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Endocrinology Vol. 140, No. 8 3562-3572
Copyright © 1999 by The Endocrine Society

ARTICLES

Intracellular Signaling in Rat Cultured Vascular Smooth Muscle Cells: Roles of Nuclear Factor-{kappa}B and p38 Mitogen-Activated Protein Kinase on Tumor Necrosis Factor-{alpha} Production1

Tadashi Yamakawa, Satoru Eguchi, Takeshi Matsumoto, Yuko Yamakawa, Kotaro Numaguchi, Ichiro Miyata, Cherilynn M. Reynolds, Evangeline D. Motley and Tadashi Inagami

Department of Biochemistry, Vanderbilt University School of Medicine (T.Y., S.E., T.M., T.K., Y.Y., K.N., T.I.), Nashville, Tennessee 37232; and the Department of Anatomy and Physiology, Meharry Medical College (C.M.R., E.D.M.), Nashville, Tennessee 37208

Address all correspondence and requests for reprints to: Tadashi Inagami, Ph.D., Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232. E-mail: inagamit{at}ctrvax.vanderbilt.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lipopolysaccharide (LPS) is responsible for initiating host responses leading to septic shock, and tumor necrosis factor-{alpha} (TNF{alpha}) is thought to be its primary mediator. In addition, TNF{alpha} is one of the major components of the pathogenesis of insulin resistance in various conditions. It has been shown that LPS induced TNF{alpha} production in rat vascular smooth muscle cells (VSMC). However, little is known about the signaling pathway by which VSMC in culture produce TNF{alpha}. We investigated the possible signaling components involved in this pathway. LPS elicited phosphorylation of p42/44 mitogen-activated protein kinase (MAPK) and p38 MAPK, degradation of inhibitor of {kappa}B (I{kappa}B), and an increase in nuclear binding activity of activating protein-1 and nuclear factor-{kappa}B (NF-{kappa}B). Different types of NF-{kappa}B inhibitors, pyrrolidine dithiocarbamate and MG132, which specifically abolished I{kappa}B degradation and subsequent NF-{kappa}B activation by LPS, suppressed TNF{alpha} secretion from VSMC. Although PD98059, a specific MAPK kinase inhibitor and SB203580, a specific p38 MAPK inhibitor, had no effect on NF-{kappa}B activity, SB203580 suppressed TNF{alpha} secretion; however, PD98059 did not. A cotransfection assay showed that transfection of dominant negative I{kappa}B or pretreatment with SB203580 suppressed the TNF{alpha} gene promotor-dependent transcription. TNF{alpha} messenger RNA expression induced by LPS was inhibited by pyrrolidine dithiocarbamate, MG132, and SB203580, but not by PD98059. These observations indicate that TNF{alpha} production in VSMC is stimulated by LPS, and its transcription and translation are dependent on NF-{kappa}B activation through proteasome-mediated I{kappa}B degradation. It is likely that p38 MAPK may play a critical role in regulating transcription of the TNF{alpha} gene in VSMC, unlike in other cell lines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TUMOR NECROSIS FACTOR-{alpha} (TNF{alpha}) is a multifunctional cytokine that acts as a central regulator of inflammation and immunity (1). Gram-negative sepsis may account for up to half of the cases of septic shock (2), a syndrome characterized by refractory hypotension leading to inadequate organ perfusion, multiorgan failure, and, frequently, death (3). The endotoxin [lipopolysaccharide (LPS)] of Gram-negative bacteria is responsible for initiating host responses leading to septic shock (3), and TNF{alpha} is thought to be its primary mediator (4). In addition, TNF{alpha} is a candidate mediator of insulin resistance (5).

In vascular smooth muscle cells (VSMC), LPS have been reported to induce the expression of a variety of genes and their products. For examples, LPS induces the expression of kininogen (6), the secretion of adrenomedullin (7), and nitric oxide synthesis (8). However, the intracellular mechanisms of induction and secretion of these genes and their products remains unknown. An earlier report by Warner et al. (9) is the only report until the present study that deals with TNF{alpha} production in VSMC. However, they did not present any possible mechanisms that could be responsible for TNF{alpha} production. Thus, no information is available to date on the regulation of TNF{alpha} production in VSMC, whereas the intracellular mechanisms of LPS-induced TNF{alpha} production have been extensively studied in myeloid cells.

To investigate the mechanisms of LPS-induced TNF{alpha} production, the present study was undertaken to clarify the possible role of p42/44 mitogen-activated protein kinase (MAPK)/activating protein-1 (AP-1), p38 MAPK, or nuclear factor-{kappa}B (NF-{kappa}B) activation in TNF{alpha} secretion in cultured rat VSMC. We found that LPS induces p42/44 and p38 MAPK activation, c-Fos and c-Jun induction, and subsequent AP-1 activation, as well as inhibitor of {kappa}B (I{kappa}B) degradation and ensuing NF-{kappa}B activation. We have obtained several lines of evidence indicating that p38 MAPK as well as the NF-{kappa}B signaling pathway are critical for LPS-induced TNF{alpha} production in VSMC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
DMEM, FCS, penicillin, and streptomycin were obtained from Life Technologies (Gaithersburg, MD). LPS (Escherichia coli, serotype 055:B5), pyrrolidine dithiocarbamate (PDTC), and antibodies to smooth muscle {alpha}-actin were purchased from Sigma Chemical Co. (St. Louis, MO). MG132 (Z-Leu-Leu-Leu-H) was purchased from American Peptide Co. (Sunnyvale, CA). PD98059 (MEK 1/2 inhibitor), polyclonal antibodies to dually phosphorylated p42/44 MAPK (P-Thr202 and P-Tyr204) and p38 MAPK (P-Thr180 and P-Tyr182) were purchased from New England Biolabs, Inc. (Beverly, CA). Monoclonal antibody to annexin II was purchased from Transduction Laboratories, Inc. (Lexington, KY). SB203580 was purchased from Calbiochem (San Diego, CA). Rabbit polyclonal antibodies against p50, p52, and p65 subunits of NF-{kappa}B and c-Fos, c-Jun subunits of AP-1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). NF-{kappa}B and AP-1 consensus oligonucleotides were purchased from Promega Corp. (Madison, WI).

Cell culture
VSMC were prepared from the thoracic aorta of 12-week-old Sprague Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) by the explant method as previously described (10). Subcultured VSMC from passages 3–15 that were used in the experiments showed more than 99% positive immunostaining against smooth muscle {alpha}-actin antibodies and were negative for mycoplasma infection. For subsequent experiments, cells at about 80% confluence in culture wells were used 1 day after serum depletion.

Rat TNF{alpha} enzyme immunoassay
After stimulation for specified durations, the culture medium of VSMC was collected and centrifuged for 5 min at 12,000 rpm. TNF{alpha} production was quantified by determining TNF{alpha} immunoreactivity in culture supernatant using a sandwich-type enzyme linked immunosorbent assay (ELISA) kit for rat TNF{alpha} (BioSource International, Camarillo, CA) according to the manufacturer’s instructions.

Cytotoxic assay for TNF{alpha}
The secretion of TNF{alpha} into the culture medium was also quantified by an in vitro cytotoxic assay using actinomycin D-treated NCTC 929 cells obtained from American Type Culture Collection (Manassas, VA) as previously described (11) with slight modifications. Briefly, NCTC 929 cells were placed in a 96-well microculture plate at 2 x 104 cells/well and incubated for 24 h at 37 C. After the incubation, medium was replaced with serum-free DMEM containing 1.5 mM actinomycin D and rat TNF{alpha} standards or samples and further incubated for 24 h. Viable cell numbers were estimated by CellTiter 96AQ, a nonradioactive cell proliferation assay kit (Promega Corp.), according to the manufacturer’s instructions.

Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared from VSMC as described previously (12). After washing with PBS, 5 x 106 cells were harvested and centrifuged at 2000 x g for 5 min. The pellet was resuspended in buffer A [10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), and 0.1% Nonidet P-40], supplemented with the following protease inhibitors: 0.2 mM phenylmethylsulfonylfluoride, 2 µM aprotinin, and 1.0 µg/ml antipain, pepstatin, and leupeptin. The pellet was mixed well and centrifuged at 5000 rpm for 1 min. The nuclear pellet was then resuspended in buffer C [20 mM HEPES-KOH (pH 7.9), 420 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 20% glycerol, and the mixture of protease inhibitors described above]. After 30 min at 4 C under constant agitation, nuclear debris were centrifuged at 15,000 x g for 15 min. The supernatant was distributed into 15-µl aliquots and stored at -80 C. EMSA were performed with a commercial kit following the instructions of the manufacturer (Promega Corp.). Briefly, NF-{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG C-3') and AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3') consensus oligonucleotides were 32P end labeled by incubation for 10 min at 37 C with 10 U T4 polynucleotide kinase (Promega Corp.) in a reaction containing 10 µCi [{gamma}-32P]ATP (3000 Ci/mM; NEN, Boston, MA). Ten micrograms of nuclear proteins were equilibrated for 10 min in a binding buffer containing 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 50 µg/ml poly(dI-dC). For competition assays, a cold probe was added to this buffer 10 min before the addition of the labeled probe. The labeled probe (0.35 pM) was added to the reaction and incubated for 20 min at room temperature. The reaction was stopped and run on a nondenaturing, 4% polyacrylamide gel at 100 V for 120 min. The gel was dried and exposed to x-ray film.

Immunoblotting
VSMC were stimulated with agonists for specified durations. After treatment, cells were washed with ice-cold PBS. Cells were lysed with ice-cold lysis buffer, pH 7.4 containing 500 mM HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, the mixture of protease inhibitors described above, and 1 mM sodium orthovanadate. Solubilized proteins were centrifuged at 14,000 x g for 30 min, and supernatants were stored at -80 C. Proteins (25 µg) were separated on SDS-PAGE. Proteins in the gel were electorphoretically transferred to a nitrocellulose membrane. The membrane was treated with indicated primary antibodies. After incubation with secondary antirabbit or antimouse antibodies, immunoreactive proteins were detected by ECL (Amersham, Arlington Heights, IL). For repeated immunoblotting, membranes were stripped in 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 0.1 M 2-mercaptoethanol for 30 min at 50 C.

Isolation of total RNA and quantitation of TNF{alpha} messenger RNA (mRNA) by Northern blot hybridization
VSMC were pretreated with 1 µg/ml anisomycin, a protein synthesis inhibitor that has been shown to enhance TNF{alpha} mRNA induction in cultured human VSMC for 30 min and then stimulated with agonist in serum-free DMEM. After the indicated time, total RNA was isolated by the standard method. Total RNA (20 µg) was size separated by electrophoresis on 1% agarose/formaldehyde gels and then transferred to Hybond-N membranes (Amersham). The RNA was immobilized on nylon filters by UV transillumination for 5 min. The membranes were prehybridized for 2 h at 65 C in 1 M NaCl, 10% dextran, 1% SDS, and 0.1 mg/ml denatured salmon sperm DNA. Hybridization was carried out at 65 C overnight with the same solution and {alpha}-32P-labeled denatured mouse TNF{alpha} complementary DNA (cDNA) probe as previously described (11). The membranes were washed with 2 x SSC (1 x = 150 mM NaCl and 15 mM sodium citrate) for 5 min at room temperature and then with 0.2 x SSC-0.1% SDS at 65 C for 30 min. The membranes were then exposed to x-ray film.

RT-PCR
RT of 1.5 µg RNA was performed with 50 µM oligo(deoxythymidine)12–18, followed by the addition of a reaction mixture containing 5 x first strand buffer [250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, and 0.1 M DTT], SuperScript II ribonuclease H-reverse transcriptase (Life Technologies, Gaithersburg, MD), and 10 mM deoxynucleotide triphosphates mix [10 mM each of deoxy (d)-ATP, dGTP, dCTP, and dTTP] in a final volume of 20 µl. The mixture was incubated at 42 C for 50 min, followed by termination at 70 C for 15 min. Then, the mixture was treated by ribonuclease H for 20 min at 37 C. Amplification of TNF{alpha} or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (as a control) was performed with an automatic thermocycler (DNA Thermal Cycler, Perkin Elmer), a reaction mixture containing 10 x PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH 9.0), and 1% Triton X-100], 15 mM MgCl2, 10 mM deoxynucleotide triphosphate mixture, 5 U Taq DNA polymerase/µl with 25 µM primer (sense and antisense), cDNA, and H2O. Amplification was performed at 94 C for 30 sec, 52 C for 30 sec, and 72 C for 60 sec followed by a 5-min extension at 72 C.

The TNF{alpha} product was amplified for 25 cycles, and the GAPDH product was amplified for 20 cycles. Both PCR products were viewed under UV light after 1.5% agarose gel electrophoresis and staining in ethidium bromide. The TNF{alpha} primer sets were 5'-ATG AGC ACG GAA AGC ATG ATC-3' (sense) and 5'-AGT AGA CCT GCC CGG ACT CCG-3' (antisense). The GAPDH primers sets were 5'-GCC GCC TGG TCA CCA GGG CTG-3' (sense) and 5'-ATG GAC TGT GGT CAT GAG CCC-3' (antisense).

Transient transfection and luciferase assays
VSMC were transfected using diethylaminoethyl-dextran (Promega Corp.). Briefly, VSMC (1–2 x 106/100 mm-dish) were seeded 24 h before transfection in 10 ml DMEM with 10% FCS. Cells were transfected with 10 µg of either pTNF{alpha}(-615)Luc, a reporter plasmid that contains a segment of the TNF{alpha} promotor (-615 to -36) (13), or pCMV4I{kappa}B{Delta}N, a deletion mutant of I{kappa}B encoding amino acids 37–317 (14). Five micrograms of pcDNAß-gal were always included as a monitor for transfection efficiency. After the transfection, cells were incubated for 48 h, then stimulated by LPS and harvested for luciferase assays. Luciferase assays were performed with a commercial kit (Promega Corp.).

Statistical methods
The results are expressed as the mean ± SD of the average responses in multiple experiments, each performed with different cell preparations. Data were analyzed by ANOVA followed by multiple range testing or by t tests for paired components.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Using a highly sensitive ELISA for rat TNF{alpha}, we measured the production of TNF{alpha} in the culture supernatant of VSMC stimulated by LPS. As shown in Fig. 1AGo, the production of TNF{alpha} in response to LPS (50 µg/ml) was detected as early as 4 h and gradually increased up to 24 h. The concentration-dependent increase in the secretion was observed from 10–100 µg/ml LPS (Fig. 1BGo). To confirm that VSMC secrete this bioactive form of TNF{alpha}, we determined the cytotoxic activity of the medium on NCTC929 cells, an established bioassay for active TNF{alpha}. The culture supernatant of VSMC showed cytotoxic activity only when stimulated by LPS (data not shown).



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  		Figure 1. Effect of LPS on TNF{alpha} secretion. VSMC were stimulated with LPS (100 µg/ml) for the indicated durations (A) or at various concentrations for 24 h (B). The TNF{alpha} concentration in the supernatant was measured by ELISA. Nine samples were studied in each group. **, P < 0.01 vs. time zero or control.

 
To investigate the mechanism leading to TNF{alpha} production in VSMC, we measured the activity of several signaling molecules, including MAPKs referred to as p42/44 MAPK (ERK1/2), p38 MAPK, AP-1, I{kappa}B, and NF-{kappa}B. The regulatory tripeptide motif-Thr-X-Tyr is a feature shared by all MAPKs. As the enzymatic activity of MAPKs is tightly controlled by the phosphorylation of these Thr and Tyr (15), we used phosphospecific antibodies directed against phosphorylated Thr202 and Tyr204 of ERKs and Thr180 and Tyr182 of p38 MAPK to detect their activation. As shown in Fig. 2AGo, LPS (100 µg/ml) induced phosphorylation of p42/44 MAPKs, which peaked at 30 min and was sustained up to 4 h. Phosphorylation of p42/44 MAPKs was dependent on the concentration of LPS (Fig. 2GoB); the increase in the activity became detectable at 1 µg/ml and reached a maximal level at 100 µg/ml. p38 MAPK was also phosphorylated by LPS stimulation with an almost similar time course and dose response (Fig. 2Go, A and B).



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  		Figure 2. LPS-induced p42/44 MAPK and p38 MAPK phosphorylation. VSMC were stimulated with LPS (100 µg/ml) for the indicated durations (A) or at the indicated concentrations for 30 min (B). Whole cell lysates were separated by SDS-PAGE and immunoblotted by the indicated antibodies. Results shown are representative of three separate experiments.

 
AP-1 consists of both homodimers of the Jun family (c-Jun, JunB, and JunD) and heterodimers of the Jun and the Fos family (c-Fos, FosB, Fra1, and Fra2). Nuclear translocation of both c-jun and c-fos was induced by LPS at 30 min to 2 h (data not shown). To examine whether induction of these protooncogenes by LPS leads to an enhanced AP-1-binding activity, EMSA was performed on nuclear extracts from VSMC treated with LPS. The AP-1-binding activity of the nuclear extracts was markedly enhanced after 60-min treatment with LPS and was sustained up to 4 h (Fig. 3Go). The reaction was proven to be specific, as the addition of an excess of cold AP-1, but not cold NF-{kappa}B, oligonucleotide eliminated the signal of the retarded bands.



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  		Figure 3. LPS-induced AP-1-binding activity. VSMC were stimulated with LPS (100 µg/ml) for the indicated durations and collected for the extraction of nuclear proteins. AP-1-binding activity was analyzed by EMSA as described in Materials and Methods. Results shown are representative of three separate experiments.

 
To address whether LPS treatment of VSMC leads to I{kappa}B degradation and nuclear NF-{kappa}B translocation, VSMC were incubated with LPS for indicated durations, and whole cell lysates and nuclear extracts were subjected to Western blot analyses with anti-I{kappa}B antibody and anti-NF-{kappa}B antibody, respectively. The amount of I{kappa}B in the whole cell lysate was markedly decreased by LPS from 20–60 min and recovered at 120 min (Fig. 4AGo). To ascertain uniformity of protein loading in each lane, the same membrane was stripped and immunoblotted by anti-annexin II, which is an abundant intracellular protein (16) and is not degraded by LPS. As shown in the lower panel of Fig. 4AGo, little variance was seen in the amount of annexin II in each lane. I{kappa}B degradation depends on the concentration of LPS; the degradation was detectable with as little as 1 µg/ml LPS (Fig. 4BGo). The amount of NF-{kappa}B in the nuclear extracts was inversely increased by LPS stimulation (Fig. 4CGo). Marked induction of NF-{kappa}B-binding activity of the nuclear extracts was also observed after 30 min of the LPS stimulation, which persisted up to 4 h (Fig. 5AGo). The specificity of the reaction was confirmed using unlabeled oligonucleotides. The signal of the retarded bands was eliminated by excess NF-{kappa}B oligonucleotide, but not by excess AP-1 oligonucleotide.



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  		Figure 4. LPS-induced I{kappa}B degradation and NF-{kappa}B translocation. VSMC were stimulated with LPS (100 µg/ml) for the indicated durations (A and C) or at the indicated concentrations for 30 min (B). Cell extracts (A and B) or nuclear extracts (C) were separated by SDS-PAGE and immunoblotted by the indicated antibodies. Results shown are representative of three separate experiments.

 


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  		Figure 5. LPS-induced NF-{kappa}B activation. A, Nuclear extracts were prepared from either control or LPS (100 µg/ml) for the indicated periods. NF-{kappa}B-binding activity was analyzed by EMSA. B, Supershift analysis of the NF-{kappa}B family members, p50, p52, and p65. Results are representative of three individual experiments.

 
NF-{kappa}B/Rel transcription factors consist of five distinct DNA-binding subunits, referred to as p50, p52, p65/Rel A, c-Rel, and Rel-B (17). To determine the identity of the proteins in the LPS-induced NF-{kappa}B-binding complex, we performed a supershift assay using selective antibodies specifically for the NF-{kappa}B/Rel proteins. Incubation with antibodies to RelA (p65) elicited a supershifted complex; however, antibody to either NF-{kappa}B-1(p50) or NF-{kappa}B-2(p52) did not alter the gel shift bands, suggesting that LPS induced a binding complex containing the homodimer of p65, or the heterodimer of p65 and c-Rel or of p65 and Rel B (Fig. 5BGo). These results showed that LPS activates p42/44 MAPK, p38 MAPK, and AP-1 in VSMC. NF-{kappa}B was also activated by LPS via the degradation of I{kappa}B.

To investigate which pathways are involved in LPS-induced TNF{alpha} production, we examined the effects of selective inhibitors of individual steps on the production of TNF{alpha}. PDTC and MG132 are believed to block the I{kappa}B/NF-{kappa}B pathways through different mechanisms. PDTC, a thiol compound, scavenges reactive oxygen intermediates, thereby impeding the release of I{kappa}B from NF-{kappa}B (18). MG132, selectively inhibits a proteasome that specifically degrades ubiquitinated I{kappa}B after its phosphorylation induced by LPS. Pretreatment of VSMC with PDTC and MG132 completely blocked the LPS-induced degradation of I{kappa}B (Fig. 6Go, A and B), whereas PDTC slightly stimulated the phosphorylation of p42/44 MAPKs and p38 MAPK in response to LPS. As shown in Fig. 6CGo, nuclear translocation of NF-{kappa}B stimulated by LPS was completely suppressed by pretreatment with MG132. In this regard, PDTC and MG132 markedly inhibited the LPS-induced nuclear NF-{kappa}B-binding activity (Fig. 7Go, A and B), whereas they enhanced the AP-1-binding activity.



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  		Figure 6. Effects of PDTC (A) and MG132 (B) on I{kappa}B degradation, MAPK phosphorylation, and NF-{kappa}B translocation. VSMC were pretreated with or without PDTC (150 µM) for 2 h or MG132 (50 µM) for 1 h and stimulated with LPS (100 µg/ml). C, Effect of MG132 on NF-{kappa}B translocation. VSMC were pretreated with or without MG132 and stimulated with LPS, and nuclear proteins were extracted. Whole cell lysates or nuclear extracts were separated by SDS-PAGE and probed by immunoblot analysis with the indicated antibodies. Results shown were representative of three separate experiments.

 


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  		Figure 7. Effects of PDTC (A) and MG132 (B) on NF-{kappa}B and AP-1 activation. VSMC were pretreated with or without PDTC for 2 h or MG132 for 1 h at the indicated concentrations and stimulated with LPS (100 µg/ml) for 1 h. Nuclear proteins were prepared, and NF-{kappa}B-binding activity and AP-1-binding activity were analyzed by EMSA. Results shown are representative of three separate experiments.

 
PD98059, a specific MEK inhibitor, suppressed LPS-induced p42/44 MAPK phosphorylation without affecting p38 MAPK phosphorylation. By contrast, SB203580, a specific p38 MAPK inhibitor suppressed p38 MAPK phosphorylation without affecting p42/44 MAPK phosphorylation (data not shown). Figure 8AGo showed that PD98059 inhibited the AP-1-binding activity stimulated by LPS. However, they had no effect on I{kappa}B degradation (data not shown) or subsequent NF-{kappa}B activation induced by LPS (Fig. 8BGo). SB203580 had no effect on NF-{kappa}B activation, even though this compound inhibited AP-1-binding activity. Thus, all of the inhibitors proved to be good tools in investigating the contribution of each signaling pathway to TNF{alpha} production in VSMC.



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  		Figure 8. Effects of the selective MEK-1 inhibitor, PD98059, and the p38 MAPK inhibitor, SB203580 on AP-1 (A) and NF-{kappa}B (B) binding activation. VSMC were pretreated with or without PD98059 (25 µM) or SB203580 (10 µM) for 1 h and stimulated by LPS (100 µg/ml). AP-1-binding activity and NF-{kappa}B-binding activity were analyzed by EMSA. The results shown are representative of three separate experiments.

 
The results in Fig. 9Go show the effects of these inhibitors on TNF{alpha} secretion. PDTC and MG132 completely suppressed TNF{alpha} secretion by LPS. SB203580, but not PD98059, also inhibited the secretion of TNF{alpha}. The combination of PDTC and SB203580 further reduced LPS-induced TNF{alpha} secretion compared with that of PDTC or MG132 alone, indicating that both I{kappa}B/NF-{kappa}B and p38 MAPK play crucial roles in LPS-induced TNF{alpha} secretion in VSMC.



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  		Figure 9. Effects of PDTC, MG132, PD98059, and SB203580 on TNF{alpha} secretion. VSMC were pretreated with or without PDTC (150 µM) for 2 h, MG132 (50 µM) for 1 h, or PD98059 (25 µM) or SB203580 (10 µM) for 1 h and stimulated with LPS (100 µg/ml) for 24 h. Supernatants were harvested, and TNF{alpha} concentration were measured by ELISA. Nine samples were studied in each group. **, P < 0.01 vs. control.

 
LPS has been shown to exert its effects at the level of transcription as well as translation to stimulate TNF{alpha} production in macrophages (19). To confirm the mechanisms of transcriptional regulation of TNF{alpha} by NF-{kappa}B activation induced by LPS, we used a promotor-luciferase construct, pTNF{alpha}(-615)Luc, containing a 5'-promotor of the TNF{alpha} gene (13). The results showed that LPS enhanced luciferase activity approximately 2-fold compared with that in control cells that were transfected with the empty vector. The LPS-induced enhancement of promotor activity of pTNF{alpha}-(-615)Luc was significantly suppressed by transfection of the dominant negative I{kappa}B, I{kappa}B{Delta}N, confirming that degradation of I{kappa}B is essential for NF-{kappa}B activation and subsequent TNF{alpha} induction by LPS in VSMC (Fig. 10AGo). In addition, SB203580, but not PD98059, decreased LPS-induced luciferase activity, suggesting that p38 MAPK might be involved in TNF{alpha} transcription or translation (Fig. 10BGo).



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  		Figure 10. Roles of I{kappa}B and MAPKs in the transcription of TNF{alpha}. A, VSMC were cotransfected with either pTNF{alpha}(-615)Luc or I{kappa}B{Delta}N, and 48 h later VSMC were stimulated by LPS for 1 h, at which time cell lysates were assayed for luciferase activity. B, VSMC were transfected with pTNF{alpha}(-615)Luc, and 48 h later VSMC were stimulated by LPS for 1 h pretreated with or without PD98059 or SB203580. Luciferase activity was measured in triplicate. Nine samples were studied in each group. **, P < 0.01 vs. control or pCMV5-transfected cells.

 
To confirm the roles of I{kappa}B/NF-{kappa}B and p38 MAPK on LPS-induced TNF{alpha} production in VSMC, we examined the levels of TNF{alpha} mRNA expression by Northern blotting with pretreatment of anisomycin (9), a protein synthesis inhibitor that has been shown to increase mRNA expression. The results in Fig. 11AGo show that LPS-induced TNF{alpha} mRNA expression was inhibited by pretreatment with PDTC and MG132, although anisomycin itself had no effect on TNF{alpha} mRNA levels. SB203580, but not PD98059, also inhibited TNF{alpha} mRNA expression induced by LPS (Fig. 11BGo). Without anisomycin, we could not detect TNF{alpha} mRNA by Northern blotting. This might be due to the fact that newly synthesized negative regulatory protein may enhance TNF{alpha} mRNA degradation. As anisomycin has been known to activate p38 MAPK and JNK (20, 21), we may not be completely able to eliminate the possibility that anisomycin might modulate the signaling pathway through the activation of both kinases. To clarify this issue, we performed RT-PCR for analyzing the levels of TNF{alpha} mRNA expression stimulated by LPS in the absence of anisomycin. As shown in the Fig. 12AGo, no RT-PCR amplification signal was detected by ethidium bromide staining in nonstimulated controls for TNF{alpha}. After 1 h of LPS, the band for TNF{alpha} was detected. The stimulation was blocked by pretreatment with PDTC or MG132. SB203580 also markedly blocked the intensity of the bands for TNF{alpha} induced by LPS, but PD98059 reduced it slightly (Fig. 12BGo). The expression of the housekeeping gene GAPDH was identical in all lanes. Although data from RT-PCR are semiquantitative, the results of RT-PCR in the absence of anisomycin are essentially in agreement with the data from Northern blotting with the pretreatment of anisomycin. Taken together, these results suggest that TNF{alpha} secretion is mediated by NF-{kappa}B activation through I{kappa}B degradation, and TNF{alpha} synthesis and secretion are also stimulated by p38 MAPK, but not by p42/44 MAPKs activation or AP-1 activation, in cultured VSMC.



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  		Figure 11. Effects of PDTC, MG132, PD98059, and SB203580 on the levels of TNF{alpha} mRNA expression. VSMC were pretreated with or without PDTC (150 µM) for 2 h, MG132 (50 µM) for 1 h (A), or PD98059 (25 µM) or SB203580 (10 µM) for 1 h (B) and stimulated with LPS (100 µg/ml) for 1 h. All VSMC were pretreated with 1 µg/ml anisomycin for 2 h. Total RNA was extracted, and Northern blotting analysis was performed. The results shown are representative of three separate experiments.

 


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  		Figure 12. Effects of PDTC and MG132 (A) and PD98059 and SB203580 (B) on the levels of RT-PCR amplification products of specific mRNA encoding TNF{alpha} and the housekeeping gene GAPDH in VSMC. VSMC were pretreated with or without PDTC (150 µM) for 2 h, MG132 (50 µM) for 1 h (A), or PD98059 (25 µM) or SB203580 (10 µM) for 1 h (B) and stimulated with LPS (100 µg/ml) for 1 h before RNA extraction. The TNF{alpha} PCR product was amplified for 25 cycles, whereas the GAPDH product was amplified for 20 cycles. The results shown are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The signal transduction mechanism by which TNF{alpha} is produced in VSMC is unknown. In this study, we have demonstrated that NF-{kappa}B inhibitors, PDTC and MG132, inhibited TNF{alpha} mRNA expression and secretion, whereas neither inhibitor had an effect on the activation of p42/44 MAPKs and AP-1 stimulation by LPS. Furthermore, an I{kappa}B dominant negative mutant suppressed the transcription of the TNF{alpha} gene. PD98059 inhibited p42/44 MAPKs and AP-1 activity; however, it had no effect on the TNF{alpha} mRNA expression and transcription of the TNF{alpha} gene. Thus, it is reasonable to speculate that LPS-induced TNF{alpha} production is mediated by NF-{kappa}B, but not by the p42/44 MAPKs-AP-1 pathway. On the other hand, SB203580 inhibited TNF{alpha} mRNA expression and transcription of the TNF{alpha} gene; however, it had no effect on NF-{kappa}B activity. Therefore, p38 MAPK may be involved in the transcriptional regulation of the TNF{alpha} gene in VSMC.

During sepsis, insulin sensitivity was impaired (22). Growing lines of evidence indicate that the cytokine, TNF{alpha}, plays a role as one of the mediators of insulin resistance (5). As TNF{alpha} is the primary mediator of endotoxin shock (4), it is likely that insulin resistance during sepsis might be in part caused by TNF{alpha}. However, TNF{alpha} production and its signaling mechanism in the vascular tissue have not been clearly identified. In the present study, we have demonstrated that rat VSMC is able to secrete TNF{alpha} in response to LPS, and TNF{alpha} mRNA levels were also moderately elevated by LPS. An earlier report on TNF{alpha} production by VSMC (9) is in agreement with the present results. This report had been the only one until the present study that dealt with TNF{alpha} production in VSMC. As discussed below, we have further demonstrated the signaling mechanisms leading to TNF{alpha} production in VSMC.

p42/44 MAPKs phosphorylate Elk-1 and activate a serum response element, leading to c-Fos induction, which is a component of AP-1 (23). The AP-1 site is present in the TNF{alpha} promotor, and AP-1/c-Jun plays an important role in the transcriptional regulation of TNF{alpha} (24, 25). It has been shown that LPS activates p42/44 MAPKs and subsequent TNF{alpha} production via the Ras/Raf-1 pathway in RAW 264.7 macrophage cells by using a dominant negative form of Ras (26). Therefore, it was reasonable to speculate that a tyrosine kinase may mediate the LPS-induced TNF{alpha} production through the Ras/MAPK/AP-1 pathway. Indeed, we observed that LPS induces p42/44 MAPKs activation, c-Fos and c-Jun induction, and subsequent AP-1 activation in VSMC. However, the MEK inhibitor PD98059, which selectively inhibits LPS-induced p42/44 MAPKs phosphorylation and AP-1 activation in the present study, had no effect on LPS-induced TNF{alpha} mRNA expression. Moreover, NF-{kappa}B inhibitors, PDTC and MG132, inhibited NF-{kappa}B activation and blocked TNF{alpha} secretion. Note that PDTC had showed only slight activation of p42/44 MAPKs stimulated by LPS. These results suggest that p42/44 MAPKs and AP-1 are not involved in the major pathway of LPS-induced TNF{alpha} production in rat VSMC.

Several investigators have shown that NF-{kappa}B activation is critical for TNF{alpha} gene expression in macrophages stimulated by LPS (27, 28). {kappa}B-like motifs were found in the TNF{alpha} promotor and are activated by TNF{alpha}, interleukin-1, and LPS (28, 29). In the present study, we have shown that LPS induced the degradation of I{kappa}B and subsequent activation of NF-{kappa}B in VSMC. Furthermore, despite the slight increase in p42/44 MAPK phosphorylation by PDTC and AP-1 activation by PDTC and MG132, both inhibitors suppressed TNF{alpha} expression as well as its secretion and NF-{kappa}B activation. As for the enhanced activation of AP-1 by MG132, c-Jun is an in vivo substrate for multiubiquitination and is degraded by the ubiquitin-proteasome pathway (30). Thus, the enhancement of AP-1 activity may be due to the inhibitory effect of MG132 on c-Jun degradation. Taken together, our results indicate that NF-{kappa}B, not AP-1, is an essential transcription factor for TNF{alpha} gene expression in VSMC. These results are compatible with the findings of Hambleton in macrophages (31).

I{kappa}B is phosphorylated by cytokines and is then degraded by the ubiquitin-proteasome pathway (32). Our observation that MG132 inhibited I{kappa}B degradation indicates that a similar mechanism is activated by LPS in VSMC. As to the mechanism of induction of I{kappa}B degradation, recent studies have identified a large, multisubunit complex in cytoplasmic extracts of HeLa cells that can phosphorylate at Ser32 and Ser36 (I{kappa}B kinase) (33). In addition, the I{kappa}B kinase complex is phosphorylated and activated by MEKK-1 (34). Further studies are needed to examine whether MEKK-1 plays a role in the phosphorylation of I{kappa}B kinase and subsequent I{kappa}B degradation in VSMC.

It was reported that p38 MAPK was also activated in response to LPS in monocytes and macrophages (35). In the present study, p38 MAPK in VSMC was activated by LPS. SB203580 partially inhibited LPS-induced TNF{alpha} production, TNF{alpha} mRNA expression, and expression of a reporter gene that was driven by the TNF{alpha} promotor. However, neither LPS-induced DNA binding of NF-{kappa}B nor LPS-induced I{kappa}B degradation was modulated by SB203580. In addition, a blockade of both NF-{kappa}B and p38 MAPK signaling pathways further reduced LPS-induced TNF{alpha} production (Fig. 9Go). These results suggest that p38 MAPK partially regulates the transcription of LPS-induced TNF{alpha} production, and NF-{kappa}B is not a direct target for p38 MAPK. The p38 MAPK signaling pathway activates the transcription factors cAMP response element (CRE)-binding protein (CREB), activation transcription factor-1 (ATF1) (36), and ATF2 (37). The promotor region of TNF{alpha} contains a CRE site as well as {kappa}B3 sites. CREB and ATF2 have been reported to bind to the CRE site. In monocytic cells, the CRE site as well as {kappa}B3 sites are important for the induction of TNF{alpha} by LPS (38). Taken together, it is likely that the transcriptional activation of TNF{alpha} by LPS may be mediated by p38 MAPK through the activation of ATF2 and/or CREB in VSMC, although several researchers have shown that p38 MAPK is involved in the translational regulation of LPS-induced TNF{alpha} production in monocytes and glial cells (39, 40). Thus, our results suggest that the mechanisms of the transcriptional regulation of TNF{alpha} induced by LPS could be different from those in myeloid and nonmyeloid cells.

As shown in Fig. 8Go, the present observation of partial suppression of LPS-induced AP-1 activation by PD98059 suggests that there might be pathways other than p42/44 MAPK that activate AP-1 in VSMC. LPS is reported to activate JNK (37, 41). Recently, it has also been reported that JNK activation itself could account for AP-1 activation, as factors critically involved in the transcription of c-fos and c-jun, such as TCF/Elk-1, c-Jun, and ATF2, are targets of phosphorylation by JNK (23, 42, 43). Thus, LPS-induced AP-1 activation might be regulated by both p42/44 MAPK and JNK in VSMC. The contributions of JNK need to be evaluated in cultured VSMC.

The TNF{alpha} promotor activation by LPS was only 2-fold as shown in Fig. 10Go. A few explanations can be considered. First, we examined LPS-induced promotor activity of TNF{alpha} by using pTNF{alpha}(-615)Luc containing a segment of the TNF{alpha} promotor (-615 to -36) (13), because all {kappa}B or AP-1 sites are present in this segment. In monocytic cells, the LPS-induced increase in luciferase activity in pTNF{alpha}(-1311)Luc was only 10% higher than that in pTNF{alpha}(-615)Luc, suggesting that the upstream region between -615 and -1311 may not make a major contribution, although there have been no data reported in VSMC (38). Thus, further upstream region of -615 of the TNF{alpha} gene could be necessary for full activation on TNF{alpha} transcription in VSMC. Second, LPS-induced TNF{alpha} mRNA expression is easy to detect by Northern blotting in macrophages (data not shown), but not in VSMC. In addition, LPS-stimulated TNF{alpha} production in macrophages is 2 orders of magnitude higher than that in VSMC (31). Thus, the promotor activity of pTNF{alpha}(-615)Luc in VSMC is expected to be lower than that in macrophages, although posttranscriptional regulation as well as the transcriptional control have been reported to be important for TNF{alpha} production (39, 40), suggesting that other factors play a significant role. Even in macrophage cell lines, U937 and THP-1 cells, induction of promotor activity of TNF{alpha} increased only 3- to 15-fold when stimulated with PMA (24, 13) or LPS (38). Taken together, a 2-fold increase in promotor activity by pTNF{alpha}(-615)Luc seems not to be inordinately low.

In the present study, LPS-induced TNF{alpha} production in VSMC requires much higher concentrations of LPS than that used in myeloid cells. This may be due to a lack of CD14 and the LPS-binding protein (LBP) in our experimental system. The membrane-bound 55-kDa glycoprotein, CD14, serves as a receptor for the complexes of LPS and LBP (44). The LPS-LBP complex acts as a potent agonist for macrophages; it is effective at much lower concentrations of LPS and induces more rapid release of cytokines than LPS alone (45, 46). CD14 is not present in VSMC (47), and in the present study, VSMC were stimulated under serum-free conditions. Thus, it is likely that LBP was absent in the present system. When LPS signaling occurs by CD14-independent mechanisms, it usually requires higher LPS concentrations. The inhibitory effects of anti-CD14 antibodies on LPS-induced myeloid cell activation are most pronounced at low LPS concentrations, but can be overcome by increasing the concentrations of LPS (48), suggesting the presence of a signaling pathway independent of CD14. Thus, in VSMC, TNF{alpha} production in response to LPS may be operating through a CD14-independent signaling mechanism. The contribution of such as alternative pathway has yet to be evaluated in cultured VSMC.

In conclusion, our results suggest that TNF{alpha} is produced from cultured VSMC in response to LPS, and this process is mediated by NF-{kappa}B activation through I{kappa}B degradation. Surprisingly, these mechanisms in VSMC are very similar to those in macrophages. On the other hand, p38 MAPK, which is not involved in the transcriptional regulation of TNF{alpha} in other cell lines, transcriptionally regulates TNF{alpha} gene expression in VSMC. This raises the possibility that a new pathway for TNF{alpha} production may be present in VSMC.


   Acknowledgments
 
We thank T. Fitzgerald and E. Price for their excellent technical assistance, and T. Stack for her secretarial assistance. The authors also gratefully acknowledge Dean W. Ballard and James S. Economou for providing pCMV4I{kappa}B{Delta}N and pTNF{alpha}Luc, respectively.


   Footnotes
 
1 This work was supported in part by NIH Grants HL-35323, HL-58205, HL-03320, and DK-20593. Back

Received November 19, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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