Metal-catalyzed oxidation of extracellular matrix components perturbs hepatocyte survival with activation of intracellular signaling pathways

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Experimental Cell Research 291 (2003) 451– 462

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Metal-catalyzed oxidation of extracellular matrix components perturbs hepatocyte survival with activation of intracellular signaling pathways Ranjit K. Giri,a,b Harmeet Malhi,a,b Brigid Joseph,a,b Jithender Kandimalla,a,b and Sanjeev Guptaa,b,c,d,* a

Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY, USA b Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA c Cancer Research Center, Albert Einstein College of Medicine, Bronx, NY, USA d Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA Received 15 May 2003, revised version received 16 July 2003

Abstract To investigate whether oxidative manipulation of extracellular matrix components could affect cell survival, we studied primary rat hepatocytes cultured on dishes coated with collagen type 1, which was oxidized with a metal-based system. Culture of hepatocytes on oxidized collagen led to decreased cellular catalase activity along with impaired cell survival. The fraction of polyploid hepatocytes decreased early followed by greater reaccumulation of polyploid cells. Cells cultured on oxidized collagen showed greater susceptibility to additional oxidant stress induced by tert.-butyl-hydroperoxide. The capacity of hepatocytes for growth factor-induced DNA synthesis was unaffected by culture on oxidized collagen. In response to culture on oxidized matrix, AP-1, Egr-1, CREB, and NF-␬B transcription factor activity was rapidly increased. This change in transcription factor activity was ameliorated by treatment of collagen with a free radical spin trap, N-tert.-butyl-␣-phenylnitrone, prior to oxidation. Moreover, culture of hepatocytes with aminoguanidine, an antioxidant drug, decreased cell injury. These findings established that exposure of primary hepatocytes to oxidized extracellular matrix components rapidly activates cell signaling events with loss of hepatocyte subpopulations. Such cell– extracellular matrix interactions may play roles in organ homeostasis and oncogenetic progression. © 2003 Elsevier Inc. All rights reserved. Keywords: Hepatocyte; Extracellular matrix; Culture; Oxidation; Injury

Introduction The aerobic microenvironment generates multiple reactive oxygen species (ROS), including superoxide anions, hydrogen peroxide, and hydroxyl radicals, which exert deleterious effects on cellular proteins, lipids, DNA, and other molecules [1]. Intracellular ROS generated by physiologically regulated intermediary metabolism, ischemia–reperfusion injury, microbial or viral infection, radiation, drug therapy, etc., may also be responsible for cellular perturba-

* Corresponding author. Ullmann 625, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. Fax: ⫹1-718430-8975. E-mail address: [email protected] (S. Gupta). 0014-4827/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0014-4827(03)00405-1

tions [2– 6]. The consequences of such oxidative stress extend to enhanced cell proliferation or growth arrest, replicative senescence, and cell death [3,5]. Another form of oxidant stress may be transmitted to cells by extracellular matrix (ECM) components [7–10]. For instance, oxidation of ECM perturbed attachment of renal mesangial cells in culture, along with release of extracellular soluble signals in mesangial cells, as well as macrophages [7–9]. Among parenchymal cells, hepatocytes are especially dependent on ECM for survival, proliferation, and gene expression [11–13], with specific interactions requiring cellular ligands, e.g., integrins, and specific receptor-binding domains on collagen, fibronectin, and other ECM components that anchor epithelial cells [14,15]. The onset and

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progression of hepatic fibrosis in chronic liver disease are associated with significant pro-oxidant activity [4], and persistent injury often enhances the susceptibility to liver cancer. Therefore, investigation of interactions between oxidized ECM and hepatocytes will help define relevant mechanisms in pathophysiological states. The intermediaries in ROS-mediated processes have remained under extensive scrutiny. Multiple potent transcription factors appear to regulate cell signaling following oxidative stress and have major roles in regulating cell proliferation and injury responses [16]. In the liver, injury and oxidative stress activate several transcription factors, including activator protein (AP) 1, early growth response (Egr-1) factor, nuclear factor ␬B (NF-␬B), signal transducer and activator of transcription (STAT), cyclic AMP-responsive element-binding protein (CREB), heat shock factor (HSF), mitogen-activated protein kinases [major subfamilies of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 kinases], of which the latter two constitute stress-activated protein kinases [6,17– 23]. Such activation of intracellular signaling pathways could interfere with both survival and proliferation of isolated cells after transplantation, which has attracted significant interest for cell therapy in several models [24]. For instance, in cell transplantation studies in a rat model of Wilson’s disease, where copper accumulation induces serious oxidative liver injury, we found that transplanted cell proliferation was significantly delayed compared with that in normal animals [25,26]. To establish connections between ECM oxidation and perturbation of primary rat hepatocytes, we addressed aspects of cell survival, proliferation, and cell signaling. The ECM was oxidized with a heavy metal-catalyzed system in an effort to reproduce copper toxicosis-type changes [7,8]. Our results shown here indicate that ECM oxidation plays a role in regulating survival of hepatocytes.

Materials and methods Cells Primary hepatocytes were isolated from adult F344 rats with in situ liver perfusion using 0.03% collagenase, as described previously [25,26]. Hepatocytes were pelleted in 45% Percoll (Amersham Biosciences Corp., Piscataway, NJ, USA) in Hanks’ balanced salt solution under 1000g for 10 min at 4°C. The viability of isolated cells was assessed by trypan blue dye exclusion. The initial viability of isolated hepatocytes was 85–90% and increased to 100% after cells were pelleted through Percoll. Matrix oxidation and cell culture Tissue culture plates were coated with type 1 collagen isolated from the tail of F344 rats, as described previously

[27]. Dishes containing collagen were incubated for 1 h at room temperature with a metal-catalyzed oxidation (MCO) system using 2 mM FeCl3, 2.4 mM EDTA, and 25 mM ascorbate (Sigma Chemical Co., St. Louis, MO, USA), as described [7,8]. Oxidative modification of collagen by MCO was quantitated by incubation with 2,4-dinitrophnylhydrazine (DNPH, Sigma) and formation of stable protein hydrazones, which were measured spectrophotometrically, as described [8]. Carbonyl content was normalized to cellular protein content measured with the Bio-Rad assay using gamma globulin standards (Bio-Rad Laboratories, Richmond, CA, USA). Cells were cultured in RPMI-1640 medium (Life Technologies, Grand Island, NY, USA) containing 100 units/ml penicillin, 100 ␮g/ml streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, GA, USA). The medium was changed 2 h after plating cells. Cell morphology was observed under phase contrast microscopy. Assessment of cell attachment and viability The efficiency of cell attachment to culture dishes was assessed 1 h after cell plating. Cells were washed with cold phosphate-buffered saline, pH 7.4 (PBS) and released with trypsin-EDTA, and cell numbers were counted in a Neubaur hemocytometer. Cell viability was analyzed by thiazolyl blue dye (MTT, Sigma) utilization, as described previously [28]. In aliquots, total protein was measured by the Bio-Rad assay. To assess capacity for withstanding further injury, cultured cells were incubated with 100 ␮M tert.-butylhydroperoxide (Sigma) for 1 h at 37°C [29]. At intervals, cells were incubated with 1 mg/ml MTT in serum-free RPMI-1640 medium for 1 h at 37°C for analysis of MTT utilization. To verify that cellular perturbations following MCO were not due to additional mechanisms, the radical spin trap N-tert.-butyl-␣-phenylnitrone (PBN) (Sigma) was used as described previously [8]. Collagen-coated wells were incubated with PBN before exposure to the MCO system, also containing PBN. The collagen was washed thoroughly and assayed for its carbonyl content [30]. In additional studies, cells were cultured with aminoguanidine or N-acetyl-L-cysteine (Sigma). Measurement of DNA synthesis Hepatocytes were plated at a density of 2 ⫻ 104/cm2. To stimulate DNA synthesis, 20 ng/ml recombinant human hepatocyte growth factor (hHGF) was added to cell cultures. Cells were pulsed with 1 ␮Ci [3H]thymidine (Sigma) for 1 h, washed twice with cold PBS, and trichloroacetic acidprecipitable DNA was extracted as described previously [31]. [3H]Thymidine incorporation was measured by liquid scintillation counting and counts were normalized against DNA content measured by a sensitive fluorometric assay.

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Measurement of catalase activity Cell extracts were obtained by scraping and sonicating cultured cells in phosphate buffer containing 25 mM KH2PO4 and 40 mM Na2HPO4 · 2H2O, as described [3]. Time in seconds was measured for decrease in optical density of phosphate buffer containing 12.5 mM H2O2 from 0.45 to 0.40 after adding cell extract. The catalase activity was derived from the formula 17/time in seconds ⫽ units/ assay mixture. Results were normalized to cellular protein content (Bio-Rad). Flow cytometric (FACS) analysis Cultured cells were removed from dishes by scraping and after pelleting at 300g for 5 min; cells were stored at ⫺70°C in 10% DMSO. Nuclei were isolated with a detergent-trypsin method and stained with 0.04% propidium iodide, as described previously [2,3]. Data were acquired using the FACSTAR Plus machine and analyzed with Cell Quest Software. Preparation of nuclear extracts The procedure described by Dignam et al. [32] was used with slight modifications. Briefly, 5 ⫻ 106 cultured hepatocytes were washed with cold PBS and resuspended in 400 ␮l cell lysis buffer [10 mM Hepes, pH 7.9, 100 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethysulfonyl fluoride (PMSF), 0.5% Nonidet P-40, and 1 mg/ml protease inhibitor cocktail (Calbiochem, La Jolla, CA, USA)]. Cells were kept on ice for 15 min followed by vigorous vortexing for 5–10 s. The homogenate was centrifuged at 10,000g for 30 s in a microcentrifuge. The supernatant was discarded and the nuclear pellet was resuspended in 50 ␮l nuclear extraction buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.1 mM EGTA, 0.5 mM DTT, 5% glycerol, 0.5 mM PMSF, and 1 mg/ml protease inhibitor cocktail). The contents of the tube were mixed intermittently for 15– 60 min and nuclear extracts were recovered by centrifuging at 10,000g for 10 min at 4°C. Electrophoretic mobility shift assay (EMSA) The oligonucleotides used as probes were as follows: AP-1, 5⬘-(CGC TTG ATG ACT CAG CCG GAA)-3⬘ and 3⬘-(GCG AAC TAC TGA GTC GGC CTT)-5⬘; NF-␬B, 5⬘-(AGT TGA GGG GAC TTT CCC AGG C)-3⬘ and 3⬘(TCA ACT CCC CTG AAA GGG TCC G)-5⬘; CREB, 5⬘-(AGA GAT TGC CTG ACG TCA GAG AGC TAG)-3⬘ and 3⬘-(TCT CTA ACG GAC TGC AGT CTC TCG ATC)5⬘. The oligonucleotides were generated in an automated synthesizer. The probes were 5⬘ end-labeled with 100 ␮Ci [␥-32P]ATP using T4 polynucleotide kinase (Invitrogen, Carlsbad, CA, USA). The labeled single-stranded sense

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oligonucleotide and labeled antisense oligonucleotide were mixed and incubated at 65°C for 5 min followed by annealing at room temperature for 15 min. DNA-binding reaction mixture contained 4 ␮g nuclear proteins, 32P-labeled double-stranded oligonucleotide probe (⬃50,000 cpm), and 2 ␮g poly (dI– dC) (Sigma). To verify specificity of DNA– protein interactions, unlabeled double-stranded oligonucleotide probes were added in 50-fold excess. Supershift assays used incubation of nuclear extracts with 2 ␮g transcription factor antibodies (Santa Cruz Biotechnologies Inc., Santa Cruz, CA, USA) for 20 min before addition of radiolabeled probes. DNA–protein complexes were analyzed in 4% nondenaturing polyacrylamide gels, which were vacuum dried for autoradiography. The intensity of the bands was measured by densitometry with the AlphaImager 2000 instrument (Alpha Innotech Corp., San Leandro, CA, USA). Western blotting Nuclear extracts were prepared as above and equal amounts of proteins were subjected to SDS–PAGE in 12% gels using standard procedures. The transblots were probed with an antibody against Egr-1 (sc-110, Santa Cruz Biotechnology, Inc.), according to the manufacturer’s recommendations, followed by visualization of signals with enzymatic chemiluminiscence. Immunostaining Cells were fixed in ethanol for 20 min, rehydrated, and stained with antibodies against NF␬B p65-kDa subunit (F-6 clone, sc-8008, Santa Cruz Biotechnologies Inc.) and I␬B␣ (C-21 clone, sc-371, Santa Cruz Biotechnologies Inc.) at 1:50 and 1:200 dilutions, respectively. For NF␬B staining, cells were blocked with goat serum and antibody binding was visualized with a 1:1000 dilution of peroxidase-conjugated anti-mouse goat IgG (A3682, Sigma). For I␬B␣ staining, cells were blocked with mouse serum and antibody binding was demonstrated with a 1:1000 dilution of peroxidase-conjugated anti-rabbit mouse IgG (clone RG-96, Sigma). To obtain gene expression profiles, five or six random areas containing at least 25–50 cells were analyzed in each condition. Statistical analysis Data are expressed as means ⫾ SD. The data were analyzed with SigmaStat 2.0 software (Jandel Corp., San Rafael, CA, USA). The significance of differences was determined by Student’s t test, ␹2 test, and one-way analysis of variance (ANOVA) using the Tukey test for pairwise comparisons of mean responses in treatment groups, as appropriate.

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Fig. 1. Hepatocellular perturbations following culture on oxidized collagen. (A) Progressive decline in cultured cell mass as assessed by MTT utilization. However, the decline in cell mass was more pronounced when hepatocytes were cultured on oxidized collagen, with only 26 ⫾ 4% (vs 72 ⫾ 2%) cells after 24 h, 18 ⫾ 7% (vs 44 ⫾ 5%) cells after 48 h and 12 ⫾ 4% (vs 28 ⫾ 4%) cells remaining after 72 h (P ⬍ 0.001, ANOVA). The inset shows that cell attachment was not perturbed by oxidized collagen. (B) DNA synthesis in cells cultured for 48 h. No unscheduled DNA synthesis was observed in cells cultured on oxidized collagen without mitogenic stimulation. DNA synthesis rates following hHGF stimulation were similar, ranging from four- to sixfold greater DNA synthesis in cells cultured on nonoxidized or oxidized collagen (P ⫽ n.s.). (C) Flow cytometric analysis of cell ploidy in hepatocytes cultured on untreated collagen and oxidized collagen. The panels at the top demonstrate flow cytometric profiles, with the table at the bottom indicating the fraction of diploid (2N) and tetraploid (4N) cells in the samples analyzed. Cell culture on oxidized matrix resulted in fewer polyploid cells initially, followed by greater accumulation of polyploid cells in later cultures. Data from 72-h cultures were similar to those from 48-h cultures. The studies were repeated four to eight times and typical experiments are shown.

Results Exposure to the MCO system led to oxidization of collagen in culture dishes, as shown by 2 ⫾ 0.3-fold greater OD374 values between ECM samples treated with DNPH or hydrochloric acid alone without DNPH, representing significantly greater carbonyl content (P ⬍ 0.001, t test) [30]. Oxidized ECM impaired viability of cultured hepatocytes with depletion of specific cell subpopulations Initial experiments were undertaken to analyze the effects of oxidized ECM on cell attachment. Equivalent numbers of cells attached after 1 h in culture dishes with or without oxidized ECM (19.7 ⫾ 4%, nonoxidized collagen,

vs 19.3 ⫾ 1%, oxidized collagen, P ⫽ n.s.). However, cell viability was progressively impaired in cells cultured on oxidized ECM (Fig. 1A). The viability of cells on nonoxidized collagen decreased significantly with time, such that after 72 h only 28 ⫾ 4% of the cell mass after 3 h remained viable, which was expected with primary hepatocytes. However, the viability of hepatocytes cultured on oxidized collagen declined far more and the number of cells surviving after 24, 48, and 72 h ranged from only 36 to 42% of controls (P ⬍ 0.001, t test). The morphological appearance of hepatocytes on oxidized matrix was not different from that of cells cultured on nonoxidized ECM. Although cell viability was perturbed, the capacity for DNA synthesis in cells cultured on oxidized ECM was

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unchanged. We did not observe unscheduled DNA synthesis in cells cultured on oxidized ECM. hHGF-stimulated DNA synthesis was of similar magnitude, indicating the ability of cells cultured on oxidized ECM to synthesize DNA (Fig. 1B). To determine whether specific cell subpopulations were perturbed following exposure to oxidized collagen, we analyzed cell ploidy after culture for various periods (Fig. 1C). Initially, primary hepatocytes showed predominantly diploid and tetraploid cells, averaging 30 and 65%, respectively, with up to 5% cells showing octaploid or greater DNA content. After culture on nonoxidized collagen, cells exhibited predominantly diploid DNA content, indicating selective loss of polyploid cells. Cell culture on oxidized collagen showed even greater depletion of tetraploid cells after 24 h, whereas subsequently, after 48 and 72 h of cell culture, more tetraploid cells were present (P ⬍ 0.001, ␹2 test). These findings indicate that oxidized collagen decreased survival of polyploid cells initially and promoted polyploidization in surviving cells subsequently. Perturbation in survival of cultured cells was a result of matrix oxidization In one set of experiments, cells were cultured after exposing collagen to MCO subsequent to PBN, the free radical spin trap agent, which antagonizes protein oxidation, including that of ECM [7–9]. PBN is known to attenuate ECM oxidation induced by the MCO system used here. When hepatocytes were cultured on oxidized collagen without prior incubation of matrix with PBN, cell viability declined (Fig. 2A). However, survival of hepatocytes cultured on collagen protected with PBN prior to MCO treatment either did not decline or improved significantly. Similarly, culture of cells on collagen treated with PBN, even in the absence of MCO, resulted in superior cell survival (not shown). The difference in the overall magnitude of cell survival seen in Figs. 1A and 2A was likely accounted for by variable properties of primary hepatocytes following collagenase perfusion of different donor livers. In a second set of studies, we measured catalase activity in cultured hepatocytes because oxidative hepatic injury decreases catalase activity [3,5]. The experimental design concerned cell culture on MCO-treated collagen with and without prior incubation with PBN. The data showed that after culture on oxidized matrix, cellular catalase activity was significantly decreased after 48 and 72 h in cell culture (Fig. 2B). The catalase activity in cells was unchanged after 24 h of culture on nonoxidized collagen versus oxidized collagen (0.11 ⫾ 0.03 and 0.12 ⫾ 0.01 catalase unit/␮g protein, respectively, P ⫽ n.s.). Remarkably, incubation of collagen with PBN prior to MCO restored catalase activity. PBN improved catalase content even in cells cultured on collagen without oxidative preparation (not shown). To examine the effects of additional oxidant injury in hepatocytes, cells were cultured on ECM and exposed to an

Fig. 2. Oxidation of ECM was responsible for perturbation of cultured cells. (A) MTT incorporation analysis showing the effect of the radical spin trap PBN on cell injury. Cells were cultured on untreated collagen, collagen oxidized with MCO, and collagen incubated with various amounts of PBN before treatment with MCO. Data were normalized to cell content in control dishes with untreated collagen. PBN pretreatment improved cell viability significantly (P ⬍ 0.05, t test). (B) Catalase activity in cells shown in (A) with decrease in catalase activity following culture on oxidized matrix and its restoration in the presence of PBN pretreatment. (C) Studies with additional oxidant injury using tert.-butyl-hydroperoxide showed greater loss of hepatocytes cultured on oxidized collagen compared with nonoxidized collagen. Prior incubation of cells with PBN again reversed the oxidant injury.

hour-long burst of oxidant injury induced by tert.-butylhydroperoxide, followed by the analysis of cell viability by MTT utilization (Fig. 2C). This produced greater oxidant

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injury in hepatocytes cultured on oxidized collagen compared with nonoxidized collagen. Moreover, PBN markedly attenuated the combined injury following cell culture on oxidized collagen and tert.-butyl-hydroperoxide exposure. Moreover, PBN improved cell survival on even nonoxidized collagen, suggesting the likelihood of spontaneous ROS-induced perturbations in hepatocyte cultures. Taken together, these studies established that ECM oxidation showed direct effects on cultured hepatocytes with depletion of cellular antioxidant mechanisms. ECM oxidization activated intracellular signaling To study potential pathways by which ECM oxidation transmitted intracellular signals, we analyzed transcription factor activations by EMSA. These studies used cells cultured for up to 5 days over either nonoxidized collagen or oxidized collagen in the presence or absence of PBN. We chose to analyze changes in the expression of AP-1, NF␬B and I␬B, Egr-1, and CREB because of their significant roles in mediating oxidative and other types of hepatic injury [16 –22]. Early activated genes, such as c-fos and c-jun, which homo- or heterodimerize to constitute AP-1, as well as Egr-1 and CREB are activated in hepatocytes rapidly following induction of proliferation or cytotoxic events. Similarly, hepatocytes rapidly begin to regulate the coordinate expression of NF␬B, and its partner I␬B, following the onset of injury. Therefore, we reasoned that oxidized ECM must mediate its intracellular effects by recruiting specific signaling pathways. AP-1 was expressed in hepatocytes during 1, 3, and 5 days of cell culture (Fig. 3). It was noteworthy that basal AP-1 DNA binding activity was relatively lower after 1 day of culture (relative expression ratio 1) compared with cells cultured for 3 and 5 days (relative expression ratios 3 to 4). The magnitude of AP-1 activity was greater in cells cultured on oxidized collagen relative to nonoxidized collagen. Prior incubation of collagen with PBN decreased AP-1 DNA binding activity, especially when collagen was not subjected to oxidation with MCO. In this situation, AP-1 activity declined to a mean of 0.4 relative expression level in three experiments compared with the baseline expression (P ⬍ 0.001, t test). On the other hand, the overall AP-1 activity was affected less by PBN when collagen was oxidized by MCO (see further analysis below). The identity of AP-1 complexes was verified by “supershift” analysis using antic-jun and anti-c-fos antibodies (Fig. 3B). It appeared that AP-1 complexes contained both c-jun and c-fos proteins, although c-fos constituted greater amounts of the shifted bands. The pattern of CREB DNA binding activity resembled AP-1 expression, with greater expression at all times in cells on oxidized collagen (Fig. 4A). Although pretreatment of collagen with PBN significantly decreased CREB activity, we did not observe such decrease in CREB activity when cells were cultured on oxidized collagen after PBN treat-

Fig. 3. Regulation of AP-1 expression in cultured hepatocytes. (A) EMSA showing AP-1 expression in hepatocytes cultured for 3 days. The experimental conditions of samples in various lanes are as shown. The position of the AP-1 complex is indicated. The bottom portion of the gel shows radioactivity in the free probe. The abundance of AP-1 was greater in cells on oxidized collagen compared with nonoxidized collagen (mean twofold increase). Also, prior incubation of collagen with the spin trap agent PBN decreased AP-1 expression when the ECM was not oxidized and less so when the ECM was oxidized with MCO (approximately twofold mean decline, P ⬍ 0.05). (B) “Supershift” analysis with specific anti-c-jun and anti-c-fos, where the complexes were shifted (SS), particularly with the latter antibody (lane 4). Incubation of extracts with an excess of cold probe abrogated AP-1 complexes (lane 3), further verifying that the activity visualized was specific to AP-1. The nuclear extracts analyzed were from cells cultured for 3 days under the conditions indicated.

ment. On the other hand, Western blots showed little Egr-1 expression in cells 1 day after culture but expression increased markedly subsequently (Fig. 4B). Egr-1 expression was upregulated by 5- to 10-fold in cells cultured on oxidized collagen 3 and 5 days after cell culture. However, Egr-1 upregulation was suppressed when cells were cultured on collagen pretreated with PBN prior to metal-catalyzed oxidation. Analysis of NF␬B DNA binding activity showed abundant complexes in cultured hepatocytes (Fig. 5). However, unlike AP-1 and Egr-1 expression, the relative magnitude of basal NF␬B activity did not change between 1 day and 3 or 5 days of cell culture. On the other hand, similar to AP-1 and Egr-1 expression, cells cultured on oxidized collagen exhibited greater NF␬B activation. While PBN pretreatment decreased AP-1 and Egr-1 expression in cultured cells, decreases in NF␬B activity following PBN pretreatment of collagen were not observed in cells cultured for 3 and 5 days, although cells cultured for 1 day showed 20 –30% lower NF␬B activity. The NF␬B bands were supershifted by the p65 NF␬B antibody, which was consistent with the presence of active NF␬B molecules in our cell lysates (Fig. 5b). We immunostained cultured cells with NF␬B and I␬B

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Fig. 4. CREB and Egr-1 expression in cultured cells. (A) CREB expression in cells cultured for 3 days. CREB complexes were not visualized after incubation of extracts with an excess of cold probe (not shown). CREB expression in cells on oxidized collagen (lanes 1 and 2) was somewhat greater (mean 1.6-fold increase, P ⫽ n.s.). However, CREB expression decreased significantly after preincubation of ECM with PBN (lanes 3 and 4) (⬎5-fold mean decrease, P ⬍ 0.05). Similar findings were observed in cells cultured for 1 and 5 days. (B) Western blot analysis showing Egr-1 expression in cells cultured for 1 (lanes 1–3), 3 (lanes 4 – 6), and 5 (lanes 7–9) days. Egr-1 expression increased by 3- to 6-fold subsequent to 1 day in cell culture and was downregulated by 3- to 4-fold when cells were cultured on ECM pretreated with 250 ␮M of the radical spin trap agent, PBN.

antibodies to demonstrate the relationship in the expression of these molecules (Fig. 5C). Immunostainable NF␬B was observed in large numbers of cells (20 –50%) cultured on either oxidized or nonoxidized collagen, although the fraction of cells with NF␬B expression was greater in 3- and 5-day cultures, compared with 1-day cultures. We analyzed the distribution of nuclear and cytoplasmic NF␬B activity, since NF␬B activation requires I␬B ubiquitination and degradation followed by nuclear translocation of NF␬B [17,19]. Morphometric analysis showed transition toward nuclear NF␬B expression during cell culture. In cells cultured on nonoxidized collagen, NF␬B was expressed in 9 ⫾ 3, 18 ⫾ 5, and 21 ⫾ 8% after 1, 3, and 5 days of cell culture, respectively (P ⬍ 0.001 vs 1 day, t tests). In contrast, after culture on oxidized collagen, nuclear localization of NF␬B was more frequent, in 23 ⫾ 6, 40 ⫾ 8 and 37 ⫾ 12% of cells at corresponding times (P ⬍ 0.001 vs nonoxidized collagen, t tests). Moreover, the intensity of I␬B expression decreased in cells cultured on oxidized collagen. However, prior incubation of collagen with PBN did not alter these NF␬B and I␬B immunostaining patterns. The cumulative analysis of transcription factor perturbations is shown in Table 1. The data are from densitometric analysis of EMSA bands in three experiments. The ranges of increased AP-1, NF␬B, and CREB DNA binding activity in cells cultured on oxidized matrix were 1.3- to 3-fold, 1.1to 3-fold, and 1.1- to 1.6-fold, respectively. As indicated earlier, PBN pretreatment was effective in decreasing AP-1 activation, Egr-1 activation, as well as CREB activation (in cells cultured on nonoxidized collagen), but was ineffective

in decreasing NF-␬B activation. Taken together, AP-1, EGR-1, and NF-kB, and CREB to a lesser extent, were key intracellular signaling molecules in cells cultured on oxidized matrix. Additional mechanisms for preventing ECM induced cell perturbations To determine whether pharmacological manipulation could prevent deleterious consequences following cell signaling from oxidized ECM, we examined the effects of aminoguanidine and N-acetyl-L-cysteine, which exhibit cytoprotective activity against oxidative injury [33,34]. Cells were cultured in the presence or absence of these drugs and cell viability was assessed by MTT incorporation analysis, including the tert.-butyl-hydroperoxide assay of additional oxidative injury. The studies established that N-acetyl-Lcysteine did not prevent cytotoxicity under these conditions and, in fact, worsened cell viability (Fig. 6). However, aminoguanidine exhibited protective activity and cells cultured with aminoguanidine showed resistance to further oxidative injury with tert.-butyl-hydroperoxide, indicating the pharmacological potential for circumventing ECM signaling-associated perturbations.

Discussion These studies advance our insights into interactions between oxidized ECM and hepatocytes. Moreover, the find-

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Table 1 Relative transcription factor expression in cultured cellsa Transcription factor

Expression in cells on oxidized ECM vs untreated ECM (%)

Effect of PBN in cells on untreated ECM (% control)

Effect of PBN in cells on oxidized ECM (% control)

AP-1 NF␬B CREB

1.93 ⫾ 0.93 1.73 ⫾ 1.10 1.63 ⫾ 0.47

0.43 ⫾ 0.06b 0.97 ⫾ 0.25 0.17 ⫾ 0.06b

0.60 ⫾ 0.03c 0.83 ⫾ 0.29 1.07 ⫾ 0.06

a

Data show densitometric analysis of EMSA bands. P ⬍ 0.001, t tests, versus NF␬B expression. c P ⬍ 0.001, t test, versus CREB expression. b

ings establish that multiple intracellular signaling pathways are activated in cultured hepatocytes exposed to oxidized ECM. We found that catalase activity was decreased in cells on oxidized ECM, which offers further evidence for intracellular perturbations. Similar to our observations, cadmiummediated injury in hepatic stellate cells results in lipid peroxidation with decreases in catalase, glutathione peroxidase, and superoxide dismutase activities [35]. Moreover, in advanced atherosclerotic plaques in humans, decreased antioxidant levels correlate with increased oxidation of ECM proteins [36]. The oxidant nature of injury in our cells was substantiated by studies using the radical spin trap PBN, which attenuated ECM oxidation and consequential cellular injury, including that induced by additional challenges with tert.-butyl-hydroperoxide [29]. The loss of polyploid hepatocytes after culture on oxidized collagen is relevant for oncogenetic pathways because polyploid hepatocytes exhibit oxidative damage and are predisposed to undergo apoptosis [2,3,5]. Also, oxidative injury in polyploid cells leads to replicative arrest [3,5]. The fact that we observed reaccumulation of polyploid cells in our cultures after initial depletion of these cells indicates that oxidized ECM exerted persistent effects on cells. Such ECM-mediated changes in intact organs could exert potentially powerful effects on selective losses and activations of specific cell subpopulations, which in the setting of ongoing injury might favor the eventual emergence of transformed cell foci. In contrast with renal mesangial cells, attachment of primary hepatocytes was unperturbed by ECM oxidation [7], which could have implications in cell metastases. Previously, the efficacy of PBN in preventing ECM oxidation-

dependent cellular perturbations was established in mesangial cells [8,9]. In addition, oxidation of collagen types 1 and 4 led to increased polymorphonuclear leukocyte adherence to ECM along with greater cell migration and ROS production [37], which could exacerbate inflammatory consequences of diseases, such as vascular lesions in atherosclerosis or hepatitis [4]. We did not study which specific components of oxidized collagen accounted for effects in hepatocytes, although tyrosine residues in fibroblast-derived ECM are targets of extracellular ROS-mediated crosslinking [38]. It was noteworthy that catalase inhibited such reactions. This was decreased in our cells on oxidized collagen. A variety of cell membrane-associated ligands, cytoarchitectural proteins, or cell– cell transmission of paracrine signals could potentially participate in mediating hepatocellular toxicity on oxidized ECM. Activation of intracellular signaling in cells cultured on ECM, as shown by increased expression of AP-1, CREB, Egr-1, and NF-␬B, further indicated that matrix oxidation was responsible for perturbations observed under our culture conditions. Interactions among various transcription factors regulate proliferation and cell injury in many cell types, including hepatocytes [20]. AP-1 consists of jun/jun homodimers or jun/fos heterodimers with monomer composition varying in relation to the activation of specific signal transduction pathways [39]. AP-1 in our cells was constituted by both c-fos and c-jun isoforms (see supershift in EMSA by anti-c-fos). In other situations with oxidative hepatic injury, c-jun might be the major AP-1 component [6], although the significance of these differences is unclear. Our findings concerning activation of CREB and Egr-1 in cells on oxidized ECM are in agreement with further coordinated regulation of transcription factors. For instance,

Fig. 5. NF␬B and I␬B expression in cultured hepatocytes. (A) EMSA showing NF␬B expression in cells cultured for 3 days. The position of NF␬B complexes is indicated. Note that NF␬B was expressed at greater levels in cells cultured on oxidized collagen, approximately twofold mean increase, although this was not statistically significant. Interestingly, pretreatment of the ECM with PBN did not significantly decrease NF␬B expression. Similar findings were observed in cells cultured for 1 and 5 days. (B) Supershift analysis with EMSA indicating a shift in NF␬B complexes with anti-p65 NF␬B (lane 5), as expected, along with abrogation of complexes in the presence of excess cold probe (lane 3) to verify the specificity of complexes. (C) Representative immunostaining profiles of hepatocytes cultured for 3 days. (a)–(c) were subjected to NF␬B staining and (d)–(f) to I␬B staining. (a) and (d) show cells cultured on nonoxidized collagen. (b), (c), (e), and (f) show cells cultured on oxidized collagen. (a) shows some cells with nuclear localization of NF␬B (arrows and inset). In contrast, cells on oxidized matrix showed both cytoplasmic and nuclear NF␬B staining (arrow in (b) and inset showing higher-magnification view of the same area). Less I␬B staining is evident in (e) compared with (d), which was consistent with the NF␬B findings. Primary antibodies were omitted in (c) and (f), resulting in no immunostaining. Original magnification: ⫻600.

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Fig. 6. Reversal of cytotoxicity following cell culture on oxidized ECM. The data were obtained from studies of cultured hepatocytes on unoxidized collagen and oxidized collagen in the presence of 10 mM N-acetyl-L-cysteine or aminoguanidine. After culture for 48 h, cells were incubated with tert.-butylhydroperoxide for 1 h followed by analysis of MTT utilization. While cells cultured with N-acetyl-L-cysteine showed worsening of injury, cells cultured with 5 mM aminoguanidine were protected from oxidative injury. P values shown are against cells cultured on untreated collagen.

along with CREM and ATF-1, CREB constitutes one of three mammalian transcriptional factors capable of binding to cAMP-responsive elements [21]. As an early-activated gene, Egr-1 plays significant roles in liver cell proliferation and survival [18]. These transcription factors can both promote and suppress cellular gene expression. For example, CREB and Egr-1 genes have been shown to subserve synergistic roles in regulating gene expression in gastric epithelial cells [40]. It is noteworthy that CREB can downregulate early response genes, including c-fos, although we did not observe this in our cells [21]. Similarly, microenvironmental factors, including growth factors, hormones, and neurotransmitters, rapidly activate Egr-1 expression [42], which helps converge multiple signaling cascades, including in cell growth control and apoptosis. Egr-1 biosynthesis is strongly stimulated by activation of ERK following mitogen activation, and genes encoding growth factors, e.g., insulin-like growth factor II, platelet-derived growth factor, transforming growth factor, etc., are targets of Egr-1 [43]. Interestingly, Egr-1 is known to interact with and modulate the activity of c-jun, which is essential for liver development [44,45]. Moreover, Egr-1 activation is an early event during liver regeneration induced by partial hepatectomy [21]. cAMP-responsive signaling also plays a major role in the liver and multiple CREB-regulated genes are expressed after partial hepatectomy [21]. These findings lend further

support to the possibility of ECM oxidization playing contributory roles in oncogenesis. The well-established role of NF-␬B in regulating a variety of proinflammatory genes is in agreement with our findings [17,19]. In quiescent cells, I-␬B binds and inactivates NF-␬B by sequestering it in the cytoplasm, whereas degradation of I-␬B leads to mobilization of NF-␬B to the cell nucleus and transcriptional activation. In liver injury, NF-␬B activation is often an early feature, similar to our findings here. Oxidative stress has been thought to be a potent stimulus for NF-␬B activation, although regulation and counterregulatory activation of NF-␬B are complex [46]. Nonetheless, our data showing greater NF-␬B expression in cells cultured on oxidative matrix are in agreement with the role of this transcription factor in oxidative injury, as well as interactions with other transcription factors, e.g., AP-1 [47]. Taken together, our transcription factor analysis suggests that the confluence of AP-1, CREB, Egr-1, NF-␬B, and perhaps other transcription factors exerts synergistic roles in mediating oxidized ECM-induced cytotoxicity. In this context, it is noteworthy that while pretreatment of collagen with the spin trap agent PBN decreased AP-1 and Egr-1 expression, and to some extent CREB expression, NF-␬B expression was unaffected. This probably suggests that NF-␬B in cells might have been regulated indirectly, as part of counterregulatory loops that activate and inactivate mul-

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tiple transcription factors, following activation of master switches, e.g., Egr-1 or other transcription factors, although Egr-1 expression was delayed in hepatocytes on oxidized ECM, which obviously will require further analysis. Our findings are relevant to obtaining further insights into disease mechanisms. ECM-associated processes may amplify oxidative stress during acute or chronic liver injury, including by modulation of bound growth factors. Similarly, oxidized ECM could deleteriously affect hepatocyte survival and amplify disease processes. Insights into the regulation of ECM-dependent oxidative hepatic injury should be relevant for improving hepatocyte survival in culture conditions. Primary hepatocytes undergo a variety of perturbations, including inhibition of gene expression, which is modulated by specific ECM components [11–13]. Limited survival of cultured primary hepatocytes hampers efforts ranging from development of suitable in vitro assays for drug or toxicological testing to manipulations for cell and gene therapies. Therefore, incorporation of antioxidants, e.g., aminoguanidine and the spin trap PBN as shown here, or other drugs, to prevent ECM oxidation will be appropriate for enhancing cell survival in culture. Similarly, it should be worthwhile examining whether survival of transplanted hepatocytes will be improved by decreasing exposure to oxidizing stimuli, especially in situations such as Wilson’s disease, acute liver failure, and chronic hepatitis, where significant oxidative stress exists. Greater susceptibility of polyploid cells to ECM-mediated oxidant stress could translate into the loss of significant fractions of transplanted cells and prolongation of time required for liver repopulation. Incorporation of specific drugs in cell transplantation strategies could be one way to address whether liver repopulation could be accelerated in such situations.

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16] [17]

Acknowledgments The work was supported in part by NIH Grants R01 DK46952, P33 DK41296, and P30 CA13330. We thank Dr. J. Mattana for helpful advice.

[18]

[19]

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