calpain and 2-aminoethoxydiphenyl-borate

Transient receptor potential melastatin 7 (TRPM7) is involved in both normal physiological processes and pathology of various diseases. The purpose of this study was to explore the function and underlying mechanisms of TRPM7 channels in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) apoptosis induced by thapsigargin in vitro. In this study, using a combination of Western blotting, RT-PCR, and nuclear morphology analysis, we investigated the influence and potential function of TRPM7 channels on the apoptosis induced by thapsigargin in RA FLSs. Chemical inhibitors (Gd(3+) and 2-APB) and specific siRNA for TRPM7 were used to study the role of TRPM7 in RA FLSs apoptosis. The expression of TRPM7 was significantly potentiated in RA FLSs. Co-incubation of RA FLSs with Gd(3+), 2-APB, or TRPM7-siRNA increased cell apoptosis. Furthermore, we found that suppression of TRPM7 channels also increased the expression CHOP and calpain and decreased the expression caspase-3. We conclude that suppression of TRPM7 channels may increase RA FLSs apoptosis in vitro, and this is associated with endoplasmic reticulum (ER) stress. Therefore, inhibition of TRPM7 could activate ER stress and induce RA FLSs apoptosis.

Topics: Animals; Apoptosis; Arthritis, Rheumatoid; Boron Compounds; Calpain; Caspase 3; Disease Models, Animal; Endoplasmic Reticulum Stress; Fibroblasts; Gadolinium; Male; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Synovial Membrane; Transcription Factor CHOP; TRPM Cation Channels

We demonstrated a role for the Mg(2+) transporter TRPM7, a bifunctional protein with channel and α-kinase domains, in aldosterone signaling. Molecular mechanisms underlying this are elusive. Here we investigated the function of TRPM7 and its α-kinase domain on Mg(2+) and pro-inflammatory signaling by aldosterone. Kidney cells (HEK-293) expressing wild-type human TRPM7 (WThTRPM7) or constructs in which the α-kinase domain was deleted (ΔKinase) or rendered inactive with a point mutation in the ATP binding site of the α-kinase domain (K1648R) were studied. Aldosterone rapidly increased [Mg(2+)]i and stimulated NADPH oxidase-derived generation of reactive oxygen species (ROS) in WT hTRPM7 and TRPM7 kinase dead mutant cells. Translocation of annexin-1 and calpain-II and spectrin cleavage (calpain target) were increased by aldosterone in WT hTRPM7 cells but not in α-kinase-deficient cells. Aldosterone stimulated phosphorylation of MAP kinases and increased expression of pro-inflammatory mediators ICAM-1, Cox-2 and PAI-1 in Δkinase and K1648R cells, effects that were inhibited by eplerenone (mineralocorticoid receptor (MR) blocker). 2-APB, a TRPM7 channel inhibitor, abrogated aldosterone-induced Mg(2+) responses in WT hTRPM7 and mutant cells. In 2-APB-treated ΔKinase and K1648R cells, aldosterone-stimulated inflammatory responses were unchanged. These data indicate that aldosterone stimulates Mg(2+) influx and ROS production in a TRPM7-sensitive, kinase-insensitive manner, whereas activation of annexin-1 requires the TRPM7 kinase domain. Moreover TRPM7 α-kinase modulates inflammatory signaling by aldosterone in a TRPM7 channel/Mg(2+)-independent manner. Our findings identify novel mechanisms for non-genomic actions of aldosterone involving differential signaling through MR-activated TRPM7 channel and α-kinase.

Topics: Adenosine Triphosphate; Aldosterone; Annexin A1; Binding Sites; Boron Compounds; Calpain; Eplerenone; Gene Expression Regulation; HEK293 Cells; Humans; Ion Transport; Magnesium; NADPH Oxidases; Phosphorylation; Protein Binding; Protein Kinases; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Reactive Oxygen Species; Signal Transduction; Spectrin; Spironolactone; TRPM Cation Channels

Peroxiredoxin-5 (PRDX5) is an antioxidant enzyme which differs from the other peroxiredoxins with regards to its enzymatic mechanism, its high affinity for organic peroxides and peroxynitrite and its wide subcellular distribution. In particular, the mitochondrial isoform of PRDX5 confers a remarkable cytoprotection toward oxidative stress to mammalian cells. Mitochondrial dysfunction and disruption of Ca²⁺ homeostasis are implicated in neurodegeneration. Growing evidence supports that endoplasmic reticulum (ER) could operate in tandem with mitochondria to regulate intracellular Ca²⁺ fluxes in neurodegenerative processes. Here, we overexpressed mitochondrial PRDX5 in SH-SY5Y cells to dissect the role of this enzyme in 1-methyl-4-phenylpyridinium (MPP)⁺-induced cell death. Our data show that mitochondria-dependent apoptosis triggered by MPP⁺, assessed by the measurement of caspase-9 activation and mitochondrial DNA damage, is prevented by mitochondrial PRDX5 overexpression. Moreover, PRDX5 overexpression blocks the increase in intracellular Ca²⁺, Ca²⁺-dependent activation of calpains and Bax cleavage. Finally, using Ca²⁺ channel inhibitors (Nimodipine, Dantrolene and 2-APB), we show that Ca²⁺ release arises essentially from ER stores through 1,4,5-inositol-trisphosphate receptors (IP3 R). Altogether, our results suggest that the MPP⁺ mitochondrial pathway of apoptosis is regulated by mitochondrial PRDX5 in a process that could involve redox modulation of Ca²⁺ transporters via a crosstalk between mitochondria and ER.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Adenosine Triphosphate; Animals; Apoptosis; Boron Compounds; Calcium; Calpain; Caspase 3; Caspase 9; Cell Line, Tumor; DNA, Mitochondrial; Dopamine Agents; Endoplasmic Reticulum; Enzyme Inhibitors; Gene Expression Regulation; Humans; Hydro-Lyases; Mice; Mitochondria; Neuroblastoma; Peroxiredoxins; Reactive Oxygen Species; RNA, Small Interfering; Subcellular Fractions; Transfection; Tyrosine

Ca(2+) release from internal stores is critical for mediating both normal and pathological intracellular Ca(2+) signaling. Recent studies suggest that the inositol 1,4,5-triphosphate (IP(3)) receptor mediates Ca(2+) release from internal stores upon cholinergic activation of the neuromuscular junction (NMJ) in both physiological and pathological conditions. Here, we report that the type I IP(3) receptor (IP(3)R(1))-mediated Ca(2+) release plays a crucial role in synaptic gene expression, development, and neuromuscular transmission, as well as mediating degeneration during excessive cholinergic activation. We found that IP(3)R(1)-mediated Ca(2+) release plays a key role in early development of the NMJ, homeostatic regulation of neuromuscular transmission, and synaptic gene expression. Reducing IP(3)R(1)-mediated Ca(2+) release via siRNA knockdown or IP(3)R blockers in C2C12 cells decreased calpain activity and prevented agonist-induced acetylcholine receptor (AChR) cluster dispersal. In fully developed NMJ in adult muscle, IP(3)R(1) knockdown or blockade effectively increased synaptic strength at presynaptic and postsynaptic sites by increasing both quantal release and expression of AChR subunits and other NMJ-specific genes in a pattern resembling muscle denervation. Moreover, in two mouse models of cholinergic overactivity and NMJ Ca(2+) overload, anti-cholinesterase toxicity and the slow-channel myasthenic syndrome (SCS), IP(3)R(1) knockdown eliminated NMJ Ca(2+) overload, pathological activation of calpain and caspase proteases, and markers of DNA damage at subsynaptic nuclei, and improved both neuromuscular transmission and clinical measures of motor function. Thus, blockade or genetic silencing of muscle IP(3)R(1) may be an effective and well tolerated therapeutic strategy in SCS and other conditions of excitotoxicity or Ca(2+) overload.

Topics: Action Potentials; Animals; Boron Compounds; Calcium; Calcium Signaling; Calpain; Carbachol; Caspase 3; Caspase 9; Cell Line, Transformed; Cholinergic Agonists; Cholinesterase Inhibitors; Disease Models, Animal; Electromyography; Electroporation; Exercise Test; Gene Expression Regulation; Green Fluorescent Proteins; Histone Deacetylases; Histones; In Vitro Techniques; Inositol 1,4,5-Trisphosphate Receptors; Male; Membrane Potentials; Mice; Mice, Transgenic; Muscle, Skeletal; Myasthenic Syndromes, Congenital; Neostigmine; Nerve Tissue Proteins; Neuromuscular Junction; Neurotoxicity Syndromes; Patch-Clamp Techniques; Receptors, Cholinergic; RNA, Small Interfering; Sciatic Nerve; Time Factors

Transient receptor potential melastatin-7 (TRPM7) channels have recently been identified to be regulated by vasoactive agents acting through G protein-coupled receptors in vascular smooth muscle cells (VSMC). However, downstream targets and functional responses remain unclear. We investigated the subcellular localization of TRPM7 in VSMCs and questioned the role of TRPM7 in proinflammatory signaling by bradykinin. VSMCs from Wistar-Kyoto rats were studied. Cell fractionation by sucrose gradient and differential centrifugation demonstrated that in bradykinin-stimulated cells, TRPM7 localized in fractions corresponding to caveolae. Immunofluorescence confocal microscopy revealed that TRPM7 distributes along the cell membrane, that it has a reticular-type intracellular distribution, and that it colocalizes with flotillin-2, a marker of lipid rafts. Bradykinin increased expression of calpain, a TRPM7 target, and stimulated its cytosol/membrane translocation, an effect blocked by 2-APB (TRPM7 inhibitor) and U-73122 (phospholipase C inhibitor), but not by chelerythrine (PKC inhibitor). Expression of proinflammatory mediators VCAM-1 and cyclooxygenase-2 (COX-2) was time-dependently increased by bradykinin. This effect was blocked by Hoe-140 (B2 receptor blocker) and 2-APB. Our data demonstrate that in bradykinin-stimulated VSMCs: 1) TRPM7 is upregulated, 2) TRPM7 associates with cholesterol-rich microdomains, and 3) calpain and proinflammatory mediators VCAM-1 and COX2 are regulated, in part, via TRPM7- and phospholipase C-dependent pathways through B2 receptors. These findings identify a novel signaling pathway for bradykinin, which involves TRPM7. Such phenomena may play a role in bradykinin/B2 receptor-mediated inflammatory responses in vascular cells.

Topics: Animals; Benzophenanthridines; Boron Compounds; Bradykinin; Bradykinin B2 Receptor Antagonists; Calpain; Caveolae; Cells, Cultured; Cyclooxygenase 2; Enzyme Inhibitors; Estrenes; Inflammation Mediators; Magnesium; Mesenteric Arteries; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Protein Kinase C; Protein Transport; Pyrrolidinones; Rats; Rats, Inbred WKY; Receptor, Bradykinin B2; Signal Transduction; TRPM Cation Channels; Type C Phospholipases; Up-Regulation; Vascular Cell Adhesion Molecule-1

Cisplatin drives specific types of tumour cells to apoptosis. In this study we investigate the involvement of intracellular calcium ([Ca(2+)](i)) in triggering apoptosis in two different cell lines. As cisplatin is used for the treatment of several forms of cancer we choose HeLa-S3 and U2-OS as two examples of tumour cell lines.. Cisplatin (1 nM-10 microM) was applied to HeLa-S3 and U2-OS cells and [Ca(2+)](i) measured with fluo-4, using laser scanning microscopy. Inositol-1,4,5-trisphosphate (IP(3)) receptors were visualized with immunostaining. Membrane conductances were measured with patch-clamp techniques. Levels of calpain and caspases were assessed by western blots and apoptotic cells were stained with Hoechst 33342 and counted.. Cisplatin increases [Ca(2+)](i) concentration-dependently in HeLa-S3 but not in U2-OS cells. This elevation of [Ca(2+)](i) depended on extracellular Ca(2+) but was reduced by the IP(3) receptor blocker, 2-APB. This effect was not due to a Ca(2+) release triggered by Ca(2+) entry. Immunostaining showed IP(3)-receptors (type 1-3) at the cellular membrane of HeLa-S3 cells, but not in U2-OS cells. Electrophysiological experiments showed an increased membrane conductance with cisplatin only when Ca(2+) was present extracellularly. Increase of [Ca(2+)](i) was related to the activation of calpain but not caspase-8 and triggered apoptosis in HeLa-S3 but not in U2-OS cells.. Our observations on the activation of IP(3)-receptors, calcium entry and apoptotic rate by cisplatin in specific carcinogenic cells might open new possibilities in the treatment of some forms of cancer.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Boron Compounds; Calcium; Calcium Signaling; Calpain; Caspase 8; Cell Line, Tumor; Cisplatin; Dose-Response Relationship, Drug; Electrophysiology; HeLa Cells; Humans; Inositol 1,4,5-Trisphosphate Receptors; Microscopy, Confocal