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* State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China; and
Vascular Biology Research Center, Institute of Molecular Medicine and Division of Hematology, University of Texas-Houston Health Science Center, Houston, Texas, USA
1Correspondence: Y.Z., State Key Laboratory of Virology College of Life Sciences Wuhan University Wuhan 430072, China; E-mail: yingzhu{at}whu.edu.cn or wu9988{at}vip.sina.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In this study, we identified another viral protein, the spike of SARS-CoV, which played an important role in virus-stimulated COX-2 expression after screening all genes from the SARS-CoV genome. We found that an upstream calcium-dependent PKC isozyme PKC
that modulates the downstream ERK/NF-
B pathway through an influx of extracellular Ca2+ is induced by the spike protein of SARS-CoV. The ERK/NF-B was identified to be involved in the activation of COX-2 promoter and production of COX-2 protein in HEK293T cells. We also demonstrated that another unusual pathway, the calcium-independent PI3K/PKC
/JNK/CREB pathway, functioned in cooperation with the calcium-dependent pathway to induce COX-2 expression upon stimulation by spike protein. This pathway can be blocked by PKC-specific, small interfering RNA, PI3K/JNK kinase-specific inhibitors as well as dominant negative JNK. PKC-specific siRNA also attenuated the phosphorylation of JNK. Our results provide evidence that helps us understand the function of SRAS-CoV spike protein in SARS pathogenesis.—Liu, M., Yang, Y., Gu, C., Yue, Y., Wu, K. K., Wu, J., Zhu, Y. Spike protein of SARS-CoV stimulates cyclooxygenase-2 expression via both calcium-dependent and calcium-independent protein kinase C pathways.
Key Words: COX • SARS-CoV • spike protein • transcriptional activation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. SARS-CoV is a member of the coronaviridae family of enveloped, positive-stranded RNA viruses containing genes encoding polymerase (polymerase-related proteins), spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins (3)
. Phylogenetic analysis indicated that the new virus is not related to known groups 1, 2, and 3 of coronaviruses, but represents a fourth group, group 4 in the genus coronavirus (4)
. Based on available information about other coronaviruses, the spike glycoprotein is the main virulence factor of SARS-CoV, with multiple functions involved in specific receptor binding, cell membrane fusion, and protease susceptibility (5)
. SARS clinically consists of an incubation phase and respiratory phase. In these two phases, acute inflammatory reaction is the most obvious symptom (6)
, characterized by the invasion of activated leukocytes into the injured tissue; its process is critically dependent on the rapid expression of proinflammatory genes (7)
.
Cyclooxygenase (COX) is a rate-limiting enzyme in the biosynthetic pathway of prostaglandins and thromboxanes from arachidonic acid. Prostaglandins play important roles in many biological processes. Altered prostanoid production is associated with a variety of illnesses, including acute and chronic inflammation, cardiovascular disease, and colon cancer (8)
. Two isoforms of COX were described: COX-1 and COX-2. COX-1 is constitutively expressed in almost all tissues (9)
; COX-2 is the inducible form of the enzyme, which is expressed in response to inflammatory and other physiological stimuli and growth factors and is involved in the production of those prostaglandins that mediate pain and support the inflammatory process (10)
. As an immediate early response gene, the promoter region of COX-2 contains a classical TATA box, an E-box, and a series of binding sites for transcription factors such as nuclear factor B (NF-B), NFAT/AP-1, nuclear factor IL-6/CCAAT enhancer binding protein (C/EBP), and cyclic AMP response element binding proteins (CREB), which regulate COX-2 gene expression in transcriptional and post-transcriptional levels (11)
. It has been shown that several types of viral proteins such as LMP-1 of Epstein-Barr virus (EBV), gp120 of HIV, and HBx of human hepatitis virus (HBV) are capable of stimulating COX-2 through a wide range of cellular signal transduction cascades, including Ras-Raf-mitogen-activated protein kinase, c-Jun N-terminal kinase, NF-B, and JAK1-STAT signaling pathways (12
13)
.
In our previous study, we identified two viral proteins—the nucleoprotein and spike from SARS-CoV—whose expression was involved in up-regulating COX-2 after screening all genes from SARS-CoV genome. In this study, we focused on the spike and delineated the molecular mechanism by which the spike activated COX-2 promoter and protein production. We postulated that spike protein (S protein) of SARS-CoV might be subjected to signaling effects in human cells in addition to cell infection and viral replication. There is a precedent for such "innocent bystander" effects, whereby viral envelope proteins can induce cell dysfunction and death by triggering specific signaling pathways (14)
. Because COX-2 is normally induced during inflammatory processes and SARS-CoV induces fever, edema, and diffuse alveolar damage in severely affected individuals, we investigated the roles of SARS-CoV S protein in regulating the expression of COX-2 and the signaling pathways through which it induces COX-2 transcriptional activation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
Human embryo kidney cell line (HEK293T), African green monkey kidney cell line (COS-7), and human airway epithelial cell line (A549) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS, 100 µg/ml penicillin, and 100 µg/ml streptomycin at 37°C in a 5% CO2 incubator.
Plasmid construct
The luciferase reporter vector (pGL3) containing a COX-2 promoter region (–891/+9), its truncation mutants, or site-specific mutants was reported earlier (15)
. A spike construct was created by RT-polymerase chain reaction (RT-PCR) amplification of the spike open reading frame from SARS-CoV (WHU), a viral strain isolated from a SARS patient in China in 2003 (16)
. To create S-encoding vectors, the spike gene was amplified using the following primers: 5'-AGCTGGATCCACATGTTTATTTTCTTATTATTTCTTAC-3'(sense) 5'-AGCTGAATTCACTGGCTGTGCAGTAATTGATCC-3'(antisense), in which EcoRI and BamHI sites were introduced, respectively. The polymerase chain reaction (PCR) product was cloned into EcoRI and BamHI sites of pCMV-tag2B to generate plasmid pCMV-S, in which S was tagged by FLAG. The resulting construct was confirmed by DNA sequencing. Mutants of ERK1 and ERK2 were gifts from Dr. Cobb (University of Texas Southwestern Medical Center) and mutants of JNK were gifts from Dr. Karin (University of California at San Diego, San Diego, CA, USA). PKC RNAi vectors were constructed by ligating the corresponding pairs of oligonucleotide to pSilencer 2.0 (Ambion, Inc., Augstin, TX, USA) based on the sequences described by Storz et al. (17)
.
Luciferase assay
HEK293T cells were cotransfected with reporter plasmids and each protein expression plasmid. Cells were lysed with luciferase cell culture lysis reagent (Promega, Madison, WI, USA). Thirty microliters of the cell lysates and 100 µl of luciferase assay substrate (Promega) were mixed and light intensity was detected by the luminometer (Turner T20/20). Assays were performed in triplicate and expressed as means ± SD relative to vector control as 100%.
Western blot analysis
Whole-cell lysates were prepared by lysing HEK293T with PBS pH7.4 containing 0.01% Triton-100, 0.01% EDTA, and 10% cocktail protease inhibitor (Roche). The cytosolic and nuclear protein fractions were separated as follows. Cells were washed with ice-cold PBS, collected by centrifugation, and pellets were resuspended in hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.5 mM DTT, 10% protease cocktail inhibitor) for 15 min on ice and vortexed for 10 s. Nuclei were pelleted by centrifugation at 13,000 rpm for 1 min. Supernatants containing cytosolic proteins were collected. Lysates were centrifuged at 13,000 rpm for 10 min. The supernatants were boiled for 5 min with equal volumes of 2 x gel loading buffer. One hundred micrograms of cultured cell lysates were electrophoresed in 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. Nonspecific IgGs were blocked with 5% nonfat dried milk before being incubated with an antibody used in this study. Protein bands were detected using SuperSignal Chemiluminescent (Pierce, Rockford, IL, USA).
Electrophoretic mobility shift assay (EMSA)
Cell nuclear extracts were prepared from HEK293T cells according to the method described previously (15)
. NF-B oligonucleotides and CRE oligonucleotides were synthesized by Takara (Takara Biotech Co. Ltd., Shiga, Japan) based on the cognate human COX-2 promoter sequence: wild-type NF-B: 5'-CAGTCATTTCGTCACATG-3', mutant type NF-B: 5'-CAGTCATTTGAGCTCATG-3', wild-type CRE: 5'-GAGGTGAGGGGATTCCCTTAG-3', mutant type CRE: 5'-GAGGTGAATTCATTCCCTTAG-3'.
The complementary oligonucleotides were annealed and purified according to the manufacturer’s protocol. Each probe was end-labeled with [
32P] ATP using T4 polynucleotide kinase (Takara Biotech). EMSA was performed by incubating 2 µg of nuclear extract with a labeled probe (15,000 cpm; 
10 fmol) in binding buffer (4 mM Tris-HCl, 12 mM HEPES-KOH, pH7.9, 60 mM KCl, 12% glycerol, 0.5 mM EDTA, and 1 mM DTT) containing 1 µg of poly(dI-dC) for 25 min at room temperature. To assure the specific binding of transcription factors to the probe, the probe was chased by 100-fold molar excess of cold wild-type or mutant oligonucleotide. Samples were electrophoresed on 5% nondenaturing polyacrylamide gel with running buffer (x0.25 TBE containing 22.5 mM Tris borate, 0.5 mM EDTA), and the gels were dried and subjected to autoradiography.
Transient transfection
Cells were seeded onto 24-well plates at a density of 1.0 x 105 or 4.0 x 105 cells per 24-well plate or 6-well plate and grown to the confluence reaching 80% at the time of transfection. Cells were transfected with 0.1 µg or 0.4 µg of plasmid pCMV-S together with 0.45 µg or 1.2 µg plasmid containing luciferase reporter, which was under the control of COX-2 promoter (COX-2-Luc) using SofastTM transfection reagent (Xiamen Sunma Biotechnology Co. Ltd., China) according to the protocol provided by the manufacturer. In all cotransfection experiments, appropriate vector DNA was used to ensure similar DNA concentrations in all conditions. After 24 h, cells were serum-starved for 24 h before being harvested.
Semiquantitative RT-PCR analysis
The semiquantitative RT-PCR analysis was performed to determine COX-2 mRNA levels. Total RNA was isolated using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and cellular RNA samples were reverse-transcribed with a random primer, then amplified by PCR; ß-actin primer set was used as an internal control. The primers and reaction conditions were shown as ß-actin, 5'-ATGATATCGCCGCGCTCG-3' and 5'-CGCTCGGTGAGGATCTTCA-3', COX-2, 5'-TACAATGCTGACTATGGCTAC-3' and 5'-ACTGATGCGTGAAGTGCTG-3'. PCR products were analyzed by electrophoresis on 1% agarose gel containing ethidium bromide.
Measurement of [Ca2+]i
Intracellular calcium was measured using Fura-2. Cells (2x104/dish) were transfected with pCMV-flag2B or pCMV-S plasmids. After 24 h, Cells were loaded with Fura-2 by incubation in a solution of 5 µM Fura-2 AM and Pluronic-F127 (0.05%) in D-Hanks buffer (140 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 1 mg/ml glucose, 0.44 mM KH2PO4, 0.37 mM Na2HPO4, 20 mM HEPES, pH7.4) at 37°C for 45 min. Fura-2 fluorescence was excited alternately (2 Hz) at 340 nm and 380 nm using a Cairn monochromator (Faversham, Kent, UK) and measured at 510 ± 20 nm using a photomultiplier tube. [Ca2+]i was calculated from the ratio of the fluorescence intensity (r=F340/380) using the formula described by Grynkiewicz et al. (18)
: [Ca2+]i = Kd [(R-Rmin)/(Rmax-R)](Fmin/Fmax), where with 0.1% aqueous Triton X-100 and 3 mM EGTA, respectively, Fmax and Fmin are values of F380 after treatment with Triton X-100 and EGTA, respectively, and Kd = 244 nM.
Statistical analysis
All experiments were reproducible and were carried out in duplicate or quadruplicate. Each set of experiments was repeated at least three times with similar results, and a representative one is shown. The results are presented at the means ±SD. Student’s t test for paired samples was used to determine statistical significance. Differences were considered statistically significant at a value of P 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). Results indicated that COX-2 expression levels were significantly increased by SARS-CoV infection (lane 2) or by PMA, a known COX-2 inducer, as positive control (lane 3). Mammalian cells were cotransfected with plasmid pCMV-S and a reporter plasmid carrying the luciferase reporter gene under the control of the COX-2 gene promoter. Results revealed that S protein stimulated the activity of COX-2 promoter in HEK293T, COS-7, and A549 cell lines (Fig. 1B
). Activation of the COX-2 promoter was in an S protein concentration-dependent manner, because the level of luciferase activity increased as the concentration of plasmid carrying the S gene increased in all three cell types (Fig. 1B
).
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COX-2 mRNA was analyzed using semiquantitative RT-PCR. Results showed that the level of COX-2 mRNA increased in the presence of S protein or 100 nM PMA (Fig. 1C
).
The expression of COX-2 protein regulated by S protein was determined by Western blot analysis using antibodies against COX-2 and Flag-tagged S protein. Results from Western blot analysis revealed a basal level of COX-2 protein detected in HEK293T cells (Fig. 1D
, lane 2); the level of COX-2 increased significantly in the presence of SARS-CoV S protein (Fig. 1D
, lane 1) or in the presence of PMA (Fig. 1D
, lane 3). These results demonstrated that the S protein of SARS-CoV activated the expression of COX-2 protein in mammalian cells.
NF-B and CREB recognition sites were involved in the induction of COX-2 by the S protein
Activation of COX-2 gene relies on many consensus cis-elements, including NF-B and CREB binding sites in the COX-2 promoter (Fig. 2
A). As S protein activates the expression of COX-2, we investigated the roles of these cis-regulatory elements in regulating COX-2 by S protein. Two binding sites for NF-B and CRE were identified in the promoter of COX-2 (19)
.
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To define the role of NF-B and CRE recognition sites in activating COX-2 by S protein, two NF-B binding site mutants (Mut1 and Mut2) and one CRE binding site mutant (Mut3) were generated by site-specific mutagenesis (Fig. 2A
). Reporter plasmids were then constructed in which the luciferase gene was under the control of COX-2 WT, Mut1, Mut2, and Mut3 promoters, respectively. 293T cells were cotransfected with pCMV-S and plasmids containing the luciferase reporter gene under the control of different COX-2 promoters. Results from luciferase assays showed that Mut1 and Mut3 reduced COX-2 promoter activity to the basal level whereas Mut2 partially reduced COX-2 activity. (Fig. 2B
). These results indicated that both NF-B sites, especially the proximal site, and the CRE recognition site were required for the activation of COX-2 expression by S protein.
S protein-induced COX-2 expression required NF-B and CREB activation
NF-B is a ubiquitously expressed transcription factor that regulates the induction of genes involved in immune and inflammatory cell function. NF-B is composed of homo- and heterodimers of Rel family members, typically p65:p50 heterodimers, which are held in the cytosol by the inhibitory IB proteins. NF-B activation results from the phosphorylation of IB, which targets it for ubiquitylation and degradation by the 26S proteasome. After the destruction of IB, NF-B can then translocate to the nucleus to activate target genes. We examined the effect of S protein in the translocation of p50, a subunit of NF-B, from cytosol to nucleus. Results from Western bolt analysis using antip50 antibody revealed that the level of p50 protein was decreased in cytosol and increased in nucleus at 48 h after S protein stimulation (Fig. 3
).
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ERK, JNK, PI3K, and PKC but not P38 MAP kinase were involved in S protein-induced COX-2 activation
It is well known that members of the MAP kinase family regulate several transcription factors that are important in the proinflammatory response (20)
. Regulation of COX-2 expression was shown to depend on different kinase activators in various cell types (21
22
23
24)
. Based on our findings that S protein activates COX-2 expression and that such activation was dependent on NF-B and CREB, we investigated the roles of kinases on the activation of COX-2 by S protein and the molecular mechanisms underlying this event.
Cells transfected with pCMV-S and the reporter plasmid COX-2-Luc were treated with PD98059 (ERK-specific inhibitor), SB203580 (P38 MAP kinase-specific inhibitor), SP600125 (JNK-specific inhibitor), LY294002 (PI3K-specific inhibitor), and GF109203 (PKC-specific inhibitor), respectively. Results from luciferase activity assays showed that the level of COX-2 promoter activity was significantly decreased in the presence of PD98059, SP600125, LY294002, GF109203, but was not affected by treatment of SB203580 (Fig. 4
A). The kinase inhibitors did not affect the expression of S protein significantly in the inhibitor-treated cells. Western blot analysis revealed a corresponding reduction of COX-2 protein (Fig. 4B
).
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We further determined the role of ERK and JNK in the activation of COX-2 induced by S protein by introducing three dominant kinase-inactive mutants—mERK1, mERK2, and mJNK—whose expression can block kinase activities by competing with endogenous kinases (25
26)
. Cells were cotransfected with pCMV-S, COX-2-Luc reporter, and each of three kinase mutants, respectively. Results from luciferase activity assays showed that all three mutants reduced COX-2 promoter activity (Fig. 4C
). We also used Western blot to detect expression of mERK1, mERK2, and mJNK; results showed that protein expression levels increased slightly in all groups, with the dose increase of plasmids DNA transfected. Protein expression levels did not show a remarkable dose-dependent increase compared with that of DNA-transfected plasmids. The results may provide an explanation for the incomplete inhibition by three kinase mutants. Taken together, these data suggested that ERK, JNK, PI3K, and PKC, but not P38 MAP kinase, were involved in S protein-induced COX-2 activation.
S protein stimulated phosphorylation of ERK1/2 and JNK resulted in the binding of NF-kappaB and CREB to COX-2 promoter
To further investigate the role of S protein in the activation of signal transduction pathways, we determined the phosphorylation status of ERK1/2 and JNK in human cells by Western blot analysis using antibodies specifically recognizing phosphorylated ERK1/2 and JNK. Results showed that the levels of phosphorylated ERK1/2 (P44/42) and phosphorylated JNK increased in a time-dependent fashion in the presence of S protein (Fig. 5
A). Phosphorylation of ERK1/2 (P44/42) was blocked by GF109203 but not LY294002 (Fig. 5B
). By contrast, the level of phosphorylation of JNK was significantly reduced by both GF109203 and LY294002 (Fig. 5B
). These results suggested that S protein-induced ERK phosphorylation depends on PKC but not PI3K whereas JNK phosphorylation depends on both.
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To investigate the roles of ERK1/2 and JNK in S protein-increased NF-B and CREB binding to COX-2 promoter, we evaluated the effect of kinase inhibitor on NF-B and CREB binding by EMSA. Results showed that S-induced binding of NF-B to COX-2 promoter (Fig. 5C
, lane 3), which was inhibited by a competitive probe (Fig. 5C
, lane 4), GF109203 (Fig. 5C
, lane 5), and PD98059 (Fig. 5C
, lane 6) were not affected by the JNK inhibitor SP600125 (Fig. 5C
, lane 7). These data suggested S protein-induced NF-B binding is mediated via the PKC/ERK pathway.
Results from EMSA showed that S induced binding of CREB to the COX-2 promoter (Fig. 5D
, lane 3), which was inhibited by the presence of competitive probe (Fig. 5D
, lane 8), SP600125 (Fig. 5D
, lane 4), GF109203 (Fig. 5C
, lane 5), but not PD98059 (Fig. 5D
, lane 6). These data suggested that S-induced CREB binding to COX-2 promoter is mediated via PI3K/PKC/JNK pathway and is independent of ERK.
PKC and PKC were involved in signal transduction pathways induced by S protein
As S-activated ERK and JNK were PKC dependent, we extended our study to identify the specific isozymes of PKC involved in the activation of COX-2 regulated by the S protein using an RNA interference (RNAi) approach. Small interference RNA (siRNA) molecules that specifically knock down genes encoding each PKC isozyme were designed and used to study the function of PKC, based on a previous study (17)
. Results indicated that PKC-specific and PKC-specific siRNA (siPKC and siPKC), but not PKCß1-specific and PKC
-specific siRNA (siPKCß1 and siPKC) or irrelevant siRNA (siCtrl), were able to significantly inhibit the induction of COX-2 in transiently transfected 293T cells (Fig. 6
A). These results suggested that PKC and PKC played important roles in the activation of COX-2 by the S protein in human cells.
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Expression of PKC, PKC, PKCß1, and PKC were determined before and after knockdown by specific siRNA using Western blot in HEK293T cells. Results showed that protein expression of different PKC isoforms were decreased significantly in the presence of PKC isoform-specific siRNAs (siPKC, siPKC, siPKCß1, and siPKC). siCtrl, a similar vector (pSilencer 2.0 from Ambion) containing an irrelevant sequence that does not show significant homology to any human gene sequence, was used as a negative control (Fig. 6B
). The results indicated that various PKC isoform-specific siRNAs worked efficiently in the assays.
The effects of PKC and PKC on the phosphorylation of ERK and JNK were also evaluated by a similar RNAi approach. Results from Western blot analysis showed that phosphorylated ERK1/2 levels stimulated by the S protein were significantly decreased by treatment of PKC-specific siRNA (siPKC) (Fig. 6C
, upper panel). Similarly, phosphorylated JNK activated by the S protein was inhibited by PKC-specific siRNA (siPKC) (Fig. 6C
, lower panel). These results suggested that PKC was the upstream kinase for S-induced ERK/NF-B activation and PKC was the upstream kinase for JNK/CREB activation.
Induction of COX-2 expression by S protein was associated with extracellular Ca2+ entry
PKC belongs to the conventional subfamily of PKC whose activities are calcium dependent (17)
. Since PKC was involved in S-induced COX-2 expression, we investigated the role of Ca2+ in the induction of COX-2 expression by measuring intracellular calcium using Fura-2 fluorescence. Results showed that the concentration of cytosolic [Ca2+]i in 293T cells was 100 nM in the absence of S protein regardless of the status of EGTA and exogenous Ca2+ (Fig. 7
A). However, after introducing S protein into the cells, the concentration of cytosolic [Ca2+]i was elevated to >300 nM, reduced to a basal level when treated with 2 mM EGTA, and increased rapidly after adding 2 mM exogenous Ca2+ (Fig. 7A
).
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The effect of Ca2+ on S-induced COX-2 expression regulated by the S protein was also studied. Human cells were cotransfected with pCMV-S and reporter followed by treatment with EGTA or BAPTA/AM, a cell-permeable Ca2+ chelator widely used as an intracellular calcium sponge. Results from luciferase activity analysis showed that up to 2 mM BATPA/AM had no effect on the regulation of COX-2 promoter activity by S protein (Fig. 7B
). EGTA had no effect on COX-2 expression at 1 mM, but inhibited COX-2 expression when the concentration reached 2 mM (Fig. 7C
). In addition, Western blot analysis revealed that S-induced COX-2 protein expression was inhibited by EGTA (Fig. 7D
). These results suggested that the spike protein elevated intracellular Ca2+ by triggering extracellular Ca2+ entry to the cytosol, thereby activating PKC and ERK.
To confirm another requirement of extracellular Ca2+ for ERK vs. JNK activation, we investigated the role of extracellular Ca2+ entry in regulating the MAPK pathway. Results showed that S protein-stimulated phosphorylated ERK was reduced in the presence of EGTA. However, phosphorylated JNK was not altered by EGTA (Fig. 8
). These results suggested that extracellular Ca2+ influx was an upstream signal of the PKC/ERK/NF-B pathway.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. We postulated that a novel mechanism existed in the cells for spike-activated COX-2 expression based on the fact that localizations of the nucleoprotein and spike are completely different. The nucleoprotein of SARS-CoV is localized in both nucleus and cytoplasm, whereas the spike is localized along the typical secretion pathway from endoplasmic reticulum to plasma membrane (28)
.
The spike gene of SARS-CoV encodes viral coat protein of 1255 amino acids with a molecular mass of 125 kDa. As previous studies have claimed that the envelope protein of Epstein-Barr virus (latent membrane protein 1) and HIV (Gp120) induce COX-2 expression after introduction into host cells (29
30)
, we evaluated the effect of S protein on COX-2 expression by transient transfection. Our results show that transfection of S protein resulted in COX-2 promoter activation and protein production.
Many viral proteins can induce multiple signaling pathways, which may cross-talk with each other or converge on common downstream effectors (31)
. In nasopharyngeal carcinoma cells, the latent membrane protein-1 of EBV induced COX-2 expression through MAPK, the JNK/AP-1, and janus-activated kinase (JAK)/STAT signaling pathways and required the transcription factor NF-B (13)
.
Our results indicate that S protein activates COX-2 promoter activity by enhancing NF-B and CREB binding to their specific enhancer elements on COX-2 promoter. Both NF-B and CREB have been claimed to mediate COX-2 activation induced by diverse stimulating agents. We demonstrated in this study that NF-B and CREB are essential for S protein-induced COX-2 expression since mutations of binding sites for NF-B and CREB eliminated S protein-induced activation of COX-2 promoter activity. Based on our finding that S protein activates COX-2 expression, we further investigated the roles of different MAP kinases on the activation of COX-2 by S protein. The results suggested that ERK, JNK, PI3K, and PKC, but not p38 MAP kinase, were involved in the activation of COX-2 regulated by S protein. An active ERK was required for the binding of NF-B to the COX-2 promoter in response to S protein stimulation; the PI3K/PKC/JNK/CREB pathway is another important signaling in the regulation of COX-2 by S protein.
Although PI3K/JNK/CREB and ERK/NFB signaling pathways appear to be independent, both were regulated by members of PKC family. Using an RNA interference approach, we demonstrated that the induction of COX-2 expression by S protein of SARS-CoV required the conventional isozyme PKC and the novel isoform PKC, since PKC- and PKC-specific siRNAs inhibited COX-2-Luc reporter expression activated by the S protein but siRNA specific for PKCß1 and PKC did not influence S protein induced COX-2 promoter activities.
It has been demonstrated that PKC is calcium dependent and PKC is calcium independent, suggesting these two PKC isoforms play different roles in transmitting S-induced COX-2 expression. We provided evidence that the S-activated ERK was dependent on PKC and the S-regulated JNK required PKC, because PKC-specific siRNA significantly affected the level of ERK1/2 phosphorylation and PKC-specific siRNA decreased the level of JNK phosphorylation. These results suggested S-activated COX-2 expression was mediated via two signaling pathways led by PKC and PKC, respectively.
The role of calcium in the regulation of COX-2 by S protein was also investigated in this study. Transient transfection of human cells with S protein induced a rapid increase in the level of intracellular calcium due to the transporting calcium influx from outside the cell. Treatment of cells with 2 mM of EDTA resulted in the chelating of extracellular calcium influx, affected the expression of COX-2, and inhibited the level of ERK phosphorylation. However, the level of JNK phosphorylation was not affected by treatment of the EDTA. These results suggested that an influx of extracellular calcium was required for induction of COX-2.
It has been reported that the elevated Ca2+ in cytosol stimulated PKC through diacylglycerol (DAG), then induced translocation of PKC to the plasma membrane, where it becomes active and regulates the ERK/NF-B pathway (32)
. Although the PKC pathway is mediated by an influx of extracellular Ca2+, the mechanism of this influx remains unclear. It was reported that endoplasmic reticulum (ER) stress opens up the calcium channel on the plasma membrane to allow extracellular Ca2+ entry from outside the cell (33)
. The ER stress response is suggested to contribute to several types of human disease. ER stress response is induced by overexpression of exogenous membrane or secreted proteins, such as virus proteins (34)
. Hepatitis C virus and Japanese encephalitis virus infection have been reported to initiate endoplasmic reticulum stress (35)
. Generally, ER stress is characterized by enhanced expression of ER-resident chaperones, such as glucose-regulated protein (GRP), Bip/GRP78, and CHOP (36)
. In our study we found that Bip/GRP78 and CHOP mRNA levels were enhanced in the presence of S protein (data not shown), suggesting ER stress was induced in response to SARS-CoV S protein stimulation. The spike protein of SARS-CoV is a glycosylated protein and is localized along the secretion pathway from ER to the plasma membrane (28)
. It is possible that excessive S proteins pass through the secretion pathway during viral infection and result in ER stress. We speculated that ER stress was the main reason for the influx of intracellular Ca2+, which was required to trigger the PKC/ERK/NF-B signaling pathway and to induce COX-2 expression.
In this study, we investigated the role of SARS-CoV S protein in regulating COX-2 gene expression. S protein regulated COX-2 gene expression through two independent signaling pathways: the calcium-dependent PKC/ERK/NF-B signaling pathway and the calcium-independent PI3K/PKC/JNK/CREB pathway. The influx of extracellular Ca2+ induced by S protein activates the PKC kinase and PKC/ERK/NF-B pathways. After activation, NF-B binds to the COX-2 promoter and activates transcription. S protein may also induce kinase PI3K through a mechanism that is unclear at this time, which in turn activates PKC, a downstream kinase of PI3K (37)
and the JNK/CREB signaling pathway. Activated CREB binds to the COX-2 promoter and regulates expression of the COX-2 gene. Our results provide a novel possible explanation for the critical role of the S protein of SARS-CoV in inducing preliminary inflammation and tissue damage leading to devastating SARS.
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ACKNOWLEDGMENTS |
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Received for publication July 20, 2006. Accepted for publication December 25, 2006.
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