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Proinflammatory cytokines up-regulate synthesis and secretion of urinary trypsin inhibitor in human hepatoma HepG2 cells
Hepatology Research 29 (2004) 243–248
Proinflammatory cytokines up-regulate synthesis and secretion of urinary trypsin inhibitor in human hepatoma HepG2 cells Shi De Lin, Yasuhiro Takikawa∗ , Ryujin Endo, Kazuyuki Suzuki The First Department of Internal Medicine, Iwate Medical University, 19-1 Uchimaru, Morioka 020-8505, Japan Received 16 January 2004; received in revised form 11 March 2004; accepted 2 April 2004 Available online 28 May 2004
Abstract Background and Aims: The urinary trypsin inhibitor (UTI), a wide range protein inhibitor synthesized by hepatocytes, is considered to play an important role not only in the protection of organ injury during severe inflammation but also in the inhibition of tumor invasion and metastasis. However, the precise mechanisms underlying control of its synthesis, secretion, and processing remain unclarified. The aim of this study is to determine whether human hepatoma HepG2 cells secrete UTI in free form and whether its synthesis and secretion are regulated by proinflammatory cytokines. Methods: Cultured HepG2 cells were stimulated using different concentrations of interleukin-6 (IL-6), tumor necrosis factor-␣ (TNF-␣) and interleukin-1 (IL-1). The concentration of free UTI in the medium was measured by ELISA and the intracellular UTI precursor was identified by western blotting. UTI mRNA expression was studied by reverse transcription polymerase chain reaction (RT-PCR). Results: HepG2 cells constantly secreted free UTI and this secretion was significantly up-regulated by IL-6, IL-1, and TNF-␣. IL-6, IL-1, and TNF-␣ enhanced the synthesis of the intracellular UTI precursor protein, and IL-1 up-regulated UTI mRNA expression. Conclusions: HepG2 cells constantly secrete free UTI. The proinflammatory cytokines, IL-1, IL-6, and TNF-␣, up-regulate UTI synthesis and secretion by up-regulating UTI mRNA expression. © 2004 Elsevier B.V. All rights reserved. Keywords: Urinary trypsin inhibitor; Interleukin-6; Tumor necrosis factor-␣; Interleukin-1; HepG2
1. Introduction The urinary trypsin inhibitor (UTI), also designated as bikunin, is a glycoprotein with an apparent molecular mass of approximately 40 kDa. UTI is synthesized by hepatocytes as a precursor in which UTI is linked to ␣1 -microglobulin [1–3]. In hepatocytes, different types of UTI-containing proteins are formed by assembly of UTI with one or two of the three evolutionary related heavy chains (H1, H2, and H3); these proteins comprise inter-␣-inhibitor (I␣I) family members, including I␣I, pre-␣-inhibitor (p␣I), inter-␣-like inhibitor (I␣LI) and free UTI. I␣I, p␣I, and I␣LI are composed of H1+H2+UTI, H3+UTI, and H2+UTI, respectively [4,5]. During inflammation, UTI is cleaved from I␣I family proteins by limited proteolysis in the peripheral circulation or at the inflammation site [6–8]. ∗ Corresponding author. Tel.: +81-19-651-5111x2314; fax: +81-19-652-6664. E-mail address: [email protected] (Y. Takikawa).
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The biological functions of UTI are still unknown; previous studies have demonstrated that UTI inhibits a wide range of proteases and regulates cytokine production and activities [9,10]. Clinically, UTI shows protective effects on organ injury and I␣I shows the effects of hemodynamic stability and improved survival during sepsis [11,12]. Recent studies also demonstrated that free UTI, but not other UTI-containing proteins, suppresses tumor invasion and metastasis through its inhibitory action against tumor cell-associated plasmin [13,14]. In response to acute systemic injury, the human liver dramatically increases or decreases the synthesis of some serum proteins. There are contradictory results regarding serum protein levels and the corresponding mRNA gene expressions of UTI and UTI-containing proteins in pathological conditions [15–18]. In a previous study we noted that plasma and urine UTI levels increase in acute hepatitis patients and decrease in fulminant hepatitis patients and concluded that UTI secretion is enhanced according to the severity of hepatitis as long as the liver retains its protein synthesis function
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[19]. Furthermore, we demonstrated that the plasma UTI level in hepatocellular carcinoma patients is higher than that in liver cirrhosis patients [20]. However, it remains unclarified whether UTI secretion by hepatocytes increases according to the severity of inflammation and whether hepatoma cells secrete free UTI. In this study, therefore, we determined whether HepG2 cells, a hepatoma cell line, secrete free UTI and whether the secretion is enhanced by representative proinflammatory cytokines, tumor necrosis factor-␣ (TNF-␣), interleukin-1 (IL-1) and interleukin-6 (IL-6) [21].
2. Materials and methods 2.1. Materials All the materials used were obtained from Sigma (St. Louis, MO) unless otherwise indicated. 2.2. Cell and culture conditions HepG2 cells were propagated at 37 ◦ C in 5% CO2 in D-MEM/F-12 medium (Gibco BRL, Grand Island, NY, USA) containing 10% (v/v) fetal calf serum (Hyclone, Logan, UT, USA), 100 U/ml penicillin and passaged every 5–7 days. The HepG2 cells were cultured until they reached 80–100% confluence. Thereafter, the cells were rinsed three times in 10 ml of PBS, and cultured in a medium lacking fetal calf serum and stimulated with different concentrations of TNF-␣ (R&D Systems, Minneapolis, MN, USA), IL-1 (R&D Systems, Minneapolis, MN, USA) and IL-6 (R&D Systems, Minneapolis, MN, USA). Aliquots of the culture medium were collected at different culture time for UTI determination. The cells were rinsed again in 10 ml of PBS three times, lysed in 1 ml of lysis buffer [33 mM Tris, 165 mM NaCl, pH 7.5, 1% Triton X-100, 10% glycerol, 50 l of 100 mM phenylmethanesulphonylfluoride (PMSF) and 50 l of protease inhibitor] for UTI detection. After centrifugation at 4 ◦ C for 10 min at 14,000 × g, the cell lysates were subjected to western blotting. The viability of the cells was not decreased until harvest and the cells showed no morphological change during serum deprivation and stimulation in every experiment. 2.3. Western blot analysis Cell lysates containing forty micrograms of protein were resolved by SDS-PAGE using 4–20% polyacrylamide gradient gel (TEFCO, Tokyo, Japan) and the fractioned proteins were subsequently transferred to nitrocellulose membranes (Gloucester, MA, USA). After blocking with Tris-based saline buffer containing 5% dry milk and 0.1% Tween 20 for 1 h, membranes were blotted with the corresponding antibodies. The primary and secondary antibodies used were as follows: rabbit anti-I␣I (Dako, Glostrup, Denmark), goat
anti-rabbit IgG conjugated with horseradish peroxidase (Cell Signaling Technology, MA, USA). The membranes were developed using a chemiluminescence detection system (Perkin-Elmer Life Science, Boston, MA, USA) and thereafter exposed to Kodak BioMax Light Film (Rochester, NY, USA). 2.4. Enzyme-linked immunosorbent assay (ELISA) UTI concentration in the culture medium was measured by the one-step sandwich enzyme-linked immunosorbent assay (Mochida Pharmaceutical Co., Tokyo, Japan) that we previously reported [19] with minor modifications. To rule out any effect of I␣I family proteins that may exist in the medium during UTI measurement, the medium was pretreated by the modified method of Jonsson-berling et al. [22]. Briefly, 50 l of a medium sample was added to 150 l of physiological saline and then mixed with 50 l of 40% perchloric acid. It was then vortexed for 5 s and centrifuged at 15,000 × g for 10 min at 4 ◦ C. The supernatant (100 l) was neutralized with 0.8 M NaOH and its pH was adjusted to 8.0 by adding 1 M Tris–HCl (pH 8.0). The acid-treated samples were used directly for assay. 2.5. Reverse transcription polymerase chain reaction When the HepG2 cells reached 40–50% confluence, they were cultured in a medium lacking serum for 24 h and stimulated with cytokines for different durations. Total RNA was extracted from HepG2 cells using Qiagen RNeasy minikit (Qiagen Company) according to the manufacturer’s instruction. Reverse transcription was performed using 1 g of total RNA using random primers in a final volume of 20 l (Omniscript Reverse Transcriptase kit, Qiagen Company). PCR was performed using one cycle of denaturation at 95 ◦ C for 2 min and 30 cycles consisting of denaturation at 95 ◦ C for 1 min, primer annealing at 58 ◦ C for 1 min, extension at 72 ◦ C for 1 min; an additional cycles at 95 ◦ C for 1 min, 60 ◦ C for 1 min, 72 ◦ C for 7 min was performed. The specific oligonucleotide primers used for UTI detection were as follows: antisense 5 -GATGAAGGCTCGGCAGGGGC-3 , sense 5 -GTCCGGAGGGCTGTGCTACC-3 [21]. The amount of template RNA was normalized using human GAPDH primers as internal controls. The primer sequences for GAPDH were 5 -GTCAACGGATTTGGTCTGTATT-3 and 5 -AGTCTTCTGGGTGGCAGTGAT-3 , and the cycling conditions were 1 min at 94 ◦ C, 1 min at 56 ◦ C, and 2 min at 72 ◦ C. The UTI RT-PCR products were analyzed on a 2% agarose gel. 2.6. Statistics Statistical significance was determined by Student’s t-test. A P value of less than 0.05 was used as the criterion of statistical significance.
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medium showed a molecular weight of about 43 kDa, which was slightly lower than that of the intracellular UTI precursor and was in agreement with the report by Jonsson-berling et al. [22]. 3.2. IL-6, TNF-α, and IL-1β significantly up-regulated UTI secretion
Fig. 1. Free UTI secreted by HepG2 cells. After the HepG2 cells reached 80–100% confluence, the medium was changed and serum removed. Aliquots (15 l) of the medium collected at 12 and 24 h were run in 4–20% SDS-PAGE and the fractionated proteins were subsequently transferred to a nitrocellulose membrane for immunoblotting with anti-I␣I. The sequential change in UTI concentration in the medium was measured by the method of ELISA. Histograms are means ± S.D. The results are the means of at least three experiments.
After confirming that HepG2 cells secrete free UTI, we next investigated whether UTI secretion of UTI by HepG2 cells is regulated by proinflammatory cytokines. The UTI concentration in the medium was too low to be measured within 12 h after serum deprivation. Therefore, to investigate the effects of IL-6, TNF-␣, and IL-1 on UTI secretion before 12 h, the HepG2 cells were stimulated with IL-6, TNF-␣ or IL-1 24 h after serum deprivation. The cultured HepG2 cells in the presence of IL-6, TNF-␣ or IL-1 at the concentrations of 10, 20, and 10 ng/ml, respectively, significantly enhanced UTI secretion 12 h after stimulation (Fig. 2). To further investigate whether UTI up-regulations by IL-6, TNF-␣ and IL-1 are dose-dependent, we also observed the
3. Results 3.1. HepG2 cells constantly secrete free UTI into medium After serum deprivation, we demonstrated by ELISA that free UTI concentration increases in a time-dependent manner in the culture medium after 12 h (Fig. 1). Before 12 h, UTI concentration in the medium was too low to be detected by our method, indicating that HepG2 cells secrete UTI. Free UTI in the medium was also confirmed by western blotting (Fig. 1). Using an antibody against I␣I, free UTI in the
Fig. 2. Effects of TNF-␣, IL-6 and IL-1 on UTI secretion by HepG2 cells. After the HepG2 cells reached 80–100% confluence, the medium was changed and the serum removed. The cells were cultured for 24 h before stimulation with TNF-␣, IL-6, or IL-1. The effects of TNF-␣, IL-6, and IL-1 at concentrations of 10, 20, and 10 ng/ml, respectively on UTI secretion were compared with the control. Histograms are means ± S.D. The results are the means of at least three experiments. (*) P < 0.05; (**) P < 0.01.
Fig. 3. The effects of IL-6 and TNF-␣ on UTI secretion by HepG2 cells. After the HepG2 cells reached 80–100% confluence, the medium was changed and serum removed. The change in UTI concentration in the medium was measured by ELISA. The effects of different concentrations of (a) IL-6 and (b) TNF-␣ on free-UTI secretion at the time of 12 h are presented. Histograms are means ± S.D. The results are the means of at least three experiments.
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Fig. 4. TNF-␣, IL-1, and IL-6 up-regulate the intracellular UTI synthesis. After the HepG2 cells reached 80–100% confluence, the medium was changed and the serum removed. The HepG2 cells were stimulated with (A) 20 ng/ml TNF-␣, (B) 10 ng/ml IL-1 or (C) 10 ng/ml IL-6 for different durations as indicated. Cell lysates extract from untreated or cytokine-treated HepG2 cells were used for western blotting anti-I␣I, as described in the text. One representative result of three individual experiments is presented.
effects of different concentrations of IL-6 and TNF-␣ on UTI secretion at 12 h. IL-6 and TNF-␣ up-regulated UTI secretion in a dose-dependent manner (Fig. 3). 3.3. TNF-α, IL-1β, and IL-6 enhanced intracellular UTI precursor synthesis As indicated in Fig. 4, the UTI precursor band appeared at about 45 kDa when anti-I␣I antibodies were used, which was consistent with previous reports [23,24]. To determine whether IL-1, IL-6, and TNF-␣ up-regulate intracellular UTI precursor synthesis, we analyzed the change in the expression level of intracellular UTI protein in HepG2 cells in response to cytokine treatment. As shown in Fig. 4, UTI protein levels were notably up-regulated by incubation with 10 ng/ml IL-1 or 20 ng/ml TNF-␣. The up-regulation induced by IL-1 lasted from 2 to 8 h, whereas that induced by TNF-␣ was only observed at 2 h. When the Hep G2 cells
were incubated with 10 ng/ml IL-6, the expression level of the intracellular UTI protein increased with time until 8 h after stimulation and kept the expression level until 12 h. We also observed that the intracellular UTI protein expression level remained unchanged from 12 to 48 h by treatment with IL-1, IL-6 or TNF-␣ and that it was not changed during 72 h serum deprivation (data not shown). 3.4. UTI mRNA expression is up-regulated by IL-1β Among the three cytokines used in this experiment, IL-1 exhibited the most effective and persistent up-regulation of UTI synthesis and secretion (Figs. 2 and 4). Thus, we next examined by RT-PCR the change in UTI mRNA expression level after IL-1 stimulation. As shown in Fig. 5, UTI mRNA levels were distinctly up-regulated after 15-min treatment with 10 ng/ml IL-1. This result correlates with the UTI protein up-regulation observed by western blotting.
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Fig. 5. UTI mRNA expression in HepG2 cells treated with 10 ng/ml IL-1. Total RNA was isolated as described in the text from untreated or IL-1-treated HepG2 cells for the stated duration and subjected to RT-PCR analysis with primers specific for UTI. The PCR product of GAPDH amplification was used as control. PCR products were electrophoresed on a 2% agarose gel and stained with ethidium bromide. Because three individual experiments showed same results, one representative result is presented.
4. Discussion Although recent evidence indicates that hepatoma cells synthesize and secrete some I␣I family proteins, whether hepatoma cells secrete free UTI as well as the UTI secretion pattern remains unclarified. Bourguignon et al. [25] did not find free UTI in the medium of cultured human hepatoma HepG2 cells by western blotting. However, Thogerson et al. [24] found free UTI using the same method; although the HepG2 did not produce some UTI containing proteins, cultured HepG2 cells secreted free UTI. Our results demonstrate that HepG2 cells secrete free UTI in a time-dependent manner by both ELISA and western blotting. Although the roles of UTI in hepatoma cell proliferation, invasion and metastasis remain unclarified, Kobayashi et al. [26] reported that some tumor cells have UTI binding sites and that UTI inhibits murine Lewis lung carcinoma cell invasion and metastasis. UTI exists in serum both as a subunit of I␣I family proteins and in the free form. In our method, the samples are treated with perchloric acid to precipitate I␣I and related UTI-containing proteins that probably exist in the medium and we successfully used this method to determine plasma UTI concentration [19]. Jonsson-berling et al. [22] had used this method to pretreat serum and noted that no UTI was released from I␣I and related proteins during precipitation with perchloric acid. Therefore, we adopted this method for the determination of free-form UTI in the medium. We further demonstrated by western blotting that HepG2 cells secrete free UTI. The secreted free UTI did not contain ␣1-microglobulin and showed a molecular weight of about 43 kDa, which was slightly lower than that of the intracellular UTI precursor; this was in agreement with report by Thogerson et al. [24]. UTI and ␣1 -microglobulin are cosynthesized in hepatocytes by an ␣1 -microglobulin/bikunin precursor gene. After the UTI and ␣1 -microglobulin precursor is assembled with different H chain to synthesize I␣I family proteins via a chondroitin sulfate chain, ␣1 -microglobulin is released. Although some studies have been carried out to investigate the behaviors of UTI and UTI-containing proteins during
inflammation, controversy persists over whether UTI is a positive acute phase protein (APP) or a negative APP. In vivo and clinical studies have revealed that under pathological conditions such as acute inflammation, the serum ␣1 -microglobulin level is relatively constant, I␣I and I␣LI behave as a negative APP and p␣I behaves as a positive APP [17,18,27]. As indicated above, during inflammation, the UTI liberated from serum UTI-containing proteins increases through limited proteolysis in peripheral circulation or inflammatory site. However, because of its small molecular mass, UTI is quickly excreted into urine [28], the plasma UTI levels increases only slightly and urinary UTI excretion increases markedly in pathological conditions [8,29–31]. The UTI mRNA expression levels in Hep3B cells were found to be down-regulated 24 h after treatment with IL-6 and IL-1 in a previous study [17]. However, some other studies demonstrated that the UTI mRNA expression level and intracellular protein level increase or remain unchanged in hepatocytes and renal proximal tubular epithelial cells during inflammation [16,32]. In yet another study, it was noted that part of the UTI mRNA response to an inflammatory or a tumor process is unstable [23]. All these findings indicate that serum UTI concentration dose not directly reflect the change in the type of free UTI secreted by hepatocytes and it is difficult to accurately quantify the change in UTI mRNA expression level due to its instability. In this study, we used a new and sensitive method to detect free UTI secreted by HepG2 cells. This method enabled us to directly observe the change in the type of free UTI secreted by HepG2 cells and investigate the effects of cytokines on such change. We chose the proinflammatory cytokines, TNF-␣, IL-6, and IL-1, because they are known as the major mediators of acute protein induction in the liver [21,33]. We confirmed that the cells used in our experiment responded to TNF-␣ and IL-6 based on the observation that TNF-␣induced IB-␣degradation and IL-6 induced STAT-3 activation (data not shown). We demonstrated that IL-6, IL-1, and TNF-␣ up-regulate the secretion of UTI from 4 to 12 h in HepG2 cells. These effects were in accordance with changes of intracellular protein and the UTI mRNA expres-
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sion levels. Whether there is any difference in the regulation of UTI between normal hepatocytes and hepatoma cells remains to be clarified, although our results strongly suggested that free UTI is an early-response positive APP. The combined effects of the above cytokines or the interactions of cytokines with other major mediators of acute protein induction such as dexamethasone remain to be further studied. The major findings of the present study are that HepG2 cells synthesize and secrete free UTI, and that the synthesis and secretion of free UTI are up-regulated by proinflammatory cytokines. Our results suggest that during inflammation, the increased plasma and urine UTI levels are not only the result of the UTI release from serum UTI-containing proteins, but also that of the enhanced synthesis and secretion of free UTI by hepatocytes. The structural and functional differences of UTI secreted in free form and that cleaved from I␣I and related molecules are not currently understood and require further studies.
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[14] Kobayashi H, Gotoh J, Hirashima Y, Fujie M, Sugino D, Terao T. Inhibitory effect of a conjugate between human urokinase and urinary trypsin inhibitor on tumor cell invasion in vitro. J Biol Chem 1995;270:8361–6. [15] Pierzchalski P, Rokita H, Koj A, Fries E, Akerstrom B. Synthesis of ␣1-microglobulin in cultured rat hepatocytes is stimulated by interlukin-6 leukemia inhibitory factor, dexamethasone and retinoic acid. FEBS 1992;298:165–8. [16] Yoshida K, Suzuki Y, Yamamoto K, Sinohara H. Guinea pig alpha 1-microglobulin/bikunin: cDNA sequencing, tissue expression and expression during acute phase. Comp Biochem Physiol B Biochem Mol Biol 1999;122:165–72. [17] Daveau M, Rouet P, Scotte M, et al. Human inter-␣-inhibitor family in inflammation: simultaneous synthesis of positive and negative acute-phase proteins. Biochem J 1993;292:485–92. [18] Daveau M, Jean L, Soury E, et al. Hepatic and extrahepatic transcription of inter-␣-inhibitor family genes under normal or acute inflammatory conditions in rat. Arch Biochem Biophys 1998;350:315–23. [19] Lin SD, Endo R, Sato A, Takikawa Y, Shirakawa K, Suzuki K. Plasma and urine levels of urinary trypsin inhibitor in patients with acute and fulminant hepatitis. J Gastroenterol Hepatol 2002;17:140– 7. [20] Lin SD, Endo R, Kuroda H, Kondo K, Miura Y, Takikawa Y, Kato A, Suzuki K. Plasma and urine levels of urinary trypsin inhibitor in patients with chronic liver diseases and hepatocellular carcinoma. J Gastroenterol Hepatol 2004;19:327–32. [21] Andus T, Bauer J, Gerok W. Effects of cytokines on the liver. Hepatology 1991;13:364–75. [22] Jonsson-berling BM, Ohlsson K, Rosengren M. Radioimmunological quantitation of the urinary trypsin inhibitor in normal blood and urine. Biol Chem Hoppe-Seyler 1989;370:1157–61. [23] Bourguignon J, Borghi H, Sesboue R, et al. Immunohistochemical distribution of inter-␣-trypsin inhibitor chains in normal and malignant human lung tissue. J Histochem Cytochem 1999;47:1625–32. [24] Thogersen IB, Enghild JJ. Biosynthesis of bikunin proteins in the human carcinoma cell line HepG2 and in primary human hepatocytes. J Biol Chem 1995;270:18700–9. [25] Bourguignon J, Sesboue R, Diarra-Mehrpour M, Daveau M, Martin JP. Human inter-␣-inhibitor: synthesis and maturation in hepatoma HepG2 cells. Biochem J 1989;261:305–8. [26] Kobayashi H, Gotoh J, Fujie M, Terao T. Characterization of the cellular binding site for the urinary trypsin inhibitor. J Biol Chem 1994;269:20642–7. [27] Akerstrom B, Logdberg L, Berggard T, Osmark P, Lindqvist A. ␣1-Microglobulin: a yellow-brown lipocalin. Biochim Biophys Acta 2000;1482:172–84. [28] Lindstrom KE, Blom A, Johnsson E, Haraldsson B, Fries E. High glomerular permeability of bikunin despite similarity in charge and hydrodynamic size to serum albumin. Kidney Int 1997;51:1053–8. [29] Noie T, Sugawara Y, Harihara T, Takayama K, Kubota K, Ohashi Y. Kinetics of urinary trypsin inhibitor in patients undergoing parting hepatectomy. Scand J Gastroenterol 2001;6:410–6. [30] Franck C, Pedersen Z. Trypsin-inhibitory activities of acid-stable fragments of the inter-alpha-trypsin inhibitor in inflammatory and uraemic conditions. Scand J Clin Lab Invest 1983;43:151–5. [31] Smith JM, Balabanian MB, Freeman RM. Serum levels of a component reacting with antiserum to urinary antitrypsin in health and disease with emphasis on high levels in renal failure. J Lab Clin Med 1976;88:904–11. [32] Janssen U, Thomas G, Glant T, Philips A. Expression of inter-␣-trypsin inhibitor and tumor necrosis factor-stimulated gene 6 in renal proximal tubular epithelial cells. Kidney Int 2001;60:126–36. [33] Castell JV, Gomez-Lechon MJ, David M, Fabra R, Trullenque R, Heinrich PC. Acute-phase response of human hepatocytes: regulation of acute-phase protein synthesis by interleukin-6. Hepatology 1990;12:1179–86.
Proinflammatory cytokines up-regulate synthesis and secretion of urinary trypsin inhibitor in human hepatoma HepG2 cells Shi De Lin, Yasuhiro Takikawa∗ , Ryujin Endo, Kazuyuki Suzuki The First Department of Internal Medicine, Iwate Medical University, 19-1 Uchimaru, Morioka 020-8505, Japan Received 16 January 2004; received in revised form 11 March 2004; accepted 2 April 2004 Available online 28 May 2004
Abstract Background and Aims: The urinary trypsin inhibitor (UTI), a wide range protein inhibitor synthesized by hepatocytes, is considered to play an important role not only in the protection of organ injury during severe inflammation but also in the inhibition of tumor invasion and metastasis. However, the precise mechanisms underlying control of its synthesis, secretion, and processing remain unclarified. The aim of this study is to determine whether human hepatoma HepG2 cells secrete UTI in free form and whether its synthesis and secretion are regulated by proinflammatory cytokines. Methods: Cultured HepG2 cells were stimulated using different concentrations of interleukin-6 (IL-6), tumor necrosis factor-␣ (TNF-␣) and interleukin-1 (IL-1). The concentration of free UTI in the medium was measured by ELISA and the intracellular UTI precursor was identified by western blotting. UTI mRNA expression was studied by reverse transcription polymerase chain reaction (RT-PCR). Results: HepG2 cells constantly secreted free UTI and this secretion was significantly up-regulated by IL-6, IL-1, and TNF-␣. IL-6, IL-1, and TNF-␣ enhanced the synthesis of the intracellular UTI precursor protein, and IL-1 up-regulated UTI mRNA expression. Conclusions: HepG2 cells constantly secrete free UTI. The proinflammatory cytokines, IL-1, IL-6, and TNF-␣, up-regulate UTI synthesis and secretion by up-regulating UTI mRNA expression. © 2004 Elsevier B.V. All rights reserved. Keywords: Urinary trypsin inhibitor; Interleukin-6; Tumor necrosis factor-␣; Interleukin-1; HepG2
1. Introduction The urinary trypsin inhibitor (UTI), also designated as bikunin, is a glycoprotein with an apparent molecular mass of approximately 40 kDa. UTI is synthesized by hepatocytes as a precursor in which UTI is linked to ␣1 -microglobulin [1–3]. In hepatocytes, different types of UTI-containing proteins are formed by assembly of UTI with one or two of the three evolutionary related heavy chains (H1, H2, and H3); these proteins comprise inter-␣-inhibitor (I␣I) family members, including I␣I, pre-␣-inhibitor (p␣I), inter-␣-like inhibitor (I␣LI) and free UTI. I␣I, p␣I, and I␣LI are composed of H1+H2+UTI, H3+UTI, and H2+UTI, respectively [4,5]. During inflammation, UTI is cleaved from I␣I family proteins by limited proteolysis in the peripheral circulation or at the inflammation site [6–8]. ∗ Corresponding author. Tel.: +81-19-651-5111x2314; fax: +81-19-652-6664. E-mail address: [email protected] (Y. Takikawa).
1386-6346/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.hepres.2004.04.003
The biological functions of UTI are still unknown; previous studies have demonstrated that UTI inhibits a wide range of proteases and regulates cytokine production and activities [9,10]. Clinically, UTI shows protective effects on organ injury and I␣I shows the effects of hemodynamic stability and improved survival during sepsis [11,12]. Recent studies also demonstrated that free UTI, but not other UTI-containing proteins, suppresses tumor invasion and metastasis through its inhibitory action against tumor cell-associated plasmin [13,14]. In response to acute systemic injury, the human liver dramatically increases or decreases the synthesis of some serum proteins. There are contradictory results regarding serum protein levels and the corresponding mRNA gene expressions of UTI and UTI-containing proteins in pathological conditions [15–18]. In a previous study we noted that plasma and urine UTI levels increase in acute hepatitis patients and decrease in fulminant hepatitis patients and concluded that UTI secretion is enhanced according to the severity of hepatitis as long as the liver retains its protein synthesis function
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[19]. Furthermore, we demonstrated that the plasma UTI level in hepatocellular carcinoma patients is higher than that in liver cirrhosis patients [20]. However, it remains unclarified whether UTI secretion by hepatocytes increases according to the severity of inflammation and whether hepatoma cells secrete free UTI. In this study, therefore, we determined whether HepG2 cells, a hepatoma cell line, secrete free UTI and whether the secretion is enhanced by representative proinflammatory cytokines, tumor necrosis factor-␣ (TNF-␣), interleukin-1 (IL-1) and interleukin-6 (IL-6) [21].
2. Materials and methods 2.1. Materials All the materials used were obtained from Sigma (St. Louis, MO) unless otherwise indicated. 2.2. Cell and culture conditions HepG2 cells were propagated at 37 ◦ C in 5% CO2 in D-MEM/F-12 medium (Gibco BRL, Grand Island, NY, USA) containing 10% (v/v) fetal calf serum (Hyclone, Logan, UT, USA), 100 U/ml penicillin and passaged every 5–7 days. The HepG2 cells were cultured until they reached 80–100% confluence. Thereafter, the cells were rinsed three times in 10 ml of PBS, and cultured in a medium lacking fetal calf serum and stimulated with different concentrations of TNF-␣ (R&D Systems, Minneapolis, MN, USA), IL-1 (R&D Systems, Minneapolis, MN, USA) and IL-6 (R&D Systems, Minneapolis, MN, USA). Aliquots of the culture medium were collected at different culture time for UTI determination. The cells were rinsed again in 10 ml of PBS three times, lysed in 1 ml of lysis buffer [33 mM Tris, 165 mM NaCl, pH 7.5, 1% Triton X-100, 10% glycerol, 50 l of 100 mM phenylmethanesulphonylfluoride (PMSF) and 50 l of protease inhibitor] for UTI detection. After centrifugation at 4 ◦ C for 10 min at 14,000 × g, the cell lysates were subjected to western blotting. The viability of the cells was not decreased until harvest and the cells showed no morphological change during serum deprivation and stimulation in every experiment. 2.3. Western blot analysis Cell lysates containing forty micrograms of protein were resolved by SDS-PAGE using 4–20% polyacrylamide gradient gel (TEFCO, Tokyo, Japan) and the fractioned proteins were subsequently transferred to nitrocellulose membranes (Gloucester, MA, USA). After blocking with Tris-based saline buffer containing 5% dry milk and 0.1% Tween 20 for 1 h, membranes were blotted with the corresponding antibodies. The primary and secondary antibodies used were as follows: rabbit anti-I␣I (Dako, Glostrup, Denmark), goat
anti-rabbit IgG conjugated with horseradish peroxidase (Cell Signaling Technology, MA, USA). The membranes were developed using a chemiluminescence detection system (Perkin-Elmer Life Science, Boston, MA, USA) and thereafter exposed to Kodak BioMax Light Film (Rochester, NY, USA). 2.4. Enzyme-linked immunosorbent assay (ELISA) UTI concentration in the culture medium was measured by the one-step sandwich enzyme-linked immunosorbent assay (Mochida Pharmaceutical Co., Tokyo, Japan) that we previously reported [19] with minor modifications. To rule out any effect of I␣I family proteins that may exist in the medium during UTI measurement, the medium was pretreated by the modified method of Jonsson-berling et al. [22]. Briefly, 50 l of a medium sample was added to 150 l of physiological saline and then mixed with 50 l of 40% perchloric acid. It was then vortexed for 5 s and centrifuged at 15,000 × g for 10 min at 4 ◦ C. The supernatant (100 l) was neutralized with 0.8 M NaOH and its pH was adjusted to 8.0 by adding 1 M Tris–HCl (pH 8.0). The acid-treated samples were used directly for assay. 2.5. Reverse transcription polymerase chain reaction When the HepG2 cells reached 40–50% confluence, they were cultured in a medium lacking serum for 24 h and stimulated with cytokines for different durations. Total RNA was extracted from HepG2 cells using Qiagen RNeasy minikit (Qiagen Company) according to the manufacturer’s instruction. Reverse transcription was performed using 1 g of total RNA using random primers in a final volume of 20 l (Omniscript Reverse Transcriptase kit, Qiagen Company). PCR was performed using one cycle of denaturation at 95 ◦ C for 2 min and 30 cycles consisting of denaturation at 95 ◦ C for 1 min, primer annealing at 58 ◦ C for 1 min, extension at 72 ◦ C for 1 min; an additional cycles at 95 ◦ C for 1 min, 60 ◦ C for 1 min, 72 ◦ C for 7 min was performed. The specific oligonucleotide primers used for UTI detection were as follows: antisense 5 -GATGAAGGCTCGGCAGGGGC-3 , sense 5 -GTCCGGAGGGCTGTGCTACC-3 [21]. The amount of template RNA was normalized using human GAPDH primers as internal controls. The primer sequences for GAPDH were 5 -GTCAACGGATTTGGTCTGTATT-3 and 5 -AGTCTTCTGGGTGGCAGTGAT-3 , and the cycling conditions were 1 min at 94 ◦ C, 1 min at 56 ◦ C, and 2 min at 72 ◦ C. The UTI RT-PCR products were analyzed on a 2% agarose gel. 2.6. Statistics Statistical significance was determined by Student’s t-test. A P value of less than 0.05 was used as the criterion of statistical significance.
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medium showed a molecular weight of about 43 kDa, which was slightly lower than that of the intracellular UTI precursor and was in agreement with the report by Jonsson-berling et al. [22]. 3.2. IL-6, TNF-α, and IL-1β significantly up-regulated UTI secretion
Fig. 1. Free UTI secreted by HepG2 cells. After the HepG2 cells reached 80–100% confluence, the medium was changed and serum removed. Aliquots (15 l) of the medium collected at 12 and 24 h were run in 4–20% SDS-PAGE and the fractionated proteins were subsequently transferred to a nitrocellulose membrane for immunoblotting with anti-I␣I. The sequential change in UTI concentration in the medium was measured by the method of ELISA. Histograms are means ± S.D. The results are the means of at least three experiments.
After confirming that HepG2 cells secrete free UTI, we next investigated whether UTI secretion of UTI by HepG2 cells is regulated by proinflammatory cytokines. The UTI concentration in the medium was too low to be measured within 12 h after serum deprivation. Therefore, to investigate the effects of IL-6, TNF-␣, and IL-1 on UTI secretion before 12 h, the HepG2 cells were stimulated with IL-6, TNF-␣ or IL-1 24 h after serum deprivation. The cultured HepG2 cells in the presence of IL-6, TNF-␣ or IL-1 at the concentrations of 10, 20, and 10 ng/ml, respectively, significantly enhanced UTI secretion 12 h after stimulation (Fig. 2). To further investigate whether UTI up-regulations by IL-6, TNF-␣ and IL-1 are dose-dependent, we also observed the
3. Results 3.1. HepG2 cells constantly secrete free UTI into medium After serum deprivation, we demonstrated by ELISA that free UTI concentration increases in a time-dependent manner in the culture medium after 12 h (Fig. 1). Before 12 h, UTI concentration in the medium was too low to be detected by our method, indicating that HepG2 cells secrete UTI. Free UTI in the medium was also confirmed by western blotting (Fig. 1). Using an antibody against I␣I, free UTI in the
Fig. 2. Effects of TNF-␣, IL-6 and IL-1 on UTI secretion by HepG2 cells. After the HepG2 cells reached 80–100% confluence, the medium was changed and the serum removed. The cells were cultured for 24 h before stimulation with TNF-␣, IL-6, or IL-1. The effects of TNF-␣, IL-6, and IL-1 at concentrations of 10, 20, and 10 ng/ml, respectively on UTI secretion were compared with the control. Histograms are means ± S.D. The results are the means of at least three experiments. (*) P < 0.05; (**) P < 0.01.
Fig. 3. The effects of IL-6 and TNF-␣ on UTI secretion by HepG2 cells. After the HepG2 cells reached 80–100% confluence, the medium was changed and serum removed. The change in UTI concentration in the medium was measured by ELISA. The effects of different concentrations of (a) IL-6 and (b) TNF-␣ on free-UTI secretion at the time of 12 h are presented. Histograms are means ± S.D. The results are the means of at least three experiments.
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Fig. 4. TNF-␣, IL-1, and IL-6 up-regulate the intracellular UTI synthesis. After the HepG2 cells reached 80–100% confluence, the medium was changed and the serum removed. The HepG2 cells were stimulated with (A) 20 ng/ml TNF-␣, (B) 10 ng/ml IL-1 or (C) 10 ng/ml IL-6 for different durations as indicated. Cell lysates extract from untreated or cytokine-treated HepG2 cells were used for western blotting anti-I␣I, as described in the text. One representative result of three individual experiments is presented.
effects of different concentrations of IL-6 and TNF-␣ on UTI secretion at 12 h. IL-6 and TNF-␣ up-regulated UTI secretion in a dose-dependent manner (Fig. 3). 3.3. TNF-α, IL-1β, and IL-6 enhanced intracellular UTI precursor synthesis As indicated in Fig. 4, the UTI precursor band appeared at about 45 kDa when anti-I␣I antibodies were used, which was consistent with previous reports [23,24]. To determine whether IL-1, IL-6, and TNF-␣ up-regulate intracellular UTI precursor synthesis, we analyzed the change in the expression level of intracellular UTI protein in HepG2 cells in response to cytokine treatment. As shown in Fig. 4, UTI protein levels were notably up-regulated by incubation with 10 ng/ml IL-1 or 20 ng/ml TNF-␣. The up-regulation induced by IL-1 lasted from 2 to 8 h, whereas that induced by TNF-␣ was only observed at 2 h. When the Hep G2 cells
were incubated with 10 ng/ml IL-6, the expression level of the intracellular UTI protein increased with time until 8 h after stimulation and kept the expression level until 12 h. We also observed that the intracellular UTI protein expression level remained unchanged from 12 to 48 h by treatment with IL-1, IL-6 or TNF-␣ and that it was not changed during 72 h serum deprivation (data not shown). 3.4. UTI mRNA expression is up-regulated by IL-1β Among the three cytokines used in this experiment, IL-1 exhibited the most effective and persistent up-regulation of UTI synthesis and secretion (Figs. 2 and 4). Thus, we next examined by RT-PCR the change in UTI mRNA expression level after IL-1 stimulation. As shown in Fig. 5, UTI mRNA levels were distinctly up-regulated after 15-min treatment with 10 ng/ml IL-1. This result correlates with the UTI protein up-regulation observed by western blotting.
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Fig. 5. UTI mRNA expression in HepG2 cells treated with 10 ng/ml IL-1. Total RNA was isolated as described in the text from untreated or IL-1-treated HepG2 cells for the stated duration and subjected to RT-PCR analysis with primers specific for UTI. The PCR product of GAPDH amplification was used as control. PCR products were electrophoresed on a 2% agarose gel and stained with ethidium bromide. Because three individual experiments showed same results, one representative result is presented.
4. Discussion Although recent evidence indicates that hepatoma cells synthesize and secrete some I␣I family proteins, whether hepatoma cells secrete free UTI as well as the UTI secretion pattern remains unclarified. Bourguignon et al. [25] did not find free UTI in the medium of cultured human hepatoma HepG2 cells by western blotting. However, Thogerson et al. [24] found free UTI using the same method; although the HepG2 did not produce some UTI containing proteins, cultured HepG2 cells secreted free UTI. Our results demonstrate that HepG2 cells secrete free UTI in a time-dependent manner by both ELISA and western blotting. Although the roles of UTI in hepatoma cell proliferation, invasion and metastasis remain unclarified, Kobayashi et al. [26] reported that some tumor cells have UTI binding sites and that UTI inhibits murine Lewis lung carcinoma cell invasion and metastasis. UTI exists in serum both as a subunit of I␣I family proteins and in the free form. In our method, the samples are treated with perchloric acid to precipitate I␣I and related UTI-containing proteins that probably exist in the medium and we successfully used this method to determine plasma UTI concentration [19]. Jonsson-berling et al. [22] had used this method to pretreat serum and noted that no UTI was released from I␣I and related proteins during precipitation with perchloric acid. Therefore, we adopted this method for the determination of free-form UTI in the medium. We further demonstrated by western blotting that HepG2 cells secrete free UTI. The secreted free UTI did not contain ␣1-microglobulin and showed a molecular weight of about 43 kDa, which was slightly lower than that of the intracellular UTI precursor; this was in agreement with report by Thogerson et al. [24]. UTI and ␣1 -microglobulin are cosynthesized in hepatocytes by an ␣1 -microglobulin/bikunin precursor gene. After the UTI and ␣1 -microglobulin precursor is assembled with different H chain to synthesize I␣I family proteins via a chondroitin sulfate chain, ␣1 -microglobulin is released. Although some studies have been carried out to investigate the behaviors of UTI and UTI-containing proteins during
inflammation, controversy persists over whether UTI is a positive acute phase protein (APP) or a negative APP. In vivo and clinical studies have revealed that under pathological conditions such as acute inflammation, the serum ␣1 -microglobulin level is relatively constant, I␣I and I␣LI behave as a negative APP and p␣I behaves as a positive APP [17,18,27]. As indicated above, during inflammation, the UTI liberated from serum UTI-containing proteins increases through limited proteolysis in peripheral circulation or inflammatory site. However, because of its small molecular mass, UTI is quickly excreted into urine [28], the plasma UTI levels increases only slightly and urinary UTI excretion increases markedly in pathological conditions [8,29–31]. The UTI mRNA expression levels in Hep3B cells were found to be down-regulated 24 h after treatment with IL-6 and IL-1 in a previous study [17]. However, some other studies demonstrated that the UTI mRNA expression level and intracellular protein level increase or remain unchanged in hepatocytes and renal proximal tubular epithelial cells during inflammation [16,32]. In yet another study, it was noted that part of the UTI mRNA response to an inflammatory or a tumor process is unstable [23]. All these findings indicate that serum UTI concentration dose not directly reflect the change in the type of free UTI secreted by hepatocytes and it is difficult to accurately quantify the change in UTI mRNA expression level due to its instability. In this study, we used a new and sensitive method to detect free UTI secreted by HepG2 cells. This method enabled us to directly observe the change in the type of free UTI secreted by HepG2 cells and investigate the effects of cytokines on such change. We chose the proinflammatory cytokines, TNF-␣, IL-6, and IL-1, because they are known as the major mediators of acute protein induction in the liver [21,33]. We confirmed that the cells used in our experiment responded to TNF-␣ and IL-6 based on the observation that TNF-␣induced IB-␣degradation and IL-6 induced STAT-3 activation (data not shown). We demonstrated that IL-6, IL-1, and TNF-␣ up-regulate the secretion of UTI from 4 to 12 h in HepG2 cells. These effects were in accordance with changes of intracellular protein and the UTI mRNA expres-
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sion levels. Whether there is any difference in the regulation of UTI between normal hepatocytes and hepatoma cells remains to be clarified, although our results strongly suggested that free UTI is an early-response positive APP. The combined effects of the above cytokines or the interactions of cytokines with other major mediators of acute protein induction such as dexamethasone remain to be further studied. The major findings of the present study are that HepG2 cells synthesize and secrete free UTI, and that the synthesis and secretion of free UTI are up-regulated by proinflammatory cytokines. Our results suggest that during inflammation, the increased plasma and urine UTI levels are not only the result of the UTI release from serum UTI-containing proteins, but also that of the enhanced synthesis and secretion of free UTI by hepatocytes. The structural and functional differences of UTI secreted in free form and that cleaved from I␣I and related molecules are not currently understood and require further studies.
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