Novel PKC signaling is required for LPS-induced soluble Flt-1 expression in macrophages Ming-Cheng Lee,* Shu-Chen Wei,† Jyy-Jih Tsai-Wu,‡ C. H. Herbert Wu,* and Po-Nien Tsao§,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
Abstract: In vitro activation of macrophages by LPS induces rapid release of vascular endothelial growth factor (VEGF) and soluble fms-like tyrosine kinase-1 receptor (sFlt-1), which are thought to be the effectors to cause sepsis. However, the signal pathway that controls the VEGF and sFlt-1 expressions in LPS-activated macrophages remains unclear. In this study, we demonstrated that phosphorylation of protein kinase C (PKC)␦ 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 LPSinduced VEGF expression at the protein and the transcription levels. In contrast, Go¨6976, 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. J. Leukoc. Biol. 84: 835– 841; 2008. Key Words: sepsis 䡠 inflammation 䡠 vascular endothelial growth factor
INTRODUCTION Upon bacterial infection, macrophages are activated by LPS, the principal component of the outer membrane of all Gramnegative bacteria, and secrete a variety of inflammatory cytokines and growth factors, including TNF-␣, IL-1, IL-6, IL-8, and vascular endothelial growth factor (VEGF), to orchestrate the immune response. However, over-reaction to LPS can cause severe sepsis [1–3]. Hence, identifying effectors that 0741-5400/08/0084-835 © Society for Leukocyte Biology
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 –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: ␣, I, II, and ␥; the novel PKCs: ␦, ε, , and ; and the atypical PKCs: , , , 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. Go¨6976 and the myristoylated PKC pseudosubstrate
1 Correspondence: Department of Pediatrics, National Taiwan University Hospital, Taipei 100, Taiwan. E-mail:
[email protected] Received October 15, 2007; revised April 9, 2008; accepted May 2, 2008. doi: 10.1189/jlb.1007691
Journal of Leukocyte Biology Volume 84, September 2008 835
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.
MATERIALS AND METHODS Reagents Escherichia coli LPS (0111:B4) and GF109203X (a general PKC inhibitor) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Go¨6976 (a classical PKC inhibitor), rottlerin (a specific inhibitor for PKC␦ 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 (GibcoBRL, 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 EcoRIPKC␦-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 g/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
836
Journal of Leukocyte Biology Volume 84, September 2008
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 l sample RNA (200 ng) and 20 l cocktail mix, including 12.5 l AmpliTaq Gold威 DNA polymerase mix (2⫻), 0.625 l RT enzyme mix (40⫻; Applied Biosystems, Foster City, CA, USA), 0.025 l TaqMan威 probes (100 M; carboxyfluorescein-labeled), 0.5 l primer mix (90 M), and 6.35 l diethylpyrocarbonate-double-distilled H2O. The probe sequences for GAPDH, sFlt-1, and VEGF were 5⬘-CAGAAGACTGTGGATGGCCCCTC-3⬘, 5⬘-CCGCAGTGCTCACCTCTAACGAGAACTTCT3⬘, 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威 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.
RESULTS LPS-induced phosphorylation of multiple PKC isoforms in murine macrophage RAW264.7 cells Figure 1 shows the phosphorylation levels of different types of PKCs upon LPS stimulation in the murine macrophage RAW264.7 cell line. Of the conventional-type PKCs, no significant change was observed in the phosphorylation levels of Thr638 or Thr641 in PKC␣ and PKCII, respectively. Elevated phosphorylation of Ser660 of PKCII 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 PKC was increased.
Activation of the PKC signal pathway is required for LPS-induced sFlt-1 secretion in RAW264.7 cells RAW264.7 cells were treated with a general PKC inhibitor, GF109203X, for 1 h followed by incubation with LPS for 24 h, and the secreted sFlt-1 was determined with ELISA. The amount of secreted sFlt-1 in the media was suppressed by GF109203X in a dose-dependent manner (0.05–5 M; Fig. 2A), suggesting that PKC signaling pathways are involved in the LPS-induced sFlt-1 secretion in RAW264.7 cells. Next, to determine whether GF109203X inhibits LPS-induced sFlt-1 secretions by regulating gene transcription of http://www.jleukbio.org
Fig. 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 foldchange compared with no LPS treatment (*, P⬍0.05).
sFlt-1, the effects of GF109203X on sFlt-1 mRNA transcription of LPS-activated RAW264.7 cells were assessed by realtime qRT-PCR assay. We found that the mRNA level of sFlt-1 declined almost to baseline levels (Fig. 2B), suggesting that inhibition of PKC signaling attenuates sFlt-1 mRNA transcription, leading to decreased sFlt-1 secretion in LPS-stimulated RAW264.7 cells.
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). As a fraction of the VEGF was bound to sFlt-1, and this VEGF kit detected only the free-form VEGF, the increase of VEGF in the GF109203X-pretreated, LPS-activated RAW264.7 could be a result of a decrease in sFlt-1 expression. To determine if VEGF transcription was up-regulated after inhibition of PKC signaling, a qRT-PCR assay was used to assess the effects of GF109203X on LPS-induced VEGF mRNA expression. We found that LPS induced an increase of VEGF mRNA expression approximately twofold that of the control (P⬍0.01) and that GF109203X pretreatment further enhanced this expression ⬃3.4-fold
Fig. 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 M) 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 M), 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).
Lee et al. PKC␦ enhances LPS-induced sFlt-1 expression
837
Fig. 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 M) 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 M), 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).
(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 Go¨6976 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 Go¨6976 or a pseudosubstrate peptide of atypical PKC, but it appeared to be suppressed by rottlerin (Fig. 4, A–C). Further, the LPS-induced VEGF secretion was augmented by rottlerin but not by Go¨6976 or a pseudosubstrate peptide of atypical PKC (Fig. 4, D–F). In addition, rottlerin decreased LPSinduced 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.
Overexpression of dominant-negative mutant K376R of PKC␦ suppresses LPS-induced sFlt-1 secretion in RAW264.7 cells To further confirm the role of PKC␦ in LPS-induced sFlt-1 and VEGF secretions, a kinase-inactive form of PKC␦ (K376Rdominant 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 LPSinduced 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 LPSinduced VEGF expression in transcription and protein levels (Fig. 5, C and D). The effects of a dominant-negative mutant were compatible with that of rottlerin. This result indicates that 838
Journal of Leukocyte Biology Volume 84, September 2008
PKC␦ indeed plays a vital role in regulation of LPS-induced sFlt-1 and VEGF expressions in RAW264.7 macrophages.
General PKC inhibitor GF109203X also regulates LPS-induced sFlt-1 and VEGF expression in human primary macrophages To investigate whether the effects of GF109203X on LPSinduced sFlt-1 and VEGF expressions were also found in human monocytes/macrophages, human monocytes/macrophages were pretreated with GF109203X, followed by LPS stimulation for 48 h. The amounts of sFlt-1 and VEGF in conditioned media were measured by human-specific ELISA. We found that compared with the control, LPS indeed induced a twofold increase in sFlt-1 secretion in human monocytes/ macrophages (P⬍0.03), and GF109203X pretreatment prevented the LPS-induced sFlt-1 secretion (P⬍0.03; Fig. 6A). Furthermore, LPS also dramatically induced VEGF secretion (P⬍0.001), and similar to RAW264.7 macrophages, GF109203X pretreatment significantly enhanced LPS-induced VEGF secretion twofold compared with LPS treatment alone (P⬍0.01; Fig. 6B). These results indicated that a regulatory role of the PKC signal pathway in LPS-induced sFlt-1 and VEGF secretion also exists in human primary macrophages.
DISCUSSION In the present study, we showed that the phosphorylation levels of PKCII, -, -␦, -, -, and - 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. http://www.jleukbio.org
Fig. 4. Effects of different PKC isoform inhibitors on LPS-induced sFlt-1 and VEGF expressions in RAW264.7 cells, which were pretreated with (A) Go¨6976 (1–100 nM), an inhibitor for the conventional PKCs; (B) rottlerin (0.03–3 M), an inhibitor for the novel-type PKCs; or (C) a pseudosubstrate (PS) peptide inhibitor of atypical PKC (40 M) 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 M), 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).
The PKC family consists of more than 10 isoforms, which are classified into three major groups. It has been reported that conventional (␣, I, and II), novel (␦ and ε), and atypical PKC isoforms (, , 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 (␣, I, II, ␦, , , and ) are expressed in the resting RAW264.7 cells. Further, we discovered that the phosphorylation levels of PKCII, -, -␦, -, -, and - were increased after LPS stimulation. Phosphorylation of the novel-type PKC␦ was increased immediately, and the increase in the conventional-type PKCII and the atypical-type PKC 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. Lee et al. PKC␦ enhances LPS-induced sFlt-1 expression
839
Fig. 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 g/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 g/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).
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 M, respectively [14, 20]. We found that pretreatment of RAW264.7 cells with 50 nM, 500 nM, and 5 M 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 LPSinduced 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.
Go¨6976 is a potent inhibitor for the conventional PKCs. It has been demonstrated that nanomolar concentrations of Go¨6976 inhibit the Ca2⫹-dependent PKC isozymes, ␣ and I (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 ␦, ε, 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 M; ␣, , and ␥, 30 – 42 M; and, ε, , and , 80 –100 M. Further, it has also been noted that rottlerin can inhibit calmodulin (CaM) kinase III (IC50⫽5.3 M) at a concentration similar to that for the PKC␦ IC50 [14, 23, 24]. In this study, we found that pretreatment with 3 M 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 Go¨6976 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 dominantnegative 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 addi-
Fig. 6. Effects of GF109203X on LPSinduced sFlt-1 and VEGF secretion in primary human macrophages, which were pretreated with 5 M 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).
840
Journal of Leukocyte Biology Volume 84, September 2008
http://www.jleukbio.org
tion, 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 [25–29]. 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.
ACKNOWLEDGMENTS This work was supported by grants from the National Science Council (NSC95-2314-B-002-079). We thank the second Core Lab of the Department of Medical Research of National Taiwan University Hospital for technical assistance.
REFERENCES 1. Guha, M., Mackman, N. (2001) LPS induction of gene expression in human monocytes. Cell. Signal. 13, 85–94. 2. Cavaillon, J. M., Adib-Conquy, M. (2005) Monocytes/macrophages and sepsis. Crit. Care Med. 33, S506 –S509. 3. Huckle, W. R., Roche, R. I. (2004) Post-transcriptional control of expression of sFlt-1, an endogenous inhibitor of vascular endothelial growth factor. J. Cell. Biochem. 93, 120 –132. 4. Bernard, G. R., Vincent, J. L., Laterre, P. F., LaRosa, S. P., Dhainaut, J. F., Lopez-Rodriguez, A., Steingrub, J. S., Garber, G. E., Helterbrand, J. D., Ely, E. W., Fisher Jr., C. J. (2001) Efficacy and safety of recombinant human activated protein C for severe sepsis. N. Engl. J. Med. 344, 699 –709. 5. Panacek, E. A., Marshall, J. C., Albertson, T. E., Johnson, D. H., Johnson, S., MacArthur, R. D., Miller, M., Barchuk, W. T., Fischkoff, S., Kaul, M., Teoh, L., Van Meter, L., Daum, L., Lemeshow, S., Hicklin, G., Doig, C. (2004) Efficacy and safety of the monoclonal anti-tumor necrosis factor antibody F(ab⬘)2 fragment afelimomab in patients with severe sepsis and elevated interleukin-6 levels. Crit. Care Med. 32, 2173–2182. 6. van der Flier, M., van Leeuwen, H. J., van Kessel, K. P., Kimpen, J. L., Hoepelman, A. I., Geelen, S. P. (2005) Plasma vascular endothelial growth factor in severe sepsis. Shock 23, 35–38. 7. Pickkers, P., Sprong, T., Eijk, L., Hoeven, H., Smits, P., Deuren, M. (2005) Vascular endothelial growth factor is increased during the first 48 hours of human septic shock and correlates with vascular permeability. Shock 24, 508 –512. 8. Rafat, N., Hanusch, C., Brinkkoetter, P. T., Schulte, J., Brade, J., Zijlstra, J. G., van der Woude, F. J., van Ackern, K., Yard, B. A., Beck, G. (2007) Increased circulating endothelial progenitor cells in septic patients: correlation with survival. Crit. Care Med. 35, 1677–1684. 9. Shapiro, N. I., Yano, K., Okada, H., Fischer, C., Howell, M., Spokes, K. C., Ngo, L., Angus, D. C., Aird, W. C. (2007) A prospective, observational study of soluble Flt-1 and vascular endothelial growth factor in sepsis. Shock, Epub ahead of print. 10. Nolan, A., Weiden, M. D., Thurston, G., Gold, J. A. (2004) Vascular endothelial growth factor blockade reduces plasma cytokines in a murine model of polymicrobial sepsis. Inflammation 28, 271–278.
11. Tsao, P. N., Chan, F. T., Wei, S. C., Hsieh, W. S., Chou, H. C., Su, Y. N., Chen, C. Y., Hsu, W. M., Hsieh, F. J., Hsu, S. M. (2007) Soluble vascular endothelial growth factor receptor-1 protects mice in sepsis. Crit. Care Med. 35, 1955–1960. 12. Yano, K., Liaw, P. C., Mullington, J. M., Shih, S. C., Okada, H., Bodyak, N., Kang, P. M., Toltl, L., Belikoff, B., Buras, J., Simms, B. T., Mizgerd, J. P., Carmeliet, P., Karumanchi, S. A., Aird, W. C. (2006) Vascular endothelial growth factor is an important determinant of sepsis morbidity and mortality. J. Exp. Med. 203, 1447–1458. 13. Aksoy, E., Goldman, M., Willems, F. (2004) Protein kinase C ε: a new target to control inflammation and immune-mediated disorders. Int. J. Biochem. Cell Biol. 36, 183–188. 14. Way, K. J., Chou, E., King, G. L. (2000) Identification of PKC-isoformspecific biological actions using pharmacological approaches. Trends Pharmacol. Sci. 21, 181–187. 15. Itaya, H., Imaizumi, T., Yoshida, H., Koyama, M., Suzuki, S., Satoh, K. (2001) Expression of vascular endothelial growth factor in human monocyte/macrophages stimulated with lipopolysaccharide. Thromb. Haemost. 85, 171–176. 16. Li, W., Yu, J. C., Shin, D. Y., Pierce, J. H. (1995) Characterization of a protein kinase C-␦ (PKC-␦) ATP binding mutant. An inactive enzyme that competitively inhibits wild type PKC-␦ enzymatic activity. J. Biol. Chem. 270, 8311– 8318. 17. Lin, W. W., Chen, B. C. (1998) Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages. Br. J. Pharmacol. 125, 1601–1609. 18. Kim, B. C., Jeon, W. K., Hong, H. Y., Jeon, K. B., Hahn, J. H., Kim, Y. M., Numazawa, S., Yosida, T., Park, E. H., Lim, C. J. (2007) The antiinflammatory activity of Phellinus linteus (Berk. & M.A. Curt.) is mediated through the PKC␦/Nrf2/ARE signaling to up-regulation of heme oxygenase-1. J. Ethnopharmacol. 113, 240 –247. 19. Hirschfeld, M., Ma, Y., Weis, J. H., Vogel, S. N., Weis, J. J. (2000) Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine Toll-like receptor 2. J. Immunol. 165, 618 – 622. 20. Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., et al. (1991) The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 266, 15771–15781. 21. Kiriakidis, S., Andreakos, E., Monaco, C., Foxwell, B., Feldmann, M., Paleolog, E. (2003) VEGF expression in human macrophages is NF-Bdependent: studies using adenoviruses expressing the endogenous NF-B inhibitor IB␣ and a kinase-defective form of the IB kinase 2. J. Cell Sci. 116, 665– 674. 22. Martiny-Baron, G., Kazanietz, M. G., Mischak, H., Blumberg, P. M., Kochs, G., Hug, H., Marme, D., Schachtele, C. (1993) Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976. J. Biol. Chem. 268, 9194 –9197. 23. Gschwendt, M., Muller, H. J., Kielbassa, K., Zang, R., Kittstein, W., Rincke, G., Marks, F. (1994) Rottlerin, a novel protein kinase inhibitor. Biochem. Biophys. Res. Commun. 199, 93–98. 24. Kontny, E., Kurowska, M., Szczepanska, K., Maslinski, W. (2000) Rottlerin, a PKC isozyme-selective inhibitor, affects signaling events and cytokine production in human monocytes. J. Leukoc. Biol. 67, 249 –258. 25. De Ponti, C., Carini, R., Alchera, E., Nitti, M. P., Locati, M., Albano, E., Cairo, G., Tacchini, L. (2007) Adenosine A2a receptor-mediated, normoxic induction of HIF-1 through PKC and PI-3K-dependent pathways in macrophages. J. Leukoc. Biol. 82, 392– 402. 26. Bian, Z. M., Elner, S. G., Elner, V. M. (2007) Regulation of VEGF mRNA expression and protein secretion by TGF-2 in human retinal pigment epithelial cells. Exp. Eye Res. 84, 812– 822. 27. Textor, B., Sator-Schmitt, M., Richter, K. H., Angel, P., Schorpp-Kistner, M. (2006) c-Jun and JunB are essential for hypoglycemia-mediated VEGF induction. Ann. N. Y. Acad. Sci. 1091, 310 –318. 28. Hoshi, S., Nomoto, K., Kuromitsu, J., Tomari, S., Nagata, M. (2002) High glucose induced VEGF expression via PKC and ERK in glomerular podocytes. Biochem. Biophys. Res. Commun. 290, 177–184. 29. Shih, S. C., Mullen, A., Abrams, K., Mukhopadhyay, D., Claffey, K. P. (1999) Role of protein kinase C isoforms in phorbol ester-induced vascular endothelial growth factor expression in human glioblastoma cells. J. Biol. Chem. 274, 15407–15414.
Lee et al. PKC␦ enhances LPS-induced sFlt-1 expression
841
