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Department of Medicine (J.-G.S., R.N., C.E.), University of Göttingen, D-37099 Göttingen, Germany; Institute of Pathology (F.D.), University of Magdeburg, D-39120 Magdeburg, Germany; Dipartimento di Biologia Sperimentale, Ambientale e Applicata (I.D.), Università di Genova, I-16132 Genova, Italy; and Children’s Hospital (B.K., T.B.), Department of Biochemistry, University of Hamburg, D-20246 Hamburg, Germany
Address all correspondence and requests for reprints to: Dr. Thomas Braulke, Children’s Hospital-Biochemistry, Universitätsklinikum Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. E-mail: braulke{at}uke.uni-hamburg.de.
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Introduction |
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In adult rats the liver is considered the main source of both circulating IGFs (9, 10) and distinct IGFBPs (11). Several studies in rats have demonstrated that biosynthesis of individual components of the IGF axis is located in different liver cells (12, 13, 14, 15, 16). Thus, IGFBP-1 and -4 are synthesized by hepatocytes, whereas IGFBP-3 is secreted from nonparenchymal cells, e.g. Kupffer cells, endothelial cells, and hepatic stellate cells (HSC), but not by hepatocytes. On the other hand, IGF-I is predominantly released from hepatocytes (17, 18, 19, 20).
Several lines of evidence indicate that HSC (also termed fat-storing cells, Ito cells, and perisinusoidal or parasinusoidal lipocytes) are one of the principal effectors involved in the repair process after liver tissue damage as well as in fibrogenesis. In response to both acute and chronic liver injuries, HSC proliferate and undergo phenotypic transformation from a quiescent, vitamin-A rich phenotype (early cultivated HSC) to a myofibroblast-like phenotype (activated HSC) characterized by increased proliferation, motility, contractility, and synthesis of extracellular matrix components (21, 22, 23). Recent studies have demonstrated that due to a high level of IGF-I receptor expression, early cultivated HSC are highly susceptible to the mitogenic activity of IGF-I, whereas activated HSC become less sensitive to IGF-I through down-regulation of the IGF-I receptor (18, 24). Indeed, in vitro proliferation of early cultivated HSC is stimulated to a higher magnitude by IGF-I than that of activated HSC (18, 25), indicating a role for IGF-I in the initiation of HSC proliferation in early steps of liver tissue repair and fibrogenesis. The role of IGFBPs in these processes is still unclear. In the present study, acute liver injury was induced by a single intragastric dose of carbon tetrachloride (CCl4) in adult rats, resulting in necrosis of perivenous hepatocytes within 24 and 48 h, followed by a resolution of the necrotic material and an almost complete normalization of liver histology 96 h after intoxication (26). In this study we report an accumulation of IGFBP-1 in perivenous areas of the liver acinus in response to CCl4 administration. Treatment of HSC on d 2–3 of culture with human IGFBP-1 (hIGFBP-1) purified from amniotic fluid as well as coculture with hIGFBP-1-overexpressing cells significantly stimulated the proliferative effects of IGF-I. These data indicate a role for IGF-I/IGFBP-1 as a mitogenic signal for the initiation of HSC proliferation during acute liver injury.
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Materials and Methods |
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Antibodies
Antibodies to rat IGFBP-1 were provided by Drs. Ling and Shimasaki (Whittier Institute for Diabetes and Endocrinology, La Jolla, CA). Rabbit polyclonal antiserum against mouse fibulin-2 was provided by Dr. Timpl (Max Planck Institut für Biochemie, Martinsried, Germany). The antiserum against hIGFBP-1 was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). The monoclonal antibody ED2 was supplied by Serotec (Oxford, UK). The antiserum directed against human albumin and that against smooth muscle 
-actin were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Rabbit IgG against human von Willebrand factor and monoclonal antibody to desmin were obtained from Dako (Hamburg, Germany). Biotinylated mouse monoclonal antibodies against rabbit and goat IgG were obtained from Dianova (Hamburg, Germany), and horseradish peroxidase-conjugated streptavidin was purchased from Zymed Laboratories (San Francisco, CA).
cDNA probes
The following cDNA probes were used for Northern blot analysis: a 407-bp fragment of rat IGFBP-1 cDNA clone pRBP-1–501 (6), a 700-bp fragment of rat IGF-I cDNA provided by Drs. Schwander and Margot (Basel, Switzerland), a 700-bp fragment of mouse albumin cDNA (27). To quantify Northern blots, an oligonucleotide (5'-AAC GAT CAG AGT AGT GGT ATT TCA CC-3') complementary to 28S rRNA was used.
Animals
Adult male Wistar rats were purchased from Charles River Breeding Laboratories (Sulzfeld, Germany) and kept under standard conditions with free access to food and water. All animals received human care in compliance with institutional and NIH guidelines. To induce acute liver injury, 32 adult male Wistar rats (200–250 g body weight) were given a single intragastric dose of 150 µl CCl4, dissolved in an equal volume of olive oil, per 100 g body weight. Control animals were treated with the solvent alone. Animals were killed at intervals of 3, 6, 9, 12, 24, 48, 72, and 96 h after intoxication. At each time point, four animals were killed. Livers from two of the four rats were processed for histology, whereas sera from the other two rats were collected, and their livers were used for RNA isolation and studies by in situ hybridization. No differences in food intake were noted between control and treated animals.
Processing of liver tissue and immunohistochemical analyses
The method used has been described in detail previously (28). In brief, before removal of the liver under anesthesia, the portal vein was cannulated, and the liver was perfused with Ringer’s solution containing 0.5% (vol/vol) procaine and 4% (wt/vol) dextran for 2 min, followed by fixation using a cocktail of Ringer’s solution, 4% (wt/vol) dextran, 0.3% (vol/vol) glutaraldehyde, and 3% (vol/vol) formaldehyde freshly prepared from paraformaldehyde. Sections of about 2–3 mm thickness were dehydrated and embedded in paraffin. Liver sections were used for immunohistochemistry essentially as described previously (28). The antiserum against rat IGFBP-1 was administered in a final dilution of 1:50 (vol/vol). Antibody detection was performed with the LSAB+ kit (Dako) and the DAB+ kit (Dako) according to the instructions of the manufacturer. For desmin staining, sections were deparaffinized with xylol, boiled for 20 min in 1 mM EDTA buffer (pH 8.0), and digested thereafter with protease (0.5 U/ ml; Ventana, Illkirch, France). The monoclonal antibody against desmin was used in a final concentration of 1:100 (vol/vol). Bound primary antibodies were detected using the biotin streptavidin system with amplifier (IView, Ventana). Tissue sections were counterstained with hematoxylin, dehydrated, and coverslipped with Eukit (Merck & Co.).
In situ hybridization
Prehybridization, hybridization, and washing procedures including removal of nonspecifically bound probe by ribonuclease A digestion were performed for both antisense and sense strand 35S-labeled RNA probes as described previously (29, 30). Sections from all time points after CCl4 administration were processed simultaneously using the same batches of probes and reagents. After exposing the slides for 2–6 wk, autoradiographic detection was performed as described in detail previously (29).
Tissue extraction
Protein extraction was essentially performed as described previously (31). In brief, approximately 20 mg liver tissue were placed in 1.5-ml plastic tubes and mixed with 0.4 ml 20 mmol/liter Tris-HCl buffer, pH 7.4, containing 2% Triton X-100 and 0.1 ml Laemmli buffer containing 2% SDS. The tissue homogenates prepared by an Ultraturrax (Janke und Kunkel, Staufen, Germany) were boiled for 5 min and then incubated overnight at 4 C. Homogenates were centrifuged for 5 min at 10,000 x g, and supernatants were stored at –70 C until further analysis.
Isolation and culture of liver cells
Rat liver HSC were isolated and purified as described previously (32). Cells were plated onto 24-well Falcon plates (BD Biosciences, Heidelberg, Germany) at a density of 2 x 105 cells/well in 1 ml culture medium. Cells were cultured in DMEM supplemented with 10% (vol/vol) fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1% (vol/vol) L-glutamine. Culture medium was replaced 2 d after plating and then every other day. Cells were kept in culture at 37 C in a 5% CO2 atmosphere and 100% humidity. The purity of the HSC was assessed on d 2 and 7 of culture by immunofluorescence microscopy for the presence of desmin, smooth muscle -actin, von Willebrand factor, ED2, and albumin as stated previously (32). HSC cultures showed less than 1% contamination with Kupffer cells, whereas endothelial cells and hepatocytes were not detectable. A significant contamination of HSC with rat myofibroblasts (<5%) was excluded by immunostaining with antibodies against fibulin-2, which has recently been identified as a specific marker for rat myofibroblasts (33, 34). Isolation and cultivation of rat hepatocytes were performed essentially as previously described (35).
Cell culture and transfection of BHK-21 cells
The human hepatoma (HepG2) cells were obtained from American Type Culture Collection (Manassas, VA). HepG2 cells were maintained in DMEM supplemented with 10% (vol/vol) fetal calf serum and antibiotics. The hIGFBP-1 cDNA clone p19, consisting of the entire coding region (36), was digested with EcoRI, and a 1.4-kb fragment was subcloned in the expression vector pBHE. Transfection of BHK-21 cells using the calcium phosphate technique was performed essentially as previously described (37). Selection was performed with 0.5 mg/ml G418. Stable colonies were isolated, and screening for IGFBP-1 was performed by ligand and immunoblotting of conditioned medium (CM) as described below. For coculture experiments, HSC were grown on 96-well plates (Nunc, Wiesbaden, Germany) separated from transfected and nontransfected BHK cells by a 0.2-µm pore size Anopore membrane (Nunc). IGFBP-1 was quantified by an ELISA according to the manufacturer’s instructions.
[125I]IGF-I ligand blot and immunoblot of CM
CM were analyzed for the presence of IGFBPs by [125I]IGF-I ligand blot according to the method described by Hossenlopp et al. (38) with slight modifications (35, 39). Identification of IGFBPs by immunoblotting was performed essentially as previously described (35).
Dephosphorylation of IGFBP-1
CM from HepG2 cells, IGFBP-1-overexpressing BHK cells, and rat hepatocytes were dialyzed overnight against 50 mM Tris-HCl and 0.1 mM EDTA (pH 8.5). CM equivalent to 0.3 ml or 200 ng hIGFBP-1 from human amniotic fluid were digested with 40 U alkaline phosphatase at 37 C for 90 min, followed by nondenaturing PAGE in the absence of sodium dodecyl sulfate (40, 41).
Isolation of total RNA and Northern blot analysis
Total RNA was isolated from rat liver as described recently (18, 36). For IGFBP-1, total RNA was evaluated using digoxigenin-labeled antisense RNAs generated by in vitro transcription with RNA polymerase (Roche) and the T3 promoter of pBluescript SK+ plasmid as described previously (35). Fragments of the rat IGF-I plasmid (700 bp) were [32P]deoxy-CTP labeled by means of random priming, and mouse albumin cDNA was labeled by nick translation. Prehybridization, hybridization, and washing of membranes were essentially performed as previously described (18, 42).
Determination of DNA synthesis
DNA synthesis of HSC was assessed by incorporation of BrdU as previously described (18). In brief, HSC on d 2–3 of culture were labeled for 24 h with 10 µmol/liter BrdU in the presence or absence of IGF-I and/or hIGFBP-1 at the indicated concentrations. Incorporated BrdU was measured with an ELISA kit (Roche) according to the instructions of the supplier. IGF-I-stimulated HSC was expressed as a percentage of the control value, which was set 100%.
Statistical analysis
All experiments were replicated, and representative autoradiograms of at least three independent experiments are shown. Autoradiographs of ligand and Northern blots were scanned using a laser densitometer (Epson GT-8000, Biometra, Göttingen, Germany). The relative densities of the bands were expressed as a percentage of the control. The BrdU assays were performed in duplicate or triplicate using at least three separate HSC isolations. The mean ± SD are indicated. A paired t test was used to evaluate differences between the sample of interest and its respective control. For statistical analysis of time-course experiments, multiple measurement ANOVA, followed by Newman-Keuls posttest analysis, were performed. P < 0.05 was considered statistically significant.
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). Control animals demonstrated constant steady state mRNA levels for IGF-I throughout the time period studied. Densitometric evaluation of the 7.5-kb IGF-I mRNA species revealed that 3 h after CCl4 intoxication, the IGF-I mRNA level was slightly, but significantly, increased compared with control values. After a transient decrease 12–48 h after CCl4 intoxication, IGF-I mRNA transcripts were increased again and were slightly elevated 72–96 h after CCl4 application compared with the respective control animals. A similar biphasic expression pattern was observed for albumin-specific transcripts that showed an early increase 3 and 6 h after CCl4 intoxication, a decrease 24–48 h after intoxication, and again an increase in specific transcripts in the recovery phase (Fig. 1) (43).
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). Densitometric evaluation of three independent experiments revealed that this increase was approximately 12-fold compared with control livers.
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). In liver extracts from control rats and CCl4-treated animals, IGFBP species in the molecular range of 24, 30, and 41–45 kDa were detected, which have been identified as IGFBP-4, -1, and -3, respectively (18, 35). As the livers were perfused with Ringer’s solution before removal, IGFBPs extracted from these liver tissues represent intracellular and extracellular matrix-associated/bound IGFBPs rather than secreted IGFBPs, which might explain the low levels of IGFBPs detected in liver extracts. In control rats, levels of hepatic IGFBPs, specifically IGFBP-1, remained unchanged throughout the observation period (data not shown). After CCl4 intoxication, hepatic IGFBP-1 levels significantly increased and reached maximum values at 12 h (2.5 ± 0.2-fold increase compared with control animals), followed by a decrease between 24 and 72 h, reaching normal levels 96 h after intoxication. A similar time course was observed for IGFBP-3. For IGFBP-4, no significant change in protein levels was detectable throughout the observation period.
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summarizes the results of the in situ hybridization study investigating the expression of IGFBP-1 mRNA in normal and acutely damaged livers. Six to 12 h after CCl4 administration, in situ hybridization for IGFBP-1 revealed a strong expression in the perivenous region (zone 3 of the liver acinus; Fig. 4, A and B). Twenty-four to 48 h after CCl4 administration, the IGFBP-1-specific mRNA signal appeared in the midzonal (zone 2) and periportal (zone 1) regions of the acinus (Fig. 4C). Seventy-two and 96 h after intoxication, the IGFBP-1 transcript level returned to normal (data not shown). In control liver, transcripts for IGFBP-1 were homogeneously distributed over parenchymal cells (Fig. 4D). In situ hybridization with a sense IGFBP-1 riboprobe was negative (Fig. 4E), indicating that the hybridization signal observed with the antisense riboprobe was specific for IGFBP-1.
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Six to 12 h after CCl4 intoxication, IGFBP-1 immunoreactivity was detected around single or groups of hepatocytes undergoing acidophilic degeneration and necrosis (Fig. 5, A and B). Twenty-four and 48 h after treatment, prominent IGFBP-1 staining was found in perivenous areas of hepatocellular necrosis (zones 2 and 3), particularly around groups of necrotic hepatocytes (Fig. 5, C and D). Concomitantly with the disappearance of necrotic cell debris between 72 and 96 h after CCl4 application, the distribution of IGFBP-1 immunoreactivity was similar to that observed in normal liver (Fig. 5, E and F), showing a homogeneous staining over parenchymal cells (Fig. 5G). For specificity control the anti- IGFBP-1 antibody was omitted. Under these conditions no immunostaining was observed (Fig. 5H).
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) after treatment, desmin-immunoreactive cells were detected in perivenous areas of the acinus. Seventy-two hours after CCl4 application, the number of desmin-positive cells further increased, appearing in the perivenous and midzonal region of the acinus (Fig. 6B). Thus, during acute liver injury induced by CCl4 the increase in desmin-immunoreactive cells, most likely HSC, followed the increase in IGFBP-1 in a similar anatomical distribution.
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). The most pronounced effect was observed at an IGFBP-1 concentration of 10 nmol/liter, resulting in a 2.1 ± 0.1-fold increase in BrdU incorporation compared with HSC stimulated with IGF-I alone (Fig. 7B). Preincubation of HSC with increasing concentrations of IGFBP-1 for 1 h before addition of 100 nmol/liter IGF-I to the IGFBP-1-containing medium resulted in additional increases in the proliferative rate compared with the effect of simultaneous addition of both IGFBP-1 and IGF-I (Fig. 7C). Thus, preincubation of HSC with 100 nmol/liter IGFBP-1, followed by addition of 100 nmol/liter IGF-I, resulted in a 3.4 ± 0.4-fold increase in DNA synthesis compared with HSC incubated with 100 nmol/liter IGF-I alone (Fig. 7C).
In a second approach, the proposed cellular interactions of HSC with IGFBP-1-secreting parenchymal cells were mimicked by cocultivation of HSC with IGFBP-1-overexpressing BHK cells. The expression of IGFBP-1 in G418 resistant BHK cells was evaluated by [125I]IGF-I ligand and Western blotting. The IGFBP-1 concentrations were quantified in selected clones using an hIGFBP-1-specific ELISA. The amounts of IGFBP-1 secreted varied from 10.9 ± 1.8 to 133.5 ± 12.5 ng/ml·24 h depending on the clone (Table 1). BHK/IGFBP-1 with the highest expression (clone 9) was used for further experiments. The secretion of human IGFBP-1 into the medium is demonstrated by [125I]IGF-I ligand blotting (Fig. 8A) and Western immunoblotting (Fig. 8B). Cocultivation of BHK/IGFBP-1 (clone 9) or nontransfected BHK cells separated by an Anopore membrane from HSC on d 2–3 of culture in the absence of IGF-I did not affect the DNA synthesis of HSC (data not shown). When 100 nmol/liter IGF-I were added after a 12-h preincubation of HSC with BHK/IGFBP-1 cells, DNA synthesis of HSC was stimulated 1.8 ± 0.2-fold, whereas BrdU incorporation of HSC cocultured with nontransfected BHK cells was similar to that of HSC treated with 100 nmol/liter IGF-I alone (Fig. 8C). Taken together, preincubation of HSC with IGFBP-1 appears to have a priming effect, resulting in an increased susceptibility of HSC for the mitogenic effects of IGF-I.
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). hIGFBP-1 prepared from amniotic fluid contained a nonphosphorylated isoform that was resistant to treatment with alkaline phosphatase. For comparison, IGFBP-1 secreted from the human hepatoma cell line HepG2 cells consisted of five phosphoisoforms of IGFBP-1 (Fig. 9) (40, 41). After incubation with alkaline phosphatase, two bands were detectable. IGFBP-1 secreted from rat hepatocytes in primary culture contained lesser phosphorylated isoforms that were reduced to a single band after digestion with alkaline phosphatase. Analysis of CM from BHK/IGFBP-1 demonstrated a single protein band resistant to alkaline phosphatase digestion with a more basic pI than nonphosphorylated IGFBP-1 from amniotic fluid or HepG2 medium (Fig. 9). Thus, BHK/IGFBP-1 cells secrete a nonphosphorylated form of hIGFBP-1, confirming previous findings that hIGFBP-1 transfected into mouse hepatoma (Hepa 1–6) cells is not phosphorylated (47).
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Discussion |
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The temporal cellular IGFBP-1 expression pattern after CCl4 application is followed by an increase in desmin-immunoreactive cells, most likely HSC rather than rat liver myofibroblasts (45), in a similar anatomical distribution. To examine the physiological significance of these observations and the underlying mechanisms, the ability of IGFBP-1 to modulate IGF-I-stimulated DNA synthesis was studied in cultured HSC. These experiments were focused on HSC on d 2–3 of culture because these early cultured HSC have been reported to be highly sensitive to the mitogenic effects of IGF-I, whereas HSC become refractory to IGF-I during the process of activation (18, 24). The simultaneous addition of hIGFBP-1 and IGF-I resulted in a 2-fold higher rate of DNA synthesis compared with HSC treated with IGF-I alone. The potentiating effect was even more pronounced when HSC were preincubated with hIGFBP-1 before the addition of IGF-I. Similarly, IGF-I-stimulated DNA synthesis in HSC was potentiated when HSC were cocultivated with hIGFBP-1-overexpressing BHK cells, but not with nontransfected BHK cells.
Recent studies suggested a role of components of the IGF axis as hepatocyte-derived mitogenic signals for HSC (49, 50). This assumption is deduced from several experimental findings, such as 1) high concentrations of IGF-I in CM of hepatocytes (17, 18, 19, 20), 2) mitogenic effects of IGF-I on human and rat HSC (18, 25, 49, 51, 52, 53, 54, 55), 3) the presence of high affinity IGF-I receptors on the surface of early cultured HSC (18, 24), 4) potentiation of the mitogenic effect of IGF-I by dilutions of CM from hepatocytes (49, 50), and 5) the increase in IGFBP-1 after partial hepatectomy (56, 57, 58). Because CM from primary cultures of rat hepatocytes contain IGFBP-1, -2, and -4 (18, 35), it is likely that the effect of IGF-I on HSC proliferation reported in this study is potentiated by at least one of these IGFBPs. The present data indicate that IGFBP-1 may be a potential IGFBP candidate leading to a potentiation of the mitogen-activated proliferation of HSC. However, it cannot be excluded that IGFBP-2, IGFBP-4, or a combination of these IGFBPs might also contribute to stimulate mitogenic effects of IGF-I in vitro and in vivo.
IGFBP-1 has been shown to either inhibit or potentiate the actions of IGF, which appear to be due to the existence to several IGFBP-1 phosphoisoforms (7). Highly phosphorylated IGFBP-1 species have been found in normal plasma and in CM of human endometrial stromal cells and HepG2 cells, which exhibit a higher affinity for IGF-I than the nonphosphorylated IGFBP-1 isoforms (40, 41, 46, 59). In the present study subequimolar concentrations of hIGFBP-1 from amniotic fluid have been found sufficient to enhance IGF-I-stimulated DNA synthesis in HSC. Furthermore, hIGFBP-1 secreted from overexpressing BHK cells in coculture with HSC also potentiated IGF-I-stimulated DNA synthesis, but to a lesser extent. The calculated concentrations of IGFBP-1 secreted by the BHK/IGFBP-1 clone 9 under the conditions of HSC coculture reach approximately 250 nmol/liter within 12 h of preincubation. Therefore, the differences in the extent of potentiation appear to be the result of lower local concentrations of IGFBP-1 affecting HSC due to the diffusion length and the barrier through the separating membranes rather than of absolute hIGFBP-1 concentrations. In addition, the BHK/IGFBP-1 cells secrete a second 24-kDa IGFBP, presumably IGFBP-4, which might display an inhibitory effect on IGF-I-stimulated DNA synthesis of HSC. Finally, molecular differences between the purified hIGFBP-1 from amniotic fluid and IGFBP-1 from overexpressing BHK cells may contribute to the different response to HSC. Nondenaturing PAGE revealed that hIGFBP-1 from amniotic fluid and hIGFBP-1 in CM from BHK/IGFBP-1 cells are nonphosphorylated, but obviously contain additional modifications affecting the pI. These data suggest that the nonphosphorylated hIGFBP-1 from amniotic fluid or from overexpressing BHK cells is responsible for the potentiation of IGF-I-stimulated DNA synthesis of HSC in vitro. This was confirmed by experiments showing that highly phosphorylated IGFBP-1 isoforms isolated from CM of HepG2 cells inhibit IGF-I-stimulated DNA synthesis in HSC, whereas nonphosphorylated isoforms elevate the effects of IGF-I (Scharf, J.-G., B. Kübler, and T. Braulke, unpublished results). Because IGFBP-1 secreted by primary cultures of rat hepatocytes contain weakly phosphorylated IGFBP-1 isoforms, it can be speculated that rat IGFBP-1 expressed during CCl4-induced acute liver injury may affect HSC via similar mechanisms in vivo.
hIGFBP-1 can associate with the cell surface. IGFBP-1 of both species has been shown to contain an RGD sequence in the carboxyl terminus (6), facilitating the binding to 5ß1 integrin receptors on the cell surface (41). The binding of IGFBP-1 to 5ß1 integrin promotes cell migration (41) and wound healing in vivo (60, 61). Because no data on integrin expression in rat HSC at early stages of transformation are available, it can be speculated that binding of IGFBP-1/IGF-I complexes to 5ß1 integrins might result in continuous release and presentation of IGF-I to IGF-I receptors, thereby leading to a stimulation of IGF-I-dependent mitogenic effects by IGFBP-1. Alternatively, the IGF-I-independent binding of IGFBP-1 to a cell surface receptor, presumably the 5ß1 integrin receptor, might affect the sensitivity of HSC for IGF-I (e.g. by an increase in IGF-I receptor expression) or by activation of a signal transduction pathway. It has been reported that IGFBP-1 stimulates the cell migration of the human placental extravillous trophoblast by binding of its RGD domain to the 5ß1 integrin, leading to activation of focal adhesion kinase and stimulation of the MAPK pathway (62). Furthermore, cross-talks of IGF-IR signaling pathways with those of other growth factors, such as platelet-derived growth factor, TGF, fibroblast growth factor, or ILs dysregulated in response to carbon tetrachloride (63), may contribute to HSC activation during acute liver injury.
A rapid and high induction of IGFBP-1 gene expression after CCl4 administration, as observed in the present paper, has been demonstrated after partial hepatectomy as well (56, 57, 58). Of interest, IGFBP-1-deficient mice demonstrate normal development, but have abnormal liver regeneration characterized by liver necrosis and both reduced and delayed hepatocyte DNA synthesis (64). IGFBP-1-deficient mice also displayed increased hepatic injury induced by the Fas agonist mediated by integrin receptor signaling and matrix metalloprotease-9 (MMP-9) and TGFß activation (65). Vice versa, the expression of MMP-9 and TGFß was down-regulated by IGFBP-1 treatment. It is tempting to speculate that the up-regulated expression of IGFBP-1 and the activation of HSC during acute CCl4-induced liver injury use the same integrin/MMP-9/TGFß signaling pathway. Taken together, these findings support the concept that IGFBP-1 plays a central role as a hepatic survival factor involved in the regulation of mitogenic pathways in vivo.
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Acknowledgments |
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Footnotes |
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Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; CM, conditioned medium; h, human; HSC, hepatic stellate cell; IGFBP, IGF-binding protein; MMP, matrix metalloprotease.
Received November 12, 2003.
Accepted for publication March 29, 2004.
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-fetoprotein and albumin. II. The structures of the -fetoprotein and albumin genes in the mouse. J Biol Chem 256:1960–19675ß1 integrin by means of its Arg-Gly-Asp sequence. Proc Natl Acad Sci USA 90:10553–105575ß1 integrin via mitogen-activated protein kinase pathway. J Clin Endocrinol Metab 86:2484–2493This article has been cited by other articles:
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) and from animals treated with CCl4 (
). The relative densities of bands were expressed as the percent increase or decrease in IGF-I mRNA compared with the respective mRNA level at 0 h (n = 3). Statistically significant differences are indicated (P < 0.001 for IGF-I after CCl4 by ANOVA; P = 0.002 for IGF-I in controls by ANOVA, posttest Newman-Keuls analysis: *, P < 0.05; **, P < 0.01, for individual values compared with the respective control).
) proteins. The relative densities of bands were expressed as the percent increase in IGFBPs compared with the respective protein level at 0 h (n = 3). Statistically significant differences are indicated (P < 0.001 for IGFBP-1 by ANOVA; P < 0.001 for IGFBP-3 by ANOVA; P = 0.0062 for IGFBP-4 by ANOVA, posttest Newman-Keuls analysis: *, P < 0.01, **, P < 0.001, for individual values compared with the respective control).
