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J. Biol. Chem., Vol. 279, Issue 19, 20363-20368, May 7, 2004

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Angiotensin II Differentially Regulates Interleukin-1--inducible NO Synthase (iNOS) and Vascular Cell Adhesion Molecule-1 (VCAM-1) Expression

ROLE OF p38 MAPK^*

Bingbing Jiang, Shanqin Xu, Xiuyun Hou, David R. Pimentel, and Richard A. Cohen

From the Whitaker Cardiovascular Institute, Vascular Biology Unit, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118

Received for publication, December 24, 2003 , and in revised form, February 17, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Angiotensin II is implicated in pathophysiological processes associated with vascular injury and repair, which include regulating the expression of numerous NF-B-dependent genes. The present study examined the effect of angiotensin II on interleukin-1-induced NF-B activation and the subsequent expression of inducible NO synthase (iNOS) and vascular cell adhesion molecule-1 (VCAM-1) in cultured rat vascular smooth muscle cells. Neither NF-B activation nor iNOS or VCAM-1 expression was induced in cells treated with angiotensin II alone. However, when added together with interleukin-1, angiotensin II, through activation of the AT₁ receptor, inhibited iNOS expression and enhanced VCAM-1 expression induced by the cytokine. The inhibitory effect of angiotensin II on iNOS expression was associated with a down-regulation of the sustained activation of extracellular signal-regulated kinase (ERK) and NF-B by interleukin-1, whereas the effect on VCAM-1 was independent of ERK activation. The effect of angiotensin II on iNOS was abolished by inhibition of p38 mitogen-activated protein kinase (MAPK) with SB203580, but not by inhibition of PI3 kinase with wortmannin or stress-activated protein kinase/c-Jun NH₂-terminal kinase (JNK) with JNK inhibitor II. Thus, angiotensin II, by a mechanism that requires the participation of p38 MAPK, differentially regulates the expression of NF-B-dependent genes in response to interleukin-1 stimulation by controlling the duration of activation of ERK and NF-B.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Angiotensin II (Ang II)¹ is an important vasoactive peptide that physiologically regulates vascular tone and maintains normal vessel structure and function. However, increased levels of Ang II have been implicated in pathophysiological processes that include atherosclerosis, cardiac hypertrophy, nephropathy, vascular injury, and remodeling (1, 2). Angiotensin-converting enzyme inhibitors and Ang II type-1 receptor (AT₁) antagonists alleviate atherosclerotic lesions in patients (3-7) and experimental animal models (8) and also prevent neointimal proliferation after balloon injury in rats (9). It is possible that Ang II may influence the response of vascular cells to inflammatory agents, including cytokines such as interleukin-1 (IL-1). IL-1 is an important cytokine known to activate nuclear factor (NF)-B and to induce the expression of numerous NF-B-dependent genes, including those encoding inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and vascular cell adhesion molecule-1 (VCAM-1) (10-12). These inflammation-related effects have pivotal roles in various models of vascular injury and repair. Although reports in the literature are conflicting, Ang II has been suggested to regulate the expression of NF-B-dependent genes, including up-regulation of the expression of VCAM-1, intracellular adhesion molecule-1, and monocyte chemoattractant protein-1 (6, 13, 14), and the down-regulation of iNOS (15-18). However, the signaling mechanism(s) by which Ang II affects NF-B activation and how Ang II differentially regulates the expression of different NF-B-dependent genes remains largely unknown.

In previous studies, we examined the involvement of the extracellular signal-regulated kinases (ERK) in regulating the IL-1 signaling pathway in rat vascular smooth muscle cells (VSMCs) (12, 19, 20). In those studies we described an early but transient increase in NF-B activation in response to IL-1, followed by a more persistent activation that was sustained for up to 24 h. We showed that inhibition of ERK effectively attenuated the more persistent activation of NF-B, but had no effect on the early activation. The different duration of NF-B activation was associated with changes in the selective induction of NF-B-dependent genes, with VCAM-1 expression being regulated solely by the early activation of NF-B, whereas both iNOS and COX-2 expression were dependent on persistent activation (12). In a separate study, we showed that platelet-derived growth factor (PDGF) and epidermal growth factor each enhanced the IL-1-induced iNOS gene expression in VSMCs and showed that this effect was by means of an ERK-dependent mechanism responsible for the persistent activation of NF-B (20). These results suggested that vasoactive agents that regulate ERK activity might modulate the duration of NF-B activation and the expression of selective NF-B-dependent genes induced by IL-1.

In the present study, we have examined the interrelationship between IL-1 and Ang II with regard to ERK activation, temporal control of NF-B, and the expression of iNOS and VCAM-1, two genes representative of those that respond to the persistent or transient activation of NF-B, respectively. The study provides strong evidence that IL-1-induced expression of VCAM-1 and iNOS is differentially regulated by Ang II through ERK-dependent temporal control of NF-B activation. In addition, p38 MAPK plays an important role in mediating the effect of Ang II. This novel mechanism reveals a potentially important integration of the signaling by vasoactive agents and IL-1 that may be critical in regulating inflammatory responses.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Dulbecco's modified Eagle's medium/Ham's F12 medium (DMEM/F12) and fetal calf serum were purchased from Invitrogen. Recombinant human IL-1 (specific activity: 1.9 x 10⁷ units/mg) was kindly provided by Dr. Aurigemma, National Cancer Institute. PDGF (BB isoform) was obtained from Upstate Biotechnology; Ang II was from Sigma; SB203580, JNK inhibitor II, and wortmannin were from Calbiochem; and antibodies against iNOS and COX-2 were obtained from Transduction Laboratories. Antibodies against phospho-p44/42 MAPK (Thr²⁰²-Tyr²⁰⁴), p44/42 MAPK, phospho-p38 MAPK (Thr¹⁸⁰-Tyr¹⁸²), p38 MAPK, phospho-SAPK/JNK (Thr¹⁸³-Tyr¹⁸⁵), SAPK/JNK, MEK-1/2, I-B, and phospho-NF-B p65 (Ser⁵³⁶) were from Cell Signaling. Antibody against VCAM-1 was obtained from Santa Cruz Biotechnology; NF-B consensus oligonucleotide was from Promega; and [-³²P]ATP was obtained from PerkinElmer Life Sciences. All other materials used were commercial products of the highest grade available.

Cell Culture—Rat VSMCs were isolated from the thoracic aorta and cultured as described previously (21). Cells were used between passages 5 and 9. When confluent, the cells were washed with serum-free medium and then maintained in DMEM/F12 with 0.1% fetal calf serum for 24-48 h. The medium was refreshed just before treatment. The cells were then incubated with or without additions (IL-1, PDGF, Ang II, inhibitors, or vehicle) for designated times as indicated under "Results."

Adenoviral Constructs and Infection—Adenovirus expressing an I-B mutant (S32A/S36A) (Adv-IBM) and adenovirus expressing a dominant-negative MEK-1 (S218A/S222A) (Adv-MEK1dn) were generated as described previously (12). When confluence was reached, the cells were washed once with serum-free medium and then cultured for 24 h in DMEM/F12 with 0.1% fetal calf serum either with or without adenovirus. The medium was refreshed and the cells were further incubated with or without additions as indicated in the text.

Western Blot Analysis and EMSA—Whole-cell lysates were prepared and Western blot analysis was performed as described previously (22). Protein content of the cell lysates was determined with BCA protein assay reagent (Pierce), with bovine serum albumin used as a standard. The images were obtained and analyzed by using a Model GS-700 imaging densitometer (BioRad). Nuclear extracts were prepared and DNA-binding activity was assessed by EMSA using an NF-B consensus oligonucleotide as described previously (22).

Reverse Transcription (RT)-PCR—Total RNA was extracted from cells by using TRIzol reagent as described previously (22). The first-strand cDNA was synthesized from 1 µg of total RNA using oligo (dT)₂₀-M4 adaptor primers and avian myeloblastosis virus reverse transcriptase (Takara Shuzo Co.). Synthetic gene-specific primer sets used in PCR were: (i) iNOS forward 20-mer, 5'-GCT ACA CTT CCA ACG CAA CA-3', and reverse 20-mer, 5'-TGG GTG GGA GGG GTA GTG AT-3', which amplified a 430-bp sequence between +2081 and +2510 of rat iNOS cDNA; (ii) VCAM-1 forward 21-mer, 5'-ACA CCT CCC CCA AGA ATA CAG-3', and reverse 21-mer, 5'-GCT CAT CCT CAA CAC CCA CAG-3', which amplified a 477-bp sequence between +659 and +1135 of rat VCAM-1 cDNA; and (iii) GAPDH forward 20-mer, 5'-GCC ATC AAC GAC CCC TTC AT-3', and reverse 20-mer, 5'-CGC CTG CTT CAC CAC CTT CT-3', which amplified a 702-bp sequence between +88 and +789 of rat GAPDH cDNA. PCR was performed using the following schedule: denaturation, annealing, and extension at 94, 57, and 72 °C for 40 s, 30 s, and 1 min, respectively, for 26 cycles. PCR products were electrophoresed on 1.2% agarose gels containing ethidium bromide and visualized by UV-induced fluorescence.

Immunofluorescent Staining—VSMCs were cultured on four-well Lab-Tek II chamber slides (Nalge Nunc Intl) under the same conditions described above. After treatment, the cells were washed with cold PBS, fixed for 8 min in methanol at -20 °C, and air dried at room temperature. The staining of NF-B p65 was performed as described previously (12). The images observed under a fluorescence microscope were recorded on a linked computer using Openlab software version 2.2.5 (Improvision).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Ang II Inhibits IL-1-induced iNOS but Not VCAM-1 Expression—Treatment of VSMCs with IL-1 for 24 h induced the expression of both iNOS and VCAM-1 (Fig. 1A). In contrast, 100 nM Ang II, when added alone, did not induce iNOS, nor did it affect the basal level of VCAM-1 expression compared with untreated cells. However, Ang II added together with IL-1 markedly suppressed iNOS expression by the cytokine. Interestingly, Ang II did not attenuate IL-1-induced VCAM-1 expression. In fact, at the concentration used, a small but significant increase in VCAM-1 expression was noted (Fig. 1A). This effect of Ang II on IL-1-induced iNOS expression was in striking contrast to that of PDGF, which significantly increased IL-1-induced iNOS expression, but was without effect on VCAM-1 expression (Fig. 1B). The ability of 24-h treatment with Ang II to modulate IL-1-induced gene expression was dose-dependent, with the most marked effects observed between 10-100 nM (Fig. 1C). As shown in Fig. 1D, Ang II reduced IL-1-induced iNOS mRNA levels, but had little effect on IL-1-enhanced VCAM-1 mRNA.

View larger version (46K): [in this window] [in a new window]   FIG. 1. Ang II inhibits iNOS but enhances VCAM-1 expression induced by IL-1. A-C, VSMCs were treated for 24 h with or without IL-1 (3 ng/ml) in the absence or presence of either Ang II (A, 100 nM; C, 0.1-100 nM) or PDGF (B, 10 ng/ml). Whole-cell lysates (20 µg protein/lane) were used for Western blot analysis of iNOS and VCAM-1. Bar graphs in A-C represent densitometric analyses of the immunoblots, with the arbitrary units from IL-1-treated cells as 1-fold. Data shown are mean ± S.D. (A and B, n = 3; C, n = 2). Statistical analysis was performed by Student's t test. C, *, p < 0.05 versus cells treated with IL-1 alone. D, VSMCs were treated for 16 h with or without IL-1 (3 ng/ml) in the absence or presence of Ang II (100 nM). Total RNA was extracted and used for RT-PCR to determine mRNA levels of iNOS, VCAM-1, and GAPDH. The results shown in D are representative of two separate experiments.

Ang II Inhibits Prolonged but Not Transient NF-B Activation Induced by IL-1

View larger version (75K): [in this window] [in a new window]   FIG. 2. Ang II inhibits prolonged but not transient activation of NF-B induced by IL-1. A, Ang II inhibits IL-1-induced persistent nuclear translocation of NF-B without influence on early translocation. VSMCs were untreated or treated with IL-1 (3 ng/ml) in the absence or the presence of Ang II (100 nM) for 1 or 16 h, as indicated. Immunofluorescent staining of NF-B p65 was performed as described under "Experimental Procedures." B, VSMCs treated with Ang II alone do not show NF-B DNA-binding activity. VSMCs were untreated or treated with 1 or 100 nM of Ang II for 1 or 16 h. Nuclear extracts were used for EMSA. Nuclear extracts from cells treated with IL-1 (3 ng/ml) for 1 or 16 h, or tumor necrosis factor- (10 ng/ml) for 1 h were used as positive controls. C, Ang II attenuates IL-1-induced phosphorylation of NF-B p65 on Ser536. VSMCs were untreated or treated with IL-1 (3 ng/ml) in the absence or in the presence of Ang II (0.1-100 nM) for 24 h. Whole-cell lysates (20 µg of proteins/lane) were used for Western blot analysis. p-p65, phosphorylated NF-B p65. Bar graph shows the densitometric analysis of the immunoblots for p-p65, with the arbitrary units from untreated cells as 1-fold. Data shown are mean ± S.D. Statistical analysis was performed by Student's t test. *, p < 0.01 versus cells treated with IL-1 alone. The results shown in A-C are representative of two separate experiments each.

An additional criterion for the activation of NF-B is the phosphorylation of NF-B p65 on Ser⁵³⁶ (26), an event measurable by Western blot analysis. The data in Fig. 2C show that Ang II attenuated the IL-1-induced phosphorylation of NF-B p65 (Ser⁵³⁶) at 24 h in a dose-dependent manner. Using a broad range of concentrations and time points, Ang II added alone had no effect on p65 phosphorylation (data not shown). Thus, the data in Fig. 2 establish clearly that Ang II attenuates the prolonged activation of NF-B initiated by IL-1, yet under our experimental conditions, has no effect on NF-B activation when added without the cytokine.

Ang II Reduces Prolonged ERK Activation Induced by IL-1—Previously we have shown that prolonged activation of ERK is required for IL-1 to induce persistent activation of NF-B and subsequent iNOS expression (12, 19, 20). Therefore, we examined whether the IL-1-induced prolonged activation of ERK was influenced by Ang II. As shown in Fig. 3, A and B, the prolonged phosphorylation of ERK (24 h after IL-1 addition) was suppressed by the addition of Ang II (100 nM), whereas the addition of PDGF (10 ng/ml) clearly enhanced ERK phosphorylation. The effective concentrations for Ang II to suppress ERK phosphorylation were 10 and 100 nM (Fig. 3C), which is consistent with the results observed for Ang II to inhibit IL-1-induced p65 phosphorylation and iNOS induction. Fig. 3D summarizes the inhibitory effect of Ang II on the phosphorylation of both ERK1 and ERK2 induced by IL-1. Although Ang II down-regulated the IL-1-induced prolonged phosphorylation of ERK observed at the 24 h time point (see Fig. 3, A, C-E), the phosphorylation levels of ERK at earlier time-points were obviously higher than those treated with IL-1 alone (Fig. 3E). Fig. 4A compares the effects of ERK inhibition caused by overexpression of a dominant-negative form of MEK-1 with that caused by Ang II on the expression of iNOS and VCAM-1 induced by IL-1. IL-1-induced iNOS expression was inhibited by either overexpression of MEK1dn or by Ang II. In contrast, VCAM-1 expression was not influenced by MEK1dn, but was enhanced by Ang II (Fig. 4A), which is consistent with VCAM-1 expression being regulated through an ERK-independent mechanism. However, both iNOS and VCAM-1 expression were down-regulated by overexpression of an I-BM that inhibited both early (transient) and prolonged (persistent) NF-B activation, as reported previously (12), indicating that NF-B activation was essential for the expression of both genes. When losartan was added to block the AT₁ receptors, Ang II was unable to influence IL-1-induced ERK phosphorylation as well as iNOS and VCAM-1 expression (Fig. 4B). This result indicates that the effect of Ang II on IL-1-induced responses was mediated through the AT₁ receptors. The AT₂ receptor antagonist, PD123319, had no effect upon the actions of Ang II. COX-2 expression also was inhibited by Ang II through an AT1-dependent pathway, consistent with our previous data showing that the expression of both iNOS and COX-2 are dependent upon the prolonged activation of NF-B by IL-1. Also included in Fig. 4B are data showing that activation of NF-B, as measured by phosphorylation of p65, is attenuated by Ang II through an AT₁-dependent pathway, and that addition of Ang II alone had no effect on p65 phosphorylation, in agreement with the observations made previously in Fig. 2.

View larger version (60K): [in this window] [in a new window]   FIG. 3. Ang II attenuates and PDGF enhances prolonged activation of ERK induced by IL-1. A-C, VSMCs were treated for 24 h with or without IL-1 (3 ng/ml) in the absence or the presence of either Ang II (A, 100 nM; C, or 0.1-100 nM) or PDGF (B, 10 ng/ml). E, VSMCs were treated with IL-1 (3 ng/ml) in the absence or the presence of Ang II (100 nM) for the indicated times. Ctl, control cells without treatment. Whole-cell lysates (20 µg of proteins/lane) were used for Western blot analysis of phosphorylated ERK (p-ERK) and total ERK. The numbers under the blots in C and E and the bar graph in D (mean ± S.D., n = 3) are the densitometric analyses of the immunoblots, with the arbitrary units from untreated cells as 1-fold. Statistical analysis was performed by one-way analysis of variance. *, p < 0.05 and **, p < 0.01 versus untreated cells; , p < 0.05 versus cells treated with IL-1 alone. Results shown in A and B are representative of three separate experiments; in C and E, results are representative of two separate experiments each.

View larger version (46K): [in this window] [in a new window]   FIG. 4. AT1 receptors mediate the effect of Ang II on persistent ERK activation and on the ERK-dependent expression of iNOS and COX-2 induced by IL-1. A, VSMCs were uninfected or infected for 24 h with AdvMEK1dn (lanes 3 and 4, 0.5 or 1.0 x 1010 viral particles/ml, respectively), AdvIBM (lanes 5 and 6, 1.3 or 2.6 x 1010 viral particles/ml, respectively), or control virus, AdvLacZ (lane 7, 1.0 x 1010 viral particles/ml) and then treated for 24 h with IL-1 alone (lanes 2-7, 3 ng/ml) or IL-1 plus Ang II (lane 8, 100 nM). B, VSMCs were untreated or treated with IL-1 (3 ng/ml), Ang II (100 nM), or both for 24 h. Losartan (AT1 antagonist, 10 µM) or PD123319 (AT2 antagonist, 10 µM) was added 1 h before IL-1 and Ang II. Whole-cell lysates were used for Western blot analysis. The overexpression of MEK1dn and I-BM was confirmed by immunoblotting with antibodies against MEK-1/2 and I-B, respectively (shown in A; small arrows indicate the mutants). Results shown are representative of two separate experiments each.

View larger version (64K): [in this window] [in a new window]   FIG. 5. Ang II activates multiple signaling pathways in cultured rat VSMCs. VSMCs were untreated or treated with 100 nM of Ang II (A), 3 ng/ml of IL-1 or both (B) for the indicated times. Whole-cell lysates (20 µg of proteins/lane) were used for Western blot analysis of various protein kinases, as indicated. Results shown are representative of two separate experiments each. p-ERK, phosphorylated ERK; p-p38 MAPK, phosphorylated p38 MAPK; p-p54/p46 JNK, phosphorylated p54/p46 SAPK/JNK; and p-Akt, phosphorylated Akt.

View larger version (39K): [in this window] [in a new window]   FIG. 6. p38 MAPK mediates the inhibitory effects of Ang II on prolonged ERK activation and iNOS expression induced by IL-1. A, VSMCs were treated for 24 h with IL-1 (3 ng/ml) in the absence or the presence of Ang II (100 nM). The selective inhibitors SB203580 (10 µM), JNK inhibitor II (1 µM), or wortmannin (100 nM) were added 1 h prior to IL-1 and Ang II. B, VSMCs were treated for 24 h with IL-1 (3 ng/ml), Ang II (100 nM), or both, in the absence or the presence of SB203580 (10 µM), which was added 1 h prior to IL-1 and Ang II. Whole-cell lysates were used for Western blot analysis of the indicated proteins. Results shown are representative of three experiments each.

View larger version (50K): [in this window] [in a new window]   FIG. 7. p38 MAPK mediates Ang II inhibition of prolonged but not early NF-B DNA-binding activity induced by IL-1. VSMCs were either untreated or treated with IL-1 (3 ng/ml) in the absence or the presence of Ang II (100 nM) for 1 or 16 h, as indicated. SB203580 (10 µM) was added 1 h prior to IL-1 and Ang II for 16-h treatment. Nuclear proteins were extracted and used for EMSA. Results shown are representative of three experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although Ang II modulated the prolonged NF-B activation in response to IL-1, Ang II added alone did not activate NF-B in rat VSMCs under our culture conditions. This result was documented in cells treated solely with Ang II by the absence of (i) NF-B DNA-binding activity as determined by EMSA, (ii) NF-B p65 nuclear localization measured by immunofluorescent staining, and (iii) inducible p65 phosphorylation using Western blot analysis. This inability of rat VSMCs to respond to Ang II by activation of NF-B was a consistent observation made in several separate preparations of our cells. In contrast to our findings, several groups have published data showing that Ang II activated NF-B directly in human and rat VSMCs (23-25). The reason for this discrepancy is unclear, but one notable difference between our results and the other reports is that there was no detectable NF-B DNA-binding activity in untreated VSMCs used in the present study. It is possible that Ang II may enhance NF-B activation in cells that have a detectable basal level of activation caused by an unknown factor(s). Another possibility is that, in the human cell studies, the required growth factors for their culture might affect the response to Ang II. In addition, several cancer cell lines have been observed to be capable of constitutively expressing IL-1 (27-29), which may account for a basal and persistent activation of NF-B in these cell lines. However, there is no evidence currently available indicating that VSMCs in culture could be transformed to a phenotype that constitutively expresses cytokines such as IL-1. We do find that Ang II does indeed enhance the expression of some NF-B-dependent genes induced by IL-1, as shown for VCAM-1.

Among the multiple signaling pathways activated by Ang II, p38 MAPK seemed to be a major component mediating the inhibitory effect of Ang II on IL-1 induction of iNOS. Inhibition of p38 MAPK by SB203580 restored not only iNOS expression but also the persistent activation of ERK and NF-B. IL-1 alone only slightly activated p38 MAPK in VSMCs, consistent with our previous report, in which we also have shown that SB203580 did not influence IL-1-induced nitrite accumulation (21). It has been shown in mesangial cells that IL-1 activates p38 MAPK in a serum-dependent manner by an unknown mechanism and that the activation of p38 MAPK prevents iNOS induction by IL-1 (30). Such p38 MAPK-mediated inhibition of iNOS expression seems cell-type-dependent, because it has been reported, in contrast, that in murine astrocytes and macrophages, bovine cartilage-derived chondrocytes, and rat pancreatic islets, p38 MAPK activation is required for iNOS induction (31-34). It has been reported recently that the different isoforms (, , , and ) of p38 MAPK play different roles in iNOS induction (35), which may suggest the involvement of the different p38 MAPK isoforms in the different cell types studied. The data in the present study suggest the possibility that one of the mechanisms for p38 MAPK inhibiting iNOS induction may relate to the down-regulation of prolonged activation of ERK, leading to the reduction of persistent activation of NF-B.

In contrast to iNOS, VCAM-1 expression induced by IL-1 was ERK-independent and did not require the persistent activation of NF-B, a distinction we have reported recently (12), and which is further emphasized by the findings in the present study. Although Ang II down-regulated the IL-1-induced prolonged activation of ERK and NF-B, Ang II actually enhanced IL-1-induced VCAM-1 expression. By contrast, PDGF enhanced the IL-1-induced prolonged activation of ERK and NF-B, but it did not influence IL-1-induced VCAM-1 expression. These findings are consistent with the earlier and transient activation of NF-B being required for VCAM-1 expression in rat VSMCs in response to cytokine stimulation.

Thus, based on the present study and previous findings (12, 19, 20), we suggest the hypothesis summarized schematically in Fig. 8. Upon IL-1 stimulation, an early but transient activation of NF-B is mediated through a mechanism requiring I-B degradation that is not affected by the ERK pathway. In contrast, prolonged or persistent activation of NF-B by IL-1 is initiated, at least partially, by an I-B degradation-mediated process that is attenuated when the ERK pathway is inhibited. In both cases, the activation of I-B kinases is a prerequisite. Growth factors such as PDGF may not trigger I-B kinase activation, but may enhance IL-1-induced persistent activation of NF-B by enhancing prolonged ERK phosphorylation. When the IL-1-induced ERK cascade is attenuated, either pharmacologically or by endogenous substances such as Ang II, expression of the genes requiring persistent NF-B activation may be prevented, as in the case of iNOS and COX-2, whereas expression of the genes dependent solely upon the transient NF-B activation may not be negatively affected, as in the case of VCAM-1. Although other signaling pathways and nuclear factors may also be involved, such ERK-dependent and ERK-independent regulation of NF-B activation apparently has a pivotal role in controlling the pattern of cytokine-inducible gene expression and may represent a novel mechanism by which growth factors, Ang II, and other vasoactive agents regulate cytokine effects and influence inflammatory processes.

View larger version (38K): [in this window] [in a new window]   FIG. 8. Involvement of growth factors and Ang II in modulation of IL-1-induced bimodal activation of NF-B. MG132, proteasome inhibitor; I-BM, I-B mutant (S32A/S36A); PD98059, MEK-1 inhibitor; U0126, MEK-1/2 inhibitor; MEK1dn, dominant-negative MEK-1; SB203580, p38 MAPK inhibitor.

	FOOTNOTES

To whom correspondence should be addressed. Tel.: 617-414-1009; Fax: 617-638-7113; E-mail: bjiang{at}bu.edu.

¹ The abbreviations used are: Ang II, angiotensin II; AT₁, Ang II type-1 receptor; IL-1, interleukin-1; NF-B, nuclear factor-B; iNOS, inducible NO synthase; COX-2, cyclooxygenase-2; VCAM-1, vascular cell adhesion molecule-1; ERK, extracellular signal-regulated kinase; VSMC, vascular smooth muscle cells; PDGF, platelet-derived growth factor; MAPK, mitogen-activated protein kinase; DMEM/F12, Dulbecco's modified Eagle's medium/Ham's F12 medium; SAPK/JNK, stress-activated protein kinase/c-Jun NH₂-terminal kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; RT-PCR, reverse transcription-PCR; EMSA, electrophoretic mobility shift assay; GADPH, glyceraldehyde-3-phosphate dehydrogenase.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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