(Hypertension. 1996;27:709-714.)
© 1996 American Heart Association, Inc.
Articles |
Mechanisms of Interleukin-1ß Regulation of Nitric Oxide Synthase in Cardiac Myocytes
From the Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, Mich.
Correspondence to Dr Margot C. LaPointe, Hypertension and Vascular Research Division, Henry Ford Hospital, 2799 W Grand Blvd, Detroit, MI 48202. E-mail mclapointe@aol.com.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Abstract Cytokines and endotoxin stimulate inducible NO synthase (iNOS) in different types of cells; however, little is known about regulatory mechanisms. Using the Griess reagent for nitrite levels, Western blots for iNOS protein, Northern blots for iNOS mRNA, and transient transfection studies to monitor transcription, we determined potential mechanisms involved in interleukin-1ß stimulation of iNOS in cultured neonatal ventricular myocytes. When myocytes were treated with interleukin-1ß (5 ng/mL), nitrite levels increased, and this effect was inhibited 80% by the specific iNOS inhibitor aminoguanidine. Neither interferon gamma nor tumor necrosis factor–
alone stimulated nitrite
production. Bacterial endotoxin alone stimulated nitrites and
potentiated the effect of interleukin. To determine whether a tyrosine
kinase–mediated signaling pathway was involved in interleukin
action, we used the inhibitor genistein, which blocked
interleukin-stimulated nitrites, iNOS protein, and iNOS mRNA. To
determine the effect of activation of protein kinase C, we treated
cells with the phorbol ester phorbol 12-myristate 13-acetate
(PMA). PMA decreased both interleukin-stimulated nitrites and iNOS
protein by 40%. To determine the involvement of cyclic
nucleotides, cells were treated with either dibutyryl cAMP
or cGMP. cAMP (1 mmol/L) stimulated iNOS mRNA, protein, and nitrite
production, whereas cGMP had no effect. To test for a direct
effect of interleukin on transcription of the iNOS gene, we transfected
the full-length mouse iNOS 5' regulatory sequences (-1592 to
+160) coupled to a luciferase reporter gene (-1592iNOSLuc).
Interleukin stimulated luciferase activity 1.8±0.2-fold. To determine
whether interleukin also affects iNOS mRNA stability,
interleukin-stimulated iNOS mRNA was allowed to decay in the
presence of the transcription inhibitor actinomycin D. iNOS
mRNA t1/2 (
1 hour) was not affected by interleukin.
Thus, our data suggest that (1) interleukin-1ß is the primary
cytokine in myocyte iNOS regulation and acts predominantly at
the transcriptional level; (2) interleukin stimulation of iNOS mRNA and
protein is coupled to a tyrosine kinase–mediated signaling
pathway; and (3) protein kinase C and cAMP can modify interleukin
signaling by decreasing and increasing iNOS, respectively.
Key Words: cytokines • nitric oxide • ventricular myocytes • tyrosine kinase • cyclic AMP
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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The iNOS was originally characterized in macrophages and shown to be maximally induced by endotoxin (bacterial LPS) and IFN. Since then, iNOS has been characterized in a number of different cell types in the cardiovascular system, including endothelial cells, vascular smooth muscle cells, fibroblasts, and cardiac myocytes.1 2 3 Regulation of iNOS seems to be cell-type specific, with different types of cells responding to different cytokines and using a number of signaling mechanisms.1 PKC has been implicated in the stimulation of iNOS in a murine macrophage cell line4 and rat hepatocytes.5 In contrast, PKC inhibits the induction of iNOS in mesangial cells,6 whereas it is not involved in iNOS regulation in vascular smooth muscle cells.7 Signaling pathways that activate tyrosine kinase4 and protein kinase A8 also modulate the induction of iNOS. At the molecular level, the transcription factors nuclear factor–
B (NFB)
9 and interferon regulatory factor 110 are
involved in the induction of NOS in macrophages.
IL is a major regulator of iNOS in neonatal11 and
adult12 cardiac myocytes. Depending on the cell type, IL
signaling may involve the sphingomyelin-ceramide pathway,
serine-threonine kinases, tyrosine kinases, and the transcription
factors NFB and activator protein 1
(AP1).13 14 15 Because the iNOS isoform
in cardiac myocytes
may participate in a number of
pathophysiological conditions, including
myocarditis, contractile dysfunction, allograft rejection, and
ischemic injury,1 2 3 it is important to
understand
the mechanisms of its induction. In this report we further characterize
the major cytokine stimulators of iNOS in myocytes, IL
signaling, and transcriptional effects.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Preparation of Ventricular Myocytes
This protocol was approved by the Henry Ford Hospital Committee for Care and Use of Experimental Animals. Hearts from 1- to 2-day-old neonatal Sprague-Dawley rats (Charles River, Kalamazoo, Mich) were minced and digested with trypsin and DNAse to prepare primary cultures of ventricular myocytes as described previously.16 Ventricular myocytes were plated at a density of 1x105 cells/cm2 in Dulbecco's modified Eagle's medium (Gibco) plus 10% fetal bovine serum (HyClone) and cultured as described previously.16 Medium lacking serum but supplemented with insulin, transferrin, and selenium was added 24 hours before cells were treated with test compounds. Unless otherwise specified, treatments were for 24 hours.
Nitrite Production
Nitrite production, an index of NO
production,
was measured in media samples by the Griess reaction.17
Nanomoles of nitrite were determined by comparison with a standard
curve of NaNO2 using an enzyme-linked immunosorbent
assay plate reader at 505 nm. NOx from triplicate wells were averaged
for each experiment. Controls (untreated cells) were assigned a value
of 1, and the values for all treatments were normalized to 1
(x-fold increase versus control). Data were expressed as mean±SE.
Differences in mean values among treatment groups were analyzed
by a Student's t test or one-way ANOVA, with pairwise
multiple comparisons made by the Student-Newman-Keuls method. A value
of P<.05 was considered significant.
Western Blot Analysis
Approximately 3x106
myocytes were lysed in
buffer containing 10 mmol/L Tris (pH 7.5), 10 mmol/L EDTA, 0.4%
deoxycholate, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 1
mmol/L phenylmethylsulfonyl fluoride, and 1x protease
inhibitor cocktail (1000X=5 mg/mL each pepstatin A,
leupeptin, antipain, and chymostatin). Cellular debris was removed by
centrifugation in a microfuge. The protein
concentration of the supernatant was determined using Coomassie protein
assay reagent (Pierce) with bovine serum albumin as the
standard. Fifty micrograms of cytosolic protein was separated out by
electrophoresis on an 8% SDS-polyacrylamide gel and then
transferred to an Immobilon-P PVDF membrane (Millipore). The membrane
was incubated in blocking buffer (Tris-buffered saline [TBS] with
5% nonfat dried milk and 0.2% NP-40) and then with 0.4 mg/mL rabbit
polyclonal antibody raised against mouse macrophage iNOS (Santa
Cruz Biotechnology) overnight at 4°C. The blot was washed in TBS plus
0.25% sodium deoxycholate, 0.2% NP-40, and 0.1% SDS and incubated
with a 1:2000 dilution of the secondary antibody (goat anti-rabbit
IgG alkaline phosphatase conjugate; BioRad) in blocking buffer.
Colorimetric detection of the reaction product was
accomplished with substrate and developing reagents from BioRad
according to the manufacturer's protocol. Laser densitometry was used
to quantitate the protein bands.
Northern Blot Analysis
Total RNA was extracted from
ventricular myocytes
and analyzed for iNOS mRNA as described
previously.11 GAPDH mRNA was used to normalize for
variations in loading of samples on gels. mRNA levels were quantified
by laser densitometry.11
Transient Transfection and Luciferase Assay
Freshly isolated
ventricular myocytes were
transiently transfected by electroporation as described
previously.11 Aliquots of 25 to 50 µg of
-1592iNOSLuc18 (containing the 5' flanking sequences
of the mouse iNOS gene extending from -1592 to +160) and 1 µg
of RSV-ß-galactosidase were transfected per
12x106 cells. Cells (1x106)
were plated onto each well of a six-well plate. Luciferase
(Luciferase Assay System, Promega) and galactosidase (Galacto-Light
chemiluminescent assay; Tropix) activities were measured in an OptoComp
1 luminometer (MGM Instruments) using the manufacturers' protocols.
Luciferase activity was normalized to ß-galactosidase or protein
and reported as x-fold increase versus untreated controls
(controls=1). The increase in response to IL was identical when
luciferase activity was normalized either to ß-galactosidase or
protein. Our previous studies have shown no effect of 24-hour IL
treatment on total protein in ventricular
myocytes.11 Also, we did not detect any effect of IL on
RSV-ß-galactosidase activity in these experiments. For each
experiment, duplicate aliquots of cell lysates from triplicate wells
were assayed. Data were expressed as mean±SE and analyzed for
statistical significance as described above.
Chemicals
The cytokines IL, TNF, and IFN were obtained from
Bachem. Endotoxin (bacterial LPS), aminoguanidine, cAMP, cGMP, and the
phorbol ester (PMA) were obtained from Sigma Chemical Co. Genistein was
purchased from Calbiochem. C2 and C16 ceramide were obtained from Fluka
and Biomol, respectively, and sphingomyelinase from Sigma. Routine
laboratory supplies and chemicals were obtained from Fisher
Scientific.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Effects of Cytokines and LPS on NO Production
We have previously shown that IL stimulates nitrite production in neonatal ventricular myocytes.11 We extended these studies to include other cytokines and LPS and combinations thereof to determine the major inducers of iNOS in myocytes. Fig 1
, top, shows
that IL and LPS (both at 5 ng/mL) induced nitrite production
12- and 5.8-fold, respectively, and that the effect of IL was
potentiated by LPS (15.8±1.1 versus 12.2±1.5; P<.01
IL
plus LPS versus IL alone). These results were confirmed at the protein
level by Western blot analysis (Fig 1, bottom). Neither TNF (25
ng/mL) nor IFN (10 ng/mL) stimulated nitrite production
(M.C.L., J.R.S., unpublished observations, 1995). Fig 1, top,
also
shows that the stimulatory effect of IL was decreased 80% by the
specific iNOS inhibitor aminoguanidine (100
µmol/L).19 Western blot analysis of iNOS protein
indicated that aminoguanidine had no effect on protein levels (Fig
1,
bottom).
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Possible Signal Transduction Mechanisms and Cross
Talk
Since induction of iNOS by LPS in macrophages involves a
tyrosine kinase–linked signaling cascade,4 we next
studied whether the tyrosine kinase inhibitor genistein (50
and 100 µmol/L) could inhibit the effects of IL in myocytes. Fig
2 shows that genistein inhibited IL-stimulated nitrite
production (Fig 2A), iNOS protein (Fig 2B), and
iNOS mRNA (Fig 2C), suggesting that the tyrosine
kinase–mediated pathway is
directly linked to regulation of iNOS gene expression.
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We have
previously shown that the PKC inhibitor
staurosporine has no effect on IL stimulation of
nitrites,11 suggesting that PKC is not part of the
signaling pathway involved in IL regulation of iNOS. We tested whether
direct activation of PKC could modulate IL stimulation of iNOS.
Stimulation of PKC with the phorbol ester PMA decreased IL-stimulated
nitrite production (Fig 3A) and iNOS protein
(Fig 3B) by 40%, but had no effect on iNOS mRNA (Fig
3C). Thus, our
data suggest that PKC may affect translation or degradation of iNOS
protein.
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In vascular smooth muscle cells, cAMP alone increases nitrites
and
stimulates iNOS mRNA.7 cGMP, an intracellular mediator of
NO actions, has also been implicated in potentiating IL stimulation of
nitrites.20 Thus, we tested whether cAMP and cGMP mediate
IL action or are able to modulate IL stimulation of nitrites. Fig
4 shows that 1 mmol/L cAMP stimulated nitrites 3.4-fold
(Fig 4A) and iNOS protein and mRNA (Fig 4B).
Treatment with cAMP and IL
synergistically stimulated nitrite production (21-fold versus
16-fold stimulation; P<.01). In contrast, cGMP (1 mmol/L)
had no effect on nitrite production and iNOS protein levels
(M.C.L., J.R.S., unpublished observations, 1995). Thus, cAMP and the
pathway it stimulates synergize with the IL signaling pathway.
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Both TNF and IL have been shown to activate the sphingomyelinase-ceramide phospholipid signaling pathway.14 To test its involvement in the regulation of iNOS, cells were treated with 10-6 and 10-5 mol/L of C2 and C16 ceramide or 10-5 to 10-1 U/mL sphingomyelinase, concentrations used in studies of EL4 thymoma cells.21 None of these treatments stimulated nitrites (M.C.L., J.R.S., unpublished observations, 1995).
IL Stimulates Transcription of iNOS
To test for a direct
effect of IL on transcription of the iNOS
gene, we transfected ventricular myocytes with a plasmid
containing the full-length mouse iNOS regulatory sequences upstream
from a luciferase reporter gene (-1592iNOSLuc). Fig 5
shows that IL stimulated the iNOS promoter
1.8±0.2-fold (n=9; P<.001). A promoterless
luciferase
vector did not respond to IL (M.C.L., J.R.S., unpublished observations,
1995).
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iNOS mRNA t1/2
Since the magnitude of IL
activation of the iNOS promoter was not
as great as the increases in nitrites and mRNA, we questioned whether
IL was stabilizing iNOS mRNA. Because our previous studies showed
maximal induction of iNOS mRNA 3 to 24 hours after IL
treatment,11 we treated myocytes with IL for 24 hours and
then with the transcription inhibitor ActD (5 µg/mL) in
the presence or absence of IL. At 0, 1, 3, and 6 hours after addition
of ActD, total RNA was extracted, followed by Northern blot
analysis. Fig 6A shows that iNOS mRNA decayed
with a t1/2 of approximately 1 hour after the addition of
ActD and that this was not affected by the addition of IL. Fig
6B shows
iNOS mRNA levels after the aforementioned treatments. Thus, IL seems to
act primarily at the transcriptional level and not through
stabilization of iNOS mRNA.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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Two important findings of our study are that the tyrosine kinase inhibitor genistein prevents IL induction of iNOS mRNA and protein and nitrite production and that the specific iNOS inhibitor aminoguanidine inhibits nitrite production in ventricular myocytes. Moreover, the effect of IL occurs primarily at the transcriptional level.
Of the cytokines tested in our study, only IL and LPS stimulated iNOS synthesis and nitrite production. LPS potentiated the effects of IL. Whether LPS is acting through a signaling mechanism distinct from IL or inducing production of cytokines, as occurs in macrophages,22 was not investigated in our study. Our data are consistent with a recently published study on iNOS expression in adult cardiac myocytes: Either IL or LPS stimulated iNOS mRNA, whereas combined treatment with IL, TNF, and IFN gave maximal stimulation.12
Because IL appeared to be the primary cytokine involved in
upregulation of iNOS, we studied its signaling mechanisms, possible
second messengers, and cross talk with other intracellular signals. Our
data indicate that a tyrosine kinase–mediated signal is required
for IL action in neonatal ventricular myocytes. Such a
mechanism has recently been reported for cardiac
myocytes23 and pancreatic ß-cells.24
Since the IL receptor has neither intrinsic tyrosine kinase activity
nor has it been shown to couple to a nonreceptor tyrosine
kinase,13 25 it is unclear how its effects are
transduced
and how they result in activation of transcription factors, such as
NFB13 26 and AP1.15 Studies suggest
that
the effect of IL is transmitted through the
sphingomyelinase-ceramide–activated kinase
cascade.14 This pathway activates
stress-activated protein kinases (SAPKs), also known as jun
kinases (JNKs), through tyrosine phosphorylation. Two
targets for SAPKs are the transcription factors JUN and ATF-2, which
can individually activate target genes or form heterodimers for
transactivation of genes at AP1/ATF sites in responsive
genes.27 28 Although we were unable to demonstrate
the
involvement of either sphingomyelinase or ceramide in the induction of
iNOS, we have preliminary evidence from Western blot analysis
for the presence of JNKs in cardiac myocytes and are exploring whether
they are tyrosine-phosphorylated in response to IL
(M.C.L., J.R.S., unpublished observations, 1995).
Our data also suggest that other intracellular signals, including those from PKC and cAMP, can modulate IL stimulation of iNOS. Previously we showed that the kinase inhibitor staurosporine did not affect IL stimulation of nitrites, a result confirmed by other investigators.23 29 Thus, PKC is not a component of the IL signaling pathway. On the other hand, our studies suggest that PKC can modulate IL-stimulated NO production. Although we did not determine the mechanism involved in this effect, our Western and Northern blot data suggest that PKC may modulate IL regulation of iNOS at the translational or posttranslational level. Such mechanisms are involved in transforming growth factor–ß suppression of NO production in macrophages.30 Two other possible roles of PKC can be excluded. It is unlikely that PKC is directly inactivating iNOS enzyme activity, because the iNOS protein does not have a consensus PKC phosphorylation site. Also, it is unlikely that PKC is downregulating IL receptors, because we have previously shown that phenylephrine, which is known to activate PKC, potentiates IL-stimulated nitrite production by myocytes.11 This potentiation is most likely unrelated to PKC and thus is not contradictory to our present results; instead this potentiation may be related to the high dose of phenylephrine used (50 µmol/L), subsequent activation of ß-adrenergic receptors, and increases in cAMP.
Unlike the effects of the phorbol ester PMA, cAMP by itself was able to stimulate iNOS mRNA, protein, and nitrite production. Moreover, it synergized with IL. Such effects have previously been reported in cultured vascular smooth muscle8 and mesangial31 cells. In the latter case, cAMP increased the t1/2 of IL-stimulated iNOS mRNA. The mechanism by which cAMP stimulates nitrite production was not addressed in our study. Since steady-state levels of iNOS mRNA were increased by cAMP, one could postulate either a direct effect on transcription or an indirect effect occurring via stimulation of an intermediate regulatory factor or a posttranscriptional effect. If cAMP directly stimulates transcription, then the DNA sequence and transcriptional regulation of the rat gene must be different from those of the mouse macrophage gene, which does not contain binding sites for cAMP-stimulated transcription factors.20
We used two approaches, transient transfection of the iNOS promoter and mRNA stability studies, to show that IL regulates iNOS primarily by a transcriptional mechanism. After transfection of -1592iNOSLuc into ventricular myocytes, stimulation with IL activated the luciferase reporter gene 1.8-fold. In a mouse macrophage cell line, -1592iNOSLuc was activated 150-fold by LPS and IFN.20 Such a discrepancy could be attributable to species-specific differences in regulatory elements in the mouse and rat iNOS genes, or it could reflect different transcriptional mechanisms for IL versus LPS plus IFN. It is also possible that other regions of the iNOS gene, in addition to the -1592 to +160 region used in these studies, contain key regulatory elements. From a methodological point of view, it could also be that traces of LPS in our plasmid preparations desensitized iNOS gene expression to subsequent cytokine stimulation, as suggested by Bogdan et al.32
A second line of evidence indicating a transcriptional effect of IL was our study on mRNA t1/2. Our data suggest that IL did not increase mRNA stability. After treatment with IL for 24 hours, the addition of ActD resulted in rapid decay of iNOS mRNA (t1/2=1 hour). This was not significantly changed by combined treatment with IL and ActD. However, if IL stabilization of mRNA required transcription, then our studies would not have detected a change in mRNA t1/2. Nonetheless, our determination of t1/2 was consistent with studies in which a number of different cell types and inducers of iNOS mRNA were used, including mesangial cells (t1/2=1 hour),30 vascular smooth muscle cells (t1/2=2 hours),8 adult cardiac myocytes (t1/2=4 hours),12 and mouse macrophages (t1/2=3 hours).33
In summary, our studies indicate that IL, acting through a tyrosine kinase–mediated pathway, is the primary cytokine in myocyte iNOS regulation and exerts its effect primarily at the transcriptional level. The IL signaling pathway can be modified by cross talk from other pathways, including those that activate PKC and cAMP.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants HL-28982 and HL-03188 from the National Institutes of Health. We thank Dr Charles J. Lowenstein for kindly providing the -1592iNOSLuc plasmid.
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References |
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TopAbstractIntroductionMethods Results Discussion References |
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R. Schuette and M. C. LaPointe Phorbol ester stimulates cyclooxygenase-2 expression and prostanoid production in cardiac myocytes Am J Physiol Heart Circ Physiol, August 1, 2000; 279(2): H719 - H725. [Abstract] [Full Text] [PDF]
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E. Isenovic and M. C. LaPointe Role of Ca2+-Independent Phospholipase A2 in the Regulation of Inducible Nitric Oxide Synthase in Cardiac Myocytes Hypertension, January 1, 2000; 35(1): 249 - 254. [Abstract] [Full Text] [PDF]
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S. Yasuda and W. Y. W. Lew Angiotensin II exacerbates lipopolysaccharide-induced contractile depression in rabbit cardiac myocytes Am J Physiol Heart Circ Physiol, May 1, 1999; 276(5): H1442 - H1449. [Abstract] [Full Text] [PDF]
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 A. Kumar, R. Brar, P. Wang, L. Dee, G. Skorupa, F. Khadour, R. Schulz, and J. E. Parrillo Role of nitric oxide and cGMP in human septic serum-induced depression of cardiac myocyte contractility Am J Physiol Regulatory Integrative Comp Physiol, January 1, 1999; 276(1): R265 - R276. [Abstract] [Full Text] [PDF]
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M. C. LaPointe and E. Isenovic Interleukin-1ß Regulation of Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Involves the p42/44 and p38 MAPK Signaling Pathways in Cardiac Myocytes Hypertension, January 1, 1999; 33(1): 276 - 282. [Abstract] [Full Text] [PDF]
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Q. He and M. C. LaPointe Interleukin-1ß Regulation of the Human Brain Natriuretic Peptide Promoter Involves Ras-, Rac-, and p38 Kinase–Dependent Pathways in Cardiac Myocytes Hypertension, January 1, 1999; 33(1): 283 - 289. [Abstract] [Full Text] [PDF]
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 D. A.M. Norman, M. H. Yacoub, and P. J.R. Barton Nuclear factor NF-{kappa}B in myocardium: developmental expression of subunits and activation by interleukin-1{beta} in cardiac myocytes in vitro Cardiovasc Res, August 1, 1998; 39(2): 434 - 441. [Abstract] [Full Text] [PDF]
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M. C. LaPointe and J. R. Sitkins Phospholipase A2 Metabolites Regulate Inducible Nitric Oxide Synthase in Myocytes Hypertension, January 1, 1998; 31(1): 218 - 224. [Abstract] [Full Text] [PDF]
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 K.-i. Kinugawa, T. Shimizu, A. Yao, O. Kohmoto, T. Serizawa, and T. Takahashi Transcriptional Regulation of Inducible Nitric Oxide Synthase in Cultured Neonatal Rat Cardiac Myocytes Circ. Res., December 19, 1997; 81(6): 911 - 921. [Abstract] [Full Text]
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S. Yasuda and W. Y. W. Lew Lipopolysaccharide Depresses Cardiac Contractility and ß-Adrenergic Contractile Response by Decreasing Myofilament Response to Ca2+ in Cardiac Myocytes Circ. Res., December 19, 1997; 81(6): 1011 - 1020. [Abstract] [Full Text]
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C. F. McTiernan, B. H. Lemster, C. Frye, S. Brooks, A. Combes, and A. M. Feldman Interleukin-1ß Inhibits Phospholamban Gene Expression in Cultured Cardiomyocytes Circ. Res., October 19, 1997; 81(4): 493 - 503. [Abstract] [Full Text]
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R. A. Kelly, J.-L. Balligand, and T. W. Smith Nitric Oxide and Cardiac Function Circ. Res., September 1, 1996; 79(3): 363 - 380. [Full Text]
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