Scabronine G-Methylester Enhances Secretion of Neurotrophic Factors Mediated by an Activation of Protein Kinase C-z

Glial cells release neurotrophic factors that maintain neurons functionally. Previously, we have shown that the scabronines isolated from Sarcodon scabrosus enhanced the secretion of neurotrophic factors from 1321N1 human astrocytoma cells. In the present study, we examined the mechanism of newly synthesized scabronine G-methylester (ME)-induced secretion of neurotrophic factors from 1321N1 cells. The dramatic neuronal differentiation of rat pheochromocytoma cells (PC-12) was observed by scabronine G-ME-conditioned medium of 1321N1 cells. Scabronine G-ME increased the secretion of nerve growth factor (NGF) and interleukin-6 (IL-6) from 1321N1 cells with the enhancement of their mRNA expressions. Scabronine G-ME concentration-dependently inhibited the carbachol-induced inositol phosphate accumulation in 1321N1 cells, which was reversed by GF109203X, an inhibitor of protein kinase C (PKC) isoforms. Furthermore, GF109203X inhibited the scabronine G-ME-induced mRNA expressions of both NGF and IL-6 and the differentiation of PC-12 cells, showing that scabronine G-ME activated PKC. Although scabronine G-ME enhanced activities of neither conventional nor novel types of PKCs, it translocated PKC-z to membranes in intact cells and cell-free condition. Furthermore, recombinant PKC-z activity was also increased by scabronine G-ME, suggesting the involvement of PKC-z in the effect of scabronine G-ME. Concerning the downstream effectors of the PKC-z, scabronine G-ME translocated nuclear factor-kB to nucleus, and enhanced its transcriptional activity. In addition, scabronine G-ME caused the degradation of inhibitor of nuclear factor-kB concentration-dependently, which was inhibited by GF109203X. These results suggest that scabronine G-ME potentially enhances the secretion of neurotrophic factors from 1321N1 cells mediated via the activation of PKC-z. Neurotrophic factors are essential for neurons to maintain and organize themselves functionally. Glial cells support neurons by releasing neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin 3 (Althaus and Richter-Landsberg, 2000), interleukin-6 (IL-6) (Schwaninger et al., 1997) and glia-derived neurotrophic factor (Lin et al., 1993). NGF has pleiotropic effects on the promotion of neuronal differentiation and survival (prevention of apoptosis) in various neurons (Levi-Montalcini, 1987). IL-6 promotes neuronal survival and differentiation of rat pheochromocytoma cells (PC-12) (Satoh et al., 1988), whereas it induces the proliferation of glial cells (Balasingam et al., 1994). It has been reported that C6 rat glioma cells synthesize NGF upon the stimulation by b-adrenergic agonists forskolin or cyclic AMP analogs (Fukumoto et al., 1994; Colangelo et al., 1996), indicating that the accumulated cyclic AMP increases NGF mRNA expression. On the other hand, NGF mRNA expression is dramatically enhanced by phorbol ester in astrocytes (Jehan et al., 1995), suggesting the involvement of protein kinase C (PKC). The phorbol ester-induced enhancement of NGF synthesis was assumed to be through the activation of activator protein-1 (AP-1) known as Fos/Jun homoor heterodimer complex (Jehan et al., 1995; Colangelo et al., 1996). In fact, an AP-1 consensus sequence exists within downstream of the TATA box at the junction of the exon I/intron I region of rat and mouse NGF gene (D’Mello and Heinrich, 1991). It has been shown that nuclear factor kB (NF-kB) regulates gene expression in NGF synthesis induced by lipopolysaccharide, IL-1b, and tumor necrosis facThis work was partly supported by Grants-in-Aid 08672496 and 09470497 (N.N.) from Scientific Research from the Ministry of Education, Science, Sport and Culture of Japan, and from Japan Society for the Promotion of Science Grant 01532 (Yu.O.). ABBREVIATIONS: NGF, nerve growth factor; IL-6, interleukin-6; PC-12, pheochromocytoma cells; PKC, protein kinase C; AP-1, activator protein-1; NF-kB, nuclear factor-kB; ME, methylester; EMEM, Eagle’s minimal essential medium; I-kB, inhibitor of nuclear factor-kB; PMA, phorbol-12-myristate-13-acetate; ELISA, enzyme-linked immunosorbent assay; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphatebuffered saline; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; PC-PLC, phosphatidylcholine-specific phospholipase C; RT, reverse transcription; PCR, polymerase chain reaction. 0026-895X/01/5905-1287–1297$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 5 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 299/899547 Mol Pharmacol 59:1287–1297, 2001 Printed in U.S.A. 1287 at A PE T Jornals on O cber 9, 2017 m oharm .aspeurnals.org D ow nladed from tor-a (Heese et al., 1998a,b). Galve-Roperh et al. (1997) showed that mitogen-activated protein kinase cascade was involved in NGF synthesis induced by sphingomyelinase and ceramide. In addition, we have shown that an increase in intracellular Ca induced by maitotoxin stimulates the synthesis of NGF in C6-BU-1 glioma cells (Obara et al., 1999b). Thus, the expression of NGF mRNA is assumed to be regulated through multiple signaling pathways. However, the detailed mechanism of NGF synthesis remains unclear. IL-6 gene transcription is also induced by various stimulations such as IL-1, tumor necrosis factor-a, platelet-derived growth factor, and interferons (Sehgal, 1992). Cyclic AMP, Ca, and diacylglycerol are essential for IL-6 gene transcription (Sehgal, 1992). Furthermore, NF-kB binding site, located at the upstream of IL-6 gene, contributes to the regulation of IL-6 mRNA expression (Sehgal, 1992). Molecular cloning has revealed the existence of 10 discrete isoforms of PKC, representing the products of separate genes (a, g, d, e, z, h, u, and l/i) and alternative splicing of one gene (bI and bII) (Zhou et al., 1994). With respect to structural features and diacylglycerolor Ca dependence, the members of the PKC family have been classified into three groups. The group that is dependent on both diacylglycerol and Ca has been considered conventional/classic PKCs (a, bI, bII, and g), whereas one that is dependent on diacylglycerol but not Ca has been regarded as novel/nonclassic PKCs (d, e, h, and u). Both conventional/classic PKC and novel/nonclassic PKC groups have been shown to be sensitive to phorbol esters. The third group of PKC isoform is considered as atypical PKCs (z and l/i), which require neither Ca nor diacylglycerol. In addition, the atypical PKC family is neither activated nor down-regulated by phorbol esters. PC-12 cells have been used as an in vitro model of neuronal differentiation. In response to NGF, these cells extend neurites and develop the characteristics of sympathetic neurons (Greene and Tischler, 1976). Human astrocytoma cells (1321N1) have been used as a model of glial cells to examine receptor-mediated events (Nakahata and Harden, 1987; Nakahata et al., 1989). We have previously reported that 1321N1 cells released a new neurotrophic factor that causes the differentiation of PC-12 cells (Obara et al., 1998). Recently, we isolated new cyathane diterpenoids from S. scabrosus, termed scabronines (Kita et al., 1998; Ohta et al., 1998). Scabronine A and G have been shown to promote the secretion of neurotrophic factors, including NGF from 1321N1 cells, causing the enhancement of differentiation of PC-12 cells (Obara et al., 1999a). The present study was undertaken to examine the effect of scabronine G-methylester (ME), the newly synthesized scabronine derivative, and its mechanism of the secretion of neurotrophic factors from 1321N1 cells. Experimental Procedures Materials. NGF was obtained from Sigma (St. Louis, MO). The NGF enzyme-linked immunosorbent assay (ELISA) kit was purchased from Roche Molecular Biochemicals (Mannheim, Germany). The IL-6 ELISA kit was purchased from Cosmobio (Tokyo, Japan). The total RNA extraction kit was from Amersham Pharmacia Biotech (Piscataway, NJ). The reverse transcription-polymerase chain reaction (RT-PCR) kit and LipoTAXI transfection kit were from Toyobo Co., Ltd (Osaka, Japan). Protein kinase C enzyme assay system and [g-P]ATP were from Amersham Pharmacia Biotech (Buckinghamshire, England). Phorbol-12-myristate-13-acetate (PMA) and GF109203X were from Wako Pure Chemicals (Tokyo, Japan). Gö6976 was from Calbiochem (La Jolla, CA). [H]inositol (23.4 Ci/mmol) was from PerkinElmer Life Science Products (Boston, MA). Anion exchange column (AG1X8) was from Bio-Rad (Hercules, CA). Purification of Scabronine G. Scabronine G was purified from the fruit bodies of the mushroom S. scabrosus, as described previously (Kita et al., 1998; Ohta et al., 1998). The melting point of scabronine G was 68.5–69.5°C. Its mass spectrometry analysis (highresolution electron impact ionization-mass spectrometry) showed m/z 330.1829 (M, calcd 330.1831 for C20H2604). Methyl Esterification of Scabronine G. Scabronine G-methylester was synthesized from scabronine G as follows: scabronine G was dissolved in acetic ester, and then diazomethane in ether was added into the solution until the color of the solution turned yellow. After the reaction, scabronine G-ME was isolated by column chromatography. Cell Culture. 1321N1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal calf serum (Cell Culture Laboratory, Cleveland, OH), penicillin (50 units/ml), and streptomycin (50 mg/ml) in an incubator containing 5% CO2 at 37°C. PC-12 cells were grown in DMEM supplemented with 10% fetal calf serum, 5% horse serum (ICN Biochemicals, Costa Mesa, CA), and the above-mentioned antibiotics in an incubator containing 5% CO2 at 37°C. Evaluation of Neurite Outgrowth. After PC-12 cells were incubated with drugs, cells were fixed with 1% glutaraldehyde (Wako, Tokyo, Japan) in phosphate-buffered saline (PBS), and cell morphology was observed under a phase-contrast microscope. The neurite outgrowth of PC-12 cells observed under a phase contrast microscope was regarded as a sign of neuronal differentiation (Obara et al., 1998). The differentiation of PC-12 cells was scored as follows: cells without neurite outgrowth were scored 0, cells bearing neurites as long as one cell diameter were scored 1, cells bearing neurites 2 to 3 times longer length than their diameter were scored 2, and cells bearing neurites that were extremely long or forming a synapse were scored 3. The mean differentiation score was obtained for seventy PC-12 cells in each well. Data are expressed as means 6 S.E.M. of the values of three to four wells. Enzyme Immunoassay. Enzyme immunoassay of NGF was carried out as described in the previous report with slight modifications (Obara et al., 1999a). Briefly, 1321N1 cells were seeded onto six-well plates. The medium was substituted with DMEM supplemented with 0.5% fetal calf serum, and the cells were further cultivated overnight. Drugs in DMEM supplemented with 0.5% fetal calf serum were added to the well. The cells were cultivated for 24 h in the presence of the drugs, and 1 ml of the condition medium was collected. The NGF content in the medium was measured by using a sandwich ELISA according to the instructions of NGF ELISA kit. The same samples as NGF ELISA were used for IL-6 sandwich ELISA with horseradish peroxidase-conjugated anti-IL-6 monoclonal antibody according to the instructions of IL-6 ELISA kit. Semiquantitative RT-PCR. Total RNA from 1321N1 cells was extracted by using a total RNA extraction kit, and semiquantitative RT-PCR was carried out by using a RT-PCR kit. NGF mRNA expression was examined as described previously (Obara et al., 1999a). In brief, the sense primer (59-CTT CAG CAT TCC CTT GAC AC-39, 316–335 of human NGF cDNA) and the antisense primer (59-AGC CTT CCT GCT GAG CAC ACA-39, 889–909) were complementary to conserved regions of the cDNA from both mouse and human NGF. The NGF cDNA of 594 base pairs was amplified 35 cycles (94°C for 60 s, 57°C for 30 s, and 72°C for 50 s). For analysis of IL-6 mRNA, the sense primer (59-AAA TTC GGT ACA TCC TCG AC-39 of human IL-6 cDNA) and the antisense primer (59-CAG GAA CTG GAT CAG GAC TT-39), which were complementary to conserved regions of cDNA from human IL-6, were used. The IL-6 cDNA of 295 base pairs was amplified 39 cycles (95°C for 60 s, 56°C for 60 s, and 74°C for 60 s). 1288 Obara et al. at A PE T Jornals on O cber 9, 2017 m oharm .aspeurnals.org D ow nladed from Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) transcripts with 450or 700-base pair length were used as a positive control. The number of cycles that yielded a quantitative amount of product was determined in a preliminary experiment. Measurement of Inositol Phosphates. Accumulation of inositol phosphates was measured as follows: 1321N1 cells were cultivated in 12-well plates for 2 days at the density of 2 3 10 cells/ml/well, and were labeled with 2 mCi/ml [H]inositol overnight. After washing twice with Eagle’s minimum essential medium (EMEM)-HEPES buffer (pH 7.4), the cells were preincubated in EMEM-HEPES containing 10 mM LiCl for 10 to 14 min, and they were incubated with drugs for an additional 10 to 14 min. Reaction was terminated by the addition of 1 ml of ice-cold 5% trichloroacetic acid after aspiration of the medium. The trichloroacetic acid extracts were washed three times with diethyl ether to remove trichloroacetic acid. Diethyl ether in the sample was removed by keeping at 47°C for 30 min. Total [H]inositol phosphates were separated by anion exchange column (AG-1X8, formate form, 100–200 mesh) as described previously (Nakahata et al., 1989). PKC Kinase Assay. PKC kinase activity was examined using by protein kinase C enzyme assay system with slight modifications. Briefly, 1321N1 cells were cultivated on 35-mm dish for 2 days at a density of 4 3 10 cells/ml, the medium was replaced with DMEM supplemented with 0.5% fetal calf serum, and the cells were further cultivated overnight. The cells were incubated with drugs at 37°C for appropriate time in EMEM-HEPES. Then, after aspiration of the medium, the cells were scraped and homogenized by sonication in homogenization buffer (10 mM Tris-HCl, 150 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 10 mg/ml aprotinin, pH 7.4 measured at 4°C). The cell homogenates were then incubated with the substrate and [g-P]ATP (40 mCi/ml) for 15 min at 37°C in a final volume of 55 ml according to the instructions of protein kinase C enzyme assay system except that PMA in the buffer was removed. Synthetic peptide (Arg-Lys-Arg-Thr-Leu-Arg-Arg-LeuOH), which was a part of epidermal growth factor receptor peptide (amino acids 651–658), was used as the substrate for PKC. After terminating the reaction by addition of stop regent (10 ml), each sample (35 ml) was spotted onto phosphocellulose paper. The papers were washed twice for 2 min in 75 mM H3PO4, and then washed twice for 2 min in distilled water. The radioactivity on each binding paper was determined by scintillation counting. For PKC-z kinase assay in cell-free condition, human recombinant PKC-z (10 ng) (Upstate Biotechnology, Lake Placid, NY), PKC-a-derived pseudosubstrate peptide (2 mg) (Life Technologies, Rockville, MD) and phosphatidylserine (5 mg) (Avanti, Alabaster, AL) were dissolved in 40 ml of kinase buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, and 1 mM sodium orthovanadate). PKC-a-derived pseudosubstrate peptide {[Ser] PKC (19–31); Arg-Phe-Ala-Arg-Lys-Gly-Ser-Leu-Arg-GlnLys-Asn-Val} was used as a substrate for PKC-z (Kochs et al., 1993). Drugs in 10 ml of the buffer were added to the tube and incubated at room temperature for 10 min. Then, the tube was incubated at 30°C for 15 min after addition of 5 ml of [P]ATP (0.2 mCi/tube) solution. The following procedure was the same as described above. SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting for PKC and I-kBa. Samples for PKC detection were prepared Fig. 1. Chemical structures of scabronine G and scabronine G-ME. Fig. 2. Effects of scabronines on glial cell-mediated morphological changes in PC-12 cells. A, morphological changes of PC-12 cells by the 1321N1 cell culture medium conditioned by scabronines. 1321N1 cells (5 3 10 cells/ml) were incubated for 2 days in DMEM containing 5% fetal calf serum in the presence of scabronine G (100 mM) or scabronine G-ME (100 mM). Then PC-12 cells were cultivated for 2 days in the conditioned 1321N1 cell culture medium. Phase-contrast microscopy of PC-12 cells after addition of DMEM supplemented with 5% fetal calf serum, 1321N1 cell culture medium conditioned by 0.1% dimethyl sulfoxide as a vehicle of the scabronines (control), scabronine G (100 mM), scabronine G-ME (100 mM), and NGF (50 ng/ml). Scale bar, 50 mm. B, evaluation of differentiation scale of PC-12 cells. The differentiation of PC-12 cells was evaluated as described under Experimental Procedures and experiments were performed as shown in A. Values represent the means 6 S.E.M. for three wells. Scabronine G and scabronine G-ME significantly accelerated differentiation compared with control (*P , 0.05). Scabronine G-ME also significantly promoted the differentiation of PC-12 cells compared with scabronine G (†P , 0.05). Neurotrophic Factor Secretion via PKC-z by Scabronines 1289 at A PE T Jornals on O cber 9, 2017 m oharm .aspeurnals.org D ow nladed from as follows: 1321N1 cells were seeded onto a 35-mm dish at a density of 4 3 10 cells/ml. The medium was replaced with DMEM supplemented with 0.5% fetal calf serum, and the cells were cultivated overnight. Cells were incubated with drugs in serum-free EMEMHEPES for various periods. After aspiration of the medium, the homogenization buffer (the same as described in the previous section) was added to terminate the reaction. The cells were homogenized and centrifuged at 100,000g for 60 min at 4°C. The supernatant was used as cytosolic fraction. The pellets were dissolved in the same buffer supplemented with 0.5% Triton X-100. After incubation for 30 min at 4°C, it was centrifuged at 100,000g for 30 min at 4°C. The supernatant was regarded as membrane fraction (Trilivas et al., 1991). Those fractions were dissolved in Laemmli sample buffer (final concentration, Tris-HCl 75 mM, SDS 2%, glycerol 15%, 2-mercaptoethanol 3%, pH 6.8), and boiled at 95°C for 5 min. For the preparation of samples for I-kBa detection, 1321N1 cells were seeded onto six-well plates at a density of 2 3 10 cells/ml. The medium was replaced with DMEM supplemented with 0.5% fetal calf serum and the cells were cultivated overnight. Cells were incubated with drugs in serum-free EMEM-HEPES for various periods. After aspiration of the medium, Laemmli sample buffer was added to terminate the reaction. The sample was boiled at 95°C for 5 min. The electrophoresis was performed on 8% acrylamide gels. Proteins were transferred electrically from the gel onto polyvinylidene difluoride membrane (Millipore Corporation, Bedford, MA) by the semidry blotting method. The blots were blocked for 2 h with 1% bovine serum albumin in Tris-buffered saline at 25°C, and incubated with anti-PKC-a (Life Technologies, Gaithersbrug, MD) (1:2000 dilution), anti-PKC-z (Santa Cruz Biochemicals, Santa Cruz, CA) (1: 2000 dilution), or anti-I-kBa (Santa Cruz Biochemicals) (1:1000 dilution) antibodies overnight at 4°C. The blots were washed several times and incubated with a 1:2000 dilution of alkaline phosphataseconjugated goat anti-rabbit IgG antibody (New England Biolabs, Beverly, MA) in Tris-buffered saline containing 1% bovine serum albumin at 25°C for 2 h. Blots were developed using a chemiluminescence assay kit, and visualized by the exposing the chemiluminescence from the membrane to the Hyperfilm enhanced chemiluminescence. The density of the bands corresponding to I-kBa was analyzed by densitometry (Advanced American Biotechnology, Fullerton, CA) and the data were expressed as percentage of control. Immunostaining of NF-kB. 1321N1 cells were seeded onto 24well plates at a density of 2 3 10 cells/ml. The medium was replaced with DMEM supplemented with 0.5% fetal calf serum and the cells Fig. 3. Effects of scabronines on the synthesis and secretion of NGF from 1321N1 cells. A, effect of scabronines on NGF secretion from 1321N1 cells. After incubation with compounds (100 mM) for 24 h, NGF released from 1321N1 cells was measured using NGF ELISA kit. Values are the means 6 S.E.M. of three determinations. Scabronine G and scabronine G-ME significantly increased NGF secretion, compared with control (*P , 0.05). Scabronine G-ME also significantly increased NGF secretion compared with scabronine G (†P , 0.05). B, effects of scabronines on NGF mRNA expression in 1321N1 cells. The cells were stimulated by the compounds for 4 h, and then total RNA from 1321N1 cells was reverse transcribed, followed by PCR as described under Experimental Procedures. The amount of G3PDH mRNA in each cell condition was also shown. Vehicle, lane 1; scabronine G (100 mM), lane 2; scabronine G-ME (100 mM), lane 3. Data are representative of three separate experiments. Fig. 4. Effects of scabronines on the synthesis and secretion of IL-6 from 1321N1 cells. A, effects of scabronines on IL-6 secretion from 1321N1 cells. After incubation with the indicated concentrations of the compounds for 24 h, IL-6 released from 1321N1 cells was measured using IL-6 ELISA kit. Values are the means 6 S.E.M. of three determinations. Scabronine G-ME significantly increased IL-6 secretion, compared with control (*P , 0.05) and scabronine G (†P , 0.05). B, effects of scabronines on IL-6 mRNA expression in 1321N1 cells. The cells were stimulated by the compounds for 4 h, and then total RNA from 1321N1 cells was reverse transcribed, followed by PCR as described under Experimental Procedures. The G3PDH mRNA in each cell condition was also shown. Vehicle, lane 1; scabronine G (100 mM), lane 2; scabronine G-ME (100 mM), lane 3. Data are representative of two separate experiments. 1290 Obara et al. at A PE T Jornals on O cber 9, 2017 m oharm .aspeurnals.org D ow nladed from were cultivated overnight. Cells were incubated with drugs in serum-free EMEM-HEPES for various periods. After the incubation, the cells were fixed with 1% glutaraldehyde/PBS for 15 min. Then, they were permeated with 0.5% Triton X-100/PBS for 5 min after washing with PBS. After blocking with skim milk (0.5%) for 1 h at 37°C, cells were incubated with a mouse anti-human NF-kB (p65) monoclonal antibody (2 mg/ml) (Roche Molecular Biochemicals) at 4°C overnight, followed by goat fluorescein-labeled anti-rabbit IgG antibody (6.25 mg/ml) (Kirkegaard & Perry Laboratories, Gaithersburg, MD) at 37°C for 30 min. Then cells were visualized under a confocal laser microscope (DMRB/E, TCS NT; Leica, Wetzlar, Germany). Transfection and Reporter Assay. The pNF-kB-SEAP reporter plasmid (CLONTECH, Palo Alto, CA) containing the four tandem copies of the NF-kB consensus sequence and secreted alkaline phosphatase was transfected into 1321N1 cells using LipoTAXI transfection kit. The pTAL-SEAP plasmid (CLONTECH), which lacks the NF-kB consensus sequence, was used as a negative control to measure the background signals. Briefly, 1321N1 cells were seeded onto 12-well plates at 3 3 10 cells/ml and cultivated for a day. The plasmid (0.7 mg/well) and transfection reagent (10 ml/well) were mixed gently in DMEM (90 ml) and incubated for 30 min at room temperature. After the addition of DMEM (200 ml), this entire mixture was transferred to the cell-cultured dish, and the cells were incubated for 5 h at 37°C. DMEM (300 ml) containing 10% fetal calf serum was added to the cell-cultured dish, and cells were incubated overnight. Next day, the medium was replaced with the normal fresh medium and incubated for 36 h. One night before the experiment, medium was substituted with DMEM containing 0.5% fetal calf serum. 1321N1 cells were incubated with drugs at 37°C for 6 h in Krebs-Ringer buffer-HEPES (130 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 4.0 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, 11.5 mM glucose, 20 mM HEPES, pH 7.4), and then medium was collected. After centrifugation to remove contaminating cells, 75 ml of sample was incubated in 20 ml of substrate solution (p-nitrophenyl phosphate disodium salt, 0.1 mg/ml of 1 M diethanolamine-HCl, pH 9.5) until the color of the solution turned light yellow. The absorbance at 415 nm subtracted with the value of background was regarded as the relative NF-kB activity. Statistical Methods. Data were expressed as mean values with 6 S.E.M., and the significant difference was determined using analysis of variance.