Published online before print May 29, 2008
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,1
Departments of Pediatrics,
Internal Medicine, and
Medical Research, National Taiwan University Hospital, and
* Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
1 Correspondence: Department of Pediatrics, National Taiwan University Hospital, Taipei 100, Taiwan. E-mail: tsaopn{at}ntu.edu.tw
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played a key role in the VEGF and sFlt-1 signaling pathway of LPS-activated macrophages. PKC is a family of serine-threonine kinases, which are classified into three major groups based on homology and cofactor requirements: conventional PKCs, novel PKCs, and atypical PKCs. In the murine RAW264.7 cells, as well as in primary human monocytes/macrophages, pretreatment with a general PKC inhibitor GF109203X or with a novel PKC inhibitor rottlerin or overexpression of a kinase-inactive form of PKC (K376R) eliminated LPS-induced sFlt-1 expression and augmented LPS-induced VEGF expression at the protein and the transcription levels. In contrast, Gö6976, an inhibitor for the conventional PKCs, or myristoylated PKC
pseudosubstrate peptide, an inhibitor for the atypical PKCs, failed to exert the same effects. These data suggest that PKC signaling is involved in LPS-induced sFlt-1 expression and serves as a negative mediator in LPS-induced VEGF expression in macrophages. A novel strategy controlling the LPS-induced PKC pathways, especially the PKC isoform, may be developed based on this study.
Key Words: sepsis • inflammation • vascular endothelial growth factor
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IL-1β, IL-6, IL-8, and vascular endothelial growth factor (VEGF), to orchestrate the immune response. However, over-reaction to LPS can cause severe sepsis [1
2
3
]. Hence, identifying effectors that block or attenuate this overwhelming inflammatory response is a key to effective sepsis therapy [4
, 5
]. VEGF is not only involved in angiogenesis but also in an inflammatory reaction. Previous studies showed that the level of VEGF in circulation is elevated in severe sepsis [6 7 8 9 ]. VEGF blockage reduces the plasma levels of IL-6 and IL-10 in the cecal ligation and puncture (CLP)-induced sepsis mouse model [10 ]. In addition, Shapiro et al. [9 ] showed that the circulatory soluble fms-like tyrosine kinase-1 receptor (sFlt-1) level was increased in septic patients and that the increase was correlated with disease severity. Furthermore, our group [11 ] and Yano et al. [12 ] have demonstrated that treatment with exogenous sFlt-1 or adenovirus-mediated overexpression of sFlt-1 attenuates inflammatory response and decreases mortality in experimental sepsis through antagonization of LPS-induced VEGF secretion [11 , 12 ]. These findings indicate that increasing the plasma sFlt-1:VEGF ratio during sepsis may be an effective therapeutic adjuvant for treatment of septic shock as well as severe immune responses caused by microbe-mediated diseases. Therefore, elucidation of the mechanisms for LPS-induced VEGF and sFlt-1 expression may lead to advances in therapeutic opportunities in the treatment of sepsis.
Multiple signaling molecules are initiated upon LPS binding to TLRs in macrophages, leading predominantly to activation of MAPKs (ERK1/2, p38, and JNK) and protein kinase Cs (PKCs). In turn, these signaling proteins activate a variety of transcription factors that coordinate the induction of genes encoding inflammatory mediators [1
, 13
]. The PKC family consists of more than 10 serine-threonine kinases, which are classified into three major PKC groups based on homology and cofactor requirements: the conventional PKCs: , βI, βII, and 
; the novel PKCs: , 
, 
, and 
; and the atypical PKCs: µ, , 
, and 
. However, identification of the physiological functions of the individual PKC isoforms has been restricted by the availability of inhibitors with specificity [14
].
Recently, a set of PKC inhibitors has been grouped to test possible involvement of specific PKC in the signaling pathways of interest. GF109203X is a general PKC inhibitor, and rottlerin and the kinase-inactive form of PKC (K376R) are specific inhibitors for PKC, which belongs to the novel type of PKCs. Gö6976 and the myristoylated PKC pseudosubstrate peptide are specific inhibitors for the conventional and atypical PKCs, respectively. In this study, we used these PKC inhibitors with selective specificities to explore the involvement of PKC activation in the expression of sFlt-1 and VEGF in LPS-activated macrophages. We found that PKC, a novel type of PKC, is involved in the attenuation of LPS-induced sFlt-1 secretion and augmentation of LPS-induced VEGF secretion in murine macrophage RAW264.7 cells and human monocytes/macrophages. In addition, a dominant-negative inhibitor of PKC, K376R, has the same effect. We further showed that these changes were caused by alternation of sFlt-1 and vegf gene transcriptions. These data show that PKC is required for LPS-induced sFlt-1 (an anti-inflammatory agent) expression in macrophages. Thus, modulating the LPS-induced PKC pathway may offer an alternative treatment strategy for sepsis.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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of the novel PKCs), and the myristoylated PKC pseudosubstrate peptide (a specific inhibitor for the atypical PKCs) were purchased from Calbiochem (La Jolla, CA, USA).
Cell culture
Murine macrophage-like cells, RAW264.7, were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (Gibco-BRL, Grand Island, NY, USA), supplemented with 10% FBS and 4 mM glutamine at 37ºC in a humidified atmosphere of 5% CO2. Cells were pretreated with PKC inhibitors for 1 h and then stimulated with 1 ug/ml LPS for 24 h. Cells pretreated with DMSO were used as a control. PBMCs of voluntary donors were isolated using Ficoll-Paque centrifugation [15
], suspended in serum-free DMEM, and seeded onto six-well, noncoated culture dishes. After 12 h of incubation, the nonadherent cells were removed by three washes of PBS, and the adherent monocytes/macrophages were cultured using DMEM, supplemented with 10% FBS for 24 h before LPS treatment.
Plasmid construction and stable transfection of the dominant-negative mutant PKC—K376R
Mutation of the invariant lysine to an arginine at the ATP-binding site (aa 376) of PKC resulted in loss of the kinase activity [16
]. For construction of the K376R mutant plasmid, cDNA from RAW264.7 cells was prepared for PCR amplification of the 5'-end fragment of PKC by using primers: EcoRI-PKC-forward (F; 5'-GGGAGAATTCATCATGGCACC-3') and K376R-BmgBI-reverse (R; 5'-ACACCACGTCCTTCTTCAGACACCTGATTGCAAA-3'); and the 3'-end fragment of PKC by using primers: K376R-BmgBI-F (5'-TTTGCAATCAGGTGTCTGAAGAAGGACGTGGTGT-3') and EcoRI-PKC-R (5'-CTGGGAATTCAATTAAATGTC-3'). These two PCR products were digested by restriction enzyme BmgBI and linked with T4 ligase. The ligation mixture was subjected to PCR amplification of PKC-K376R by using primers EcoRI-PKC-F and EcoRI-PKC-R. Then, the PCR product was digested with restriction enzyme EcoRI and cloned into pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA). Plasmid DNA was prepared using a Qiagen EndoFree plasmid kit (Qiagen, Hilden, Germany). For stable transfections, cells were seeded in six-well plates and transfected with pcDNA 3.1 vector or pcDNA3.1-PKC-K376R using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. Forty-eight hours after transfection, cells with stable chromosomal integration of the plasmid were selected with 800 µg/ml G418 (Calbiochem Novabiochem, San Diego, CA, USA) for 7 days.
ELISA measurement of sFlt-1 and VEGF levels
After 24 h LPS stimulation, the conditioned culture media of RAW264.7 cells were collected and stored at –20ºC until analysis. The concentrations of sFlt-1 and free-form VEGF in each conditioned medium were analyzed using the mouse or human-specific ELISA kit (R&D Systems, Minneapolis, MN, USA). All experiments were performed in triplicate. Data are expressed as mean ± SEM.
Real-time quantitative RT-PCR (qRT-PCR)
Each tube of reaction mixture contained 5 µl sample RNA (200 ng) and 20 µl cocktail mix, including 12.5 µl AmpliTaq Gold® DNA polymerase mix (2x), 0.625 µl RT enzyme mix (40x; Applied Biosystems, Foster City, CA, USA), 0.025 µl TaqMan® probes (100 µM; carboxyfluorescein-labeled), 0.5 µl primer mix (90 µM), and 6.35 µl diethylpyrocarbonate-double-distilled H2O. The probe sequences for GAPDH, sFlt-1, and VEGF were 5'-CAGAAGACTGTGGATGGCCCCTC-3', 5'-CCGCAGTGCTCACCTCTAACGAGAACTTCT-3', and 5'-AGCCTGCACAGCGCATCAGCG-3', respectively. The respective forward and reverse primer sequences for GAPDH, sFlt-1, and VEGF were: 5'-TGCACCACCAACTGCTTAG-3' and 5'-GGATGCAGGGATGATGTTC-3'; 5'-GGGAAGACATCCTTCGGAAGA-3' and 5'-TCCGAGAGAAAATGGCCTTTT-3' [3
]; and 5'-CATCTTCAAGCCGTCCTGTGT-3' and 5'-CTCCAGGGCTTCATCGTTACA-3'. To measure the expression of the sFlt-1 and VEGF genes, real-time RT-PCR was performed using an ABI 7700 sequence detection system according to the same PCR profile for all probes: 30 min at 48°C (for room temperature) and then 10 min at 95°C (activation of AmpliTaq Gold® DNA polymerase), followed by 40 cycles of 15 s at 95°C and 1 min at 60°C (PCR). For each gene, the relative concentration of RNA to GAPDH mRNA was determined using the equation: 2–
comparative threshold (CT), where CT = (CT mRNA–CT GAPDH RNA).
Statistical analysis
The Student’s t-test was used to analyze the results, and a P value of less than 0.05 was considered statistically significant.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and PKCβII, respectively. Elevated phosphorylation of Ser660 of PKCβII was detected at 15–45 min after LPS stimulation. Among the novel-type PKCs, the phosphorylation level of Thr538 of PKC was slightly increased at 15 min poststimulation and that of Thr505 of PKC oscillated. For the atypical-type PKCs, the phosphorylation level of Thr410 of PKC and Thr403 of PKC declined at 5 and 15 min after LPS stimulation. In contrast, the phosphorylation level of Ser916 of PKCµ was increased.
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Figure 1. Phosphorylation of multiple PKC isoforms induced by LPS in murine macrophage RAW264.7 cells, which were stimulated with LPS for various times, after which, they were lysed, and the phosphorylation of PKC isoforms was detected by Western blotting using the phospho-PKC (p-PKC) antibody sample kit. Actin was used as sample loading control. Relative levels of phosphorylation were determined by densitometry and normalized by the corresponding actin amount. The results were shown as the mean + SEM of three indepedent experiments and presented as the fold-change compared with no LPS treatment (*, P<0.05).
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Figure 2. GF109203X suppressed sFlt-1 expression in LPS-activated RAW264.7 cells (A), which were pretreated with various concentrations of the general PKC inhibitor GF109203X (0.05–5 µM) for 1 h and subsequently stimulated with LPS for 24 h. The quantity of sFlt-1 in the conditioned media was measured using ELISA. Data are presented as mean ± SEM (n=3). (B) After pretreatment with GF109203X (5 µM), RAW264.7 cells were stimulated with LPS for 8 h, and total RNA was harvested for determining sFlt-1 expression by real-time qRT-PCR assays. The data were normalized by GAPDH expression and presented as mean ± SEM (n=3).
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Inhibition of the general PKC pathway increases the level of free-form VEGF in culture medium of the LPS-activated RAW264.7 cell
To investigate the role of PKC signaling in LPS-induced VEGF secretion, RAW264.7 cells were pretreated with GF109203X for 1 h followed by incubation with LPS for 24 h, and the amount of free-form VEGF in the culture media was determined with ELISA. We found that the GF109203X pretreatment apparently increased VEGF levels in the conditional culture medium (Fig. 3A
).
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Figure 3. GF109203X increased VEGF expression in LPS-activated RAW264.7 cells (A), which were pretreated with various concentrations of the general PKC inhibitor GF109203X (0.05–5 µM) for 1 h followed by LPS stimulation for 24 h. The quantity of VEGF in the conditioned media was measured using ELISA. Data are presented as mean ± SEM (n=3). (B) After pretreatment with GF109203X (5 µM), RAW264.7 cells were stimulated with LPS for 8 h, and total RNAs were harvested for determining VEGF expression by real-time qRT-PCR assays. The data were normalized by GAPDH expression and presented as mean ± SEM (n=3).
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3.4-fold (P<0.05; Fig. 3B
). These results indicate that inhibition of PKC signaling indeed augments LPS-induced VEGF transcription.
Inhibition of the novel-type but not the conventional or atypical PKC isoforms regulates LPS-induced sFlt-1 and VEGF expressions in RAW264.7 cells
A number of pharmacological agents have been developed that selectively inhibit specific PKC isoforms, such as Gö6976 for the conventional PKCs, rottlerin for the novel-type PKC, and pseudosubstrate peptide inhibitor for the atypical PKC. These inhibitors were used in this study to identify the roles of different groups of PKC isoforms in the regulation of sFlt-1 and VEGF expressions in LPS-activated RAW264.7 cells. We found that LPS-induced sFlt-1 secretion was not altered by Gö6976 or a pseudosubstrate peptide of atypical PKC, but it appeared to be suppressed by rottlerin (Fig. 4A
4B
4C
). Further, the LPS-induced VEGF secretion was augmented by rottlerin but not by Gö6976 or a pseudosubstrate peptide of atypical PKC (Fig. 4D
4E
4F)
. In addition, rottlerin decreased LPS-induced sFlt-1 mRNA expression (Fig. 4G)
and increased the mRNA level of LPS-induced VEGF (Fig. 4H)
. These results suggest that the novel PKC isoform is critical for regulating LPS-induced sFlt-1 and VEGF expressions in murine RAW264.7 macrophages.
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Figure 4. Effects of different PKC isoform inhibitors on LPS-induced sFlt-1 and VEGF expressions in RAW264.7 cells, which were pretreated with (A) Gö6976 (1–100 nM), an inhibitor for the conventional PKCs; (B) rottlerin (0.03–3 µM), an inhibitor for the novel-type PKCs; or (C) a pseudosubstrate (PS) peptide inhibitor of atypical PKC (40 µM) or DMSO as vehicle alone and subsequently stimulated with LPS for 24 h. ELISA was then used to measure the amounts of sFlt-1 in the collected conditioned culture media. (D–F) The quantity of VEGF in the cultured media was also measured. Data are presented as mean + SEM (n=3). (G and H) RAW264.7 cells were pretreated with the novel PKC inhibitor, rottlerin (3 µM), or DMSO as vehicle alone for 1 h and subsequently stimulated with LPS for 8 h. Total RNAs were harvested for real-time qRT-PCR assays of sFlt-1 and VEGF. The data were normalized by GAPDH expression and presented as mean + SEM (n=3).
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suppresses LPS-induced sFlt-1 secretion in RAW264.7 cells in LPS-induced sFlt-1 and VEGF secretions, a kinase-inactive form of PKC (K376R-dominant mutant) was constructed and transfected into RAW264.7 cells. After LPS stimulation for 24 h, conditioned media were tested by ELISA assay. We found that LPS-induced sFlt-1 expression was suppressed in RNA (Fig. 5A
) and protein levels (Fig. 5B)
in the K376R-transfected cells compared with the vector control. Furthermore, overexpression of dominant-negative mutant K376R also augmented LPS-induced VEGF expression in transcription and protein levels (Fig. 5C
and 5D)
. The effects of a dominant-negative mutant were compatible with that of rottlerin. This result indicates that PKC indeed plays a vital role in regulation of LPS-induced sFlt-1 and VEGF expressions in RAW264.7 macrophages.
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Figure 5. Effects of overexpressing dominant-negative mutant K376R of PKC on LPS-induced sFlt-1 and VEGF expressions in RAW264.7 macrophages. RAW264.7 cells containing the stably transfected pcDNA3.1 vector (3.1) or a dominant-negative mutant of PKC (K376R) were used. (A and C) These cells were treated with or without LPS (1 µg/ml) for 15 h, and the total RNAs were collected for sFlt-1 and VEGF measurement by real-time qRT-PCR assays. The data were normalized by GAPDH expression and presented as mean ± SEM (n=3). (B and D) These cells were treated with or without LPS (1 µg/ml) for 24 h, and the quantity of sFlt-1 and VEGF in the conditioned media was measured by ELISA. Data are presented as mean ± SEM (n=3).
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Figure 6. Effects of GF109203X on LPS-induced sFlt-1 and VEGF secretion in primary human macrophages, which were pretreated with 5 µM GF109203X or DMSO as vehicle alone for 1 h and subsequently stimulated with LPS for 48 h. Human-specific ELISA kits were then used to detect sFlt-1 (A) and VEGF (B) in the collected conditioned media. Data are presented as mean ± SEM (n=3).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, -, -, -, and -µ are alternated by LPS stimulation in murine macrophage RAW264.7 cells. A general PKC inhibitor (GF109203X), a selective inhibitor for novel PKC (rottlerin), and overexpression of a dominant-negative mutant of PKC (K376R) inhibited LPS-induced sFlt-1 expression and augmented LPS-induced VEGF expression in RAW264.7 cells at transcriptional and protein levels. In addition, the effects of GF109203X on LPS-induced sFlt-1 and VEGF expressions were observed in primary human macrophages. These results indicate that PKC signaling is required for LPS-induced sFlt-1 expression and that it also exerts a negative regulatory effect on LPS-induced VEGF secretion in the murine RAW264.7 macrophages as well as in human macrophages.
The PKC family consists of more than 10 isoforms, which are classified into three major groups. It has been reported that conventional (, βI, and βII), novel ( and ), and atypical PKC isoforms (µ, , and ) are expressed in murine macrophage RAW264.7 cells [17
] and that the phosphorylated isoforms of these PKCs are detectable in the resting RAW264.7 cells [18
]. In this study, the phospho-PKC antibody sampler kit was used to confirm that these PKC isoforms (, βI, βII, , µ, , and ) are expressed in the resting RAW264.7 cells. Further, we discovered that the phosphorylation levels of PKCβII, -, -, -, -, and -µ were increased after LPS stimulation. Phosphorylation of the novel-type PKC was increased immediately, and the increase in the conventional-type PKCβII and the atypical-type PKCµ was relatively delayed. Together, these findings suggest that the signal pathways of all three PKC groups are alternated in LPS-stimulated RAW264.7 cells. As the LPS preparations may contain other bacterial components that can stimulate TLR2 [19
], we cannot rule out the possibility that TLR2-mediated signaling is also involved in LPS-induced sFlt-1 and VEGF expressions. We will address this issue in detail in future studies.
It has been reported that the increased VEGF circulation detected in the mouse septic model and in human sepsis is associated with disease severity [6 , 8 , 9 , 11 ]. Moreover, it has been demonstrated that suppression of VEGF function by adenovirus-mediated sFlt-1 overexpression or by treatment with exogenous sFlt-1 attenuates the levels of proinflammatory cytokines in plasma and decreases the mortality caused by endotoxemia in the CLP-induced septic mouse [11 , 12 ]. These results suggest that using sFlt-1 to decrease VEGF plasma levels enhances the efficacy of sepsis therapy. In the septic mouse model, the plasma sFlt-1 is also elevated during endotoxemia, declining to near-basal levels within 5 h after LPS injection [11 ]. However, the regulatory mechanisms of sFlt-1 expression in sepsis remain unexplored. In this study, a general PKC inhibitor, GF109203X, was used to assess the role of PKC activation on LPS-induced sFlt-1 expression in RAW264.7 cells.
GF109203X inhibits PKCs by acting as a competitive inhibitor of the ATP-binding site for PKCs. The IC50 values of GF109203X for inhibition of classical, novel, and atypical PKCs are 20 nM, 100–200 nM, and 5.8 µM, respectively [14
, 20
]. We found that pretreatment of RAW264.7 cells with 50 nM, 500 nM, and 5 µM GF109203X apparently decreased LPS-induced sFlt-1 secretions by 11%, 36%, and 90%, respectively. Similar results were also found in primary human macrophages. Moreover, we also used real-time qRT-PCR to demonstrate that GF109203X dramatically suppressed LPS-induced transcription of the Flt-1 gene. These results are the first set of evidence that activation of the PKC signal pathway is involved in the LPS-induced sFlt-1 release in macrophages.
Previous studies have revealed that VEGF expression in LPS-activated monocytes and macrophages is blocked by specific inhibition of MAPK signal pathways [15 , 21 ]. However, the relationship between the LPS-activated PKC signal pathway and VEGF expression in macrophages is unknown. In this study, we found that treatment with GF109203X not only dramatically increased LPS-induced VEGF secretion in RAW264.7 cells but also significantly augmented the vegf transcription. The effect of GF109203X on LPS-induced VEGF expression was also confirmed in human macrophages. These results suggest that PKC signaling acts as a negative mediator in LPS-induced VEGF secretion in macrophages.
Gö6976 is a potent inhibitor for the conventional PKCs. It has been demonstrated that nanomolar concentrations of Gö6976 inhibit the Ca2+-dependent PKC isozymes, and βI (IC50=2.3–6.2 nM), whereas even micromolar concentrations of this inhibitor have no effect on the kinase activity of the Ca2+-independent PKC isoforms , , and [22
]. Rottlerin is a specific PKC inhibitor that can inhibit PKCs by competition for the ATP-binding site. The IC50 for the PKC isoforms are: , 3–6 µM; , β, and , 30–42 µM; and, , , and , 80–100 µM. Further, it has also been noted that rottlerin can inhibit calmodulin (CaM) kinase III (IC50=5.3 µM) at a concentration similar to that for the PKC IC50 [14
, 23
, 24
]. In this study, we found that pretreatment with 3 µM rottlerin, which is an effective concentration to inhibit the novel-type PKC, significantly eliminated LPS-induced sFlt-1 secretion and augmented LPS-induced VEGF secretion in RAW264.7 cells. As the effects of rottlerin on LPS-induced sFlt-1 and VEGF were similar to GF109203X analogs, and Gö6976 had no such effects, we ruled out the possibility that CaM-dependent kinase III was involved in the regulation of this process. This result was further confirmed by an experiment using a dominant-negative PKC mutant to specifically inhibit PKC. These findings suggest that the novel PKC isoform serves as an important mediator in the regulatory mechanism of LPS-induced sFlt-1 and VEGF secretions in macrophages. In addition, a MTT assay showed that these inhibitors had no significant effects on cell viability (data not shown).
It has been reported that PKC molecules are required for the VEGF expression induced by various treatments in different cell types [25 26 27 28 29 ]. The disparity between these results and our current findings is probably a result of differences in the experimental cell types or stimulators used.
In summary, we demonstrated that the novel PKC isoform is required for LPS-induced sFlt-1 (an anti-inflammatory agent) expression in mouse and human macrophages. Thus, controlling the LPS-induced PKC pathway—especially the novel PKC isoform—may offer an alternative treatment strategy in sepsis and/or tumor therapy.
Received October 15, 2007; revised April 9, 2008; accepted May 2, 2008.
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: a new target to control inflammation and immune-mediated disorders Int. J. Biochem. Cell Biol. 36,183-188[CrossRef][Medline] (PKC-) ATP binding mutant. An inactive enzyme that competitively inhibits wild type PKC- enzymatic activity J. Biol. Chem. 270,8311-8318/Nrf2/ARE signaling to up-regulation of heme oxygenase-1 J. Ethnopharmacol. 113,240-247[CrossRef][Medline]
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