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Endocrinology Vol. 142, No. 1 241-248
Copyright © 2001 by The Endocrine Society

ARTICLES

Interleukin-6 Stimulates Hepatic Insulin-Like Growth Factor Binding Protein-4 Messenger Ribonucleic Acid and Protein1

Laura Fernández-Celemín and Jean-Paul Thissen

Unité de Diabétologie et Nutrition, Université Catholique de Louvain, 54 B-1200 Brussels, Belgium

Address all correspondence and requests for reprints to: Jean-Paul Thissen, M.D., Unité de Diabétologie et Nutrition, UCL/DIAB 5474, Avenue Hippocrate, 54, Brussels B-1200, Belgium. E-mail: thissen{at}diab.ucl.ac.be


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sepsis and bacterial lipopolysaccharide (LPS) injection decrease circulating concentrations of insulin-like growth factor (IGF)-I and induce an increase in IGFBP-1 and IGFBP-4 that may have impact upon IGF-I anabolic actions. Although the mechanisms responsible for the IGFBP-1 increase in response to LPS have already been unraveled, the cause for the IGFBP-4 elevation is still unknown. The aim of this study was to characterize the regulation of IGFBP-4 by proinflammatory cytokines and glucocorticoids. In rat primary cultured hepatocytes, interleukin (IL)-6 strongly stimulated IGFBP-4 messenger RNA (mRNA) and protein levels in a dose- and time-dependent way (mRNA levels: 9-fold, P < 0.01 and protein levels: ~3-fold at 24 h, with IL-6 10 ng/ml). Interleukin (IL)-1ß and tumor necrosis factor (TNF)-{alpha} blunted the IL-6 stimulation of IGFBP-4 mRNA (66% and 46% decrease, respectively) and protein levels (82% and 68% decrease, respectively). In contrast, dexamethasone induced IGFBP-4 mRNA and protein and potentiated the effect of IL-6 on IGFBP-4 mRNA (2.5-fold, P < 0.01 vs. IL-6 alone). Both actinomycin and cycloheximide prevented the IL-6 induction of IGFBP-4 mRNA suggesting that the IL-6 effect on IGFBP-4 gene occurs probably at the transcriptional level and needs an ongoing protein synthesis. Administration of IL-6 to rats caused a 3-fold increase in liver IGFBP-4 mRNA (P < 0.001) reflected in serum levels of IGFBP-4 (P < 0.05). In conclusion, our results show that IL-6 stimulates hepatic IGFBP-4 gene expression and production in vitro and in vivo, thereby suggesting another mechanism by which cytokines could control IGF-I action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEPSIS AND ENDOTOXIN (Escherichia coli lipopolysaccharide or LPS) administration are associated with low circulating concentrations of insulin-like growth factor (IGF)-I and changes in several of its binding proteins (IGFBPs) (1, 2, 3). These IGFBP alterations are thought to have impact upon IGF-I actions, among them the ability of IGF-I to inhibit protein breakdown (4, 5). The decline of circulating IGF-I biological activity might therefore play a role in the protein hypercatabolism observed in sepsis. Proinflammatory cytokines such as interleukin (IL)-1ß, tumor necrosis factor (TNF)-{alpha} or IL-6 and stress hormones as glucocorticoids, which are both massively released in acute sepsis, are thought to mediate some of the changes in IGF-I and IGFBPs. Indeed, the inhibition of liver IGF-I production caused by LPS injection can be reproduced in vitro as in vivo by IL-1ß and TNF-{alpha} (6, 7, 8, 9). The increase in IGFBP-1 observed in sepsis may be also reproduced in vivo by IL-1ß, TNF-{alpha} and dexamethasone (6, 7, 10) and in vitro by IL-1ß, IL-6, and dexamethasone (11, 12, 13). However, the mediators responsible for the increase in circulating IGFBP-4 caused by LPS injection have not been identified yet.

IGFBP-4 is the most abundant serum IGFBP after IGFBP-3 in adult rat and human circulation (14) and seems so far to exert only inhibitory effects on IGF action in all cell types tested in vitro (15, 16, 17, 18, 19). Several groups reported an increase in circulating IGFBP-4 after LPS injection in rats (2, 20, 21). Thus, the up-regulation of IGFBP-4 production could contribute to impair IGF-I action during sepsis. Because the production of IGFBP-4 by the liver, which is the main contributor to the circulatory levels, is increased by LPS injection (20, 22), we decided to investigate the regulation of its expression and production in primary cultured hepatocytes. Although thyroid hormones have been reported to stimulate IGFBP-4 production and gene expression (23), the regulation of IGFBP-4 by other hormones, in particular proinflammatory cytokines and glucocorticoids, has not yet been investigated. The present study was thus conducted to characterize the regulation of IGFBP-4 by proinflammatory cytokines and glucocorticoids in primary cultured hepatocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
For in vitro experiments, recombinant murine interleukin-6 (rmIL-6) was a kind gift of J. C. Renauld and J. Van Snick (ICP, Université Catholique de Louvain, Brussels, Belgium) and recombinant rat interleukin-6 was purchased through R&D Systems (Abingdon, UK). Both were used with similar results on IGFBP-4. Recombinant rat interleukin-1ß and tumor necrosis factor-{alpha} (rrIL-1ß, rrTNF-{alpha}) were purchased through R&D Systems (Abingdon, UK). Collagenase (type B) was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Insulin, hydrocortisone, dexamethasone, ethanolamine, sodium selenite, L-ornithine, L-lactic acid, EGTA and HEPES were purchased from Sigma (St. Louis, MO). NUNCLON plastic dishes and DMEM/Ham’s F-12 culture medium were purchased from Life Technologies, Inc. (Paisley, Scotland, UK). For in vivo experiments, recombinant human (rh)IL-6 was kindly supplied by Novartis Pharma AG (Basel, Switzerland).

Hepatocytes isolation and cell culture
Matrigel was prepared from Engelbreth-Holm-Swarm sarcoma propagated in C57BL/6J female mice and stored at -20 C as previous described (24). After thawing on ice, 170 µl of Matrigel were evenly spread on 60-mm plastic dishes and allowed to form a gel at 37 C. The hepatocytes were isolated from male Wistar rats (Katholieke Universiteit of Leuven, Leuven, Belgium) about 6 weeks of age (216 ± 2 g; mean ± SEM) that were maintained under standardized conditions of light (12-h light, 12-h dark cycle) and temperature (22 ± 2 C), with free access to animal chow and water. Hepatocytes were isolated by a nonrecirculating collagenase perfusion through the portal vein of rats anesthetized with pentobarbital (60 mg/kg). Hepatocytes were purified by differential centrifugation at 50 x g at 4 C. The centrifugation lasted 1 min and was performed three times immediately after isolation. Supernatants containing most of dead and nonparenchymatous cells were discarded each time. The percentage of contaminating cells (endothelial cells, Ito cells, Kupffer cells, fibroblasts, biliary duct cells) in the hepatocyte preparation was less than 5% as determined by immunocytochemistry with a vimentin antibody. An aliquot of the final cell suspension was diluted with trypan blue (1:6) to count the cells and to determine the viability, which was always over 80% (86 ± 2%; mean ± SEM). Cells were seeded at a density of 1.8 x 106 per dish in 3 ml serum-free DMEM/Ham’s F-12 medium supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), hydrocortisone (5 x 10-5 M), insulin (1 mg/ml), L-ornithine (4 x 10-1 M), L-lactic acid (2.25 mg/ml), sodium selenite (2.5 x 10-5 M), ethanolamine (1 x 10-3 M). Hepatocytes were cultured for 48 h before the beginning of the experimental period. Cultures were maintained at 37 C in a humidified incubator in an atmosphere containing 5% CO2. Twenty-four hours after the plating, the medium was changed. After this 48-h period, the experiments were started by incubating the cells anew in serum-free DMEM/Ham’s F-12 medium supplemented as previously described, and containing cytokines and dexamethasone at different concentrations as showed in the figures. Cells were collected 0, 6, 10, or 24 h after exposition to experimental conditions. The maximum duration of the experimental period was 24 h, and the total duration of the culture was 72 h. Each value represents a pool of four 60-mm plates. Results are presented as mean values of three or four separate experiments each performed with a different rat.

In vivo experiment
Male Wistar rats (Katholieke Universiteit of Leuven, Leuven, Belgium) about 7 weeks of age (231 ± 2 g; mean ± SEM) were maintained 1 week under standardized conditions of light (12-h light, 12-h dark cycle) and temperature (22 ± 2 C), with free access to animal chow and water. The morning of the eighth day, rats were injected ip with rhIL-6 (40 µg/kg) diluted in PBS containing 0.2% BSA. An equivalent volume of vehicle (1 µl/g of body weight) was injected to control rats. Rats were killed by decapitation at different times after injection. Livers were flash-frozen in liquid nitrogen and stored at -80 C. Blood was collected and centrifuged 15 min at 1800 rpm at 4 C and sera were stored at -20 C.

RNA extraction and Northern blot analysis
For in vitro experiments, the cells were washed twice with PBS 1x at pH 7.4, collected with guanidine thiocyanate 4 M (900 µl/60-mm plate), and stored immediately at -70 C. The four plates representing the same point were pooled at this step and cells were homogenized by Ultraturrax. For in vivo experiments, RNA was extracted from 200 mg of rat liver, previously pestled in liquid nitrogen, and homogenized by Ultraturrax in 9 ml of guanidine thiocyanate 4 M.

Total hepatocyte and liver RNA was extracted using the guanidine thiocyanate and cesium chloride method (25). Twenty micrograms of total RNA from each sample were denatured in formaldehyde-MOPS and subjected to electrophoresis on 1% agarose gels. Homogeneity of the loading was assessed by UV transillumination of the gels after staining with ethidium bromide. The RNA was transferred to nylon membranes (Hybond-N, Amersham Pharmacia Biotech, Buckinghamshire, UK) by capillary overnight blotting and levels of IGFBP-4 messenger RNA (mRNA) were determined by hybridization with a specific riboprobe. A 221-bp complementary DNA rat IGFBP-4 exon fragment ligated into the plasmid vector Bluescript (Stratagene, La Jolla, CA) was linearized with BamHI. A 355-bp complementary DNA rat IGFBP-1 exon fragment ligated into the plasmid vector Bluescript was linearized with XhoI. Both plasmids were kindly provided by Billie Moats-Staats (UNC, Chapel Hill, NC). Specific [32P]-labeled RNA antisense probe were generated from linearized plasmids with uridine 5'-[{alpha}-32P]-triphosphate (Amersham Pharmacia Biotech, 800 Ci/mmol) using the T7 (IGFBP-4) or T3 (IGFBP-1) RNA polymerase. Blots were prehybridized for 3 to 4 h at 65 C in buffer containing NaCl (0.8 M), Na phosphate (50 mM), EDTA (0.5 mM), Denhardt (BSA, Ficoll and polyvinylpyrrolidone 2 g/liter of each), salmon sperm DNA (300 mg/liter) and formamide (50%). They were then hybridized with the radiolabeled antisense RNA probe (1 x 106 cpm/ml of buffer) for 16–18 h in a shaking waterbath at 65 C. After hybridization, blots were quickly washed once with 300 ml of washing buffer (Na phosphate 20 mM, NaCl 50 mM, SDS 0.1%, EDTA 1 mM) at 75 C (for IGFBP-4) or at 70 C (IGFBP-1) and then more extensively twice for 30 min in a shaking waterbath at 75 C (IGFBP-4) or 70 C (IGFBP-1). When needed, a supplementary third washing was done in the same way. Membranes were finally exposed to XAR-5 Kodak film for 3–6 h at -70 C. The mRNA levels were quantified by densitometric scanning of the hybridization signal (LKB Ultroscan XL laser densitometry, LKB Bromma, Sweden) with the use of a software (Gel Scan, Amersham Pharmacia Biotech, Uppsala, Sweden). Densitometric values were normalized by assigning to the mRNA levels observed after 48 h of culture (corresponding to the beginning of the experimental period) an arbitrary value of 100% (=basal levels). This group neither served for statistical analysis nor appeared in the figures with the exception of the in vitro time-course (Fig. 2Go).



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  		Figure 2. Time-dependent effect of IL-6 on IGFBP-4 mRNA and protein in primary cultured hepatocytes. After 48 h of culture, hepatocytes were incubated for 6, 10, or 24 h with IL-6 (10 ng/ml). A, Northern blot: Cells were collected after 6, 10, or 24 h and total RNA was extracted. 20 µg of RNA were electrophoresed in each lane and Northern blot analysis was performed as indicated in Materials and Methods. Upper panel, autoradiography; lower panel: densitometric analysis. Data are expressed as the mean ± SEM of three separate experiments. *, P < 0.05; **, P < 0.01 vs. time-matched control. B, Ligand blot, Culture medium was collected at the same time as mRNA (6, 10, or 24 h), concentrated and electrophoresed, and ligand blot analysis was performed as described in Materials and Methods. Upper panel, autoradiography; lower panel, densitometric analysis. Data are expressed as the mean ± SEM of two separate experiments.

 
Western ligand blotting
To determine the culture medium content of IGFBP-4, 2 ml of pooled media from four plates were concentrated 10-fold in a Centricon microconcentrator-10 (Amicon, Beverly, MA) by centrifugation at 5000x g at 10 C during 90 min. A volume of sample corresponding to 200 µl of culture medium or to 1.5 µl of serum was diluted with Laemmli buffer, incubated 4 min at 95 C and applied to each lane for electrophoresis in a 12.5% SDS-PAGE polyacrylamide gel as previously described (26). The molecular size of IGFBPs was estimated using a prestained SDS-PAGE standard (Bio-Rad Laboratories, Inc., Hercules, CA). The samples were then transferred onto Immobilon-P membranes (Millipore Corp., Bedford, MA), probed with 500 000 cpm/ml of [125I]rhIGF-II kindly provided by Eli Lilly & Co. (Indianapolis, IN) and visualized by autoradiography at -70 C. The autoradiograms were scanned using an Ultroscan XL laser densitometry (LKB-Bromma, Bromma, Sweden) and the results were expressed as arbitrary scanning densitometric units. For each sample, density signal of the 28-kDa band was determined. Ligand blot autoradiographies were cropped to show only binding proteins in the 20–30 kDa range because higher molecular weight IGFBP-bands were absent. The lack of slower migrating IGFBPs such as IGFBP-3 emphasizes the absence of significant amount of contaminating cells in our hepatocyte population. Densitometric values were normalized by assigning to the protein levels observed in control (untreated) group after 72 h of culture (corresponding to the end of the experimental period) an arbitrary value of 100% (=control levels).

Western immunoblotting
To confirm that the 28-kDa band identified by ligand blot corresponds indeed to IGFBP-4, we performed immunoblots with a specific antibody against IGFBP-4. Conditioned medium was concentrated, subjected to electrophoresis, and transferred as described above with the exception that for immunoblot DTT (100 mM) was included in the Laemmli buffer. The membrane was blocked for 30 min with 5% nonfat dried milk in buffer [50 mM Na-phosphate (pH 7.4), 150 mM NaCl, 0.05% Tween-20], followed by overnight incubation at 4 C with a specific anti-IGFBP-4 antibody at a titer of 1:5000. The antibody was raised in rabbits against a synthetic 20-amino acid peptide (81–100) from rat IGFBP-4 and was a kind gift from Steven Chernausek (Children’s Hospital Medical Center, Cincinnati, OH). The membranes were then rinsed four times in PBS containing 0.05% Tween-20 and incubated for 1 h at RT with horseradish peroxidase-labeled swine antirabbit (DAKO Corp. A/S, Glostrup, Denmark) as secondary antibody at a dilution of 1:5000. After further washing, the antigen-antibody complex was identified by the ECL (chemiluminescence) detection system as recommended by the manufacturer (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Statistical analysis
Statistical significance between samples was determined by one or two-way ANOVA followed by the Newman-Keuls multiple comparisons test. For mRNA analysis in cultured hepatocytes, each point is the mean ± SEM of values obtained in three or four separated experiments each performed in one rat. Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of IGFBP-4 by IL-6 in vitro
Dose response and time-course of IL-6 stimulation of IGFBP-4 mRNA and protein levels. Among the three major proinflammatory cytokines (IL-1ß, TNF-{alpha}, and IL-6) released by LPS, only IL-6 stimulated IGFBP-4 production. Interleukin-6 increased IGFBP-4 mRNA levels in a dose-dependent way (Fig. 1AGo). Ten nanograms per milliliter of IL-6, which is the dose used throughout the experiments, produced a statistically significant increase in IGFBP-4 gene transcripts levels (9.7-fold; P < 0.01 vs. control).



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  		Figure 1. Dose-dependent effect of IL-6 on IGFBP-4 mRNA and protein in primary cultured hepatocytes. After 48 h of culture, hepatocytes were incubated for 24 h with 0.1, 1, or 10 ng/ml of IL-6. A, Northern blot: Cells were collected at the end of the 24 h and total RNA was extracted. 20 µg of RNA were electrophoresed in each lane and Northern blot analysis was performed as indicated in Materials and Methods. Upper panel, autoradiography; lower panel: densitometric analysis. Data are expressed as the mean ± SEM of three separate experiments. *, P < 0.05; **, P < 0.01 vs. control. B, Ligand blot and immunoblot: Culture medium was collected at the same time as the mRNA, concentrated electrophoresed and ligand blot and immunoblot analysis were performed as described in Materials and Methods. Upper panel, autoradiography of ligand blot (top) and immunoblot (bottom); lower panel, densitometric analysis of the ligand blot results. Data are expressed as the mean ± SEM of two separate experiments.

 
To investigate whether the changes observed in IGFBP-4 mRNA were associated to changes in IGFBP-4 protein production, IGFBP-4 protein levels were assessed in the culture media by Western ligand blotting and immunoblotting. The protein levels reflect the accumulation from the beginning of the experimental period, when cells were challenged with cytokines and glucocorticoids, until the end. The 28-kDa IGFBP identified by ligand blot of hepatocyte conditioned medium is IGFBP-4. This conclusion is suggested by ligand blot results showing the same motility of this 28-kDa IGFBP and the pure IGFBP-4 (a kind gift of Steven Chernausek) (data not shown), and it is further demonstrated by immunoblot with an IGFBP-4 antibody where IGFBP-4 appears as a 32-kDa band (Fig. 1BGo, bottom). The apparent discrepancy in motility between ligand blot and immunoblot may be due to differences in disulfide bonds reduction (27).

IGFBP-4 protein levels in the culture media followed the mRNA pattern of induction, increasing 3.5, 6, and 7.7 times (vs. control) in response to 0.1, 1, and 10 ng/ml of IL-6 respectively (Fig. 1BGo).

The IGFBP-4 stimulation by IL-6 was also time-dependent. At 10 ng/ml, IL-6 increased IGFBP-4 mRNA levels as early as 6 h (2-fold the control levels), and this stimulatory effect became significant after 10 h (3.5-fold; P < 0.05) and after 24 h (9.2-fold; P < 0.01) (Fig. 2AGo). The stimulation of IGFBP-4 protein levels caused by IL-6 (10 ng/ml) was maximal at 24 h, the last time point examined (2.8-fold the control levels at that time) (Fig. 2BGo).

Potentiation of the IL-6 effect on IGFBP-4 by dexamethasone. Given the synergy between glucocorticoids and IL-6 on several acute phase reactants produced by hepatocytes, we investigated, in a second step, the effect of dexamethasone on IGFBP-4. Dexamethasone alone induced IGFBP-4 mRNA levels 3.4-fold at 10-8 M and 9.6-fold at 10-6 M. The half maximal increase of the IGFBP-4 mRNA levels was obtained with a concentration between 10-8 M and 10-6 M. In addition, dexamethasone potentiated IGFBP-4 mRNA induction by IL-6 (dexamethasone 10-6 M alone: 9.6-fold; IL-6 alone: 13.6-fold; and dexamethasone and IL-6: 34-fold; P < 0.01 vs. dexamethasone 10-6 M alone and P < 0.05 vs. IL-6 alone) (Fig. 3AGo). These changes in IGFBP-4 mRNA were reflected in the IGFBP-4 protein content of the culture media that increased 5.5 times after exposure to dexamethasone, 7.7 times after exposure to IL-6 and 10.9 times after exposure to IL-6 and dexamethasone combined (Fig. 3BGo).



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  		Figure 3. Effect of IL-6 and dexamethasone on IGFBP-4 and IGFBP-1 mRNA and protein levels. After 48 h of culture, hepatocytes were incubated for 24 h with different concentrations of dexamethasone in the presence or absence of IL-6 (10 ng/ml). A, Northern blot: Cells were collected after the 24 h and total RNA was extracted. Twenty micrograms of RNA were electrophoresed in each lane and Northern blot analysis was performed as indicated in Materials and Methods. Upper panel, autoradiography of IGFBP-4 and IGFBP-1 Northern blot. The Northern blot of IGFBP-1 was performed in two different experiments with similar results. Lower panel, densitometric analysis of IGFBP-4 Northern blot in which data are expressed as the mean ± SEM of four separate experiments. **, P < 0.01 vs. dose-matched control; °, P < 0.05 vs. IL-6 alone. B, Ligand blot and immunoblot: After 48 h in culture, hepatocytes were incubated in 10-6 M of dexamethasone in the presence or absence of IL-6 (10 ng/ml). Culture medium was collected at the same time as mRNA, concentrated, electrophoresed, and ligand blot and immunoblot analysis were performed as described in Materials and Methods. Upper panel, Autoradiography of ligand blot (top) and immunoblot (bottom); lower panel, densitometric analysis of the ligand blot results. Data are expressed as the mean ± SEM of two separate experiments.

 
In contrast to IGFBP-4, IGFBP-1, another IGF binding protein, was strongly stimulated by dexamethasone but not by IL-6 (Fig. 3AGo).

Inhibition of the IL-6 effect on IGFBP-4 by proinflammatory cytokines. We then tested the effect of other cytokines which take part in inflammatory reactions together with IL-6 and might influence its actions on IGFBP-4. Hepatocytes were exposed to different doses of IL-1ß or TNF-{alpha} for 24 h in the presence or absence of IL-6. The IL-6-induced stimulation of IGFBP-4 gene expression levels was blunted by IL-1ß and TNF-{alpha}. This effect was dose-dependent with a maximal inhibition of the IL-6 action on IGFBP-4 mRNA levels observed at 100 ng/ml of IL-1ß or TNF-{alpha} (66% decrease, P < 0.05 and 46% decrease, NS). However, IL-1ß and TNF-{alpha} alone failed to inhibit the basal expression of IGFBP-4 (Fig. 4Go, A and C). In agreement with the changes induced in IGFBP-4 mRNA by IL-1ß and TNF-{alpha}, these cytokines completely blunted the effects of IL-6 on IGFBP-4 protein levels. IL-1ß diminished the IL-6 stimulation of IGFBP-4 by 82% and TNF-{alpha} by 68% (Fig. 4Go, B and D).



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  		Figure 4. Inhibition of the IL-6 effect on IGFBP-4 mRNA and protein by IL-1ß (A and B) and TNF-{alpha} (C and D) in primary cultured hepatocytes. After 48 h of culture, hepatocytes were incubated for 24 h with 0.1, 1, 10, or 100 ng/ml of IL-1ß or TNF-{alpha} in the presence or absence of IL-6 (10 ng/ml). A and C, Northern blot: Cells were collected after the 24 h and total RNA was extracted. 20 µg of RNA were electrophoresed in each lane and Northern blot analysis was performed as indicated in Materials and Methods. Upper panel: autoradiography; lower panel: densitometric analysis. Data are expressed as the mean ± SEM of three separate experiments. **, P < 0.01, ***P < 0.001 vs. control; °P < 0.05 vs. IL-6 alone. B and D, Ligand blot and immunoblot: After 48 h of culture, hepatocytes were incubated for 24 h in 100 ng/ml of IL-1 ß or TNF-{alpha} in the presence or absence of IL-6 (10 ng/ml). Culture medium was collected after 24 h, concentrated, electrophoresed, and ligand blot and immunoblot analysis were performed as described in Materials and Methods. Upper panel, Autoradiography of ligand blot (top) and immunoblot (bottom); lower panel, densitometric analysis of the ligand blot results. Data are expressed as the mean ± SEM of two separate experiments.

 
Effect of actinomycin and cycloheximide on the IL-6 stimulation of IGFBP-4 mRNA. To determine whether the effect of IL-6 on IGFBP-4 mRNA resulted from an increase in the transcriptional rate of the gene and involved an ongoing protein synthesis, hepatocytes were incubated with IL-6 in the presence of actinomycin or cycloheximide. Both actinomycin (2 µg/ml) and cycloheximide (10 µg/ml) prevented the stimulatory effect of IL-6 on IGFBP-4 mRNA levels after 10 h (P < 0.001) (Fig. 5Go).



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  		Figure 5. Effect of cycloheximide and actinomycin on the IL-6 stimulation of IGFBP-4 mRNA in primary cultured hepatocytes. After 48 h of culture, hepatocytes were incubated for 10 h with 10 ng/ml of IL-6 in the presence or absence of cycloheximide (10 µg/ml) or actinomycin (2 µg/ml). Northern blot, Cells were collected after 10 h, and total RNA was extracted. Twenty micrograms of RNA were electrophoresed in each lane and Northern blot analysis was performed as indicated in Materials and Methods. Upper panel, autoradiography; lower panel, densitometric analysis. Data are expressed as the mean ± SEM of three separate experiments. ***P < 0.001 vs. control.

 
Regulation of IGFBP-4 by IL-6 in vivo
In the following step, we investigated whether IL-6 was also able to induce liver IGFBP-4 production in vivo.

Time-course of IL-6 effect on IGFBP-4 hepatic mRNA. IL-6 injection (40 µg/kg), induced a 3-fold stimulation of IGFBP-4 gene transcripts between 2 and 4 h (P < 0.001). Twenty-four hours after the injection, the values of IGFBP-4 gene transcript were not different from control ones (Fig. 6AGo).



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  		Figure 6. Time-course induction of IGFBP-4 hepatic mRNA and serum protein content after in vivo administration of rhIL-6. Male Wistar rats were injected ip with 40 µg rhIL-6/kg body weight. A, Northern blot: After total liver RNA was extracted, 20 µg of RNA were electrophoresed in each lane, and Northern blot analysis was performed as indicated in Materials and Methods. Upper panel, Autoradiography; lower panel, densitometric analysis. Data are expressed as the mean ± SEM of 4 rats in each group. *, P < 0.05; ***, P < 0.001 vs. time 0 h. B, Ligand blot: Ligand blot analysis was performed on serum as described in Materials and Methods. Upper panel, Autoradiography; lower panel, densitometric analysis. Data are expressed as the mean ± SEM of 4 rats in each group. *, P < 0.05 vs. time 0 h.

 
Time-course of IL-6 effect on IGFBP-4 serum levels. To investigate whether the increase in IGFBP-4 mRNA levels was reflected by an increase in serum IGFBP-4, we analyzed the serum levels of IGFBP-4. In animals injected with IL-6, serum IGFBP-4 protein increased by 50% between 8 and 12 h following IL-6 administration (P < 0.05). Again, at 24 h the IGFBP-4 protein values were similar to control ones (Fig. 6BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results show that IL-6 stimulates hepatic IGFBP-4 gene expression and protein production in vitro and in vivo. This effect of IL-6 is blunted by other proinflammatory cytokines like IL-1ß and TNF-{alpha}, whereas it is potentiated by dexamethasone. The stimulation by IL-6 of IGFBP-4 gene occurs probably at transcriptional level and needs an ongoing protein synthesis.

The physiological relevance of our findings is supported firstly by the dose-response curve of the IL-6 effect on IGFBP-4 expression. Indeed, 1 ng/ml of IL-6, a concentration close to peripheral circulating concentrations of IL-6 in sepsis or in chronic inflammatory situations (28, 29, 30), is able to stimulate IGFBP-4 expression and production in a significant way. Furthermore, taking into account the possible paracrine interactions between hepatocytes and Kupffer cells, the cytokine-producing cells in the liver, local concentrations of IL-6 may be expected to be even higher than those reached in the peripheral circulation after LPS injection. Secondly, IL-6 administrated in vivo, at a dose meant to trigger the acute phase response in rat liver (31), was able to increase liver IGFBP-4 gene and protein production.

The peak response of IGFBP-4 mRNA occurs between 2 and 4 h after IL-6 injection, whereas in vitro the peak is observed after 10 h of incubation. One possibility for explaining the faster response of IGFBP-4 in vivo than in vitro may reside in the different pattern of hepatocytes exposure to IL-6 (transient vs. continuous). Such a discrepancy has already been reported for an acute-phase protein, the {alpha}-2 macroglobulin, after IL-6 stimulation (32, 33).

The stability with time of IGFBP-4 protein levels in conditioned medium despite progressive decrease of its mRNA is not unexpected. Indeed, the decrease in IGFBP-4 mRNA inhibits any further increase in IGFBP-4 protein levels but cannot diminish them unless there is a proteolytic activity in the conditioned media.

The effect of IL-6 on IGFBP-4 gene expression was specific for this IGF binding protein. In fact, IL-6 alone failed to stimulate the mRNA of IGFBP-1, another IGFBP produced by hepatocytes, whereas dexamethasone strongly induced it as previously reported (10, 11). This indicates that the IGFBP-4 stimulation by IL-6 does not reflect a generalized stimulation of IGFBPs gene expression.

The proinflammatory cytokines IL-1ß and TNF-{alpha} markedly antagonized the stimulation of IGFBP-4 mRNA and protein by IL-6, although these cytokines alone did not inhibit the IGFBP-4 basal gene expression. The fact that IL-1ß and TNF-{alpha} inhibit to a larger extent the IGFBP-4 protein production than the mRNA suggests an additional mechanism of inhibition of IGFBP-4 by these cytokines. Although production of specific IGFBP-4 proteases in hepatocytes have not yet been described, the possibility remains that IL-1ß and TNF-{alpha} increase IGFBP-4 proteolysis as described for other cytokines (34).

A similar inhibition by IL-1ß of the IL-6 stimulation of the production of fibrinogen and {alpha}-2-macroglobulin by human hepatocytes (35) has been reported and more recently it has been shown that the stimulation by IL-6 of angiogenin, a potent inducer of neovascularization in vivo, is also abolished by IL-1ß (36).

In contrast to these cytokines, dexamethasone increased IGFBP-4 mRNA and protein, and potentiated the effect of IL-6 on IGFBP-4 transcript levels but not on IGFBP-4 protein production. Again, it is possible that the discrepancy between IGFBP-4 mRNA and protein levels in response to IL-6 and dexamethasone combination results from IGFBP-4 proteases induced by dexamethasone (37). The synergy between glucocorticoids and IL-6 has been reported for several other genes, notably the acute-phase proteins (APPs) (38) and is probably physiologically relevant in sepsis, when both hormones are present in high concentrations. The relationship between the two hormones is reinforced by the fact that IL-6 increases the production of glucocorticoids through stimulation of the hypothalamic-pituitary-adrenal axis (39, 40). The mechanism responsible for the synergy may reside in the ability of glucocorticoids to increase the levels of IL-6 receptors at the hepatocyte cell surface (41, 42). Furthermore, IL-6 and glucocorticoids may also synergize at the intracellular level. Indeed, it has been shown that IL-6-activated STAT-3 can associate with ligand-bound glucocorticoid receptor to form a transactivating/signaling complex, which can function through either an IL-6 responsive element or a glucocorticoid-responsive element (43, 44).

Acute phase proteins (APPs) are grouped typically into two classes. Class I APPs, are stimulated by IL-1ß or TNF-{alpha}, synergistically or not with IL-6-type cytokines and/or glucocorticoids, like C-reactive protein (CRP) in humans, haptoglobin in rats, serum amyloid A (SAA) (rat and human) among others (45). Class II APPs are stimulated by IL-6 or IL-6-type cytokines, with or without glucocorticoids, but without a requirement for IL-1ß or TNF-{alpha}, like fibrinogen chains, {alpha}2-macroglobin in rats, haptoglobin in humans among others (46). The features of the IGFBP-4 regulation by cytokines and glucocorticoids evidenced by our experiments typify regulation of class II APPs.

Typically, in critically ill patients, an early proinflammatory response, characterized by a rapid release of TNF-{alpha} and IL-1ß and to a minor extent IL-6, is followed by a more sustained antiinflammatory response involving IL-6 and glucocorticoids (47). Therefore, our observations suggest that induction of IGFBP-4 in critical states is probably associated with the antiinflammatory response.

The IL-6-induction of IGFBP-4 mRNA was suppressed in the presence of actinomycin, suggesting that IL-6 induces IGFBP-4 gene expression by stimulating its transcription. This would not be surprising taking in consideration that the effect of IL-6 on the expression of other hepatic genes, particularly the APPs, is mediated primarily at the level of transcription (48, 49). Cycloheximide abolished the ability of IL-6 to increase IGFBP-4 mRNA. Although data obtained using metabolic inhibitors must be interpreted with caution, this may suggest that the up-regulation of IGFBP-4 by IL-6 is protein-synthesis dependent.

Taken together, these results support the conclusion that IL-6 increases IGFBP-4 mRNA probably at the level of transcription and this process needs an ongoing protein synthesis. Indeed, during the acute-phase response, the major transcription factors transducing the IL-6 stimulation on class II APPs are STAT-3 and C/EBPß (50). Once activated, these factors translocate to the nucleus, where they form a transcription complex with IL-6 responsive elements, and this process is dependent on new protein synthesis and thus inhibitable by cycloheximide (51). Alternatively, our data are also compatible with a model in which IL-6 increases the expression of a gene that regulates the stability of IGFBP-4 mRNA.

The DNA sequence of the upstream region of the rat IGFBP-4 gene as described by Gao et al. (52), was inspected for specific sequences susceptible to confer response to transcription factors activated by IL-6 (MatInspector V2.2-GenBank, TRANSFAC 3.5). Sequences matching exactly the consensus that enable potential cis-elements like STAT-3 and C/EBPß to land, are actually present in the 5' flanking upstream region of the rat IGFBP-4 gene.

Regarding glucocorticoid responsiveness, the consensus sequence is not present in the promoter region of the IGFBP-4 gene, although dexamethasone stimulates IGFBP-4 mRNA and protein. However, regions containing sequences resembling the motif of glucocorticoid responsive element (GRE) that may serve as an enhancer element for glucocorticoids are present.

Taken altogether, our data suggest that the stimulation of IGFBP-4 by IL-6 is likely to happen in vivo at the late stage of the acute inflammatory response, when the production of IL-6 and glucocorticoids takes place after that of TNF-{alpha} and IL-1ß.

The regulation of IGFBP-4 by inflammatory and hormonal stimuli common to many acute-phase proteins of class II suggests that this IGFBP plays a role in physiologic response to inflammation and infection.


   Acknowledgments
 
We gratefully acknowledge Mrs. Josiane Verniers, Pascale Lause, and Ms. Anne-Laure Hubert for expert technical assistance.


   Footnotes
 
1 This work was supported by grants from the Fund for Scientific Medical Research (Belgium), from the Fonds Spéciaux de Recherche (Université Catholique de Louvain, UCL) and from the Danone Institute (Brussels). Back

Received June 7, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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