Justicidin A Inhibits the Transport of Tumor Necrosis Factor- to Cell Surface in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Exposure of macrophages to lipopolysaccharide (LPS) induces release of tumor necrosis factor(TNF), which is initially synthesized as a 26-kDa pro-TNFfollowed by proteolytic processing to a 17-kDa secreted form. In this study, justicidin A, an arylnaphthalide lignan isolated from Justicia procumbens, was found to inhibit LPS-stimulated TNFrelease from RAW 264.7 macrophages in a concentrationand time-dependent manner, and the underlying mechanism was investigated. In the presence of justicidin A, challenge with LPS increased the steady-state level of the 26-kDa membrane-bound form of TNFprotein, whereas justicidin A had little effect on the expression of TNFmRNA and on the synthesis of pro-TNFprotein. Results of the pulse-chase experiment, revealed that the conversion of pro-TNFto mature TNFwas inhibited by justicidin A. Moreover, justicidin A suppressed the transport of TNFto cell surface as analyzed by flow cytometry. The immunofluorescence analysis demonstrated that large amounts of LPS-induced TNFaccumulated primarily within Golgi complex. These results indicate that justicidin A inhibits TNFrelease at the step of transport of pro-TNFto cell surface, and this leads to the accumulation of TNFin Golgi complex in RAW 264.7 macrophages. Tumor necrosis factor (TNF) is an important pro-inflammatory cytokine with a broad range of immune and inflammatory functions. TNFis generally considered the principal mediator of septic shock, and its overexpression is associated with autoimmune diseases such as rheumatoid arthritis and Crohn’s disease (Beutler, 1999). TNFis initially synthesized as a 26-kDa pro-TNF, a type II integral membrane protein, and then is proteolytically cleaved to release a mature 17-kDa secreted form (Kriegler et al., 1988; Perez et al., 1990). Both forms of TNFare biological active, in which the 26-kDa pro-TNFpresents at restricted sites and acts by cell-cell contact, whereas the 17-kDa TNFexists systemically and acts in paracrine and autocrine modes (Kriegler et al., 1988). Pro-TNFis processed by TNFconverting enzyme (TACE), which is a metalloproteinase ADAM 17 that belong to the ADAM family (Black et al., 1997; Moss et al., 1997). The cleavage of pro-TNFoccurs primarily at the cell surface (Black et al., 1997; Glaser et al., 1999). On the other hand, there is evidence that the earlier proteolytic processing occurs in Golgi complex (Solomon et al., 1997; Watanabe et al., 1998). It is well documented that the synthesis of TNFis regulated at the stages of transcriptional, post-transcriptional, and translational levels (Han et al., 1990; Biragyn and Nedospasov, 1995; Lewis et al., 1998; Raabe et al., 1998; Anderson, 2000). On exposure to LPS, the steady-state levels of TNFmRNA and the translation rate of TNFprotein increased in activated macrophages. The synthesized TNFprotein initially accumulates primarily within the Golgi complex and then travels through the secretory pathway to the cell surface (Shurety et al., 2000). A transient appearance of TNFon the cell surface (Solomon et al., 1997; Shurety et al., 2000) is followed by either degradation or endocytosis of this protein (Shurety et al., 2001). However, the mechanisms of processing, trafficking, and secretion of TNFin cells in response to LPS is far from clear. Justicidin A, an arylnaphthalide lignan isolated from the acanthaceous plant Justicia procumbens (Fukamiya and Lee, 1986), has been shown to have strong antiviral activity (Asano et al., 1996), potent cytotoxic effects (Day et al., 1999, 2002), and enhanced TNFgeneration in LPS-stimulated This work was supported by research grants from the National Science Council (NSC-89–2320-B-075A-010) and Taichung Veterans General Hospital (TCVGH-917316D), Taiwan, Republic of China. ABBREVIATIONS: TNF, tumor necrosis factor; TACE, TNFconverting enzyme; ADAM, a disintegrin and metalloprotease; LPS, lipopolysaccharide; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; TBST, Tris-buffered saline-Tween 20; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; RIPA, radioimmunoprecipitation assay. 0026-895X/04/6505-1063–1069$20.00 MOLECULAR PHARMACOLOGY Vol. 65, No. 5 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 2918/1144257 Mol Pharmacol 65:1063–1069, 2004 Printed in U.S.A. 1063 at A PE T Jornals on M ay 8, 2017 m oharm .aspeurnals.org D ow nladed from RAW 264.7 cells at lower concentrations ( 1 M justicidin A) (Day et al., 2002). Surprisingly, we found that at concentrations greater than 5 M, justicidin A significantly inhibited LPS-stimulated TNFsecretion in the present study. This inhibitory effect probably occurs at the post-translational level because justicidin A did not affect the expression of TNFmRNA and the synthesis of pro-TNFprotein. Moreover, justicidin A suppressed the transport of TNFto cell surface and resulted in the accumulation of large amounts of LPS-induced TNFin Golgi complex. Materials and Methods Materials. Justicidin A was isolated and purified as described previously (Fukamiya and Lee, 1986) and dissolved in DMSO. The concentration of DMSO in all experiments was 0.1%. The structure of justicidin A (purity 99%) was characterized by the spectral data of infrared, nuclear magnetic resonance, and mass spectroscopy. Dulbecco’s modified Eagle medium, penicillin, streptomycin, and fetal calf serum were purchased from Invitrogen (Carlsbad, CA). Polyvinylidene difluoride membrane was obtained from Millipore (Bedford, MA). TNFand IL-6 ELISA kits, and the antibodies against mouse TNFand FITC-conjugated anti-mouse TNFwere obtained from R&D Systems (Minneapolis, MN). ECL Western blotting reagent, Hybond-N nylon membranes, and protein A Sepharose were obtained from Amersham Biosciences (Kent, UK). REzol C&T reagent was obtained from Protech Technology (Taiwan). Express Hyb hybridization solution was obtained from BD Biosciences Clontech (Palo Alto, CA). Random primer fluorescein labeling kit and [S]methionine were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). Mouse TNFprimer set was obtained from Stratagene (La Jolla, CA). All other reagents and chemicals were purchased from Sigma (St. Louis, MO). Cell Culture and Cytokines Assay. The RAW 264.7 murine macrophage-like cell line was obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml of penicillin, and 100 g/ml of streptomycin and maintained in humidified incubator with 5% CO2. Cells were passaged every 3 to 4 days with 1:10 split ratio, and the passage number never exceeds 10. For TNFand IL-6 assay, cells were seeded in 96-well plates at 2 10 cells and allowed to adhere overnight. Before stimulation with 1 g/ml of LPS (Escherichia coli, serotype 0111:B4) for 4 h, cells were pretreated with indicated concentration of drug at 37°C for 1 h in a final volume of 200 l. The cell-free culture medium was collected, and TNFand IL-6 content was analyzed using ELISA kit according to the manufacturer’s guidelines. Western Blot Analysis. Cells were washed with PBS twice and harvested in Laemmli SDS sample buffer. Protein extracts were separated by 15% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. Membranes were blocked for 1 h at room temperature in TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween-20) containing 5% nonfat milk. Membranes were washed with TBST buffer and then incubated for 1 h with a polyclonal anti-TNFantibody (1:500). After wash with TBST buffer, a horseradish peroxidase labeled anti-goat IgG (1: 10,000) was added at room temperature for 1 h. The blots were developed using ECL Western blotting reagents. The band intensity was detected by Luminescent Image Analyzer (Fujifilm LAS-3000) using MultiGauge software. Northern Blot Analysis. Total cellular RNA was isolated using REzol C&T reagent (Protech Technology, Taiwan) according to the manufacturer’s instructions. For Northern blot analysis, 20 g of total RNA was denatured in glyoxal/DMSO mixture at 55°C for 1 h, separated by electrophoresis on a 1% agarose gel containing 100 mM sodium phosphate buffer, pH 6.8, and then transferred to Hybond-N nylon membranes. After UV cross-linking, the membranes were prehybridized and then hybridized with TNFcDNA probe using ExpressHyb hybridization solution (BD Biosciences, San Jose, CA) according to the manufacturer’s instructions. TNFcDNA probe was made by RT-PCR amplification using the mouse TNFprimer set and labeled with random primer fluorescein labeling kit. After hybridization, the membranes were washed and subsequently probed with antifluorescein-HRP conjugate antibody and visualized using nucleic acid chemiluminescence reagent. To control for equal loading of RNA, the GAPDH probe was used as an internal control to normalize the TNFmRNA expression. Metabolic Labeling and Immunoprecipitation. Cells were treated with or without justicidin A for 1 h before stimulation or nonstimulation with 1 g/ml of LPS for another 30 min in methionine-free media. After 15-min labeling with 75 Ci/ml [S]methionine, cells were lysed in RIPA buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS, 0.15 M NaCl, and 25 mM Tris-HCl, pH 7.4). The RIPA extracts of cells and the culture media were incubated with TNFantibody and protein A Sepharose beads at 4°C for 1 h. Beads were pelleted and washed three times with RIPA buffer, and then boiled in Laemmli SDS sample buffer. Samples were separated on 15% SDS-PAGE. For pulse-chase experiments, cells were labeled for 15 min and chased with fresh media containing unlabeled methionine for the indicated time intervals. The radioactivity of S-labeled TNFwas detected by PhosphorImager (445SI; Amersham Biosciences) using ImageQuant software. Flow Cytometry. Cells were grown on a six-well plate for 2 days then treated with or without justicidin A for 1 h before stimulation or nonstimulation with 1 g/ml of LPS for another 2 h. After being washed with PBS, cells were stained with FITC-conjugated antiTNFat 4°C for 1 h and counted on a flow cytometer (BD Biosciences). Immunofluorescence Microscopy. Cells were grown on glass coverslips and then treated with indicated drugs for 1 h before stimulation or nonstimulation with 1 g/ml of LPS for 4 h. Cells were then fixed in 4% paraformaldehyde, permeabilized, and subsequently blocked with blocking buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.2% saponin, 2% fetal calf serum, and 1% bovine serum albumin) for 1 h at room temperature. After being incubated with indicated primary antibodies diluted in blocking buffer at 4°C overnight, the FITC-conjugated or rhodamine-conjugated secondary antibody was added. Coverslips were mounted on glass slides in a solution of 50% glycerol in PBS, then examined using a laser scanning confocal microscope (TCS NT; Leica, Wetzlar, Germany). Statistical Analysis. Statistical analyses were performed by the Bonferroni t-test method after analysis of variance. A p value less than 0.05 was considered significant for all tests. Data are expressed as means S.D. of the indicated number of independent experi-