Low-density lipoprotein (LDL) as a transporter for 2,2',4,4',5,5'-hexabromobiphenyl (HBB) into the cell.

This study investigated whether HBB could associate with LDL and, thereby, be carried into the cells. Wild type (K1) and a mutant strain (ldlA-7) of CHO cells which lacked functional receptors for LDL, were used to conduct kinetic studies in vitro designed to determine whether (\sp{14}C) HBB associated with LDL reconstituted with (\sp3H) cholesteryl linoleate, could be transported into the cell and, if so, to investigate the mechanism involved. LDL, reconstituted with a fluorescent probe, labeled K1 cells but not ldlA-7 cells indicating that K1 cells had functional receptors but ldlA-7 did not. Kinetic data confirmed this conclusion. The shapes of the kinetic curves for the entry of radiolabeled LDL showed that the rate of entry into the mutant cell was linear and slow. On the other hand, the entry of the LDL into K1 cells was initially rapid, ceased for a short period--presumably while the receptors were recycled from inside to the surface of the cell--and then resumed at a rapid rate. While free (\sp{14}C) HBB appeared to enter both cell types by diffusion, the rate of entry was reduced by about 75% when HBB was associated with LDL. This reduction in rate of entry and the observation that the ratio of cellular \sp3H and \sp{14}C remained constant as the amount of each isotope increased supported the view that (\sp{14}C) HBB and (\sp3H) LDL, when associated, entered both cell types at the same rate--a rate that was 4 times greater than the rate of entry of LDL alone into K1 cells and 15 times greater than the entry into the mutant cells. It was postulated that the increase in the rate of entry of LDL when associated with HBB was a function of an increase in hydrophobicity or charge. The findings that LDL associated with HBB entered both cell types at the same rate and that monensin, which blocks the recycling of LDL receptors in the cell, did not inhibit the entry of the LDL-HBB complex in K1 cells, suggested that the major route of entry for the complex occurred via a non-receptor pathway. (Abstract shortened with permission of author.).Ph.D.ToxicologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105917/1/9226927.pdfDescription of 9226927.pdf : Restricted to UM users only

Distinct Contributions of Orai1 and TRPC1 to Agonist-Induced [Ca²⁺]_i Signals Determine Specificity of Ca²⁺-Dependent Gene Expression

<div><p>Regulation of critical cellular functions, including Ca²⁺-dependent gene expression, is determined by the temporal and spatial aspects of agonist-induced Ca²⁺ signals. Stimulation of cells with physiological concentrations of agonists trigger increases [Ca²⁺]_i due to intracellular Ca²⁺ release and Ca²⁺ influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca²⁺]_i increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca²⁺]_i increases in human submandibular gland (HSG) cells. However, only Orai1-mediated Ca²⁺ entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca²⁺ store depletion in these cells, it is not clear how the cells decode individual Ca²⁺ signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca²⁺]_i signals and activation of NFAT in single cells. We report that Orai1-mediated Ca²⁺ entry generates [Ca²⁺]_i oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca²⁺ entry generates sustained [Ca²⁺]_i elevation at high [CCh] and contributes to frequency of [Ca²⁺]_i oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca²⁺ dependent gene expression pathways, consistent with the different patterns of [Ca²⁺]_i signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display “all-or-none” activation that is exclusively driven by local [Ca²⁺]_i generated by Orai1, independent of global [Ca²⁺]_i changes or TRPC1-mediated Ca²⁺ entry. In contrast, Ca²⁺ entry via TRPC1 primarily regulates NFκB-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca²⁺ signals following agonist stimulation of cells, which determine the functional specificity of the channels in activating different Ca²⁺-dependent gene expression pathways.</p> </div

TRPC1 and Orai1 contribute distinct [Ca²⁺]_i changes in individual HSG cells following CCh stimulation.

<p>Ca²⁺ oscillations in single cells following relatively low [CCh] (1 µM) stimulation of control HSG cells, with and without extracellular Ca²⁺ (<b>A</b> and <b>B</b>); or cells expressing siSTIM1 (<b>C</b>), Orai1E106Q (<b>D</b>), siOrai1 (<b>E),</b> shTRPC1 (<b>F</b>) or STIM1-KK/EE (<b>G</b>). Each trace is representative of ≥50 cells in at least 3 separate experiments (all traces shown were obtained from a single experiment). (<b>H</b>) Different oscillatory patterns seen in control cells as well as the changes induced by loss of TRPC1 or Orai1 function. Cells showing the various oscillatory patterns were counted and shown as percentage (%) of total cells (N). Cells showing sustained, non-oscillatory responses, or no responses were excluded from the data.</p

“All-or-none” mode of NFAT activation by Orai1-mediated Ca²⁺ entry. [

<p>Ca²⁺]_i responses induced by 300 nM CCh in control cells (<b>A</b>), cells expressing STIM1-KK/EE (<b>B</b>) or Orai1E106Q (<b>D</b>), and cells in Ca²⁺-free media (<b>C</b>). Each trace is representative of data obtained from ≥50 cells in at least 3 separate experiments. Insets in A and B show corresponding responses induced by 1 µM CCh over a 10 min time period. (<b>E</b>) Average change in the amplitude of [Ca²⁺]_i at t = 350 s (F_t−F₀). (<b>F</b>) Number of oscillations between the 300 and 600 s time points in control cells, cells expressing STIM1-KK/EE or cells in Ca²⁺-free media following stimulation with 1 µM or 300 nM CCh. (<b>G</b>) Panels showing nuclear translocation of NFAT in control cells and cells expressing STIM1-KK/EE, following stimulation with 300 nM CCh with 1 and 10 mM extracellular CaCl₂. (<b>H</b>) Histogram showing the proportion of cells (%) showing nuclear translocation of NFAT following stimulation with 300 nM CCh with 1 and 10 mM extracellular CaCl₂. Trace shows the <b>[</b>Ca²⁺]_i responses induced by 300 nM CCh in the presence of 1 and 10 mM extracellular CaCl₂, and is representative of data obtained from ≥50 cells in at least 3 separate experiments.</p

Contribution of Orai1 and TRPC1 to [Ca²⁺]_i signals in individual cells stimulated at high [CCh].

<p>[Ca²⁺]_i increases induced by high [CCh] (100 µM) in control cells (<b>A</b>), and in cells transfected with siSTIM1 (<b>B</b>), Orai1E106Q (<b>C</b>), siOrai1 (<b>D</b>), shTRPC1 (<b>E</b>), or STIM1-KK/EE (<b>F</b>). Traces are from a single experiment and are representative of ≥50 cells in at least 3 separate experiments.</p

Contribution of Orai1 and TRPC1 channels to the activation NFAT- and NFκB-driven luciferase activities.

<p>Luciferase activities driven by NFAT and NFκB following stimulation with CCh (100 µM) or PMA+CCh (10 ng/ml and 100 µM, respectively) in cells transfected with siOrai1 or shTRPC1. Histogram shows % decrease in CCh-stimulated luciferase activities in shTRPC1- or siOrai1-treated cells (relative to that in mock-transfected control cells). Data were obtained in at least 3 separate experiments for each transcription factor.</p

SOCE-driven [Ca²⁺]_i increases in HSG cells stimulated with relatively low [CCh].

<p>Averaged [Ca²⁺]_i responses induced by relatively low [CCh] (1 µM) in control HSG cells, with and without extracellular Ca²⁺ (<b>A, B</b>), and cells expressing siSTIM1 (<b>C</b>), Orai1E106Q (<b>D</b>), siOrai1 (<b>E</b>), shTRPC1 (<b>F</b>), or STIM1-KK/EE (<b>G</b>). Data for each trace were obtained from ≥50 cells in at least 3 separate experiments. (<b>H)</b> Average data showing amplitude of [Ca²⁺]_i increase at t = 250 s (F_t−F₀). *** indicates a significant difference (P<0.001, n ≥ 80 cells).</p

Effect of high [CCh] on NFAT nuclear translocation.

<p>Translocation of NFAT into the nucleus in control cells (<b>A</b>), and cells transfected with siOrai1 (<b>B</b>), Orai1E106Q (<b>C</b>), shTRPC1 (<b>D</b>) and STIM1EE (<b>E</b>). Traces show changes in GFP fluorescence intensities within the nucleus (black) and cytoplasm (red), following stimulation with 100 µM CCh. Each trace is representative of data obtained from at least 3 separate experiments (≥ 30 cells).</p

Germ line gain of function with SOS1 mutation in hereditary gingival fibromatosis

Made available in DSpace on 2019-09-12T16:53:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2007Office of Intramural Research (OIR) NIH HHSMutation of human SOS1 is responsible for hereditary gingival fibromatosis type 1, a benign overgrowth condition of the gingiva. Here, we investigated molecular mechanisms responsible for the increased rate of cell proliferation in gingival fibroblasts caused by mutant SOS1 in vitro. Using ectopic expression of wild-type and mutant SOS1 constructs, we found that truncated SOS1 could localize to the plasma membrane, without growth factor stimuli, leading to sustained activation of Ras/MAPK signaling. Additionally, we observed an increase in the magnitude and duration of ERK signaling in hereditary gingival fibromatosis gingival fibroblasts that was associated with phosphorylation of retinoblastoma tumor suppressor protein and the up-regulation of cell cycle regulators, including cyclins C, D, and E and the E2F/DP transcription factors. These factors promote cell cycle progression from G1 to S phase, and their up-regulation may underlie the increased gingival fibroblast proliferation observed. Selective depletion of wild-type and mutant SOS1 through small interfering RNA demonstrates the link between mutation of SOS1, ERK signaling, cell proliferation rate, and the expression levels of Egr-1 and proliferating cell nuclear antigen. These findings elucidate the mechanisms for gingival overgrowth mediated by SOS1 gene mutation in humans.NIH, NIDCR, Sect Human & Craniofacial Genet, Bethesda, MD 20892 USA; NHGRI, NIH, Bethesda, MD 20892 USA; Universidade de Taubaté (Unitau), Dept Dent, Periodont Res & Grad Studies Div, BR-12020 Sao Paulo, Brazi

Targeting the Ca2+ Sensor STIM1 by Exosomal Transfer of Ebv-miR-BART13-3p is Associated with Sjögren's Syndrome

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease that is associated with inflammation and dysfunction of salivary and lacrimal glands. The molecular mechanism(s) underlying this exocrinopathy is not known, although the syndrome has been associated with viruses, such as the Epstein Barr Virus (EBV). We report herein that an EBV-specific microRNA (ebv-miR-BART13-3p) is significantly elevated in salivary glands (SGs) of pSS patients and we show that it targets stromal interacting molecule 1 (STIM1), a primary regulator of the store-operated Ca2+ entry (SOCE) pathway that is essential for SG function, leading to loss of SOCE and Ca2+-dependent activation of NFAT. Although EBV typically infects B cells and not salivary epithelial cells, ebv-miR-BART13-3p is present in both cell types in pSS SGs. Importantly, we further demonstrate that ebv-miR-BART13-3p can be transferred from B cells to salivary epithelial cells through exosomes and it recapitulates its functional effects on calcium signaling in a model system