G protein-coupled receptor Ca2+-linked mitochondrial reactive oxygen species are essential for endothelial/leukocyte adherence

Abstract

Receptor-mediated signaling is commonly associated with multiple functions, including the production of reactive oxygen species. However, whether mitochondrion-derived superoxide (mROS) contributes directly to physiological signaling is controversial. Here we demonstrate a previously unknown mechanism in which physiologic Ca(2+)-evoked mROS production plays a pivotal role in endothelial cell (EC) activation and leukocyte firm adhesion. G protein-coupled receptor (GPCR) and tyrosine kinase-mediated inositol 1,4,5-trisphosphate-dependent mitochondrial Ca(2+) uptake resulted in NADPH oxidase-independent mROS production. However, GPCR-linked mROS production did not alter mitochondrial function or trigger cell death but rather contributed to activation of NF-kappaB and leukocyte adhesion via the EC induction of intercellular adhesion molecule 1. Dismutation of mROS by manganese superoxide dismutase overexpression and a cell-permeative superoxide dismutase mimetic ablated NF-kappaB transcriptional activity and facilitated leukocyte detachment from the endothelium under simulated circulation following GPCR- but not cytokine-induced activation. These results demonstrate that mROS is the downstream effector molecule that translates receptor-mediated Ca(2+) signals into proinflammatory signaling and leukocyte/EC firm adhesion.

FIG. 1.

Thrombin-evoked cytosolic Ca²⁺ mobilization precedes mitochondrial Ca²⁺ uptake. MPMVECs loaded with the cytosolic Ca²⁺ indicator Fluo-4/AM (green) and the mitochondrial Ca²⁺ indicator Rhod-2 (red) were stimulated with 500 mU/ml thrombin. (A) Time-lapse confocal microscopy revealed rapid Ca²⁺ mobilization, followed by mitochondrial Ca²⁺ uptake. Representative tracings of Ca²⁺ mobilization and mitochondrial Ca²⁺ uptake in response to 500 mU/ml (B) or 1 mU/ml (C) thrombin. (D and E) Inhibition of thrombin-mediated cytosolic Ca²⁺ mobilization by Hir and BAPTA abrogates mitochondrial Ca²⁺ uptake.

FIG. 2.

Thrombin-mediated Ca²⁺ signaling stimulates mROS generation. MPMVECs loaded with Fluo-4/AM (green) and the mROS indicator MitoSOX Red (red) were stimulated with 500 mU/ml thrombin. (A) Live imaging of thrombin-evoked cytosolic Ca²⁺ mobilization and mROS production. (B to E) Representative tracings of cytosolic Ca²⁺ and mROS generation in response to 500 mU/ml (B) or 1 mU/ml (C) thrombin, 500 mU/ml thrombin plus 2 U/ml Hir (D), or 10 μM Iono (E). (F) Thrombin-induced mROS production in response to 500 mU/ml thrombin in MPMVECs pretreated with RR (10 μM or 20 μM). (G and H) mROS generation by thrombin in gp91^phox−/− MPMVECs (G) or in MnSOD-overexpressing WT MPMVECs (H). (I) Quantitation of peak MitoSOX Red fluorescence following thrombin or Iono addition.

FIG. 3.

BCR-mediated Ca²⁺ signaling via InsP₃Rs triggers mROS production in DT40 lymphocytes. DT40 WT cells and cells lacking all three InsP₃R isoforms (TKO) were loaded with Fluo-4/AM (green) and the mROS indicator MitoSOX Red (red), stimulated with 1.5 μg/ml anti-BCR antibody (anti-IgM), and simultaneously visualized for cytosolic Ca²⁺ and mROS generation. (A) Single-cell analysis of DT40 WT cells after anti-IgM cross-linking. (B) Mean increase in mROS production observed in DT40 WT cells. (C) Normal expression levels of mitochondrial complex protein I (NADH-ubiquinol oxidoreductase; OxPhos I) and complex IV (cytochrome c oxidase; OxPhos IV) in both DT40 WT and TKO cells. (D to G) mROS production following anti-IgM cross-linking in TKO cells (D) or WT cells in Ca²⁺-free bath (E) or following pretreatment with the SOD mimetic MnTBAP (50 μM) (F) or DPI (10 μM) (G). (H) Low- and high-magnification images in DT40 WT cells after anti-IgM stimulation. (I) Quantitation of peak MitoSOX Red fluorescence following anti-IgM cross-linking.

FIG. 4.

Effect of thrombin-triggered Ca²⁺ signaling on mitochondrial function, morphology, and apoptosis. MPMVECs were loaded with the potentiometric dye TMRE and Fluo-4/AM (green) and imaged by confocal microscopy. Representative tracings reveal cytosolic Ca²⁺ and ΔΨ_m in response to Iono (10 μM) (A), Tg (5 μM) (B), thrombin (1 mU/ml [C], 500 mU/ml [D], or 1,000 mU/ml [E]), or thrombin (500 mU/ml) inhibited by 2.0 U/ml Hir (F). (G and H) Increased mROS generation (complex III inhibitor AA; 20 μM) (G) or dissipation of the mitochondrial proton gradient (mitochondrial uncoupler carbonylcyanide-p-trifluoromethoxyphenyldrazone [FCCP]; 2 μM) (H) resulted in rapid mitochondrial depolarization independent of cytosolic Ca²⁺. (I) Paracrine O₂·⁻ (100 μM xanthine-20 mU/ml xanthine oxidase) triggered rapid Ca²⁺ mobilization and ΔΨ_m loss. (J) Transmission electron microscopy of untreated MPMVECs or MPMVECs following stimulation with thrombin (1 U/ml) or inactivated thrombin (2.0 U/ml Hir). (K) Annexin V and TOTO-3 staining of thrombin-treated (1 U/ml) or superoxide-treated (100 μM xanthine-20 mU/ml xanthine oxidase) MPMVECs. (L) Quantitation of percentages of annexin V-positive cells.

FIG. 5.

Activation of NF-κB and expression of ICAM-1 by GPCR-linked mROS. (A) Protein binding to the NF-κB consensus sequence following thrombin stimulation in the presence or absence of BAPTA and MnTBAP. (B) NF-κB transcriptional activity in response to thrombin or TNF-α (10 ng/ml) as assessed by luciferase reporter assay. Luciferase reporter constructs were normalized to β-galactosidase activity. (C) Overexpression of MnSOD abolished thrombin-induced NF-κB activity. (D) Global O₂·⁻ production is assessed by HE fluorescence in response to both thrombin (500 mU/ml) and AA (20 μM) following 10 min of stimulation. (E) Quantitation of nuclear HE fluorescence increase in response to AA and thrombin. (F) VCAM-1 and ICAM-1 expression following thrombin or TNF-α stimulation in the presence of MnTBAP (50 μM), BAPTA (25 μM), or the proteosomal inhibitor MG132 in HPMVECs.

FIG. 6.

Thrombin-mediated mROS is requisite for leukocyte firm adherence via ICAM-1. Cell Tracker Red-labeled HPMVECs were treated with thrombin (500 mU/ml) or TNF-α (10 ng/ml) with or without MnTBAP (50 μM) pretreatment. An equivalent number of Cell Tracker Green-labeled J774.1 macrophages were added to HPMVECs for each experiment and subjected to simulated circulation to assess leukocyte firm adhesion. (A) Live cell images were acquired under high-power field via confocal microscopy prior to and following 6 min of 2.5 dynes/cm² shear stress. (B) Quantitation of adherent leukocytes following thrombin or TNF-α challenge in the absence or presence of MnTBAP. Thrombin-mediated leukocyte/EC binding was effectively reduced by MnTBAP, overexpression of MnSOD, and an anti-ICAM-1 blocking antibody. (C) Proposed model of local versus global leukocyte/EC firm adhesion following GPCR-linked mROS production and TNFR signaling, respectively.