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Originally published In Press as doi:10.1074/jbc.M204994200 on June 4, 2002

J. Biol. Chem., Vol. 277, Issue 33, 29584-29592, August 16, 2002
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p38 MAPK-mediated Transcriptional Activation of Inducible Nitric-oxide Synthase in Glial Cells

ROLES OF NUCLEAR FACTORS, NUCLEAR FACTOR kappa B, cAMP RESPONSE ELEMENT-BINDING PROTEIN, CCAAT/ENHANCER-BINDING PROTEIN-beta , AND ACTIVATING TRANSCRIPTION FACTOR-2*

Narayan R. BhatDagger §, Douglas L. Feinstein, Qin ShenDagger , and Aruna N. BhatDagger

From the Dagger  Department of Neurology, Medical University of South Carolina, Charleston, South Carolina 29425 and the  Department of Anesthesiology, University of Illinois, Chicago, Illinois 60612

Received for publication, May 21, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous studies have shown that mitogen-activated protein kinase (MAPK) cascades signal the induction of inducible nitric-oxide synthase (iNOS) in glial cells (Bhat, N. R., Zhang, P., Lee, J. C., and Hogan E. L. (1998) J. Neurosci. 18, 1633-1641; Bhat, N. R., Zhang, P., and Bhat, A. N. (1999) J. Neurochem. 72, 472-478). This study further investigates the role of p38 MAPK in the transcriptional activation of the iNOS gene by transient transfection with constitutively active upstream kinases in the pathway (i.e. MAPK kinase 3 (MKK3b(E)) and MAPK kinase 6 (MKK6b(E)). Expression in C-6 glial cells of either MKK3b(E) or MKK6b(E) resulted in an induction of the activity of a cotransfected rat iNOS promoter-reporter (iNOS-luciferase (Luc)) gene and an enhancement of cytokine-induced expression of iNOS mRNA, both of which were inhibitable by the p38 MAPK inhibitor SB203580. The MKK constructs also induced cAMP response element-mediated (CRE-Luc) and nuclear factor kappa B-dependent (nuclear factor kappa B-Luc) transcriptional activities. Transfection with dominant negative (dn) forms of CRE-binding protein (CREB) and CCAAT/enhancer-binding protein (C/EBP), the two CRE-binding transcription factors targeted by the p38 MAPK pathway, resulted in opposite effects; dnCREB enhanced and dnC/EBP inhibited iNOS-Luc parallel to their effects on CRE-Luc. In addition, the induction, by MKK3b(E) and MKK6b(E), of iNOS promoter activity was enhanced by a wild-type activating transcription factor (ATF-2), whereas a phosphorylation-defective form of ATF-2 had a suppressive effect. The results of these molecular studies provide evidence for an important role for the p38 MAPK pathway in the transcriptional activation of the iNOS gene in rat glial cells involving the transcription factors nuclear factor kappa B, C/EBP, and ATF-2.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO),1 a short lived, highly reactive free radical, is produced from L-arginine by three forms of nitric-oxide synthase (NOS): the two constitutively expressed forms, endothelial NOS and neuronal NOS, and the inducible form (iNOS) expressed mainly in inflammatory and immune cells (1, 2). Although NO derived from neuronal NOS may play a role in neuronal degeneration and death (3) besides performing a role in neurotransmission, that produced by iNOS, the high output isoform of NOS, is thought to contribute significantly to the pathogenesis of inflammatory demyelinating diseases such as multiple sclerosis, neurodegeneration in diseases such as Alzheimer's disease and Parkinson's disease, and the pathology associated with viral and bacterial infections (4-6). In these and other inflammatory conditions, iNOS is expressed mainly by activated astrocytes and microglia, the two glial cell types involved in intracerebral immune regulation (7-10). Several in vitro studies have documented the induction of iNOS in glial cells treated with proinflammatory cytokines, microbial and viral products, and abnormal protein aggregates (6, 11-13). The mechanisms by which these agents induce the expression of iNOS have been the focus of intense study in the hope that strategies can be developed to inhibit NO synthesis and thus suppress inflammatory reactions.

We (14, 15) and others (16) have shown recently that mitogen-activated protein kinase (MAPK) cascades are involved in cytokine and lipopolysaccharide (LPS)-mediated iNOS induction in primary glial cells. MAPKs are a family of Ser/Thr-specific signaling kinases activated by a variety of intracellular stimuli through a cascade of protein phosphorylations. The three well studied mammalian MAPKs are: extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. ERK is commonly activated by receptor Tyr kinases and G protein-coupled receptors, whereas JNK and p38 MAPK are potently activated by a variety of environmental stress and proinflammatory signals. Once activated, the MAPKs phosphorylate their respective substrates including several nuclear and cytoplasmic targets to regulate diverse cellular responses including cell proliferation, differentiation, survival, the inflammatory response, and even cell death (17-22). The upstream transduction chain of ERK is well known and consists of Ras-Raf-MAPK or ERK kinase (MKK/MEK)1/2. By contrast, the upstream regulators of JNK and p38 MAPK are less well defined. The immediate upstream transducers for these stress-activated protein kinases are MKK4/7-MEKK1 and MKK3/6-MEKK1, respectively. There is growing evidence that a number of additional MKKs (including MEKK2-5, mixed lineage kinases, or MLK1-3, apoptosis signal-regulating kinase 1 or ASK1, transforming growth factor-beta -activated kinase-1 or TAK1, p21-activated kinase or PAK and other Ste20 homologs) can function as upstream elements within the JNK and p38 MAPK pathways (18, 20). Each of the three MAPK modules has the potential to elicit transcriptional activation through phosphorylation of sets of transcription factors (22-25). For example, ERKs target c-Myc, Elk-1, and Ets-2; JNKs phosphorylate c-Jun, Elk-1, and ATF-2; p38 MAPKs prefer Elk-1, CHOP/C/EBP, MEF2C, ATF-2, and CREB. Additionally, they may have cytoplasmic targets including downstream kinases, i.e. the 90-kDa ribosomal S6 protein kinase (p90rsk) or mitogen-activated protein kinase-activated protein kinase-1 (MAPKAP K-1) (for ERKs), MAPKAP K2/K-3 (for p38), and MAPK-interacting kinases MNK1/2 and mitogen- and stress-activated kinase-1 (MSK-1) (for both ERK and p38 MAPK). These downstream kinases are capable of phosphorylating both cytoplasmic targets and nuclear factors such as SRF and CREB.

A prominent role for p38 MAPK-mediated signaling in the transcriptional control of iNOS gene expression has been suggested in previous studies using a specific inhibitor of the kinase, SB203580 (14, 15). This finding is in line with accumulating evidence in favor of a major function of the kinase pathway in inflammatory cell signaling and the synthesis of a variety of proinflammatory molecules including cytokines, chemokines, and cell adhesion molecules (26, 27). DNA sequence analysis indicates that the promoter regions of iNOS (as well as other proinflammatory molecules) contain consensus binding sites for numerous transcription factors including NFkappa B, C/EBP, CRE, Oct-1, AP-1, GAS, and IRE, of which many act as substrates of p38 MAPK or its downstream kinases (28-30). A detailed understanding of the relevant intracellular and nuclear signaling pathways including those mediated by MAPK cascades is helpful in identifying the control mechanisms whereby the expression of iNOS and other proinflammatory molecules are regulated. In the present study, we have employed a molecular approach, i.e. transient transfection with active forms of MKK3 and MKK6, the two known intracellular activators of p38 MAPK, to characterize further the signaling cascade involving the p38 MAPK pathway with respect to the regulation of iNOS gene expression in a glial cell model, i.e. C-6 glia. The results provide evidence for p38 MAPK-mediated nuclear signaling via NFkappa B, C/EBP, and ATF-2 in the regulation of iNOS promoter activity.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Calf serum and Dulbecco's modified Eagle's medium were from Invitrogen. Bacterial LPS, forskolin, and an antibiotic-antimycotic mixture were from Sigma. The recombinant cytokines IFN-gamma , IL-1, and TNF-alpha were from Prepro Tech (Rocky Hill, NJ). Anti-phosphospecific p38 MAPK (Tyr-182) antibody was purchased from New England Biolabs (Beverly, MA). Anti-total p38 MAPK was from Santa Cruz Biotechnology (Santa Cruz, CA). SB203580 was purchased from Calbiochem. 6-well, 12-well, and 100-mm culture dishes were purchased from Corning-CoStar (Cambridge, MA). The transfection reagent PolyFect was from Qiagen (Valencia, CA). Luciferase assay kits were from Promega (Madison, WI).

Plasmids-- Constructs containing active forms of MKK3 (pcDNAMKK3b(E)) and MKK6 (pcDNAMKK6b(E)) were a generous gift from Dr. J. Han (Scripps Clinic). Wild type and phosphorylation-defective ATF-2 (ATF-269,71Thr-Ala) were from Dr. Roger Davis (University of Massachusetts). Dominant negative (dn)CREB was from Dr. R. H. Goodman (Oregon Health Science University), and dnC/EBP-beta was from Dr. R. M. Pope (Northwestern University). The reporter constructs pCRE-Luc and pNFkappa B-Luc were purchased from Stratagene (La Jolla, CA).

The rat iNOS-Luc construct was made as described before (31). Briefly, a 2,168-bp fragment of the rat iNOS promoter was amplified from Harlan Sprague-Dawley liver genomic DNA by PCR using primers derived from the published rat iNOS promoter sequence (32) (forward, 5'-CAG CCA AGT ATT CCA AAG CAA-3', corresponding to bases 1108-1127; reverse, 5'-AGT CCA GTC CCC TCA CCA A-3', corresponding to bases 3259-3277), and its identity was confirmed by DNA sequence analysis. The 2.2-kb fragment was ligated into the pGL3 basic luciferase vector (Promega) and used for transient transfections. Renilla luciferase used as the internal control was from Promega.

Transient Transfections and Reporter Gene (Luciferase) Assays-- Transient transfections were carried out using a kit from Qiagen (PolyFect) according to manufacturer's protocols. Subconfluent cultures of C-6 glial cells were transfected with different expression plasmids together with each reporter plasmid (iNOS-Luc, NFkappa B-Luc, and pCRE-Luc) and a plasmid expressing the enzyme Renilla luciferase from Renilla reniformis as an internal control. In all cases, the total amount of plasmid DNA was adjusted with corresponding empty vector. Where indicated, cells, 48 h after transfection, were treated with different stimuli for 6 h. Firefly and Renilla luciferase activities present in cellular lysates were assayed by the use of the Dual Luciferase Reporter System (Promega), with a luminometer as specified by the manufacturer. The data represent firefly luciferase activity normalized by Renilla luciferase activity present in each sample expressed as -fold induction relative to control.

Reverse Transcription-PCR of iNOS-- Reverse transcription-PCR analysis of iNOS mRNA was carried out as described previously (14, 15) parallel to glyceraldehyde 3-phosphate dehydrogenase controls. The products were separated on agarose gels impregnated with ethidium bromide and photographed under UV light.

Cell Culture-- C-6 glial cells were maintained as stock cultures in Dulbecco's modified Eagle's medium with 10% calf serum. Whenever required for experiments, the cells were subcultured in 12-well plates and used either for transfections or for the measurement of nitric oxide synthesis after cytokine treatment.

Western Blot Analysis-- Western blot was performed for the analysis of p38 MAPK activation (using antibodies specific for the phosphorylated form of the kinase) and iNOS. Briefly, protein samples (cell extracts, 20 µg of protein equivalents) were separated by SDS-PAGE and blotted onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% bovine serum albumin, 1% milk powder in 10 mM Tris-HCl containing 150 mM NaCl, and 0.5% Tween 20 (TBST) for 1 h and incubated overnight with suitably diluted primary antibodies. After extensive washing with TBST, the membrane was incubated with anti-IgG-alkaline phosphatase conjugate. The blot was finally developed with the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (50 µg/ml) along with nitro blue tetrazolium (100 µg/ml) in sodium glycinate buffer (pH 9.6) in the presence of 4 mM MgCl2

Measurement of Nitrite Production-- NO production was determined by measurement of nitrite in the medium as described before (14). An aliquot of the spent medium was mixed with an equal volume of 1:1 mixture of 1% sulfanilamide in water and 0.1% N-1-naphthylethylenediamine dihydrochloride in 5% phosphoric acid. The absorbance was then read at 570 nm. Sodium nitrite dissolved in the culture medium was used as the standard.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In previous studies, we have shown that combinations of cytokines and LPS stimulate iNOS induction in primary glial cultures in a p38 MAPK-dependent manner (14, 15). We tested whether a similar type of regulation occurs in the rat C-6 glioma cell line in which iNOS regulation and inflammatory gene expression have been studied extensively. Cell cultures were treated with LPS and the cytokines TNF-alpha and IFN-gamma individually and in combinations, and the production of NO in the form of nitrite was determined. As shown in Fig. 1A, incubation with the combination of LPS, TNF-alpha , and IFN-gamma (LTI) yielded the maximal induction of NO production. To confirm a role for p38 MAPK in C-6 cell iNOS induction, the cultures were exposed to LTI in the presence of increasing concentrations of SB203580. A dose-dependent inhibitory effect of the drug is depicted in Fig. 1B, which indicates that as in the case of primary astrocytes and microglia (14), iNOS expression in C-6 glia depends on the activity of the p38 MAPK pathway.


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  		Fig. 1.   LPS and cytokine induction of nitrite production in C-6 glial cells and its inhibition by SB203580. A, C-6 glia were subcultured into 12-well plates and treated with 1 µg/ml LPS, 10 ng/ml IFN-gamma , and 10 ng/ml TNF-alpha individually and in the indicated combinations for 72 h. Nitrite levels in the medium were determined using Greiss reagent as described under "Experimental Procedures." The values given here and elsewhere in other figures are the average ± S.D. of triplicate determinations. B, cultures were treated with a combination of LPS, IFN-gamma , and TNF-alpha (LTI) in the presence of different concentrations of the kinase inhibitor, and the nitrite levels in the medium were determined after 72 h. The values are expressed as the average ± S.D. of triplicate determinations. #, p <=  0.05 compared with LTI.

We next examined the activation of a rat iNOS promoter construct in C-6 glia in response to cytokine treatment and the effect of the MAPK inhibitor on that activity. A plasmid containing a 2.2-kb portion of the rat iNOS promoter attached to the luciferase gene (iNOS-Luc) was introduced into subconfluent cultures of C-6 glia by transient transfection. After 40 h, the cultures were treated with LPS and cytokines, individually and in combinations, for a further 6 h (Fig. 2A). In contrast to NO production, the iNOS promoter activity was substantially (2-fold) stimulated upon incubation with LPS alone. However, that activity was enhanced further when LPS was supplemented with other cytokines, and maximal stimulation (3-fold) of the iNOS promoter was achieved using a combination of LPS with TNF-alpha and IFN-gamma . The activation of the iNOS promoter was inhibited by SB203580 with a similar dose-response curve (Fig. 2B).


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  		Fig. 2.   Induction of iNOS-Luc activity by LPS and cytokines and its inhibition by SB203580. A, the cultures were transfected with the plasmid containing iNOS-Luc as described under "Experimental Procedures," and after 48 h, triplicate wells were treated with various combinations of LPS with the two cytokines for 6 h. Luciferase activity in the cell lysates was determined, normalized with the internal control (Renilla luciferase), and expressed as -fold induction over control. B, cultures were transfected with iNOS-Luc plasmid and after 48 h treated with LTI in the presence of the indicated amounts of the kinase inhibitor for 6 h. The luciferase activity in the cell lysates was determined. Data represent the percent change compared with a control (LTI-stimulated) set at 100%. A, n = 3; #, p <=  0.05 compared with basal; *, p <=  0.05 compared with LTI. B, n = 3; #, p <=  0.05 compared with LTI.

p38 MAPK is activated by its immediately upstream kinases MKK3 and MKK6 upon their activation in response to extracellular stimuli. Introduction of active forms of these intermediate kinases is therefore expected to mimic cellular responses elicited by extracellular signals acting through this kinase cascade. In cotransfection experiments, we tested the effects of expression of the active MKK3 (MKK3b(E)) and MKK6 (MKK6b(E)) on iNOS promoter activity. Transfection of C-6 cells with either MKK3b(E) or MKK6b(E) (but not their corresponding wild type, nonactive forms, data not shown) resulted in p38 MAPK phosphorylation (Fig. 3A), reflecting its activation state and therefore confirming the intracellular activity of the transfected kinases. A dose-dependent induction of the iNOS-Luc activity by transfected MKK3b(E) and MKK6b(E) but not the wild type forms, is shown in Fig. 3B. Although transfection with the active MKK3b(E) kinase strongly induced iNOS promoter activity, by itself it was minimally active in inducing the expression of the endogenous iNOS enzyme (Fig. 4, A and B). However, incubation of kinase-transfected cells with LPS resulted in a detectable level of nitrite and iNOS expression, whereas exposure to LPS in combination with IFN-gamma led to a further increase in iNOS induction. Similar results were obtained with cells transfected with MKK6b(E) (data not included). These results suggest that post-transcriptional events are necessary for iNOS protein expression, but they are not activated by MKK3 or MKK6.


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  		Fig. 3.   Induction of iNOS promoter activity by transfected MKK3b(E) and MKK6b(E). A, immunoblot analysis of p38 MAPK phosphorylation in active MKK3- and MKK6-transfected cultures. The cultures were transfected with pcDNA (empty vector), MKK3b(E), and MKK6b(E). After a 24-h incubation in the CO2 incubator, cell extracts were analyzed by Western blot using antibodies specific for the phosphorylated form of p38 MAPK and total p38 MAPK. B, dose response of MKK3/6 induction of iNOS promoter activity. Triplicate wells were transfected with increasing concentrations of MKK3 and MKK6, the total amount of the DNA being kept constant with pcDNA, the empty vector. The activation of cotransfected iNOS-Luc was determined after 48 h as above. Values in B are the average ± S.D. of triplicate determinations.


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  		Fig. 4.   Induction of iNOS enzyme expression and nitrite production in C-6 glia transfected with MKK3b(E). A, cells were transfected with either pcDNA (empty vector) or MKK3b(E). After 2 days, the two sets of cultures were treated with LPS, IFN-gamma , or a combination of the two for an additional 3 days. The medium from each culture well was then tested for nitrite production, and the cell pellets were analyzed for iNOS protein by immunoblot using a specific antibody. Nitrite values are expressed as the average ± S.D. of triplicate determinations. #, p <=  0.05 compared with the empty vector. B, sets of cultures transfected with either the control vector (pcDNA) or MKK3b(E) for 40 h were treated with a combination of LPS and IFN-gamma in the presence and absence of 15 µM SB203580. The cultures were incubated for an additional 3 days, and the cell extracts were subjected to immunoblot using anti-iNOS antibodies.

To test the role, if any, of the p38 pathway in the post-transcriptional regulation of iNOS involving mRNA stabilization, we determined the degradation rate of iNOS mRNA in the presence and absence of SB203580. To induce iNOS, the cultures were treated with the cytokine/LPS combination, LTI for 6 h, a time point at which the expression of iNOS mRNA peaks (data not shown). As seen in Fig. 5A, this induction is inhibitable by SB203580. After a 6-h induction of iNOS mRNA in response to LTI, the cultures in two sets were exposed to actinomycin D (an inhibitor of RNA synthesis) alone or to actinomycin D plus SB203580 for various lengths of time, and the iNOS mRNA levels were determined by reverse transcription-PCR. The data shown in Fig. 5, B and C, indicate that the rate of decay of LTI-induced iNOS mRNA is essentially unaltered in the presence of the kinase inhibitor, suggesting that the p38 MAPK pathway may not participate in stabilization of iNOS mRNA. Similar results were obtained with cells transfected with MKK3b(E) and treated with the cytokines (data not shown).


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  		Fig. 5.   Noninvolvement of p38 MAPK pathway in iNOS mRNA stability. A, control cultures and those treated with LTI in the presence and absence of 15 µM SB203580 for 6 h were extracted for RNA and subjected to reverse transcription-PCR using primers specific for iNOS and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). B, cultures were treated with LTI for 6 h and divided into two sets. One set received 3 µg/ml actinomycin D and the other, actinomycin D plus SB203580. After the indicated times, RNA was extracted and analyzed for iNOS and glyceraldehyde 3-phosphate dehydrogenase mRNA by reverse transcription-PCR. C, graphical representation of the results in B after densitometric quantitation by Quantity One software (Bio-Rad). Similar results were obtained in repeat experiments where the cultures treated for 6 h with LTI were washed and then exposed to the inhibitors.

As expected, the activation of iNOS promoter activity elicited by the two active MKKs was susceptible to inhibition by SB203580 (Fig. 6, top panel). The iNOS promoter contains several regulatory elements including NFkappa B, CRE, and C/CAAT box, which act as binding sites for specific transcription factors that are presumed targets (direct or indirect) of p38 MAPK. Therefore, we tested the effect of active MKKs on NFkappa B- and CRE-dependent transcriptional activities parallel to iNOS promoter activation. As shown in Fig. 6, middle and bottom panels, the kinases stimulate the activities of both CRE-Luc and NFkappa B-Luc, respectively, and parallel to iNOS-Luc. Again, these activities are susceptible to inhibition by SB203580, suggesting that the relevant transcription factors, i.e. CREB or CREB-related proteins and NFkappa B, lie downstream from the p38 MAPK signal.


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  		Fig. 6.   Inhibition of MKK3/6-induced iNOS-Luc, CRE-Luc, and NFkappa B-Luc by SB203580. Three sets (six wells each) of cultures were transfected with iNOS-Luc (top panel), CRE-Luc (middle panel), and NFkappa B-Luc (bottom panel) plasmids along with control vector, active forms of MKK3 or MKK6. After 16 h, triplicate dishes from each set were treated with 15 µM SB203580. After an additional incubation for 30 h, luciferase activities were determined and normalized with the cellular protein content. n = 3; #, p <=  0.05 compared with the empty vector; *, p <=  0.05 compared with active kinases, MKK3 or MKK6.

The transcription factor CREB is a target of the p38 MAPK pathway via downstream kinases, MAPKAP kinases and MNK1/2 (23), after which phospho-CREB protein binds to CRE motifs present in target gene promoters to induce transcriptional activities. To determine the possible role of CREB in mediating transcriptional activation of the iNOS promoter in response to activated MKK3 and MKK6, we cotransfected cells with the active kinases together with dnCREB followed by determination of the iNOS-Luc activity. As shown in Fig. 7A, dnCREB significantly potentiated iNOS activation by MKKs. These results suggest that CREB normally negatively regulates iNOS promoter activity in C-6 cells. Interestingly, similar effects of dnCREB on the activation of CRE-Luc promoter by MKKs were observed, namely a potentiation of MKK activation by dnCREB (Fig. 7B). In fact, dnCREB was found to increase CRE-Luc activity markedly in MKK3/6-transfected cells. That the dnCREB was functional was indicated by a parallel experiment in which the forskolin-stimulated CRE-Luc activity was inhibited by dnCREB (Fig. 7C).


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  		Fig. 7.   Effect of dnCREB on MKK3b(E)- and MKK6b(E)-induced iNOS-Luc and CRE-Luc activities. Cultures were transfected with combinations of either iNOS-Luc (A) or CRE-Luc (B) with dnCREB or the corresponding control vector, and the luciferase activity was determined after 48 h. In another set of experiments (C), two sets of cultures were transfected with CRE-Luc plasmid with either dnCREB or the control vector and after 48 h. One set was treated with 10 µM forskolin for 6 h before determination of the luciferase activity. Values given are the average ± S.D. of triplicate determinations. #, p <=  0.05 compared with empty vector; *, p <=  0.05 compared with MKK3b(E) or MKK6b(E) (A and B) or with forskolin (C).

The CRE site present in the rat iNOS promoter has the potential to bind transcription factors in addition to CREB which are also potential direct targets of MAPKs including C/EBP and ATF-2. To examine the role of C/EBP in iNOS induction, we introduced a dominant negative form of C/EBP and assayed iNOS promoter activity in cells cotransfected with active MKK3. As shown in Fig. 8, dnC/EBP elicits a significant inhibitory effect on MKK3-induced activation of the iNOS promoter activity. Similar results were obtained with MKK6 cotransfected with dnC/EBP (data not shown). These findings suggest that C/EBP normally acts to increase activation of the iNOS promoter.


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  		Fig. 8.   A dominant negative form of C/EBP inhibits MKK3b(E)-induced activation of iNOS-Luc and CRE-Luc. The cultures were transfected with MKK3b(E) and the control vector in combination with either dnCREB or the corresponding empty vector for 48 h, and the luciferase activity was determined. n = 3; #, p <=  0.05 compared with empty vector; *, p <=  0.05 compared with MKK3b(E).

A possible role for ATF-2 in mediating iNOS promoter activation by MKKs was examined by transfecting cells with either a wild type or a mutant (phosphorylation-defective) form of ATF-2 (33). Transfection with wild type ATF-2 enhanced, whereas mutant ATF-2 inhibited the MKK3-induced iNOS promoter activity (Fig. 9), thereby implicating a role for this transcription factor in iNOS gene expression. The inhibitory effect of the mutant was more prominent upon MKK3 but not MKK6 induction of iNOS-Luc.


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  		Fig. 9.   ATF-2 enhancement of MKK3b(E)- and MKK6b(E)-induced activation of iNOS promoter activity. Cultures were transfected with combinations of MKK3b(E) and MKK6b(E) with either wild type (wt) ATF-2 or a phosphorylation mutant. After 48 h, the activities of cotransfected iNOS-Luc (upper panel) and CRE-Luc (lower panel) were determined. n = 3; #, p <=  0.05 compared with empty vector; *, p <=  0.05 compared with MKK3b(E) or MKK6b(E); @, p <=  0.05 compared with MKK3b(E).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Excessive production of NO, mainly by activated glial cells, has been linked to several inflammatory and degenerative diseases of the central nervous system (3-6). A greater understanding of the signaling mechanisms involved in the regulation of glial iNOS has therefore potential therapeutic implications. Using a pharmacological approach, we had shown previously that MAPK signaling, in particular p38 MAPK, mediates endotoxin and cytokine stimulation of iNOS expression in cultured glial cells (14, 15). The present studies extend these observations and examine further the role of p38 MAPK in the transcriptional activation of iNOS gene using a transiently transfected rat iNOS promoter-reporter construct. Stimulation of cultured C-6 glial cells with cytokines (TNF-alpha and IFN-gamma ) and LPS results in an induced production of NO, which is mostly accounted for by a stimulation of iNOS promoter activity. The induction of iNOS-Luc and the production of NO were similarly inhibited by SB203580, suggesting the role of this kinase pathway in iNOS expression at the transcriptional level. Transfection of glial cells with constitutively active forms of MKK3 and MKK6, the two prominent and specific intracellular activators of p38 MAPK, potently induced iNOS promoter activity, which was susceptible to inhibition by SB203580. An investigation of the roles of specific transcription factor targets of the kinase pathway indicated the participation of ATF-2, C/EBP, and NFkappa B in the stimulation of iNOS promoter activity.

The upstream steps in cytokine and LPS activation of p38 MAPK pathway are complex and involve initial recruitment by activated receptors of a variety of signal transducers. Thus, the TNF receptor-associated factor-2 and the receptor-interacting protein kinase recruited to the activated TNF receptor are involved in the downstream signaling to activate MAPK cascades and NFkappa B, via the MKKK homologs, MEKK1 and NFkappa B-activating kinase, respectively (34). The receptors for LPS and IL-1beta belong to the newly defined family of Toll-like receptors, which, via an adapter protein (MyD88), activate a Ser/Thr kinase termed IL-1 receptor-associated kinase (35). Upon autophosphorylation, this kinase interacts with TNF receptor-associated factor-6 to activate MKKK members and hence, NFkappa B and MAPK cascades. Activation of p38 MAPK is specifically and most commonly catalyzed by the two intermediate dual specificity kinases, MKK3 and MKK6, which are downstream from MEKK1 and related MKKKs. Experimental manipulation of these two intermediate kinases, therefore, provides a way of analyzing the function of the p38 MAPK irrespective of the activating signals and upstream events. We find that expression of constitutively active forms of MKK3 and MKK6 brings about an induction of the activity of iNOS promoter in C-6 glial cells. These results seem to be in agreement with the findings in mesangial cells where the expression of dominant negative forms of MKK3 and MKK6 interfere with iNOS expression in response to IL-beta treatment (36). It should be noted that activation of the iNOS promoter by itself does not necessarily lead to the expression of iNOS enzyme. Thus, although LPS (alone or with TNF-alpha ) did not induce significant nitrite production, these two combinations significantly increased promoter activation, suggesting that IFN-gamma itself is necessary for iNOS expression and activity but not for promoter activation. Consistent with this is the fact that IFN-gamma alone did not induce the iNOS promoter, did not increase promoter activation caused by LPS, and only slightly increased promoter activation caused by TNF-alpha (Fig. 2). Similarly, although transfection with constitutively active forms of MKK3 or MKK6 substantially activated the promoter, they were unable to induce either iNOS protein expression or nitrite production (Fig. 4) unless cells were incubated with LPS and IFN-gamma . Taken together, these results indicate that combinations of signals are required for the full expression of the iNOS gene, most likely involving both transcriptional and post-transcriptional regulatory steps. The results on mRNA degradation rates indicate that although the p38 signal may not be required for the stability of iNOS mRNA under the conditions used, it may still play a role in translational control. The results also suggest that MAPK activation caused by MKK3b(E) (or MKK6b(E)) activates signals that can also be induced by IFN-gamma and are necessary for iNOS protein expression (and nitrite production) but not for promoter activation.

Although the p38 MAPK pathway can elicit post-transcriptional control during the expression of certain cytokine genes (37), the primary effect on iNOS seems to involve transcriptional activation (14). The transcriptional control of iNOS is complex, displaying species-, tissue-, and signal-specific variations that could reflect the presence of discrete cis regulatory and enhancer elements (28, 29). The rat iNOS promoter contains several transcription factor binding sites potentially involved in cytokine and LPS signaling (38). These include NFkappa B/Rel, C/EBP, CREB/ATF, IRF-1, STAT, and AP-1, some of which become targets of the p38 MAPK pathway either directly (e.g. C/EBP, ATF-2) or indirectly (NFkappa B, CREB). There are four binding sites for members of the C/EBP family of transcription factors, two of which are located with less than 200 bp upstream from the transcription start site and close to one of the two NFkappa B sites. The C/EBP family of transcription factors, in particular, C/EBP-alpha , C/EBP-beta (also known as NF-IL-6), and C/EBP-delta , regulate genes involved in acute phase response, inflammation, as well as growth and differentiation depending on the stimulus and cell system (39, 40). C/EBP regulation can occur both at the level of their expression and via their phosphorylation. For example, C/EBP-beta and -delta , the two members involved in the cytokine-dependent regulation of acute phase proteins in the liver, are inducible by LPS and cytokines (41). Transactivation via C/EBP may also be regulated by phosphorylation, and there is evidence for p38 kinase mediated phosphorylation of C/EBP family members (42). The transcription factor CREB pre-exists in cells as an inactive protein that can be activated after its phosphorylation at Ser-133 catalyzed by a number of kinases including protein kinase A, Ca2+-calmodulin-dependent kinases, and several downstream targets of MAPKs i.e. MAPKAP kinase-1/2, MSK-1, and MNK (43). Activated CREB binds to a coactivator termed CREB-binding protein and its paralog p300, through a kinase-inducible domain, and the complex formed then elicits transcriptional activation of the target genes (44). Our results point to a positive regulation of iNOS promoter by C/EBP and ATF-2 and a repressor-like effect of CREB. Also, we have found that the active forms of MKK3 and MKK6 stimulate NFkappa B-dependent transcriptional activity parallel to iNOS promoter activation.

We find that activated MKK3/6 stimulate CRE- and NFkappa B-dependent transcription parallel to iNOS-Luc activity and that the p38 MAPK inhibitor suppresses this activation. Because activation at CRE sites in many promoters is often associated with transcriptional activation by CREB, we examined the effect of a cotransfected dnCREB on MKK induction of the iNOS reporter gene. Contrary to our expectation, the dnCREB construct failed to block MKK-induced iNOS-Luc and CRE-Luc activities but rather enhanced these two activities, suggesting a negative regulation by CREB. This negative effect on iNOS promoter, despite being at odds with studies implicating CREB in the induction of iNOS in other systems (45, 46), seems to reflect the known dual regulatory roles of cAMP/protein kinase A pathway in iNOS induction. Thus, cAMP/protein kinase A signal, which, prominently targets CREB, is known to elicit both an inhibitory and stimulatory influence on the iNOS (47). To explain these conflicting effects, it has been suggested that there might exist cAMP-responsive cell-specific suppressors and/or activators of iNOS expression (47). Gavrilyuk et al. (31) have recently identified a 27-bp region (bp -187 to -160) as critical for mediating the suppressive effect of cAMP on glial iNOS promoter activation by cytokines. This 27-bp promoter region is highly conserved among species and contains one of the two CRE sites, which could be shared by members of the C/EBP family. It is to be noted that the rat but not mouse iNOS promoter contains the typical CRE site, although cAMP inhibition occurs in mouse cells as well. This may be because although in both mouse and human the CRE site (TGACGTA) differs by 1 bp from the canonical CRE sequence (TGACGGTA), the C/EBP site is maintained. This C/EBP site may be shared by both CREB and C/EBP as has been shown for cyclooxygenase-2 and phosphoenolpyruvate carboxykinase promoters (48, 49). Interestingly, transient transfection of a plasmid encoding a wild type CREB represses LPS-induced cyclooxygenase-2 reporter activity in RAW 264.7 cells (48), an effect very similar to our finding with the rat iNOS promoter. Relevant to our findings, the binding of a 27-bp region of the iNOS promoter to nuclear factors present in glial cells can be blocked by CREB-specific antibodies, suggesting thereby the role of CREB or a CREB-like factor(s) in mediating the cAMP suppressive effect on iNOS expression (31). We find that in contrast to CREB, C/EBP positively regulates iNOS promoter as revealed by the results showing an inhibition of MKK3-mediated promoter activation by cotransfected dnC/EBP. In this regard, the importance of the C/EBP2 site in the mouse promoter activation by LPS and cytokines has been confirmed by deletional/mutational analysis (46, 50). Although studies with other cell types have suggested that CREB and C/EBP synergize (through heterodimerization) to induce iNOS (45), it is likely that the CRE site in the rat promoter is competed for by these two factors and that they elicit opposite effects.

Our results with NFkappa B-Luc suggest the involvement of NFkappa B signal in MKK-mediated induction of the iNOS promoter. NFkappa B plays a key role in transcriptional activation of iNOS in response to LPS and proinflammatory cytokines in most cell types including glia (46, 51-53). The activation of NFkappa B involves its release from a complex with Ikappa B followed by nuclear translocation (54). Ikappa B is phosphorylated by a cytokine-activated protein kinase termed Ikappa B kinase, thereby marking it for destruction. Ikappa B kinase, itself, is targeted by upstream kinases, including MEKK members, NIK, MEKK1, and TPL-2 (55). The role of NFkappa B in MAPK signaling to induce iNOS has been examined in some studies with contrasting results. Thus, in mouse astrocytes treated with a cytokine combination (IL-1 plus TNF-alpha ), inhibition of p38 MAPK resulted in a blockade of iNOS gene expression without an effect on NFkappa B (16). But in a mouse macrophage cell line, SB203580 was able to inhibit LPS-stimulated DNA binding activity of NFkappa B parallel to its ability to inhibit iNOS expression (56). We have found that both MKK3 and MKK6 increase NFkappa B-mediated transcriptional activation in C-6 glial cells and that SB203580 suppresses this activation, suggesting an involvement of NFkappa B pathway in mediating p38 MAPK induction of iNOS promoter activity. However, we do not yet know whether the kinase directly activates the transcription factor or acts to influence its function at later steps involving modulatory phosphorylations of the DNA-binding subunits and/or their interactions with other nuclear factors.

We have found that overexpression of ATF-2 can stimulate the iNOS promoter in MKK3- and MKK6-transfected cells and that a phosphorylation mutant of ATF-2 has an opposite effect, suggesting thereby a role for this transcription factor in mediating p38 MAPK induction of glial iNOS. ATF-2 acts as a direct target of p38 MAPK, which phosphorylates it on Thr-69 and Thr-71 (23). Activated ATF-2 binds to CRE-like elements (T(G/T)ACGTCA) in the promoters of target genes to induce their expression (57). It is possible that ATF-2 binds to CRE and/or C/EBP sites in stimulating iNOS gene expression. It may also modulate binding of NFkappa B to its kappa B sites on the iNOS promoter as has been shown previously in the case of IL-1 induction of iNOS gene expression in an insulin-producing cell line (58).

Fig. 10 summarizes schematically the regulation of glial iNOS promoter including the positive and negative regulatory inputs in terms of known transcription factors potentially targeted by the p38 MAPK pathway. Further studies are under way to dissect the promoter to test directly the role of specific cis regulatory elements by deletional analysis. These analyses would potentially identify yet to be defined regulatory elements and interacting proteins that respond to MAPK signaling. Such studies would also have practical utility because targeting of transcription factors represents an interesting approach for interfering selectively with the cytokine-induced overproduction of NO in inflammatory conditions.


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  		Fig. 10.   Schematic representation of p38 MAPK-mediated transcriptional activation of iNOS in glial cells.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants NS41035 (to N. R. B.) and NS31556 (to D. L. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Neurology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel.: 843-792-7593; Fax: 843-792-8626; E-mail: bhatnr@musc.edu.

Published, JBC Papers in Press, June 4, 2002, DOI 10.1074/jbc.M204994200

    ABBREVIATIONS

The abbreviations used are: NO, nitric oxide; ATF-2, activating transcription factor-2; C/EBP, CCAAT/enhancer-binding protein; CREB, cAMP response element-binding protein; dn, dominant negative; ERK, extracellular signal-regulated kinase; IFN, interferon; IL, interleukin; iNOS, inducible nitric-oxide synthase; JNK, -Jun N-terminal kinase; LPS, lipopolysaccharide; LTI, combination of LPS, TNF-alpha , and IFN-gamma ; Luc, luciferase; MAPK, mitogen-activated protein kinase; MAPKAP, mitogen-activated protein kinase-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MEKK, MEK kinase; MKK, MAP kinase kinase; MSK, mitogen- and stress-activated kinase; NFkappa B, nuclear factor kappa B; NOS, nitric-oxide synthase; TNF, tumor necrosis factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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