- Email: [email protected]
36 Mutant IκBα
Journal of Surgical Research 82, 48 –55 (1999) Article ID jsre.1998.5503, available online at http://www.idealibrary.com on
Complement Component C3 Production in IL-1b-Stimulated Human Intestinal Epithelial Cells Is Blocked by NF-kB Inhibitors and by Transfection with Ser 32/36 Mutant IkBa 1 M. Ryan Moon, M.D., Alexander A. Parikh, M.D., Timothy A. Pritts, M.D., Josef E. Fischer, M.D., Sarah Cottongim,* Csaba Szabo,* Andrew L. Salzman, M.D.,* and Per-Olof Hasselgren, M.D. 2 *Department of Surgery, University of Cincinnati Medical Center, Division of Critical Care, Children’s Hospital Medical Center, Cincinnati, Ohio 45267; and the Shriners Burn Institute, Cincinnati, Ohio Submitted for publication November 17, 1997
ture medium were measured after 24 h. Cytosolic IkBa was determined by Western blot analysis. Results. TLCK, genistein, and LLnL each inhibited IL-1b-induced C3 production in a dose-dependent fashion. These responses were associated with decreased C3 mRNA levels. In contrast, PDTC did not influence C3 production or C3 mRNA in the Caco-2 cells. Transfection of the Caco-2 cells with the Ser 32/36 mutant IkBa resulted in maintained IkBa levels and decreased IL-b-induced C3 production. Conclusions. IL-1b-stimulated C3 production in the enterocyte may be regulated by NF-kB. © 1999 Academic
Background. Recent studies suggest that interleukin-1b (IL-1b) stimulates the production of the acute phase protein complement component C3 in human intestinal epithelial cells. The transcription factor NF-kB activates different genes involved in the response to cytokines. It is not known if IL-1b-induced C3 production in the enterocyte is regulated by NF-kB. Materials and methods. Cultured Caco-2 cells, a human intestinal epithelial cell line, were treated with one of the NF-kB inhibitors, tosyl-lys-chloromethylketone (TLCK), genistein, or pyrrolidine dithiocarbamate (PDTC), or with N-acetyl-leu-leu-norleucinal (LLnL), a proteasome inhibitor known to block the degradation of IkB, the cytosolic inhibitor of NF-kB. Following this treatment, the Caco-2 cells were stimulated with IL-1b, and C3 levels in the culture medium were measured after 24 h by ELISA. C3 mRNA levels were determined after 4 h by Northern blot analysis. In other experiments, Caco-2 cells were transfected with a mutant IkBa in which serines 32 and 36 were substituted by alanine. This mutation prevents IkBa phosphorylation and subsequent NF-kB nuclear translocation. After transfection, the cells were stimulated with IL-1b, and C3 levels in the cul-
Press
Key Words: enterocyte; interleukin-1; complement component C3; NF-kB. INTRODUCTION
Recent studies suggest that the intestinal mucosa participates in the metabolic response to injury, inflammation, and sepsis [1]. We have previously shown that protein synthesis in the intestinal mucosa is increased during sepsis and endotoxemia [2– 4]. This response may reflect both increased cell proliferation [5] and stimulated synthesis of specific proteins, including certain gut peptides [6, 7]. The acute phase proteins are another class of proteins locally synthesized in intestinal mucosa [8]. Among the acute phase proteins, complement component C3 is particularly important because of its role in both local and systemic defense [9 –11]. Experiments in our laboratory provided evidence that the production of C3 is increased in jejunal mucosa of endotoxemic mice, and immunohistochemistry demonstrated that the enterocyte may be a source of C3 under these conditions [12]. In other studies we
Presented at the 31st Annual Meeting of the Association for Academic Surgeons, Dallas, Texas, November 6 – 8, 1997 1 Supported in part by Grant 8510 from the Shriners of North America. M.R.M. was supported by a research fellowship from the Surgical Infection Society (Zeneca ICI Fellowship Award). A.A.P. was supported by a research fellowship from the Shriners of North America. T.A.P. was supported by NIH Training Grant 1T32GM008478. 2 To whom correspondence should be addressed at University of Cincinnati Medical Center, Department of Surgery, 231 Bethesda Avenue, Mail Location 558, Cincinnati, OH 45267-0558. Fax: (513) 558-2958.
0022-4804/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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MOON ET AL.: C3 PRODUCTION IN HUMAN ENTEROCYTES
found that C3 production in the enterocyte is stimulated by interleukin-1b (IL-1b), and that this stimulation may be regulated at the transcriptional level [13]. The molecular mechanisms of IL-1b–induced C3 production in the enterocyte, however, are not fully understood. Nuclear factor kappa B (NF-kB) is a transcription factor that activates different genes involved in the inflammatory response [14]. NF-kB is a member of the Rel family of transcriptional activator proteins [15] and can be induced by several pathogens and mediators, including IL-1, tumor necrosis factor (TNF), and endotoxin in different cell types [16]. In recent studies in our laboratory, NF-kB was activated by IL-1b in cultured Caco-2 cells [17], but the role of NF-kB in the regulation of IL-1b-induced C3 production in the enterocyte is not known. In the present study, we tested the hypothesis that IL-1b-stimulated production of C3 in the human enterocyte is regulated by NF-kB. This was done by treating cultured Caco-2 cells with substances that have been shown to inhibit NF-kB in different cell types (i.e., tosyl-lys-chloromethylketone, TLCK; genistein; and pyrrolidine dithiocarbamate, PDTC) as well as with N-acetyl-leu-leu-norleucinal (LLnL), a proteasome inhibitor that has been shown to block the breakdown of the inhibitor of NF-kB, IkB [18]. In other experiments, the involvement of NF-kB was determined by transfecting cultured Caco-2 cells with a Ser 32/36 mutant IkBa that is resistant to phosphorylation and therefore prevents activation of NF-kB. MATERIALS AND METHODS Materials. Caco-2 cells were from American Type Culture Collection (Rockville, MD). Dulbecco’s modified Eagle’s medium (DMEM), nonessential amino acids, low-endotoxin bovine serum (FBS), L-glutamine, penicillin, streptomycin, and TRIZOL were purchased from Gibco-BRC (Grand Island, NY). Human recombinant IL-1b was generously provided by Amgen (Boulder, CO). PDTC, TLCK, genistein, LLnL, and all other chemicals, unless otherwise noted, were purchased from Sigma (St. Louis, MO). Cell culture. Caco-2 cells, a human colon adenocarcinoma cell line that has enterocyte-like features in culture [19], were grown at 37°C in 5% CO 2 in DMEM supplemented with 10% FBS, nonessential amino acids, 6 mM glutamine, 10 mM Hepes, 10 mg/ml apotransferrin, 1 mM pyruvate, 24 mM NaHCO 3, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells, between passages 5 and 25, were seeded at a density of 100,000 cells/cm 2 onto 24-well tissue culture plates (Falcon-Becton Dickinson, Franklin Lakes, NJ) for the determination of C3 protein or into 25-cm 2 tissue culture flasks ( FalconBecton Dickinson) for the determination of C3 mRNA. The cells were grown for 72 h to 90% confluence before use. Experimental conditions. Prior to the experiments, the Caco-2 cells were washed three times with serum-free DMEM and were then pretreated for 1 h in serum-free medium containing one of the NF-kB inhibitors PDTC, genistein, TLCK, or LLnL, followed by incubation for 24 h in fresh medium with 0.5 ng/ml of rIL-1b. The concentration of IL-1b used here was based on a recent report in which we found that IL-1b stimulated C3 production in cultured Caco-2 cells with a maximal effect noted at 0.5 ng/ml [13]. After
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treatment of the cultured cells for 24 h, the culture medium was collected for C3 determination, and the cells were tested for viability (see below). When C3 mRNA levels were determined, the cultured cells were incubated with one of the NF-kB inhibitors, followed by incubation with 0.5 ng/ml of rIL-1b for 4 h, whereafter the cells were harvested for mRNA determination as described below. Determination of cell viability. Cell viability was determined by measuring mitochondrial respiration, assessed by the mitochondrialdependent reduction of 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) to formazan as described previously [20]. Cells were incubated with MTT (200 mg/ml) at 37°C for 1 h. The supernatant was then removed and the cells were solubilized in 500 ml of DMSO. Reduction of MTT to formazan was quantitated by measurement of OD 550 using a microplate reader (Molecular Devices, Sunnyvale, CA). Determination of C3. C3 in the culture medium was quantified by enzyme-linked immunosorbent assay (ELISA). Purified human C3 (Calbiochem, San Diego, CA) was used for calibration of the standard curve. Wells of a 96-microtiter plate were coated with 100 ml of a 1:500 dilution of goat antibody to human C3 in carbonate/ bicarbonate buffer (pH 9.5), incubated for 2 h, and washed three times with 0.05% Tween-20 in phosphate buffered saline (PBS). Aliquots of cell culture media were added in triplicate wells in a volume of 100 ml and incubated for 1 h at 37°C. After three washes, 100 ml of a 1:2000 dilution of peroxidase-conjugated goat antibody to human C3 was added to each well and incubated for 1 h at 37°C. The developing reagent consisted of 0.4 mg/ml of o-phenylenediamine in 0.2 M Na 2HPO 4 (pH 5.0 with 0.1 M citric acid), containing 0.8 ml 30% H 2O 2 2/ml. Color was developed for 15 min, and absorbance was measured at 490 nm. The lower limit of the ELISA for detection of C3 was 4 ng/ml. Northern blot analysis. Total RNA was isolated and extracted from cell monolayers by a modification of the acid guanidinium thiocyanate-phenol-chloroform method using a commercially available reagent (TRIZOL) as previously described [21]. Aliquots containing 15 mg of total RNA were fractionated by electrophoresis on a 1% agarose gel containing 5% formaldehyde and were transferred to a nylon membrane (Magna NT, Micro Separations Inc., Westboro, MA). The blots were auto-crosslinked by ultraviolet light (UV Stratalinker 1800, Stratagene, La Jolla, CA) and prehybridized for 4 h at 42°C in a mixture containing 50% formamide, 5X Denhardt’s solution, 5X SSPE (1X SSPE 5 0.15 M NaCl, 0.01 M Na 2PO 4, 0.001 M EDTA), 0.3% sodium dodecyl sulfate (SDS), and 0.25 mg/ml herring sperm DNA. Oligonucleotide primers were constructed from the published cDNA sequence of C3 [22]. The cDNA samples were synthesized by polymerase chain reaction using a Perkin-Elmer DNA thermal cycler. The sequence of the C3 primers was (a) 59TCGGATGACAAGGTCACCCT-39 (coding sense) corresponding to bases 4627– 4646 of the cloned full-length sequence, and (b) 59GACAACCATGCTCTCGGTGA-39 (anti coding sense) which anneals to bases 5015–5034. The membranes were then hybridized overnight at 42°C with the C3 cDNA probe labeled by random priming with [ 32P]dCTP (Pharmacia Biotech, Uppsala, Sweden). The membranes were then serially washed with 2X sodium chloride-sodium citrate (SSC) (1X SSC 5 0.15 M NaCl, 15 mM Na-citrate) and 0.1% SDS at room temperature for 15 min twice, followed by 0.2X SSC and 0.1% SDS at 50°C. After washing, exposure was carried out overnight using a series 400 Phosphoimager (Molecular Dynamics, Sunnyvale, CA) and signals were detected by autoradiography and quantitated by densitometry. Transfection of Caco-2 cells with a Ser 32/36 mutant IkBa. The CMV-MAD-3 dominant negative IkBa mutant was a generous gift from Drs. Thomas Henkel and Patrick Baeuerle (Medigene GmbH, Martinsried, Germany). Briefly, the IkBa construct contained double point mutations substituting serines 32 and 36 by alanine residues, which prevents phosphorylation of IkBa [23]. The construct was cloned into a CMV-MAD-3 expression vector to overexpress the dominant negative mutant. An expression vector containing the CMV-
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JOURNAL OF SURGICAL RESEARCH: VOL. 82, NO. 1, MARCH 1999 ences, Buckingham, England), and exposed on radiographic film (Eastman-Kodak). Statistical analysis. Results are expressed as means 6 SEM. ANOVA followed by Tukey’s test was used for statistical analysis. All experiments were repeated at least three times for evidence of reproducibility.
RESULTS
FIG. 1. The effect of TLCK on IL-1b-induced C3 production in Caco-2 cells. Cultured Caco-2 cells were pretreated with different concentrations of TLCK for 1 h, followed by incubation with 0.5 ng/ml of IL-1b for 24 h. Ctr, cells cultured without additions to the medium; TLCK, cells treated with 200 mM TLCK alone; 0 –200 indicates the concentration of TLCK (mM). n $ 3 for each concentration. *P , 0.05 v IL-1b 1 0 TLCK by ANOVA.
MAD-3 plasmid alone was used as a control for transfections. Caco-2 cells were seeded onto 6-well culture dishes and grown to 50% confluency prior to transfection. The Lipofectin (Gibco, Grand Island, NY) transfection method was used as described elsewhere [24]. Briefly, the DNA was diluted to a concentration of 2 mg/well. Lipofectin (6 mg/well) was incubated with serum-free OPTIMEM at room temperature for 15 min. The Lipofectin mixture was then incubated with DNA complexes for 30 min at room temperature. The Caco-2 cells were washed three times with serum-free medium, and the lipid-DNA complexes were added to the cells. After incubation at 37°C for 24 h, the culture medium was changed to DMEM supplemented with 10% FBS and incubated for an additional 24 h at 37°C. Neomycin (Geneticin, Gibco) resistance conferred by the CMVMAD-3 plasmid was utilized for selection of transfected clones. Western blot analysis. The culture medium was aspirated and the Caco-2 cells were lysed in ice-cold buffer containing 50 mM Tris (pH 8.0), 5 mM EDTA, 1% Triton X-100, 110 mM NaCl, and 100 mg/ml PMSF. Protein concentrations were determined by a Bio-Rad Bradford protein assay kit (Bio-Rad Laboratories). Aliquots of the cell lysates containing 50 mg of protein were boiled in equal volumes of loading buffer (125 mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, and 10% 2-mercaptoethanol) for 3 min and then separated by electrophoresis on an 8 –16% Tris-glycine gradient gel (Novex, San Diego, CA). The proteins were then transferred to nitrocellulose membranes using the Novex Xcell II Blot Module. The membranes were blocked with 10% nonfat dried milk in Trisbuffered saline, pH 7.6 (TBS) containing 0.05% Tween-20 (TTBS) for 1 h and then incubated with a polyclonal rabbit anti-mouse antibody to IkBa (Santa Cruz Laboratories, CA) for 45 min. After washing twice with TTBS, the blots were incubated with a peroxidaseconjugated goat anti-rabbit IgG secondary antibody for 15 min. The blots were washed in TTBS for 5 min three times, incubated in enhanced chemiluminescence reagents (ECL, Amersham Life Sci-
TLCK is a serine protease inhibitor with chymotrypsin-like specificity that has been shown to inhibit the activation of NF-kB in a variety of cell types, including Caco-2 cells [25]. When the Caco-2 cells were preincubated with increasing concentrations of TLCK for 1 h, the IL-1b-induced C3 production was reduced in a dose-dependent manner, with a maximal effect noticed at a TLCK concentration of 200 mM (Fig. 1). Treatment with TLCK alone had no effect on C3 production. Cell viability was unaffected by TLCK up to a concentration of 200 mM (Table 1), but was reduced at higher concentrations. Genistein inhibits tyrosine kinase activity [26] (and may have other effects as well) and has recently been shown to inhibit activation of NF-kB in different cell types [27]. When the Caco-2 cells were preincubated with increasing concentrations (10 –75 mM) of genistein for 1 h before the addition of IL-1b, a dosedependent reduction in IL-1b-induced C3 production was noticed (Fig. 2). Cell viability was unaffected by genistein (Table 1) and genistein alone had no effect on C3 production. Because genistein was solubilized in DMSO prior to addition to the tissue culture, control experiments were performed in which the same concentration of DMSO was added to the cells 1 h before the addition of IL-1b. DMSO did not decrease IL-1binduced C3 production, suggesting that the inhibition of C3 production was caused by genistein itself. PDTC, the pyrrolidine derivative of dithiocarbamate, belongs to a family of metal chelators with antioxidant properties that has been shown to inhibit TABLE 1 Mitochondrial Respiration in Cultured Caco-2 Cells Group
Absorbance for MTT
Control IL-1b TLCK (200 mM) PDTC (200 mM) Genistein (75 mM) LLnL (200 mM)
0.53 6 0.01 0.50 6 0.01 0.49 6 0.03 0.44 6 0.02 0.43 6 0.03 0.46 6 0.02
Note. Cultured Caco-2 cells were pretreated with TLCK (200 mM), PDTC (200 mM), genistein (75 mM), or LLnL (200 mM) followed by stimulation with 0.5 ng/ml of IL-1b for 24 h. Cell viability was assessed by measuring the reduction of MTT to formazan as described under Materials and Methods. Results are means 6 SEM with n $ 3 for each experimental condition.
MOON ET AL.: C3 PRODUCTION IN HUMAN ENTEROCYTES
FIG. 2. The effect of genistein on IL-1b -induced C3 production in Caco-2 cells. Cultured Caco-2 cells were pretreated with different concentrations of genistein for 1 h, followed by incubation with 0.5 ng/ml of IL-1b for 24. Ctr, cells cultured without additions to the medium; genistein, cells treated with 75 mM genistein alone; 0 –75 indicates the concentration of genistein (mM). n $ 3 for each concentration. *P , 0.05 v IL-1b 1 0 genistein by ANOVA.
NF-kB activation in different cell types, including Jurkat T cells [28 –30], pre-B cells [28, 30], and human monocytes [30]. When the Caco-2 cells were preincubated with increasing concentrations of PDTC for 1 h before the addition of IL-1b, C3 production was not affected (Fig. 3). PDTC alone had no effect on C3 production and cell viability was unaffected by PDTC (Table 1). LLnL is a peptide-aldehyde inhibitor of the chymotryptic site of the 20S proteasome, an enzyme complex that degrades ubiquitin-conjugated proteins [31]. Because the ubiquitin-proteasome pathway has been implicated in the processing and regulation of NF-kB in various cell types [18], we next tested the effect of LLnL on enterocyte IL-1b-induced C3 production. When the Caco-2 cells were preincubated with increasing concentrations of LLnL (10 –500 mM) for 1 h before the addition of IL-1b, C3 production was reduced in a dose-dependent manner (Fig. 4). Treatment of the Caco-2 cells with LLnL alone had no effect on C3 production, and cell viability was unaffected by LLnL (Table 1). In previous studies in our laboratory, IL-1b-induced both C3 mRNA expression and C3 production, suggesting regulation of C3 production at the transcriptional level [13]. We next examined whether the NF-kB inhibitors used here decreased C3 mRNA levels. Preincubation of the Caco-2 cells with TLCK (200 mM),
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FIG. 3. The effect of PDTC on IL-1b-induced C3 production in Caco-2 cells. Cultured Caco-2 cells were pretreated with different concentrations of PDTC for 1 h, followed by incubation with 0.5 ng/ml of IL-1b for 24 h. Ctr, cells cultured without additions to the medium; PDTC, cells treated with 500 mM PDTC alone; 0 –500 indicates the concentration of PDTC (mM). n $ 3 for each concentration.
genistein (75 mM), or LLnL (200 mM) for 1 h reduced the IL-1b-induced increase in C3 mRNA levels (Figs. 5 and 6). In contrast, treatment of the cells with PDTC did not reduce C3 mRNA levels. In Caco-2 cells that were transfected with the Ser
FIG. 4. The effect of LLnL on IL-1b-induced C3 production in Caco-2 cells. Cultured Caco-2 cells were pretreated with different concentrations of LLnL for 1 h, followed by incubation with 0.5 ng/ml of IL-1b for 24 h. Ctr, cells cultured without additions to the medium; LLnL, cells treated with 500 mM LLnL alone; 0 –500 indicates the concentration of LLnL (mM). n $ 3 for each concentration. *P , 0.05 v IL-1b 1 0 LLnL by ANOVA.
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FIG. 6. C3 mRNA determined by Northern blot analysis (upper panel) in cultured Caco-2 cells treated with IL-1b (0.5 ng/ml) alone or following treatment for 1 h with LLnL (200 mM). Quantitation of the blots is shown in the lower panel.
FIG. 5. C3 mRNA determined by Northern blot analysis (upper panel) in cultured Caco-2 cells treated with IL-1b (0.5 ng/ml) alone or following treatment for 1 h with PDTC (500 mM), genistein (75 mM), or TLCK (200 mM). Quantitation of the blots is shown in the lower panel.
32/36 IkBa mutant, the IL-1b-induced degradation of IkBa was inhibited (Fig. 7) concomitant with reduced C3 production (Fig. 8). DISCUSSION
In the present study, treatment of cultured Caco-2 cells with TLCK, genistein, or LLnL resulted in a dosedependent decrease in IL-1b-induced C3 production. Because these substances have been shown in other reports to inhibit NF-kB activity in various cell types, the results support (but do not prove) the concept that C3 production in stimulated enterocytes is regulated by NF-kB. A more definitive link between enterocyte C3 production and NF-kB activity was found in the experiments in which IL-1b-induced C3 production was reduced in Caco-2 cells transfected with the Ser 32/36 IkBa mutant. NF-kB responsive elements in the C3 gene [22] further support a role of NF-kB in the regulation of C3 production in the enterocyte. The present results are important because they define some of the intracellular mechanisms of IL-1b-induced C3
production in the human enterocyte. An understanding of the regulation of enterocyte C3 production is important from a clinical standpoint considering the essential role of C3 in both local and systemic responses to sepsis and inflammation [32]. The finding that TLCK decreased IL-1b-induced production of C3 protein and C3 mRNA suggests that a serine protease may be involved in the regulation of C3 by IL-1b, potentially through the NF-kB pathway. Although recent studies in our laboratory suggest that serine protease activity is required for NF-kB activation in the enterocyte [17, 25], the exact mechanism by which TLCK inhibits NF-kB activation is not clear. Most likely, however, the effect of TLCK reflects inhib-
FIG. 7. IL-1b-induced degradation of IkBa in cells transfected with CMV-MAD-3 Ser 32/36 IkBa mutant. Caco-2 cells were transfected with CMV-MAD-3 (control, upper row) or with CMV-MAD-3 Ser 32/36 IkBa (mutant, lower row) followed by stimulation with IL-1b for up to 60 min. IkBa levels were determined by Western blotting.
MOON ET AL.: C3 PRODUCTION IN HUMAN ENTEROCYTES
FIG. 8. IL-1b-induced C3 production in Caco-2 cells transfected with CMV-MAD-3 Ser 32/36 IkBa mutant. Caco-2 cells were transfected with CMV-MAD-3 alone (sham) or with CMV-MAD-3 Ser 32/36 (mutant) followed by stimulation with IL-1b for 24 h. n 5 3 for each group. *P , 0.05 v cells not treated with IL-1b; †P , 0.05 vs IL-1b-stimulated nonmutant cells by ANOVA.
ited breakdown of IkBa secondary to inhibited phosphorylation of the inhibitory protein [33, 34]. It should be noted that while TLCK blocks NF-kB activation in certain cell types by inhibiting the phosphorylation of IkB, it is possible that TLCK induces other metabolic changes as well. PDTC belongs to a family of metal chelators that are known antioxidants [35]. In previous studies, PDTC, as well as other antioxidants, inhibited NF-kB activation in human T and B cells [36], and in monocytes [37], supporting the role of oxygen free radicals in the activation of NF-kB. In the present study, treatment of the Caco-2 cells with PDTC did not decrease C3 production or C3 mRNA levels. This suggests that oxygen-derived radicals may not be involved in the activation of NF-kB in the enterocyte. Indeed, in recent studies in our laboratory, PDTC did not block NF-kB activation in Caco-2 cells [25], indicating that the regulation of NF-kB activation may be different in this cell type than in other cell types. Evidence for oxygen radicalindependent NF-kB activation was reported recently in other epithelial cells as well [38] and distinct signal transduction pathways are probably responsible for the induction of NF-kB in epithelial and lymphoid cells [39]. Recent studies suggest that tyrosine kinase activity is required for NF-kB activation in human monocytes [27]. Genistein is an isoflavone compound isolated from Pseudomonas, with specific protein tyrosine kinase inhibitory properties [26]. The present finding that genistein blocked C3 production and decreased C3 mRNA levels in the Caco-2 cell suggests that tyrosine kinase activity may be necessary for enterocyte C3 production. Because in a recent study we found that
53
genistein did not inhibit NF-kB activity in IL-1bstimulated Caco-2 cells [25] it is possible that the inhibitory effect of genistein on C3 production noted here was not secondary to NF-kB inhibition. The exact relationship among genistein-induced inhibition of C3 production in Caco-2 cells, tyrosine kinase activity, and NF-kB activity remains to be established. LLnL is a peptide-aldehyde compound that inhibits the activity of the mammalian proteasome [31]. Recent studies have demonstrated that IkB degradation is blocked by proteasome inhibitors and that the ubiquitin-proteasome proteolytic pathway is required for NF-kB activation in various cell types [18]. In the present study, preincubation of the Caco-2 cells with LLnL blocked C3 production and decreased C3 mRNA levels, suggesting that the proteasome pathway is involved in the regulation of enterocyte C3 production, possibly via the NF-kB pathway. Although recent studies in our laboratory demonstrated that LLnL inhibits IkBa degradation and subsequent NF-kB activation in the enterocyte (unpublished observations), further studies are needed to determine whether the inhibited C3 production seen following treatment of the Caco-2 cells with LLnL is caused by inhibited NF-kB activity. In a recent study, transfection of Hela cells with a Ser 32/36 IkBa mutant diminished activation of NF-kB [23]. In the present study, transfection with the Ser 32/36 IkBa mutant reduced the degradation of the inhibitory protein IkBa in IL-1b-stimulated cultured Caco-2 cells, probably resulting in inhibited translocation of NF-kB to the nucleus. The finding that the degradation of the mutant IkBa was not completely blocked may reflect partial expression of the transfected plasmid. The observation that transfection of Caco-2 cells with Ser 32/36 IkBa mutant reduced IL1b-induced C3 production strongly supports the concept that C3 production in Caco-2 cells is regulated by NF-kB. Although the different substances that were used in the present study (TLCK, PDTC, genistein, and LLnL) have been shown in a number of previous reports to inhibit NF-kB activation in various cell types [18, 25, 27, 28, 33, 36, 37], they are probably not specific in their action as NF-kB inhibitors but may have other metabolic effects as well. The observation that multiple substances, inhibiting NF-kB activation by different mechanisms, reduced IL-1b-induced C3 production supports our interpretation of the results, i.e., that the inhibited C3 production most likely reflected inhibited NF-kB activation. The strongest support for that argument, however, was found in the experiment in which Caco-2 cells were transfected with a Ser 32/36 mutant IkBa. In these cells, the degradation of IkBa, which is a required step in the activation of NF-kB [28, 29], was inhibited concomitant with reduced IL-1b-induced C3
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production, more firmly linking C3 production with NF-kB activation. It should be noted that although the present results may be consistent with the concept that NF-kB is involved in the regulation of IL-1b-induced C3 production in human enterocytes, the results need to be interpreted with caution for several reasons. First, although the Caco-2 cells used in the present experiments take on the characteristics of small intestinal epithelial cells during culture, they are transformed cells that are derived from a colon cancer cell line [19]. The role of NF-kB in the regulation of C3 production needs to be established in other cell lineages as well before a generalized conclusion regarding enterocyte NF-kB activity and C3 production can be made. Second (as discussed above), the inhibitors used to block NF-kB in our experiments may not be completely specific, but may affect other metabolic processes in the cell as well. The apparently conflicting results of inhibited C3 production (present study) and unaffected NF-kB activity [25] after treatment of the Caco-2 cells with genistein need to be resolved in future experiments.
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Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen, M. Lipofectin: A highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA 84: 7413, 1987.
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Parikh, A. A., Moon, M. R., Fischer, J. E., Szabo, C., Hasselgren, P. O., and Salzman, A. L. IL-1b induction of NF-kB activation in human intestinal epithelial cells is independent of oxyradical signaling. Submitted for publication.
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Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuay, M., and Fukami, Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 262: 5592, 1987.
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Yoza, B. K., Hu, J. Y. Q., and McCall, C. E. Protein-tyrosine kinase activation is required for lipopolysaccharide induction of
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interleukin 1b and NF-kB activation, but not NF-kB nuclear translocation. J. Biol. Chem. 271: 18306, 1996. Henkel, T., Machleidt, T., Alkalay, I., Kronke, M., BenNeriah, Y., and Baeurle, P. A. Rapid proteolysis of IkBa is necessary for activation of transcription factor NF-kB. Nature 365: 182, 1993. Alkalay, I., Yaron, A., Hatzubai, A., Jung, S., Avraham, A., Gerlitz, O., Paashut-Lavon, I., and Ben-neriah, Y. In vivo stimulation of IkB phosphorylation is not sufficient to activate NFkB. Mol. Cell. Biol. 15: 1294, 1995. Schreck, R., Meie, B., Mannel, D. N., Droge, W., and Bauerle, P. A. Dithiocarbamates as potent inhibitors of nuclear factor kB activation in intact cells. J. Exp. Med. 175: 1181, 1992. Rock, K. L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D., and Goldberg, A. L. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78: 761, 1994. Quezado, Z. M. N., Hoffman, W. D., Winkelstein, J. A., Yatsiu, I., Koev, C. A., Cork, L. C., Elin, R. J., Eichacker, P. Q., and Natanson, C. The third component of complement protects against Escherichia coli endotoxin-induce shock and multiple organ failure. J. Exp. Med. 179: 569, 1994. Finco, T. S., Beg, A. A., and Baldwin, A. S. Inducible phosphorylation of IkBa is not sufficient for its dissociation from NF-kB
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and is inhibited by protease inhibitors. Proc. Natl. Acad. Sci. USA 91: 11884, 1994. Finco, T. S., and Baldwin, A. S. Mechanistic aspects of NF-kB regulation: the emerging role of phosphorylation and proteolysis. Immunity 3: 263, 1995. Schreck, R., Rieber, P., and Bauerle, P. A. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kB transcription factor and HIV-1. EMBO J. 10: 2247, 1991. Menon, S. D., Qin, S., Guy, G. R., and Tan, Y. H. Differential induction of nuclear NF-kB by protein phosphatase inhibitors in primary and transformed human cells. J. Biol. Chem. 268: 26805, 1993. Frandenberger, M., Pforte, A., Sternsdorf, T., Passlick, B., Bauerle, P. A., and Ziegler-Heitbrock, H. W. Constitutive nuclear NF-kB in cells of the monocyte lineage. Biochem. J. 304: 87, 1994. Bonizzi, G., Dejardin, E., Piette, B., Piette, J., Merville, P., and Bours, V. Interleukin-1b induces nuclear factor kB in epithelial cells independently of the production of reactive oxygen intermediates. Eur. J. Biochem. 242: 544, 1996. Bonizzi, G., Piette, J., Merville, P., and Bours, V. Distinct signal transduction pathways mediate nuclear factor-kB induction by IL-1b in epithelial and lymphoid cells. J. Immunol. 159: 5264, 1997.
Complement Component C3 Production in IL-1b-Stimulated Human Intestinal Epithelial Cells Is Blocked by NF-kB Inhibitors and by Transfection with Ser 32/36 Mutant IkBa 1 M. Ryan Moon, M.D., Alexander A. Parikh, M.D., Timothy A. Pritts, M.D., Josef E. Fischer, M.D., Sarah Cottongim,* Csaba Szabo,* Andrew L. Salzman, M.D.,* and Per-Olof Hasselgren, M.D. 2 *Department of Surgery, University of Cincinnati Medical Center, Division of Critical Care, Children’s Hospital Medical Center, Cincinnati, Ohio 45267; and the Shriners Burn Institute, Cincinnati, Ohio Submitted for publication November 17, 1997
ture medium were measured after 24 h. Cytosolic IkBa was determined by Western blot analysis. Results. TLCK, genistein, and LLnL each inhibited IL-1b-induced C3 production in a dose-dependent fashion. These responses were associated with decreased C3 mRNA levels. In contrast, PDTC did not influence C3 production or C3 mRNA in the Caco-2 cells. Transfection of the Caco-2 cells with the Ser 32/36 mutant IkBa resulted in maintained IkBa levels and decreased IL-b-induced C3 production. Conclusions. IL-1b-stimulated C3 production in the enterocyte may be regulated by NF-kB. © 1999 Academic
Background. Recent studies suggest that interleukin-1b (IL-1b) stimulates the production of the acute phase protein complement component C3 in human intestinal epithelial cells. The transcription factor NF-kB activates different genes involved in the response to cytokines. It is not known if IL-1b-induced C3 production in the enterocyte is regulated by NF-kB. Materials and methods. Cultured Caco-2 cells, a human intestinal epithelial cell line, were treated with one of the NF-kB inhibitors, tosyl-lys-chloromethylketone (TLCK), genistein, or pyrrolidine dithiocarbamate (PDTC), or with N-acetyl-leu-leu-norleucinal (LLnL), a proteasome inhibitor known to block the degradation of IkB, the cytosolic inhibitor of NF-kB. Following this treatment, the Caco-2 cells were stimulated with IL-1b, and C3 levels in the culture medium were measured after 24 h by ELISA. C3 mRNA levels were determined after 4 h by Northern blot analysis. In other experiments, Caco-2 cells were transfected with a mutant IkBa in which serines 32 and 36 were substituted by alanine. This mutation prevents IkBa phosphorylation and subsequent NF-kB nuclear translocation. After transfection, the cells were stimulated with IL-1b, and C3 levels in the cul-
Press
Key Words: enterocyte; interleukin-1; complement component C3; NF-kB. INTRODUCTION
Recent studies suggest that the intestinal mucosa participates in the metabolic response to injury, inflammation, and sepsis [1]. We have previously shown that protein synthesis in the intestinal mucosa is increased during sepsis and endotoxemia [2– 4]. This response may reflect both increased cell proliferation [5] and stimulated synthesis of specific proteins, including certain gut peptides [6, 7]. The acute phase proteins are another class of proteins locally synthesized in intestinal mucosa [8]. Among the acute phase proteins, complement component C3 is particularly important because of its role in both local and systemic defense [9 –11]. Experiments in our laboratory provided evidence that the production of C3 is increased in jejunal mucosa of endotoxemic mice, and immunohistochemistry demonstrated that the enterocyte may be a source of C3 under these conditions [12]. In other studies we
Presented at the 31st Annual Meeting of the Association for Academic Surgeons, Dallas, Texas, November 6 – 8, 1997 1 Supported in part by Grant 8510 from the Shriners of North America. M.R.M. was supported by a research fellowship from the Surgical Infection Society (Zeneca ICI Fellowship Award). A.A.P. was supported by a research fellowship from the Shriners of North America. T.A.P. was supported by NIH Training Grant 1T32GM008478. 2 To whom correspondence should be addressed at University of Cincinnati Medical Center, Department of Surgery, 231 Bethesda Avenue, Mail Location 558, Cincinnati, OH 45267-0558. Fax: (513) 558-2958.
0022-4804/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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MOON ET AL.: C3 PRODUCTION IN HUMAN ENTEROCYTES
found that C3 production in the enterocyte is stimulated by interleukin-1b (IL-1b), and that this stimulation may be regulated at the transcriptional level [13]. The molecular mechanisms of IL-1b–induced C3 production in the enterocyte, however, are not fully understood. Nuclear factor kappa B (NF-kB) is a transcription factor that activates different genes involved in the inflammatory response [14]. NF-kB is a member of the Rel family of transcriptional activator proteins [15] and can be induced by several pathogens and mediators, including IL-1, tumor necrosis factor (TNF), and endotoxin in different cell types [16]. In recent studies in our laboratory, NF-kB was activated by IL-1b in cultured Caco-2 cells [17], but the role of NF-kB in the regulation of IL-1b-induced C3 production in the enterocyte is not known. In the present study, we tested the hypothesis that IL-1b-stimulated production of C3 in the human enterocyte is regulated by NF-kB. This was done by treating cultured Caco-2 cells with substances that have been shown to inhibit NF-kB in different cell types (i.e., tosyl-lys-chloromethylketone, TLCK; genistein; and pyrrolidine dithiocarbamate, PDTC) as well as with N-acetyl-leu-leu-norleucinal (LLnL), a proteasome inhibitor that has been shown to block the breakdown of the inhibitor of NF-kB, IkB [18]. In other experiments, the involvement of NF-kB was determined by transfecting cultured Caco-2 cells with a Ser 32/36 mutant IkBa that is resistant to phosphorylation and therefore prevents activation of NF-kB. MATERIALS AND METHODS Materials. Caco-2 cells were from American Type Culture Collection (Rockville, MD). Dulbecco’s modified Eagle’s medium (DMEM), nonessential amino acids, low-endotoxin bovine serum (FBS), L-glutamine, penicillin, streptomycin, and TRIZOL were purchased from Gibco-BRC (Grand Island, NY). Human recombinant IL-1b was generously provided by Amgen (Boulder, CO). PDTC, TLCK, genistein, LLnL, and all other chemicals, unless otherwise noted, were purchased from Sigma (St. Louis, MO). Cell culture. Caco-2 cells, a human colon adenocarcinoma cell line that has enterocyte-like features in culture [19], were grown at 37°C in 5% CO 2 in DMEM supplemented with 10% FBS, nonessential amino acids, 6 mM glutamine, 10 mM Hepes, 10 mg/ml apotransferrin, 1 mM pyruvate, 24 mM NaHCO 3, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells, between passages 5 and 25, were seeded at a density of 100,000 cells/cm 2 onto 24-well tissue culture plates (Falcon-Becton Dickinson, Franklin Lakes, NJ) for the determination of C3 protein or into 25-cm 2 tissue culture flasks ( FalconBecton Dickinson) for the determination of C3 mRNA. The cells were grown for 72 h to 90% confluence before use. Experimental conditions. Prior to the experiments, the Caco-2 cells were washed three times with serum-free DMEM and were then pretreated for 1 h in serum-free medium containing one of the NF-kB inhibitors PDTC, genistein, TLCK, or LLnL, followed by incubation for 24 h in fresh medium with 0.5 ng/ml of rIL-1b. The concentration of IL-1b used here was based on a recent report in which we found that IL-1b stimulated C3 production in cultured Caco-2 cells with a maximal effect noted at 0.5 ng/ml [13]. After
49
treatment of the cultured cells for 24 h, the culture medium was collected for C3 determination, and the cells were tested for viability (see below). When C3 mRNA levels were determined, the cultured cells were incubated with one of the NF-kB inhibitors, followed by incubation with 0.5 ng/ml of rIL-1b for 4 h, whereafter the cells were harvested for mRNA determination as described below. Determination of cell viability. Cell viability was determined by measuring mitochondrial respiration, assessed by the mitochondrialdependent reduction of 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) to formazan as described previously [20]. Cells were incubated with MTT (200 mg/ml) at 37°C for 1 h. The supernatant was then removed and the cells were solubilized in 500 ml of DMSO. Reduction of MTT to formazan was quantitated by measurement of OD 550 using a microplate reader (Molecular Devices, Sunnyvale, CA). Determination of C3. C3 in the culture medium was quantified by enzyme-linked immunosorbent assay (ELISA). Purified human C3 (Calbiochem, San Diego, CA) was used for calibration of the standard curve. Wells of a 96-microtiter plate were coated with 100 ml of a 1:500 dilution of goat antibody to human C3 in carbonate/ bicarbonate buffer (pH 9.5), incubated for 2 h, and washed three times with 0.05% Tween-20 in phosphate buffered saline (PBS). Aliquots of cell culture media were added in triplicate wells in a volume of 100 ml and incubated for 1 h at 37°C. After three washes, 100 ml of a 1:2000 dilution of peroxidase-conjugated goat antibody to human C3 was added to each well and incubated for 1 h at 37°C. The developing reagent consisted of 0.4 mg/ml of o-phenylenediamine in 0.2 M Na 2HPO 4 (pH 5.0 with 0.1 M citric acid), containing 0.8 ml 30% H 2O 2 2/ml. Color was developed for 15 min, and absorbance was measured at 490 nm. The lower limit of the ELISA for detection of C3 was 4 ng/ml. Northern blot analysis. Total RNA was isolated and extracted from cell monolayers by a modification of the acid guanidinium thiocyanate-phenol-chloroform method using a commercially available reagent (TRIZOL) as previously described [21]. Aliquots containing 15 mg of total RNA were fractionated by electrophoresis on a 1% agarose gel containing 5% formaldehyde and were transferred to a nylon membrane (Magna NT, Micro Separations Inc., Westboro, MA). The blots were auto-crosslinked by ultraviolet light (UV Stratalinker 1800, Stratagene, La Jolla, CA) and prehybridized for 4 h at 42°C in a mixture containing 50% formamide, 5X Denhardt’s solution, 5X SSPE (1X SSPE 5 0.15 M NaCl, 0.01 M Na 2PO 4, 0.001 M EDTA), 0.3% sodium dodecyl sulfate (SDS), and 0.25 mg/ml herring sperm DNA. Oligonucleotide primers were constructed from the published cDNA sequence of C3 [22]. The cDNA samples were synthesized by polymerase chain reaction using a Perkin-Elmer DNA thermal cycler. The sequence of the C3 primers was (a) 59TCGGATGACAAGGTCACCCT-39 (coding sense) corresponding to bases 4627– 4646 of the cloned full-length sequence, and (b) 59GACAACCATGCTCTCGGTGA-39 (anti coding sense) which anneals to bases 5015–5034. The membranes were then hybridized overnight at 42°C with the C3 cDNA probe labeled by random priming with [ 32P]dCTP (Pharmacia Biotech, Uppsala, Sweden). The membranes were then serially washed with 2X sodium chloride-sodium citrate (SSC) (1X SSC 5 0.15 M NaCl, 15 mM Na-citrate) and 0.1% SDS at room temperature for 15 min twice, followed by 0.2X SSC and 0.1% SDS at 50°C. After washing, exposure was carried out overnight using a series 400 Phosphoimager (Molecular Dynamics, Sunnyvale, CA) and signals were detected by autoradiography and quantitated by densitometry. Transfection of Caco-2 cells with a Ser 32/36 mutant IkBa. The CMV-MAD-3 dominant negative IkBa mutant was a generous gift from Drs. Thomas Henkel and Patrick Baeuerle (Medigene GmbH, Martinsried, Germany). Briefly, the IkBa construct contained double point mutations substituting serines 32 and 36 by alanine residues, which prevents phosphorylation of IkBa [23]. The construct was cloned into a CMV-MAD-3 expression vector to overexpress the dominant negative mutant. An expression vector containing the CMV-
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JOURNAL OF SURGICAL RESEARCH: VOL. 82, NO. 1, MARCH 1999 ences, Buckingham, England), and exposed on radiographic film (Eastman-Kodak). Statistical analysis. Results are expressed as means 6 SEM. ANOVA followed by Tukey’s test was used for statistical analysis. All experiments were repeated at least three times for evidence of reproducibility.
RESULTS
FIG. 1. The effect of TLCK on IL-1b-induced C3 production in Caco-2 cells. Cultured Caco-2 cells were pretreated with different concentrations of TLCK for 1 h, followed by incubation with 0.5 ng/ml of IL-1b for 24 h. Ctr, cells cultured without additions to the medium; TLCK, cells treated with 200 mM TLCK alone; 0 –200 indicates the concentration of TLCK (mM). n $ 3 for each concentration. *P , 0.05 v IL-1b 1 0 TLCK by ANOVA.
MAD-3 plasmid alone was used as a control for transfections. Caco-2 cells were seeded onto 6-well culture dishes and grown to 50% confluency prior to transfection. The Lipofectin (Gibco, Grand Island, NY) transfection method was used as described elsewhere [24]. Briefly, the DNA was diluted to a concentration of 2 mg/well. Lipofectin (6 mg/well) was incubated with serum-free OPTIMEM at room temperature for 15 min. The Lipofectin mixture was then incubated with DNA complexes for 30 min at room temperature. The Caco-2 cells were washed three times with serum-free medium, and the lipid-DNA complexes were added to the cells. After incubation at 37°C for 24 h, the culture medium was changed to DMEM supplemented with 10% FBS and incubated for an additional 24 h at 37°C. Neomycin (Geneticin, Gibco) resistance conferred by the CMVMAD-3 plasmid was utilized for selection of transfected clones. Western blot analysis. The culture medium was aspirated and the Caco-2 cells were lysed in ice-cold buffer containing 50 mM Tris (pH 8.0), 5 mM EDTA, 1% Triton X-100, 110 mM NaCl, and 100 mg/ml PMSF. Protein concentrations were determined by a Bio-Rad Bradford protein assay kit (Bio-Rad Laboratories). Aliquots of the cell lysates containing 50 mg of protein were boiled in equal volumes of loading buffer (125 mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, and 10% 2-mercaptoethanol) for 3 min and then separated by electrophoresis on an 8 –16% Tris-glycine gradient gel (Novex, San Diego, CA). The proteins were then transferred to nitrocellulose membranes using the Novex Xcell II Blot Module. The membranes were blocked with 10% nonfat dried milk in Trisbuffered saline, pH 7.6 (TBS) containing 0.05% Tween-20 (TTBS) for 1 h and then incubated with a polyclonal rabbit anti-mouse antibody to IkBa (Santa Cruz Laboratories, CA) for 45 min. After washing twice with TTBS, the blots were incubated with a peroxidaseconjugated goat anti-rabbit IgG secondary antibody for 15 min. The blots were washed in TTBS for 5 min three times, incubated in enhanced chemiluminescence reagents (ECL, Amersham Life Sci-
TLCK is a serine protease inhibitor with chymotrypsin-like specificity that has been shown to inhibit the activation of NF-kB in a variety of cell types, including Caco-2 cells [25]. When the Caco-2 cells were preincubated with increasing concentrations of TLCK for 1 h, the IL-1b-induced C3 production was reduced in a dose-dependent manner, with a maximal effect noticed at a TLCK concentration of 200 mM (Fig. 1). Treatment with TLCK alone had no effect on C3 production. Cell viability was unaffected by TLCK up to a concentration of 200 mM (Table 1), but was reduced at higher concentrations. Genistein inhibits tyrosine kinase activity [26] (and may have other effects as well) and has recently been shown to inhibit activation of NF-kB in different cell types [27]. When the Caco-2 cells were preincubated with increasing concentrations (10 –75 mM) of genistein for 1 h before the addition of IL-1b, a dosedependent reduction in IL-1b-induced C3 production was noticed (Fig. 2). Cell viability was unaffected by genistein (Table 1) and genistein alone had no effect on C3 production. Because genistein was solubilized in DMSO prior to addition to the tissue culture, control experiments were performed in which the same concentration of DMSO was added to the cells 1 h before the addition of IL-1b. DMSO did not decrease IL-1binduced C3 production, suggesting that the inhibition of C3 production was caused by genistein itself. PDTC, the pyrrolidine derivative of dithiocarbamate, belongs to a family of metal chelators with antioxidant properties that has been shown to inhibit TABLE 1 Mitochondrial Respiration in Cultured Caco-2 Cells Group
Absorbance for MTT
Control IL-1b TLCK (200 mM) PDTC (200 mM) Genistein (75 mM) LLnL (200 mM)
0.53 6 0.01 0.50 6 0.01 0.49 6 0.03 0.44 6 0.02 0.43 6 0.03 0.46 6 0.02
Note. Cultured Caco-2 cells were pretreated with TLCK (200 mM), PDTC (200 mM), genistein (75 mM), or LLnL (200 mM) followed by stimulation with 0.5 ng/ml of IL-1b for 24 h. Cell viability was assessed by measuring the reduction of MTT to formazan as described under Materials and Methods. Results are means 6 SEM with n $ 3 for each experimental condition.
MOON ET AL.: C3 PRODUCTION IN HUMAN ENTEROCYTES
FIG. 2. The effect of genistein on IL-1b -induced C3 production in Caco-2 cells. Cultured Caco-2 cells were pretreated with different concentrations of genistein for 1 h, followed by incubation with 0.5 ng/ml of IL-1b for 24. Ctr, cells cultured without additions to the medium; genistein, cells treated with 75 mM genistein alone; 0 –75 indicates the concentration of genistein (mM). n $ 3 for each concentration. *P , 0.05 v IL-1b 1 0 genistein by ANOVA.
NF-kB activation in different cell types, including Jurkat T cells [28 –30], pre-B cells [28, 30], and human monocytes [30]. When the Caco-2 cells were preincubated with increasing concentrations of PDTC for 1 h before the addition of IL-1b, C3 production was not affected (Fig. 3). PDTC alone had no effect on C3 production and cell viability was unaffected by PDTC (Table 1). LLnL is a peptide-aldehyde inhibitor of the chymotryptic site of the 20S proteasome, an enzyme complex that degrades ubiquitin-conjugated proteins [31]. Because the ubiquitin-proteasome pathway has been implicated in the processing and regulation of NF-kB in various cell types [18], we next tested the effect of LLnL on enterocyte IL-1b-induced C3 production. When the Caco-2 cells were preincubated with increasing concentrations of LLnL (10 –500 mM) for 1 h before the addition of IL-1b, C3 production was reduced in a dose-dependent manner (Fig. 4). Treatment of the Caco-2 cells with LLnL alone had no effect on C3 production, and cell viability was unaffected by LLnL (Table 1). In previous studies in our laboratory, IL-1b-induced both C3 mRNA expression and C3 production, suggesting regulation of C3 production at the transcriptional level [13]. We next examined whether the NF-kB inhibitors used here decreased C3 mRNA levels. Preincubation of the Caco-2 cells with TLCK (200 mM),
51
FIG. 3. The effect of PDTC on IL-1b-induced C3 production in Caco-2 cells. Cultured Caco-2 cells were pretreated with different concentrations of PDTC for 1 h, followed by incubation with 0.5 ng/ml of IL-1b for 24 h. Ctr, cells cultured without additions to the medium; PDTC, cells treated with 500 mM PDTC alone; 0 –500 indicates the concentration of PDTC (mM). n $ 3 for each concentration.
genistein (75 mM), or LLnL (200 mM) for 1 h reduced the IL-1b-induced increase in C3 mRNA levels (Figs. 5 and 6). In contrast, treatment of the cells with PDTC did not reduce C3 mRNA levels. In Caco-2 cells that were transfected with the Ser
FIG. 4. The effect of LLnL on IL-1b-induced C3 production in Caco-2 cells. Cultured Caco-2 cells were pretreated with different concentrations of LLnL for 1 h, followed by incubation with 0.5 ng/ml of IL-1b for 24 h. Ctr, cells cultured without additions to the medium; LLnL, cells treated with 500 mM LLnL alone; 0 –500 indicates the concentration of LLnL (mM). n $ 3 for each concentration. *P , 0.05 v IL-1b 1 0 LLnL by ANOVA.
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JOURNAL OF SURGICAL RESEARCH: VOL. 82, NO. 1, MARCH 1999
FIG. 6. C3 mRNA determined by Northern blot analysis (upper panel) in cultured Caco-2 cells treated with IL-1b (0.5 ng/ml) alone or following treatment for 1 h with LLnL (200 mM). Quantitation of the blots is shown in the lower panel.
FIG. 5. C3 mRNA determined by Northern blot analysis (upper panel) in cultured Caco-2 cells treated with IL-1b (0.5 ng/ml) alone or following treatment for 1 h with PDTC (500 mM), genistein (75 mM), or TLCK (200 mM). Quantitation of the blots is shown in the lower panel.
32/36 IkBa mutant, the IL-1b-induced degradation of IkBa was inhibited (Fig. 7) concomitant with reduced C3 production (Fig. 8). DISCUSSION
In the present study, treatment of cultured Caco-2 cells with TLCK, genistein, or LLnL resulted in a dosedependent decrease in IL-1b-induced C3 production. Because these substances have been shown in other reports to inhibit NF-kB activity in various cell types, the results support (but do not prove) the concept that C3 production in stimulated enterocytes is regulated by NF-kB. A more definitive link between enterocyte C3 production and NF-kB activity was found in the experiments in which IL-1b-induced C3 production was reduced in Caco-2 cells transfected with the Ser 32/36 IkBa mutant. NF-kB responsive elements in the C3 gene [22] further support a role of NF-kB in the regulation of C3 production in the enterocyte. The present results are important because they define some of the intracellular mechanisms of IL-1b-induced C3
production in the human enterocyte. An understanding of the regulation of enterocyte C3 production is important from a clinical standpoint considering the essential role of C3 in both local and systemic responses to sepsis and inflammation [32]. The finding that TLCK decreased IL-1b-induced production of C3 protein and C3 mRNA suggests that a serine protease may be involved in the regulation of C3 by IL-1b, potentially through the NF-kB pathway. Although recent studies in our laboratory suggest that serine protease activity is required for NF-kB activation in the enterocyte [17, 25], the exact mechanism by which TLCK inhibits NF-kB activation is not clear. Most likely, however, the effect of TLCK reflects inhib-
FIG. 7. IL-1b-induced degradation of IkBa in cells transfected with CMV-MAD-3 Ser 32/36 IkBa mutant. Caco-2 cells were transfected with CMV-MAD-3 (control, upper row) or with CMV-MAD-3 Ser 32/36 IkBa (mutant, lower row) followed by stimulation with IL-1b for up to 60 min. IkBa levels were determined by Western blotting.
MOON ET AL.: C3 PRODUCTION IN HUMAN ENTEROCYTES
FIG. 8. IL-1b-induced C3 production in Caco-2 cells transfected with CMV-MAD-3 Ser 32/36 IkBa mutant. Caco-2 cells were transfected with CMV-MAD-3 alone (sham) or with CMV-MAD-3 Ser 32/36 (mutant) followed by stimulation with IL-1b for 24 h. n 5 3 for each group. *P , 0.05 v cells not treated with IL-1b; †P , 0.05 vs IL-1b-stimulated nonmutant cells by ANOVA.
ited breakdown of IkBa secondary to inhibited phosphorylation of the inhibitory protein [33, 34]. It should be noted that while TLCK blocks NF-kB activation in certain cell types by inhibiting the phosphorylation of IkB, it is possible that TLCK induces other metabolic changes as well. PDTC belongs to a family of metal chelators that are known antioxidants [35]. In previous studies, PDTC, as well as other antioxidants, inhibited NF-kB activation in human T and B cells [36], and in monocytes [37], supporting the role of oxygen free radicals in the activation of NF-kB. In the present study, treatment of the Caco-2 cells with PDTC did not decrease C3 production or C3 mRNA levels. This suggests that oxygen-derived radicals may not be involved in the activation of NF-kB in the enterocyte. Indeed, in recent studies in our laboratory, PDTC did not block NF-kB activation in Caco-2 cells [25], indicating that the regulation of NF-kB activation may be different in this cell type than in other cell types. Evidence for oxygen radicalindependent NF-kB activation was reported recently in other epithelial cells as well [38] and distinct signal transduction pathways are probably responsible for the induction of NF-kB in epithelial and lymphoid cells [39]. Recent studies suggest that tyrosine kinase activity is required for NF-kB activation in human monocytes [27]. Genistein is an isoflavone compound isolated from Pseudomonas, with specific protein tyrosine kinase inhibitory properties [26]. The present finding that genistein blocked C3 production and decreased C3 mRNA levels in the Caco-2 cell suggests that tyrosine kinase activity may be necessary for enterocyte C3 production. Because in a recent study we found that
53
genistein did not inhibit NF-kB activity in IL-1bstimulated Caco-2 cells [25] it is possible that the inhibitory effect of genistein on C3 production noted here was not secondary to NF-kB inhibition. The exact relationship among genistein-induced inhibition of C3 production in Caco-2 cells, tyrosine kinase activity, and NF-kB activity remains to be established. LLnL is a peptide-aldehyde compound that inhibits the activity of the mammalian proteasome [31]. Recent studies have demonstrated that IkB degradation is blocked by proteasome inhibitors and that the ubiquitin-proteasome proteolytic pathway is required for NF-kB activation in various cell types [18]. In the present study, preincubation of the Caco-2 cells with LLnL blocked C3 production and decreased C3 mRNA levels, suggesting that the proteasome pathway is involved in the regulation of enterocyte C3 production, possibly via the NF-kB pathway. Although recent studies in our laboratory demonstrated that LLnL inhibits IkBa degradation and subsequent NF-kB activation in the enterocyte (unpublished observations), further studies are needed to determine whether the inhibited C3 production seen following treatment of the Caco-2 cells with LLnL is caused by inhibited NF-kB activity. In a recent study, transfection of Hela cells with a Ser 32/36 IkBa mutant diminished activation of NF-kB [23]. In the present study, transfection with the Ser 32/36 IkBa mutant reduced the degradation of the inhibitory protein IkBa in IL-1b-stimulated cultured Caco-2 cells, probably resulting in inhibited translocation of NF-kB to the nucleus. The finding that the degradation of the mutant IkBa was not completely blocked may reflect partial expression of the transfected plasmid. The observation that transfection of Caco-2 cells with Ser 32/36 IkBa mutant reduced IL1b-induced C3 production strongly supports the concept that C3 production in Caco-2 cells is regulated by NF-kB. Although the different substances that were used in the present study (TLCK, PDTC, genistein, and LLnL) have been shown in a number of previous reports to inhibit NF-kB activation in various cell types [18, 25, 27, 28, 33, 36, 37], they are probably not specific in their action as NF-kB inhibitors but may have other metabolic effects as well. The observation that multiple substances, inhibiting NF-kB activation by different mechanisms, reduced IL-1b-induced C3 production supports our interpretation of the results, i.e., that the inhibited C3 production most likely reflected inhibited NF-kB activation. The strongest support for that argument, however, was found in the experiment in which Caco-2 cells were transfected with a Ser 32/36 mutant IkBa. In these cells, the degradation of IkBa, which is a required step in the activation of NF-kB [28, 29], was inhibited concomitant with reduced IL-1b-induced C3
54
JOURNAL OF SURGICAL RESEARCH: VOL. 82, NO. 1, MARCH 1999
production, more firmly linking C3 production with NF-kB activation. It should be noted that although the present results may be consistent with the concept that NF-kB is involved in the regulation of IL-1b-induced C3 production in human enterocytes, the results need to be interpreted with caution for several reasons. First, although the Caco-2 cells used in the present experiments take on the characteristics of small intestinal epithelial cells during culture, they are transformed cells that are derived from a colon cancer cell line [19]. The role of NF-kB in the regulation of C3 production needs to be established in other cell lineages as well before a generalized conclusion regarding enterocyte NF-kB activity and C3 production can be made. Second (as discussed above), the inhibitors used to block NF-kB in our experiments may not be completely specific, but may affect other metabolic processes in the cell as well. The apparently conflicting results of inhibited C3 production (present study) and unaffected NF-kB activity [25] after treatment of the Caco-2 cells with genistein need to be resolved in future experiments.
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Wang, Q., Meyer, T. A., Boyce, S., Wang, J. J., Sun, X., Tiao, G., Fischer, J. E., and Hasselgren, P. O. Endotoxemia in mice stimulates the production of complement C3 and serum amyloid A in mucosa of small intestine. Am. J. Physiol. 275: R 1584, 1998.
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Moon, M. R., Parikh, A. A., Szabo, C., Fischer, J. E., Salzman, A. L., and Hasselgren, P. O. Complement C3 production in human intestinal epithelial cells is regulated by IL-1b and TNFa. Arch. Surg. 132: 1289, 1997.
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Thanos, D., and Manlatis, T. NF-kB: A lesson in family values. Cell 80: 529, 1995.
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Zhang, Y., Broser, M., and Rom, W. N. Activation of the interleukin 6 gene by Mycobacterium tuberculosis or lipopolysaccharide is mediated by nuclear factor NF-IL6 and NF-kB. Proc. Natl. Acad. Sci. USA 91: 2225, 1994.
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Beg, A. A., Finco, T. S., Nantermet, P. V., and Baldwin, A. S. Tumor necrosis factor and interleukin-1 lead to phosphorylation of IkBa: A mechanism for NF-kB activation. Mol. Cell. Biol. 13(6): 3301, 1993.
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Parikh, A. A., Salzman, A. L., Kane, C., Fischer, J. E., and Hasselgren, P. O. IL-6 production in human intestinal epithelial cells following stimulation with IL-1b is associated with activation of the transcription factor NF-kB. J. Surg. Res. 69: 139, 1997.
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Palombella, V. J., Rando, O. J., Goldberg, A. L., and Maniatis, T. The ubiquitin-proteasome pathway is required for processing the NF-kB 1 precursor protein and the activation of NF-kB. Cell 78: 773, 1994.
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Pinto, M., Robine-Leon, S., Appay, M. D., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix, B., Simon-Assman, P., Haffen, K., Fogh, J., and Zweibaum, A. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol. Cell 47: 323, 1983.
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Salzman, A. L., Denenberg, A. G., Ueta, I., O’Connor, M., Linn, S. C., and Szabo, C. Induction and activity of nitric oxide synthase in cultured human epithelial monolayers. Am. J. Physiol. 270: G565, 1996.
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Chomzynski, P., and Sacchi, N. Single step method of RNA isolation by guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156, 1987.
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Vik, D. P., Amiguet, P., Moffat, G. J., Fey, M., Amiguet-Barras, F., Westel, R. A., and Tack, B. F. Structural features of the human C3 gene: Intron/exon organization, transcriptional start site, and promoter region sequence. Biochemistry 30: 1080, 1991.
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Traenckner, E. B, Pahl, H. L., Henkel, T., Schmidt, K. N., Wilk, S., and Baeuerle, P. Phosphorylation of human IkB-a proteolysis and NF-kB activation in response to diverse stimuli. EMBO J. 14: 2876, 1995.
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Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen, M. Lipofectin: A highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA 84: 7413, 1987.
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Parikh, A. A., Moon, M. R., Fischer, J. E., Szabo, C., Hasselgren, P. O., and Salzman, A. L. IL-1b induction of NF-kB activation in human intestinal epithelial cells is independent of oxyradical signaling. Submitted for publication.
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Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuay, M., and Fukami, Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 262: 5592, 1987.
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