Protein Kinase C eta  Mediates Lipopolysaccharide-induced Nitric-oxide Synthase Expression in Primary Astrocytes*

Ching-Chow ChenDagger §, Jia-Kae WangDagger , Wei-Chyuan ChenDagger , and Shwu-Bin Lin

From the Dagger  Institutes of Pharmacology and  Medical Technology, College of Medicine, National Taiwan University, No.1, Jen-Ai Road, 1st Section, Taipei 10018, Taiwan

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The signaling pathway involved in protein kinase C (PKC) activation and role of PKC isoforms in lipopolysaccharide (LPS)-induced nitric oxide (NO) release were studied in primary cerebellar astrocytes. LPS caused a dose- and time-dependent increase in NO release and inducible NO synthase (iNOS) expression. The tyrosine kinase inhibitor, genestein, the phosphatidylcholine-phospholipase C inhibitor, D609, and the phosphatidate phosphodrolase inhibitor, propranolol, attenuated the LPS effects, whereas the PI-PLC inhibitor, U73122, had no effect. The PKC inhibitors (staurosporine, Ro 31-8220, Go 6976, and calphostin C) also inhibited LPS-induced NO release and iNOS expression. However, long term (24 h) pretreatment of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA) did not affect the LPS response. Previous results have shown that TPA-induced translocation, but not down-regulation, of PKCeta occurs in astrocytes (Chen, C. C., and Chen, W. C. (1996) Glia 17, 63-71), suggesting possible involvement of PKCeta in LPS-mediated effects. Treatment with antisense oligonucleotides for PKCeta or delta , another isoform abundantly expressed in astrocytes, demonstrated the involvement of PKCeta , but not delta , in LPS-mediated effects. Stimulation of cells for 1 h with LPS caused activation of nuclear factor (NF)-kB in the nuclei as detected by the formation of a NF-kB-specific DNA-protein complex; this effect was inhibited by genestein, D609, propranolol, or Ro 31-8220 or by PKCeta antisense oligonucleotides, but not by long term TPA treatment. These data suggest that in astrocytes, LPS might activate phosphatidylcholine-phospholipase C and phosphatidylcholine-phospholipase D through an upstream protein tyrosine kinase to induce PKC activation. Of the PKC isoforms present in these cells, only activation of PKCeta by LPS resulted in the stimulation of NF-kB-specific DNA-protein binding and then initiated the iNOS expression and NO release. This is further evidence demonstrating that different members of the PKC family within a single cell are involved in specific physiological responses.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Nitric oxide (NO),1 a bioactive free radical, is involved in various physiological and pathological processes in many systems (1). Low concentrations of NO play a role in neurotransmission and vasodilation. However, when secreted at higher concentrations, NO is implicated in the pathogenesis of stroke and other degenerative diseases, such as demyelinating conditions and ischemic and traumatic injury (2). NO is formed enzymatically from L-arginine by nitric-oxide synthase (NOS). NOS enzymes are classified into two groups. One type (cNOS) is constitutively present in several cell types (e.g. neurons and endothelial cells) and is regulated predominantly at the post-transcriptional level by calmodulin in a Ca2+-dependent manner (2). In contrast, the inducible form (iNOS), expressed in various cell types, including vascular smooth muscle cells, macrophages, hepatocytes, and astrocytes, is induced in response to proinflammatory cytokines and bacterial lipopolysaccharide (LPS) (3-6). Cellular NO release following iNOS induction in astrocytes and microglia has been implicated in oligodendrocyte degeneration in demyelinating diseases and in neuronal death during trauma (7-9).

The mechanism of the signal transduction cascade involved in the induction of iNOS in response to LPS and cytokines is an active area of investigation. Although LPS-produced iNOS induction in primary astrocytes has been reported (6, 10), the molecular events involved are not understood. Previous reports have shown a potential role for tyrosine kinase in LPS-produced iNOS induction (11, 12). The murine iNOS promotor contains 24 transcriptional factor binding sites, including those for NF-kB and activator protein-1 (13, 14). Proteins of the NF-kB family appear to be essential for the enhanced iNOS gene expression seen in macrophages exposed to LPS (15), and the p65 NF-kB also seems to be responsible for iNOS induction in astrocytes (16). In the present study, the intracellular signaling pathway by which LPS induces iNOS expression in primary astrocytes was studied. The results show that LPS might activate phosphatidylcholine-phospholipases C and D (PC-PLC and PC-PLD) via tyrosine phosphorylation to produce PKC and NF-kB activation, iNOS expression, and, finally, NO release. Of the PKC isoforms alpha , delta , theta , eta , and zeta  expressed in astrocytes (17, 18), only PKCeta is involved in the regulation of LPS-induced NF-kB activation, iNOS expression, and NO release.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Affinity-purified rabbit polyclonal antibody to iNOS was obtained from Transduction Laboratories (Lexington, KY). Basal modified Eagle's medium, fetal calf serum, glutamine, gentamycin, penicillin, and streptomycin were purchased from Life Technologies, Inc. Rabbit polyclonal antibodies to PKCdelta and eta  and the NF-kB probe were purchased from Santa Cruz Biotechnology. TPA was from L. C. Services Corp. (Woburn, MA). LPS (from Escherichia coli serotype 0127:B8), staurosporine, pyrolidine dithiocarbamate, sulfanilamide, and N-(1-naphthyl)-ethylenediamine were from Sigma. Genestein, calphostin C, Go 6976, and Ro 31-8220 were from Calbiochem (San Diego, CA). D609, U73122, and U73343 were from RBI (Natick, MA). T4 polynucleotide kinase was from New England Biolabs (Beverly, MA). Poly(dI·dC) was from Amersham Pharmacia Biotech. Reagents for SDS-polyacrylamide gel electrophoresis were from Bio-Rad. [tau -32P]ATP (3000 Ci/mmol) was from NEN Life Science Products. The horseradish peroxidase-labeled donkey anti-rabbit second antibody and ECL detecting reagent were purchased from Amersham Pharmacia Biotech.

Primary Cultures of Astrocytes-- Glial cell cultures were prepared from the cerebellum of 8-day Wistar rats as described previously (17). Briefly, the cerebella were dissected and dissociated by mechanical chopping and trypsinization to obtain a cell suspension. Cells were plated at a density of 105 cells/well in poly-L-lysine-precoated 12-well plates for the nitrite assay and at a density of 107 cells/10-cm dish for iNOS and PKCeta and delta  isoform expression tests and the NF-kB gel shift assay. Cultures were maintained in basal modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 50 µg/ml gentamycin, which was changed twice each week. Cells grown in an atmosphere of 5% CO2/95% humidified air at 37 °C were used after 10-12 days in culture, at which time they consisted of confluent glial cells, which stained positively for glial fibrillary acidic protein (17).

Determination of NO Concentration-- NO production in culture supernatant was evaluated by measuring nitrite, its stable degradation product, using the Griess reagent. The basal modified Eagle's medium was changed to phenol red-free medium before the cells were stimulated with LPS (1 µg/ml) for 24 h, and then the isolated supernatant was centrifuged and mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochloride, 2% phosphoric acid) and incubated at room temperature for 10 min before the absorbance was measured at 550 nm in a microplate reader. Sodium nitrite (NaNO2) was used as a standard. In pretreatment experiments, cells were incubated with genestein (a tyrosine kinase inhibitor), U73122 (a PI-PLC inhibitor), D609 (a PC-PLC inhibitor), propranolol (a phosphatidate phosphohyrolase inhibitor), staurosporine, calphostin C, Go 6976, or Ro 31-8220 (PKC inhibitors) for 30 min or with TPA for 24 h before the addition of LPS.

Preparation of Cell Extracts and Western Blot Analysis of iNOS and PKCeta and delta -- Following treatment with LPS, or pretreatment with inhibitors, TPA or antisense oligonucleotides (see below) followed by LPS, the cells were harvested and collected. For studies of iNOS expression or PKCeta and delta  expression (antisense oligonucleotides treatment), cell homogenates were prepared and subjected to SDS-polyacrylamide gel electrophoresis using 7.5% (iNOS) or 10% (PKC isoform) running gels, and then the proteins were transferred to nitrocellulose paper, and immunoblot analyses were performed as described previously (17). Briefly, the membrane was incubated successively at room temperature with 0.1% milk in Tris-buffered saline/Tween 20 (TTBS) for 1 h, with rabbit antibodies specific for iNOS or PKCeta or delta for 1 h and with horseradish peroxidase-labeled anti-rabbit antibody for 30 min. After each incubation, the membrane was washed extensively with TTBS. The immunoreactive band was detected with ECL detecting reagents and developed with Hyperfilm-ECL.

Synthesis of PKCeta and delta  Antisense Oligonucleotides and Treatment of Cells with Oligonucleotides-- Phosphorothioate oligodeoxynucleotides were synthesized in trityl-on mode using an Applied Biosystems model 391 DNA synthesizer, as described previously (19), using A, G, C, and T phosphoramidites, controlled pore glass supports, and sulfuring reagent purchased from Glen Research Corp. (Sterling, VA). The oligodeoxynucleotides were deblocked and cleaved from the solid support using concentrated ammonia water by a standard procedure. After evaporation of the ammonia, the deprotected oligodeoxynucleotides were purified on Sep-Pak C18 cartridges (Millipore, Milford, MA), as reported previously (20). Control sequences (scrambled versions of the antisense oligonucleotides) with the same base composition as the PKCeta antisense oligonucleotides were also synthesized. The sequences of the PKCeta and delta  antisense oligonucleotides and scrambled PKCeta controls are listed in Table I.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Sequences of antisense oligonucleotides and controls used in this study

Following attachment on day 1 of culture, cells were treated with PKCeta or delta  antisense oligonucleotides or control oligonucleotides (10 µM) for 9 days (the medium was changed once) before challenge with 1 µg/ml LPS for 24 h, and then the protein levels of PKCeta and delta  were determined by Western blotting using antibodies specific for PKCeta or delta .

Preparation of Nuclear Extracts and the Electrophoretic Mobility Shift Assay (EMSA)-- Control cells or cells pretreated with genestein, D609, propranolol or Ro 31-8220 for 30 min, TPA for 24 h, or antisense oligonucleotides for 9 days were treated with 1 µg/ml LPS for 1 h. Nuclear extracts were then isolated as described previously (21). Briefly, cells were washed with ice-cold phosphate-buffered saline (PBS) and pelleted. The cell pellet was resuspended in hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM NaF, and 1 mM NaVO4) and incubated for 15 min on ice, and then the cells were lysed by the addition of 0.5% Nonidet P-40, followed by vigorous vortexing for 10 s. The nuclei were pelleted and resuspended in extraction buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, and 1 mM Na3VO4), and the tube vigorously shaken at 4 °C for 15 min on a shaking platform. The nuclear extracts were then centrifuged, and the supernatants were aliquoted and stored at -80 °C.

A double-stranded oligonucleotide probe containing NF-kB sequences (5'-AGTTGAGGGGACTTTCCCAGGGC-3', Santa Cruz) was purchased and end-labeled with [tau -32P]ATP using T4 polynucleotide kinase. The nuclear extract (6-10 µg) was incubated with 1 ng of the 32P-labeled NF-kB probe (40,000-60,000 cpm) in 10 µl of binding buffer containing 1 µg of poly(dI·dC), 15 mM HEPES, pH 7.6, 80 mM NaCl, 1 mM EGTA, 1 mM DTT, and 10% glycerol at 30 °C for 20 min. DNA/nuclear protein complexes were separated from the DNA probe by electrophoresis on a native 6% polyacrylamide gel, and then the gels were vacuum-dried and subjected to autoradiography with an intensifying screen at -80 °C.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Signaling Pathways for LPS-induced NO Production and 130-kDa iNOS Expression-- Exposure of cells to LPS resulted in concentration- and time-dependent nitrite production and expression of the 130-kDa iNOS (Fig. 1). Using a 24-h exposure period, maximum nitrite release (65 ± 9 nmol/105 cells/24 h; n = 3) was obtained at 10 µg/ml LPS, and the basal nitrite release was 2 ± 1 nmol/105 cells/24 h (n = 3) (Fig. 1A). When cells were treated with 1 µg/ml LPS for various times, nitrite release was significant at 12 h (8 ± 5 nmol/105 cells/24 h; n = 3) and maximal at 48 h (Fig. 1B). In the following NO release experiments, the cells were treated with 1 µg/ml LPS for 24 h.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Concentration- and time-dependent LPS-induced stimulation of nitrite release and iNOS expression in astrocytes. Cells were incubated at 37 °C with various concentrations of LPS for 24 h (A) or with 1 µg/ml LPS for various time intervals (B), and then the medium was removed and analyzed for nitrite release. The results are expressed as the mean ± S.E. of three independent experiments performed in triplicate. In C, cells were incubated with the indicated concentrations of LPS for 24 h or with 1 µg/ml of LPS for the indicated time intervals, and then cell lysates were subjected to Western blotting using iNOS-specific antibody as described under "Experimental Procedures."

In order to study the intracellular signaling pathway involved in the LPS-induced NO production and iNOS expression, the tyrosine kinase inhibitor genestein was used. When cells were pretreated for 30 min with 30 µM genestein, LPS-induced nitrite production was inhibited by 35% and iNOS expression decreased (Fig. 2A). When cells were pretreated for 30 min with 10 µM U73122 or U73343 (an inactive analog of U73122), 50 µM D609, or 50 µM propranolol, LPS-induced nitrite production was inhibited 50 and 38% by D609 and propranolol, respectively, whereas U73122 and U73343 had no effect (Fig. 2B); iNOS protein expression was also inhibited by D609 or propranolol but not by U73122 (Fig. 2C).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of genestein, U73122, U73343, D609, or propranolol on LPS-induced nitrite release and iNOS expression in astrocytes. Cells were pretreated with 10 or 30 µM genestein (A), 10 µM U73122 or U73343, 50 µM D609, or 50 µM propranolol (B) for 30 min before incubation with 1 µg/ml of LPS for 24 h; the medium was then removed and analyzed for nitrite release. The results are expressed as the mean ± S.E. for a minimum of three independent experiments performed in triplicate. *, p < 0.05 as compared with LPS alone. For iNOS expression studies, cells were pretreated with 30 µM genestein (A) or 10 µM U73122, 50 µM D609, or 50 µM propranolol (C) for 30 min before incubation with 1 µg/ml LPS for 24 h, and then cell lysates were subject to Western blotting using iNOS-specific antibody as described under "Experimental Procedures."

Inhibitory Effect of PKC Inhibitors and Lack of Effect of Long Term TPA Treatment on LPS-Induced NO production and iNOS Expression-- LPS-induced nitrite production and iNOS expression were both inhibited by D609 and propranolol, indicating the involvement of the PC-PLC and PC-PLD pathways. Both pathways can increase diacylglycerol levels and then activate PKC. To determine whether activation of PKC by LPS was involved in the regulation of LPS-induced NO production, PKC inhibitors were used. Pretreatment of cells for 30 min with 100 nM staurosporine, 0.5 µM Ro 31-8220, 3 µM Go 6976, or 100 nM calphostin C inhibited LPS-induced nitrite production by 48, 73, 47, or 26%, respectively; iNOS protein expression was also inhibited by Ro 31-8220 (Fig. 3A). When cells were treated with 1 µM TPA for 24 h, NO release (10 ± 1 nmol/105 cells/24 h; n = 4) was seen; under these conditions, the LPS-induced nitrite production was 66 ± 5 nmol/105 cells/24 h (n = 7), which was greater than with LPS alone (48 ± 5 nmol/105 cells/24 h; n = 3), although not statistically significant by the t test (Fig. 3B). Similar results were obtained for iNOS expression (Fig. 3B). Previous studies have shown that in macrophages, MDCK cells and astrocytes, PKCeta is translocated but not down-regulated by TPA treatment (21). The inhibition of the LPS-induced effects by PKC inhibitors, but not by long term TPA treatment, suggested the possible involvement of PKCeta in LPS-induced NO production.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of PKC inhibitors or long term TPA treatment on LPS-induced nitrite release and iNOS expression in astrocytes. Cells were pretreated with 100 nM staurosporine, 100 nM calphostin C, 500 nM Ro 31-8220, or 3 µM Go 6976 for 30 min (A) or with 1 µM TPA for 24 h (B) before incubation with 1 µg/ml LPS for 24 h; the medium was then removed and analyzed for nitrite release. The results are expressed as the mean ± S.E. for a minimum of three independent experiments performed in triplicate. *, p < 0.05 as compared with LPS alone. For iNOS expression studies, cells were pretreated with 500 nM Ro 31-8220 for 30 min or 1 µM TPA for 24 h before incubation with 1 µg/ml LPS for 24 h, and then cell lysates were subjected to Western blotting using iNOS-specific antibody as described under "Experimental Procedures."

Because PKCeta is involved in the LPS-mediated NO production and iNOS expression, the effect of direct TPA-mediated activation of PKC on NO release and iNOS expression was examined. As shown in Fig. 4, 1 µM TPA also induced a time-dependent increase in nitrite release. However, the increase was much smaller than that induced by LPS (compare with Fig. 1B). Similar time-dependent results were seen for TPA-induced iNOS expression (Fig. 4).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4.   Time-dependent TPA-induced stimulation of nitrite release and iNOS expression in astrocytes. Cells were incubated at 37 °C with 1 µM TPA for various intervals, and then the medium was removed and analyzed for nitrite release. The results are expressed as the mean ± S.E. of one typical experiment performed in triplicate, and similar results were obtained in three experiments. In iNOS expression studies, cells from the nitrite assay were subjected to electrophoresis and Western blotting with iNOS-specific antibody as described under "Experimental Procedures."

Inhibitory Effect of PKCeta Antisense Oligonucleotides but Not PKCdelta Antisense Oligonucleotides on LPS-Induced NO Production and iNOS Expression-- To further study the involvement of PKCeta and the lack of involvement of other isoforms in the LPS-induced NO release and iNOS expression, PKCeta antisense and scrambled control oligonucleotides and antisense oligonucleotides for PKCdelta , another isoform abundantly expressed in astrocytes (17), were used. Following treatment of primary astrocyte cultures with PKCeta or PKCdelta antisense oligonucleotides for 9 days, the expression levels of PKCeta or delta  were determined by Western blotting. As shown in Fig. 5A, 10 µM of PKCeta or delta  antisense oligonucleotides caused a specific reduction in the level of the corresponding immunoreactive isoform protein, e.g. PKCeta antisense oligonucleotides specifically inhibited the expression of PKCeta protein but had no effect on the expression of PKCdelta . Because cerebellar astrocytes grow confluently after 10-12 days in culture, PKCeta or delta  antisense oligonucleotides were added for entire culture periods, and the reduction in the level of PKCeta and PKCdelta was similar to those shown in Fig. 5A.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of PKCdelta or PKCeta antisense oligonucleotides or control oligonucleotides on PKCdelta and eta  expression and on LPS-induced nitrite release and iNOS expression in astrocytes. In A, cells were pretreated with PKCdelta or PKCeta antisense oligonucleotides or control oligonucleotides (Anti-eta C) (10 µM) for 9 days. Whole cell proteins were prepared and electrophoresed and then blotted and immunodetected with PKCdelta - or PKCeta -specific antibodies as described under "Experimental Procedures." In B, cells were pretreated with PKCdelta or PKCeta antisense oligonucleotides or control oligonucleotides (10 µM) for 9 days before incubation with 1 µg/ml of LPS for 24 h; the medium was then removed and analyzed for nitrite release. The results are expressed as the mean ± S.E. of one typical experiment performed in triplicate. *, p < 0.05 as compared with LPS alone. Similar results were obtained in three independent experiments. In iNOS expression studies, cells from the nitrite release assay were subjected to electrophoresis and Western blotting with iNOS-specific antibody as described under "Experimental Procedures."

When cells were treated for 9 days with PKCeta or delta  antisense oligonucleotides, LPS-induced nitrite production was inhibited 39% by the PKCeta antisense oligonucleotides, whereas the PKCdelta antisense oligonucleotides, although they inhibited the expression of PKCdelta , had no effect (Fig. 5B). The control sequences for PKCeta , which did not affect the expression of PKCeta , also had no effect on the LPS response. Similar results were seen for LPS-induced iNOS expression (Fig. 5B).

Induction of NF-kB in the Nuclei of LPS-stimulated Astrocytes and the Inhibitory Effect of PKCeta Antisense Oligonucleotides-- In resting cells, the NF-kB heterodimer is held in the cytosol by binding to IkB (22); after stimulation of the cells with various agents, the cytosolic NF-kB/IkB complex dissociates and free NF-kB translocates to the nuclei. pyrolidine dithiocarbamate, an antioxidant that acts as a specific inhibitor of NF-kB activation (23), blocks the ability of astrocytes to produce nitrite production and the nuclear binding activity for NF-kB normally seen in response to LPS.2 Thus, activation of NF-kB is critical in the induction of iNOS by LPS in astrocytes. We performed an EMSA using oligonucleotides containing NF-kB recognition site-like sequences present in the iNOS gene (13) and nuclear extracts prepared from LPS-stimulated cells. In nuclear extracts of unstimulated astrocytes, one faint NF-kB-specific DNA-protein complex was identified, the intensity of which markedly increased following exposure of the cells to 1 µg/ml LPS for 10 min and was even greater after 1 h of treatment (Fig. 6A). For the EMSA, cells were treated with LPS for 1 h.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 6.   Kinetics of NF-kB-specific DNA-protein complex formation in nuclear extracts of astrocytes stimulated with LPS (A) and the effect of various inhibitors or long term TPA pretreatment (B and C). Cells were treated with 1 µg/ml of LPS for 10 min or 1 h (A) or pretreated with 30 µM genestein, 50 µM D609, 50 µM propranolol, or 500 nM Ro 31-8220 for 30 min or with 1 µM TPA for 24 h (B and C) before incubation with 1 µg/ml of LPS for 1 h, and then nuclear extracts were prepared. NF-kB DNA-protein binding activity was determined by EMSA as described under "Experimental Procedures."

After pretreatment of cells for 30 min with 30 µM genestein, 50 µM D609, 50 µM propranolol, or 0.5 µM Ro 31-8220, the LPS-induced activation of NF-kB-specific DNA-protein complex formation was inhibited (Fig. 6, B and C). However, overnight pretreatment with TPA, which, in astrocytes, down-regulates conventional and new PKC isoforms, but not PKCeta (17, 18, 21), did not affect the LPS-induced NF-kB activation (Fig. 6C). Following exposure of cells to 1 µM TPA for 1 h, activation of NF-kB-specific DNA-protein complex formation was also seen (Fig. 7A). When cells were treated with 10 µM of PKCeta antisense oligonucleotide for 9 days, the LPS-induced NF-kB activation was inhibited, whereas PKCeta control sequences had no effect (Fig. 7B).


View larger version (60K):
[in this window]
[in a new window]
 
Fig. 7.   NF-kB-specific DNA-protein complex formation in nuclear extracts of astrocytes stimulated with TPA (A) and effect of PKCeta antisense or control oligonucleotides on LPS-induced NF-kB-specific DNA-protein complex formation (B). Cells were treated with 1 µg/ml of LPS or 1 µM TPA for 1 h (A) or pretreated with 10 µM PKCeta antisense or control oligonucleotides for 9 days before incubation with 1 µg/ml of LPS for 1 h (B), and then nuclear extracts were prepared. NF-kB-specific DNA-protein binding activity in nuclear extracts was determined by EMSA as described under "Experimental Procedures."

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In both cultured astrocytes and C6 glioma cells, LPS induces NO production (6, 24). In macrophages, activation by LPS requires the presence of a 53-kDa glycoprotein, mCD14 (25), which is attached to the cell surface by a glycosylphosphatidylinositol moiety (26). A second form of CD14, soluble CD14, is found at high concentrations in the serum (27), and can confer LPS responsiveness upon cells that lack mCD14, including astrocytoma cells (28). Complexes formed between LPS and either mCD14 or soluble CD14 are thought to lead to transfer of bound LPS to a distinct signaling molecule, which may be either transmembrane or intracellular (29). The formation of LPS/CD14 complexes is accelerated by LPS-binding protein (30), which is also present at high concentrations in normal serum and thus can contribute to the serum effects (31). Recent studies have demonstrated the presence of CD14 mRNA and protein in rat astrocytes and that LPS-produced iNOS induction requires membrane and soluble forms of CD14 (32). Thus, in astrocytes, formation of a LPS/LPS-binding protein complex might allow binding to, and activation of, CD14, and then trigger signal transduction to initiate the expression of iNOS and NO release.

Although PKC has been shown to be involved in LPS-induced iNOS expression and NO production in macrophages (33, 34), it has been reported to be unnecessary for iNOS induction by LPS in astrocytes (11). However, in the present study, four PKC inhibitors, calphostin C, Go 6976, Ro 31-8220, and staurosporine, inhibited LPS-stimulated NO production and iNOS expression, indicating that PKC activation is an obligatory event in the LPS-mediated regulation of NO release and iNOS expression in astrocytes. PKC is activated by the physiological activator diacylglycerol, which can be generated either directly by the action of PLC or indirectly by a pathway involving the production of phosphatidic acid by PLD, followed by a dephosphorylation reaction catalyzed by phosphatidate phosphohydrolase. Normally, the PLC involved in the production of diacylglycerol is PI-PLC, but PC-PLC may also be involved (36, 37). The PC-PLC inhibitor, D609, and the phosphatidate phosphohydrolase inhibitor, propranolol, both inhibited LPS-induced iNOS expression and NO production, whereas the PI-PLC inhibitor, U73122, had no effect. Thus, LPS may act through the PC-PLC and PC-PLD pathways, but not the PI-PLC pathway, to induce PKC activation in astrocytes; this contrasts with the situation in RAW 264.7 macrophages in which the PI-PLC and PC-PLC pathways, but not the PC-PLD pathway, are involved (38). The mechanism involved in the activation of PC-PLC and PC-PLD is still unknown but may involve tyrosine phosphorylation (37, 39). Genestein also inhibited LPS-induced iNOS induction and NO production in astrocytes. The tyrosine kinase involved might be p53/p56lyn, because, in monocytes, LPS activates this kinase, which is associated with CD14 (40, 41). Thus, in astrocytes, the LPS/LPS-binding protein complex binds to CD14 and then might activate PC-PLC and PC-PLD via an upstream protein tyrosine phosphorylation to elicit PKC activation and, finally, iNOS expression and NO production.

Although PKC inhibitors attenuated LPS-induced iNOS expression and NO production, long term TPA pretreatment, which down-regulates PKCalpha , delta , and theta , but not PKCeta , in astrocytes (17, 18), had no effect, indicating the possible involvement of PKCeta in LPS-mediated effects. To confirm the involvement of PKCeta , PKCeta antisense oligonucleotides and the scrambled controls or antisense oligonucleotides for PKCdelta , which is abundantly expressed in astrocytes and down-regulated by TPA, were used. The specificity of the PKCeta and PKCdelta antisense oligonucleotides was demonstrated (Fig. 5A), and the results showed inhibition of LPS-stimulated iNOS expression and NO production by PKCeta antisense oligonucleotides but not by PKCdelta antisense or control oligonucleotides. Thus, a crucial role for PKCeta in the LPS-induced stimulation of NO production and iNOS expression has been demonstrated. The PKC family, which consists of phospholipid-dependent serine/threonine kinases, is believed to play a major role in cellular functions. Molecular cloning has shown that it consists of at least 12 isoforms with different tissue expressions (36), which have been shown to be related to specialized cell functions (36). Primary cerebellar astrocytes express the new types of PKCdelta , theta , and eta , which are not expressed in neuronal granule cells (17, 18).3 PKCdelta and theta , but not PKCeta , have been shown to be involved in the regulation of receptor-mediated PI hydrolysis (17, 18). However, in this study, PKCeta , but not other isoforms, was shown to be involved in the LPS-induced iNOS expression and NO production. This is further evidence that different members of the PKC family within a single cell elicit specific physiological responses. However, PKCeta , which is abundantly expressed in RAW 264.7 macrophages (21), was not involved in the LPS-induced iNOS expression and NO production (38).

The transcriptional factor, NF-kB, is critical in the induction of iNOS by LPS in macrophages (13, 14). In astrocytes, the NF-kB blocker, pyrolidine dithiocarbamate, inhibits LPS-induced NO production and iNOS expression,2 indicating that NF-kB is also critical in the induction of iNOS by LPS in these cells. LPS increased the levels of the NF-kB-specific DNA-protein complex in nuclear extracts (Fig. 6A); this activation was inhibited by genestein, D609, propranolol, or Ro 31-8220 but not by long term TPA treatment. Furthermore, PKCeta antisense oligonucleotides, but not the scrambled controls, attenuated this activation, indicating the involvement of PKCeta in the LPS-stimulated up-regulation of iNOS in astrocytes. Direct activation of PKC by TPA does induce NF-kB activation, NO production, and iNOS expression in astrocytes (Figs. 4 and 7A). Similar findings have been reported in peritoneal macrophages, hepatocytes, and human umbilical vein endothelial cells (42-44) but not in RAW 264.7 macrophages, in which TPA alone did not induce NF-kB activation and NO production (38, 45). Although the role of PKCeta has been clearly demonstrated, other LPS-activated components are also involved in the co-stimulation of NF-kB and iNOS expression, because direct activation of PKC by TPA induced less NF-kB activation and NO production than LPS stimulation (Figs. 1B, 4, and 7A).

Glial cells, including astrocytes, microglia, and oligodendrocytes, are involved in lesion and plaque formation in multiple sclerosis and experimental allergic encephalomyelitis, a model for multiple sclerosis. Multiple sclerosis is a central nervous system disorder with immune-mediated destruction of myelin and the myelin-producing cells, oligodendrocytes. The presence of iNOS in tissues of patients with multiple sclerosis and in animals with experimental allergic encephalomyelitis suggests that NO may play a role in the central nervous system autoimmune diseases (35, 46, 47). Rodent astrocytes and microglia express a high level of iNOS and release significant amounts of NO within hours of LPS stimulation (Refs. 6, 7, and 10 and the present study). Astrocytes are the major cell population in the central nervous system; therefore, induction of iNOS in astrocytes may be an important source of NO in central nervous system inflammatory disorders associated with neuronal and oligodendrocytes death (9).

In summary, the signaling pathway involved in the LPS-induced activation of PKC in primary astrocytes was explored, and the PKC isoform eta , but not other isoforms, was found to be involved in the regulation of LPS-induced NF-kB activation, iNOS expression, and NO release. This is the first study showing the involvement of these two mechanisms in LPS-stimulated NO release in such cells.

    FOOTNOTES

* This work was supported by a research grant from the National Science Council of Taiwan.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. Tel: 886-2-23970800, ext. 8321; Fax: 886-2-23947833; E-mail: ccchen{at}ha.mc.ntu.edu.tw.

1 The abbreviations used are: NO, nitric oxide, iNOS, inducible nitric- oxide synthase; LPS, lipopolysaccharide; PC-PLC, phosphatidylcholine-phospholipase C; PC-PLD, phosphatidylcholine-phospholipase D; PI-PLC, phosphoinositide-phospholipase C; NF-kB, nuclear factor kB; EMSA, electrophoretic mobility shift assay; PKC, protein kinase C; TPA, 12-O-tetradecanoyl phorbol-13-acetate.

2 C.-C. Chen, J.-K. Wang, D.-Y. Lin, and W.-C. Chen, unpublished data.

3 W.-C. Chen, and C.-C. Chen, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1991) Pharmacol. Rev. 43, 109-142[Medline] [Order article via Infotrieve]
  2. Jaffrey, S. E., and Snyder, S. H. (1995) Annu. Rev. Cell Dev. Biol. 11, 417-440[CrossRef][Medline] [Order article via Infotrieve]
  3. Nunokawa, Y., Ishida, N., and Tanaka, S. (1993) Biochem. Biophys. Res. Commun. 191, 89-94[CrossRef][Medline] [Order article via Infotrieve]
  4. Lyons, C. R., Orloff, G. J., and Cumningham, J. M. (1992) J. Biol. Chem. 267, 6370-6374[Abstract/Free Full Text]
  5. Geller, D. A., Lowenstein, C. J., Shapiro, R. A., Nussler, A. K., Di Silvio, M., Wang, S. C., Nakayama, D. K., Simmons, R. L., Snyder, S. H., and Billiar, T. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3491-3495[Abstract]
  6. Galea, E., Feinstein, D. L., and Reis, D. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10945-10949[Abstract]
  7. Merrill, J. E., Ignarro, L. J., Sherman, M. P., Melinek, J., and Lance, T. E. (1993) J. Immunol. 151, 2132-2142[Abstract/Free Full Text]
  8. Iadecola, C. (1997) Trends Neurosci. 20, 132-139[CrossRef][Medline] [Order article via Infotrieve]
  9. Mitrovic, B., Ignarro, L. J., Montestruque, S., Smoll, A., and Merril, J. E. (1994) Neuroscience 61, 575-585[CrossRef][Medline] [Order article via Infotrieve]
  10. Murphy, S., Simmons, M. L., Agullo, L., Garcia, A., Feinstein, D. L., Galea, E., Reis, D. J., Minc-Golomb, D., and Schwartz, J. P. (1993) Trends. Neurosci. 16, 323-328[CrossRef][Medline] [Order article via Infotrieve]
  11. Simmons, M., and Murphy, S. (1994) Glia 11, 227-234[Medline] [Order article via Infotrieve]
  12. Kong, L. Y., McMillian, M. K., Maronpot, R., and Hong, J. S. (1996) Brain Res. 729, 102-109[CrossRef][Medline] [Order article via Infotrieve]
  13. Lowenstein, C. J., Alley, E. W., Raval, P., Snowman, A. M., Synder, S. H., Russell, S. W., and Murphy, W. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9730-9734[Abstract]
  14. Xie, Q. W., Wishnan, R., and Nathan, C. (1993) J. Exp. Med. 177, 1779-1784[Abstract]
  15. Xie, Q. W., Kashiwabara, Y., and Nathan, C. (1994) J. Biol. Chem. 269, 4705-4708[Abstract/Free Full Text]
  16. Feinstein, D. L., Galea, E., Aquino, D. A., Li, G. C., Xu, H., and Reis, D. J. (1996) J. Biol. Chem. 271, 17724-17732[Abstract/Free Full Text]
  17. Chen, C. C., Chang, J., and Chen, W. C. (1995) Mol. Pharmacol. 48, 39-47[Abstract]
  18. Chen, C. C., and Chen, W. C. (1996) Glia 17, 63-71[CrossRef][Medline] [Order article via Infotrieve]
  19. Lin, S. B., Huang, S. S., Choo, K. B., Chen, P. J., and Au, L. C. (1995) J. Biochem. 117, 1100-1104[Abstract]
  20. Lin, S. B., Chang, G. W., Teh, G. W., Lin, K. I., and Au, L. C. (1993) BioTechniques 14, 795-798[Medline] [Order article via Infotrieve]
  21. Chen, C. C., Wang, J. K., and Chen, W. C. (1997) FEBS Lett. 412, 30-34[CrossRef][Medline] [Order article via Infotrieve]
  22. Baeuerle, P. A., and Henkel, T. (1994) Annu. Rev. Immunol. 12, 141-149[CrossRef][Medline] [Order article via Infotrieve]
  23. Schreck, R., Meiser, B., Mannel, D. N., Droge, W., and Baeverle, P. A. (1992) J. Exp. Med. 175, 1181-1194[Abstract]
  24. Feinstein, D. L., Galea, E., Roberts, S., Berquist, H., Wang, H., and Reis, D. J. (1994) J. Neurochem. 62, 315-321[Medline] [Order article via Infotrieve]
  25. Wright, S. D., Ramos, R. A., Tobias, P. S., Ulevitch, R. J., and Mathison, J. C. (1990) Science 249, 1431-1433[Medline] [Order article via Infotrieve]
  26. Haziot, A., Chen, S., Ferrero, F., Low, M. G., Silber, R., and Goyert, S. M. (1988) J. Immunol. 141, 547-552[Abstract/Free Full Text]
  27. Bazil, V., Horejst, V., Baudys, M., Kristofova, H., Strominger, J. L., Kostka, W., and Hilgert, I. (1986) J. Immunol. 16, 1583-1589
  28. Frey, E. A., Miller, D. S., Jahr, T. G., Sundan, A., Bazil, V., Espevik, T., Fintay, B. B., and Wright, S. D. (1992) J. Exp. Med. 176, 1665-1671[Abstract]
  29. Ulevitch, R. J., and Tobias, P. S. (1995) Annu. Rev. Immunol. 13, 437-457[CrossRef][Medline] [Order article via Infotrieve]
  30. Hailman, E., Lichtenstein, H. S., Wurfel, M. M., Miler, D. S., Johnson, D. A., Kelley, M., Busse, L. A., Zukowski, M. H., and Wright, S. D. (1994) J. Exp. Med. 179, 269-277[Abstract]
  31. Pugin, J., Schurer-Maly, C. C., Leturcq, D., Moriarty, A., Ulevitch, R. J., and Tobias, P. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2744-2748[Abstract]
  32. Galea, E., Reis, D. J., Fox, E. S., Xu, H., and Feinstein, D. L. (1996) J. Neuroimmunol. 64, 19-28[CrossRef][Medline] [Order article via Infotrieve]
  33. Severn, A., Wakelam, M. J. O., and Liew, F. R. (1992) Biochem. Biophys. Res. Commun. 199, 461-466[CrossRef]
  34. Paul, A., Pendreigh, R. H., and Plevin, R. (1995) Br. J. Pharmacol. 114, 482-488[Abstract]
  35. Bagasra, O., Michaels, F. H., Zheng, Y. M., Boroski, L. E., Spitsin, S. V., Fu, Z. F., Tawadros, R., and Koprowski, H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12041-12045[Abstract]
  36. Nishizuka, Y. (1995) FASEB J. 9, 484-496[Abstract/Free Full Text]
  37. Exton, J. H. (1994) Biochim. Biophys. Acta 1212, 26-42[Medline] [Order article via Infotrieve]
  38. Chen, C. C., Wang, J. K., and Lin, S. B. (1998) J. Immunol., in press
  39. Dubyak, G. R., Schomisch, S. J., Kusner, D. J., and Xie, M. (1993) Biochem. J. 292, 121-128[Medline] [Order article via Infotrieve]
  40. Stefanova, I., Corcoran, M. L., Horak, E. M., Wahl, L. M., Bolen, J. B., and Horak, I. D. (1993) J. Biol. Chem. 268, 20725-20728[Abstract/Free Full Text]
  41. Beaty, C. D., Franklin, T. L., Uehara, Y., and Wilson, C. B. (1994) Eur. J. Immunol. 24, 1278-1284[Medline] [Order article via Infotrieve]
  42. Hortelano, S., Genaro, A. M., and Bosca, L. (1993) FEBS Lett. 320, 135-139[CrossRef][Medline] [Order article via Infotrieve]
  43. Diaz-Guerra, M. J. M., Velasco, M., Martin-Sanz, P., and Bosca, L. (1996) J. Biol. Chem. 271, 30114-30120[Abstract/Free Full Text]
  44. Johnson, D. R., Douglas, I., Jahnke, A., Ghosh, S., and Pober, J. S. (1996) J. Biol. Chem. 271, 16317-16322[Abstract/Free Full Text]
  45. Diaz-Guerra, M. J. M., Bodelon, O. G., Velasco, M., Whelan, R., Parker, P. J., and Bosca, L. (1996) J. Biol. Chem. 271, 32028-32033[Abstract/Free Full Text]
  46. Lin, R. F., Lin, T. S., Tilton, R. G., and Gross, A. H. (1993) J. Exp. Med. 178, 643-648[Abstract]
  47. Gross, A. H., Misko, T. P., Lin, R. F., Hicky, W. F., Trotter, J. L., and Tilton, R. G. (1994) J. Clin. Invest. 93, 2684-2690[Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.