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production through inhibition of Egr-1 expression
Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Submitted 27 August 2004 ; accepted in final form 8 November 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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- production, the detailed mechanism(s) remains unclear. This study was undertaken to elucidate the molecular mechanism(s) underlying this inhibitory effect in monocytes in vitro and in rats given lipopolysaccharide (LPS). TNF- production by monocytes stimulated with LPS (100 ng/ml) was inhibited by UTI at concentrations higher than 100 U/ml. Expression of early growth response factor-1 (Egr-1) and phosphorylation of extracellular signal-regulated protein kinases 1/2 in monocytes stimulated with LPS were inhibited by UTI. UTI (50,000 U/kg iv) inhibited LPS (5 mg/kg iv)-induced increases in lung tissue levels of Egr-1, TNF- mRNA, and TNF- in rats. UTI inhibited LPS-induced hypotension by inhibiting pulmonary induction of inducible nitric oxide synthase (iNOS). We previously demonstrated that anti-TNF- antibody and aminoguanidine, a selective inhibitor of iNOS, reduced LPS-induced hypotension in this animal model. Furthermore, we also reported that reduction of LPS-induced coagulation abnormalities in rats did not affect inflammatory responses and hypotension in this animal model. Taken together, these observations strongly suggested that UTI inhibited LPS-induced production of TNF- by inhibiting activation of the extracellular signal-regulated protein kinases 1/2-Egr-1 pathway in monocytes, which might at least partly contribute to reduction of hypotension through inhibition of iNOS induction in rats given LPS.
septic shock; lipopolysaccharide; early growth response factor-1; inducible nitric oxide synthase
(19).
The excessive production of TNF- in endotoxemia contributes to the induction of the inducible form of nitric oxide (NO) synthase (iNOS) in various cells, including endothelial cells and smooth muscle cells, resulting in excessive formation of NO, which in turn causes hypotension and peripheral vasodilatation (25). TNF- also contributes to the development of various types of organ failure, such as acute respiratory distress syndrome, by activating neutrophils (10). Activated neutrophils damage endothelial cells by releasing a wide variety of inflammatory mediators such as neutrophil elastase and oxygen free radicals that are capable of damaging endothelial cells (27).
Urinary trypsin inhibitor (UTI) is one of the Kunitz-type protease inhibitors found in urine (9). UTI is synthesized from inter--trypsin inhibitor through proteolytic cleavage by neutrophil elastase at the site of inflammation (6). Various serine proteases such as trypsin, chymotrypsin, neutrophil elastase, and plasmin are inhibited by UTI (13). Based on the multivalent nature of protease inhibition, UTI appears to prevent organ injury by inhibiting the activity of these proteases (14, 15).
UTI has been shown to prevent LPS-induced hypotension and improve the survival rate in various animal models of sepsis (18, 24, 29). Although Aosasa et al. (1) previously reported that UTI inhibited TNF- production in LPS-stimulated monocytes, the detailed molecular mechanism(s) underlying this inhibitory effect remains to be elucidated.
In the present study, we analyzed the molecular mechanism(s) by which UTI inhibits TNF- production in isolated human monocytes stimulated with LPS in vitro to determine whether UTI reduces LPS-induced hypotension by inhibiting TNF- production in rats administered LPS.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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UTI was a generous gift from Mochida Pharmaceutical (Tokyo, Japan). One unit of UTI defines the amount of UTI that inhibits the activity of 2 µg of trypsin and is equivalent to 0.4 µg of UTI. LPS (Escherichia coli, serotype 055:B5) was purchased from Difco (Detroit, MI). Antibodies against human extracellular signal-regulated protein kinases (ERK)1/2 and phosphorylated ERK1/2 were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against early growth response factor-1 (Egr-1) and PU.1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All reagents used were of analytical grade.
In Vitro Experiments
Monocyte preparation and incubation. Peripheral blood mononuclear cells were isolated from the buffy coat obtained from a single healthy volunteer blood donor in one experiment as described previously (26). Each experiment was repeated three times using independent mononuclear cell preparations from different donors. Mononuclear cells were adjusted to an appropriate volume and cultured in RPMI 1640 (Invitrogen, Grand Island, NY) plus 1% supplemented calf serum (Hyclone, Logan, UT) at 37°C in a humidified 5% CO2 incubator. Various concentrations of UTI were added to cells at 30 min before LPS (100 ng/ml) stimulation.
Measurement of TNF-.
Human monocytes (5 x 105 cells/assay) were stimulated with LPS in the presence or absence of UTI. Concentrations of TNF- in culture media were determined using an ELISA kit for human TNF- (Biosource International, Camarillo, CA).
Western blot analysis. Human monocytes (2 x 106 cells/assay) were stimulated with LPS for various times in the presence or absence of UTI. Whole cell lysates were collected as described previously (31). Samples containing equal amounts of protein were separated using SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Membranes were incubated with appropriate antibodies against human ERK1/2, phosphorylated ERK1/2, PU.1, and Egr-1 at 4°C overnight and subsequently with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Specific proteins were visualized using an enhanced chemoluminescence system (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). Because the level and the DNA-binding activity of PU.1 in monocytic cells were not affected by LPS stimulation, PU.1 was used as a control for Egr-1 expression (12). ERK1/2 was detected as a control for phosphorylated ERK1/2.
Cell viability. Isolated monocytes were stimulated with LPS in the presence or absence of UTI (1,000 U/ml). The number of monocytes was counted at 6 h after stimulation with LPS. Cell viability was evaluated using the trypan blue dye exclusion method (26).
Detection of DNA-binding activities of p65, c-Fos, and Egr-1. Human monocytes (1 x 107 cells/assay) were stimulated with LPS for 1 h in the presence or absence of UTI. Nuclear extracts were prepared as described previously (31). DNA-binding activities of p65 and c-Fos were evaluated using an ELISA-based kit (Trans AM, Active Motif, Carlsbad, CA) as described previously (31). Double-stranded oligonucleotides containing the sequences corresponding to the Egr-1 consensus site (5'-GGATCCAGCGGGGGCGAGCGGGGGCGA-3' 3'-CCTAGGTCGCCCCCGCTCGCCCCCGCT-5') and Sp1 consensus site (5'-ATTCGATCGGGGCGGCGGGGCGAGC-3' 3'-TAAGCTAGCCCCGCCCCGCTCG-5') were 3' end labeled with digoxigenin. Specific binding of Egr-1 and Sp1 to their DNA consensus oligonucleotides was analyzed by the electrophoretic mobility shift assay as described previously (4, 31). Since DNA-binding activity of Sp1 in monocytic cells was not affected by LPS stimulation, DNA-binding of Sp1 was used as a control for DNA-binding of Egr-1 (4).
In Vivo Experiments
Animal model of LPS-induced hypotension. A nonlethal rat model of LPS-induced hypotension was produced as described previously (8). The study protocol was approved by the Kumamoto University School of Medicine Animal Care and Use Committee, and the care and handling of the animals were in accordance with the guidelines of the National Institutes of Health. Specific pathogen-free male Wistar rats weighing 220–280 g were obtained from Kyudo (Kumamoto, Japan). Hypotension was induced in rats by intravenous administration of 5 mg/kg LPS. UTI (50,000 U/kg) was administered intravenously 30 min before or 15 min after LPS administration. Control animals were treated similarly except that they received saline instead of LPS. Animals given either saline or UTI survived for 7 days after LPS administration.
Measurement of lung levels of TNF-.
Lung tissue levels of TNF- were measured using an ELISA kit for rat TNF- (Genzyme, Cambridge, MA) as described previously (8).
Isolation of RNA, and Northern blotting.
Total RNA from rat lungs was prepared by the acid-guanidium-phenol-chloroform extraction procedure (7). Northern blotting of iNOS mRNA and TNF- mRNA was performed as previously described (7).
Western blot analysis of Egr-1 in the lung tissue. Whole cell extracts were prepared from lung tissue by homogenization in 10 mM Tris·HCl buffer (pH 7.5) containing 400 mM NaCl, 1 mM dithiotheriotol, and 10% glycerol. The Tris buffer also contained phosphatase and protease inhibitors. Protein concentrations in the extracts were determined using a protein assay reagent (Bio-Rad, Hercules, CA). Ten micrograms of protein were charged in each well, separated by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. Western blot analysis was performed as described above using an anti-rat Egr-1 antibody.
Measurement of lung level of iNOS activity. The lungs were removed from rats after perfusion with saline through the right cardiac ventricle and homogenized on ice in HEPES buffer (pH 7.5, 30 mM). Lung levels of iNOS activity were measured as described previously (8).
Measurement of plasma levels of NO2–/ NO3–.
NO
and NO
are the primary oxidized products of NO, generated on reaction with water, and therefore the total concentration of NO/NO in plasma was used as an indicator of NO production in vivo (7).
Measurement of mean arterial blood pressure. The right femoral artery was cannulated and connected to a pressure transducer for measurement of mean arterial blood pressure. Mean arterial blood pressure equals the diastolic pressure plus one-third of the pulse pressure, the difference between the systolic and diastolic pressure.
Data Analysis
Data are presented as means ± SD. The results were compared using analysis of variance and Scheffé’s post hoc test or t-test. A level of P < 0.05 was accepted as statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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, and Activation of Nuclear Factor-
B and Activator Protein-1 in Isolated Monocytes Stimulated With LPS In Vitro
TNF- production by isolated monocytes began to increase at 2 h after LPS stimulation, peaking after 4 h. TNF- production by isolated monocytes observed at 4 h after LPS stimulation was inhibited by UTI at concentrations of 100 and 1,000 U/ml (Fig. 1). The number of living cells was not decreased in the presence of UTI (1,000 U/ml) at 6 h after stimulation with LPS (data not shown). Nuclear factor (NF)-B and activator protein (AP)-1 are important transcription factors in the induction of TNF- transcription in response to LPS (5, 30). LPS-induced increases in the specific binding of p65 and c-Fos to DNA, reflecting activation of NF-B and AP-1, respectively, were unaffected by UTI (data not shown).
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Egr-1 has been demonstrated to be an important transcription factor promoting TNF- gene expression in monocytes stimulated with LPS (4). Intracellular levels of Egr-1 were increased in monocytes after LPS stimulation, peaking at 30 min after LPS stimulation (data not shown). UTI inhibited the increase of Egr-1 expression at 30 min after LPS stimulation (P < 0.05) (Fig. 2A). Egr-1 binding to DNA was significantly increased in nuclear extracts at 1 h after LPS stimulation compared with that seen in unstimulated cells. UTI significantly inhibited LPS-induced increase in the binding of Egr-1 to DNA at 1 h after LPS stimulation (P < 0.05) (Fig. 2B).
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by increasing the expression of Egr-1 (3). Phosphorylation of ERK1/2 in monocytes was significantly increased after LPS stimulation, peaking after 15 min (data not shown). UTI inhibited the phosphorylation of ERK1/2 in monocytes at 15 min after LPS stimulation (P < 0.05) (Fig. 2C).
Effect of UTI on Increases in Lung Tissue Levels of Egr-1, TNF- mRNA and TNF- in Rats Given LPS
We attempted to determine whether UTI inhibits TNF- production by inhibiting Egr-1 expression, thereby preventing hypotension in rats given LPS (5 mg/kg iv). The lung is one of the major organs expressing a large amount of iNOS, an enzyme responsible for the development of septic shock (23). Lung levels of Egr-1 in rats were increased after LPS administration, peaking after 30 min followed by a decrease to the preadministration level (data not shown). Although intravenous administration of UTI at a dose of 25,000 U/kg did not inhibit LPS-induced increases in lung levels of Egr-1 at 30 min after LPS administration (data not shown), UTI at a dose of 50,000 U/kg did inhibit them (P < 0.05) (Fig. 3). Lung tissue levels of TNF- mRNA began to increase at 30 min after LPS administration, peaked at 60 min, and gradually decreased to near basal levels at 180 min after LPS administration (Fig. 4A). UTI (50,000 U/kg iv) inhibited increases in lung tissue levels of TNF- mRNA observed from 30 to 180 min after LPS administration (Fig. 4A). Lung tissue levels of TNF- began to increase at 30 min after LPS administration, peaked at 90 min, and gradually decreased thereafter to near basal levels at 180 min after LPS administration (Fig. 4B). UTI (50,000 U/kg iv) inhibited increases in lung tissue levels of TNF- from 30 to 120 min after LPS administration (Fig. 4B).
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The expression of iNOS activity and iNOS mRNA in the lung began to increase at 90 and 60 min after LPS administration, respectively, and they continued to increase until 180 min after LPS administration (7). UTI significantly inhibited both increases in lung tissue levels of iNOS mRNA (P < 0.05) and iNOS activity (P < 0.05) observed at 180 min after LPS administration (Fig. 5, A and B).
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/NO began to increase at 90 min after LPS administration and continued to increase until 180 min, as shown in our previous study (7). Intravenously administered UTI significantly inhibited the increases in plasma levels of NO/NO at 180 min after LPS administration (P < 0.05) (Fig. 5C). Mean arterial blood pressure was markedly decreased at 90 min after LPS administration. Hypotension was sustained for 180 min after LPS administration (Fig. 6A). Pretreatment with UTI significantly reduced the LPS-induced hypotension (P < 0.05) (Fig. 6A). Although UTI did not reduce LPS-induced hypotension when injected later than 30 min after LPS administration (data not shown), it inhibited increases in lung tissue levels of Egr-1 (Fig. 3) and TNF- (Fig. 4C), as well as hypotension (Fig. 6B), when injected 15 min after LPS administration.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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in LPS-stimulated monocytes in vitro. TNF- production in monocytes is regulated by various transcription factors, including NF-B, AP-1, and Egr-1 (5, 30). UTI did not inhibit the activation of NF-B and AP-1 in monocytes stimulated with LPS, as shown in the present study, which is consistent with previous observations by Aosasa et al. (1). UTI inhibited both expression of Egr-1 and phosphorylation of ERK1/2 in monocytes stimulated with LPS. Because ERK1/2 has been shown to regulate LPS-induced Egr-1 expression and inhibition of ERK1/2 phosphorylation reduces TNF- production by monocytes (22), it is possible that UTI inhibits TNF- production by inhibition of Egr-1 expression through inhibition of phosphorylation of ERK1/2 in LPS-stimulated monocytes.
The precise mechanism(s) by which UTI inhibited LPS-induced phosphorylation of ERK1/2 remains unclear at present. Kobayashi et al. (11) reported that UTI inhibited transforming growth factor-1
-induced ERK1/2 phosphorylation by inhibiting the increase in calcium influx in an ovarian cancer cell line. Because the increase in calcium influx was shown to be potentially involved in LPS-induced ERK1/2 activation and the subsequent TNF- production in monocytes (20), it is likely that UTI inhibits LPS-induced ERK1/2 activation by inhibiting an increase in calcium influx.
We previously demonstrated that anti-rat TNF- antibody inhibited inflammatory responses leading to hypotension in rats given 5 mg/kg LPS (8), suggesting that inhibition of TNF- production by UTI through inhibition of Egr-1 expression might contribute to reduction of hypotension. Furthermore, we previously showed that aminoguanidine, a selective inhibitor of iNOS, significantly inhibited the increases in lung iNOS activities and plasma levels of nitric oxide metabolites, as well as hypotension (8), suggesting that iNOS might play a causative role in LPS-induced hypotension in the present study. These observations strongly suggested that UTI might inhibit LPS-induced production of TNF- by inhibiting activation of the ERK1/2-Egr-1 pathway in monocytes, thereby contributing to the reduction of hypotension through inhibition of iNOS induction in rats given LPS.
TNF- plays critical roles in the development of various organ failures, including septic shock and disseminated intravascular coagulation observed in sepsis (16). We previously demonstrated that serum levels of fibrin degradation products were increased in rats given 5 mg/kg LPS (26). Thus inhibition of Egr-1 expression by UTI in vivo might be observed in the presence of coagulation abnormalities in the present study. We also previously demonstrated that coagulation abnormalities increased TNF- production in rats subjected to hepatic reperfusion (17), suggesting that mechanism(s) underlying inhibition by UTI of Egr-1 expression in vivo might be different from that observed in vitro. However, since reduction of coagulation abnormalities by administration of an inactive derivative of the activated form of coagulation factor X (an anti-coagulant that selectively inhibits thrombin generation) did not attenuate inflammatory responses, including the increase in TNF- production and the subsequent hypotension in rats given 5 mg/kg of LPS (7), coagulation abnormalities per se might not affect inflammatory responses in rats given 5 mg/kg of LPS in the present study. These observations strongly suggested that UTI might directly inhibit TNF- production by inhibiting Egr-1 expression in vivo.
In normal human subjects, the physiological concentration of UTI in serum ranges from 6 to 50 U/ml (3), and the concentration is increased to 150 U/ml after administration of 5,000 U/kg of UTI (21). Because 100 U/ml of UTI inhibited TNF- production in LPS-stimulated monocytes in vitro, as shown in the present study, it is possible that the therapeutic dose (5,000 U/kg) of UTI inhibits monocytic TNF- production in patients with sepsis.
In the present study, 50,000 U/kg of UTI, 10 times higher than the therapeutic dose of UTI in the clinical setting, was required to inhibit inflammatory responses and hypotension in rats given LPS. Yamaguchi et al. (28) also reported that ischemia-reperfusion-induced proinflammatory cytokine production in rats was inhibited by 50,000 U/kg of UTI but not by 5,000 U/kg. Why such a large dose of UTI was necessary to reduce LPS-induced pathological events in rats is unclear at present, but structural differences between human and rat UTI might explain the low sensitivity of rats to human UTI (3).
In the present study, posttreatment with UTI reduced LPS-induced hypotension, suggesting that UTI might be effective in attenuating shock responses in the clinical setting. Because our rat model of LPS-induced hypotension is nonlethal, whether UTI improves the outcome of rats given LPS is not known. This point should be clarified by examining the effect of UTI in a lethal rat model of LPS-induced hypotension in a future study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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production of monocytes. Shock 15: 101–105, 2001.[Web of Science][Medline]
expression by inducing Elk-1 phosphorylation and Egr-1 expression. Blood 98: 1429–1439, 2001.1 signaling cascade. J Biol Chem 278: 7790–7799, 2003. production. Shock 13: 183–189, 2000.[Web of Science][Medline]
production via Egr-1. Am J Physiol Cell Physiol 282: C1205–C1211, 2002. promoter in human monocytic cells: regulation by Egr-1, c-Jun and NF-B transcription factors. J Biol Chem 272: 17795–17801, 1997. production by inhibiting activation of both nuclear factor-B and activator protein-1 in human monocytes. J Pharmacol Exp Ther 305: 298–305, 2003.This article has been cited by other articles:
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  H. Harada, V. N. Lama, L. N. Badri, T. Ohtsuka, D. Petrovic-Djergovic, H. Liao, Y. Yoshikawa, K. Iwanaga, C. L. Lau, and D. J. Pinsky Early growth response gene-1 promotes airway allograft rejection Am J Physiol Lung Cell Mol Physiol, July 1, 2007; 293(1): L124 - L130. [Abstract] [Full Text] [PDF]
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, control; 
, UTI without LPS; 
, LPS; 
, LPS + UTI. *P < 0.01 vs. control; 
P < 0.01 vs. LPS. B: human monocytes were preincubated with various concentrations of UTI for 30 min and then stimulated with LPS (100 ng/ml). Four hours after stimulation, supernatants were collected and TNF-
