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Expression of matrix metalloproteinase-13 and tissue inhibitor of metalloproteinase-1 in acute liver injury
Journal of Hepatology 1999; 30:419-424 Printed in Denmark • All rights reserved Munksgaard. Copenhagen
Copyright © European Association for the Study of the Liver 1999 Journal of Hepatology ISSN 0168-8278
Expression of matrix metalloproteinase-13 and tissue inhibitor of metaHoproteinase-1 in acute liver injury Yutaka Yata, Terumi Takahara, Kei Furui, Li Ping Zhang and Akiharu Watanabe Third Department of lnternal Medicine, Toyama Medical and Pharmaceutical University, Japan
Background~Aims: Matrix metalloproteinase-13, one of the principal neutral proteinases capable of cleaving native fibrillar collagens, is important in the degradation and remodeling of extracellular matrix. However, its precise expression in liver injury has not been characterized. We examined the kinetics of the expression of matrix metalloproteinase-13 and one of its specific inhibitors, tissue inhibitor of metalloproteinase-1, in acute liver injury in rats. Methods: Acute liver injury was induced by administration of carbon tetrachloride or two different doses of D-galactosamine hydrochioride in Wistar rats. Hepatic matrix metalloproteinase-13 and tissue inhibitor of metalloproteinase-1 mRNA levels were then examined by Northern blotting. Results: All rats survived after liver injury induced by carbon tetrachloride or low doses of D-galactosamine hydrochloride. However, rats died 5 days after induction of liver injury by high doses of D-galactosamine hydrochloride. In carbon tetrachloride-induced liver injury, matrix metalloproteinase-13 mRNA was transiently increased between 6 h and 1 day after injury. Tissue inhibitor of metalloproteinase-1 mRNA expression was increased between 6 h and 3 days after the peak of matrix metalloproteinase-13 expression.
Similar patterns of matrix metalloproteinase-13 and tissue inhibitor of metalloproteinase-1 expression were observed in low-dose D-galactosamine hydrochloride-induced liver injury. In contrast, in high-dose D-galactosamine hydrochloride-induced liver injury, tissue inhibitor of metalloproteinase-1 expression peaked before matrix metalloproteinase-13 expression, which was increased 2 days after injury. Both mRNA levels continued to increase until death. Conclusions: Transient expression of matrix metalloproteinase-13, followed by that of tissue inhibitor of metalloproteinase-1, was observed during recovery from acute liver injury induced by carbon tetrachloride and low-dose D-galactosamine hydrochloride. In contrast, disordered expression of matrix metailoproteinase-13 was observed in fatal liver injury caused by high-dose D-galactosamine hydrochloride. These results indicate that matrix metalloproteinase13 plays an important role in the early phase of recovery from liver injury.
HE MEMBERSof the collagenase subfamily, interstitial collagenase (matrix metalloproteinase-1; MMP-1), neutrophil collagenase (MMP-8), and collagenase-3 (MMP-13) are the principal neutral proteinases capable of cleaving native fibrillar collagens and they apparently play important roles in the degradation of extracellular matrix (ECM) (1). Rat collagen-
ase, now named MMP-13, has been found to show exceptionally wide substrate specificity and acts in the degradation and remodeling of ECM (1-3). It has also been reported that urokinase type plasminogen activator (u-PA), which activates latent MMPs, plays an important role in the early phases of liver regeneration through remodeling of ECM (4). However, the precise kinetics of MMP expression during liver repair are not known. Many hepatotoxins, such as carbon tetrachloride (CC14), D-galactosamine hydrochloride (Gal-N), and dimethylnitrosamine (DMN), cause acute liver injury in rats, mice, and rabbits (5-7). Excessive doses of
T
Received 29 May; revised 50tober; accepted 13 October 1998
Correspondence: Terumi Takahara, Third Department of Internal Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 9304)194, Japan. Tel" 81 764 34 2281. Fax: 81 764 34 5027.
Key words: Carbon tetrachloride; D-galactosamine hydrochloride; Liver repair; Matrix metalloproteinase-13; Stellate cell; Tissue inhibitor of metalloproteinase-1.
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Y. Yata et al. these h e p a t o t o x i n s i n d u c e f a t a l liver injury, w h i c h is h i s t o l o g i c a l l y s i m i l a r to h u m a n f u l m i n a n t h e p a t i c failure, while low doses cause t r a n s i e n t a c u t e liver i n j u r y (8). I n this study, we e x a m i n e d the m e s s e n g e r R N A ( m R N A ) levels o f M M P - 1 3 a n d one o f its specific inh i b i t o r s , tissue i n h i b i t o r o f m e t a t l o p r o t e i n a s e - 1 ( T I M P - 1 ) , using m o d e l s o f t r a n s i e n t o r fatal liver inj u r i e s to clarify the expressions o f these genes d u r i n g r e p a i r after a c u t e liver injury.
Materials and Methods Rat models of fiver injury Twenty-eight male Wistar rats weighing approximately 200 g each were subjected to CC14 intoxication. To produce acute CCl4-induced injury, 50% CC14in olive oil was given to rats at a volume of 0.2 ml/ 100 g body weight, using a gastric tube as previously described (5). Four rats in each group were killed 6 h, 12 h, or 1, 2, 3, 5 or 7 days after treatment. Four control rats were given only olive oil and killed at 2 days. Fifty-six similar rats were subjected to two models of GaI-N intoxication. Gal-N was dissolved in physiologic saline to obtain a 20% solution, as previously described (5). The GaI-N solution was intraperitoneally injected at a volume of 0.25 ml/100 g body weight as a low-dose model or 0.5 ml/100 g body weight as a high-dose model. Four rats in each group were killed 6 h, 12 h, or 1, 2, 3, 5 or 7 days after intoxication. Four control rats were treated with saline alone and killed at 2 days. The livers were immediately removed. A portion was processed for routine histologic studies and the remainder was snap-frozen in liquid nitrogen. Specimens were stored at -80°C until analyzed.
RNA extraction and Northern blot analysis Expression of MMP-13 and TIMP-1 mRNA was analyzed by Northern hybridization. Total RNA was isolated directly from frozen liver specimens, according to the method of Chirgwin et al. (9), as previously described (10,11). Briefly, the tissues were homogenized in 4 M guanidinium isothiocyanate, followed by cesium chloride density gradient centrifugation. Poly (A) + RNA was obtained by oligo (dT) cellulose column chromatography (12) and stored at -80°C. For Northern analysis, poly (A)+RNA was fractionated on 0.9% agarose gels containing 2.2 M formaldehyde. RNA was then transfered onto nylon filters (Zeta Probe; BioRad, Richmond, CA, USA), and immobilized by heating at 80°C under vacuum for 30 min. The filters were hybridized with cDNA probes labeled with 32P-dCTP and washed with solutions of increasing temperature and decreasing ionic strength, the final wash stringency being 0.1 xstandard saline citrate (SSC)/0.1% sodium dodecyl sulfate (SDS) at 52°C. In some experiments, the initial probe was removed by washing the filters in solution containing 0.1 X SSC/0.1% SDS at 95°C for 10 min, followed by prehybridization and hybridization with other cDNAs. After washing, the filters were kept moist and exposed to X-ray film (X-omat; Eastman Kodak, Rochester, NY, USA) at -80°C. After autoradiography, the relative quantities of mRNAs were determined by a Bioimage Analyzer BAS 2000 (Fuji Film Ltd., Tokyo, Japan). Relative values of MMP-13 and TIMP-1 levelswere corrected for differences in specific activities and in the abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression (13).
Probes Complementary DNAs (cDNAs) of MMP-13 (14), TIMP-1 (15), TNF-a (16), TGF-fll (17), and GAPDH (18) were used. The cDNA of MMP-13 and TNF-c~ were generous gifts from Dr. Partridge and Dr. Decker, respectively. These cDNAs were labeled with 32P-dCTP using a random primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN, USA).
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Stat&tical analys& All densitometry values are reported as the mean ---SD. Statistical analysis was performed using Fisher's protected least significant difference, and p-values less than 0.05 were considered significant.
Results Histology CCl4-induced liver injury. A s p r e v i o u s l y r e p o r t e d , ball o o n i n g o f h e p a t o c y t e s a n d necrosis with i n f l a m m a t o r y cells o c c u r r e d a d j a c e n t to the central veins a n d in the lobules 6 h after CC14 i n t o x i c a t i o n , a n d z o n a l necrosis was o b s e r v e d at 2 d a y s (19). These signs o f t r a n s i e n t i n j u r y were i m p r o v e d at 5 days, a n d a l m o s t resolved at 7 days. Gal-N-induced liver injur): W i t h l o w - d o s e G a I - N intoxication, s p o t t y necrosis with i n f l a m m a t o r y cells was o b s e r v e d in the lobules 6 h after intoxication, as prev i o u s l y r e p o r t e d (20). T h e p e a k o f h e p a t i c necrosis was o b s e r v e d at 2 days. These signs o f t r a n s i e n t i n j u r y were i m p r o v e d at 5 days, a n d a l m o s t resolved at 7 days. W i t h h i g h - d o s e G a l - N i n t o x i c a t i o n , h e p a t i c necrosis was o b s e r v e d at 6 h a n d r a p i d l y p r o g r e s s e d until m a s s ive necrosis o c c u r r e d at 3 days. A l l rats died at 5 days, as p r e v i o u s l y d e s c r i b e d (20), a n d their livers showed massive necrosis.
Expression o f M M P - 1 3 and TIMP-1 m R N A CCl4-induced liver injury. In acute CC14-induced liver injury, M M P - 1 3 m R N A levels were t r a n s i e n t l y increased at 6 h (5.7-fold) a n d decreased at 2 days, b u t were b a r e l y d e t e c t a b l e in c o n t r o l livers. T I M P - 1 exp r e s s i o n was increased between 6 h (2.2-fold) a n d 3 d (2.1-fold) with a p e a k at 2 d a y s (4.3-fold) (Fig. l a , b). T h e p e a k o f T I M P - 1 e x p r e s s i o n o c c u r r e d after t h a t o f MMP-13. Gal-N-induced liver injury. W i t h low doses o f G a l - N , the p a t t e r n s o f M M P - 1 3 a n d T I M P - 1 expression were similar to t h o s e seen after CC14 intoxication. M M P - 1 3 m R N A expression increased between 6 h (1.5-fold) a n d 1 d a y (2.5-fold), with a p e a k at 1 day. T I M P - 1 m R N A expression increased between 6 h (1.5-fold) a n d 2 d a y s (2.3-fold), with a p e a k at 2 days, M M P - 1 3 m R N A was n o t d e t e c t e d in c o n t r o l liver (Fig. 2a, b). O n the o t h e r h a n d , in the h i g h - d o s e G a l - N m o d e l , m i n i m a l increased expression o f M M P - 1 3 was d e t e c t e d f r o m 6 h to 3 days c o m p a r e d with control. However, T I M P - 1 m R N A b e g a n increasing f r o m 6 h (1.5-fold) a n d also c o n t i n u e d to increase until death. T h e greatest increase in T I M P - 1 expression was 5.3-fold at 3 d a y s (Fig. 3a, b). T h e p e a k expression o f M M P - 1 3 was prec e d e d by t h a t o f T I M P - 1 in this lethal injury.
Expression o f TGF-fll and TNF-a T G F - f l l m R N A expression was increased f r o m 6 h until 3 d a y s in the CC14 m o d e l , with a p e a k at 2 days. In
MMP-13 and TIMP-1 in acute liver injury
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Fig. 1. Northern blot analysis in CCl4-induced liver injury. (a) Messenger RNA from rat livers obtained at 6 h, 12 h, and 1, 2, 3, 5, and 7 days was extracted and subjected to Northern analysis as described in Materials and Methods. Twenty micrograms of poly (A) ÷ RNA per lane were electrophoresed and the filters were hybridized with cDNA probes for MMP-13, TIMP-1, TGF-fll and TNF-a. These probes hybridized to mRNAs o f l . 8 kb, 0.9 kb, 2.5 kb, and 1.5 kb, respectively. MMP-13 mRNA increased transiently 6 h after injury and was decreased at 2 days. TIMP-1 mRNA expression increased between 6 h and 3 days with a peak at 2 days. TGF-fll mRNA increased transiently between 6 h and 3 days with a peak at 2 days. (b) Densitometric analysis using four individual rats per time point showed that relative mRNA levels of MMP-13 were increased up to 5.7-fold (6 h) compared with controls and then decreased. Relative mRNA levels of TIMP-1 were increased up to 4.3-foM (2 days) and then decreased. (mean±SD of four separate experiments). *p
the low-dose Gal-N model, TGF-fll mRNA expression was also increased with a peak at 1 day. In the high-
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Fig. 2. Northern blot analysis in low-dose Gal-N-induced liver injury, (a) Poly (A) + RNA (20 pg per lane) were electrophoresed and the filters were hybridized with cDNA probes for MMP-13, TIMP-1, TGF-fll and TNF-a. These probes hybridized to mRNAs o f l . 8 kb, 0.9 kb, 2.5 kb and 1.5 kb, respectively. MMP-13 mRNA expression increased between 6 h and 1 day, with a peak at 1 day. TIMP-1 mRNA expression increased between 6 h and 2 days, with a peak at 2 days. TGF-fll mRNA increased between 12 h and 2 days, with a peak at 1 day. TNF-ct mRNA increased transiently at 6 h. (b) Densitometric analysis using four individual rats showed that relative mRNA levels of MMP13 increased up to 2.5-fold (1 day) at the peak and then decreased. Relative mRNA levels of TIMP-1 increased up to 2.3-foM (2 days) and then decreased. (mean+_SD of four separate experiments). *p
dose GaI-N model, TGF-fll m R N A expression was increased from 12 h onward and continued to increase until death. This pattern of expression was similar to that of TIMP-1 (Fig. la, 2a, 3a). TNF-a m R N A expression was transiently increased at 6 h in the CC14 and low-dose GaI-N models. How421
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Fig. 3. Northern blot analysis in high-dose Gal-N-induced liver injury. (a) MMP-13 m R N A expression gradually increased from 12 h to 3 days with a peak at 3 days. TIMP1 m R N A expression also increased from 6 h to 3 clays, with a peak at 3 days. TGF-fll m R N A increased from 12 h to 3 days, with a peak at 2 day's. TNF-a m R N A increased transiently at 6 h. In this" model, all rats died after 5 day's. (b) Densitometric analysis using four individual rats demonstrated that relative levels of MMP-13 increased up to 1.4fold (3 days) at the peak, compared with control. Relative m R N A levels of TIMP-1 also increased up to 5.3-Jold of control levels (3 days). (mean +_SD of four separate experiments). *p
ever, in the high-dose Gal-N model its expression was dramatically increased by 6 h and continued to be detectable until death (Fig. 2a, 3a). Discussion
We examined the expressions of MMP-13 and TIMP1 in acute liver injury induced by CC14 and two differ422
ent doses of Gal-N. In spite of the use of different hepatotoxins, the patterns of expression of MMP-13 and TIMP-t were similar in transient liver injury induced by CC14 and low doses of Gal-N. MMP-13 mRNA levels increased transiently and the peak of TIMP-1 expression occurred after that of MMP-13. Recently, it was reported that rat collagenase has a high degree of functional and sequence homology with human collagenase-3, and that these enzymes belong to the collagenase-3 subfamily (MMP-13) and are distinct from human interstitial collagenase (MMP- 1) (1). It was also reported that MMP-13 play a significant role in the degradation and remodeling of ECM constituents (2,3). Moreover, active MMP-13 is specifically inhibited in a 1:1 stoichiometric fashion by the three homologous TIMPs (TIMP-1, -2, -3) (1). MMP-13 may be inhibited by a2-macroglobulin like other MMPs (21). Previous studies reported that matrix degradation occurring in the very early stages of liver regeneration after partial hepatectomy is due to plasminogen activator, which is important for hepatocyte proliferation and ECM remodeling (4,22,23). Current studies concerning burn wounds (24) and colonic anastomosis (25,26) showed simultaneous expression of collagenase and TIMP-1. These results suggest that collagenase and TIMP-1 play important roles in wound healing after burn injury or colonic anastomosis. Furthermore, increased collagenase activity during the early stages of cirrhosis is thought to be important for hepatic regeneration (27). Taking these results together, increased expression of MMP-13 may be important during liver repair after transient liver injury. In contrast, in fatal liver injury induced by high doses of Gal-N, the pattern of mRNA expression was different from that with CCI4 or low doses of Gal-N. MMP-13 mRNA levels increased but after the peak of TIMP-I expression. Expression of TIMP-1 was increased significantly compared to that of MMP-13, and its peak expression preceded that of MMP-13. Okada et at. have reported that MMPs and TIMPs are sequentially detected during normal wound healing in rat skin (28). These results may suggest that ordered regulation of MMP-13 and TIMP-1 is required for repair after acute liver injury. However, it is not clear that these changes in expression of these genes are seen in other models of transient or fatal hepatotoxicity. Further study is needed using other animal models to determine whether disordered expression is present in fatal liver injury. Additionally, MMPs except MMP-13 can degrade interstitial collagens (21,29). Besides TIMP-1, TIMP-2 and -3 inhibit MMP-13. Further studies including other MMP and
M M P - 1 3 and TIMP-1 in acute liver injury
TIMP families are also needed to show the real role of these genes in liver injuries. The present different patterns of MMP-13 and TIMP-1 gene expression may be induced by a network of inflammatory cytokines. In our fatal liver injury model, TGF-fll m R N A expression was detectable from 12 h onward and continued to increase until death, although its expression was transient in Cfl4 and low-dose Gal-N-induced liver injury. TGF-fll upregulates TIMP-1 expression as well as other inflammatory cytokines such as interleukin (IL)-lfl, IL-6, and IL-11 (30,31). In addition, TGF-fll inhibits proliferation of hepatocytes and other cells (32-35). Thus, TGF-fll may inhibit liver repair after fatal liver injury through its anti-proliferative actions. On the other hand, TN F - a was expressed transiently by 6 h and decreased rapidly in CC14 and Gal-N liver injury. Concerning the role of TNF-a in hepatic regeneration, it is known that T N F - a causes hepatic regeneration by induction of NF-xB (36,37). MMP-13 is also known to be up-regulated by TNF-a (2,38), in agreement with our findings. TNF-t~ may play an important role in hepatic regeneration after acute injury, as suggested by previous reports (36,37). Iredale et al. (39) also examined the expression of MMPs and TIMPs in acute CC14 liver injury. They reported that the expression of TIMP-1 increased gradually after intoxication, although interstitial collagenase m R N A was not significantly changed over time. However, they only examined gene expression 24 and 72 h after intoxication. Thus, they might not have detected the early interstitial collagenase response. As it has been reported that rat cells express only MMP-13 (1), interstitial collagenase, which was detected by Iredale et al. (39) in acute CC14 liver injury, might now be identified with MMP-13. In the present study we have shown a significant increase in TIMP-1 in fatal liver injury. It has been reported that serum concentrations of TIMP-1 are greatly increased in patients with fulminant hepatitis (40). Histologically, stellate cells proliferate in the livers of patients with massive and submassive hepatic necrosis due to fulminant hepatitis (41). In fact, TIMP1 is produced by cultured stellate cells as previously described (42). Herbst et al. also reported that TIMP1 m R N A was mainly expressed on stellate cells in fibrotic rat and human livers (43). Since TIMP-1 inhibits MMP-13 stoichiometrically (44), increased expression of TIMP-1 relative to MMP-13 may cause failure of liver repair. In our study we did not examine the cell types which produce MMP-13 and TIMP-1. The cell types which produce MMP-13 have not been determined yet.
Further study is needed to clarify the cell types which express MMP-13 in rat liver. On the other hand, TIMP-1 is produced by stellate cells (42), hepatocytes (45), bile duct cells and endothelial cells (46). However, the early expression of TIMP-1 after acute liver injury may originate from activated stellate cells (39). In conclusion, transiently increased expressions of MMP-13 and TIMP-1 were observed during repair after acute liver injury. Further study is needed to clarify the roles of MMP-13 and TIMP-1 using other models such as transgenic mice.
Acknowledgements We thank Drs. Partridge and Decker for the kind gift of cDNA probes of MMP-13 and TNF-a. We also thank Miss M. Maekawa for her technical assistance in preparing liver sections.
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Tschesche H, et al. Synthesis of tissue inhibitor of metalloproteinase-1 (TIMP-1) in human hepatoma cells (HepG2). Up-regulation by interleukin-6 and transforming growth factor beta 1. FEBS Lett 1992; 313: 143-7. 32. Armendariz-Borunda J, Katai H, Jones CM, Seyer JM, Kang AH, Raghow R. Transforming growth factor beta gene expression is transiently enhanced at a critical stage during liver regeneration after CC14 treatment. Lab Invest 1993; 69: 283-94. 33. Nakamura T, Tomita Y, Hirai R, Yamaoka K, Kaji K, Ichihara A. Inhibitory effect of transforming growth factor-beta on DNA synthesis of adult rat hepatocytes in primary culture. Biochem Biophys Res Commun 1985; 133: 1042-50. 34. Russell WE. Transforming growth factor beta (TGF-beta) inhibits hepatocyte DNA synthesis independently of EGF binding and EGF receptor autophosphorylation. J Cell Physiol 1988; 135: 253-61. 35. Win KM, Charlotte E Mallat A, Cherqui D, Martin N, Mavier E e t al. Mitogenic effect of transforming growth factor-beta 1 on human Ito cells in culture: evidence for mediation by endogenous platelet-derived growth factor. Hepatology 1993; 18: 137-45. 36. Fausto N, Laird AD, Webber EM. Role of growth factors and cytokines in hepatic regeneration. FASEB J 1995; 9: 1527-36. 37. Yamada Y, Fausto N. Deficient liver regeneration after carbon tetrachloride injury in mice lacking type 1 but not type 2 tumor necrosis factor receptor. Am J Pathol 1998; 152: 1577-89. 38. Reboul P, Pelletier JE Tardif G, Cloutier JM, Pelletier JM. The new collagenase, collagenase-3, is expressed and synthesized by human chondrocytes but not by synoviocytes. J Clin Invest 1996; 97:2011-9. 39. Iredale JP, Benyon RC, Arthur M J, Ferris WE Alcolado R, Winwood P J, et al. Tissue inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver injury and fibrosis. Hepatology 1996; 24: 176-84. 40. Tsuchida T, Takahara, T, Hioki O, Higuchi K, Watanabe A, lwata K. Change of serum tissue inhibitor of metalloproteinases-1 in fulminant hepatitis patients. Nippon Shokakibyo Gakkai Zasshi 1992; 89: 1835. 41. Enzan H, Himeno H, Iwamura S, Saibara T, Onishi S, Yamamoto Y, et al. Sequential changes in human Ito cells and their relation to postnecrotic liver fibrosis in massive and submassive hepatic necrosis. Virchows Arch 1995; 426: 95-101. 42. lredale JP, Murphy G, Hembry RM, Friedman SL, Arthur MJ. Human hepatic lipocytes synthesize tissue inhibitor of metalloproteinases-1. Implications for regulation of matrix degradation in liver. J Clin Invest 1992; 90: 282-7. 43. Herbst H, Wege T, Milani S, Pellegrini G, Orzechowski HD, Bechstein WO, et al. Tissue inhibitor of metalloproteinase-1 and 2 RNA expression in rat and human liver fibrosis. Am J Pathol 1997; 150: 1647-59. 44. Murphy G, Docherty AJ. The matrix metalloproteinases and their inhibitors. [review]. Am J Respir Cell Mol Biol 1992; 7: 1205. 45. Roeb E, Graeve L, Hoffmann R, Decker K, Edwards DR, Heinrich PC. Regulation of tissue inhibitor of metalloproteinases-1 gene expression by cytokines and dexamethasone in rat hepatocyte primary cultures. Hepatology 1993; 18: 1437~42. 46. Nakatsukasa H, Ashida K, Higashi T, Ohguchi S, Tsuboi S, Hino N, et al. Cellular distribution of transcripts for tissue inhibitor of metalloproteinases 1 and 2 in human hepatocellular carcinomas. Hepatology 1996; 24: 82-8.
Copyright © European Association for the Study of the Liver 1999 Journal of Hepatology ISSN 0168-8278
Expression of matrix metalloproteinase-13 and tissue inhibitor of metaHoproteinase-1 in acute liver injury Yutaka Yata, Terumi Takahara, Kei Furui, Li Ping Zhang and Akiharu Watanabe Third Department of lnternal Medicine, Toyama Medical and Pharmaceutical University, Japan
Background~Aims: Matrix metalloproteinase-13, one of the principal neutral proteinases capable of cleaving native fibrillar collagens, is important in the degradation and remodeling of extracellular matrix. However, its precise expression in liver injury has not been characterized. We examined the kinetics of the expression of matrix metalloproteinase-13 and one of its specific inhibitors, tissue inhibitor of metalloproteinase-1, in acute liver injury in rats. Methods: Acute liver injury was induced by administration of carbon tetrachloride or two different doses of D-galactosamine hydrochioride in Wistar rats. Hepatic matrix metalloproteinase-13 and tissue inhibitor of metalloproteinase-1 mRNA levels were then examined by Northern blotting. Results: All rats survived after liver injury induced by carbon tetrachloride or low doses of D-galactosamine hydrochloride. However, rats died 5 days after induction of liver injury by high doses of D-galactosamine hydrochloride. In carbon tetrachloride-induced liver injury, matrix metalloproteinase-13 mRNA was transiently increased between 6 h and 1 day after injury. Tissue inhibitor of metalloproteinase-1 mRNA expression was increased between 6 h and 3 days after the peak of matrix metalloproteinase-13 expression.
Similar patterns of matrix metalloproteinase-13 and tissue inhibitor of metalloproteinase-1 expression were observed in low-dose D-galactosamine hydrochloride-induced liver injury. In contrast, in high-dose D-galactosamine hydrochloride-induced liver injury, tissue inhibitor of metalloproteinase-1 expression peaked before matrix metalloproteinase-13 expression, which was increased 2 days after injury. Both mRNA levels continued to increase until death. Conclusions: Transient expression of matrix metalloproteinase-13, followed by that of tissue inhibitor of metalloproteinase-1, was observed during recovery from acute liver injury induced by carbon tetrachloride and low-dose D-galactosamine hydrochloride. In contrast, disordered expression of matrix metailoproteinase-13 was observed in fatal liver injury caused by high-dose D-galactosamine hydrochloride. These results indicate that matrix metalloproteinase13 plays an important role in the early phase of recovery from liver injury.
HE MEMBERSof the collagenase subfamily, interstitial collagenase (matrix metalloproteinase-1; MMP-1), neutrophil collagenase (MMP-8), and collagenase-3 (MMP-13) are the principal neutral proteinases capable of cleaving native fibrillar collagens and they apparently play important roles in the degradation of extracellular matrix (ECM) (1). Rat collagen-
ase, now named MMP-13, has been found to show exceptionally wide substrate specificity and acts in the degradation and remodeling of ECM (1-3). It has also been reported that urokinase type plasminogen activator (u-PA), which activates latent MMPs, plays an important role in the early phases of liver regeneration through remodeling of ECM (4). However, the precise kinetics of MMP expression during liver repair are not known. Many hepatotoxins, such as carbon tetrachloride (CC14), D-galactosamine hydrochloride (Gal-N), and dimethylnitrosamine (DMN), cause acute liver injury in rats, mice, and rabbits (5-7). Excessive doses of
T
Received 29 May; revised 50tober; accepted 13 October 1998
Correspondence: Terumi Takahara, Third Department of Internal Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 9304)194, Japan. Tel" 81 764 34 2281. Fax: 81 764 34 5027.
Key words: Carbon tetrachloride; D-galactosamine hydrochloride; Liver repair; Matrix metalloproteinase-13; Stellate cell; Tissue inhibitor of metalloproteinase-1.
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Y. Yata et al. these h e p a t o t o x i n s i n d u c e f a t a l liver injury, w h i c h is h i s t o l o g i c a l l y s i m i l a r to h u m a n f u l m i n a n t h e p a t i c failure, while low doses cause t r a n s i e n t a c u t e liver i n j u r y (8). I n this study, we e x a m i n e d the m e s s e n g e r R N A ( m R N A ) levels o f M M P - 1 3 a n d one o f its specific inh i b i t o r s , tissue i n h i b i t o r o f m e t a t l o p r o t e i n a s e - 1 ( T I M P - 1 ) , using m o d e l s o f t r a n s i e n t o r fatal liver inj u r i e s to clarify the expressions o f these genes d u r i n g r e p a i r after a c u t e liver injury.
Materials and Methods Rat models of fiver injury Twenty-eight male Wistar rats weighing approximately 200 g each were subjected to CC14 intoxication. To produce acute CCl4-induced injury, 50% CC14in olive oil was given to rats at a volume of 0.2 ml/ 100 g body weight, using a gastric tube as previously described (5). Four rats in each group were killed 6 h, 12 h, or 1, 2, 3, 5 or 7 days after treatment. Four control rats were given only olive oil and killed at 2 days. Fifty-six similar rats were subjected to two models of GaI-N intoxication. Gal-N was dissolved in physiologic saline to obtain a 20% solution, as previously described (5). The GaI-N solution was intraperitoneally injected at a volume of 0.25 ml/100 g body weight as a low-dose model or 0.5 ml/100 g body weight as a high-dose model. Four rats in each group were killed 6 h, 12 h, or 1, 2, 3, 5 or 7 days after intoxication. Four control rats were treated with saline alone and killed at 2 days. The livers were immediately removed. A portion was processed for routine histologic studies and the remainder was snap-frozen in liquid nitrogen. Specimens were stored at -80°C until analyzed.
RNA extraction and Northern blot analysis Expression of MMP-13 and TIMP-1 mRNA was analyzed by Northern hybridization. Total RNA was isolated directly from frozen liver specimens, according to the method of Chirgwin et al. (9), as previously described (10,11). Briefly, the tissues were homogenized in 4 M guanidinium isothiocyanate, followed by cesium chloride density gradient centrifugation. Poly (A) + RNA was obtained by oligo (dT) cellulose column chromatography (12) and stored at -80°C. For Northern analysis, poly (A)+RNA was fractionated on 0.9% agarose gels containing 2.2 M formaldehyde. RNA was then transfered onto nylon filters (Zeta Probe; BioRad, Richmond, CA, USA), and immobilized by heating at 80°C under vacuum for 30 min. The filters were hybridized with cDNA probes labeled with 32P-dCTP and washed with solutions of increasing temperature and decreasing ionic strength, the final wash stringency being 0.1 xstandard saline citrate (SSC)/0.1% sodium dodecyl sulfate (SDS) at 52°C. In some experiments, the initial probe was removed by washing the filters in solution containing 0.1 X SSC/0.1% SDS at 95°C for 10 min, followed by prehybridization and hybridization with other cDNAs. After washing, the filters were kept moist and exposed to X-ray film (X-omat; Eastman Kodak, Rochester, NY, USA) at -80°C. After autoradiography, the relative quantities of mRNAs were determined by a Bioimage Analyzer BAS 2000 (Fuji Film Ltd., Tokyo, Japan). Relative values of MMP-13 and TIMP-1 levelswere corrected for differences in specific activities and in the abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression (13).
Probes Complementary DNAs (cDNAs) of MMP-13 (14), TIMP-1 (15), TNF-a (16), TGF-fll (17), and GAPDH (18) were used. The cDNA of MMP-13 and TNF-c~ were generous gifts from Dr. Partridge and Dr. Decker, respectively. These cDNAs were labeled with 32P-dCTP using a random primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN, USA).
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Stat&tical analys& All densitometry values are reported as the mean ---SD. Statistical analysis was performed using Fisher's protected least significant difference, and p-values less than 0.05 were considered significant.
Results Histology CCl4-induced liver injury. A s p r e v i o u s l y r e p o r t e d , ball o o n i n g o f h e p a t o c y t e s a n d necrosis with i n f l a m m a t o r y cells o c c u r r e d a d j a c e n t to the central veins a n d in the lobules 6 h after CC14 i n t o x i c a t i o n , a n d z o n a l necrosis was o b s e r v e d at 2 d a y s (19). These signs o f t r a n s i e n t i n j u r y were i m p r o v e d at 5 days, a n d a l m o s t resolved at 7 days. Gal-N-induced liver injur): W i t h l o w - d o s e G a I - N intoxication, s p o t t y necrosis with i n f l a m m a t o r y cells was o b s e r v e d in the lobules 6 h after intoxication, as prev i o u s l y r e p o r t e d (20). T h e p e a k o f h e p a t i c necrosis was o b s e r v e d at 2 days. These signs o f t r a n s i e n t i n j u r y were i m p r o v e d at 5 days, a n d a l m o s t resolved at 7 days. W i t h h i g h - d o s e G a l - N i n t o x i c a t i o n , h e p a t i c necrosis was o b s e r v e d at 6 h a n d r a p i d l y p r o g r e s s e d until m a s s ive necrosis o c c u r r e d at 3 days. A l l rats died at 5 days, as p r e v i o u s l y d e s c r i b e d (20), a n d their livers showed massive necrosis.
Expression o f M M P - 1 3 and TIMP-1 m R N A CCl4-induced liver injury. In acute CC14-induced liver injury, M M P - 1 3 m R N A levels were t r a n s i e n t l y increased at 6 h (5.7-fold) a n d decreased at 2 days, b u t were b a r e l y d e t e c t a b l e in c o n t r o l livers. T I M P - 1 exp r e s s i o n was increased between 6 h (2.2-fold) a n d 3 d (2.1-fold) with a p e a k at 2 d a y s (4.3-fold) (Fig. l a , b). T h e p e a k o f T I M P - 1 e x p r e s s i o n o c c u r r e d after t h a t o f MMP-13. Gal-N-induced liver injury. W i t h low doses o f G a l - N , the p a t t e r n s o f M M P - 1 3 a n d T I M P - 1 expression were similar to t h o s e seen after CC14 intoxication. M M P - 1 3 m R N A expression increased between 6 h (1.5-fold) a n d 1 d a y (2.5-fold), with a p e a k at 1 day. T I M P - 1 m R N A expression increased between 6 h (1.5-fold) a n d 2 d a y s (2.3-fold), with a p e a k at 2 days, M M P - 1 3 m R N A was n o t d e t e c t e d in c o n t r o l liver (Fig. 2a, b). O n the o t h e r h a n d , in the h i g h - d o s e G a l - N m o d e l , m i n i m a l increased expression o f M M P - 1 3 was d e t e c t e d f r o m 6 h to 3 days c o m p a r e d with control. However, T I M P - 1 m R N A b e g a n increasing f r o m 6 h (1.5-fold) a n d also c o n t i n u e d to increase until death. T h e greatest increase in T I M P - 1 expression was 5.3-fold at 3 d a y s (Fig. 3a, b). T h e p e a k expression o f M M P - 1 3 was prec e d e d by t h a t o f T I M P - 1 in this lethal injury.
Expression o f TGF-fll and TNF-a T G F - f l l m R N A expression was increased f r o m 6 h until 3 d a y s in the CC14 m o d e l , with a p e a k at 2 days. In
MMP-13 and TIMP-1 in acute liver injury
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Fig. 1. Northern blot analysis in CCl4-induced liver injury. (a) Messenger RNA from rat livers obtained at 6 h, 12 h, and 1, 2, 3, 5, and 7 days was extracted and subjected to Northern analysis as described in Materials and Methods. Twenty micrograms of poly (A) ÷ RNA per lane were electrophoresed and the filters were hybridized with cDNA probes for MMP-13, TIMP-1, TGF-fll and TNF-a. These probes hybridized to mRNAs o f l . 8 kb, 0.9 kb, 2.5 kb, and 1.5 kb, respectively. MMP-13 mRNA increased transiently 6 h after injury and was decreased at 2 days. TIMP-1 mRNA expression increased between 6 h and 3 days with a peak at 2 days. TGF-fll mRNA increased transiently between 6 h and 3 days with a peak at 2 days. (b) Densitometric analysis using four individual rats per time point showed that relative mRNA levels of MMP-13 were increased up to 5.7-fold (6 h) compared with controls and then decreased. Relative mRNA levels of TIMP-1 were increased up to 4.3-foM (2 days) and then decreased. (mean±SD of four separate experiments). *p
the low-dose Gal-N model, TGF-fll mRNA expression was also increased with a peak at 1 day. In the high-
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Fig. 2. Northern blot analysis in low-dose Gal-N-induced liver injury, (a) Poly (A) + RNA (20 pg per lane) were electrophoresed and the filters were hybridized with cDNA probes for MMP-13, TIMP-1, TGF-fll and TNF-a. These probes hybridized to mRNAs o f l . 8 kb, 0.9 kb, 2.5 kb and 1.5 kb, respectively. MMP-13 mRNA expression increased between 6 h and 1 day, with a peak at 1 day. TIMP-1 mRNA expression increased between 6 h and 2 days, with a peak at 2 days. TGF-fll mRNA increased between 12 h and 2 days, with a peak at 1 day. TNF-ct mRNA increased transiently at 6 h. (b) Densitometric analysis using four individual rats showed that relative mRNA levels of MMP13 increased up to 2.5-fold (1 day) at the peak and then decreased. Relative mRNA levels of TIMP-1 increased up to 2.3-foM (2 days) and then decreased. (mean+_SD of four separate experiments). *p
dose GaI-N model, TGF-fll m R N A expression was increased from 12 h onward and continued to increase until death. This pattern of expression was similar to that of TIMP-1 (Fig. la, 2a, 3a). TNF-a m R N A expression was transiently increased at 6 h in the CC14 and low-dose GaI-N models. How421
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Fig. 3. Northern blot analysis in high-dose Gal-N-induced liver injury. (a) MMP-13 m R N A expression gradually increased from 12 h to 3 days with a peak at 3 days. TIMP1 m R N A expression also increased from 6 h to 3 clays, with a peak at 3 days. TGF-fll m R N A increased from 12 h to 3 days, with a peak at 2 day's. TNF-a m R N A increased transiently at 6 h. In this" model, all rats died after 5 day's. (b) Densitometric analysis using four individual rats demonstrated that relative levels of MMP-13 increased up to 1.4fold (3 days) at the peak, compared with control. Relative m R N A levels of TIMP-1 also increased up to 5.3-Jold of control levels (3 days). (mean +_SD of four separate experiments). *p
ever, in the high-dose Gal-N model its expression was dramatically increased by 6 h and continued to be detectable until death (Fig. 2a, 3a). Discussion
We examined the expressions of MMP-13 and TIMP1 in acute liver injury induced by CC14 and two differ422
ent doses of Gal-N. In spite of the use of different hepatotoxins, the patterns of expression of MMP-13 and TIMP-t were similar in transient liver injury induced by CC14 and low doses of Gal-N. MMP-13 mRNA levels increased transiently and the peak of TIMP-1 expression occurred after that of MMP-13. Recently, it was reported that rat collagenase has a high degree of functional and sequence homology with human collagenase-3, and that these enzymes belong to the collagenase-3 subfamily (MMP-13) and are distinct from human interstitial collagenase (MMP- 1) (1). It was also reported that MMP-13 play a significant role in the degradation and remodeling of ECM constituents (2,3). Moreover, active MMP-13 is specifically inhibited in a 1:1 stoichiometric fashion by the three homologous TIMPs (TIMP-1, -2, -3) (1). MMP-13 may be inhibited by a2-macroglobulin like other MMPs (21). Previous studies reported that matrix degradation occurring in the very early stages of liver regeneration after partial hepatectomy is due to plasminogen activator, which is important for hepatocyte proliferation and ECM remodeling (4,22,23). Current studies concerning burn wounds (24) and colonic anastomosis (25,26) showed simultaneous expression of collagenase and TIMP-1. These results suggest that collagenase and TIMP-1 play important roles in wound healing after burn injury or colonic anastomosis. Furthermore, increased collagenase activity during the early stages of cirrhosis is thought to be important for hepatic regeneration (27). Taking these results together, increased expression of MMP-13 may be important during liver repair after transient liver injury. In contrast, in fatal liver injury induced by high doses of Gal-N, the pattern of mRNA expression was different from that with CCI4 or low doses of Gal-N. MMP-13 mRNA levels increased but after the peak of TIMP-I expression. Expression of TIMP-1 was increased significantly compared to that of MMP-13, and its peak expression preceded that of MMP-13. Okada et at. have reported that MMPs and TIMPs are sequentially detected during normal wound healing in rat skin (28). These results may suggest that ordered regulation of MMP-13 and TIMP-1 is required for repair after acute liver injury. However, it is not clear that these changes in expression of these genes are seen in other models of transient or fatal hepatotoxicity. Further study is needed using other animal models to determine whether disordered expression is present in fatal liver injury. Additionally, MMPs except MMP-13 can degrade interstitial collagens (21,29). Besides TIMP-1, TIMP-2 and -3 inhibit MMP-13. Further studies including other MMP and
M M P - 1 3 and TIMP-1 in acute liver injury
TIMP families are also needed to show the real role of these genes in liver injuries. The present different patterns of MMP-13 and TIMP-1 gene expression may be induced by a network of inflammatory cytokines. In our fatal liver injury model, TGF-fll m R N A expression was detectable from 12 h onward and continued to increase until death, although its expression was transient in Cfl4 and low-dose Gal-N-induced liver injury. TGF-fll upregulates TIMP-1 expression as well as other inflammatory cytokines such as interleukin (IL)-lfl, IL-6, and IL-11 (30,31). In addition, TGF-fll inhibits proliferation of hepatocytes and other cells (32-35). Thus, TGF-fll may inhibit liver repair after fatal liver injury through its anti-proliferative actions. On the other hand, TN F - a was expressed transiently by 6 h and decreased rapidly in CC14 and Gal-N liver injury. Concerning the role of TNF-a in hepatic regeneration, it is known that T N F - a causes hepatic regeneration by induction of NF-xB (36,37). MMP-13 is also known to be up-regulated by TNF-a (2,38), in agreement with our findings. TNF-t~ may play an important role in hepatic regeneration after acute injury, as suggested by previous reports (36,37). Iredale et al. (39) also examined the expression of MMPs and TIMPs in acute CC14 liver injury. They reported that the expression of TIMP-1 increased gradually after intoxication, although interstitial collagenase m R N A was not significantly changed over time. However, they only examined gene expression 24 and 72 h after intoxication. Thus, they might not have detected the early interstitial collagenase response. As it has been reported that rat cells express only MMP-13 (1), interstitial collagenase, which was detected by Iredale et al. (39) in acute CC14 liver injury, might now be identified with MMP-13. In the present study we have shown a significant increase in TIMP-1 in fatal liver injury. It has been reported that serum concentrations of TIMP-1 are greatly increased in patients with fulminant hepatitis (40). Histologically, stellate cells proliferate in the livers of patients with massive and submassive hepatic necrosis due to fulminant hepatitis (41). In fact, TIMP1 is produced by cultured stellate cells as previously described (42). Herbst et al. also reported that TIMP1 m R N A was mainly expressed on stellate cells in fibrotic rat and human livers (43). Since TIMP-1 inhibits MMP-13 stoichiometrically (44), increased expression of TIMP-1 relative to MMP-13 may cause failure of liver repair. In our study we did not examine the cell types which produce MMP-13 and TIMP-1. The cell types which produce MMP-13 have not been determined yet.
Further study is needed to clarify the cell types which express MMP-13 in rat liver. On the other hand, TIMP-1 is produced by stellate cells (42), hepatocytes (45), bile duct cells and endothelial cells (46). However, the early expression of TIMP-1 after acute liver injury may originate from activated stellate cells (39). In conclusion, transiently increased expressions of MMP-13 and TIMP-1 were observed during repair after acute liver injury. Further study is needed to clarify the roles of MMP-13 and TIMP-1 using other models such as transgenic mice.
Acknowledgements We thank Drs. Partridge and Decker for the kind gift of cDNA probes of MMP-13 and TNF-a. We also thank Miss M. Maekawa for her technical assistance in preparing liver sections.
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