Dual-FRET imaging of IP3 and Ca2+ revealed Ca2+-induced IP3 production maintains long lasting Ca2+ oscillations in fertilized mouse eggs

In most species, fertilization induces Ca(2+) transients in the egg. In mammals, the Ca(2+) rises are triggered by phospholipase Czeta (PLCzeta) released from the sperm; IP3 generated by PLCzeta induces Ca(2+) release from the intracellular Ca(2+) store through IP3 receptor, termed IP3-induced Ca(2+) release. Here, we developed new fluorescent IP3 sensors (IRIS-2s) with the wider dynamic range and higher sensitivity (Kd = 0.047-1.7 muM) than that we developed previously. IRIS-2s employed green fluorescent protein and Halo-protein conjugated with the tetramethylrhodamine ligand as fluorescence resonance energy transfer (FRET) donor and acceptor, respectively. For simultaneous imaging of Ca(2+) and IP3, using IRIS-2s as the IP3 sensor, we developed a new single fluorophore Ca(2+) sensor protein, DYC3.60. With IRIS-2s and DYC3.60, we found that, right after fertilization, IP3 concentration ([IP3]) starts to increase before the onset of the first Ca(2+) wave. [IP3] stayed at the elevated level with small peaks followed after Ca(2+) spikes through Ca(2+) oscillations. We detected delays in the peak of [IP3] compared to the peak of each Ca(2+) spike, suggesting that Ca(2+)-induced regenerative IP3 production through PLC produces small [IP3] rises to maintain [IP3] over the basal level, which results in long lasting Ca(2+) oscillations in fertilized eggs

Calreticulin and integrin alpha dissociation induces anti-inflammatory programming in animal models of inflammatory bowel disease

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn’s disease, is a chronic intestinal inflammatory condition initiated by integrins-mediated leukocyte adhesion to the activated colonic microvascular endothelium. Calreticulin (CRT), a calcium-binding chaperone, is known as a partner in the activation of integrin α subunits (ITGAs). The relationship between their interaction and the pathogenesis of IBD is largely unknown. Here we show that a small molecule, orally active ER-464195-01, inhibits the CRT binding to ITGAs, which suppresses the adhesiveness of both T cells and neutrophils. Transcriptome analysis on colon samples from dextran sodium sulfate-induced colitis mice reveals that the increased expression of pro-inflammatory genes is downregulated by ER-464195-01. Its prophylactic and therapeutic administration to IBD mouse models ameliorates the severity of their diseases. We propose that leukocytes infiltration via the binding of CRT to ITGAs is necessary for the onset and development of the colitis and the inhibition of this interaction may be a novel therapeutic strategy for the treatment of IBD

Calmodulin inhibits inositol 1,4,5-trisphosphate-induced calcium release through the purified and reconstituted inositol 1,4,5-trisphosphate receptor type 1

AbstractOur previous studies have demonstrated that calmodulin binds to IP3R type 1 (IP3R1) in a Ca2+ dependent manner, which suggests that calmodulin regulates the IP3R1 channel. In the present study, we investigated real-time kinetics of interactions between calmodulin and IP3R1 as well as effects of calmodulin on IP3-induced Ca2+ release by purified and reconstituted IP3R1. Kinetic analysis revealed that calmodulin binds to IP3R1 in a Ca2+ dependent manner and that both association and dissociation phase consist of two components with time constants of ka=4.46×102 and >104 M−1 s−1, kd=1.44×10−2 and 1.17×10−1 s−1. The apparent dissociation constant was calculated to be 27.3 μM. The IP3-induced Ca2+ release through the purified and reconstituted IP3R1 was inhibited by Ca2+/calmodulin, in a dose dependent manner. We interpret our findings to mean that calmodulin binds to IP3R1 in a Ca2+ dependent manner to exert inhibitory effect on IP3R channel activity. This event may be one of the mechanisms governing the negative feedback regulation of IP3-induced Ca2+ release by Ca2+

Phospholipase C-β1 and β4 contribute to non-genetic cell-to-cell variability in histamine-induced calcium signals in HeLa cells.

A uniform extracellular stimulus triggers cell-specific patterns of Ca(2+) signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3) concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca(2+) oscillations in terms of the time constant of Ca(2+) spike amplitude decay and the Ca(2+) oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca(2+) signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca(2+) signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca(2+) oscillations, such as the time constant of the temporal changes in the Ca(2+) spike amplitude and the Ca(2+) oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca(2+) release, can cause cell-to-cell variability in the patterns of Ca(2+) signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca(2+) signals evoked by G protein-coupled receptor stimulation

Effects of PLC-β1 or PLC-β4 overexpression on Ca²⁺ and IP₃ dynamics evoked by histamine stimulation.

<p>(A) Western blotting analyses of cell lysates prepared from HeLa cells transfected with PLC-β1-IRES-mRFP (lane 2) or PLC-β4-IRES-mRFP (lane 4). Non-transfected cells were used as controls (lanes 1 and 3). (B) Representative traces of Indo-5F signal changes (F/F₀; top) and IRIS-1 signal changes (ΔR/R₀; bottom) in transfected cells stimulated with 3 µM histamine. The plasmid DNAs used to transfect the cells are shown on the left. The horizontal broken lines indicate the baseline levels of IRIS-1 and Indo-5F signals. The vertical broken lines indicate the onsets of stimulation. (B–D) Histograms for the inverse time constants for exponential decay of the Ca²⁺ oscillation amplitude (B), Ca²⁺ oscillation frequencies (C), and integrated IP₃ signals (D) in cells expressing mRFP (top row), PLC-β1 and mRFP (second row), and PLC-β4 and mRFP (third row). The means ± SD are shown at the bottom. The numbers of cells measured are shown in parentheses. Statistical analyses were performed by one-way ANOVA followed by Scheffe’s multiple comparison test. **P<0.01, vs. the values in IRES-mRFP transfected cells.</p