Coupling factor 6 attenuates CXCR4 expression through the HIF-1α and c-Src pathways and promotes endothelial apoptosis and inflammation.

Vascular endothelial cells are exposed to an acidic pH, and CXC chemokine receptor type 4 (CXCR4) is a key protective molecule against acidosis. We investigated the effect of coupling factor 6 (CF6), a novel proton import activator, on CXCR4 signaling and its molecular mechanism. CF6 decreased CXCR4 expression in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent manner. Pretreatment with small interfering RNA (siRNA) for hypoxia-inducible factor (HIF)-1α or PP1, a specific c-Src inhibitor, attenuated the CF6-induced decrease in CXCR4 without affecting CF6-induced intracellular acidosis. Chromatin immunoprecipitation revealed that CF6 enhanced the interaction between HIF-1α and the CXCR4 promoter at the hypoxia response element. CF6 also enhanced protein-protein interactions between phospho-c-Src and histone deacetylase 3 (HDAC3), but did not affect the binding of HDAC3 to the CXCR4 promoter at the hypoxia response element. Apoptotic cells, as measured by an Annexin-V-FITC Propidium Iodide Kit, were increased by CF6 in normoxia and hypoxia at 24 h; however, this increase was abolished by pretreatment with either siRNA for HIF-1α or the CXCR4 ligand. The coronary arteries and perivascular tissues obtained from CF6-overexpressing transgenic mice showed a lower expression of CXCR4 in the heart, increased wall thickness and infiltration of CD16-positive, CD206-positive or apoptotic cells. CF6 decreases CXCR4 expression through both HIF-1α- and c-Src-mediated mechanisms in vascular endothelial cells. Because CXCR4 has an important role in survival function, CF6 may have a role in the progression of arteriosclerosis via these complex mechanisms.

Coupling factor 6 enhances the spontaneous microaggregation of platelets by decreasing cytosolic cAMP irrespective of antiplatelet therapy.

The spontaneous microaggregation of platelets (SMAPs) is a marker for the prognosis of patients with cardiovascular diseases. Coupling factor 6 (CF6) binds to the plasma membrane ATP synthase and functions as a pro-atherogenic molecule in the cardiovascular system. However, the role of CF6 in SMAPs and stroke remains unknown. In 650 consecutive patients, including those with acute-onset stroke, and 20 control subjects, platelet-rich plasma (PRP) was obtained, and SMAP was measured using a laser light-scattering aggregometer. The cytosolic cyclic adenosine monophosphate (cAMP) concentration in platelets was measured using an enzyme-linked immunosorbent assay. CF6 increased SMAPs in patients and control subjects to a similar degree by binding to the α- and ß-subunits of ATP synthase and inducing intracellular acidosis. It was abolished by PRP pretreatment with antibodies against CF6, and the α- or ß-subunit of the plasma membrane ATP synthase, and the ATP synthase inhibitor efrapeptin. CF6 increased SMAPs in patient groups with and without antiplatelet therapy to a similar degree, and no difference was found among the subgroups taking aspirin, thienopyridine or cilostazol. The cytosolic cAMP concentration in platelets was decreased by CF6 in the presence of the direct adenylate cyclase activator forskolin. Pretreatment of PRP with the Gs activator cholera toxin blocked the decrease, whereas the Gi inactivator pertussis toxin and cilostazol had no influence. The CF6-induced acceleration of SMAPs was suppressed by cholera toxin but not by cilostazol or pertussis toxin. CF6 enhanced SMAPs by decreasing cytosolic cAMP. Because it was observed irrespective of antiplatelet agents, CF6 appears to be a novel target for antiplatelet therapy.

Medicinal chemistry of ATP synthase: a potential drug target of dietary polyphenols and amphibian antimicrobial peptides.

In this review we discuss the inhibitory effects of dietary polyphenols and amphibian antimicrobial/antitumor peptides on ATP synthase. In the beginning general structural features highlighting catalytic and motor functions of ATP synthase will be described. Some details on the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it an interesting drug target with respect to dietary polyphenols and amphibian antimicrobial peptides will also be reviewed. ATP synthase is known to have distinct polyphenol and peptide binding sites at the interface of α/ß subunits. Molecular interaction of polyphenols and peptides with ATP synthase at their respective binding sites will be discussed. Binding and inhibition of other proteins or enzymes will also be covered so as to understand the therapeutic roles of both types of molecules. Lastly, the effects of polyphenols and peptides on the inhibition of Escherichia coli cell growth through their action on ATP synthase will also be presented.

Upregulation of soluble vascular endothelial growth factor receptor type 1 by endogenous prostacyclin inhibitor coupling factor 6 in vascular endothelial cells: a role of acidosis-induced c-Src activation.

Vascular endothelial growth factor (VEGF) is a well-known promoter of angiogenesis, but its receptor VEGFR-1 and a soluble short form of VEGFR-1 (sFlt-1) play a negative role in the VEGF signal pathway by trapping VEGF. We recently showed that endogenous prostacyclin inhibitor coupling factor 6 (CF6) forces the clockwise rotation of F(1) motor of plasma membrane adenosine triphosphate synthase and induces intracellular acidosis and c-Src activation. We investigated the role of CF6 in regulation of sFlt-1, and its mechanism in human umbilical vein endothelial cells. The ratio of sFlt-1 to glyceraldehyde 3-phosphate dehydrogenase mRNA was increased at 24 h by 1.59+/-0.29-fold by 10(-7) M CF6 (P<0.05), concomitantly with the increases in intercellular adhesion molecule-1 and lectin-like oxidized low-density lipoprotein receptor-1 and no change in VEGF-A. When the dose of CF6 was increased to 10(-6) M, no further increase in sFlt-1 mRNA was observed. The release of sFlt-1 protein was increased by 1.72+/-0.24-fold (P<0.05) at 48 h after exposure to CF6 at 10(-7) M, and it was blocked by pretreatment with anti-CF6 antibody. The immunoreactive bands for sFlt-1 and VEGFR-1 were both increased by CF6 to similar degrees. Pretreatment with PP1, an inhibitor of c-Src, and 10(-5) Mefrapeptin, an inhibitor of F(1) motor, inhibited CF6-induced increases in expression and release of sFlt-1 (P<0.05). In mice overexpressing CF6, the plasma level of sFlt-1 was increased by 1.36+/-0.29-fold compared with that in wild-type mice (P<0.05). These indicate that CF6 might increase the expression and release of sFlt-1 in the vessels through acidosis-induced c-Src activation.

Effect of vasoconstrictor coupling factor 6 on gene expression profile in human vascular endothelial cells: enhanced release of asymmetric dimethylarginine.

BACKGROUND: Coupling factor 6 (CF6), a component of ATP synthase, inhibits phospholipase A2 and induces vasoconstriction. However, because arachidonic acid acts in the widespread fields of vascular biology, CF6 might exert profound effects in addition to vasoconstriction. We investigated the effect of CF6 on the gene expression profile in human umbilical vein endothelial cells. METHODS AND RESULTS: The increased gene expression after 24-h exposure to CF6 at 10 mol/l, assessed by cDNA microarray (n = 3), included neuregulin-1 (1.84 +/- 0.07 fold compared with control, P < 0.05) and relaxin-1 (1.74 +/- 0.20, P < 0.05), both relating to congestive heart failure, urokinase type plasminogen activator receptor (1.77 +/- 0.24, P = 0.06) and estrogen receptor beta (1.74 +/- 0.36, P = 0.08), both relating to vascular inflammation and cell infiltration, and protein arginine methyltransferase (PRMT-1; 1.73 +/- 0.20, P < 0.05). Out of these genes, the enzyme relating to the synthesis (PRMT-1) of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), was further examined concomitantly with the degradation enzyme, dimethylarginine dimethylaminohydrolase 2 (DDAH-2). The ratio of PRMT-1 to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, measured by real-time quantitative reverse transcription-polymerase chain reaction, was increased by 9 +/- 2% (n = 10, P < 0.01) at 48 h after CF6 at 10 mol/l, whereas the ratio of DDAH-2 to GAPDH was decreased by 12 +/- 2% (n = 8, P < 0.01). DDAH-2 protein and activity were decreased by 28 +/- 5% (n = 5, P < 0.01) and 19 +/- 2% (n = 6, P < 0.01) by CF6, respectively. ADMA release was enhanced by 20 +/- 8% and NOS activity was decreased by 13 +/- 1% (both n = 8, P < 0.05) by CF6. CONCLUSIONS: CF6 changes the gene expression profile to be proatherogenic and functions as a novel stimulator for ADMA release by enhancing its synthesis and suppressing its degradation.

Intracellular signaling for vasoconstrictor coupling factor 6: novel function of beta-subunit of ATP synthase as receptor.

Coupling factor 6 (CF6), a component of adenosine triphosphate (ATP) synthase, is circulating and functions as an endogenous vasoconstrictor by inhibiting cytosolic phospholipase A2. We showed a high plasma level of CF6 in human hypertension. The present study focused on the identification and characterization of a receptor for CF6 and its post-receptor signaling pathway. Incubation of human umbilical vein endothelial cells (HUVECs) with an excess of free CF6 reduced by 50% the immunoreactivity for the antibody to beta-subunit of ATP synthase at the cell surface, but unaffected that for the alpha-subunit antibody. A significant displacement of radioligand was observed at 3x10(-9) through 10(-7) M unlabeled CF6, and the Kd was 7.6 nM. Adenosine diphosphate (ADP) at 10(-7) M and beta-subunit antibody suppressed the binding of (125)I-CF6 by 81.3+/-9.7% and 32.0+/-2.0%, respectively, whereas the alpha-subunit antibody unaffected it. The hydrolysis activity of ATP to ADP was increased by 1.6-fold by CF6 at 10(-7) M, and efrapeptin at 10(-5) M, an inhibitor of ATP synthase, blocked it. CF6 at 10(-7) M decreased intracellular pH in 2',7'-bis(carboxyethyl-5 (6))-carboxyfluorescein-loaded HUVEC. Amyloride at 10(-4) M augmented the pH decrease in response to CF6, whereas efrapeptin at 10(-5) M blocked it. Arachidonic acid release was suppressed by CF6, and it was reversed by efrapeptin at 10(-5) M or beta-subunit antibody or ADP at 10(-7) M. The beta-subunit antibody suppressed coupling factor 6-induced increase in blood pressure. These indicate that membrane-bound ATP synthase functions as a receptor for CF6 and may have a previously unsuspected role in the genesis of hypertension by modulating the concentration of intracellular hydrogen.

The F1-ATPase beta-subunit is the putative enterostatin receptor.

It has been suggested that the F1-ATPase beta-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, beta-casomorphin1-7, in the absence and presence of cold enterostatin. 125I-beta-casomorphin1-7 weakly binds to the rat F1-ATPase beta-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect beta-casomorphin1-7 binding to the F1-ATPase beta-subunit. Peptides that suppressed food intake promoted beta-casomorphin1-7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced beta-casomorphin1-7 binding. Surface plasmon resonance measurements show that the beta-subunit of F1-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F1-ATPase complex. Western blot analysis showed the F1-ATPase beta-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F1-ATPase beta-subunit is the enterostatin receptor and suggests that enterostatin and beta-casomorphin1-7 bind to distinct sites on the protein.

Total synthesis of human and rat coupling factor-6 amide and pressor effects in the rat.

Mitochondrial coupling factor-6 (CF-6) is a component of the ATP synthase complex essential for energy transduction. CF-6, which is localized to the surface of endothelial cells (ECs) and released by shear stress, has been implicated as an endogenous vasoconstrictor. Previous methods of obtaining CF-6 through purification and recombinant methods were laborious and inefficient. Here, we describe the chemical synthesis of human CF-6, (33-108)-NH(2), its C-terminal fragment (55-108)-NH(2), which is termed pCF-6; the rat CF-6, (33-108)-NH(2), its C-terminal fragment pCF-6, (55-108)-NH(2); and two N-terminal fragments of the rat pro-coupling factor-6, (24-52)-NH(2) and (33-52)-NH(2). Biological activities of each peptide were initially screened with bioassays and verified by in vivo studies. Accordingly, intravenous administration of CF-6, pCF-6, rat CF-6, and rat pCF-6 produced a modest but statistically significant increase in blood pressure and heart rate in urethane anesthetized rats, whereas the N-terminal rat pro-coupling factor-6, (24-52)-NH(2) and (33-52)-NH(2) caused no significant pressor response. Thus, the biologically active site probably resides at the C-terminal portion of CF-6 peptides.

Mitochondrial coupling factor 6 as a potent endogenous vasoconstrictor.

We demonstrated recently that coupling factor 6, an essential component of the energy-transducing stalk of mitochondrial ATP synthase, suppresses the synthesis of prostacyclin in vascular endothelial cells. Here, we tested the hypothesis that coupling factor 6 is present on the cell surface and is involved in the regulation of systemic circulation. This peptide is present on the surface of CRL-2222 vascular endothelial cells and is released by these cells into the medium. In vivo, the peptide circulates in the vascular system of the rat, and its gene expression and plasma concentration are higher in spontaneously hypertensive rats (SHRs) than in normotensive controls. Elevation of blood pressure with norepinephrine did not affect the plasma concentration of coupling factor 6. Intravenous injection of recombinant peptide increased blood pressure, apparently by suppressing prostacyclin synthesis, whereas a specific Ab to coupling factor 6 decreased systemic blood pressure concomitantly with an increase in plasma prostacyclin. Interestingly, the antibody's hypotensive effect could be abolished by treating with the cyclooxygenase inhibitor indomethacin. These findings indicate that mitochondrial coupling factor 6 functions as a potent endogenous vasoconstrictor in the fashion of a circulating hormone and may suggest a new mechanism for hypertension.