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Hepatic collagen synthesis and degradation during liver regeneration after partial hepatectomy
Hepatic Collagen Synthesis and Degradation During Liver Regeneration After Partial Hepatectomy HIROKO YAMAMOTO, YOSHIKAZU MURAWAKI, AND HIRONAKA KAWASAKI
To elucidate hepatic collagen metabolism during liver regeneration after partial hepatectomy, we measured collagen content, collagen synthesis, and collagen-degrading enzyme activity in the remnant livers of rats 3, 5, 7, and 14 days after a partial hepatectomy of 68%. Hepatic collagen synthesis was significantly higher 3, 5, and 7 days after partial hepatectomy than it was in sham-operated control rats, but there was no such difference 14 days after surgery, the maximal hepatic collagen synthesis being observed 5 days after surgery. Although the collagen concentration in the remnant liver was similar to that in the control liver, the total collagen content of the remnant liver increased rapidly with liver regeneration until 7 days after partial hepatectomy. Hepatic collagenase activity was similar to the control; however, hepatic cathepsin B and cathepsin L activity and the intracellular degradation of newly synthesized collagen were markedly decreased 3, 5, and 7 days after partial hepatectomy compared with the controls. Hepatic collagen synthesis was significantly and inversely correlated with cathepsin L activity and with the intracellular degradation of newly synthesized collagen. These findings suggest that a combination of increased collagen synthesis and decreased intracellular collagen degradation contributes to the rapid supply of collagen that is observed during the early phase of liver regeneration. (HEPATOLOGY1995;21:155-161.)
The liver has a remarkable capacity for regeneration. The r e m n a n t liver after partial hepatectomy is rapidly restored by the active proliferation of liver cells. The increase in hepatocyte DNA synthesis is observed 12 hours after 68% partial hepatectomy, and the peak mitotic activity in hepatocytes occurs 24 to 36 hours after hepatectomy, whereas that in littoral and Kupffer's cells occurs later. 13 Although the mechanism responsi-
Abbreviations: Hyp, 14C-hydroxyproline;BW, body weight; HEPES, N-[2hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]; FITC, fluorescein isothiocyanate; APMA, aminophenylmercuric acetate; EDTA, ethylenediaminetetraacetic acid; TGF-fl, transforming growth factor-Beta; mRNA, messenger RNA. From the Second Department of Internal Medicine, Tottori University School of Medicine, Yonago, Japan. Received April 5, 1994; accepted July 7, 1994. Address reprint requests to: Yoshikazu Murawaki, MD, Second Department of Internal Medicine, Tottori University School of Medicine, Yonago, 683, Japan. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2101-002553.00/0
ble for hepatocyte proliferation and the regulatory factors operating during liver regeneration have been investigated, there are few reports on extracellular matrix metabolism during liver regeneration after partial hepatectomy. Prolyl hydroxylase activity and the in vivo synthesis of collagen-bound '4C-hydroxyprohne (Hyp) are reported to be increased in regenerating liver after partial hepatectomy, 4'5 indicating that hepatic collagen synthesis is enhanced during liver regeneration. However, the sequential changes taking place in hepatic collagen degradation after partial hepatectomy have not yet been demonstrated. We performed this study to elucidate the sequential changes in hepatic collagen metabolism during liver regeneration after partial hepatectomy; to this end, we examined hepatic collagen synthesis and the hepatic activities of the collagen-degrading enzymes, collagenase, cathepsin B, and cathepsin L and the intracellular degradation of newly synthesized collagen in the remnant liver at 3, 5, 7, and 14 days after partial hepatectomy of 68% and sham operation in rats. MATERIALS AND METHODS Animals and Operation. Male Wistar rats were bred in specific pathogen-free facilities. All rats were humanely handled in accordance with the guidelines for animal experimentation by the Faculty of Medicine, Tottori University. During the study, they were housed in a room at constant temperature, with 12 hours each of light and darkness, and they had free access to a commercial balanced stock diet (CE-2; Clea Japan Inc., Tokyo, Japan) and to water. Twenty-five rats underwent the 68% partial hepatectomy of 68% and 22 rats underwent the sham operation. During the operation, the rats were anesthetized by intraperitoneal injection of 50 mg/ kg body weight (BW) pentobarbital (Nembutal; Dainippon Pharmaceutical Co., Tokyo, Japan). Partial hepatectomy was performed between 9 AM and 11 AM, according to the procedure of Higgins and Anderson. GThe median and left lateral lobes were removed. The sham operation was a laparotomy with gentle manipulation of the liver lobes. After the operation, the animals had free access to food and water, but were given only water during the 12 hours before the experiments. Three, 5, 7, and 14 days after surgery, rats were anesthetized with pentobarbital and weighed; they were then killed by bleeding from the abdominal aorta. The liver was perfused with cold saline solution via the aortic cannulation, after which it was excised and subjected to biochemical analysis. Assays for Hepatic Collagen Synthesis and Intracellular Degradation of Newly Synthesized Collagen. Hepatic collagen
155
156 YAMAMOTO,MURAWAKI,AND KAWASAKI synthesis and intracellular degradation of newly synthesized collagen were measured after 3-hour in vitro incubation of liver slices with 14C-proline, as previously described. 7's In brief, liver slices (1 g) were incubated with 5 pCi L-14C-proline (>250 mCi/mmol/L: Amersham Life Science, Tokyo, Japan) in 10 ml of Earle's balanced salt solution containing 0.2 mmol/ L of L-proline, 25 mmol/L of N-[2-hydroxyethyl]piperazineN'-[2-ethanesulfonic acid] (HEPES), 0.5 mmol/L of ferrous sulfate, and 0.5 mmol/L of ascorbic acid. Incubation was performed under an atmosphere of 95% 02 and 5% CO2 in a waterbath that was shaken at 37°C for 3 hours, and the reaction was terminated by the addition of absolute ethanol to give a final concentration of 67%. The liver slices were homogenized in the medium, which was centrifuged separating it into 67% ethanol-soluble and -insoluble fractions. The latter fraction was washed twice with 20 mL of 67% ethanol and each supernatant was combined with the first 67% ethanolsoluble fraction and then evaporated. The dried fractions were hydrolyzed in 10 ml of 6N HC1 for 24 hours at 105°C, dried by evaporation to remove the HC1, and dissolved in 20 ml of distilled water. Hydrolyzates were neutralized with 1N NaOH and lyophilized. To remove 14C-glutamate and 14C-aspartate produced from ~4C-proline during the incubation, the lyophilizate of ethanolsoluble fraction was dissolved in 4 ml of 1 mol/L of ammonium acetate, pH 6.8, and was applied to a column (1 × 30 cm) containing a water-washed Dowex l-X8 resin (Nippon BioRad Laboratories KK, Tokyo, Japan) in acetate form. The column was eluted with water, and the Hyp-containing fractions were pooled and lyophilized. Because the ~4C-proline metabolites were not used in protein synthesis, the 67% ethanol-insoluble fraction was not applied to the Dowex l-X8 column. To separate ~4C-Hyp from 14C-proline, the lyophilized Hypcontaining material from the 67% ethanol-soluble fraction and the lyophilized material from the 67% ethanol-insoluble fraction were dissolved in 2 ml of0.25N HC1, applied together with 0.1 ml of a mixture of 10 mmol/L of L-proline and 10 mmol/L of L-Hyp to a 1 × 30 cm Dowex 50W-X8 ion exchange column, and eluted with 0.25N HC1. The effluent fractions corresponding to Hyp were collected from fractions 60 to 80 (each fraction, 4 mL), and the radioactivity was measured in an Aqueous Counting Scintillant (ACS II; Amersham Life Science, Tokyo, Japan) with a liquid scintillation counter (Packard Tricarb 4640; Packard Instrument Co., Warrenville, IL). Collagen synthesis was expressed as the amount of ~4C-Hyp in the 67% ethanol-insoluble fraction, and the intracellular degradation of newly synthesized collagen was expressed as the amount of '4C-Hyp in the 67% ethanol-soluble fraction. A percentage of intracellular degradation of newly synthesized collagen was calculated as the amount of 14C-Hyp in the 67% ethanol-soluble fraction relative to the total amounts of ltC-Hyp in the 67% ethanol-soluble and -insoluble fractions. Assay far CoUagenase Activity. Hepatic collagenase activity was determined in solution, using fluorescein isothiocyanate (FITC)-labeled, acid-soluble, teloptide-free collagen (Cosmo-Bio Co. Ltd., Tokyo, Japan) as described previously. 9'~°The liver enzyme solution was prepared as follows: the liver specimen (approximately 200 mg) was cut into small pieces, placed in a beaker containing 100 mL of 50 mmol/L of Tris-HC1 and 0.15 mol/L of NaC1 at pH 7.5, and washed on the magnetic stirring apparatus six times, each time for 10 minutes; replacement of the wash solution was performed
HEPATOLOGYJanuary 1995 with the same buffer. One hundred milligrams of the liver specimen was then homogenized in 0.9 ml of 50 mmol/L of Tris-HC1, 0.2 mol/L of NaC1, 10 mmol/L of CaC12, 0.02% of Na azide, and 0.05% of Brij-35 at pH 7.5 (assay buffer). The latent collagenase was activated with aminophenylmercuric acetate (APMA) by incubating 0.1 mL of 10% liver homogenate at 35°C for 2 hours in a waterbath that was shaken with 60 #L of assay buffer and 40 #L of 5 mmol/L APMA solution; this served as the enzyme solution (0.2 mL). The enzyme assays were performed in duplicate with appropriate blanks. One volume of 0.1% FITC-labeled collagen, dissolved in 0.01 mol/L of acetic acid, was gently mixed with an equal volume of 100 mmol/L of Tris-HC1, 400 mmol/L of NaC1, 20 mmol/L of CaC12, and 0.04% of Na azide, pH 7.5, immediately before the enzyme assay. The resulting substrate solution (0.2 mL) was incubated with liver enzyme solution (0.2 mL) for 18 hours at 35°C in a waterbath that was shaken. The reaction was stopped with 10 #L of 80 mmol/ L of o-phenantroline in 50% ethanol, and the digestion products were extracted with 35% ethanol by adding 0.4 mL of 70% ethanol dissolved in 170 mmol/L of Tris-HC1, 670 mmol/ L of NaC1 at pH 9.5, followed by vigorous shaking. After centrifugation at 2,000g for 10 minutes at 4°C to precipitate the residual undigested collagen, the supernatant was assayed for fluorescein intensity, with excitation and emission wavelengths set at 495 nm and 520 nm, respectively (Hitachi Fluorescence Spectrophotometer F-4010, Tokyo, Japan). Blanks were prepared by incubating the enzyme in the presence of 2 mmol/L of o-phenantroline. The substrate FITClabeled collagen solution (0.2 mL) was mixed with 0.2 mL of assay buffer, 0.4 mL of 70% ethanol, and 10 #L of 80 mmol/L of o-phenantroline solution. The resulting solution was heatdenatured at 80°C for 10 minutes, and used as the standard solution for the assay of fluorescein intensity. Assays for Cathepsin B and L. Cathepsin B and L activity in the liver was determined according to the method of Barrett and Kirschke, 11as previously described. 12All assays were performed in duplicate. Liver specimens were homogenized in 9 vol of physiological saline solution and centrifuged at 2,000g for 10 minutes at 4°C. The supernatant was used as the enzyme solution and the enzyme reaction was performed as follows: for cathepsin B, 5 #L of the enzyme solution was diluted to 500 #L with 0.1% of Brij-35 solution; this was then added to 250 #L of 0.2 mol/L of phosphate buffer, pH 6.8, containing 1.33 mmol/L of disodium ethylenediaminetetraacetic acid (EDTA) and 2.7 mmol/L of cysteine (free base). The mixture was preincubated for 1 minute at 30°C, and then 250 #L of 0.5 mmol/L of Z-Arg-Arg-MCA (Peptide Research Foundation, Osaka, Japan) was added as the substrate. For cathepsin L, 5 pL of the enzyme solution was diluted to 500 #L with 0.1% of Brij-35 solution; this was then added to 250 #L of 0.4 mol/L of acetate buffer, pH 5.5, containing 4.0 mmol/ L of disodium EDTA and 40 mmol/L cysteine (monobase). The mixture was preincubated for 1 minute at 30°C, and then 250 #L of 0.5 mmol/L of Z-Phe-Arg-MCA (Peptide Research Foundation, Osaka, Japan) was added as the substrate. After a 10-minute incubation at 30°C, both reactions were terminated with 1 mL of the stopping reagent containing 100 mmol/L of sodium monochloro-acetate, 30 mmol/L of sodium acetate, and 70 mmol/L of acetic acid, pH 4.3. The fluorescence intensity of the resulting free MCA was determined with a fluorometric spectrophotometer (Hitachi Fluorescence Spectrophotometer F-4010, Tokyo, Japan) with excitation at 370 nm and emission at 460 nm.
YAMAMOTO, MURAWAKI, AND KAWASAKI 157
HEPATOLOGYVol. 21, No. 1, 1995 TABLE 1. Body and Liver Weights in Partial Hepatectomy and Sham Operation Groups Body Weight (g) Postoperative Days
3 days Sham PH 5 days Sham PH 7 days Sham PH 14 days Sham PH
No.
At Operation
At Necropsy
Liver Weight (g)
7 6
163 ± 51 167 +_50
163 ± 53 157 _+48
5.8 _+2.3 4.3 ± 1.1
5 6
168 ± 48 179 _+54
191 +_33 184 ± 39
6.9 +_0.9 5.8 ± 1.1
5 6
144 ± 27 152 ± 30
168 _+33 165 _+34
5.6 _+ 1.2 5.3 ± 0.8
5 7
118 _+27 133 _+41
192 ± 40 204 ± 40
6.6 + 1.8 7.0 _+ 1.9
percentage of intracellular degradation, it was 16% lower 3 days after partial hepat ect om y (% degradation; sham, 74%; pH, 62%), 26% lower at 5 days (% degradation; sham, 75%; pH, 56%), and 13% lower at 7 days (% degradation; sham, 82%; pH, 71%), a p a t t e r n similar to t h a t of the activity. There was no significant difference in intracellular degradation between the group t h a t u n d e r w e n t partial hepatectomy and the group t h a t u n d e r w e n t sham operation at 14 days after surgery. The nadir in intracellular deg~'adation occurred 5 days after partial hepatectomy. We examined the correlation of collagen synthesis and collagen degradation and found t h a t hepatic collagen synthesis was significantly inversely correlated with hepatic cathepsin L and with the intracellular degradation of newly synthesized collagen (Fig. 4).
NOTE. Data given as mean +_SD.
A
O t h e r Methods. Hepatic Hyp concentration was measured
by the method of Stegemann and Stadler. 1~ All data were expressed as mean +_ SD. The significance of differences was assessed by one-way analysis of variance, and correlation between variables was evaluated by linear regression analysis. RESULTS
There was no difference in body weight at necropsy between those t h a t u n d e r w e n t partial hepatectomy and those t h a t u n d e r w e n t sham operation. The remn a n t liver weight in the group t h a t u n d e r w e n t partial h e p atecto my was 74% of t h a t in the sham-operated group 3 days after surgery, 85% at 5 days, 95% at 7 days, and 106% at 14 days (Table 1). Although hepatic collagen concentration was not altered during liver regeneration, total collagen content in the r e m n a n t liver increased rapidly with liver regeneration (Fig. 1). Hepatic collagen synthesis was 33% higher at 3 days, 79% higher at 5 days, and 41% higher at 7 days after partial hepatectomy t h a n in each control, all these differences being significant. However, t h e re was no difference 14 days after surgery (Fig. 1). Maximal hepatic collagen synthesis was observed 5 days after partial hepatectomy. As shown in Fig. 2, hepatic collagenase activity in the r e m n a n t liver did not change during liver regeneration after partial hepatectomy. By contrast, hepatic cathepsin B activity was 17% lower t h a n t h a t in the control 3 days after partial hepatectomy, 22% lower at 5 days, 19% lower at 7 days, and 18% lower at 14 days. Hepatic cathepsin L activity was 56% lower at 3 days, 44% lower at 5 days, 31% lower at 7 days, and 12% lower at 14 days after partial hepatectomy. The nadir of hepatic cathepsin L activity was observed 3 days after surgery. The intracellular degradation of newly synthesized collagen was 24% lower t h a n t h a t in the control 3 days after partial hepatectomy, 37% lower at 5 days, and 28% lower at 7 days (Fig. 3). When expressed as a
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HEPATOLOGYJanuary 1995
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framework formation that occurs during the early phase of liver regeneration. Benjamin et al 4 examined the sequential changes in prolyl hydroxylase activity in the regenerating liver after partial hepatectomy and reported that increased prolyl hydroxylase activity was first noted at 24 hours, and then increased rapidly to the peak value of threefold that of the control, at 72 hours, after which it decreased. Rojkind et al 5 measured the in vivosynthesis of collagen-bound 14C-Hyp in regenerating liver, and showed that 14C-Hyp synthesis was increased fourfold compared with the control at 5 days posthepatectomy. In our present study, hepatic collagen synthesis, mea-
suredbydeterminingtheinvitrosynthesisofprotein-
bound 14C-hydroxyproline, was significantly increased by 30% a t 3 days, 50% a t 5 days, and 32% a t 7 days after partial hepatectomy compared with the control. Thus, it is clear that collagen synthesis in the regenerating liver is enhanced until 7 days after partial hepatectomy. The extracellular matrix consists of structural glycoproteins, such as laminin and fibronectin, and proteo-
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FIG. 2. Sequential changes in (A) hepatic collagenase, (B) cathepsin B, and (C) cathepsin L activity in the partial hepatectomy and the sham operation groups. *P < .05; **P < .01 compared with sham operation group. ©, sham operation group (sham); and •, partial hepatectomy group (PH).
DISCUSSION In general, liver regeneration after a partial hepatectomy is characterized by a burst of hepatocyte mitotic activity, beginning 1 day posthepatectomy and reaching a peak at 2 days. The consequence of this cell division is the formation of hepatocyte clusters without intervening sinusoids. By 6 days, sinusoids insinuate themselves into hepatocyte clusters, and by 8 days, the normal hepatic size and architecture is achieved. 14 In this study, we found that collagen synthesis increased significantly until 7 days after partial hepatectomy, whereas intracellular collagen degradation, including the activity of the collagenolytic cathepsins and the intracellular degradation of newly synthesized collagen, decreased significantly during this period. These findings suggest that a combination of increased collagen synthesis and decreased intracellular collagen degradation contributes to the rapid supply of collagen for
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HEPATOLOGYVol. 21, No. 1, 1995
YAMAMOTO, MURAWAKI, AND KAWASAKI 159
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3 days(sham:o,PH:e) 5 days(sham:o,PH:..) 7 days(sham:,:,,Pit,,) 14 days(sham:O,Pfl:e) FIG. 4. Correlation of hepatic collagen synthesis with (A) hepatic cathepsin L activity and (B) intracellular degradation of newly synthesized collagen in the partial hepatectomy and sham operation groups.
glycans, as well as collagens. It has been reported that, like collagen synthesis, the synthesis of glycoproteins and proteoglycans is enhanced during liver regeneration. Otsu et a115have demonstrated that proteoglycan synthesis in regenerating liver increases with time after partial hepatectomy, reaches a maximal level 3 to 5 days after operation, and decreases toward the control level by 10 days. The study by light microscopic immunohistochemistry has demonstrated that, although the staining intensity of collagens and fibronectin did not change during liver regeneration, laminin staining was dramatically changed./4 Laminin appeared in the hepatic sinusoids by 24 hours posthepatectomy, reached maximal staining intensity at 48 hours, and remained at high intensity until 6 days. These findings indicate that the synthesis of all extracellular matrix components increased during liver regeneration; the peak was 3 to 5 days after partial hepatectomy. Much attention has been focused on Ito cells as the extracellular matrix-producing cells in the liver; these cells have been found to produce a variety of matrix components, such as collagens, structural glycoproteins, and proteoglycans. 16 Tanaka et all7 have demonstrated in terms of the 5-bromo-2'-deoxyuridine (BrdUrd)-labeling index that DNA synthesis in Ito cells
peaks 48 hours after partial hepatectomy and decreases to that of the control on day 4 after operation. This observation is in agreement with the findings for the aH-thymidine labeling index in classical littoral cells, is The peak DNA synthesis in nonparenchymal cells occurs 24 hours later than that in hepatocytes during liver regeneration after partial hepatectomy and corresponds with the increase in extracellular matrix synthesis. Although it is still unclear which factors stimulate collagen synthesis during liver regeneration, several cytokines and growth hormones that are produced in the liver or are increased in the blood after partial hepatectomy are considered to be possible candidates. Of these factors, transforming growth factorBeta (TGF-B) is thought to be important in this regard because it is a strong stimulator of collagen synthesis 19 and the peak expression of TGF-/~ messenger RNA (mRNA) during liver regeneration occurs 48 to 72 hours after partial hepatectomy, 2° corresponding to the period of increased collagen synthesis. Together, collagenase, collagenolytic cathepsins, and the intracellular degradation of newly synthesized collagen are known as the collagen degradation system in the liver. 7'9 Collagenase, a secretory enzyme, extracellularly cleaves collagen molecules near neutral pH at specific loci in the polypeptide chain across the helix.
160
YAMAMOTO, MURAWAKI, AND KAWASAKI
Collagenolytic cathepsins degrade collagen molecules in acidic media at the N-terminal nonhelical region containing the intramolecular cross-link. There are four distinct cathepsins (B, L, N, and S), which differ in molecular weight and in the processes required for their extraction and purification; they also differ in their action on synthetic and protein substrates. 21 The intracellular degradation of newly synthesized collagen follows an intracellular degradation pathway in that the hydroxylated pro-~ chains and procollagens are degraded in lysosomes and/or the Golgi complex before secretion; this pathway seems to play an important part in the regulation of both the quality and the quantity of collagen. 22'23 In our present study, although we found no difference in hepatic collagenase activity between the group that underwent partial hepatectomy and the group that underwent sham operation, we did find that cathepsin B and cathepsin L activity, and the intracellular degradation of newly synthesized collagen decreased in the regenerating liver after partial hepatectomy. The nadir of cathepsin L activity and the intracellular degradation occurred 3 and 5 days, respectively, after operation. Suleiman et a124 have reported that the activity of cathepsin B1 and D decreased rapidly in regenerating liver after partial hepatectomy. Thus, it appears that cathepsin activity is decreased in the regenerating liver after partial hepatectomy. In this study, we have demonstrated for the first time that the intracellular degradation of newly synthesized collagen is also decreased in the regenerating liver after partial hepatectomy. Although the mechanism responsible for this degradation is unclear, it is known that the intracellular proteolysis of a newly synthesized protein, crinophagy, is closely regulated by the intracellular concentration of free amino acids. 25 Mortimore and POSO26'27 have shown that, of 20 amino acids metabolized in the liver, 8 amino acids (Leu, Phe, Tyr, Gln, Pro, Met, Try, and His) are active inhibitors for the intracellular proteolysis. We have previously observed sequential changes in serum concentrations of free amino acids in rats during liver regeneration after partial hepatectomy and found that serum concentrations of Pro, His, Tyr, and Met were significantly higher than those in the control group until 5 days posthepatectomyY These findings indicate that the increases in these amino acids may be responsible for the decrease in intracellular degradation of newly synthesized collagen during liver regeneration. In general, a net protein gain during liver regeneration can be attained by an increased rate of synthesis and/or by a decreased rate of protein degradation. 29 In fact, Preedy et al 3° have demonstrated that increases in protein synthesis in the regenerating liver are accompanied by reciprocal decreases in protein degradation after partial hepatectomy. We have also shown here that hepatic collagen synthesis was increased during liver regeneration, whereas intracellular collagen
HEPATOLOGYJanuary 1995
degradation was decreased at t h a t time. There was a significant inverse correlation between these findings, suggesting t h a t a combination of increased collagen
synthesis and decreased intracellular collagen degradation contributes to the rapid supply of collagen observed during the early phase of liver regeneration. REFERENCES 1. Karran S, McLaren M. Physical aspects of hepatic regeneration. In: Wright R, Millward-Sadler GH, Alberti KGMM, Karran S, eds. Liver and biliary disease. London: Saunders, 1985:233250. 2. Bucher NLR. Liver regeneration: an overview. J Gastroenterol Hepatol 1991;6:615-624. 3. Michalopoulos G. Liver regeneration and growth factors: old puzzles and new perspectives. Lab Invest 1992; 67:413-415. 4. Benjamin IS, Than T, Ryan S, Rodger MC, McGee J O'D, Blumgart LH. Prolyl hydroxylase and collagen biosynthesis in rat liver following varying degrees of partial hepatectomy. Br J Exp Pathol 1978;59:333-336. 5. Rojkind M, Rojkind MH, J Cordero-Hernandez J. In vivo collagen synthesis and deposition in fibrotic and regenerating rat livers. Collagen Rel Res 1983;3:335-347. 6. Higgins GM, Anderson RM. Experimental pathology of the liver: I. Restoration of the liver of the white rat following partial surgical removal. Arch Pathol 1931; 12:186-202. 7. Murawaki Y, Kate S, Koda M, Yamada M, Hirayama C. Hepatic collagen degradation system in the early stage of ethanol-induced liver injury. In: Hirayama C, Kivirikko KI, eds. Pathobiology of hepatic fibrosis. Amsterdam: Elsevier Science Publishers, 1985:121-129. 8. Koda M, Murawaki Y, Hirayama C. Free and small peptidebound ['4C]hydroxyproline synthesis in rat liver in vitro in CC14induced hepatic fibrosis. Biochem Biophys Res Commun 1988; 151:1128-1135. 9. Murawaki Y, Yamada S, Koda M, Hirayama C. Collagenase and collagenolytic cathepsin in normal and fibrotic rat liver. J Biochem 1990; 108:241-244. 10. Murawaki Y, Koda M, Yamada S, Kawasaki H, Shima H, Burkhardt H. Serum collagenase activity in patients with chronic liver disease. J Hepatol 1993; 18:328-334. 11. Barrett AJ, Kirschke H. Cathepsin B, cathepsin H, and cathepsin L. Methods Enzymol 1981;80:535-561. 12. Yamamoto H, Murawaki Y, Kawasaki H. Collagenolytic cathepsin B and L activity in experimental fibrotic liver and human liver. Res Commun Chem Pathol Pharm 1992;76:95-112. 13. Stegemmm H, Stadler K. Determination ofhydroxyproline. Clin Chim Acta 1967; 18:267-273. 14. Martinez-Hernandez A, Delgado FM, Amenta PS. The extracellular matrix in hepatic regeneration: localization of collagen types I, III, IV, laminin, and fibronectin. Lab Invest 1991; 64:157166. 15. Otsu K, Kate S, Ohtake K, Akamatsu N. Alteration of rat liver proteoglycans during regeneration. Arch Biochem Biophys 1992; 294:544-549. 16. Knittel T, Schuppan D, Meyer zum Btischenfelde K-H, Ramadori G. Differential expression of collagen types I, III, and IV by fatstoring (Ito) cells in vitro. Gastroenterology 1992;102:17241735. 17. Tanaka Y, Mak KM, Lieber CS. Immunohistochemical detection of proliferating lipocytes in regenerating rat liver. J Pathol 1990; 160:129-134. 18. Grisham JW. Morphologic study of deoxyribonucleic acid synthesis and cell proliferation in regenerating rat liver: autoradiography with thymidine-SH. Cancer Res 1962;22:842-849. 19. Lissoos TW, Davis BH. Pathogenesis of hepatic fibrosis and the role of cytokines. J Clin Gastroenterol 1992;15:64-68. 20. Fausto N, Mead JE, Gruppuso PA, Braun L. TGF-beta in liver development, regeneration, and carcinogenesis. Ann N Y Acad Sci 1990;593:231-242.
HEPATOLOGYVol. 21, No. 1, 1995 21. Maciewicz RA, Etherington DJ. A comparison of four cathepsins (B, L, N and S) with collagenolytic activity from rabbit spleen. Biochem J 1988;256:433-440. 22. Bienkowski RS. IntraceUular degradation of newly synthesized collagen. Collagen Rel Res 1984;4:399-412. 23. McAnulty RJ, Laurent GJ. Collagen synthesis and degradation in vivo: evidence for rapid rates of collagen turnover with extensive degradation of newly synthesized collagen in tissues of the adult rat. Collagen Rel Res 1987;7:93-104. 24. Suleiman SA, Jones GL, Singh H, Labreque DR. Changes in lysosomal cathepsins during liver regeneration. Biochim Biophys Acta 1980; 627:17-22. 25. Yamazaki K, LaRusso NF. The liver and intracellular digestion: how liver cells eat! HEPATOLOGY1989; 10:877-886. 26. Mortimore GE, P6s6 AR. Lysosomal pathways in hepatic protein
YAMAMOTO, MURAWAKI, AND KAWASAKI 161
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degradation: regulatory role of amino acids. Fed Proc 1984;43: 1289-1294. Mortimore GE, P6s6 AR. Intracellular protein catabolism and its control during nutrient deprivation and supply. Annu Rev Nutr 1987;7:539-564. Murawaki Y, Ikuta Y, Yamamoto H, Kawasaki H. Serum amino acid levels and hepatic protein synthesis during liver regeneration after partial hepatectomy in rats. Res Commun Chem Pathol Pharm 1992; 77:43-54. Scornik OA, Botbol V. Role of changes in protein degradation in the growth of regenerating livers. J Biol Chem 1976;251:28912897. Preedy VR, Paska L, Sugden PH. Protein synthesis in liver and extra-hepatic tissues after partial hepatectomy. Biochem J 1990; 267:325-330.
To elucidate hepatic collagen metabolism during liver regeneration after partial hepatectomy, we measured collagen content, collagen synthesis, and collagen-degrading enzyme activity in the remnant livers of rats 3, 5, 7, and 14 days after a partial hepatectomy of 68%. Hepatic collagen synthesis was significantly higher 3, 5, and 7 days after partial hepatectomy than it was in sham-operated control rats, but there was no such difference 14 days after surgery, the maximal hepatic collagen synthesis being observed 5 days after surgery. Although the collagen concentration in the remnant liver was similar to that in the control liver, the total collagen content of the remnant liver increased rapidly with liver regeneration until 7 days after partial hepatectomy. Hepatic collagenase activity was similar to the control; however, hepatic cathepsin B and cathepsin L activity and the intracellular degradation of newly synthesized collagen were markedly decreased 3, 5, and 7 days after partial hepatectomy compared with the controls. Hepatic collagen synthesis was significantly and inversely correlated with cathepsin L activity and with the intracellular degradation of newly synthesized collagen. These findings suggest that a combination of increased collagen synthesis and decreased intracellular collagen degradation contributes to the rapid supply of collagen that is observed during the early phase of liver regeneration. (HEPATOLOGY1995;21:155-161.)
The liver has a remarkable capacity for regeneration. The r e m n a n t liver after partial hepatectomy is rapidly restored by the active proliferation of liver cells. The increase in hepatocyte DNA synthesis is observed 12 hours after 68% partial hepatectomy, and the peak mitotic activity in hepatocytes occurs 24 to 36 hours after hepatectomy, whereas that in littoral and Kupffer's cells occurs later. 13 Although the mechanism responsi-
Abbreviations: Hyp, 14C-hydroxyproline;BW, body weight; HEPES, N-[2hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]; FITC, fluorescein isothiocyanate; APMA, aminophenylmercuric acetate; EDTA, ethylenediaminetetraacetic acid; TGF-fl, transforming growth factor-Beta; mRNA, messenger RNA. From the Second Department of Internal Medicine, Tottori University School of Medicine, Yonago, Japan. Received April 5, 1994; accepted July 7, 1994. Address reprint requests to: Yoshikazu Murawaki, MD, Second Department of Internal Medicine, Tottori University School of Medicine, Yonago, 683, Japan. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2101-002553.00/0
ble for hepatocyte proliferation and the regulatory factors operating during liver regeneration have been investigated, there are few reports on extracellular matrix metabolism during liver regeneration after partial hepatectomy. Prolyl hydroxylase activity and the in vivo synthesis of collagen-bound '4C-hydroxyprohne (Hyp) are reported to be increased in regenerating liver after partial hepatectomy, 4'5 indicating that hepatic collagen synthesis is enhanced during liver regeneration. However, the sequential changes taking place in hepatic collagen degradation after partial hepatectomy have not yet been demonstrated. We performed this study to elucidate the sequential changes in hepatic collagen metabolism during liver regeneration after partial hepatectomy; to this end, we examined hepatic collagen synthesis and the hepatic activities of the collagen-degrading enzymes, collagenase, cathepsin B, and cathepsin L and the intracellular degradation of newly synthesized collagen in the remnant liver at 3, 5, 7, and 14 days after partial hepatectomy of 68% and sham operation in rats. MATERIALS AND METHODS Animals and Operation. Male Wistar rats were bred in specific pathogen-free facilities. All rats were humanely handled in accordance with the guidelines for animal experimentation by the Faculty of Medicine, Tottori University. During the study, they were housed in a room at constant temperature, with 12 hours each of light and darkness, and they had free access to a commercial balanced stock diet (CE-2; Clea Japan Inc., Tokyo, Japan) and to water. Twenty-five rats underwent the 68% partial hepatectomy of 68% and 22 rats underwent the sham operation. During the operation, the rats were anesthetized by intraperitoneal injection of 50 mg/ kg body weight (BW) pentobarbital (Nembutal; Dainippon Pharmaceutical Co., Tokyo, Japan). Partial hepatectomy was performed between 9 AM and 11 AM, according to the procedure of Higgins and Anderson. GThe median and left lateral lobes were removed. The sham operation was a laparotomy with gentle manipulation of the liver lobes. After the operation, the animals had free access to food and water, but were given only water during the 12 hours before the experiments. Three, 5, 7, and 14 days after surgery, rats were anesthetized with pentobarbital and weighed; they were then killed by bleeding from the abdominal aorta. The liver was perfused with cold saline solution via the aortic cannulation, after which it was excised and subjected to biochemical analysis. Assays for Hepatic Collagen Synthesis and Intracellular Degradation of Newly Synthesized Collagen. Hepatic collagen
155
156 YAMAMOTO,MURAWAKI,AND KAWASAKI synthesis and intracellular degradation of newly synthesized collagen were measured after 3-hour in vitro incubation of liver slices with 14C-proline, as previously described. 7's In brief, liver slices (1 g) were incubated with 5 pCi L-14C-proline (>250 mCi/mmol/L: Amersham Life Science, Tokyo, Japan) in 10 ml of Earle's balanced salt solution containing 0.2 mmol/ L of L-proline, 25 mmol/L of N-[2-hydroxyethyl]piperazineN'-[2-ethanesulfonic acid] (HEPES), 0.5 mmol/L of ferrous sulfate, and 0.5 mmol/L of ascorbic acid. Incubation was performed under an atmosphere of 95% 02 and 5% CO2 in a waterbath that was shaken at 37°C for 3 hours, and the reaction was terminated by the addition of absolute ethanol to give a final concentration of 67%. The liver slices were homogenized in the medium, which was centrifuged separating it into 67% ethanol-soluble and -insoluble fractions. The latter fraction was washed twice with 20 mL of 67% ethanol and each supernatant was combined with the first 67% ethanolsoluble fraction and then evaporated. The dried fractions were hydrolyzed in 10 ml of 6N HC1 for 24 hours at 105°C, dried by evaporation to remove the HC1, and dissolved in 20 ml of distilled water. Hydrolyzates were neutralized with 1N NaOH and lyophilized. To remove 14C-glutamate and 14C-aspartate produced from ~4C-proline during the incubation, the lyophilizate of ethanolsoluble fraction was dissolved in 4 ml of 1 mol/L of ammonium acetate, pH 6.8, and was applied to a column (1 × 30 cm) containing a water-washed Dowex l-X8 resin (Nippon BioRad Laboratories KK, Tokyo, Japan) in acetate form. The column was eluted with water, and the Hyp-containing fractions were pooled and lyophilized. Because the ~4C-proline metabolites were not used in protein synthesis, the 67% ethanol-insoluble fraction was not applied to the Dowex l-X8 column. To separate ~4C-Hyp from 14C-proline, the lyophilized Hypcontaining material from the 67% ethanol-soluble fraction and the lyophilized material from the 67% ethanol-insoluble fraction were dissolved in 2 ml of0.25N HC1, applied together with 0.1 ml of a mixture of 10 mmol/L of L-proline and 10 mmol/L of L-Hyp to a 1 × 30 cm Dowex 50W-X8 ion exchange column, and eluted with 0.25N HC1. The effluent fractions corresponding to Hyp were collected from fractions 60 to 80 (each fraction, 4 mL), and the radioactivity was measured in an Aqueous Counting Scintillant (ACS II; Amersham Life Science, Tokyo, Japan) with a liquid scintillation counter (Packard Tricarb 4640; Packard Instrument Co., Warrenville, IL). Collagen synthesis was expressed as the amount of ~4C-Hyp in the 67% ethanol-insoluble fraction, and the intracellular degradation of newly synthesized collagen was expressed as the amount of '4C-Hyp in the 67% ethanol-soluble fraction. A percentage of intracellular degradation of newly synthesized collagen was calculated as the amount of 14C-Hyp in the 67% ethanol-soluble fraction relative to the total amounts of ltC-Hyp in the 67% ethanol-soluble and -insoluble fractions. Assay far CoUagenase Activity. Hepatic collagenase activity was determined in solution, using fluorescein isothiocyanate (FITC)-labeled, acid-soluble, teloptide-free collagen (Cosmo-Bio Co. Ltd., Tokyo, Japan) as described previously. 9'~°The liver enzyme solution was prepared as follows: the liver specimen (approximately 200 mg) was cut into small pieces, placed in a beaker containing 100 mL of 50 mmol/L of Tris-HC1 and 0.15 mol/L of NaC1 at pH 7.5, and washed on the magnetic stirring apparatus six times, each time for 10 minutes; replacement of the wash solution was performed
HEPATOLOGYJanuary 1995 with the same buffer. One hundred milligrams of the liver specimen was then homogenized in 0.9 ml of 50 mmol/L of Tris-HC1, 0.2 mol/L of NaC1, 10 mmol/L of CaC12, 0.02% of Na azide, and 0.05% of Brij-35 at pH 7.5 (assay buffer). The latent collagenase was activated with aminophenylmercuric acetate (APMA) by incubating 0.1 mL of 10% liver homogenate at 35°C for 2 hours in a waterbath that was shaken with 60 #L of assay buffer and 40 #L of 5 mmol/L APMA solution; this served as the enzyme solution (0.2 mL). The enzyme assays were performed in duplicate with appropriate blanks. One volume of 0.1% FITC-labeled collagen, dissolved in 0.01 mol/L of acetic acid, was gently mixed with an equal volume of 100 mmol/L of Tris-HC1, 400 mmol/L of NaC1, 20 mmol/L of CaC12, and 0.04% of Na azide, pH 7.5, immediately before the enzyme assay. The resulting substrate solution (0.2 mL) was incubated with liver enzyme solution (0.2 mL) for 18 hours at 35°C in a waterbath that was shaken. The reaction was stopped with 10 #L of 80 mmol/ L of o-phenantroline in 50% ethanol, and the digestion products were extracted with 35% ethanol by adding 0.4 mL of 70% ethanol dissolved in 170 mmol/L of Tris-HC1, 670 mmol/ L of NaC1 at pH 9.5, followed by vigorous shaking. After centrifugation at 2,000g for 10 minutes at 4°C to precipitate the residual undigested collagen, the supernatant was assayed for fluorescein intensity, with excitation and emission wavelengths set at 495 nm and 520 nm, respectively (Hitachi Fluorescence Spectrophotometer F-4010, Tokyo, Japan). Blanks were prepared by incubating the enzyme in the presence of 2 mmol/L of o-phenantroline. The substrate FITClabeled collagen solution (0.2 mL) was mixed with 0.2 mL of assay buffer, 0.4 mL of 70% ethanol, and 10 #L of 80 mmol/L of o-phenantroline solution. The resulting solution was heatdenatured at 80°C for 10 minutes, and used as the standard solution for the assay of fluorescein intensity. Assays for Cathepsin B and L. Cathepsin B and L activity in the liver was determined according to the method of Barrett and Kirschke, 11as previously described. 12All assays were performed in duplicate. Liver specimens were homogenized in 9 vol of physiological saline solution and centrifuged at 2,000g for 10 minutes at 4°C. The supernatant was used as the enzyme solution and the enzyme reaction was performed as follows: for cathepsin B, 5 #L of the enzyme solution was diluted to 500 #L with 0.1% of Brij-35 solution; this was then added to 250 #L of 0.2 mol/L of phosphate buffer, pH 6.8, containing 1.33 mmol/L of disodium ethylenediaminetetraacetic acid (EDTA) and 2.7 mmol/L of cysteine (free base). The mixture was preincubated for 1 minute at 30°C, and then 250 #L of 0.5 mmol/L of Z-Arg-Arg-MCA (Peptide Research Foundation, Osaka, Japan) was added as the substrate. For cathepsin L, 5 pL of the enzyme solution was diluted to 500 #L with 0.1% of Brij-35 solution; this was then added to 250 #L of 0.4 mol/L of acetate buffer, pH 5.5, containing 4.0 mmol/ L of disodium EDTA and 40 mmol/L cysteine (monobase). The mixture was preincubated for 1 minute at 30°C, and then 250 #L of 0.5 mmol/L of Z-Phe-Arg-MCA (Peptide Research Foundation, Osaka, Japan) was added as the substrate. After a 10-minute incubation at 30°C, both reactions were terminated with 1 mL of the stopping reagent containing 100 mmol/L of sodium monochloro-acetate, 30 mmol/L of sodium acetate, and 70 mmol/L of acetic acid, pH 4.3. The fluorescence intensity of the resulting free MCA was determined with a fluorometric spectrophotometer (Hitachi Fluorescence Spectrophotometer F-4010, Tokyo, Japan) with excitation at 370 nm and emission at 460 nm.
YAMAMOTO, MURAWAKI, AND KAWASAKI 157
HEPATOLOGYVol. 21, No. 1, 1995 TABLE 1. Body and Liver Weights in Partial Hepatectomy and Sham Operation Groups Body Weight (g) Postoperative Days
3 days Sham PH 5 days Sham PH 7 days Sham PH 14 days Sham PH
No.
At Operation
At Necropsy
Liver Weight (g)
7 6
163 ± 51 167 +_50
163 ± 53 157 _+48
5.8 _+2.3 4.3 ± 1.1
5 6
168 ± 48 179 _+54
191 +_33 184 ± 39
6.9 +_0.9 5.8 ± 1.1
5 6
144 ± 27 152 ± 30
168 _+33 165 _+34
5.6 _+ 1.2 5.3 ± 0.8
5 7
118 _+27 133 _+41
192 ± 40 204 ± 40
6.6 + 1.8 7.0 _+ 1.9
percentage of intracellular degradation, it was 16% lower 3 days after partial hepat ect om y (% degradation; sham, 74%; pH, 62%), 26% lower at 5 days (% degradation; sham, 75%; pH, 56%), and 13% lower at 7 days (% degradation; sham, 82%; pH, 71%), a p a t t e r n similar to t h a t of the activity. There was no significant difference in intracellular degradation between the group t h a t u n d e r w e n t partial hepatectomy and the group t h a t u n d e r w e n t sham operation at 14 days after surgery. The nadir in intracellular deg~'adation occurred 5 days after partial hepatectomy. We examined the correlation of collagen synthesis and collagen degradation and found t h a t hepatic collagen synthesis was significantly inversely correlated with hepatic cathepsin L and with the intracellular degradation of newly synthesized collagen (Fig. 4).
NOTE. Data given as mean +_SD.
A
O t h e r Methods. Hepatic Hyp concentration was measured
by the method of Stegemann and Stadler. 1~ All data were expressed as mean +_ SD. The significance of differences was assessed by one-way analysis of variance, and correlation between variables was evaluated by linear regression analysis. RESULTS
There was no difference in body weight at necropsy between those t h a t u n d e r w e n t partial hepatectomy and those t h a t u n d e r w e n t sham operation. The remn a n t liver weight in the group t h a t u n d e r w e n t partial h e p atecto my was 74% of t h a t in the sham-operated group 3 days after surgery, 85% at 5 days, 95% at 7 days, and 106% at 14 days (Table 1). Although hepatic collagen concentration was not altered during liver regeneration, total collagen content in the r e m n a n t liver increased rapidly with liver regeneration (Fig. 1). Hepatic collagen synthesis was 33% higher at 3 days, 79% higher at 5 days, and 41% higher at 7 days after partial hepatectomy t h a n in each control, all these differences being significant. However, t h e re was no difference 14 days after surgery (Fig. 1). Maximal hepatic collagen synthesis was observed 5 days after partial hepatectomy. As shown in Fig. 2, hepatic collagenase activity in the r e m n a n t liver did not change during liver regeneration after partial hepatectomy. By contrast, hepatic cathepsin B activity was 17% lower t h a n t h a t in the control 3 days after partial hepatectomy, 22% lower at 5 days, 19% lower at 7 days, and 18% lower at 14 days. Hepatic cathepsin L activity was 56% lower at 3 days, 44% lower at 5 days, 31% lower at 7 days, and 12% lower at 14 days after partial hepatectomy. The nadir of hepatic cathepsin L activity was observed 3 days after surgery. The intracellular degradation of newly synthesized collagen was 24% lower t h a n t h a t in the control 3 days after partial hepatectomy, 37% lower at 5 days, and 28% lower at 7 days (Fig. 3). When expressed as a
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158
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HEPATOLOGYJanuary 1995
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framework formation that occurs during the early phase of liver regeneration. Benjamin et al 4 examined the sequential changes in prolyl hydroxylase activity in the regenerating liver after partial hepatectomy and reported that increased prolyl hydroxylase activity was first noted at 24 hours, and then increased rapidly to the peak value of threefold that of the control, at 72 hours, after which it decreased. Rojkind et al 5 measured the in vivosynthesis of collagen-bound 14C-Hyp in regenerating liver, and showed that 14C-Hyp synthesis was increased fourfold compared with the control at 5 days posthepatectomy. In our present study, hepatic collagen synthesis, mea-
suredbydeterminingtheinvitrosynthesisofprotein-
bound 14C-hydroxyproline, was significantly increased by 30% a t 3 days, 50% a t 5 days, and 32% a t 7 days after partial hepatectomy compared with the control. Thus, it is clear that collagen synthesis in the regenerating liver is enhanced until 7 days after partial hepatectomy. The extracellular matrix consists of structural glycoproteins, such as laminin and fibronectin, and proteo-
14
FIG. 2. Sequential changes in (A) hepatic collagenase, (B) cathepsin B, and (C) cathepsin L activity in the partial hepatectomy and the sham operation groups. *P < .05; **P < .01 compared with sham operation group. ©, sham operation group (sham); and •, partial hepatectomy group (PH).
DISCUSSION In general, liver regeneration after a partial hepatectomy is characterized by a burst of hepatocyte mitotic activity, beginning 1 day posthepatectomy and reaching a peak at 2 days. The consequence of this cell division is the formation of hepatocyte clusters without intervening sinusoids. By 6 days, sinusoids insinuate themselves into hepatocyte clusters, and by 8 days, the normal hepatic size and architecture is achieved. 14 In this study, we found that collagen synthesis increased significantly until 7 days after partial hepatectomy, whereas intracellular collagen degradation, including the activity of the collagenolytic cathepsins and the intracellular degradation of newly synthesized collagen, decreased significantly during this period. These findings suggest that a combination of increased collagen synthesis and decreased intracellular collagen degradation contributes to the rapid supply of collagen for
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HEPATOLOGYVol. 21, No. 1, 1995
YAMAMOTO, MURAWAKI, AND KAWASAKI 159
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3 days(sham:o,PH:e) 5 days(sham:o,PH:..) 7 days(sham:,:,,Pit,,) 14 days(sham:O,Pfl:e) FIG. 4. Correlation of hepatic collagen synthesis with (A) hepatic cathepsin L activity and (B) intracellular degradation of newly synthesized collagen in the partial hepatectomy and sham operation groups.
glycans, as well as collagens. It has been reported that, like collagen synthesis, the synthesis of glycoproteins and proteoglycans is enhanced during liver regeneration. Otsu et a115have demonstrated that proteoglycan synthesis in regenerating liver increases with time after partial hepatectomy, reaches a maximal level 3 to 5 days after operation, and decreases toward the control level by 10 days. The study by light microscopic immunohistochemistry has demonstrated that, although the staining intensity of collagens and fibronectin did not change during liver regeneration, laminin staining was dramatically changed./4 Laminin appeared in the hepatic sinusoids by 24 hours posthepatectomy, reached maximal staining intensity at 48 hours, and remained at high intensity until 6 days. These findings indicate that the synthesis of all extracellular matrix components increased during liver regeneration; the peak was 3 to 5 days after partial hepatectomy. Much attention has been focused on Ito cells as the extracellular matrix-producing cells in the liver; these cells have been found to produce a variety of matrix components, such as collagens, structural glycoproteins, and proteoglycans. 16 Tanaka et all7 have demonstrated in terms of the 5-bromo-2'-deoxyuridine (BrdUrd)-labeling index that DNA synthesis in Ito cells
peaks 48 hours after partial hepatectomy and decreases to that of the control on day 4 after operation. This observation is in agreement with the findings for the aH-thymidine labeling index in classical littoral cells, is The peak DNA synthesis in nonparenchymal cells occurs 24 hours later than that in hepatocytes during liver regeneration after partial hepatectomy and corresponds with the increase in extracellular matrix synthesis. Although it is still unclear which factors stimulate collagen synthesis during liver regeneration, several cytokines and growth hormones that are produced in the liver or are increased in the blood after partial hepatectomy are considered to be possible candidates. Of these factors, transforming growth factorBeta (TGF-B) is thought to be important in this regard because it is a strong stimulator of collagen synthesis 19 and the peak expression of TGF-/~ messenger RNA (mRNA) during liver regeneration occurs 48 to 72 hours after partial hepatectomy, 2° corresponding to the period of increased collagen synthesis. Together, collagenase, collagenolytic cathepsins, and the intracellular degradation of newly synthesized collagen are known as the collagen degradation system in the liver. 7'9 Collagenase, a secretory enzyme, extracellularly cleaves collagen molecules near neutral pH at specific loci in the polypeptide chain across the helix.
160
YAMAMOTO, MURAWAKI, AND KAWASAKI
Collagenolytic cathepsins degrade collagen molecules in acidic media at the N-terminal nonhelical region containing the intramolecular cross-link. There are four distinct cathepsins (B, L, N, and S), which differ in molecular weight and in the processes required for their extraction and purification; they also differ in their action on synthetic and protein substrates. 21 The intracellular degradation of newly synthesized collagen follows an intracellular degradation pathway in that the hydroxylated pro-~ chains and procollagens are degraded in lysosomes and/or the Golgi complex before secretion; this pathway seems to play an important part in the regulation of both the quality and the quantity of collagen. 22'23 In our present study, although we found no difference in hepatic collagenase activity between the group that underwent partial hepatectomy and the group that underwent sham operation, we did find that cathepsin B and cathepsin L activity, and the intracellular degradation of newly synthesized collagen decreased in the regenerating liver after partial hepatectomy. The nadir of cathepsin L activity and the intracellular degradation occurred 3 and 5 days, respectively, after operation. Suleiman et a124 have reported that the activity of cathepsin B1 and D decreased rapidly in regenerating liver after partial hepatectomy. Thus, it appears that cathepsin activity is decreased in the regenerating liver after partial hepatectomy. In this study, we have demonstrated for the first time that the intracellular degradation of newly synthesized collagen is also decreased in the regenerating liver after partial hepatectomy. Although the mechanism responsible for this degradation is unclear, it is known that the intracellular proteolysis of a newly synthesized protein, crinophagy, is closely regulated by the intracellular concentration of free amino acids. 25 Mortimore and POSO26'27 have shown that, of 20 amino acids metabolized in the liver, 8 amino acids (Leu, Phe, Tyr, Gln, Pro, Met, Try, and His) are active inhibitors for the intracellular proteolysis. We have previously observed sequential changes in serum concentrations of free amino acids in rats during liver regeneration after partial hepatectomy and found that serum concentrations of Pro, His, Tyr, and Met were significantly higher than those in the control group until 5 days posthepatectomyY These findings indicate that the increases in these amino acids may be responsible for the decrease in intracellular degradation of newly synthesized collagen during liver regeneration. In general, a net protein gain during liver regeneration can be attained by an increased rate of synthesis and/or by a decreased rate of protein degradation. 29 In fact, Preedy et al 3° have demonstrated that increases in protein synthesis in the regenerating liver are accompanied by reciprocal decreases in protein degradation after partial hepatectomy. We have also shown here that hepatic collagen synthesis was increased during liver regeneration, whereas intracellular collagen
HEPATOLOGYJanuary 1995
degradation was decreased at t h a t time. There was a significant inverse correlation between these findings, suggesting t h a t a combination of increased collagen
synthesis and decreased intracellular collagen degradation contributes to the rapid supply of collagen observed during the early phase of liver regeneration. REFERENCES 1. Karran S, McLaren M. Physical aspects of hepatic regeneration. In: Wright R, Millward-Sadler GH, Alberti KGMM, Karran S, eds. Liver and biliary disease. London: Saunders, 1985:233250. 2. Bucher NLR. Liver regeneration: an overview. J Gastroenterol Hepatol 1991;6:615-624. 3. Michalopoulos G. Liver regeneration and growth factors: old puzzles and new perspectives. Lab Invest 1992; 67:413-415. 4. Benjamin IS, Than T, Ryan S, Rodger MC, McGee J O'D, Blumgart LH. Prolyl hydroxylase and collagen biosynthesis in rat liver following varying degrees of partial hepatectomy. Br J Exp Pathol 1978;59:333-336. 5. Rojkind M, Rojkind MH, J Cordero-Hernandez J. In vivo collagen synthesis and deposition in fibrotic and regenerating rat livers. Collagen Rel Res 1983;3:335-347. 6. Higgins GM, Anderson RM. Experimental pathology of the liver: I. Restoration of the liver of the white rat following partial surgical removal. Arch Pathol 1931; 12:186-202. 7. Murawaki Y, Kate S, Koda M, Yamada M, Hirayama C. Hepatic collagen degradation system in the early stage of ethanol-induced liver injury. In: Hirayama C, Kivirikko KI, eds. Pathobiology of hepatic fibrosis. Amsterdam: Elsevier Science Publishers, 1985:121-129. 8. Koda M, Murawaki Y, Hirayama C. Free and small peptidebound ['4C]hydroxyproline synthesis in rat liver in vitro in CC14induced hepatic fibrosis. Biochem Biophys Res Commun 1988; 151:1128-1135. 9. Murawaki Y, Yamada S, Koda M, Hirayama C. Collagenase and collagenolytic cathepsin in normal and fibrotic rat liver. J Biochem 1990; 108:241-244. 10. Murawaki Y, Koda M, Yamada S, Kawasaki H, Shima H, Burkhardt H. Serum collagenase activity in patients with chronic liver disease. J Hepatol 1993; 18:328-334. 11. Barrett AJ, Kirschke H. Cathepsin B, cathepsin H, and cathepsin L. Methods Enzymol 1981;80:535-561. 12. Yamamoto H, Murawaki Y, Kawasaki H. Collagenolytic cathepsin B and L activity in experimental fibrotic liver and human liver. Res Commun Chem Pathol Pharm 1992;76:95-112. 13. Stegemmm H, Stadler K. Determination ofhydroxyproline. Clin Chim Acta 1967; 18:267-273. 14. Martinez-Hernandez A, Delgado FM, Amenta PS. The extracellular matrix in hepatic regeneration: localization of collagen types I, III, IV, laminin, and fibronectin. Lab Invest 1991; 64:157166. 15. Otsu K, Kate S, Ohtake K, Akamatsu N. Alteration of rat liver proteoglycans during regeneration. Arch Biochem Biophys 1992; 294:544-549. 16. Knittel T, Schuppan D, Meyer zum Btischenfelde K-H, Ramadori G. Differential expression of collagen types I, III, and IV by fatstoring (Ito) cells in vitro. Gastroenterology 1992;102:17241735. 17. Tanaka Y, Mak KM, Lieber CS. Immunohistochemical detection of proliferating lipocytes in regenerating rat liver. J Pathol 1990; 160:129-134. 18. Grisham JW. Morphologic study of deoxyribonucleic acid synthesis and cell proliferation in regenerating rat liver: autoradiography with thymidine-SH. Cancer Res 1962;22:842-849. 19. Lissoos TW, Davis BH. Pathogenesis of hepatic fibrosis and the role of cytokines. J Clin Gastroenterol 1992;15:64-68. 20. Fausto N, Mead JE, Gruppuso PA, Braun L. TGF-beta in liver development, regeneration, and carcinogenesis. Ann N Y Acad Sci 1990;593:231-242.
HEPATOLOGYVol. 21, No. 1, 1995 21. Maciewicz RA, Etherington DJ. A comparison of four cathepsins (B, L, N and S) with collagenolytic activity from rabbit spleen. Biochem J 1988;256:433-440. 22. Bienkowski RS. IntraceUular degradation of newly synthesized collagen. Collagen Rel Res 1984;4:399-412. 23. McAnulty RJ, Laurent GJ. Collagen synthesis and degradation in vivo: evidence for rapid rates of collagen turnover with extensive degradation of newly synthesized collagen in tissues of the adult rat. Collagen Rel Res 1987;7:93-104. 24. Suleiman SA, Jones GL, Singh H, Labreque DR. Changes in lysosomal cathepsins during liver regeneration. Biochim Biophys Acta 1980; 627:17-22. 25. Yamazaki K, LaRusso NF. The liver and intracellular digestion: how liver cells eat! HEPATOLOGY1989; 10:877-886. 26. Mortimore GE, P6s6 AR. Lysosomal pathways in hepatic protein
YAMAMOTO, MURAWAKI, AND KAWASAKI 161
27. 28.
29. 30.
degradation: regulatory role of amino acids. Fed Proc 1984;43: 1289-1294. Mortimore GE, P6s6 AR. Intracellular protein catabolism and its control during nutrient deprivation and supply. Annu Rev Nutr 1987;7:539-564. Murawaki Y, Ikuta Y, Yamamoto H, Kawasaki H. Serum amino acid levels and hepatic protein synthesis during liver regeneration after partial hepatectomy in rats. Res Commun Chem Pathol Pharm 1992; 77:43-54. Scornik OA, Botbol V. Role of changes in protein degradation in the growth of regenerating livers. J Biol Chem 1976;251:28912897. Preedy VR, Paska L, Sugden PH. Protein synthesis in liver and extra-hepatic tissues after partial hepatectomy. Biochem J 1990; 267:325-330.