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Regulation of the Expression of theInducible Isoform of Nitric OxideSynthase by Glucocorticoidsa

CSABA SZABÓb

Division of Critical Care, Children’s Hospital Medical Center, 3333Burnet Avenue, Cincinnati, Ohio 45229 USA

INTRODUCTION

Nitric oxide (NO), produced by the inducible NO synthase (iNOS), is an impor-tant final common mediator during various forms of inflammation and circula-tory shock.1–3 Glucocorticoids are potent antiinflammatory hormones that play acrucial role in the stress response.4–6 There is now considerable evidence demon-strating the importance of glucocorticoids in suppressing the expression of iNOSin inflammation. This chapter focuses on the physiological and pathophysiologi-cal importance of regulation of iNOS expression by steroids. Specifically, thischapter overviews (1) the effects of therapeutic administration of steroids on theexpression of iNOS in circulatory shock and selected inflammatory conditions; (2)the role of endogenous glucocorticoids in the modulation of iNOS expression inresponse to endotoxin (bacterial lipopolysaccharide, LPS); (3) the role of endoge-nous glucocorticoids in the regulation of iNOS expression during the developmentof endotoxic tolerance, and (4) the molecular mechanism of the regulation of iNOSexpression by glucocorticoids, with special reference to the difference between themurine and the human systems.

REGULATION OF iNOS EXPRESSION BY STEROIDS

In 1990, Radomski and co-workers have demonstrated in in vitro studies thatdexamethasone and other glucocorticoid hormones potently inhibit the expres-sion of iNOS in response to LPS and to proinflammatory cytokines.7 This obser-vation has subsequently been confirmed by a multitude of groups in a variety ofcell types. The authors in the original article have proposed that inhibition ofiNOS expression by glucocorticoids may be a major contributor to the antiinflam-matory effects of the glucocorticoid hormones. The initial in vitro studies havebeen followed by studies demonstrating that glucocorticoids also prevent theexpression of iNOS in vivo in response to LPS or other proinflammatory stimuli.8–10

In endotoxic shock, we have demonstrated that prevention of iNOS by glucocor-ticoids is associated with maintained mean arterial blood pressure and protection

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aThis work was supported by a grant from the National Institutes of Health(R29GM54773).

bAdditional correspondence information: Telephone: (513) 636-8714; Fax: (513) 636-4892.

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against the loss of contractile responsiveness to norepinephrine.10 We have alsodemonstrated a similar protection against the expression of iNOS and a simulta-neous protection against the development of cardiovascular failure in rodentmodels of hemorrhagic shock.11 Subsequent studies have demonstrated that thesuppression of iNOS protein expression by glucocorticoids in shock is due to sup-pression of iNOS mRNA expression.12 Protection against iNOS expression andamelioration of the inflammatory response has been demonstrated in a variety ofinflammatory models.13–17 It is generally believed that the suppression of iNOSexpression is a major contributor to the anti-inflammatory effect of steroids.Indeed, in the same inflammatory models, inhibition of NOS activity has alsobeen shown to provide a protective effect against the inflammation.10–17 The inhi-bition of iNOS expression by glucocorticoids in vivo may be due to a direct tran-scriptional effect, similar to the results of the in vitro studies (see below). It is wellestablished that the production of these intermediary cytokines, such as tumornecrosis factor alpha, interferon-γ, and interleukin-1 contribute to the process ofiNOS expression during inflammation.3 Therefore, in addition to a transcriptionaleffect, inhibition of proinflammatory cytokine production by the steroids mayalso contribute to the suppression of iNOS expression (FIG. 1).

ROLE OF ENDOGENOUS GLUCOCORTICOIDS IN THE REGULATION OFiNOS EXPRESSION

We have investigated the role of endogenous glucocorticoids in the regulationof iNOS expression by the use of adrenalectomized (ADX) animals. It is well doc-umented that ADX animals, which lack endogenous glucocorticoids, developmore severe forms of inflammation, including a more severe form of circulatoryshock in response to LPS, that can be prevented by pretreatment with exogenousglucocorticoids.18 Our data demonstrated that endogenous glucocorticoids caninhibit the expression of iNOS induced by LPS in the anesthetized rat.19 This con-clusion was supported by the findings that (i) ADX rats, but not normal rats,develop severe circulatory failure (hypotension and vascular hyporeactivity toexogenous vasoconstrictor agents) when challenged with a low dose of LPS and(ii) in ADX rats, but not in normal rats, low-dose LPS caused the induction ofiNOS activity.19 The finding that the induction of iNOS was attenuated by endoge-nous glucocorticoids19 may explain why ADX animals develop a more severe cir-culatory failure in response to low-dose endotoxin when compared to normalanimals. Our finding also provides a reasonable explanation why the cardiovas-cular failure in response to low-dose LPS in ADX animals is abolished by pre-treatment with the synthetic glucocorticoid dexamethasone. Our findings mayprovide an explanation for the clinical observation that patients with adrenocorti-cal insufficiency are more susceptible to developing severe circulatory failure inresponse to low-grade endotoxemia.

ROLE OF ENDOGENOUS GLUCOCORTICOIDS IN THE DEVELOPMENTOF ENDOTOXIN TOLERANCE

Tolerance to endotoxin develops after repeated administration of small dosesof LPS to animals and is characterized by the attenuation of the cardiovasculareffects and mortality of subsequent challenge with a high-dose endotoxin. Several

338 ANNALS NEW YORK ACADEMY OF SCIENCES

different mechanisms may contribute to the development of endotoxin tolerancein vivo. The early-phase tolerance to endotoxin is associated with a reducedrelease of proinflammatory cytokines.20 Activation of the pituitary–adrenocorticalaxis has also been demonstrated during endotoxin tolerance.21 On the basis ofthese data, we have investigated the relationship between the tolerance to LPS andthe induction of iNOS in vivo. Our data22,23 and data by other groups24,25 demon-strated that the reduced cardiovascular response to LPS that occurs after repeated

FIGURE 1. Mechanisms of the inhibitory effect of glucocorticoids on the expression ofiNOS. Glucocorticoids may inhibit the expression of iNOS by inhibiting the production ofintermediary cytokines tumor necrosis factor-α (TNF), interleukin-1β (IL-1β) and interferonγ (IFNγ), and inhibit the production of the proinflammatory lipid mediator platelet-activat-ing factor (PAF). In addition, glucocorticoids inhibit the nuclear translocation of the tran-scription factor nuclear factor κ B (NF-κB). It is unknown whether glucocorticoids alsointerfere with the activation of tyrosine kinases (TK) and mitogen-activated kinases (MAPK).Glucocorticoids induce the expression of lipocortin-1, which, in turn, inhibits the inductionof iNOS via a mechanism that is incompletely characterized. Glucocorticoids may alsoincrease iNOS mRNA degradation, thereby decreasing iNOS mRNA half-life. Straight arrowsindicate stimulation; dashed arrows indicate inhibition. See text for further explanation.

administration of small doses of LPS is associated with a reduced capacity of LPSto induce iNOS. The inhibition of iNOS expression lies on the level of iNOSexpression, since not only reduced iNOS protein but also reduced iNOS mRNAcan be demonstrated in the endotoxin-tolerant animals.25 Elevated plasma levelsof steroids plays a central role in the development of endotoxin tolerance, as itrelates to iNOS expression. This conclusion is based on the finding that thereduced ability of LPS to cause hypotension and iNOS induction in endotoxin-tol-erant rats is reversed by the glucocorticoid receptor antagonist, RU-486.22 Thus,the elevated levels of endogenous glucocorticoids in endotoxin-tolerant rats canaccount for the protection against cardiovascular failure and for the attenuation ofthe induction of iNOS by LPS.22

MECHANISM OF iNOS EXPRESSION: REGULATION BYGLUCOCORTICOIDS

Surprisingly, the cellular and molecular mechanisms of inhibition iNOSexpression by glucocorticoids are not fully characterized. In most rodent cellularsystems, glucocorticoids inhibit the expression of iNOS, and the presence of thesteroid is required before the stimulus of iNOS expression. In most studies, theinhibition of iNOS expression by glucocorticoids is associated with reduced iNOSmRNA levels. From a study in cardiac fibroblasts, it appears that dexamethasonedelays rather than prevents the accumulation of iNOS mRNA, and the mode ofdexamethasone’s action is related, at least in part, to a reduction of iNOS mRNAstability.26 We have obtained recent data that support the view that lipocortin-1, asteroid-inducible endogenous protein, contributes to the inhibition of iNOSexpression in rodent systems in vitro as well as in vivo.27,28

The intracellular signal transduction pathways leading to the expression ofiNOS include activation of the nuclear factor kappa B system, as well as activationof mitogen-activated kinases and tyrosine kinases.1–3,28 The extent to which gluco-corticoids affect these systems in relation to iNOS expression remains to be fur-ther investigated. Based on studies in pulmonary epithelial cells, Kleinert andco-workers have proposed that the inhibition of iNOS expression by dexametha-sone is related, at least in part, to inhibition of activation of the nuclear factorkappa B system.29

In contrast to the rodent systems, where glucocorticoids are potent inhibitorsof the process of iNOS expression, in many human cell types glucocorticoidsappear to be less effective, and the degree of inhibition is dependent on the celltype.15,29,30 We have recently characterized the mechanism of iNOS expression inthe DLD-1 human intestinal epithelial cell line.30,31 In this cell line, and similar tothe murine systems, the process of iNOS expression involves nuclear factor kappaB activation and tyrosine kinases.30,31 However, and in contrast to the murine sys-tems, the process is fully steroid-resistant.30,31 In this respect it is noteworthy thatwe have recently discovered that the promoter of the human iNOS gene is radi-cally different from the murine promoter: In the human iNOS promoter, a classi-cal enhancer element is present, which is located between 8.7 and 10.7 kbupstream of the transcription initiation site.31

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340 ANNALS NEW YORK ACADEMY OF SCIENCES

CONCLUSIONS

Glucocorticoids are powerful suppressors of the expression of iNOS in variousexperimental systems: Suppression of iNOS expression is an important contribu-tor to the antiinflammatory effect of these hormones. Endogenous glucocorticoidsmaintain a tonic suppression of iNOS expression in response to proinflammatorystimuli. Endogenous glucocorticoids are also crucial in the suppression of iNOSexpression during endotoxin tolerance. In contrast to the rodents, steroids are lesseffective in inhibiting the expression of iNOS in human systems.

REFERENCES

1. NATHAN, C. 1992. Nitric oxide as a secretory product of mammalian cells. FASEB J. 6:3051–3064.

2. SZABÓ, C. & C. THIEMERMANN. 1993. Potential therapeutic use of nitric oxide synthaseinhibitors. Cur. Opin. Dev. Drugs 2: 1165–1174.

3. SZABÓ, C. 1995. Alterations in the production of nitric oxide in various forms of circula-tory shock. New Horizons 3: 3–32.

4. SELYE, H. 1985. The nature of stress. Basal Facts 7: 3–11.5. PRUETT, S. B., D. K. ENSLEY & P. L. CRITTENDEN. 1993. The role of chemical-induced stress

responses in immunosuppression: A review of quantitative associations andcause–effect relationships between chemical-induced stress responses and immuno-suppression. J. Toxicol. Environ. Health 39: 163–192.

6. BAMBERGER, C. M., H. M. SCHULTE & G. P. CHROUSOS. 1996. Molecular determinants ofglucocorticoid receptor function and tissue sensitivity to glucocorticoids. Endocr. Rev.17: 245–261.

7. RADOMSKI, M. W, R. M. J. PALMER & S. MONCADA. 1990. Glucocorticoids inhibit theexpression of an inducible, but not the constitutive nitric oxide synthase in vascularendothelial cells. Proc. Natl. Acad. Sci. USA 87: 10043–10047.

8. KNOWLES, R. G., et al. 1990. Anti-inflammatory glucocorticoids inhibit the induction byendotoxin of nitric oxide synthase in the lung, liver and aorta of the rat. Biochem.Biophys. Res. Commun. 172: 1042–1048.

9. SALTER, M., R. G. KNOWLES & S. MONCADA. 1991. Widespread tissue distribution, speciesdistribution and changes in activity of calcium-dependent and calcium-independentnitric oxide synthesis. FEBS Lett. 291: 145–149.

10. SZABÓ, C., et al. 1993. Nitric oxide mediated hyporeactivity to noradrenaline precedesnitric oxide synthase induction in endotoxin shock. Br. J. Pharmacol. 108: 786–792.

11. THIEMERMANN, C., et al. 1993. Vascular hyporeactivity to vasoconstrictor agents andhaemodynamic decompensation in hemorrhagic shock is mediated by nitric oxide.Proc. Natl. Acad. Sci. USA 90: 267–271.

12. LIU, S. F., P. J. BARNES & T. W. EVANS. 1997. Time course and cellular localization oflipopolysaccharide-induced inducible nitric oxide synthase messenger RNA expres-sion in the rat in vivo. Crit. Care Med. 25: 512–518.

13. HILL, G. E., et al. 1995. Glucocorticoid reduction of bronchial epithelial inflammationduring cardiopulmonary bypass. Am. J. Respir. Crit. Care Med. 152: 1791–1795.

14. RACHMILEWITZ, D., et al. 1995. Enhanced colonic nitric oxide generation and nitric oxidesynthase activity in ulcerative colitis and Crohn’s disease. Gut 36: 718–723.

15. MURRELL, G.A., et al. 1996. Nitric oxide: An important articular free radical. J. Bone Joint.Surg. Am. 78: 265–274.

16. ARKOVITZ, M. S., et al. 1996. Selective inhibition of the inducible isoform of nitric oxidesynthase prevents pulmonary transvascular flux during acute endotoxemia. J. Ped.Surg. 31: 1009–1015.

17. KHARITONOV, S. A., et al., 1997. Nasal nitric oxide is increased in patients with asthmaand allergic rhinitis and may be modulated by nasal glucocorticoids. J. Allergy Clin.Immunol. 99: 58–64.

18. ALTURA, B. M., Ed. 1983. Handbook of Shock and Trauma. Raven Press. New York.19. SZABÓ, C., C.THIEMERMANN & J. R. VANE. 1993. Inhibition of the production of nitric

oxide and vasodilator prostaglandins attenuates the cardiovascular response to bac-terial endotoxin in adrenalectomized rats. Proc. R. Soc. B (London) 253: 233–238.

20. ZUCKERMAN, S. H., G. F. EVANS & L. D. BUTLER. 1989. Endotoxin tolerance: Independentregulation of interleukin-1 and tumor necrosis factor expression. Infect. Immun. 59:2774–2780.

21. ZUCKERMAN, S. H. & N. QURESHI. 1992. In vivo inhibition of lipopolysaccharide-inducedlethality and tumor necrosis factor synthesis by rhodobacter sphareoides diphospho-ryl lipid A is dependent on corticosterone induction. Infect. Immun. 60: 2581–2587.

22. SZABÓ, C., et al. 1994. Inhibition of nitric oxide synthase induction by endogenous glu-cocorticoids accounts for endotoxin tolerance in vivo. Proc. Natl. Acad. Sci. USA 91:271–275.

23. CHAMULITRAT, W., J. F. WANG & J. J. SPITZER. 1995. Electron paramagnetic resonanceinvestigations of nitrosyl complex formation during endotoxin tolerance. Life Sci. 57:387–395.

24. GILAD, E., et al. 1996. Effects of radiodetoxified endotoxin on nitric oxide production inJ774 macrophages and in endotoxin shock. J. Endotoxin Res. 3: 513–516.

25. CHANG, C. C., et al. 1997. Inhibition of nitric oxide synthase gene expression in vivo andin vitro by repeated doses of endotoxin. Am. J. Physiol. 271: G539–G548.

26. FARIVAR, S. & P. BRECHER. 1996. Salicylate is a transcriptional inhibitor of the induciblenitric oxide synthase in cultured cardiac fibroblasts. J. Biol. Chem. 271: 31585–31592.

27. WU, C. C., et al. 1994. Lipocortin 1 mediates the inhibitory effect of dexamethasone onthe induction of nitric oxide synthase in vitro and in vivo. Can. J. Physiol. Pharmacol.Suppl. 1: 465.

28. SZABÓ C. & C. THIEMERMANN. 1995. Mechanism of the induction of nitric oxide synthase.Adv. Pharmacol. 34: 113–153.

29. KLEINERT, H., et al. 1996. Glucocorticoids inhibit the induction of nitric oxide synthase IIby down-regulating cytokine-induced activity of transcription factor nuclear factor-kappa B. Mol. Pharmacol. 49: 15–21.

30. SALZMAN, A.L., et al. 1996. Characterization of the induction and activity of the humannitric oxide synthase in a transformed intestinal epithelial cell line. Am. J. Physiol.270: G565-573.

31. LINN, C., et al. 1997. Promoter analysis of the human inducible nitric oxide synthasegene suggests both transcriptional and post-transcriptional regulation. Am. J.Physiol. 272: G1499-1508.

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