2 is a regulator of cytoskeletal dynamics and a substrate for second messenger generation. The relative importance of these two processes needs to be determined. Second, PIP.sub.2 is turned over by multiple biosynthetic and metabolizing enzymes. The joint effect of these enzymes on PIP.sub.2 distributions needs to be determined with resolution in time and space. We found that T cells express four isoforms of the principal PIP.sub.2 -generating enzyme phosphatidylinositol 4-phosphate 5-kinase (PIP5K) with distinct spatial and temporal characteristics. In the context of a larger systems analysis of T cell signaling, these data identify the T cell/APC interface and the T cell distal pole as sites of differential PIP.sub.2 turnover. Overexpression of different PIP5K isoforms, as corroborated by knock down and PIP.sub.2 blockade, yielded an increase in PIP.sub.2 levels combined with isoform-specific changes in the spatiotemporal distributions of accessible PIP.sub.2 . It rigidified the T cell, likely by impairing the inactivation of Ezrin Moesin Radixin, delayed and diminished the clustering of the T cell receptor at the cellular interface, reduced the efficiency of T cell proximal signaling and IL-2 secretion. These effects were consistently more severe for distal PIP5K isoforms. Thus spatially constrained cytoskeletal roles of PIP.sub.2 in the control of T cell rigidity and spatiotemporal organization dominate the effects of PIP.sub.2 on T cell activation.

Mitogen activated protein kinases (MAPKs) are specific serine/threonine kinases which respond to various stimuli and control various cellular activities including gene expression, mitosis, cell differentiation, and cell survival/apoptosis. Histamine is implicated in allergic disease and asthma and SAPK/JNK and ERK1/2 are involved in certain aspects of allergic inflammation such as TH2 differentiation and proliferation and apoptosis. This study was designed to investigate the effects of histamine on ERK1/2 and SAPK/JNK phosphorylation in splenocytes. C57BL/6 splenocytes were treated with different concentrations of histamine (10-4 M to 10-11 M). Histamine at higher concentration (10-4 M) increased ERK2 phosphorylation. There was, however no significant effect seen at other concentrations (10-6 M - 10-11 M). Surprisingly, H1 receptor agonist betahistine (10-5 M), H2 agonist amthamine (10-5 M), H3 agonist methimepip (10 -6 M) and H4 agonist 4-methyl histamine (10-6 M), all increased ERK2 phosphorylation. H1R antagonist pyrilamine (10-6 M), H2R antagonist ranitidine (10-5 M), H3/H4R antagonist thioperamide (10-6 M), and H3R antagonist clobenpropit (10-5 M) inhibited histamine mediated ERK2 phosphorylation suggesting that all four histamine receptor subtypes played some role in this phosphorylation. Since TNF-alpha causes phosphorylation of ERK1/2, we investigated whether histamine acted via secretion of TNF-alpha to affect ERK1/2 phosphorylation. As a consequence, TNF-alpha knockout mice were used and we found that TNF-alpha was involved in ERK2 phosphorylation. There was complete inhibition of ERK2 phosphorylation by histamine via H2, H3 and H4 agonists, but effects of H1 agonist were inconclusive in TNF-alpha knockout splenocytes. This suggested that histamine indirectly affected the ERK2 phosphorylation via its effects on the secretion of TNF-alpha and H1 receptor played a role in this process. We performed similar experiments with stress activated protein kinases/c-jun N-terminal kinases (SAPK/JNK). C57BL/6 mice splenocytes were treated with different concentrations of histamine (10-4 M to 10 -11 M), phorbol 12 myristate 13-acetate (PMA) was used as a positive control and phosphorylation of SAPK/JNK was determined. Histamine inhibited phosphorylation of SAPK/JNK at high concentrations (10-4 M-10-8 M) and had no effect on SAPK/JNK phosphorylation at lower concentrations (10 -9 M- 10-11 M). Histamine receptor specific agonists were used to identify the histamine receptors involved in the inhibition of SAPK/JNK phosphorylation. H1R agonist betahistine (10-5 M) decreased the phosphorylation of SAPK/JNK. The decrease in SAPK/JNK phosphorylation by histamine was predominantly an H1 receptor effect. H2R agonist amthamine (10-5 M) did not show any significant effect on SAPK/JNK phosphorylation. H3R agonist methimepip (10-6 M) and H4R agonist 4-methyl histamine (10-6 M), increased SAPK/JNK phosphorylation. H1R antagonist pyrilamine (10-6 M), H2R antagonist ranitidine (10-5 M), H3/H4R antagonist thioperamide (10-6 M), and clobenpropit (10-5 M), partially reversed the histamine mediated inhibition of SAPK/JNK phosphorylation. TNF-alpha knockout mice were used to determine if histamine regulated SAPK/JNK phosphorylation via TNF-alpha. In TNF-alpha knockout mice splenocytes, histamine inhibited SAPK/JNK phosphorylation. Activation of H1 receptors inhibited SAPK/JNK phosphorylation in knockout as was the case in wild type mice. Histamine via H2 receptor inhibited SAPK/JNK phosphorylation in knockout mice, but did not show any significant effect in wild type mice. Activation of H3 receptors decreased SAPK/JNK phosphorylation in knockout mice, as opposed to an increase in wild type mice suggesting that another cytokine besides TNF-alpha was involved in SAPK/JNK phosphorylation. H4 receptor did not show any significant effect in knockout mice, but showed an increase in SAPK/JNK phosphorylation in wild type mice suggesting that TNF-alpha is required for histamine mediated effects via H4 receptors. This data showed a role of TNF-alpha in histamine-mediated effects of SAPK/JNK phosphorylation via H4 receptors.