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Cardiovascular Research 2004 62(3):578-586; doi:10.1016/j.cardiores.2004.01.031
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Tumor necrosis factor {alpha} as an endogenous stimulator for circulating coupling factor 6

Satoko Sasakia, Tomohiro Osanai*,a, Hirofumi Tomitaa, Toshiro Matsunagaa, Koji Magotab and Ken Okumuraa

aThe Second Department of Internal Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan
bDaiichi Suntory Biomedical Research CO., LTD., Osaka 618-8513, Japan

* Corresponding author. Tel.: +81-172-39-5057; fax: +81-172-35-9190. Email address: osanait{at}cc.hirosaki-u.ac.jp

Received 2 May 2003; revised 25 January 2004; accepted 27 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: We recently showed that mitochondrial coupling factor 6 (CF6) is present as a pressor substance and a prostacyclin inhibitor in systemic circulation. However, the regulation mechanism for circulating CF6 is unknown. We investigated the role of tumor necrosis factor-{alpha} (TNF-{alpha}) in the generation and release of CF6. Methods and results: We used two kinds of cells, human umbilical vein endothelial cells (HUVEC) and ECV-304. The concentration of CF6 in the medium increased with time in both ECV-304 and HUVEC. Treatment of ECV-304 and HUVEC with TNF-{alpha} enhanced the release of CF6 in a dose-dependent manner concomitantly with the decrease in CF6 content in the mitochondria at 24 h. The released CF6 was characterized to be an active full-length peptide by Western blot. The ratio of CF6 to GAPDH mRNA, measured by real-time polymerase chain reaction, was 1.7 fold increased at 1 h after exposure to TNF-{alpha} in ECV-304 and HUVEC. This enhanced gene expression and release was blocked or suppressed by 70% by stable transfection of dominant negative mutant I{kappa}B kinase {alpha} whose efficacy was confirmed by blockade of translocation of NF-{kappa}B p65 protein and of degradation of I{kappa}B{alpha} protein. Flow cytometry analysis revealed that the cell surface-associated CF6 was significantly increased at 24 h after TNF-{alpha} in a dose-dependent manner. Conclusions: TNF-{alpha} stimulates the gene expression of CF6 via activation of NF-{kappa}B signaling pathway, and promotes the release of CF6 from ECV-304 and HUVEC.

KEYWORDS Prostacyclin; Coupling factor 6; Tumor necrosis factor {alpha}; Vascular endothelial cells; Nuclear factor {kappa}B


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Coupling factor 6 is an essential component of the energy-transducing stalk of mitochondrial adenosine triphosphate (ATP) synthase [1]. We recently identified a novel function for coupling factor 6 while investigating the mechanism of suppression of prostacyclin in spontaneously hypertensive rats (SHR). Levels of circulating prostacyclin in SHR were decreased compared with those in normotensive control Wistar Kyoto rats (WKY), despite the fact that, as measured in isolated aortic strips, prostacyclin generation was elevated in SHR [2,3]. We postulated that some endogenous prostacyclin synthesis inhibitor might be acting in SHR and showed that mitochondrial coupling factor 6 is an endogenous inhibitor of prostacyclin synthesis that suppresses cytosolic phospholipase A2 in vascular endothelial cells [4]. We further reported that coupling factor 6 functions as an endogenous vasoconstrictor in the fashion of a circulating hormone [5]. It has been shown that ATP synthase is present on the surface of vascular endothelial cells [6], and more recently we identified the cell surface-associated ATP synthase as the source of circulating coupling factor 6 [7].

Identification of the regulators of this vasoconstrictor peptide promotes further understanding of vascular biology and may lead to therapeutic strategies aimed at blocking the progression of cardiovascular disorders. We recently demonstrated that mechanical forces such as shear stress stimulate the generation and release of coupling factor 6 in vascular endothelial cells. However, the regulation mechanism for circulating coupling factor 6, especially by chemical mediators, is still unknown. Tumor necrosis factor (TNF)-{alpha} is a proinflammatory cytokine [8] that plays an important role in the pathogenesis of cardiovascular disorders including reperfusion injury, left ventricular dysfunction, pulmonary edema, and endotoxin shock in human subjects [9–11]. The variety of these effects is attributable to the ability of TNF-{alpha} to activate multiple signal transductions such as nuclear factor {kappa}B (NF-{kappa}B) pathway [12]. Previously, it was shown that shear stress activates NF-{kappa}B pathway as well as the release of coupling factor 6 in vascular endothelial cells [7,13,14], suggesting that NF-{kappa}B may be present in a common signaling pathway for the release of coupling factor 6. The present study was designed to investigate the effect of TNF-{alpha} on the gene expression and release of coupling factor 6 and the involvement of NF-{kappa}B signaling pathway in its effect.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Cell culture
Primary human umbilical vein endothelial cells (HUVEC) were cultured in HuMedia supplemented with 2% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor, 1 µg/ml hydrocortisone, 5 ng/ml fibroblast growth factor, and 10 µg/ml heparin. ECV-304 (ATCC) were cultured in medium M199 containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (complete medium) at 37 °C under 5% CO2. ECV-304 was used as a cell line of HUVEC. However, it was recently shown that ECV-304 is identical with cells of the bladder carcinoma cell line T-24 [15]. The investigation conformed with the principles outlined in the Declaration of Helsinki.

2.2. Protocol
Confluent ECV-304 and HUVEC monolayers on 100-mm petri dishes were gently washed three times with phosphate buffered saline (PBS) and replaced with 5 ml serum-free medium M199 or HuMedia. One monolayer of the petri dish was kept without TNF-{alpha} (control), and the others were exposed to TNF-{alpha} at 100 and 250 U/ml. The medium was taken for the measurement of coupling factor 6 and characterization of immunoreactive substances, and the cells were used for flow cytometry analysis against the cell surface-associated coupling factor 6, measurement of coupling factor 6 in the cell homogenate, and determination for the gene expression of coupling factor 6.

2.3. Preparation of samples and radioimmunoassay (RIA)
Coupling factor 6 in the medium, the cell homogenate, and the mitochondrial fraction was measured by RIA after partial purification as previously reported [7]. The mitochondrial fraction was separated by centrifugation strategy and its purity was checked by microscopy.

The cross-reactivity of anti-coupling factor 6 antibody was examined by Western blot analysis. Briefly, the cell homogenate was subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) using 16.5% separating gel. Proteins were transferred electrophoretically to a nitrocellulose membrane. The membranes were then treated with anti-coupling factor 6 antibody (1:1000 dilution), and stained by amplified alkaline phosphatase immunoblot kits.

2.4. Characterization of immunoreactive substances in the medium
The immunoreactive species present in the medium were characterized by Western blot analysis. The culture medium obtained after exposure to TNF-{alpha} at 250 U/ml for 24 h was subjected to SDS-PAGE and stained by the immunoblot kit. The plasma membrane and the mitochondrial fractions separated by centrifugation strategy were used as control of coupling factor 6. To further characterize, the samples were applied on reverse-phase high performance liquid chromatography (HPLC) using an Inertsil ODS-2 C18 column (GL Science, Tokyo, Japan), and followed by linear gradient of acetonitrile (20–60%/30 min). Coupling factor 6 in each fraction was measured by RIA.

2.5. Flow cytometry analysis
Confluent ECV-304 and HUVEC were trypsinized, washed, and reacted with saturating concentrations of anti-coupling factor 6 antibody (dilution of 1:1000) in PBS containing 1% BSA for 30 min on ice. After washing three times with PBS, they were stained with FITC-conjugated goat anti-rabbit IgG in PBS for another 30 min, and then analyzed in a FACScan.

2.6. Expression vector
I{kappa}B kinase (IKK) {alpha} cDNA was obtained from human monocytic THP-1 cells by reverse transcription-polymerase chain reaction (RT-PCR). Full-length IKK{alpha} was subcloned into the EcoRI–NotI site of pcDNA3.1(+). Expression vector encoding the dominant negative mutant IKK{alpha} (K44M) was constructed using a QuikChange site-directed mutagenesis kit (Stratagene). Cells were transfected with expression vectors using Effectene Transfection Reagent (Qiagen). Cells were maintained in selective medium containing 200 µg/ml of geneticin until colonies appear.

2.7. Determination of coupling factor 6 mRNA by real-time quantitative RT-PCR
Total RNA was prepared from ECV-304 and HUVEC using the Trizol RNA purification system. The forward and reverse primers were 5'-TCTTCAGAGGCTCTTCAGGTTCTC-3' and 5'-GCCACTGCTGTAACACCAATGT-3', respectively. The TaqMan probe was 5'-TCATTCGGTC-AGCCGTCTCAGTCCAT-3'. It consists of an oligonucleotide with a 5'-reporter dye and a downstream 3'-quencher dye. The fluorescent reporter dye FAM (6-carboxy-fluorescein) is covalently linked to the 5'-end of the oligonucleotide. RT-PCR reaction was carried out in a reaction buffer containing 1 x TaqMan EZ buffer, 3 mM Mn(OAc)2, 300 µM dA/dC/dG/dUTP, 2.5 units rTth DNA polymerase, 200 nM primers, 100 nM TaqMan probe, and 25–50 ng total RNA. RT reaction conditions were 25 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min for 1 cycle; PCR conditions were 95 °C for 15 s, and 58 °C for 1.5 min for 40 cycles on an ABI PRISM 7700 Sequence Detector (Perkin-Elmer Applied Biosystems). Target mRNA content was determined as the average for data points and normalized with to the human housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

2.8. Immunostaining and fluorescence microscopy
The translocation of NF-{kappa}B was investigated by immunostaining. Subconfluent monolayers of ECV-304 and HUVEC were fixed in methanol at –20 °C for 5 min and incubated with 100% goat serum at 4 °C overnight. The specimens were washed three times with PBS, followed by incubation in PBS containing 1% BSA, 0.2% Triton X-100, and polyclonal anti-NF-{kappa}B p65 antibody (1:100, v/v; Santa Cruz Biotechnology) for 2 h at 37 °C. After washing three times with PBS containing 0.2% Triton X-100, the specimens were incubated with FITC-conjugated goat anti-rabbit IgG (1:200, v/v; Sigma) for 2 h at room temperature. The immunostaining was observed under an epifluorescence microscope.

2.9. Western blot analysis of I{kappa}B{alpha}
ECV-304 and HUVEC were suspended in ice-cold lysis buffer (25 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Triton X-100). The supernatant (40 µg/lane) was subjected to SDS-PAGE and stained by the immunoblot kit.

2.10 RIA of 6-keto-prostaglandin (PG)F1{alpha}
Six-keto-PGF1{alpha} was measured by RIA after partial extraction using a specific antibody to 6-keto-PGF1{alpha} as previously reported [5].

2.11. Statistical analysis
All data are shown as mean±1 S.E.M. An unpaired t test for comparison of two variables, one-way ANOVA, ANOVA for repeated measures, and two-way ANOVA for multiple comparisons were used. The level of significance was less than 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. RIA for coupling factor 6
The antiserum to coupling factor 6 detected the peptide with high affinity at a final dilution of 1:1000. Half-maximum inhibition of radioiodinated ligand binding by human recombinant coupling factor 6 was observed at 300 pg/tube. An appropriate amount of cold recombinant coupling factor 6 added to the RIA sample was precisely determined by the present RIA. Recovery rate was more than 90% when the culture sample was treated with a Sep-Pak C-18 cartridge. The intra- and inter-assay coefficients of variance were 8.0% and 10.2%, respectively. As shown in Fig. 1A, the antibody reacted to only one substance (9 kDa) which is compatible with authentic coupling factor 6.


Figure 1
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  		Fig. 1 Cross reactivity of coupling factor 6 antibody (A) and time-dependent release of coupling factor 6 in the absence or presence of TNF-{alpha} at 100 and 250 U/ml in ECV-304 (B) and HUVEC (C). *p<0.05 by two-way ANOVA.

 
As shown in Fig. 1B, in the absence of TNF-{alpha}, the concentration of coupling factor 6 in medium M199 of ECV-304 was increased gradually with time to the levels of 30.5±3.2 ng/dish at 24 h (n=16) and 41.3±4.9 ng/dish at 48 h (n=8). Treatment with TNF-{alpha} at 100 U/ml enhanced the release of coupling factor 6 to the levels of 53.0±9.6 ng/dish at 24 h (n=9) and 112.6±9.8 ng/dish at 48 h (n=3) (p<0.05 vs. without TNF-{alpha} treatment, two-way ANOVA). TNF-{alpha} at 250 U/ml further enhanced the release of coupling factor 6 to the levels of 81.6±10.3 ng/dish at 24 h (n=6) and 215.4±18.6 ng/dish at 48 h (n=6) (p<0.05 vs. treatment with TNF-{alpha} at 100 U/ml, two-way ANOVA).

Fig. 2 shows the time courses of the changes in coupling factor 6 levels in the medium (A) and the cell homogenate (B) during 24 h after treatment with TNF-{alpha}. The concentration of coupling factor 6 in the medium M199 of ECV-304 was increased gradually with time at ≥6 h in the absence and presence of TNF-{alpha}. Treatment with TNF-{alpha} at 250 U/ml enhanced the release of coupling factor 6 compared with that of the control (33.0±0.3 vs. 17.7±1.7 ng/dish at 12 h, p<0.05; 56.5±6.2 vs. 28.7±7.5 ng/dish at 24 h, p<0.05). Coupling factor 6 level in the cell homogenate was 26.0±1.8 ng/dish at baseline and was unchanged until 24 h in the absence of TNF-{alpha} (control) (n=3). In the presence of TNF-{alpha} at 250 U/ml, the level was similar to those of the control until 12 h, but it was decreased at 24 h (p<0.01 vs. baseline) to the level lower than that of the control (15.6±0.6 ng/dish with TNF-{alpha} vs. 32.0±5.8 ng/dish without TNF-{alpha} at 24 h, p<0.05). Coupling factor 6 in the mitochondrial fraction of ECV-304 was 15.0±2.1 ng/dish at baseline and was unchanged until 24 h in the absence of TNF-{alpha}. In the presence of TNF-{alpha} at 250 U/ml, the level was similar to those of the control until 12 h, but it was decreased at 24 h (p<0.05 vs. baseline) to the level lower than that of the control (6.9±0.4 ng/dish, p<0.05). At 48 h after stimulation with TNF-{alpha} at 250 U/ml, coupling factor 6 levels in the cell homogenate and the mitochondrial fraction were similar to those of the control. TNF-{alpha} at 100 U/ml did not affect the amount of coupling factor 6 in the cell homogenate at 24 h (n=3).


Figure 2
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  		Fig. 2 Time courses of coupling factor 6 levels in the medium (A) and in the cell homogenate (B) for 24 h after treatment of ECV-304 with TNF-{alpha} at 250 U/ml. *p<0.05, **p<0.01 vs. baseline.

 
As shown in Fig. 1C, in HUVEC, the concentration of the peptide was 21.6±2.2 ng/dish at 24 h and 52.5±14.4 ng/dish at 48 h (n=6). Treatment with TNF-{alpha} at 100 U/ml enhanced the release of coupling factor 6 to the levels of 61.0±7.3 ng/dish at 24 h (n=6) and 119.2±24.9 ng/dish at 48 h (n=4) (p<0.05 vs. levels without TNF-{alpha} treatment, two-way ANOVA). TNF-{alpha} at 250 U/ml further enhanced the release of coupling factor 6 to the levels of 75.5±7.1 ng/dish at 24 h (n=3) and 210.4±14.5 ng/dish at 48 h (n=3) (p<0.05 vs. levels after TNF-{alpha} at 100 U/ml, two-way ANOVA). In the time course of the change in coupling factor 6 level in the medium of HUVEC, the concentration of coupling factor 6 was increased gradually with time at ≥6 h in the absence and presence of TNF-{alpha}. Treatment with TNF-{alpha} at 250 U/ml enhanced the release of coupling factor 6 compared with that of the control (47.0±11.4 vs. 20.3±2.6 ng/dish at 12 h, p=0.08; 75.5±7.1 vs. 36.6±9.6 ng/dish at 24 h, p<0.05) as seen in ECV-304. Coupling factor 6 level in the cell homogenate was 30.2±2.2 ng/dish at baseline and was unchanged until 24 h in the absence of TNF-{alpha} (n=4). In the presence of TNF-{alpha} at 250 U/ml, the level in the cell homogenate was similar to those of the control until 12 h, but it was decreased at 24 h (p<0.05 vs. baseline) to the level lower than the control (19.6±3.6 ng/dish with TNF-{alpha} vs. 32.3±2.5 ng/dish without TNF-{alpha}, p<0.05). Coupling factor 6 in the mitochondrial fraction was 13.0±1.2 ng/dish at baseline and was unchanged until 24 h in the absence of TNF-{alpha}. In the presence of TNF-{alpha} at 250 U/ml, the level was similar to those of the control until 12 h, but it was decreased at 24 h (p<0.05 vs. baseline) to the level lower than the control (7.7±1.6 ng/dish, p<0.05). Under these conditions, neither trypan blue-positive ECV-304 nor HUVEC was detectable and protein content was unchanged.

3.2 RIA for 6-keto-PGF1{alpha}
In HUVEC grown in 24-well plates, the production of prostacyclin was examined in the serum-free medium after stimulation with TNF-{alpha} in the presence or absence of anti-coupling factor 6 antibody. Prostacyclin synthesis was 354±16 pg/well per 24 h at baseline and 545±27 pg/well per 24 h after treatment with TNF-{alpha} at 250 U/ml. Anti-coupling factor 6 antibody at 0.14 µg/ml significantly enhanced prostacyclin synthesis to 427±15 pg/well per 24 h at baseline and to 719±18 pg/well per 24 h after treatment with TNF-{alpha} at 250 U/ml (both p<0.05).

3.3. Characterization of immunoreactive substances in the medium
As shown in Fig. 3, Western blot analysis revealed a single immunoreactive band in the lanes of the medium after exposure to TNF-{alpha} at 250 U/ml for 24 h in ECV-304 (A) and HUVEC (A'), the membrane in ECV-304 (B) and HUVEC (B'), and the mitochondria in ECV-304 (C) and HUVEC (C'). The molecular weight of these immunoreactive substances was approximately 9 kDa, and was identical to that of authentic coupling factor 6. In HPLC analysis, the retention time of immunoreactive coupling factor 6 separated from the medium in both cell types was identical to that of authentic human coupling factor 6.


Figure 3
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  		Fig. 3 Characterization of immunoreactive substances. Medium after treatment with TNF-{alpha} at 250 U/ml for 24 h in ECV-304 (A) and HUVEC (A'). Membrane fraction of ECV-304 (B) and HUVEC (B'). Mitochondrial fraction of ECV-304 (C) and HUVEC (C').

 
3.4. Flow cytometry
Fig. 4 shows the representative data of FACS for the cell surface-associated coupling factor 6 in ECV-304 and HUVEC. The cell surface-associated coupling factor 6 was increased by 43±3% in ECV-304 and 45±1% in HUVEC at 24 h after treatment with TNF-{alpha} at 100 U/ml (both n=3, p<0.01 vs. baseline) and by 60±1% in ECV-304 and 57±1% in HUVEC at 24 h after TNF-{alpha} at 250 U/ml (both n=3, p<0.01 vs. TNF-{alpha} at 100 U/ml).


Figure 4
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  		Fig. 4 Representative tracings of flow cytometry for the cell surface-associated coupling factor 6 in ECV-304 (A) and HUVEC (B) treated with or without TNF-{alpha} at 100 and 250 U/ml. Solid line: without TNF-{alpha} treatment, dotted lines: with TNF-{alpha} at 100 and 250 U/ml.

 
3.5. Coupling factor 6 gene expression
The expression of coupling factor 6 mRNA was increased after 1-h treatment with TNF-{alpha} at 250 U/ml in both ECV-304 and HUVEC. GAPDH mRNA was unchanged during 12 h. As shown in Fig. 5, the ratio of coupling factor 6 mRNA to GAPDH mRNA was increased by 1.7±0.2 fold in ECV-304 and 1.7±0.1 in HUVEC at 1 h after treatment with TNF-{alpha} (n=3–5, both p<0.05 vs. baseline), and returned to the baseline at 1.5 or 3 h in both cells.


Figure 5
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  		Fig. 5 The ratio of coupling factor 6 to GAPDH mRNA after treatment with TNF-{alpha} at 250 U/ml in ECV-304 (A) and HUVEC (B). *p<0.05. GAPDH=glyceraldehyde 3-phosphate dehydrogenase.

 
3.6. Effect of dominant negative mutant IKK{alpha} (K44M)
The increase in the ratio of coupling factor 6 mRNA to GAPDH mRNA after TNF-{alpha} was suppressed by 100% in ECV-304 and by 70.3±12.5% in HUVEC after the stable transfection of IKK{alpha} (K44M) when compared with that in the cells with transfection of the expression vector (both n=3, p<0.05). The increase in coupling factor 6 release at 12 h after treatment with TNF-{alpha} at 250 U/ml was blocked (p<0.01) in ECV-304 and was suppressed from 19.0±3.8 to 5.8±0.1 ng/dish (p<0.05) in HUVEC when the cells were transfected by the dominant negative mutant IKK{alpha} (K44M).

3.7. Immunostaining of the p65 subunit of NF-{kappa}B
In quiescent ECV-304 and HUVEC, NF-{kappa}B was mainly distributed in the cytoplasm (Fig. 6A, A'), whereas at 30 min after exposure to TNF-{alpha} at 250 U/ml, NF-{kappa}B was mainly localized in the nucleus (Fig. 6B, B'). Antibody specificity was verified by the absence of NF-{kappa}B immunostaining in the control experiments in which nonimmune serum was used instead of the primary antibody. TNF-{alpha} induced the translocation of NF-{kappa}B from the cytoplasm into the nucleus only in the cells transfected by expression vector. In contrast, the stable transfection of the dominant negative mutant IKK{alpha} (K44M) blocked the NF-{kappa}B translocation induced by TNF-{alpha} at 250 U/ml (Fig. 6C, C') without affecting its presence in the cytoplasm in a quiescent condition in HUVEC (Fig. 6D').


Figure 6
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  		Fig. 6 Immunostaining of the p65 subunit of NF-{kappa}B in ECV-304 and HUVEC. TNF-{alpha} (–) in the expression vector-transfected cells in ECV-304 (A) and HUVEC (A'). TNF-{alpha} at 250 U/ml (30 min) in the expression vector-transfected ECV-304 (B) and HUVEC (B'). TNF-{alpha} at 250 U/ml (30 min) in the dominant negative mutant IKK{alpha} (K44M)-transfected ECV-304 (C) and HUVEC (C'). TNF-{alpha} (–) in the dominant negative mutant IKK{alpha} (K44M)-transfected HUVEC (D').

 
3.8. Western blot of I{kappa}B{alpha} protein
As shown in Fig. 7, the immunoreactive band for I{kappa}B{alpha} protein was exhibited in a control (A). Treatment of HUVEC with TNF-{alpha} at 250 U/ml for 30 min led to the degradation of I{kappa}B{alpha} protein (B). In contrast, the stable transfection of the dominant negative mutant IKK{alpha} (K44M) blocked the degradation of I{kappa}B{alpha} protein after treatment with TNF-{alpha} at 250 U/ml for 30 min (C). In ECV-304, treatment with TNF-{alpha} at 250 U/ml for 30 min led to the degradation of I{kappa}B{alpha} protein (D, E).


Figure 7
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  		Fig. 7 Western blot of I{kappa}B{alpha} protein in HUVEC and ECV-304. (A) TNF-{alpha} (–) in HUVEC. (B) TNF-{alpha} at 250 U/ml (30 min) in HUVEC. (C) TNF-{alpha} at 250 U/ml (30 min) in the dominant negative mutant IKK{alpha} (K44M)-transfected HUVEC. (D) TNF-{alpha} (–) in ECV-304. (E) TNF-{alpha} at 250 U/ml (30 min) in ECV-304. The data was reproducible.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The major findings of the present study were that TNF-{alpha} enhanced the gene expression and release of a novel vasoconstrictor coupling factor 6 concomitantly with the increase in cell surface-associated form and that the inhibition of NF-{kappa}B signaling pathway suppressed the gene expression of coupling factor 6 by TNF-{alpha}.

4.1. TNF-{alpha} as a promoter of the release of coupling factor 6
Since mitochondria is a major store site of coupling factor 6, cell damage would be expected to be a major mechanism for the increase in its concentration in the culture medium. However, no cell damage was verified by staining with trypan blue after treatment with TNF-{alpha} at 250 U/ml for 48 h. It is therefore proposed that mechanisms other than cell damage are responsible for the TNF-{alpha}-induced increase in the concentration of coupling factor 6 in the medium. The result clearly demonstrated that TNF-{alpha} increased the cell surface-associated coupling factor 6 and the release of coupling factor 6 into the medium concomitantly with enhancement of the gene expression of coupling factor 6. The time course study showed that the expression of coupling factor 6 mRNA reached the maximum at 1 h and returned to the baseline at ≥1.5 or 3 h, and the release of coupling factor 6 was increased at 12 h without changes in coupling factor 6 in the total intracellular content and the mitochondrial fraction. Furthermore, Western blot analysis showed that the anti-coupling factor 6 antibody reacted to the only one substance of the cell lysate which was identical with mitochondrial coupling factor 6, and that the molecular weight of the immunoreactive substance released from the cells after treatment with TNF-{alpha} was identical with that of mitochondrial coupling factor 6. HPLC analysis also showed that the retention time of the immunoreactive substance was identical to that of authentic coupling factor 6. These indicate that TNF-{alpha} induces de novo protein synthesis of coupling factor 6 and promotes the release of the entire form of coupling factor 6 through the intact cell surface. Coupling factor 6 is first synthesized as an immature form in the cytosol, then led to the mitochondria by an import signal peptide (32 amino acids) present in the upstream [16], and then becomes an active form by the enzymatic deletion of the signal peptide. Therefore, coupling factor 6 seems to be first stored in the mitochondria and then transferred to the cell surface and released. Coupling factor 6 in the mitochondrial fraction did not change until 12 h even in the cells stimulated by TNF-{alpha}. Thus, the influx of coupling factor 6 to the mitochondria and the efflux to the cell surface may be balanced with each other at least until 12 h.

It may be pointed out that the release of coupling factor 6 was increased at 24 h after treatment with TNF-{alpha} at 250 U/ml, while its intracellular content was decreased. The degree of the decrease in the intracellular content was almost equivalent to that of the increase in the medium. It also was almost equivalent to the degree of the decrease in the mitochondrial fraction. These suggest that at 24 h, the efflux of coupling factor 6 from mitochondria is enhanced. Furthermore, the cell surface-associated coupling factor 6 seems to be a source of coupling factor 6 in the culture medium, because TNF-{alpha} increased the cell surface-associated coupling factor 6 at 24 h in a dose-dependent manner and the release of coupling factor 6 into the medium at 24 h was in proportion to the amount of coupling factor 6 on the cell surface.

4.2. Regulation of coupling factor 6 through NF-{kappa}B activation
The effect of TNF-{alpha} is dependent on activation of multiple signal transduction pathways. We focused on the role of NF-{kappa}B signaling pathway in TNF-{alpha}-induced gene expression of coupling factor 6. Stable transfection of the dominant negative mutant IKK{alpha} was established by using selective medium. It was confirmed by the finding that TNF-{alpha}-induced translocation of the NF-{kappa}B active complex to the nucleus was completely abolished in the cells. The expression of coupling factor 6 mRNA reached the maximum at 1 h in the cells without dominant negative mutant, whereas the increase in its gene expression was 70% suppressed in HUVEC with dominant negative mutant. The release of coupling factor 6, a downstream consequence of the gene expression, also was suppressed in HUVEC with dominant negative mutant. The present findings therefore suggest that TNF-{alpha} stimulates the gene expression of coupling factor 6 via activation of NF-{kappa}B signaling pathway not fully but in large part in HUVEC, and that other pathways including mitogen-activated protein kinases, Jun NH2-terminal kinase, and/or signal transducer and activator of transcription [12] also may contribute to upregulation of coupling factor 6 by TNF-{alpha} in HUVEC but in small part. Further examination will be needed to elucidate this issue.

4.3. Implications of linkage between TNF-{alpha} and coupling factor 6
TNF-{alpha} is a proinflammatory cytokine and induces apoptosis of cardiomyocytes, left ventricular dysfunction, and endotoxin shock when overexpressed in human subjects [9–11]. Recently, apoptosis has become increasingly recognized as one mechanism of cell death after myocardial infarction, and TNF-{alpha} is postulated as a mediator for the paracrine apoptosis in its condition [17]. TNF-{alpha}-induced increase in coupling factor 6 would contribute to impaired coronary circulation and exacerbation of myocardial infarction. Indeed, we recently showed that coupling factor 6 is a novel risk factor for ischemic heart disease in patients with end-stage renal disease [18]. The level of circulating TNF-{alpha} is elevated in patients with advanced congestive heart failure. In addition to its negative inotropic effects [19], TNF-{alpha}-induced increase in coupling factor 6 may contribute to the exacerbation of congestive heart failure. On the other hand, previous evidence showed that TNF-{alpha} stimulates ceramide and prostacyclin generation to induce vasodilation, which is responsible for the marked hypotension seen in septic shock [20–23]. This seems inconsistent with our findings. However, treatment with anti-coupling factor 6 antibody increased the release of prostacyclin. Thus, the effect of TNF-{alpha} on the release of coupling factor 6 may provide a new implication for the pathogenesis of septic shock.

Because ECV-304 are identical with human tumor cells, the results derived from ECV-304 may not be applied to vascular biology but tumor cell biology. The results obtained with ECV-304 were almost similar with those of HUVEC. Therefore, the effect of TNF-{alpha} on the release of coupling factor 6 is also found in the vascular system.

In conclusion, the present study first provides the evidence that TNF-{alpha} is a potent endogenous stimulator for vasoconstrictor coupling factor 6. This may promote further understanding of vascular biology and presumably pathogenesis of cardiovascular disorders.


   Notes
 
Time for primary review 16 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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T. Osanai, H. Tomita, M. Kushibiki, M. Yamada, M. Tanaka, T. Ashitate, T. Echizen, C. Katoh, K. Magota, and K. Okumura
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