Phosphatidic acid as a regulator of matrix metalloproteinase-9 expression via the TNF-α signaling pathway

FEBS Letters 581 (2007) 787–793

Phosphatidic acid as a regulator of matrix metalloproteinase-9 expression via the TNF-a signaling pathway Jin-Gu Leea, Sun-Hye Leea, Dae-Weon Parka, Yoe-Sik Baeb, Sung-Su Yunc, Jae-Ryong Kima, Suk-Hwan Baeka,* a

Department of Biochemistry & Molecular Biology, Aging-Associated Vascular Disease Research Center, Yeungnam University, Daegu 705-717, Republic of Korea b Department of Biochemistry, College of Medicine, Dong-A University, Busan, Republic of Korea c Department of Surgery, College of Medicine, Yeungnam University, Daegu, Republic of Korea Received 16 January 2007; accepted 22 January 2007 Available online 30 January 2007 Edited by Robert Barouki

Abstract Phosphatidic acid (PA) is implicated in pathophysiological processes associated with cellular signaling events and inflammation, which include the expressional regulation of numerous genes. Here, we show that PA stimulation increases matrix metalloproteinase-9 (MMP-9) expression in macrophages through tumor necrosis factor (TNF)-a signaling. We performed antibody array analysis on proteins from macrophages stimulated with PA. PA was found to induce the production of TNF-a, but not of TNF receptor (TNFR)1 and TNFR2 in a time-dependent manner and stimulated significant, though delayed, MMP-9 expression. PA induced the phosphorylations of both ERK1/2 and p38, but not of c-jun amino-terminal kinase. Moreover, only ERK1/2 inhibition by U0126 suppressed PAinduced TNF-a production and MMP-9 expression. Neutralizing TNF-a, TNFR1 or TNFR2 antibodies significantly suppressed PA-induced MMP-9 expression, suggesting that the production of TNF-a in response to PA preceded the expression of MMP-9. Moreover, lipopolysaccharide-induced PA also led to TNF-a release and resulted in MMP-9 expression. Taken together, these observations suggest that PA may play a role in MMP-9 regulation through ERKs/TNF-a/TNFRs-dependent signaling pathway.  2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Phosphatidic acid; Tumor necrosis factor-a; TNF receptor; Matrix metalloproteinase-9

1. Introduction Macrophages play a central role in inflammatory responses. These include the elimination of foreign stimuli, the initiation of adaptive immune response, and regulation of the healing process. This various activities require macrophages migration * Corresponding author. Fax: +82 53 623 8032. E-mail address: [email protected] (S.-H. Baek).

Abbreviations: PA, phosphatidic acid; lysoPA, lysophosphatidic acid; DAG, diacylglycerol; PLD, phospholipase D; mTOR, mammalian target of rapamycin; IL, interleukin; ERK, extracellular signal-regulated kinase; ECM, extracellular matrix; MMP, matrix metalloproteinase; TNF, tumor necrosis factor; MAPK, mitogen activated protein kinase; C8-PA, 1,2-dioctanoyl-glycero-3-phosphate; TNFR, TNF receptor; JNK, c-jun amino-terminal kinase; LPS, lipopolysaccharide

to other tissues and their fast adaptation to changing environments. To migrate across the extracellular matrix (ECM), macrophages secrete several proteases, including matrix metalloproteinases (MMPs). Macrophages are thought to play an important role in this balance between inflammatory responses and healthy process due to their ability to produce enzymes capable of degrading the ECM. MMPs are important enzymes to breakdown ECM components like gelatin and collagen. The MMPs consist of gelatinases, collagenases, stromelysins, and membrane type MMPs. Among these, MMP-2 and MMP-9 are believed to be primarily responsible for the degradation of laminin and type IV collagen, the latter is a major component of basement membranes [1]. MMP-2 is expressed constitutively in many cell types, whereas MMP-9 is mostly induced by proinflammatory stimuli, such as, tumor necrosis factor (TNF)-a, endotoxins, and interleukin (IL)-1, probably through the activation of its transcription factor. In particular, the macrophage expression of active MMP-9 induces acute inflammatory responses like plaque disruption [2]. Additional data from human genetic studies suggest that MMP-9 is the strongest candidate for the induction of macrophage functionalities associated with inflammatory responses [1]. Phosphatidic acid (PA) is an important metabolite, and is involved in phospholipid biosynthesis and membrane remodeling. However, it is the regulatory function of PA that has attracted attention in recent years. PA has emerged as a new class of lipid mediator, like lysophosphatidic acid (lysoPA) and sphingosine-1-phosphate [3]. The synthesis and turnover of cellular PA can be regulated by several metabolic pathways, such as, lysoPA acyl transferase, diacylglycerol (DAG) kinase and phospholipase D (PLD) [3]. The generation of PA by the PLD pathway is central to several physiological functions including vesicular trafficking, secretion, phagocytosis, and barrier alterations [4]. However, some data show that PA itself plays a crucial role in the regulation of several important biological events. For instance, PA has been implicated in the regulation of cell growth [5,6] and protein phosphorylation [7,8], and in the release of cytokines [9]. PA stimulates the mammalian target of rapamycin (mTOR) signaling toward ribosomal S6 kinase 1, and the eukaryotic translation initiation factor 4E binding protein 1 [5]. Hornberger et al. [10] suggested that PA may directly interact with mTOR and modulate mechanical load-induced growth of skeletal muscle. PA has been described its function in relation to inflammation and cytokine release. We have previously shown that PA

0014-5793/$32.00  2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.01.048

788

is potent stimuli for cytokine production including TNF-a in murine macrophages [9]. Other groups have shown that PA induce IL-8 secretion, and this secretion is regulated by extracellular signal-regulated kinase (ERK)1/2 phosphorylation [11]. The production of cytokines in inflammatory cells requires activation gene transcription. These events are initiated and regulated by a various intracellular kinases, which in turn drive membrane signals to nuclear factors such as NF-jB [12]. Among signaling kinases, ERK1/2 play a prominent role in the regulation of cytokines. For example, ERK1/2 is involved in the pathways leading to cytokine production in murine macrophages stimulated with lipopolysaccharide (LPS) [13]. In addition, other evidence has indicated that PA mediate translocation of Raf-1 from the cytosol to the plasma membrane [14]. Membrane-bound Ras then activates the PA-tethered Raf-1 that initiates the ERK1/2 signaling cascades [15]. However, the involvement of these kinases in the production of TNF-a by PA has not been defined yet. We interested in the possibility that PA may contribute to TNF-a-mediated MMP-9 expression. In this study, we examined the signaling pathways that regulate PA-induced TNFa release from Raw264.7 murine macrophages and found that TNF-a release is required for MMP-9 expression. Furthermore, we found that ERK1/2 is critical for the regulation of TNFa-mediated MMP-9 expression in response to PA stimulation.

2. Materials and methods 2.1. Reagents and cells Cell culture reagents, including FBS, were purchased from Life Technologies (Grand Island, NY), C8-PA, C8-DAG and NBD-PA

J.-G. Lee et al. / FEBS Letters 581 (2007) 787–793 were from Avanti (Alabaster, AL), mouse cytokine antibody arrays from RayBiotech (Norcross, GA), TNF-a ELISA kit, and MMP-9 and TNF receptor (TNFR)1 antibodies from R&D Systems (Minneapolis, MN). TNFR2 antibody was purchased from Santa-Cruz Biotechnology (Santa-Cruz, CA), and ERK1/2, phospho-ERK1/2, p38, and phospho-p38 antibodies were from Cell Signaling Inc. (Beverley, MA). U0126 and mitogen activated protein kinase (MAPK) inhibitors were purchased from Calbiochem (San Diego, CA), and murine macrophages Raw264.7 cells (ATCC, CCL-2278) were grown in RPMI 1640 supplemented with 10% FBS, 2 mM L -glutamine, and 1% antibiotics. Culture dishes were maintained in humidified 5% CO2 at 37 C. 2.2. Gelatin zymography MMP activity was determined by gelatinase zymography using 0.1% gelatin as a substrate in 10% SDS–polyacrylamide gel. After electrophoresis, gels were washed for 30 min with 2.5% Triton X-100 in water and then incubated overnight at 37 C in 0.2% Brij 35, 5 mM CaCl2, 1 mM NaCl, and 50 mM Tris (pH 7.4), in a closed container. Gels were then stained for 1 h with 0.2% Brilliant Blue in 10% acetic acid and 50% ethanol and destained for 30 min using an aqueous mixture of 10% acetic acid, and 30% methanol. Areas of protease activity appeared as clear bands. 2.3. Cytokine antibody array After incubation for the indicated times at 37 C, conditioned media were harvested from cells treated with PA, centrifuged at 700 · g for 10 min to remove cell debris, and stored at 80 C for cytokine analysis. Conditioned media were probed for cytokine profiles using the RayBio mouse cytokine antibody array 1.1 kit according to the manufacturer’s instructions. Briefly, membranes were blocked with a blocking buffer, and then 2 ml of medium from each culture of treated cells was added and incubated at 4 C overnight. Membranes were washed and primary biotin-conjugated antibody was added and incubated at room temperature for 2 h. The membranes were then incubated with 2 ml of horseradish peroxidase-conjugated streptavidin at room temperature for 30 min. Cytokine presence was detected by enhanced ECL system.

Fig. 1. PA induces TNF-a production by Raw264.7 macrophages. (A) Macrophages were incubated in medium alone or medium containing 100 lM PA for 6 or 24 h. Conditioned media were then assayed for various cytokines and receptors using an antibody array. Cells were treated with (B) various concentrations of PA for 1 h at 37 C or (C) 100 lM PA for specified times at 37 C. TNF-a release was measured by ELISA. (D) Cells were treated with the various phospholipids for 1 h and then measured TNF-a release from conditioned medium.

J.-G. Lee et al. / FEBS Letters 581 (2007) 787–793 2.4. Western blot analysis Cells were plated in wells and treated with PA in the presence or absence of inhibitor. They were then washed with cold-PBS, and pelleted at 700 · g. Cell pellets were re-suspended in lysis buffer (50 mM Tris–HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM PMSF, and protease inhibitor cocktail), cleared by centrifugation, and supernatants were saved as cell lysates. Proteins were separated by 10% reducing SDS–PAGE and immunoblotted in 20% methanol, 25 mM Tris, and 192 mM glycine onto nitrocellulose membranes. Membranes were then blocked with 5% non-fat dry milk in TTBS (25 mM Tris–HCl, 150 mM NaCl, and 0.2% Tween-20) and incubated with the antibody for overnight. Subsequently, membranes were washed and incubated for 1 h with secondary antibody conjugated to HRP. Finally, membranes were washed and developed using an enhanced ECL system by LAS-3000 analyzer (Fujifilm, Japan). For MAPKs phosphorylation, cells were treated with PA and Western blotting was performed as described above, membranes so obtained were probed with antibody against active (phosphorylated) MAPKs. Blots were stripped and reproved with antibody recognizing total form of each MAPK to demonstrate equal loading. 2.5. Detection of soluble TNF-a Raw264.7 cells were incubated for 1 h in PA alone or containing various inhibitors. At designated times conditioned media were harvested and assayed for TNF-a in triplicate wells by ELISA as described by the manufacturer. The data shown represent at least three independent experiments.

789

the control level was observed in cells cultured for 1 h with PA concentrations ranging from 20 to 200 lM (Fig. 1B). Cells treated with 100 lM PA for 1 h also showed significantly increased TNF-a levels compared with unstimulated control cells (Fig. 1C). Additions of PA are complicated by the fact that it may be further metabolized to DAG or lyso-PA in the cells. Because the lyso derivative of C8-PA is not commercially available, we used lysoC6-PA. Addition of lysoC6-PA or C8-DAG did not cause release of TNF-a in cells (Fig. 1D). These results imply that PA acts directly on cellular targets, independently of DAG or lyso derivatives. 3.2. PA induced the expression of MMP-9 in macrophages To determine whether PA induces the expression of MMP-9 in macrophages, cells were treated with PA, and levels of MMP-9 protein in culture medium were determined. After 24 h of treatment, PA enhanced MMP-9 protein expression in a concentration-dependent manner. To determine whether MMP-9 protein released in response to PA was biologically active, we assessed the gelatinolytic activities of culture supernatants. As shown in Fig. 2A, low gelatinolytic activity was detected in control supernatant, whereas the supernatants of

2.6. Real-time RT-PCR Total RNA was extracted from cells using Tri reagent (Invitrogen, Carlsbad, CA). One microgram of total RNA was used as a template to make first strand cDNA by oligo-dT priming using the Promega reverse transcriptase system. Real-time RT-PCR was performed using LightCycler 1.5 (Roche Diagnostics, Almere, Netherlands) and SYBRGreen I as the florescent dye to enable the real-time detection of PCR products, according to the manufacturer’s instructions. The synthetic gene-specific primer sets used in PCR were: (i) TNF-a forward primer, 5 0 -TTC TGT CTA CTG AAC TTC GGG GTG ATC GGT CC-3 0 , and reverse primer, 5 0 -GTA TGA GAT AGC AAA TCG GCT GAC GGT GTG GG-3 0 , which amplified 354-bp of mouse TNF-a cDNA; (ii) MMP-9 forward primer, 5 0 -CCT ACT CTG CCT GCA CCA CTA AA-3 0 , and reverse primer, 5 0 -CTG CTT GCC CAG GAA GAC GAA-3 0 , which amplified 179-bp of mouse MMP-9 cDNA; (iii) b-actin forward primer, 5 0 -AGA GGG AAA TCG TGC GTG AC-3 0 , and reverse primer, 5 0 -CAA TAG TGA TGA CCT GGC CGT-3 0 , which amplified 137-bp of mouse b-actin cDNA. Cycling conditions were 94 C for 3 min, followed by 45 cycles of 95 C for 5 s, 62 C for 5 s, and 72 C for 12 s. For quantification, target genes were normalized versus b-actin.

3. Results 3.1. Effect of exogenous PA stimulation on TNF-a release in macrophages In order to increase cellular levels of PA, we added a watersoluble synthetic PA (C8-PA) to cell cultures. To examine the mechanisms involving cytokines in PA-induced signaling, we first used cytokine antibody arrays, and then studied TNF-a production in macrophages. In unstimulated cells, very low levels of TNF-a were detected. However, PA induced a significant increase in TNF-a release by 6 h, which increased more so at 24 h. Meanwhile the basal level of both soluble TNFR1 and TNFR2 was quite high, but the expression of neither TNFR1 nor TNFR2 was affected by PA stimulation (Fig. 1A). There are no changes of other spots between unstimulated and PA-stimulated cells. To confirm that TNF-a detected after PA stimulation represented a specific increase from antibody array, we performed TNF-a production assay using ELISA. A significant increase in TNF-a release above

Fig. 2. PA stimulates MMP-9 expression by macrophages. (A) Macrophages were plated on 6-well plates in medium containing 10% FBS. Following overnight serum starvation, cells were treated with PA at the indicated concentrations. The conditioned media were collected at 24 h and analyzed for MMP-9 expression and activity by Western blotting and zymography. (B) Cells were cultured with PA (100 lM) for the indicated times and conditioned media were collected and analyzed for MMP-9 expression and activity by Western blotting and zymography. The results shown are representative of three separate experiments. (C) Cells were cultured with PA for the indicated times and total RNA was isolated from cells and then subjected to realtime RT-PCR. The graph represents fold changes of TNF-a and MMP-9 mRNA levels normalized by b-actin mRNA after cells had been treated with PA.

790

J.-G. Lee et al. / FEBS Letters 581 (2007) 787–793

PA-treated cells had strong gelatinolytic activity and these correlated with MMP-9 protein levels. Cells treated with PA for the indicated times also showed increased MMP-9 levels and activities compared with unstimulated cells (Fig. 2B). Addition of lysoC6-PA or C8-DAG did not change expression of MMP9 in cells (data not shown). We next investigated the effects of PA on TNF-a and MMP-9 mRNA levels in macrophages. Cells were treated with PA for different times between 30 min and 9 h. The mRNAs of TNF-a and MMP-9 were also found to be increased by PA stimulation, although their kinetics differed. In particular, PA induced the expression of TNF-a mRNA earlier than MMP-9 mRNA (Fig. 2C). 3.3. PA-induced TNF-a and MMP-9 expressions via ERK1/2 pathway Because PA has been implicated in the activation of the Raf-1/MAPK pathway [14], we examined the effect of PA on MAPKs activation, using Western blot analysis with antiphospho-specific antibodies against the three MAPKs. After exposure to PA for various periods of time, macrophages were harvested and ERK1/2, p38 and c-jun amino-terminal kinase (JNK) phosphorylations were measured. As shown in Fig. 3A, PA significantly triggered the phosphorylation of both ERK1/2 and p38 MAPK, and these peaked after 30 min of stimulation and sustained up to 60 min. Both MAPKs phosphorylations were also increased by PA stimulation in a dose-dependent manner (Fig. 3B). However, JNK phosphorylation was not increased by PA stimulation (data not shown). To determine whether activation of MAPKs plays a role in the regulation of TNF-a and MMP-9, cells were pretreated with MAPK selective inhibitors for 1 h and then treated with PA for the indicated times. The viability of the cells was no effect after treatment of inhibitors. As shown in Fig. 4, inhibition of ERK1/2 phosphorylation with U0126 reduced PA-induced TNF-a production and MMP-9 expression, and in agreement with these results, we found that

Fig. 4. ERK1/2 activation is required for TNF-a and MMP-9 expression. (A) Cells were pretreated with 1 lM U0126, 10 lM SB203580, or 5 lM SP600125 for 1 h before being treated with PA (100 lM) for 1 h. Conditioned media were collected and TNF-a release was measured by ELISA. (B) Cells were pretreated with specific inhibitors for 1 h before adding 100 lM PA for 30 min (for ERK1/2 phosphorylation) or 24 h (for MMP-9). Whole cell lysates were Western blotted for phospho-ERK1/2, as indicated. Equal lane loading was confirmed by for total ERK1/2. Conditioned media were collected at 24 h and analyzed for MMP-9 expression and activity by Western blotting and zymography.

Fig. 3. (A) PA stimulates ERKs and p38 phosphorylation in macrophages. Macrophages were treated with PA (100 lM) for the indicated times (A) or with the indicated concentrations for 30 min (B). Whole cell lysates were Western blotted for phospho-MAPKs, as indicated. Equal lane loading was confirmed by detecting blots for total MAPKs. Relative amounts of phospho-ERK or phospho-p38 were determined using densitometry and then plotted. The results shown are representative of two separate experiments.

J.-G. Lee et al. / FEBS Letters 581 (2007) 787–793

U0126 inhibited MMP-9 gelatinolytic activity. However, the inhibition of p38 or JNK did not significant affect PA-induced TNF-a release and MMP-9 expression. To confirm whether the activation of ERK1/2 plays a role in the regulation of MMP-9 expression, we measured MMP-9 mRNA levels in the presence of U0126. Analysis of mRNA demonstrated that U0126 strongly inhibited TNF-a and MMP-9 mRNA (data not shown). These results suggest that ERKs play critical roles in the PA-induced TNF-a and MMP-9 expressions. 3.4. PA induces MMP-9 expression via secretion of TNF-a The above results demonstrate that PA induces the expression of TNF-a mRNA earlier than MMP-9 mRNA. Experiments were therefore undertaken to determine whether MMP-9 exerts its effects through TNF-a. In addition, because TNF-a is a key modulator of MMP-9, we focused on the PAinduced expression of TNF-a and its receptors. Stimulation with PA induced a transient increase in the mRNA levels of TNF-a. We confirmed the expressional patterns of TNFR1 and TNFR2 by Western blotting, which did not change in response to PA (data not shown). These findings confirm the antibody array data, and suggest that PA induces the expression of TNF-a, but not of TNFRs by macrophages. Next, to examine whether the PA-induced MMP-9 expression by macrophages is TNF-a-dependent, cells were pretreated with neutralizing anti-TNF-a antibody. Consistent with the previous results, MMP-9 was increased in the conditioned media of cells after PA stimulation. However, cells treated with anti-TNF-a antibody failed to express MMP-9 or TNF-a in response to PA (Fig. 5A), indicating that PA-induced MMP-9 expression by macrophages requires TNF-a signaling. In addition, an

791

isotype-matched control antibody IgG had no effect on MMP-9 expression (data not shown). To confirm the physiological function of PA, we used LPS for the increase of intracellular PA. Previously, we have reported that LPS stimulate acute increase of PA formation by PLD activity and PLD2 is predominantly expressed in Raw264.7 cells [9]. To determine whether intracellular PA formation is involved in the TNF-a release and MMP-9 expression, we used 1-butanol which is inhibitor of PLD activity. 1-Butanol significantly blocked the LPS-stimulated TNF-a release and MMP-9 expression while tert-butanol did not affect this LPS stimulatory effect (Fig. 5B), thus confirming the specificity of involvement of intracellular PA in the PLD-mediated effect. 3.5. PA induces MMP-9 expression through TNFR1/2 TNF-a can bind and signal through TNFR1 and TNFR2, and macrophages express both of these receptors [16]. We used receptor-specific neutralizing antibodies to determine the role of each receptor subtype with respect to the elicitation of MMP-9 expression using Western blotting and zymography. MMP-9 expression was reduced significantly in cells treated with neutralizing anti-TNFR1 antibody or anti-TNFR2 antibody. Moreover, both antibodies in combination had no greater effect than anti-TNFR2 treatment alone (Fig. 5C). In addition, an isotype-matched control antibody IgG had no effect on MMP-9 expression (data not shown). Similar results were obtained by MMP-9 zymography. These findings suggest that both TNFR1 and 2 are the important receptor involved in PA-induced MMP-9 expression in mouse macrophages. Furthermore, to confirm whether the LPS-stimulated MMP-9 expression is also TNF-a- or TNFR-dependent, cells were

Fig. 5. Extracellular or intracellular PA-induced MMP-9 expression was mediated via a TNF-a and TNFRs signaling pathway. (A) Cells were pretreated with neutralizing anti-TNF-a antibody (2 lg/ml) for 1 h before adding PA (100 lM). Conditioned media were collected and analyzed for TNF-a release and MMP-9 expression. (B) Cells were grown in 6-well plates and treated with 10 ng/ml LPS in the presence of 1-butanol or tertbutanol for 6 h. Conditioned media were collected and analyzed for TNF-a release and MMP-9 expression. (C and D) Cells were pretreated with neutralizing anti-TNF-a antibody or anti-TNFR1/2 antibodies for 1 h before adding PA or LPS. Conditioned media were collected at 6 h and analyzed for MMP-9 expression.

792

pretreated with neutralizing anti-TNF-a, TNFR1, or TNFR2 antibody. As shown in Fig. 5D, MMP-9 expression by LPS was reduced significantly in cells treated with neutralizing anti-TNF-a or anti-TNFR1/2 antibody.

4. Discussion In this study, we examined the PA function of TNF-a and MMP-9 regulation in murine macrophages, which are may be involved in macrophage extravasation from blood vessels and migration in damaged tissue. Our previous study showed that PA stimulates the release of inflammatory cytokines such as TNF-a by Raw264.7 cells. Although the induction of MMP-9 by PLD activation has been reported previously [17,18], it elicitation of MMP-9 expression and the intracellular signaling pathways are not well understood. In the present study, we further examine that PA-induced ERK1/2 activation regulates the expression of MMP-9 through the TNF-a and its receptor signaling. Inflammation includes the accumulation of inflammatory cells and the increased degradation of ECM components. Macrophages are the most important inflammatory effector cells and invariably accumulate at sites of inflammation [19]. Moreover, activated macrophages release cytotoxic molecules such as cytokines that cause tissue damage and are associated with the manifestations of various diseases, e.g., arthritis and sepsis. When tissue is being degraded in this manner, increased ECM turnover can result in the release of the degradation products of ECM components. TNF-a is a powerful cytokine of inflammation and can be produced by a variety of cell types, including macrophages and monocytes [12,16]. Although TNF-a has a central role in the innate immune system for protection against infections, when produced in excess, TNF-a may also orchestrate chronic inflammatory responses that lead to severe tissue damage. Thus, the regulation of inflammatory cell functions by interactions with basement membrane components may provide important information concerning how inflammatory responses are resolved. Following our initial observation that MMP-9 expression was upregulated in macrophages by PA, we hypothesized that this increased MMP-9 expression may have been triggered by an early event such as TNF-a release from cells. Because TNF-a can induce MMP-9 expression via the activation of specific cell signaling [20,21], we tested the hypothesis that PA induces the macrophage production of MMP-9 via TNF-a-dependent signaling. Our results show that PA increased the release of TNF-a before the expression of MMP-9 and TNFR1/2 is required for this MMP-9 expression. Several studies have suggested that PA regulates specific kinase cascades. For example, in vitro studies have demonstrated that there are at least two-protein kinase activities, including protein kinase C, regulated by PA [22,23]. The most characterized functional role of PA is the regulation of Raf-1/ERKs cascades [15]. ERK1/2 has been shown to serve as important regulators of inflammation. In addition, ERKs have been found to regulate MMPs including MMP-9, and a number of proinflammatory cytokines including TNF-a, IL-1b, and IL-6 in many inflammatory diseases [20,24]. In the present study, treatment of cells with PA caused a transient increase in ERK1/2 phosphorylation. Notably, the inhibition of ERK1/2 phosphorylation by U0126 dramatically reduced the

J.-G. Lee et al. / FEBS Letters 581 (2007) 787–793

expressions of MMP-9 at the protein and mRNA levels. This reduction in MMP-9 mRNA was not due to artifactual RNA degradation during RNA preparation or the reverse transcription reaction because the result was reproducible and there was no concomitant decrease in b-actin mRNA levels. These data suggest that the activity of ERK1/2 plays a role in the regulation of MMP-9 expression in macrophages by PA. Increasing cellular levels of PA (by adding exogenous C8PA) promotes TNF-a release, and TNFRs signaling was found to be critically required for MMP-9 expression. One complication of studying the functions of PA is that there is obscure how it enters cells. Our studies rely to a significant extent on adding short chain C8-PA to cells. Direct administration of PA to examine its cellular effects is potentially problems because PA may not be delivered to appropriate cellular locations. However, adding of fluorescent PA (NBD-C6-PA) is incorporated into cells very fast, which is observed under the confocal microscopy (data not shown). The other complication of PA studying is that they are convertible into lyso-PA or DAG. It is unlikely that it acts by lyso-PA, because most assays were performed in serum except MMP-9 assay, which contains biologically saturating amounts of lyso-PA, and lysoC6-PA was inactive in inducing TNF-a production. It is also unlikely that C8-PA acts after hydrolysis to C8-DAG, because there is no effect of C8-DAG in the TNF-a release. Moreover, the disposition of PA has always been an issue when PA is added to intact cells because PA used extremely high concentration about 100 lM. Although PA is present small amounts in biological membranes, however PA concentration is estimated to be 50–150 lM in plant [25,26]. This may also be relevant in PA signaling because others have observed that high concentrations of exogenous C8-PA (about 250 lM) serve as a regulator of cell motility [27,28]. Moreover, PA is wellknown lipid product by PLD activation [3]. To evaluate the significance of intracellular PA, we used an established model of LPS stimulation in Raw264.7 cells. Alcohols compete with water as hydroxyl donors in the hydrolysis of phospholipids by PLD, which results in the production of phosphatidylalcohol at the expense of PA. Treatment of macrophages with 1butanol, not tert-butanol, suppressed the LPS-stimulated TNF-a release and MMP-9 expression. These results suggest that the involvement of intracellular PA in the LPS-stimulated TNF-a release and MMP-9 expression. The biological activities of TNF-a are mediated by two main, but functionally distinct, receptors, i.e., TNFR1 and TNFR2. Although the binding of activated TNF-a to its receptors can result in the activation of various events, signaling triggered by TNFR1 and TNFR2 usually causes the activation and induction of numerous genes involved in inflammatory responses. TNFR1 is constitutively expressed in most cell types, and the majority of inflammatory responses classically attributed to TNF-a are mediated by TNFR1 [16]. Studies using TNFR knock-out mice revealed that TNFR1 plays a critical role in inflammatory response and tissue damage [29]. However, some evidence indicate that TNFR2 signaling also plays an important role in chronic inflammatory conditions [30]. Our findings showed that the PA-induced expression of MMP-9 by macrophages is mediated through both TNFR1 and TNFR2. In addition, these findings implicate that PA does not only affect on the expression of both TNFRs, there is no favorable receptor to execute TNF-a signaling which results in MMP-9 expression either.

J.-G. Lee et al. / FEBS Letters 581 (2007) 787–793

In conclusion, this study shows that ERK1/2 is essential for the induction of TNF-a release and MMP-9 expression by PA. Furthermore, based on the MMP-9 inhibitory effects of specific TNF-a or TNFR neutralizing antibodies, we conclude that TNF-a and TNFRs signaling is a major determinant of PA-induced MMP-9 expression by macrophages. Our findings provide new clues concerning the mechanism of inflammation and on its regulation. Acknowledgement: This work was supported by Korea Research Foundation Grant (KRF-2004-041-E00054) and by the Korean Science and Engineering Foundation via the Aging-Associated Vascular Disease Research center at Yeungnam University (R13-2005-005-01002-0 (2006)).

793

[15] [16] [17]

[18]

References [1] Parks, W.C., Wilson, C.L. and Lopez-Boado, Y.S. (2004) Matrix metalloproteinases as modulators of inflammation and innate immunity. Nat. Rev. Immunol. 4, 617–629. [2] Gough, P.J., Gomez, I.G., Wille, P.T. and Raines, E.W. (2006) Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice. J. Clin. Invest. 116, 59–69. [3] Wang, X., Devaiah, S.P., Zhang, W. and Welti, R. (2006) Signaling functions of phosphatidic acid. Prog. Lipid Res. 45, 250–278. [4] Testerink, C. and Munnik, T. (2005) Phosphatidic acid: a multifunctional stress signaling lipid in plants. Trends Plant Sci. 10, 368–375. [5] Chen, J. (2004) Novel regulatory mechanisms of mTOR signaling. Curr. Top. Microbiol. Immunol. 279, 245–257. [6] Foster, D.A. and Xu, L. (2003) Phospholipase D in cell proliferation and cancer. Mol. Cancer Res. 1, 789–800. [7] Fang, Y., Vilella-Bach, M., Bachmann, R., Flanigan, A. and Chen, J. (2001) Phosphatidic acid-mediated mitogenic activation of mTOR signaling. Science 294, 1942–1945. [8] Jones, J.A. and Hannun, Y.A. (2002) Tight binding inhibition of protein phosphatase-1 by phosphatidic acid. Specificity of inhibition by the phospholipid. J. Biol. Chem. 277, 15530–15538. [9] Lim, H.K., Choi, Y.A., Park, W., Lee, T., Ryu, S.H., Kim, S.Y., Kim, J.R., Kim, J.H. and Baek, S.H. (2003) Phosphatidic acid regulates systemic inflammatory responses by modulating the Akt-mammalian target of rapamycin-p70 S6 kinase 1 pathway. J. Biol. Chem. 278, 45117–45127. [10] Hornberger, T.A., Chu, W.K., Mak, Y.W., Hsiung, J.W., Huang, S.A. and Chien, S. (2006) The role of phospholipase D and phosphatidic acid in the mechanical activation of mTOR signaling in skeletal muscle. Proc. Natl. Acad. Sci. USA 103, 4741–4746. [11] Wang, L., Cummings, R., Usatyuk, P., Morris, A., Irani, K. and Natarajan, V. (2002) Involvement of phospholipases D1 and D2 in sphingosine 1-phosphate-induced ERK (extracellular-signalregulated kinase) activation and interleukin-8 secretion in human bronchial epithelial cells. Biochem. J. 367, 751–760. [12] Hanada, T. and Yoshimura, A. (2002) Regulation of cytokine signaling and inflammation. Cytokine Growth Factor Rev. 13, 413–421. [13] Dumitru, C.D., Ceci, J.D., Tsatsanis, C., Kontoyiannis, D., Stamatakis, K., Lin, J.H., Patriotis, C., Jenkins, N.A., Copeland, N.G., Kollias, G. and Tsichlis, P.N. (2000) TNF-alpha induction by LPS is regulated posttranscriptionally via a Tpl2/ERKdependent pathway. Cell 103, 1071–1083. [14] Rizzo, M.A., Shome, K., Vasudevan, C., Stolz, D.B., Sung, T.C., Frohman, M.A., Watkins, S.C. and Romero, G. (1999) Phos-

[19] [20]

[21]

[22]

[23]

[24] [25]

[26]

[27]

[28]

[29]

[30]

pholipase D and its product, phosphatidic acid, mediate agonistdependent raf-1 translocation to the plasma membrane and the activation of the mitogen-activated protein kinase pathway. J. Biol. Chem. 274, 1131–1139. Andresen, B.T., Rizzo, M.A., Shome, K. and Romero, G. (2002) The role of phosphatidic acid in the regulation of the Ras/MEK/ Erk signaling cascade. FEBS Lett. 531, 65–68. Baud, V. and Karin, M. (2001) Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol. 11, 372–377. Lee, H.S., Park, S.Y., Lee, H.W. and Choi, H.S. (2004) Secretions of MMP-9 by soluble glucocorticoid-induced tumor necrosis factor receptor (sGITR) mediated by protein kinase C (PKC)delta and phospholipase D (PLD) in murine macrophage. J. Cell. Biochem. 92, 481–490. Kato, Y., Lambert, C.A., Colige, A.C., Mineur, P., Noel, A., Frankenne, F., Foidart, J.M., Baba, M., Hata, R., Miyazaki, K. and Tsukuda, M. (2005) Acidic extracellular pH induces matrix metalloproteinase-9 expression in mouse metastatic melanoma cells through the phospholipase D-mitogen-activated protein kinase signaling. J. Biol. Chem. 280, 10938–10944. Fujiwara, N. and Kobayashi, K. (2005) Macrophages in inflammation. Curr. Drug Targets Inflamm. Allergy 4, 281–286. Zhou, M., Zhang, Y., Ardans, J.A. and Wahl, L.M. (2003) Interferon-gamma differentially regulates monocyte matrix metalloproteinase-1 and -9 through tumor necrosis factor-alpha and caspase 8. J. Biol. Chem. 278, 45406–45413. Adair-Kirk, T.L., Atkinson, J.J., Kelley, D.G., Arch, R.H., Miner, J.H. and Senior, R.M. (2005) A chemotactic peptide from laminin alpha 5 functions as a regulator of inflammatory immune responses via TNF alpha-mediated signaling. J. Immunol. 174, 1621–1629. Corbalan-Garcia, S., Sanchez-Carrillo, S., Garcia-Garcia, J. and Gomez-Fernandez, J.C. (2003) Characterization of the membrane binding mode of the C2 domain of PKC epsilon. Biochemistry 42, 11661–11668. Jose Lopez-Andreo, M., Gomez-Fernandez, J.C. and CorbalanGarcia, S. (2003) The simultaneous production of phosphatidic acid and diacylglycerol is essential for the translocation of protein kinase C epsilon to the plasma membrane in RBL-2H3 cells. Mol. Biol. Cell 14, 4885–4895. Dong, C., Davis, R.J. and Flavell, R.A. (2002) MAP kinases in the immune response. Annu. Rev. Immunol. 20, 55–72. Welti, R., Li, W., Li, M., Sang, Y., Biesiada, H., Zhou, H.E., Rajashekar, C.B., Williams, T.D. and Wang, X. (2002) Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis. J. Biol. Chem. 277, 31994–32002. Zhang, W., Qin, C., Zhao, J. and Wang, X. (2004) Phospholipase D alpha 1-derived phosphatidic acid interacts with ABI1 phosphatase 2C and regulates abscisic acid signaling. Proc. Natl. Acad. Sci. USA 101, 9508–9513. Mazie, A.R., Spix, J.K., Block, E.R., Achebe, H.B. and Klarlund, J.K. (2006) Epithelial cell motility is triggered by activation of the EGF receptor through phosphatidic acid signaling. J. Cell Sci. 119, 1645–1654. Zouwail, S., Pettitt, T.R., Dove, S.K., Chibalina, M.V., Powner, D.J., Haynes, L., Wakelam, M.J. and Insall, R.H. (2005) Phospholipase D activity is essential for actin localization and actin-based motility in Dictyostelium. Biochem. J. 389, 207– 214. Quintana, A., Giralt, M., Rojas, S., Penkowa, M., Campbell, I.L., Hidalgo, J. and Molinero, A. (2005) Differential role of tumor necrosis factor receptors in mouse brain inflammatory responses in cryolesion brain injury. J. Neurosci. Res. 82, 701–716. Holtmann, M.H. and Neurath, M.F. (2004) Differential TNFsignaling in chronic inflammatory disorders. Curr. Mol. Med. 4, 439–444.