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Biochemical and Biophysical Research Communications 272, 681–686 (2000)

doi:10.1006/bbrc.2000.2837, available online at http://www.idealibrary.com on

egulation and Significance of Hepatocyte-Derived Matrixetalloproteinases in Liver Remodeling

akahiro Haruyama, Itsuki Ajioka, Toshihiro Akaike, and Yoshifumi Watanabe1

epartment of Biomolecular Engineering, Tokyo Institute of Technology,259 Nagatsuda, Midori-ku, Yokohama 226-8501 Japan

eceived May 6, 2000

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Regulation in expression and activation of protein-ses is one of the most important mechanisms in organorphogenesis. In this study, we investigated the ex-

ression of MMPs in primary hepatocytes and theiroles in liver remodeling. A hepatocyte proliferationnitiating cytokine, TNFa, induced MMP-9 expressionn these cells while the expression of MMP-2 did nothange by zymography analysis. Interestingly, bothhe induced MMP-9 expression and hepatocyte prolif-ration by TNFa were synergistically enhanced byGF in vitro. The increased proliferation was sup-ressed by MMP inhibitor TIMP-1, suggesting thatytokine-induced MMP regulates proliferation. Thencreased expression of MMP-9 by the cytokines wasnhibited by cytochalasin D or colchicine but not byI3 kinase inhibitor wortmannin. In addition, co-timulation by TNFa and HGF of spheroidal hepato-ytes cultured in 3-dimensional collagen gel drasti-ally induced morphological changes by cell extensionnd migration in the gel, which was in parallel withhe induced expression of MMP-9 and was inhibited byIMP-1 and -2. The MMP activity was also detected inivo in the regenerating liver after partial hepatec-omy by in situ zymography. These results suggest theoles of MMPs produced by parenchymal cells in liveremodeling. © 2000 Academic Press

Organ remodeling is composed of strictly controlledultiple processes such as cell proliferation, cell death,igration and invasion, extracellular matrix (ECM)

ynthesis, and degradation. Liver regeneration afterartial hepatectomy is one of the best-studied exam-les of organ remodeling. The process is regulated byarious molecules. Among these molecules, proteinasesuch as matrix metalloproteinases (MMPs) arehought to be mainly involved in the ECM degradation,hich could also control cell proliferation via integrin

1 To whom correspondence should be addressed. Fax: 81-45-924-815. E-mail: ywatanab@bio.titech.ac.jp.

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emonstrated that proteinases play important roles inot only ECM remodeling (1) but also hepatocyte pro-

iferation (2–5).MMPs are a large family of zinc requiring matrix-

egrading enzymes. They have been implicated in in-asive cell behavior and in embryonic development andrgan morphogenesis. These enzymes are also sup-osed to be essential for liver remodeling after PH andhe expression of some MMPs and their endogenousnhibitors, TIMP-1 and -2, were investigated in theegenerating liver (6, 7). However, the regulation andunction of MMPs involved in the regenerating liver istill unclear. In terms of cell types, information haseen accumulated in the MMP-2 regulation by hepatictellate cells (8–12). In contrast, there is little infor-ation about MMPs regulated by parenchymal cells.herefore, in this article, we examined the expressionnd function of MMPs produced by primary hepato-ytes. Isolated primary hepatocytes produced constitu-ive MMP-2 and MMP-9 increased by the stimulationf liver regeneration related cytokines, TNFa andGF. The produced MMPs increased cytokine-inducedepatocyte proliferation and induced cell invasion andigration in 3-D collagen gel. These results suggests

hat the hepatocyte-derived MMPs play essential rolesn liver remodeling.

ATERIALS AND METHODS

Reagents. Recombinant human TNFa, human HGF, and humanGF were kindly provided by Asahi Chemical Inc. (Tokyo, Japan),now Brand Milk Products Co., Ltd. (Tokyo, Japan), and Hitachihemical Co. Ltd. (Tokyo, Japan), respectively. Polyclonal anti-ouse MMP-9 antibody was purchased from Santa Cruz Biotechnol-

gy (Santa Cruz, CA). Cytochalasin D, colchicine, and wortmanninere obtained from Sigma Chemical Co. (St. Louis, MO). The female

CR mice employed in the experiments in this study were purchasedrom Charles River Japan, Inc. (Kanagawa, Japan). All animal ex-eriments were conducted in accordance with local institutionaluidelines for the care and use of laboratory animals.

Cell culture. Parenchymal hepatocytes were prepared as previ-usly described (13). Briefly, a liver was perfused with a 0.0125%

0006-291X/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

collagenase solution. After the liver had been excised, parenchymalhtc(wtpctwcbfsstg

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epatocytes were separated from nonparenchymal cells by differen-ial centrifugation at 50g for 90 s. The dead parenchymal hepato-ytes were removed by density gradient centrifugation on PercollPharmacia, Uppsala, Sweden). The viable parenchymal hepatocytesere suspended in Williams’ E medium containing antibiotics and

hen plated at a density of 1 3 104 cells/well in flat-bottomed 96-welllates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) pre-coated withollagen. The hepatocytes were incubated at 37°C for 3 h in order forhem to adhere to the collagen-coated plates and then the mediumas changed to a new one before the experiments. For collagen gel

ulture, isolated hepatocytes were cultured on BSA-coated hydropho-ic dishes for 48 h in the presence of EGF (50 ng/ml), resulted in theormation of spheroids. These spheroids were transferred onto pre-olidified collagen gels. After the cells sedimented, upper collagenolution was overlaid and formed sandwich gels containing cells inhe interface. The cells in the gels were cultured with serum orrowth factor free medium.

Gelatin zymography. Supernatants from cultured cells or lysatesrom tissues were electrophoresed on 7.5% SDS-polyacrylamide gelontaining 0.1% gelatin. The gel was washed with 0.1% Triton X-100olution for 30 min and then incubated at 37°C for overnight in thectivation buffer (50 mM Tris–HCl, pH 7.4, 0.2 M NaCl, 5 mM CaCl2,nd 0.02% NaN3). After staining with CBB R-250, the gelatinolyticctivities were detected as clear bands against the blue background.n some cases, MMPs in supernatants were concentrated by gelatin-epharose (Pharmacia, Uppsala, Sweden). The contrast images ofymography were inverted to facilitate the band detection.In situ zymography was basically performed as described (2).riefly, freshly frozen liver sections on coverslips were directly over-

ayed with 0.7% agarose containing 0.1% gelatin or 0.1% BSA in thectivation buffer. The samples were incubated in a humidified cham-er at 37°C overnight and then counterstained with crystal violet.fter gentle washing, the samples were observed by a phase-contrasticroscopy.

Western blotting. After SDS–PAGE on a 7.5% gel, proteins werelectroblotted onto PVDF membranes (Amersham, IL) in cold trans-er buffer for 3 h at 60 V. The filters were incubated with anti-MMP-9ntibodies for 1 h at room temperature after blocking with 3% skimilk, washed with TBS, and then incubated with the peroxidase-

abeled second antibodies for 1 h. Bands were detected with an ECLit (Amersham, Illinois).

ESULTS

Expression of hepatocyte-derived MMPs. We firstxamined the effect of cytokines involved in liver re-eneration on the expression of MMPs in primaryepatocytes since these cytokines are thought to regu-

ate not only hepatocyte proliferation but also organemodeling. Liver regeneration is a very complicatednd exquisitely controlled process and many moleculesre involved in the mechanism. Among them, TNFa,GF, EGF, or TGFa are thought to contribute to theroliferation of parenchymal cells (14). When primaryepatocytes were stimulated with these cytokines, onlyNFa induced MMP-9 activity but not MMP-2 (Fig.A). Interestingly, the activity of MMP-9 was synergis-ically enhanced by either HGF, EGF, or TGFa. Theffect was synergistic since, as shown in Fig. 1B, thectivity was dependent on both TNFa and HGF at highoncentration. Western blot analysis confirmed thathe induction and increasing effect of MMP-9 activityas due to the increased expression of the correspond-

682

ng protein (Fig. 1C). We noticed that all the enhancingactors (HGF, EGF, or TGFa) of MMP-9 expression arelso known as cell motility factors. Thus, the effect wasupposed to be derived from cytoskeleton modification.n fact, both cytoskeleton inhibitors, cytochalasin Dnd colchicine dose-dependently inhibited the in-reased expression of MMP-9 by TNFa and HGF (Fig.). In contrast, it is noteworthy that PI3 kinase inhib-tor, wortmannin, did not have any effect on the ex-ression despite that the inhibitor blocked the signal-ng process from HGF receptor to cytoskeleton (15).

Roles of hepatocyte-derived MMPs in liver remodel-ng in vitro. Organ remodeling is performed by im-eccably orchestrated combination of cell proliferation,ell migration, cell death, ECM degradation, and pro-uction. MMPs are thought to play multiple roles inhese processes. In the following figures, we clearlyemonstrated that hepatocyte-derived MMPs partici-ate in the regulation of hepatocyte proliferation andigration. Hepatocyte proliferation was induced byGF, and TNFa also induced weaker proliferation in

itro as reported (16, 17). Intriguingly, the combinationf TNFa and HGF showed the synergistic positive ef-

FIG. 1. Cytokine-induced MMP-9 expression in primary mouseepatocytes. Freshly isolated mouse hepatocytes were cultured inhe absence or presence of cytokines for 48 h, then the supernatantsere subjected to zymography (A and B) or Western blotting (C). (A)ytokine induction of MMP-9 activity in primary hepatocytes. 1;ontrol, 2; HGF (100 ng/ml), 3; EGF (100 ng/ml), 4; TGF-a (100g/ml), 5; TNFa (100 ng/ml), 6; TNFa 1 HGF, 7; TNFa 1 EGF, 8;NFa 1 TGFa (B) Dose-dependent induction and increased expres-ion of MMP-9 in hepatocytes by TNFa and HGF. (C) The MMP-9rotein expression by TNFa and HGF by Western blotting. 1; control,; HGF (100 ng/ml), 3; TNFa (50 ng/ml), 4; TNFa 1 HGF.

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ect on in vitro hepatocyte proliferation as the combi-ation showed in the induction of MMP-9 expressionFig. 3A). In addition, the increased DNA synthesis byNFa and HGF was dose-dependently suppressed by aatural MMP inhibitor, TIMP-1 (Fig. 3B), implyinghat cell proliferation induced by TNFa and HGF isegulated by autocrinally produced MMPs. Further-ore, this cytokine combination also showed a drastic

ffect on the organ morphological changes of threeimensional cultured hepatocyte spheroids in collagenel, which is considered to mimic the in vivo state ofepatocytes. TNFa slightly induced the cell extension

n or spike formation at the perimeters of the sphe-oids. In contrast, the combination of TNFa and HGFnduced the significant cell extension and migration inhese cells while HGF alone did not show any morpho-ogical effect (Fig. 4A). The spheroidal formation wasompletely collapsed and these cells were randomlycattered The drastic morphological changes were evi-ently induced by the cytokine-induced, hepatocyte-erived MMPs since the changes were completely in-ibited by TIMP-1. In addition, in this 3-D culturedystem, we also detected the cytokine-induced expres-ion of MMP-9 but not MMP-2 by gelatin zymographyFig. 4B). The different patterns of zymography be-ween monolayer (Fig. 1) and 3-D culture (Fig. 4) ofepatocytes might suggest that cell-cell interactionpossibly via E-cadherin) or 3-D interaction with ECMs also involved in the regulation of MMPs expressionn hepatocytes. Further experiments are required fordentification of the unknown bands in the zymographyf 3-D culture.

States of MMPs in regenerating liver. To examinehether MMPs function in liver remodeling in vivo, we

hecked the states of MMP activation in the regener-ting liver, a natural organ remodeling. MMP-9 andMP-2 activities were detected in zymography of the

ysates from the regenerating livers after partial hep-tectomy (Fig. 5A). These activities were observed 3 hfter PH and gradually decreased to 48 h after. The

FIG. 2. Cytokine-induced MMP-9 expression in primary hepato-ytes is regulated by cytoskeleton signaling. Primary hepatocytesere cultured with TNFa (100 ng/ml) and HGF (50 ng/ml) for 48 h in

he presence or absence of cytochalasin D (CytD) (0.2, 1, 5 mM),olchicine (Col) (0.2, 1, 5 mM), or wortmannin (Wtn) (5, 50, 500 nM)t various concentrations, then the supernatants were subjected toymography. *TNFa (50 ng/ml) 1 HGF (100 ng/ml).

683

n the regenerating liver (Fig. 5A). In addition, in situymography showed that the activity evenly distrib-ted in the histological section of the regenerating liverFig. 5B). The results directly demonstrates the activa-ion of MMPs in the regenerating liver.

ISCUSSION

Liver regeneration is a suitable model for the studyf organ remodeling. The process is regulated by vari-us types of molecules. Cytokines such as TNFa, EGF,GFa or HGF are considered to play critical roles inhe process (14). Of these cytokines, evidence has beenccumulated that TNFa is an initiation factor in theiver regeneration (14, 18, 19) although the detail

echanism is unclear. In this study, we demonstrated

FIG. 3. Involvement of hepatocyte-derived MMPs in hepatocyteroliferation. (A) Primary hepatocytes were cultured with cytokinesTNFa; 50 ng/ml, HGF; 100 ng/ml) for 48 h, then the DNA synthesisas measured as described. (B) Hepatocytes were cultured withNFa (50 ng/ml) 1 HGF (100 ng/ml) in the presence of a MMP-

nhibitor, TIMP-1 at the indicated concentrations for 48 h, and theNA synthesis was measured.

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hat TNFa induced MMP-9 expression in primaryepatocytes and the expression was enhanced by HGF.he same combination synergistically increased hepa-ocyte proliferation which was inhibited by TIMP-1.hese data indicated that TNFa and HGF inducedMPs critically regulated hepatocyte proliferation.

ome researchers have shown the significance of pro-

FIG. 4. Hepatocyte-derived MMPs induced morphologicalhanges in 3-D cultured spheroids. Spheroidal hepatocytes pre-ultured on BSA-coated dishes were entrapped in sandwiched colla-en gel, then cultured for 5 days in the presence of cytokines. (A)hese cells were observed under a phase contrast microscopy (380).; control, B; HGF (100 ng/ml), C; TNFa (50 ng/ml), D; TNFa 1 HGF,; TNFa 1 HGF 1 TIMP-1 (5 mg/ml). (B) The cell supernatants from

he collagen gels were subjected to zymography after being concen-rated by gelatin-sepharose. 1; control, 2; HGF, 3; TNFa, 4; TNFa 1GF. The arrow indicates MMP-9.

684

). They suggested that the proteinases may processhe proform of growth factors such as HGF, result inhe production of active form which would trigger theepatocyte proliferation. However, in our system, thexplanation is not suitable since we employed recom-inant active HGF. It is more plausible that theytokine-induced function as a growth factor or co-actor to hepatocytes. In fact, it has been reported that

MP-2 might be a growth factor in another liver cellype (11). However, the possibility can not be ruled outhat the disruption of ECM by the cytokine-inducedMPs which would modify the integrin signaling may

e involved in the mechanism. Taken together, TNFas thought to function as an initiator of hepatocyteroliferation by directly increasing the proliferation16) and inducing MMPs in hepatocytes.

The detail mechanism in which TNFa and HGF in-uces MMP-9 expression in hepatocytes remains to beolved. However, some mechanisms can be speculated.ince the activation of NF-kB signaling is important

or the expression of MMP-9 (20), TNFa may directlynduce MMP-9 via the activation of NF-kB. Alterna-ively, TNFa might indirectly induce MMP-9 in hepa-ocytes via the induction of ceramide as reported innother cell type (21). The enhancement by HGF of

FIG. 5. Activities of MMPs in the regenerating liver in vivo. (A)he regenerating livers were excised out at the indicated times andhe homogenized lysates were subjected to gelatin zymography afterhe protein concentrations were adjusted. (B) The sections (10 mm)ere prepared from the regenerating liver 48 h after partial hepa-

ectomy (PH), then in situ gelatin zymography was performed asescribed. A; normal liver with gelatin zymography, B; PH liver withelatin zymography, C; PH liver with BSA zymography.

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n cytoskeleton modification since other cell motilityactors such as EGF and TGFa also showed the sameffect and the effect was inhibited by cytoskeleton in-ibitors. In fact, it has been reported that cytoskeleton

ntegrity is prerequisite for MMP-9 expression (22–24).t is, however, notable that a PI3 kinase inhibitor,ortmannin, did not inhibit the enhancing effect ofGF because the pathway is thought to be critical for

he modification of cytoskeleton by HGF (25, 26). Thisesult indicates that the effect of HGF is regulated byther pathways. One possibility is ERK pathway. Zei-ler et al. showed that only prolonged activation byGF/EGF but not transient activation by KGF/IGF-1

f MEK/ERK pathway induced cell motility and inva-ion via the induction of MMP-9 (27). Alternatively,GF enhanced MMP-9 expression via phospholipase Dctivation downstream of PKC (28, 29). Which path-ay is the dominant to regulate the effect requires

urther study.It is more likely that MMP-9 dominantly plays roles

n the systems of this study since the expression norctivation of MMP-2 in primary cultured hepatocytesid not appear to be influenced by either TNFa or HGF.n addition, the activity of MMP-2 was relatively mi-or, if any, to that of MMP-9 in stimulated hepatocytesccording to gelatin zymography (Fig. 2). MMP-2 isainly produced and activated by hepatic stellate cells

n the liver (8, 9, 11), explaining the expression ofMP-2 in vivo after PH (Fig. 5A). Therefore, it is

aturally thought that these cell-type specific MMPsMMP-2 by stellate cells and MMP-9 by hepatocytes)oordinately function in vivo. The significance ofMP-9 in organ remodeling was demonstrated in

ther system (30, 31).Spheroidal hepatocytes are thought to mimic the in

ivo milieu (32, 33). Especially, the culture of the sphe-oidal hepatocytes in collagen gel is supposed to imi-ate the in vivo environments surrounded by ECM.ell spreading and migration are essential for organ

emodeling to fill the damaged space or trigger mor-hological changes. Both MMP-2 and MMP-9 haveeen reported to regulate cell migration in other cellypes (30, 34–36). In this study, we demonstrated forhe first time that regeneration-related cytokines in-uced cell spreading and migration of parenchymalells from spheroids in ECM via the induction ofMPs. This phenomenon strongly indicates the signif-

cance of cytokine-induced MMPs in liver remodeling.he further study how these proteinases are tempo-ally and spatially regulated in vivo is necessary tolicit the detail mechanism of liver remodeling.

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