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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1506.)
© 2000 American Heart Association, Inc.

Vascular Biology

Nitric Oxide Enhances Expression and Shedding of Tumor Necrosis Factor Receptor I (p55) in Endothelial Cells

Masaki Okuyama; Seiji Yamaguchi; Minako Yamaoka; Joji Nitobe; Satoshi Fujii; Tetsuhiko Yoshimura; Hitonobu Tomoike

From the First Department of Internal Medicine (M.O., S.Y., M.Y., J.N., H.T.), Yamagata University School of Medicine, and the Institute for Life Support Technology (S.F., T.Y.), Yamagata Technopolis Foundation, Yamagata, Japan.

Correspondence to Seiji Yamaguchi, MD, First Department of Internal Medicine, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan. E-mail syamaguc{at}med id. yamagata-u.ac.jp

	Abstract

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Abstract—The biological actions of tumor necrosis factor- (TNF-) are mediated by 2 distinct receptors, TNF-RI (p55) and TNF-RII (p75). The extracellular domains of both receptors are shed in soluble form (sTNF-RI and sTNF-RII). The soluble receptors are involved in regulating TNF- activities and may have therapeutic potential as TNF-neutralizing agents. However, it remains unclear as to what kind of physiological molecule can regulate TNF receptors. Nitric oxide (NO) mediates a variety of biological and pathophysiological functions. We hypothesized that NO may modulate the expression and shedding of TNF-RI. An NO donor, diethylamine/NO complex (NOC 5), increased sTNF-RI in the supernatants of ECV304, a human umbilical vein cell line, in a dose-dependent manner. TNF-RI mRNA in these cells was upregulated by NOC 5. 8-Br-cGMP and peroxynitrate had no effect on sTNF-RI release. Genistein and herbimycin A, inhibitors of tyrosine kinase, inhibited sTNF-RI release. Herbimycin A inhibited the levels of TNF-RI mRNA enhanced by NOC 5, which downregulated the surface expression of TNF-RI, indicating that NO is also involved in the shedding process of TNF-RI. The shedding of TNF-RI was abolished by a synthetic inhibitor of matrix metalloproteinase, KB-R8301. In conclusion, NO enhanced the release of sTNF-RI from endothelial cells by a cGMP-independent mechanism. Dual pathways suggested for NO-induced sTNF-RI release include (1) enhanced expression of TNF-RI, at least partially, by a tyrosine kinase–dependent mechanism and (2) increased shedding of TNF-RI by a type of metalloproteinase.

Key Words: nitric oxide • TNF- • TNF receptor (p55) • human endothelial cells • tyrosine kinase inhibitor

	Introduction

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Tumor necrosis factor- (TNF-) is a key mediator in immune and inflammatory responses. Recently, TNF- has been detected in human cardiac disorders, including congestive heart failure^{1 2 3} and atherosclerosis.^{4 5} It is known that this proinflammatory cytokine can produce cardiac cachexia,¹ negative inotropic effects in myocytes,^{6 7} ventricular remodeling,^{8 9} and promotion of atherosclerosis.^{4 10} The biological actions of TNF- are mediated by 2 distinct receptors, TNF-RI (p55) and TNF-RII (p75), which have been identified on various cells.¹¹

The extracellular domain fragments of both receptors are shed by proteolytic cleavage from the cell surface and can be detected in soluble form (sTNF-RI and sTNF-RII) in mammalian blood and urine.¹² The soluble TNF receptors attenuate TNF- bioactivity by competing with the cell surface receptors for TNF-,^{13 14} and they may have therapeutic potential as TNF-neutralizing agents.^{15 16} Serum levels of soluble TNF receptors are increased in relation to the severity of a variety of diseases, such as congestive heart failure.^{17 18 19} Although several chemical substances have been shown to influence the expression and shedding of TNF receptors,^{20 21 22} it has not been rigorously examined as to what kind of physiological molecule can regulate TNF receptors.

Nitric oxide (NO) is a free radical produced by a variety of cell types and is involved in various biological and pathobiological processes, including vasorelaxation,²³ regulation of platelet activities,²⁴ antiatherogenic effects,²⁵ and negative inotropic effects on cardiac myocytes.⁶ NO also plays a role as a messenger molecule involved in inflammatory and immune reactions.²⁶ Thus, any alteration in NO production is critical to the progression or regression of a variety of diseases.^{27 28 29} Plasma nitrite and nitrate, the stable end products of NO production, have been reported to be elevated in patients with disorders such as congestive heart failure.^{30 31} It has simply been hypothesized that NO modulates the expression and shedding of TNF receptors.

In the present study, we examined the effects of NO on the expression and shedding of TNF-RI in ECV304 endothelial cells, which is a spontaneously transformed human umbilical vein endothelial cell (HUVEC) line.^{32 33 34} We used a diethylamine/NO complex (NOC 5), which served as an NO donor and which controls the amount of NO released by its structural modification.^{35 36} The specific objectives of this study were as follows: (1) to examine whether NO increases sTNF-RI levels in ECV304 cell supernatants and upregulates TNF-RI mRNA levels in these cells; (2) to test whether cGMP, peroxynitrate (the product of NO^· and O₂^-), or NO₂^- (the stable end product of NO) alters sTNF-RI release in cell culture; (3) to determine whether a tyrosine kinase is related to TNF-RI expression; and (4) to determine whether NO enhances the shedding of TNF-RI on the cell surface membrane and, if so, to test whether the increased shedding of TNF-RI is regulated by the activities of a metalloproteinase or a group of related metalloproteinases.

	Methods

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Cell Culture and Reagents
ECV304 is a spontaneously immortalized EC line from human umbilical vein^{32 33 34} and was obtained from the Health Science Research Resources Bank (Osaka, Japan). ECV304 cells were passaged once a week in medium 199 (Gibco BRL) supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified incubator containing 5% CO₂ in air at 37°C. HUVECs were purchased from Clonetics Co (Walkersville, Md). HUVECs were passaged in endothelial basal culture medium (Clonetics Co), which was supplemented with 3 mg/mL bovine brain extract, 10 mg/mL human epidermal growth factor, 1 mg/mL hydrocortisone, 5% fetal bovine serum, 50 mg/mL gentamicin, and 50 mg/mL amphotericin, and were grown as described above. NOC 5 (T_1/2 25 minutes) as the NO donor and peroxynitrite liquid were purchased from Dojindo Laboratories. Phorbol 12-myristate 13-acetate (PMA) and 8-Br-cGMP were obtained from Sigma Chemical Co. Herbimycin A was purchased from Research Biochemicals International, genistein was from LC Laboratories, and NaNO₂ was from Wako. Oxyhemoglobin was from Calzyme Laboratories, and KB-R8301, a synthetic hydroxamate inhibitor of matrix metalloproteinase (MMP) ([4-(N-hydroxamino)-2R-isobutyl-3S-methylsuccinyl]-L-3-(5,6,7,8-tetrahydro-1-naphthyl)alanine-N-methylamide),³⁷ was a gift from Kanebo Ltd, Osaka, Japan.

Determination of NO_x (NO₂^-/NO₃^-) and NO^·
NO concentrations in the culture media were evaluated by both the Griess reaction and electron paramagnetic resonance spectral methods. Samples were applied to a copper-coated cadmium reduction column, and NO_x was determined by the Griess method with the use of an autoanalyzer³⁸ (ENO-10, Eicom Corp). Unstable NO (ie, NO^·) was measured by an electron paramagnetic resonance spectral method with a spin-trapping technique. Fe-N-(dithiocarboxy)sarcosine was used as a spin-trapping reagent for NO^· owing to its high solubility in water.^{39 40}

Cell Stimulation and Determination of sTNF-RI Levels in Culture Media
After ECV304 cells were grown to confluence in the 24-multiwell plates (Becton Dickinson Labware), the cells were stimulated with several doses of NOC 5 in fresh medium. When necessary, reagents were added to the culture dishes 1 hour before stimulation with NOC 5. The volume of the medium in each well was 500 µL. After treatment at 37°C in the CO₂ incubator, culture supernatants were collected and centrifuged at 1500 rpm for 5 minutes at 4°C and stored at -20°C until assay for sTNF-RI. The levels of sTNF-RI in the culture supernatant were determined by using an ELISA kit purchased from R&D Systems¹⁹ and following the manufacturer’s instructions.

TNF-RI mRNA
Total cellular RNA from ECV304 cells was isolated by the acid guanidinium thiocyanate–phenol-chloroform method with RNasol (Cinna/Biotex Laboratories). RNA (15 µg per lane) was electrophoresed in 1.2% agarose gel containing 3 mol/L formaldehyde in MOPS buffer, transferred to a nylon membrane (Nytran, Schleicher and Schuell), and hybridized with a ³²P-labeled TNF-RI probe generated by the random-priming method. The probe for TNF-RI was a 304-bp cDNA fragment encompassing the HindIII site to the EcoRI site of the human TNF-RI cDNA.⁴¹ Membranes were washed sequentially in 2x SSC/0.1% SDS and 0.5x SSC/0.1% SDS at room temperature for 15 minutes and finally in 1x SSC/0.1% SDS at 65°C for 15 minutes. Blots were then exposed to film (XAR-5, Eastman Kodak Co) with an intensifying screen (Eastman Kodak Co) for 1 week at -70°C. Levels of TNF-RI mRNA were normalized to RNA loading and expressed in relative densitometric units with respect to control values.

Flow Cytometry
After being grown to confluence (2x10⁶ cells per well), ECV304 cells were stimulated with 0.1 mmol/L NOC 5 and 10 ng/mL PMA for 6 hours. After stimulation, the cells were detached by exposure to 0.5% trypsin/0.2% EDTA for 2 minutes and then rinsed twice with 10% fetal calf serum in PBS. Suspended cells were incubated with either a monoclonal antibody against mouse anti-human TNFR (p60, Genzyme) or a nonspecific mouse IgG1 (Becton Dickinson Immunocytometry Systems). The cells (5x10⁵) were incubated with the antibody (1 mg/mL) for 30 minutes on ice, washed twice, and incubated with rabbit anti-mouse IgG labeled with FITC (Dako) for 30 minutes on ice. The cells were then washed again, resuspended in 500 µL of PBS, and analyzed on a FACSCalibur (Becton Dickinson) equipped with CellQuest software. The mean fluorescence intensity of a cell population was determined in single-parameter histograms (units of detection, channel number). Cell viability was monitored by staining with trypan blue and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.

Statistical Analysis
All data are expressed as mean±SEM or as percentages of control of the indicated number of observations. Statistical comparisons between groups were performed with Students’ t test or 1-way ANOVA, followed by a post hoc test (Fisher’s t test) as appropriate. Differences among means were considered significant when P<0.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
sTNF-RI is spontaneously released into the culture supernatants of ECV304 cells. To investigate whether NO contributes to the release of sTNF-RI in ECV304 cell supernatants, cell cultures were treated with NOC 5 for 6 hours (Figure 1). In a concentration-dependent manner, NOC 5 increased sTNF-RI levels: sTNF-RI levels were significantly increased with treatments of 0.1 and 0.5 mmol/L NOC 5. When the cells were preincubated with oxyhemoglobin (1.0 mg/mL), which traps NO in the supernatants, the effect of NOC 5 on sTNF-RI levels was completely abolished (Figure 1). NO_x released from NOC 5 reached a plateau 2 hours after incubation in the culture supernatants (with 0.1 mmol/L, 76±4 µmol/L). NO^· level measured by the electron paramagnetic resonance method in the supernatant was increased 2 hours after NOC 5 treatment, from <5 to 44±2 µmol/L. At the tested concentrations of NOC 5, >98% by trypan blue staining and 97% by the MTT assay of the cells were viable.

View larger version (14K): [in this window] [in a new window]   Figure 1. NO donor–induced sTNF-RI release in cell supernatants. ECV304 cells were incubated with NOC 5 (0.01 to 0.5 mmol/L) for 6 hours. ECV304 cells were also incubated with oxyhemoglobin (Oxy-Hb; 1.0 mg/mL) in the presence of NOC 5 (0.1 mmol/L). An aliquot of culture supernatant was analyzed for sTNF-RI by ELISA. Results are expressed as mean±SEM of 3 separate experiments. *P<0.05 vs control.

NOC 5 (0.1 mmol/L) markedly upregulated expression of TNF-RI mRNA in ECV304 cells in a time-dependent manner (Figure 2). When the densitometric band intensities were normalized to 28S rRNA ethidium bromide staining, the NO donor time-dependently upregulated the expression of TNF-RI mRNA (relative densitometric units, 245±13% and 328±11% at 6 and 12 hours’ incubation, respectively; P<0.01). PMA as a positive control also upregulated TNF-RI mRNA levels in the cells.

View larger version (38K): [in this window] [in a new window]   Figure 2. Northern blot analysis showing time-dependent effect of NOC 5 on TNF-RI mRNA levels of ECV304 cells. Cells were incubated with medium alone (control), PMA (10 ng/mL), and NOC 5 (0.1 mmol/L). Lower panel shows corresponding ethidium bromide staining for 28S and 18S rRNA.

To determine whether cGMP, a second messenger of NO, can mimic the effect of NO on the release of sTNF-RI, ECV304 cells were incubated with 8-Br-cGMP for 6 hours. sTNF-RI levels were unaffected by 0.01, 0.1, and 1.0 mmol/L 8-Br-cGMP (respectively, 98±2%, 98±3%, and 93±2% compared with control; NS). Similarly, the release of sTNF-RI was not affected by 0.01, 0.1, and 1.0 mmol/L ONOO^-, which rapidly permeates cell membranes⁴² (respectively, 98±2%, 103±3%, and 95±1% of control; NS). NO₂^-, an end product of NO, also had no effect on sTNF-RI levels (NS).

As shown in Figure 3, genistein inhibited the spontaneous release of sTNF-RI in a dose-dependent manner and also inhibited the release of sTNF-RI induced by NOC 5. Similar inhibitory effects were observed for the release of sTNF-RI after treatment with herbimycin A (Figure 3). To examine the modulation of TNF-RI mRNA by tyrosine kinase, ECV304 cells were treated with herbimycin A (Figure 4). Herbimycin A downregulated TNF-RI mRNA levels in unstimulated cells. TNF-RI mRNA levels upregulated by NOC 5 were attenuated after treatment with herbimycin A (P<0.01).

View larger version (20K): [in this window] [in a new window]   Figure 3. Effects of tyrosine kinase inhibitors on release of sTNF-RI. ECV304 cells were preincubated with genistein or herbimycin A for 1 hour. After preincubation, cell cultures were incubated with or without NOC 5 (0.1 mmol/L) for 6 hours. An aliquot of culture supernatant was analyzed for sTNF-RI by ELISA. Percentage of sTNF-RI concentrations in supernatants compared with control cells is shown. Results are expressed as mean±SEM of 3 separate experiments. *P<0.05, **P<0.01 vs control.

View larger version (36K): [in this window] [in a new window]   Figure 4. ECV304 cells were preincubated with herbimycin A for 1 hour. After preincubation, cell cultures were incubated with or without NOC 5 (0.1 mmol/L) for 6 hours. Lower panel shows corresponding ethidium bromide staining for 28S and 18S rRNA.

As shown in Figure 5, cell surface expression of TNF-RI was downregulated after 6-hour incubation with 0.1 mmol/L NOC 5 (mean fluorescence intensity, 44.1±5.0 to 12.5±2.5; P<0.01), indicating enhanced shedding of TNF-RI from the cell membrane by the NO donor. As previously reported,²⁰ PMA (10 ng/mL) also downregulated cell surface expression of TNF-RI (mean fluorescence intensity, 44.1±5.0 to 14.7±1.8; P<0.01). Similar results were observed after a 2-hour incubations with NOC 5 and PMA (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 5. NO reduced TNF-RI surface expression on ECV304 cells. ECV304 cells were incubated with NOC 5 (0.1 mmol/L) and PMA (10 ng/mL) for 6 hours. Cells were stained with mouse anti-human TNFR (p60) monoclonal antibody (dotted line) and with nonspecific IgG1 (solid line).Cells were then incubated with rabbit anti-mouse IgG labeled with FITC and analyzed by flow cytometry.

To investigate whether an MMP-like enzyme is involved in the shedding of TNF-RI from ECV304 cells, they were pretreated with a synthetic inhibitor of MMP, KB-R8301 (Figure 6). The spontaneous shedding of TNF-RI was inhibited with 1 and 10 µmol/L KB-R8301. Furthermore, an NO-induced increase in sTNF-RI levels was also inhibited with KB-R8301, suggesting that a kind of metalloproteinase is involved in NO-induced shedding of TNF-RI. At 1 and 10 µmol/L KB-R8301, >98% and 97% of cells, respectively, were viable by trypan blue staining. At similar concentrations, 96% and 84%, respectively, of cells were viable as assessed by MTT assay.

View larger version (11K): [in this window] [in a new window]   Figure 6. Effect of KB-R8301 on release of sTNF-RI. ECV304 cells were preincubated with KB-R8301, a synthetic hydroxamate inhibitor of MMP (1 and 10 µmol/L), for 1 hour; cell cultures were then incubated with or without NOC 5 (0.1 mmol/L) for another 6 hours. An aliquot of culture supernatant was analyzed for sTNF-RI by ELISA. Percentage of sTNF-RI concentrations in supernatants compared with control is shown. Results are expressed as mean±SEM of 3 separate experiments. *P<0.05 vs control.

We investigated whether NO induces the release of sTNF-RI in HUVECs. HUVECs were treated with NOC 5 in the same way that ECV304 cells were treated. sTNF-RI levels were significantly increased, by 25% and 62%, respectively, with 0.1 and 0.5 mmol/L NOC 5 (P<0.01).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
A wide spectrum of TNF biological activity is mediated by 2 structurally related but functionally distinct receptors, TNF-RI (p55) and TNF-RII (p75), present on most cell types.¹¹ The responsiveness of the cell to TNF- is modified by the expression of TNF-RI and TNF-RII. Furthermore, TNF receptors can be proteolytically shed, and the shedding of these receptors may have some physiological implications. First, reduction in the number of receptors on the cell surface could temporarily render unresponsiveness to TNF-. Second, soluble TNF receptors bind circulating TNF- and thereby inhibit its ability in TNF receptor–bearing cells.¹³ It has been demonstrated that the levels of TNF receptors in tissue are modulated^{19 43 44} and that circulating levels of soluble TNF receptors are increased in a variety of diseases, such as congestive heart failure.^{17 18} Consequently, there has been some recent focus on natural physiological molecules that regulate the expression and shedding of TNF receptors.

Expression of TNF-RI in Endothelial Cells With NO
In general, TNF-RI gene expression is believed not to respond to biological substances¹¹ ; the TNF-RI mRNA level is upregulated by only a few substances, such as phorbol diesters⁴¹ and interleukin-1.⁴⁵ Thus, it has been believed that expression of TNF-RI is constitutive whereas TNF-RII is inducible.¹¹ It has remained unclear how TNF-RI can be regulated in a variety of disorders. In this study, NO enhanced mRNA transcripts of TNF-RI in ECV304 cells.

Various effects of NO have been attributed to cGMP produced by soluble guanylate cyclase. In this study, the membrane-permeable cGMP analogue 8-Br-cGMP was unable to mimic the effect of NO on the release of sTNF-RI in ECV304 cell supernatants, suggesting that NO regulates TNF-RI in ECs by a cGMP-independent mechanism. ONOO^- is an important member of the reactive oxygen and nitrogen species and can be rapidly formed from NO^· and O₂^-. The reaction of ONOO^- with biological substrates and the subsequent signal molecule have been investigated.⁴⁶ It is also possible that a radical, ONOO^-, rather than NO could be directly responsible for the sTNF-RI release. However, sTNF-RI levels in cell culture were not affected by ONOO^-, which has a high diffusibility through phospholipid membranes.⁴² The molecular mechanisms involved in the regulation of TNF-RI by NO may be related to other signaling pathways, such as protein tyrosine kinases.

The cellular signaling mechanisms responsible for the expressions of TNF-RI remain unclear. In this study, tyrosine kinase inhibitors attenuated sTNF-RI release and TNF-RI mRNA levels in ECV304 cells. Genistein inhibited spontaneous and NO-induced sTNF-RI release at 1 to 10 µg/mL. Furthermore, in addition to the inhibition of sTNF-RI release, herbimycin A (100 to 1000 nmol/L) inhibited TNF-RI mRNA transcripts in cells left untreated or treated with an NO donor. To our knowledge, this is the first study to demonstrate that expression of TNF-RI is sensitive to a tyrosine kinase inhibitor. Tyrosine protein kinases may be involved in the signaling pathway of TNF-RI gene expression. Several nonreceptor protein kinases may be activated by NO, which is suggested by the fact that reactive oxygen species have been shown to activate several tyrosine kinases in addition to c-Src.^{47 48} We do not know the specific tyrosine kinase that may be responsible for initiating downstream events. As downstream molecules of tyrosine kinase molecules, stress-activated protein kinase (SAPK; also termed c-Jun N-terminal kinase) may be activated and trigger the cascade for TNF-RI expression. Pfeilschifter and Huwiler⁴⁹ reported that in ECs and mesangial cells, an NO donor activated SAPK in a time- and concentration-dependent manner but that dibutyryl cGMP had no effect on SAPK activity. Furthermore, tyrosine kinase inhibitor attenuated NO-induced c-Jun phosphorylation. These lines of data seem to be concordant with our findings that TNF-RI was expressed in a cGMP-independent fashion and that a tyrosine kinase inhibitor attenuated TNF-RI gene expression.

The increased transcription of TNF-RI might occur due to shedding of TNF-RI per se, rather than to stimulation by NO. Cleavage of the receptors from the cell surface may affect the cytoplasmic membrane and residual receptors, resulting in initiation of a signaling pathway, ie, the tyrosine kinase–dependent pathway. Identification of the signaling for TNF-RI expression and target molecules to the signaling triggered by NO will offer new approaches for therapeutic intervention in a variety of diseases.

Shedding of TNF-RI in ECs With NO
Flow-cytometric analysis showed enhanced shedding of TNF-RI on ECV304 cells after treatment with an NO donor. Thus, enhanced shedding of TNF-RI as well as the increased expression of TNF-RI contributed to the release of sTNF-RI into cell supernatants after treatment with an NO donor. The shedding process of cell-surface cytokine receptors is of great interest, as it reduces the pathogenic effects of the cytokine. Recently, it has been shown that a synthetic, broad-spectrum MMP inhibitor curtailed the shedding of TNF receptors.^{19 50 51} In this study, both spontaneous and NO-induced releases of sTNF-RI were abrogated by a hydroxamate inhibitor of MMP, KB-R8301. Thus, release of the extracellular domain of TNF-RI (sTNF-RI) appears to be induced by a kind of metalloproteinase. Metalloproteinases are generally produced as inactive zymogens that are subsequently activated by other enzymes. Thus, NO-induced enzymatic activity could attack the signaling cascade of the specific protease at any point, not necessarily at the most distal point, and would subsequently result in the enhancement of shedding of sTNF-RI.

PMA is involved in the shedding of TNF-RI through a protein kinase C–dependent pathway.^{52 53} However, it is unlikely that NO activates protein kinase C and the subsequent activation of a protease for TNF-RI shedding. NO and NO-generating agents have been reported to inactivate protein kinase C.⁵⁴ Furthermore, NO-induced release of sTNF-RI was not attenuated by the protein kinase C inhibitor calphostin C (our unpublished data), suggesting that NO may not interact with molecules that activate protein kinase C. Thus, there may be another enzymatic cascade leading to the shedding of TNF-RI.^{55 56} Different signaling pathways activated by different stimuli may converge and may lead to the final proteolytic activity for the shedding of TNF-RI. TNF- on the cell membrane is processed by TNF-–converting enzyme, which is a member of the A disintegrin and metalloproteinase family.⁵⁶ TNF-–converting enzyme also cleaves extracellular domains of other receptors and ligands, such as TNF-RII (p75), but not TNF-RI (p55).⁵⁷ The cloning of a protease for TNF-RI shedding will cast light on the intriguing possibility of therapeutic targets for the TNF/TNF receptor system.

In summary, NO upregulates TNF-RI gene expression, at least in part, through tyrosine kinase, and NO sheds the extracellular domain of TNF-RI through a kind of metalloproteinase in human ECs. Taken together, these data indicate that NO can increase the release of a soluble form of TNF-RI.

	Acknowledgments

 
This study was supported part by grants 07670750, 08457200, and 10307016 from the Ministry of Education, Science and Culture, Japan.

	Footnotes

 
Part of this study was presented at the 71st Scientific Sessions of the American Heart Association, Dallas, Tex, November 8–11, 1998, and published in abstract form (Circulation. 1998;98:I-720).

Received July 19, 1999; accepted March 2, 2000.

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