©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
In Murine 3T3 Fibroblasts, Different Second Messenger Pathways Resulting in the Induction of NO Synthase II (iNOS) Converge in the Activation of Transcription Factor NF-B (*)

(Received for publication, September 18, 1995; and in revised form, November 29, 1995)

Hartmut Kleinert Christian Euchenhofer Irmgard Ihrig-Biedert Ulrich Förstermann (§)

From the Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, D-55101 Mainz, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transcription factor NF-kappaB is essential for the induction of nitric oxide synthase (NOS) II (iNOS) by bacterial lipopolysaccharide in murine macrophages (Xie, Q. W., Kashiwabara, Y., and Nathan, C.(1994) J. Biol. Chem. 269, 4705-4708). In 3T3 fibroblasts, agents other than cytokines are efficacious inducers of NOS II expression. In addition to cytokines such as interferon- or tumor necrosis factor-alpha, protein kinase C-stimulating agents such as tetradecanoylphorbol-13-acetate, or cyclic AMP-elevating agents such as forskolin and 8-bromo-cAMP markedly increased NOS II mRNA (measured by S1 nuclease and RNase protection analyses), NOS II protein (determined by Western blotting), and NOS activity (measured by chemiluminescence detection of NO(2)). Transforming growth factor-beta1 (which is an inhibitor of NOS II induction in other cell types) potentiated NOS II mRNA expression produced by all inducing agents listed, whereas dexamethasone, pyrrolidine dithiocarbamate and 3,4-dichloroisocoumarin (inhibitors of NF-kappaB activation) suppressed NOS II mRNA induction in response to all stimulants. In electrophoretic mobility shift assays, nuclear protein extracts from 3T3 cells stimulated with any of the inducing agents significantly slowed the migration of an NF-kappaB-binding oligonucleotide, whereas nuclear extracts from untreated control cells did not. These experiments indicate that NF-kappaB is the key control element for the induction of NOS II in response to at least three different second messenger pathways in 3T3 cells.


INTRODUCTION

Nitric oxide (NO) (^1)is a short-lived bioactive molecule participating in the physiology and/or pathophysiology of many organ systems(1) . The expression of the inducible isoform of nitric oxide synthase (NOS II or iNOS) is regulated mainly at the transcriptional level(2) . Inflammatory stimuli such as bacterial lipopolysaccharide (LPS) and cytokines induce the expression of this enzyme in many cell types. Interestingly, in some cells, agents other than cytokines are efficacious inducers of NOS II expression. For example, in rat mesangial cells, cAMP-elevating agents stimulate NOS II expression(3) . Phorbol ester induction of NOS II has been reported for rat peritoneal macrophages(4) . In murine BALB 3T3 fibroblasts, NOS II is expressed in response to forskolin, dibutyryl cAMP, or tetradecanoylphorbol-13-acetate (TPA)(5) .

Analyses of the cloned murine NOS II promoter (6, 7, 8) have revealed the presence of numerous consensus sequences for the binding of transcription factors. Of these potentially relevant transcription factors, nuclear factor-kappaB (NF-kappaB) (6, 9) and interferon regulatory factor (10, 11) have been shown to be functionally important for NOS II induction. The molecular mechanisms utilized by other second messenger pathways are still unclear. In rat mesangial cells, the inhibitor of NF-kappaB activation, pyrrolidine dithiocarbamate (PDTC), blocked NOS II expression induced by interleukin-1beta (IL-1beta), but not the expression stimulated by 8-bromo-cAMP, suggesting two different induction pathways(12) .

In the current study, we attempted to induce NOS II expression in 3T3 fibroblasts via four different second messenger pathways, namely receptor tyrosine kinase, protein kinase C, protein kinase A, and protein kinase G. We characterized the induction processes with modulators of NOS II induction such as transforming growth factor-beta1 (TGF-beta1), dexamethasone, PDTC, and 3,4-dichloroisocoumarin (DCI). The experiments indicate that all NOS II-inducing second messenger pathways are modulated in the same way and all converge in the activation of NF-kappaB as an essential transcription factor.


MATERIALS AND METHODS

Reagents

Mouse INF-, human TNF-alpha and human TGF-beta1 were purchased from Genzyme. LPS (Escherichia coli 026:B6), PDTC, DCI, dexamethasone, forskolin, 8-bromo-cAMP, 8-bromo-cGMP, isobutylmethylxanthine (IBMX) and TPA were purchased from Sigma. Isotopes were obtained from Amersham Corp. Restrictions enzymes, polynucleotide kinase, Taq polymerase, S1 nuclease, T3 RNA polymerase, dNTPs, and oligo(dT) primer were purchased from Pharmacia Biotech Inc. RNase ONE(TM) was obtained from Promega. Superscript reverse transcriptase and DNase I were obtained from Life Technologies, Inc.

Cell Culture

Murine BALB 3T3 fibroblasts and RAW 264.7 macrophages (both ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) with 10% fetal bovine serum, 2 mML-glutamine, penicillin, and streptomycin. For induction, confluent 3T3 cells were cultured for 16 h in DMEM with only 0.5% fetal calf serum and then incubated for 3-18 h (in DMEM with 0.5% fetal calf serum) with one of the following agents: INF- (100 units/ml), TNF-alpha (10 ng/ml), LPS (1 µg/ml), TPA (50 ng/ml), forskolin (100 µM), 8-bromo-cAMP (1 mM), IBMX (250 µM), or 8-bromo-cGMP (1 mM). In some experiments the following modulators of NOS II induction were added with one of the inducing agents: dexamethasone (5 µM), TGF-beta1(2 ng/ml), PDTC (100 µM), or DCI (50 µM).

NOS II Protein Preparation and Western Blotting

3T3 cells (untreated and induced for 18 h with TNF-alpha, 10 ng/ml; LPS, 1 µg/ml; TPA, 50 ng/ml; or forskolin, 100 µM) were homogenized on ice as described previously for brain tissue or endothelial cells(13, 14) . Homogenates were centrifuged at 100,000 times g for 1 h, and the soluble (cytosolic) fraction was partially purified on 2`,5`-ADP-Sepharose(13, 14) . The eluates from the affinity columns were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (7.5% gels)(15) . The proteins were transferred to nitrocellulose membranes (Schleicher & Schuell) by electroblotting (Bio-Rad). All subsequent steps were performed at room temperature. The blots were blocked in Blotto (3% non-fat dried milk in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) for 45 min. The blots were incubated for 1 h with a monoclonal anti-NOS II antibody (1 µg/ml, Transduction Laboratories, Lexington, KY) in Tris-buffered saline/Tween (TBS/T: 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) containing 50 mg/liter gelatin. The blots were washed twice with TBS/T (7 min each) and then incubated for 30 min with horseradish peroxidase-conjugated goat anti-mouse IgG diluted 1:2000 in TBS/T with 50 mg/liter gelatin. The blots were washed three times (5 min each) in TBS/T, followed by one wash (5 min) in TBS alone. The immunocomplexes were developed using an enhanced horseradish peroxidase/luminol chemiluminescence reaction (DuPont NEN) according to the manufacturer's instructions.

Cloning of a Murine NOS II and a Murine beta-Actin cDNA Fragment

Total RNA was isolated by guanidinium isothiocyanate/phenol/chloroform extraction (16) from RAW 264.7 cells induced with 1 µg/ml LPS for 16 h. Two µg of this RNA were annealed with 0.5 µg of oligo(dT) primer (Pharmacia) and reverse-transcribed with Superscript reverse transcriptase (Life Technologies, Inc.) following the manufacture's instructions. Reverse transcriptase-generated cDNA encoding for murine NOS II and murine beta-actin were amplified using PCR. Oligonucleotide primers for NOS II and beta-actin were: GACAAGAGGCTGCCCCCC (sense), and GCTGGGAGTCATGGAGCCG (antisense); GTGGGCCGCTCTAGGCACCAA (sense), and CTCTTTGATGTCACGCACGATTTC (antisense), respectively. They generated PCR fragments corresponding to the murine NOS II cDNA (17) (positions 2612-3170) and murine beta-actin (18) (positions 25-564). PCR was performed in 100 µl of Taq polymerase buffer (Pharmacia), containing 0.2 mM dNTPs, 1.5 mM MgCl 2 units of Taq polymerase, 50 pmol oligonucleotide primers, and reverse transcriptase products (0.10 of the reverse transcriptase reaction). After a initial denaturation step of 95 °C for 5 min, 30 cycles were performed (1 min at 95 °C, 1 min at 60 °C, and 1 min at 72 °C). The final extension period at 72 °C was 10 min. Amplified cDNA fragments (NOS II, 559 base pairs; beta-actin, 540 base pairs) were cloned into the EcoRV site of pCR-Script (Stratagene) using the Sure Clone ligation kit (Pharmacia), generating the cDNA clones pCR_NOS II_mouse and pCR_beta-actin_mouse. DNA sequences of the cloned PCR products were determined from plasmid templates using the dideoxy chain termination method with the T7 sequencing kit (Pharmacia).

Preparation of DNA and Antisense RNA Probes

To generate radiolabeled DNA probes for S1 nuclease protection analysis, the cDNA clones pCR_NOS II_mouse and pCR_beta-actin_mouse were restricted with NcoI or BglII, dephosporylated (with calf intestinal alkaline phosphatase, Boehringer Mannheim), extracted with phenol/chloroform, and concentrated by ethanol precipitation. Fifty to one hundred ng of this DNA were labeled with [-P]ATP using polynucleotide kinase (Pharmacia). The radiolabeled DNA was separated from unincorporated radioactivity using NucTrap probe purification columns (Stratagene). To generate radiolabeled antisense RNA probes for RNase protection assays, the cDNA clones pCR_NOS II_mouse and pCR_beta-actin_mouse were linearized with NcoI or BstEII, extracted with phenol/chloroform, and concentrated by ethanol precipitation. One half of a microgram of this DNA was in vitro transcribed using T3 RNA polymerase (Pharmacia) and [alpha-P]UTP. After a 1-h incubation, the template DNA was degraded with DNase I for 15 min. The radiolabeled RNA was purified using NucTrap probe purification columns (Stratagene).

S1 Nuclease Protection Analyses and RNase Protection Analyses

S1 nuclease protection analyses were performed as described(19, 20) . Briefly, after a denaturation step at 85 °C for 30 min, 20 µg of total RNA isolated by the guanidinium isothiocyanate/phenol/chloroform extraction method (16) were hybridized at 52 °C for 16 h with 75,000 cpm labeled NOS II DNA probe and 30,000 cpm labeled beta-actin DNA probe in hybridization buffer (40 mM Pipes, pH 6.4, 400 mM NaCl, 1 mM EDTA, 80% formamide) in a volume of 30 µl. The S1 nuclease digestion was started by adding 310 µl of digesting buffer (280 mM NaCl, 4.5 mM Zn(CH(3)COO)(2), pH 4.5, 30 µg/ml denatured salmon sperm DNA, and 300 units/ml S1 nuclease). After 20 min at 37 °C, the reaction was stopped by adding 65 µl of stop-buffer (2.5 M NH(4)-acetate, 50 mM EDTA), followed by a phenol/chloroform extraction. The reaction products were concentrated by ethanol precipitation and analyzed by electrophoresis in denaturing urea-polyacrylamide gels (8 M urea, 6% polyacrylamide gel electrophoresis). The electrophoresis buffer was 1 times TBE (1.08% Tris, pH 8.3, 0.55% boric acid, and 20 mM EDTA). The gels were electrophoresed for 2-3 h, dried, and exposed to x-ray films. The protected DNA fragments of NOS II and beta-actin were 380 and 150 nucleotides, respectively. RNase protection assays were performed with RNase ONE(TM) according to the manufacturer's instructions (Promega). Briefly, following denaturation, 20 µg of total RNA (prepared as described above) were hybridized with 100,000 cpm labeled NOS II antisense RNA probe and 10,000 cpm labeled beta-actin antisense RNA probe at 51 °C for 16 h in a volume of 30 µl. Then the mixture was digested with 5 units of RNase ONE(TM) for 1 h at room temperature in 300 µl. The reaction was stopped with 1% SDS, and the samples were concentrated and electrophoresed as described for the S1 nuclease protection analysis. The protected RNA fragments of NOS II and beta-actin were 184 and 108 nucleotides, respectively.

Electrophoretic Mobility Shift Assay (EMSA)

NF-kappaB binding activity in the nuclei of control 3T3 fibroblast- or RAW 264.7 cells, or cells incubated for 3 h with one of the NOS II-inducing agents mentioned above were determined by EMSA using the Promega gel shift assay system. Nuclear proteins were extracted from the cells by detergent lysis(21) . Ten µg of nuclear protein were incubated with 17.5 fmol of P-labeled double-stranded oligonucleotide containing a motif for NF-kappaB binding (5`-AGTTGAGGGGACTTTCCCAGGC-3`). In some experiments, 1.75 pmol of an oligonucleotide with the putative NF-kappaB binding sequence of the murine NOS II promoter (5`-CAACTGGGGACTCTCCCTTTG-3`) were added. The DNA-protein complexes were analyzed on 5% polyacrylamide gels (electrophoresis buffer: 6.7 mM Tris/HCl, pH 7.5, 3.3 mM sodium acetate, 1 mM EDTA), dried, and autoradiographed.

Measurement of NO Production by Chemiluminescence

Confluent 3T3 fibroblasts were cultured for 18 h in DMEM containing 10% fetal bovine serum. Control cells received no additions to the medium; other cells were incubated with LPS (1 µg/ml), TNF-alpha (10 ng/ml), TPA (50 ng/ml), or forskolin (100 µM). After 18 h, the cell supernatants were collected and aliquots were deproteinized with 2 volumes of ethanol. Following centrifugation, 200 µl of the supernatant were injected into a collection chamber containing 100 mM KI in 10 mM sulfuric acid. This strong reducing environment converts NO(2) (and nitrosyl compounds) back to NO. A constant stream of N(2) gas carried the NO into a nitric oxide analyzer (Sievers, Boulder, CO) where the NO was reacted with ozone, resulting in the emission of light. The light emission is proportional to the NO formed; standard amounts of NO(2) were used for calibration.


RESULTS AND DISCUSSION

Stimulation of Different Second Messenger Pathways Induced NOS II mRNA Expression

In 3T3 cells, NOS II mRNA was markedly induced with IFN- (100 units/ml) or TNF-alpha (10 ng/ml) (Fig. 1). NOS II expression was also enhanced with TPA (50 ng/ml) or the cAMP-elevating agents forskolin (100 µM) or 8-bromo-cAMP (1 mM) (Fig. 2). In contrast, 8-bromo-cGMP (1 mM) was ineffective as a stimulator of NOS II induction (Fig. 2). The phosphodiesterase inhibitor IBMX (250 µM) also produced a marked induction of NOS II mRNA in 3T3 cells (Fig. 3). This can be explained by the increase in cAMP, but not cGMP (cf. Fig. 2). LPS (up to 1 µg/ml) showed little efficacy in inducing NOS II mRNA (Fig. 3). Thus the stimulation of the receptor tyrosine kinase pathway (by INF-, TNF-alpha, and possibly LPS), the stimulation of the protein kinase C pathway (by TPA), and the stimulation of the protein kinase A pathway (by forskolin, 8-bromo-cAMP, and IBMX) all induced the transcription of NOS II mRNA in 3T3 fibroblasts.


Figure 1: Double S1 nuclease protection analysis using cDNA probes for murine NOS II and beta-actin (for standardization). RNAs were prepared from untreated 3T3 fibroblasts (control, Co) and 3T3 cells induced with human tumor necrosis factor-alpha (10 ng/ml, TNF-alpha) or murine interferon- (100 units/ml, IFN-). T, tRNA control; M, molecular weight markers (pGl(2)-Basic, Promega, restricted with HinfI). The gel is representative of three experiments with similar results. Densitometric analysis demonstrated that the NOS II signal in the TNF-alpha lane (after correction by the beta-actin signal) was 1033% of the NOS II signal in the control (Co) lane, and the NOS II signal in the IFN- lane was 440% of the NOS II signal in the control (Co) lane.




Figure 2: S1 nuclease protection analysis using cDNA probes for murine NOS II and beta-actin (for standardization). RNAs were prepared from untreated control 3T3 cells (Co) and cells stimulated with the following agents: the protein kinase C stimulator tetradecanoylphorbol-13-acetate (50 ng/ml, TPA), the adenylyl cyclase-stimulating agent forskolin (100 µM, Forsk), 8-bromo-cAMP (1 mM, cAMP), or 8-bromo-cGMP (1 mM, cGMP). T, tRNA control; M, molecular weight markers (pGl(2)-Basic restricted with HinfI). The gel is representative of three experiments with similar results. Densitometric analysis demonstrated that the NOS II signal in the TPA lane (after correction by the beta-actin signal) was 460% of the NOS II signal in the control (Co) lane; the NOS II signal in the Forsk lane was 570% of the NOS II signal in the control (Co) lane, and the NOS II signal in the cAMP lane was 1270% of the NOS II signal in the control (Co) lane. The NOS II signal in the cGMP lane was not different (53%) from the signal in the control (Co) lane (100%).




Figure 3: S1 nuclease protection analysis using cDNA probes for murine NOS II and beta-actin (for standardization). RNAs were obtained from 3T3 cells stimulated with various agents in the absence and presence of dexamethasone (5 µM). The following stimulating agents were used: bacterial lipopolysaccharide (1 µg/ml, LPS), and LPS in the presence of dexamethasone (Dex); tetradecanoylphorbol-13-acetate (50 ng/ml, TPA) and TPA in the presence of dexamethasone (Dex); the phosphodiesterase inhibitor isobutylmethylxanthine (250 µM, IBMX) and IBMX in the presence of dexamethasone (Dex). T, tRNA control; M, molecular weight markers (pGl(2)-Basic restricted with HinfI). The gel is representative of three experiments with similar results. Densitometric analysis demonstrated that the NOS II signal in the LPS + Dex lane (after correction by the beta-actin signal) was 40% of the NOS II signal in the LPS lane; the NOS II signal in the TPA + Dex lane was 28% of the NOS II signal in the TPA lane, and the NOS II signal in the IBMX + Dex lane was 15% of the NOS II signal in the IBMX lane.



Because double protected bands for NOS II mRNA were seen in some of the S1 nuclease analyses, RNase protection assays were performed on the same RNAs (using antisense RNA probes derived from the same NOS II cDNA fragment). The RNase protection assays yielded single protected bands and quantitatively similar results as obtained in the S1 nuclease protection assays (data not shown). Therefore, the double bands seen in the S1 assays are unlikely to represent two different NOS II mRNAs.

Small amounts of NOS II mRNA were detected even in the absence of cytokines or stimulants (cf. Fig. 1and Fig. 2). The same phenomenon has been described previously for murine 3T3 cells (5) and human DLD-1 epithelial cells(22) . It may either represent a low constitutive expression of this isoform or an autocrine/paracrine induction of these cells by endogenous cytokines(22) .

In 3T3 cells, INF- alone produced a marked induction of NOS II mRNA (Fig. 1). There is controversy as to whether INF- alone can induce NOS II in RAW 264.7 macrophages. While this has been described by some authors(8) , others only see an effect of INF- in the presence of LPS(6, 7) . In the current experiments, polymyxin B (10 µg/ml), an inhibitor of the induction of murine cells by LPS(23) , did not prevent the NOS II-inducing action of INF- (100 units/ml), TNF-alpha (10 ng/ml), TPA (50 ng/ml), or forskolin (100 µM) (n = 4, data not shown), suggesting that INF- alone is an effective NOS II inducer in 3T3 cells.

The signal transduction pathways effective in inducing NOS II expression vary considerably between cell types (and probably species). In many cells, stimulation of the protein kinase C pathway has little or no effect on NOS II induction by itself, but potentiates cytokine induction(24, 25, 26) . In 3T3 fibroblasts, it is an efficacious inducing pathway by itself. Stimulators of the protein kinase A pathway alone have been shown to promote NOS II expression in vascular smooth muscle cells and rat mesangial cells(12, 27, 28) . On the other hand, protein kinase A activation seems to inhibit NOS II induction in rat RINm5F insulinoma cells(26) . Thus the NOS II-inducing mechanisms seem to be cell-specific, and the stimulation pattern observed in the present study (Fig. 1Fig. 2Fig. 3) is unique to 3T3 cells.

Stimulation of Different Second Messenger Pathways in 3T3 Cells Increased NOS II Protein Expression

Similar to the NOS II mRNA expression induced by the various signal transduction pathways, expression of NOS II immunoreactive protein was stimulated by TNF-alpha (10 ng/ml), LPS (1 µg/ml), TPA (50 ng/ml), or forskolin (100 µM) (Fig. 4). Noninduced 3T3 cells showed no NOS II immunoreactivity in Western blots (n = 3, not shown).


Figure 4: Western blot analysis of the soluble (cytosolic) fraction from 3T3 fibroblasts. Protein samples were prepared from 3T3 cells as described under ``Materials and Methods,'' separated on SDS-polyacrylamide gels (7.5%), and transferred to nitrocellulose membranes. A anti-NOS II antibody was used for detection. 3T3 cells were induced with different agents before protein was prepared: tumor necrosis factor-alpha (10 ng/ml, TNF-alpha), bacterial lipopolysaccharide (1 µg/ml, LPS), tetradecanoylphorbol-13-acetate (50 ng/ml, TPA), or forskolin (100 µM, Forsk). The blot is representative of three experiments with similar results. The double (or triple) bands detected by the antibody probably correspond to the double or triple bands seen by Stuehr et al.(54) for the purified NOS II protein from macrophages.



Stimulation of Different Second Messenger Pathways in 3T3 Cells Enhanced NO(2) Production

Incubation of 3T3 fibroblasts with TNF-alpha, TPA, or forskolin markedly enhanced the NO(2) content in the supernatant of the cells (Fig. 5). LPS was a much weaker stimulant of 3T3 cell NO(2) production (Fig. 5). This indicates that NOS II protein and activity is also induced via the receptor tyrosine kinase, protein kinase C, and protein kinase A pathways.


Figure 5: Chemiluminescence determination of NO(2) in the supernatant of 3T3 cells as an indicator of NO production. The cells were kept for 18 h in culture medium alone (control, Co) or were incubated with bacterial lipopolysaccharide (1 µg/ml, LPS), tumor necrosis factor-alpha (10 ng/ml, TNF-alpha), tetradecanoylphorbol-13-acetate (50 ng/ml, TPA), or forskolin (100 µM, Forsk).



Stimulation of Three Different Second Messenger Pathways in 3T3 Cells Induced Proteins with NF-kappaB Binding Activity

NF-kappaB is a multisubunit transcription factor that can rapidly activate the expression of genes involved in immune and acute phase responses(29) . NF-kappaB is composed mainly of proteins with molecular weights of 50 kDa (p50) and 65 kDa (p65). Both types of proteins share significant homology with the proto-oncogene c-rel(30, 31, 32) . The proteins p50, p65, and c-Rel can interact with each other and, following activation, bind the NF-kappaB response element as homo- or heterodimers (33) (consensus sequence: GGGRNNYYCC)(34) . In its unstimulated form, NF-kappaB is present in the cytosol bound to the inhibitory protein I-kB. After induction of cells by a variety of agents, NF-kappaB is released from I-kB and translocated to the nucleus. Agents that have been described as NF-kappaB activators include mitogens, cytokines, and LPS, TPA, and cAMP(29, 35) . EMSA experiments shown in Fig. 6demonstrated that nuclear extracts of untreated 3T3 cells contained low concentrations of proteins that bind an oligonucleotide containing the NF-kappaB response element. Incubation of 3T3 cells either with TPA (50 ng/ml), TNF (10 ng/ml) or 8-bromo-cAMP (1 mM) markedly increased the NF-kappaB binding activity (Fig. 6). In 3T3 fibroblasts, TNF-alpha was the most efficacious inducer of NF-kappaB binding activity tested. TPA and cAMP-elevating agents (8-bromo-cAMP or forskolin) were less efficacious in inducing NF-kappaB binding activity; there were no significant differences in efficacy between these two classes of agents. This parallels the NOS II mRNA and NOS II protein expression as well as the NOS activity stimulated by these compounds. The protein-DNA interaction was totally prevented in all cases with an excess of unlabeled double-stranded oligonucleotide containing the NF-kappaB site of the murine NOS II promoter ( Fig. 6and data not shown). These data suggest that, in 3T3 cells, the receptor tyrosine kinase pathway, the protein kinase A pathway, and the protein kinase C pathway stimulate the activation of transcription factor NF-kappaB. While cytokines such as TNF-alpha can activate NF-kappaB in most cell types, there is a marked inter-cell variability for the protein kinase A and C pathways. For example, in murine RAW264.7 cells, neither the protein kinase A pathway nor the protein kinase C pathway are able to stimulate this transcription factor; they even inhibit NF-kappaB-dependent reporter gene expression in response to LPS(36) . In human Jurkat T cells, the protein kinase C pathway, but not the protein kinase A pathway activates NF-kappaB(37) . Conversely, in murine J774 macrophages, activators of protein kinase A are effective stimulators of NF-kappaB, whereas protein kinase C activators failed to stimulate this transcription factor(38) .


Figure 6: EMSA using a 5`-end-labeled consensus oligonucleotide for NF-kappaB binding (O) and nuclear extracts from 3T3 fibroblasts (3T3) and murine RAW 264.7 macrophages as positive controls (RAW). 3T3 cells were incubated for 3 h with medium alone (negative control, Co), tumor necrosis factor-alpha (10 ng/ml, TNF-alpha), tetradecanoylphorbol-13-acetate (50 ng/ml, TPA), or 8-bromo-cAMP (1 mM, cAMP). Densitometric analyses of the gels demonstrated that TPA and 8-bromo-cAMP were about equieffective in stimulation NF-kappaB binding activity, whereas TNF-alpha induced the largest increase in NF-kappaB binding activity. Nuclear extracts from LPS-induced RAW 264.7 macrophages are known to contain NF-kappaB binding activity (9) and were used as positive controls (RAW). RAW 264.7 macrophages were induced with bacterial lipopolysaccharide (1 µg/ml, LPS), and the same nuclear protein extract from RAW 264.7 cells was tested in the presence of a 100-fold excess of an oligonucleotide containing the NF-kappaB binding site of the murine NOS II promoter (competition experiment, LPS comp.). The gels are representative of three experiments yielding similar results.



Effect of Dexamethasone on NOS II mRNA Expression

Glucocorticoids such as dexamethasone have been known for some years to inhibit cytokine induction of NOS II activity in various cell types (such as endothelial cells, macrophages, and smooth muscle cells(39, 40, 41, 42) ). More recently, this inhibition has also been demonstrated at the mRNA level in several cell types(5, 43, 44) . In a recent communication, Kunz et al.(45) demonstrated in rat mesangial cells that dexamethasone prevented the induction of NOS II activity in response to IL-1beta and dibutyryl cAMP. Interestingly, NOS II mRNA levels were only reduced when dibutyryl cAMP was used as the inducing agent, but not after IL-1beta. Consequently, these authors postulated that dexamethasone acts at different levels, depending on the stimulus used to suppress NOS II induction in rat mesangial cells(45) . In the current study we examined the effect of dexamethasone (5 µM) on NOS II mRNA expression in 3T3 cells. We found that the steroid was equally effective against inductions produced by LPS, TPA or IBMX (Fig. 3). Also the NOS II mRNA inductions in response to TNF-alpha (10 ng/ml) or INF- (100 units/ml) were markedly inhibited by dexamethasone (5 µM) (n = 3, not shown).

Effect of TGF-beta1 on NOS II mRNA Expression

TGF-beta1 is an inhibitor of NOS II induction in mouse macrophages and rat vascular smooth muscle cells(42, 43, 46, 47, 48) . On the other hand, in 3T3 cells and in bovine retinal pigmented epithelial cells, TGF-beta1 has been described as a stimulator of cytokine-induced NOS II mRNA induction (43, 49) . Also in the current experiments, TGF-beta1 (2 ng/ml) potentiated NOS II mRNA production irrespective of the second messenger pathway used for induction (Fig. 7).


Figure 7: S1 nuclease protection analysis using cDNA probes for murine NOS II and beta-actin (for standardization). RNAs were obtained from 3T3 cells stimulated with various agents in the absence and presence of transforming growth factor-beta1 (2 ng/ml, TGF-beta1). TGF-beta1 alone did not produced any NOS II induction. The following stimulating agents were used: bacterial lipopolysaccharide (1 µg/ml, LPS) and LPS in the presence of TGF-beta1; tetradecanoylphorbol-13-acetate (50 ng/ml, TPA) and TPA in the presence of TGF-beta1; the phosphodiesterase inhibitor isobutylmethylxanthine (250 µM, IBMX) and IBMX in the presence of TGF-beta1. T, tRNA control; M, molecular weight markers (pGl(2)-Basic restricted with HinfI). The gel is representative of three experiments with similar results. Densitometric analysis demonstrated that the NOS II signal in the LPS + TGF-beta1 lane (after correction by the beta-actin signal) was 130% of the NOS II signal in the LPS lane; the NOS II signal in the TPA + TGF-beta1 lane was 230% of the NOS II signal in the TPA lane, and the NOS II signal in the IBMX + TGF-beta1 lane was 330% of the NOS II signal in the IBMX lane.



Inhibition of NF-kappaB Activation Blocks NOS II mRNA Induction

The activation of NF-kappaB can be blocked by thiol compounds such as PDTC or diethyldithiocarbamate, which leave the DNA binding activity of other transcription factors (e.g. SP1, Oct, and CREB) unaffected(50) . PDTC or diethyldithiocarbamate have been shown to prevent the induction of NOS II in LPS-induced murine macrophages (9, 51) and rat alveolar macrophages(52) . Eberhardt et al.(12) reported that PDTC inhibits the induction of NOS II expression in response to IL-1beta, but not to dibutyryl cAMP. They concluded that in rat mesangial cells cAMP-stimulated NOS II expression is activated through a transcription factor different from NF-kappaB. In the current series of experiments in 3T3 cells, PDTC prevented the induction of the NOS II mRNA expression in response to all inducing compounds used (Fig. 8). Also DCI, a serine protease inhibitor, which blocks NF-kappaB activation by inhibiting proteolytic degradation of I-kappaB(53) , blocked (by over 90%) NOS II mRNA expression induced by INF- (100 units/ml), TNF-alpha (10 ng/ml), TPA (50 ng/ml), and forskolin (100 µM) (n = 3, data not shown). This confirms the results of our EMSA experiments and indicates that in 3T3 fibroblasts NF-kappaB is essential for NOS II induction in response to different second messengers. Interestingly, the inhibition of NOS II induction by dexamethasone (described above) is likely to reflect its ability to inactivate NF-kappaB(20) .


Figure 8: S1 nuclease protection analysis using cDNA probes for murine NOS II and beta-actin (for standardization). RNA were obtained from unstimulated 3T3 fibroblasts (Co) and 3T3 cells stimulated with various agents in the absence and presence of pyrrolidine dithiocarbamate (100 µM, PDTC). The following stimulators were used: the adenylyl cyclase-stimulating agent forskolin (100 µM, Forsk) and forskolin in the presence of PDTC; interferon- (100 units/ml, IFN-) and IFN- in the presence of PDTC; tetradecanoylphorbol-13-acetate (50 ng/ml, TPA) and TPA in the presence of PDTC. M, molecular weight markers (pGl2-Basic restricted with HinfI). The gel is representative of four experiments with similar results. Densitometric analysis demonstrated that the NOS II signal In the Forsk + PDTC lane (after correction by the beta-actin signal) was 28% of the NOS II signal in the Forsk lane; the NOS II signal in the IFN- + PDTC lane was 5% of the NOS II signal in the IFN- lane, and the NOS II signal in the TPA + PDTC lane was 12% of the NOS II signal in the TPA lane.



In conclusion, our data demonstrate that in 3T3 cells at least three different signal transduction pathways can stimulated NOS II mRNA expression, namely the cytokine/receptor tyrosine kinase pathway, the cAMP/protein kinase A pathway, and the protein kinase C pathway. All these pathways seem to converge in the activation of the essential transcription factor NF-kappaB, which increases the transcription of the NOS II gene.


FOOTNOTES

*
This study was supported by Grants Fo 144/3-1 and Fo 144/4-1 from the Deutsche Forschungsgemeinschaft, Bonn, Germany and by a grant from the Ministry of the Environment of the State of Rhineland-Palatinate, Mainz, Germany. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work is dedicated to Dr. Ernst Mutschler, Professor of Pharmacology, Johann Wolfgang Goethe University, Frankfurt, Germany on the occasion of his 65th birthday.

§
To whom correspondence should be addressed: Dept. of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Str. 67, D-55101 Mainz, Germany. Tel.: 49-6131-17-3123; Fax: 49-6131-17-6611; uforster{at}mzdmza.zdv.uni-mainz.de.

(^1)
The abbreviations used are: NO, nitric oxide; NOS, nitric oxide synthase; DCI, 3,4-dichloroisocoumarin; EMSA, electrophoretic mobility shift assay; IBMX, 3-isobutyl-1-methylxanthine; I-kB, specific inhibitor protein of NF-kappaB; INF-, interferon-; IL-1beta, interleukin-1beta; LPS, bacterial lipopolysaccharide; NF-kappaB, nuclear factor-kappaB; PDTC, pyrrolidine dithiocarbamate; TNF-alpha, tumor necrosis factor-alpha; TGF-beta1, transforming growth factor-beta1; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; Pipes, 1,4-piperazinediethanesulfonic acid.


ACKNOWLEDGEMENTS

We greatly appreciate the technical help of Bärbel Hering with the cell culture.


REFERENCES

  1. Förstermann, U., Closs, E. I., Pollock, J. S., Nakane, M., Schwarz, P., Gath, I., and Kleinert, H. (1994) Hypertension 23, 1121-1131 [Abstract]
  2. Förstermann, U., and Kleinert, H. (1995) Naunyn Schmiedebergs Arch. Pharmacol. 352, 351-364 [Medline] [Order article via Infotrieve]
  3. Kunz, D., Mühl, H., Walker, G., and Pfeilschifter, J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5387-5391 [Abstract]
  4. Hortelano, S., Genaro, A. M., and Bosca, L. (1993) FEBS Lett. 320, 135-139 [CrossRef][Medline] [Order article via Infotrieve]
  5. Gilbert, R. S., and Herschman, H. R. (1993) J. Cell. Physiol. 157, 128-132 [Medline] [Order article via Infotrieve]
  6. Lowenstein, C. J., Alley, E. W., Raval, P., Snowman, A. M., Snyder, S. H., Russell, S. W., and Murphy, W. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9730-9734 [Abstract]
  7. Xie, Q. W., Whisnant, R., and Nathan, C. (1993) J. Exp. Med. 177, 1779-1784 [Abstract]
  8. Weisz, A., Oguchi, S., Cicatiello, L., and Esumi, H. (1994) J. Biol. Chem. 269, 8324-8333 [Abstract/Free Full Text]
  9. Xie, Q. W., Kashiwabara, Y., and Nathan, C. (1994) J. Biol. Chem. 269, 4705-4708 [Abstract/Free Full Text]
  10. Kamijo, R., Harada, H., Matsuyama, T., Bosland, M., Gerecitano, J., Shapiro, D., Le, J., Koh, S. I., Kimura, T., Green, S. J., Mak, T. W., Taniguchi, T., and Vilcek, J. (1994) Science 263, 1612-1615 [Medline] [Order article via Infotrieve]
  11. Martin, E., Nathan, C., and Xie, Q. W. (1994) J. Exp. Med. 180, 977-984 [Abstract]
  12. Eberhardt, W., Kunz, D., and Pfeilschifter, J. (1994) Biochem. Biophys. Res. Commun. 200, 163-170 [CrossRef][Medline] [Order article via Infotrieve]
  13. Schmidt, H. H. H. W., Pollock, J. S., Nakane, M., Gorsky, L. D., Förstermann, U., and Murad, F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 365-369 [Abstract]
  14. Förstermann, U., Pollock, J. S., Schmidt, H. H. H. W., Heller, M., and Murad, F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1788-1792 [Abstract]
  15. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  16. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  17. Lyons, C. R., Orloff, G. J., and Cunningham, J. M. (1992) J. Biol. Chem. 267, 6370-6374 [Abstract/Free Full Text]
  18. Alonso, S., Minty, A., Bourlet, Y., and Buckingham, M. (1986) J. Mol. Evol. 23, 11-22 [Medline] [Order article via Infotrieve]
  19. Kleinert, H., and Benecke, B. J. (1988) Nucleic Acids Res. 16, 1319-1331 [Abstract]
  20. Kleinert, H., Euchenhofer, C., Ihrig-Biedert, I., and Förstermann, U. (1996) Mol. Pharmacol. 49, 1-7 [Abstract]
  21. Osborn, L., Kunkel, S., and Nabel, G. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2336-2340 [Abstract]
  22. Chu, S. C., Wu, H. P., Banks, T. C., Eissa, N. T., and Moss, J. (1995) J. Biol. Chem. 270, 10625-10630 [Abstract/Free Full Text]
  23. Salkowski, C. A., and Vogel, S. N. (1992) J. Immunol. 149, 4041-4047 [Abstract/Free Full Text]
  24. Jun, C. D., Choi, B. M., Hoonryu, Um, J. Y., Kwak, H. J., Lee, B. S., Paik, S. G., Kim, H. M., and Chung, H. T. (1994) J. Immunol. 153, 3684-3690 [Abstract/Free Full Text]
  25. Simmons, M. L., and Murphy, S. (1994) Glia 11, 227-234 [Medline] [Order article via Infotrieve]
  26. Messmer, U. K., and Brüne, B. (1994) Cell. Signalling 6, 17-24 [CrossRef][Medline] [Order article via Infotrieve]
  27. Koide, M., Kawahara, Y., Nakayama, I., Tsuda, T., and Yokoyama, M. (1993) J. Biol. Chem. 268, 24959-24966 [Abstract/Free Full Text]
  28. Imai, T., Hirata, Y., Kanno, K., and Marumo, F. (1994) J. Clin. Invest. 93, 543-549 [Medline] [Order article via Infotrieve]
  29. Baeuerle, P. A. (1991) Biochim. Biophys. Acta 1072, 63-80 [CrossRef][Medline] [Order article via Infotrieve]
  30. Bours, V., Villalobos, J., Burd, P. R., Kelly, K., and Siebenlist, U. (1990) Nature 348, 76-80 [CrossRef][Medline] [Order article via Infotrieve]
  31. Ghosh, S., Gifford, A. M., Riviere, L. R., Tempst, P., Nolan, G. P., and Baltimore, D. (1990) Cell 62, 1019-1029 [Medline] [Order article via Infotrieve]
  32. Kieran, M., Blank, V., Logeat, F., Vandekerckhove, J., Lottspeich, F., Le, B. O., Urban, M. B., Kourilsky, P., Baeuerle, P. A., and Israel, A. (1990) Cell 62, 1007-1018 [Medline] [Order article via Infotrieve]
  33. Kunsch, C., Ruben, S. M., and Rosen, C. A. (1992) Mol. Cell. Biol. 12, 4412-4421 [Abstract]
  34. Grilli, M., Chiu, J. J., and Lenardo, M. J. (1993) Int. Rev. Cytol. 143, 1-62 [Medline] [Order article via Infotrieve]
  35. Serkkola, E., and Hurme, M. (1993) FEBS Lett. 334, 327-330 [CrossRef][Medline] [Order article via Infotrieve]
  36. Vincenti, M. P., Burrell, T. A., and Taffet, S. M. (1992) J. Cell. Physiol. 150, 204-213 [Medline] [Order article via Infotrieve]
  37. Feuillard, J., Gouy, H., Bismuth, G., Lee, L. M., Debre, P., and Korner, M. (1991) Cytokine 3, 257-265 [CrossRef][Medline] [Order article via Infotrieve]
  38. Muroi, M., and Suzuki, T. (1993) Cell. Signalling 5, 289-298 [CrossRef][Medline] [Order article via Infotrieve]
  39. Radomski, M. W., Palmer, R. M., and Moncada, S. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 10043-10047 [Abstract]
  40. DiRosa, M., Radomski, M., Carnuccio, R., and Moncada, S. (1990) Biochem. Biophys. Res. Commun. 172, 1246-1252 [Medline] [Order article via Infotrieve]
  41. Kanno, K., Hirata, Y., Imai, T., and Marumo, F. (1993) Hypertension 22, 34-39 [Abstract]
  42. Schini, V. B., Durante, W., Elizondo, E., Scott, B. T., Junquero, D. C., Schafer, A. I., and Vanhoutte, P. M. (1992) Eur. J. Pharmacol. 216, 379-383 [CrossRef][Medline] [Order article via Infotrieve]
  43. Gilbert, R. S., and Herschman, H. R. (1993) Biochem. Biophys. Res. Commun. 195, 380-384 [CrossRef][Medline] [Order article via Infotrieve]
  44. Robbins, R. A., Springall, D. R., Warren, J. B., Kwon, O. J., Buttery, L., Wilson, A. J., Adcock, I. M., Riverosmoreno, V., Moncada, S., Polak, J., and Barnes, P. J. (1994) Biochem. Biophys. Res. Commun. 198, 835-843 [CrossRef][Medline] [Order article via Infotrieve]
  45. Kunz, D., Walker, G., and Pfeilschifter, J. (1994) Biochem. J. 304, 337-340 [Medline] [Order article via Infotrieve]
  46. Ding, A., Nathan, C. F., Graycar, J., Derynck, R., Stuehr, D. J., and Srimal, S. (1990) J. Immunol. 145, 940-944 [Abstract/Free Full Text]
  47. Förstermann, U., Schmidt, H. H. H. W., Kohlhaas, K. L., and Murad, F. (1992) Eur. J. Pharmacol. Mol. Pharmacol. Sect. 225, 161-165 [CrossRef][Medline] [Order article via Infotrieve]
  48. Vodovotz, Y., Bogdan, C., Paik, J., Xie, Q. W., and Nathan, C. (1993) J. Exp. Med. 178, 605-613 [Abstract]
  49. Goureau, O., Lepoivre, M., Becquet, F., and Courtois, Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4276-4280 [Abstract]
  50. Schreck, R., Meier, B., Mannel, D. N., Droge, W., and Baeuerle, P. A. (1992) J. Exp. Med. 175, 1181-1194 [Abstract]
  51. Mülsch, A., Schray-Utz, B., Mordvintcev, P. I., Hauschildt, S., and Busse, R. (1993) FEBS Lett. 321, 215-218 [CrossRef][Medline] [Order article via Infotrieve]
  52. Sherman, M. P., Aeberhard, E. E., Wong, V. Z., Griscavage, J. M., and Ignarro, L. J. (1993) Biochem. Biophys. Res. Commun. 191, 1301-1308 [CrossRef][Medline] [Order article via Infotrieve]
  53. Machleidt, T., Wiegmann, K., Henkel, T., Schutze, S., Baeuerle, P., and Kronke, M. (1994) J. Biol. Chem. 269, 13760-13765 [Abstract/Free Full Text]
  54. Stuehr, D. J., Cho, H. J., Kwon, N. S., Weise, M. F., and Nathan, C. F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7773-7777 [Abstract]

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