- Email: [email protected]
EFFECTS OF NEUROTROPIC PYRIMIDINE HETEROCYCLIC COMPOUND, MS-430, ON CULTURED HEPATIC PARENCHYMAL AND STELLATE CELLS
Life Sciences, Vol. 61, No. 3, pp.273-2S2,1997 @@@t 01997 Ekvier Science Inc. Printed in the USA. All rights reserved 0324-3205/97$17.CO+ a)
PII S0024-3205(97)00383-4 ~••ŒÀÓ«•ˆ••Œ4M¨• •w•æ
EFFECTS OF NEUROTROPIC PYRIMIDINE HETEROCYCLIC COMPOUND, MS-430, ON CULTURED HEPATIC PARENCHYMALAND STELLATE CELLS Mie Inaol, Satoshi Mouhidal, Hitoshi Ikedal~, Akira Awaya3and Keqji Fujiwaral
Depar@ent of Internal Medicine, Saitalna Medical School) 38 Morohongb, Moroyama-cho, Iruma-gun, Saitama 350-04, ZFirstDepartment of I~ternal Medicine, Faculty of Medicine, University of Tokyo? 7+3-1 Hongo, Bunkyo-ku, Tokyo 113, jInstitute of $iologim] Science, Mitsui Pharmaceuticals?Inc.? 3-12-2 Nihonbashi,Chuo-ku,Tokyo 103, Japan. IThird
~(Receivedia finalformApril 14, 1~
Summary MS-430 is a novel sy@hetic pyrimidine derivative that stimulates regeneration of the nerve as a promo~er for various growth factors such as epidermal growth factor (EGF) and ne~e growth factor, and differentiation of astrocytes. The ef– fects of MS-430 on the liver were tested using hepatocytes and stellate cells in primary culture isolatep from rats. MS-430 enhanced EGF-induced DNA synthesis in hepatocytes w~ile it alone failed to increase the basal DNA synthesis. Albumin mRNA expression in the cells and its amount in the medium were not changed by addition $ifEGF or MS–430 alone or both. Basic fibroblast growth factor (bFGF) increas~d DNA and but not collagen synthesis by hepatic stellate cells. Addition of M!?+430 inhibited DNA synthesis by hepatic stellate cells at either presence or ab$ence of bFGF, and collagen synthesis at the presence of bFGF. However, MS~430 had no effects on basal or bFGF-stimulated TGF~ mRNA expression in the cells. These results suggest that MS–430 stimulated proliferation of hepat~cytes as a comitogen for EGF without affecting albumin synthesis, and suppressed proliferation of activated hepatic stellate cells and their collagen synthesis wit~out affecting TGF~ expression. @ Wordr: hepatoeyte,hepaticst@ate ceu liver regeneration% livertibrosis,MS-W
Liver failure occurs as acu{e and chronic types. Acute liver failure often takes a fatal course with multiple organ failure (1). Since massive or submissive liver necrosis is commonly seen in this disease (2), sufficient li~er regeneration is required for survival. However, impaired liver regeneration seems to exist u~der this state, as hepatocyte growth factor (HGF) in the serum is markedly increased in patientp with acute liver failure (3,4). Thus, the use of agents capable of enhancing the action of growth factors for hepatocytes might be a therapeutic strategy for such liver failure. Chronic liver failure develops as a consequence of progressive liver fibrosis due to repetitive necrosis and inflammation (5). Hepatic stellate cells are known to proliferate and produce extracellular matrix components in this process (6), suggesting that the suppression of these functions of hepatic stel~atecells may be therapeutic for this type of liver failure. CorrespondingAuthor: Kenji~Fujiwara,M.D., Ph.D. ThirdDepartment of InternalMedicine, Saitama Medical School, 38 Morohongo,Moroyama-cho, Iruma–gun,Saitama 350–04, Japan. Tel:81-492-76-1198, Fax:81-492-94-8404
274
MS-430on LiverCells
Vol. 61, No. 3, 1997
MS-430, 2-piperodino–5,6 -dihydro-7-methy l-6-oxo (7H) pyrrolo [2,3–d] pyrimidine maleate is a novel synthetic pyrimidine heterocyclic compound that has a variety of actions on peripheral nerves and neuron cells (7-9). MS-430 can promote regeneration of rat sciatic nerve following dissection (7) and differentiation of astrocytes in culture (8). Also, MS-430 is reported to promote neurite sprouting of PC–12 cells, pheochromocytoma-derived cell lines, when stimulated by nerve growth factor (NGF) or epidermel growth factor (EGF) (9), suggesting that MS430 can act as a growth promoting agent on epithelial cells with the receptors for these growth factors. Hepatic stellate cells are pericytes like astrocytes (10). These observations prompted us to test whether MS-430 can act similarly on hepatocytes and hepatic stellate cells. The present paper reports on the effects of MS-430 on rat hepatocytes and hepatic stellate cells in primary culture. Its possibility as a therapeutic candidate for liver failure is also discussed. Materials and Methods Animals Male Sprague-Dawley rats (Charles River Japan, Atsugi, Japan) were housed in a room at 22 t 20C under nomal laboratory li@ting conditions and maintained on a commercial Pelleted ‘iet and water ad libitum. Rats weighing 190-210 g and 290-310 g were subjected to the isolation of hepatocytes and hepatic stellate cells, respectively. All protocols confirmed to National Research Council criteria for human care of animals, and all in vitro experiments were repeated at least 3 times. Preparation of &lS-430 MS–430 (Fig 1) was synthesized as previously reported (8) by Mr. Ikuo Tomino et al. at Synthetic Chemistry and Bioscience Laboratories, Mitsui Petrochemical Industries, Ltd., Tokyo, Japan.
o COOH If
2-piperidino-5,6-dihydro-7-methyl-6-oxo pyrrolo [2,3-d] pyrimidine maleate
(7H)
Fig 1 The Chemical Structure of MS-430. Isolation of Hepatocytes and Hepatic Stelkzte CelLs Hepatocytes were isolated by Seglen’smethod (11). Briefly, the liver was minced after perfusion through the portal vein with 0.05Y0collagenase (type I: Worthington Biochemical Corp., Freefold, NJ) in Hanks’ balanced salt solution (HBSS: Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) at a flow rate of 30 mL/min for 15 min at 37”C. Hepatocytes were separated from nonparenchymal cells by centrifusion at 28g for 2 min at 4“C. Hepatic stellate cells were isolated by the method of Knook et a/. (12) with partial modifica– tions. After liver perfusion with 0.05% collagenase and 0.1% pronase E (Kaken Pharmaceutical Co. Ltd., Urayasu, Japan) in HBSS at a flow rate of 10 mL/min for 15 and 10 rein, respectively, at 37”C, the liver was minced and incubated with HBSS containing both enzymes at 37°C for 30 min. The resultant cells were collected after filtration through a nylon mesh. The recovered cells were overlaid on double–layered metrizamide (Sigma Chemical Company, St. Louis, MO) solu-
Vol. 61, No. 3, 1997
MS-430on LiverCells
275
tions at concentrations of IYZOand 18%, and centrifuged at 1400g for 17 min at room temperature. Hepatic stellate cells were collected from the top layer of 13% metrizamide solution. The viability rates of the collected hepatocytes and hepatic stellate cells were constantly higher than 90%, as tested by trypan blue exclusion. The purity of hepatocytes immediately after isolation and hepatic stellate cells 6 days after incubation on plastic dishes were 90% or more as– sessed by light microscopic examination and immunostaining of desmin (13), respectively. Experimental Design Experiment I: The isolated hepatocytes were suspended in Williams’ medium E (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) containing 10% fetal calf serum (FCS: Gibco Laborato– ries Ltd., Life Technologies Inc., Grand Island, NY) heat–inactivated at 56°C for 30 rein, 10-6M insulin (Sigma Chemical Company) and 10-5 M dexamethasone (Sipa Chemical Company). They were seeded on plastic dishes at a density of 5.0 x 104cells/cm and incubated at 37°C in an atmosphere of 5% C02 and 95% air for 2 hr. Following removal of non-adherent cells by washing with Hank’s balanced salt solution without Ca2+and Mg2+(HBSS(-); Gibco Laboratory Ltd.), adherent hepatocytes were further cultured in Williams’ medium E containing 10% heatinactivated FCS with addition of Oor 50 ng/mL of EGF (Toyobo Co. Ltd., Osaka, Japan) and MS-430 at various concentrations at 24 hr after plating. At 24 hr after MS-430 addition, the medium was collected for the determination of lactate dehydrogenase (LDH) activity and albumin concentration. The hepatocytes were subjected to Northern blot analysis of albumin mRNA and their DNA contents were determined. In another series of experiments, similarly prepared hepatocytes were subjected to the determination of DNA synthesis and protein synthesis in the cells. The same experiments were repeated 3 times. Experiment II: Isolated hepatic stellate cells were suspended in Dulbecco’s Modified Eagle Medium (DMEM: Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) containing 10% heat-inactivated FCS and seeded on plastic dishes at a density of 1.25 x 105cells/cm2.They were incubated for 6 days similarly as was done in hepatocytes. During this period, non–adherent cells were removed by washing with HBSS(-) every 2 days. On 6th day, Oor 10 ng/mL of basic fibroblast growth factor (bFGF: Toyobo Co. Ltd.) and various concentrations of MS-430 were added to the medium and the cells were further cultured. At 24 hr later, the medium was collected for the determination of LDH activity. The cells were subjected to Western and Northern blot analyses of smooth muscle a actin and transforming growth factor (TGF) ~, respectively and their DNA contents were determined. [n another series of experiments, syntheses of DNA, protein and collagen by the cells were determined at 24 hr after MS-430 addition. The same experiments were repeated 3 times. Evaluation of Cell Killing Cytotoxicity of MS-430 was evaluated as cell killing measured by LDH activity in the medium and DNA contents of adherent cells. LDH activity was determined according to the method of Wioblewski et al. (14), which meas– ures the breakdown of pyruvate (Sigma Chemical Company) to lactate detected as the increase in absorbance at 340 nm following addition of b–NADH (Sigma Chemical Company) to the culture medium. Cell killing was expressed as the percentage of LDH activity in the medium to total LDH activity in non-MS-430-treated cells when the cell membrane of those cells was de– stroyed by addition of 0.5% Triton X (Wako Pure Chemical Industries Ltd., Osaka, Japan). For the determination of DNA contents in adherent cells, the cells in dishes were washed 3 times with saline to remove non-adherent cells. The adherent cells were solubilized in 0.1 N NaOH. An aliquot of the cell solution was collected, and kept at 60”C for 45 minutes with the same volume of 0.4 glmL diaminobenzoic acid (Aldrich Chemical Company Inc., Milwaukee, WI) solution in water. DNA contents in the aliquot were measured with a fluorescence spectrophotometer (F-2000, Hitachi, Tokyo, Japan) as previously reported (15). A4easurementofAlbumin Concentration Albumin concentration in the medium was measured by enzyme-linked irnmunosorbent assay as follows. In each well of a 96-well assay plate (Becton Dickinson Labware, Lincoln Park,
276
MS-430on LiverCells
Vol. 61, No. 3, 1997
NJ), 50~g of polyclonal goat IgG antibody against rat albumin (Cappel Research Products, Durham, NC) was distributed as 500 p#mL solution in phosphate buffered saline (PBS), pH 7.4, kept overnight at 4“C, and removed by suction. After 4 washes with 10% Block Ace (blocking agent derived from skim milk; Snow Brand Milk Products Co., Tokyo, Japan) in PBS, pH 7.4, the wells were filled with 400 ~L of 70% Block Ace in PBS, pH 7.2, and kept overnight at 4°C. After removing the solution, the wells were washed 4 times with 10% Block Ace in PBS, pH 7.2 (10% BA). Then, 100 ~L of assay sample and the same volume of 10% BA were added to each well, kept overnight at 4“C, and removed by suction. After 8 washes with 10YoBA, 5 pg of peroxidase-conjugated polyclonal sheep IgG antibody against rat albumin (Cappel Research Products) was distributed as 50 @mL solution in 10% BA, kept overnight at 4“C, and removed by suction. After 8 washes with 10% BA, the wells were added with 200 pL of 2,2’-azino-di(3-ethylbenzthiazoline sulfonate) solution (Boehlinger Mannheim Biochemical, Germany), and kept at room temperature for 30 min. Then, 100 pL of 0.25 M oxalic acid was added to the solution, and the optical density of the solution was determined at 410 nm with an automated microplate reader (MR 5000; Dynatech Laboratories Inc., Chantilly, VA). Measurement of DNA Synthesis At 2 hr before cell harvest, 2 pCi/mL of [methyl-3 H]–thymidine (New England Nuclear, Boston, MA) was added to the culture medium of hepatocytes and hepatic stellate cells. The cells were washed with cold PBS, immersed in 10’%trichloroacetic acid at 4°C for 10 rein, and solubilized in 1 N NaOH at 37°C for 1 hr. An aliquot of the cell solution was collected, and treated with 100Yotrichloroacetic acid at 4“C. The resultant precipitate was further treated with 10% trichloroacetic acid, and solubilized in 0.1 N NaOH. Then, the radioactivity of the solution was measured by liquid scintillation counter. Measurement of Protein Synthesis At 24 hr before cell harvest, 1 pCi/mL of L-[3,4,5-3H]-leucine (New England Nuclear) was added to the culture medium of hepatocytes and hepatic stellate cells. The cells were washed, immersed and solubilized. Then, an aliquot of cell solution was treated with 100!ZO trichloroacetic acid similarly as was done for the measurement of DNA synthesis. The precipitate was further treated with 107otrichloroacetic acid, incubated at 70”C for 10 min and put on ice for 10 min. The resultant precipitate was solubilized in 0.1 N NaOH, and the radioactivity of the solution was measured. Measurement of Collagen Synthesis At 24 hr before cell harvest, 10 ~Ci/mL of L-[2,3-3H]-proline (New England Nuclear), 0.1 mM of ascorbic acid and 0.5 mM ~-aminopropionitrile were added to the culture medium of hepatic stellate cells. At harvest, the medium was removed and the cells were solubilized in 1 N NaOH. The medium and cell solution were precipitated with 10% trichloroacetic acid. The pellet was digested with purified bacterial collagenase (Form III, Advance Biofactures, Lymbrook, NY) at 37°C for 90 rein, and precipitated with trichloroacetic acid and tannic acid. The radioac– tivity in the supematant collected by centrifugation was measured, and collagen synthesis by the cells was expressed as total radioactivity of the supematant obtained from the medium and cells. Northern Blot Analysis of Albumin and TGFf! Total RNA was extracted from cultured hepatocytes and hepatic stellate cells by homogenization in guanidine thiocyanate and centrifugation through phenollchloroform (16). Ten pg of total RNA was subjected to electrophoresis on 1.62% agarose-formaldehyde gel, blotted to nylon filters (Hybond-N; Amersham International Plc., Little Chalfont, England) by upward capillary transfer, and fixed to filters using UV crosslinker (UVP Inc., Upland, CA). cDNA fragments of rat albumin (a gift from Dr. Yasufumi Kaneda through Japanease Cancer Research Resources Bank-Gene, Tokyo, Japan) or rat TGF@(17) were radiolabeled with [a-32P]dCTP (New England Nuclear) using Megaprime DNA labeling system (Amersham International PIc.). Prehybridized filters were hybridized at 42°C for 16 hr with sZp_labeledcDNA fragments, then washed under high-stringency conditions and exposed to XAR film (Eastman Kodak Company, Rochester, NY). Western Blot Analysis of Smooth Muscle aActin After discarding the medium, hepatic stellate cells were incubated in 0.5% sodium dodecyl
277
MS-430on LiverCells
Vol. 61, No. 3, 1997
sulfate (SDS), 60 mM Tris–HCl, pH 6.8 and 100 mM dithiothreitol, boiled for 5 rein, and destroyed by sonication. Samples containing the same amount of protein were separated by SDS polyacrylamide gel electrophoresis (12% acrylamide) under reducing conditions. The gel was electrophoretically transferred onto a nitrocellulose using Trans–Blot SD Semi–Dry Electrophoretic Transfer Cell (Bio Rad, Richmond, CA), exposed to monoclinal antibody against mouse smooth muscle a actin (BioMakor, Rehovot, Israel), and incubated with alkaline phospha– tase conjugated rabbit antibody against IgG (Boehringer Mannheim Biochemical).Then, the filters were washed and developed in 5–bromo-4–chloro–3 -indolyl phosphate/nitro blue tetrazoli– urn solution. Results The baselines of the results ranged in 3 repeated experiments, but the relations among groups were almost similar in each experiment. The following results are those in one of the 3 experiments. Cytotoxicity of MS-430 for Ilepatocytes and Hepatic Stellate Cells in Primary Culture As shown in TABLE I, si~ificant cell killing by MS-430 assessed from LDH activity in the medium was not observed at 0.1 mM or less in both hepatocytes and hepatic stellate cells irres– pective of the presence of EGF or bFGF. However, MS–430 at 1 mM killed most hepatocytes, but much less hepatic stellate cells. The similar results were obtained by trypan blue exclusion test (data not shown). Also, DNA contents in both adherent hepatocytes and stellate cells were not changed by addition of EGF, bFGF and MS-430 at concentrations of 0.1 mM or less. TABLE I Effects of MS-430 on Cell Killing of Cultured Rat Hepatocytes and Hepatic Stellate Cells MS-430 concentration in the medium (mM)
Hepatocytes
Stellate cells
EGF addition (ng/mL) 50 o
bFGF addition (ng/mL) o 10
. . . Actlwty l~m
[fya o)
0 0.001 0.01 0.1 1.0
35.955.3 34.7 ? 6.0 39.4 ? 5.3 32.4 ? 5.4 68.3 ? 21.2*
29.6 ? 2.0 30.7 ? 2.0 31.916.1 26.0 ? 8.2 89.8 ? 33.0”
12.7 t 15.9 ? 17.6 t 15.9 f 22.2 ?
0 0.001 0.01 0.1 1.0
46.3 ? 0.6 46.9 f 0.4 43.5 t 4.1 45.4 ? 1.3 41.4 ? 3.6”
45.0 t 1.2 48.4 ? 0.8 44.9 ? 3.0 43.4 ? 3.6 34.6 ? 3.2”
33.0 ? 1.3 31.9 ? 2.7 29.8 ? 1.2 29.1 t 1.8 23.3 ? 1.2**
2.8 2.8 2.8 2.8 5.5*
12.2 t 4.lb 17.6 ? 2.4 16.3 ~ 0.0 14.9 f 2.3 21.7 ? 2.3$”
35.3 ? 3.2b 35.2 ? 3.5 34.2 z 1.6 33.924.1 22.3 ~ 1,8”
‘The percent of LDH activity in the medium to that in cell lysate as described in Materials and Methods. bMean ? SD of 3 wells. *pcO.1,**p
Vol. 61, No. 3>1997
MS-430on LiverCells
278
H). Albumin mRNA expression in the cells was also unchanged (Fig 2). When MS–430 was added at 0.01 mM to the medium concomitantly with EGF addition, DNA synthesis by hepatocytes was significantly increased compared to controls (TABLE II.).However, the similar change was not seen in hepatocytes cultured without EGF addition. Cellular protein synthesis and albumin concentration in the medium were not changed by MS-430 at this concentration irrespective of the presence of EGF (TABLE II). The same result was also observed in albumin-mRNA expression (Fig 2). TABLE II Effects of MS-430 on DNA, Protein and Albumin Syntheses in Cultured Hepatocytes
MS-430 concentration in the medium (mM)
DNA synthesis (104dpm/well)
Albumin concentration in the medium (w#mL)
EGF concentration (ng/mL) o
0 0.001 0.01 0.1
Protein synthesis (105dpm/well)
3.75? 4.01~ 3.88? 2.53?
50 0.17 0.42 0.36 0.58
0
24.62? 2.69” 2.48 f 26.85* 1.78$ 2.44? 31.48? 2.53*#2.29? 22.41* 2.53* 2.28 ?
50 0.28 0.56 0.22 0.31
3.93? 3.82? 3.47? 3.39 ?
0.60” 0.30” 0.21’ 0.36”
50
0 1.o5 ? 0.90? 1.21 z 0.83?
0.27 0.08 0.37 0.02
1.092 0.85 z 1.70 ? 0.85 z
0.23a 0.09 0.82 1.12
‘Mean * SD of 3 wells. *p
~EGF
Ong/mLv
EGF 50 rig/m< - 28S
a)
- 18S - 28S
b)
=18S
o 10-310-2
10-1
0
10-3 10-210-1
Doses of MS-430(mM) Fig 2 Albumin mRNA Expression in Hepatocytes in Primary Culture. a) Northern blotting, b) Ethidium bromide staining.
MS-43(Ion LiverCells
Vol. 61, No. 3, 1997
279
Effects of MS-430 on the Function of Hepatt”cStellate Cel&in Pn”maryCulture bFGF at 10 ng/mL significantly increased DNA synthesis, but not protein synthesis and collagen synthesis by hepatic stellate cells compared to controls (TABLE III). MS-430 decreased DNA synthesis by the cells in a dose-related manner irrespective of the presence of bFGF. The similar dose-related attenuation by MS-430 was seen in protein synthesis by the cells cultured without bFGF and in collagen synthesis by the cells cultured with bFGF (TABLE III). As shown in Fig 3, bFGF decreased smooth muscle a actin expression in the cells. This decrease was significantly attenuated by addition of MS-430 at 0.001 to 0.1 mM. In the cells cul– tured without bFGF, however, MS–430 did not change smooth muscle a actin expression. bFGF significantly increased TGF~-mRNA expression in the cells (Fig 4). MS-430 did not change this increase as well as TGF(3-mRNA expression in the absence of bFGF. TABLE HI Effects of MS-430 on DNA, Protein and Collagen Syntheses in Cultured Hepatic Stellate Cells
MS-430 concentration in the medium (mM)
DNA synthesis (105dprnAvell)
Collagen Synthesis (104dprn/well)
bFGF concentration (nglmL) o
0 0.001 0.01 0.1
Protein synthesis (105dprn/well)
1.91t 1.58t 1.39? 0.52?
10 0.26 4.29? 0.02 3.70? 0.07# 3.972 0.23# 1,99*
0
0.69” 1.43 ? 0.49” 1.39? 0.11’ 1.23? 0.17*#1.06 ?
10 0.13 1.62? 0.06 1.54? 0.86 1.49? 0.33#1.39 ?
10
0
0.19 3.52 z 0.12 3.25? 0.08$ 3.20? 0.07” 3.18?
0.29 4.12 z 0.08 4.12? 0.59 3.45 ? 0.11#2.84 ?
0.48a 0.67 0.27 0.23#
Qne way analysis of ANOVA p value
<0.01
<0.01
<0.01
0.25
0.44
<0.05
aMean t SD of 3 wells. *p<0,05vs bFGF at Ong/mL, #p
Vol. 61, No. 3, 1997
MS-43(Ion LiverCells
280
factors for hepatocytes and hepatic stellate cells, respectively, in the present experiments. As a marker of hepatocyte differentiation, albumin synthesis, specific function of mature hepatocytes, was evaluated from its concentration in the medium and cellular mRNA expression. Collagen synthesis and smooth muscle a actin expression were measured in hepatic stellate cells as markers of activation.(5) Also, cellular TGFfl expression was determined, since this cytokine regulates both proliferation and activation of the stellate cells in an autocrine manner (19, 20). DNA, protein and collagen syntheses were expressed per well, since DNA contents of hepatocytes and stellate cells were not affected by EGF, bFGF and MS-430 at concentrations of 0.1 mM or less (TABLE I). As shown in TABLE II, DNA synthesis by cultured hepatocytes was markedly increased by addition of EGF, and this increase was further enhanced by concomitant addition of MS-430 at 0.01 mM. However, the enhancement was not seen in hepatocytes in the absence of EGF, suggesting that MS–430 acted as a comitogen for EGF. The concentration of MS-430 was 10 times lower than that reported to be effective for stimulating astrocytes and PC 12 cells (8,9). MS-430 at this concentration did not affect protein synthesis and albumin synthesis by the hepatocytes. It is well known that cell proliferation and differentiation progress inversely (21). From the therapeutic viewpoint, growth factors that stimulate hepatocyte proliferation might decrease the function of mature hepatocytes. In this point, MS-430 may be a promising agent as a comitogen for hepatocytes.
o
10-310-210-1 0
10-310-210-1
Doses of MS-430 (mM) Fig 3 Smooth Muscle a Actin Expression in Hepatic Stellate Cells in Primary Culture.
~ bFGF Orig/m+
bFGF 10 ng/mL=
28s
a)
18S 28S
b)
18S
o
10”310-2
10-1
0 10-3 10-210-1
Doses of MS-430 (mM) Fig 4 TGFfl Expression in Hepatic Stellate Cells in Primary Culture. a) Northern blotting, b) Ethidium bromide staining.
Vol. 61, No. 3, 1997
MS-430on LiverCells
’281
bFGF significantly increased DNA synthesis by hepatic stellate cells without affecting protein synthesis and collagen synthesis, as shown in TABLE III. MS-430 reduced DNA synthesis by the cells in a dose-related manner irrespective of the presence of bFGF. Also, MS-430 suppressed collagen synthesis by the cells in the presence of bFGF. Hepatic stellate cells are known to be activated by platelet derived growth factor, bFGF (22) and TGFf3(23) to produce a large amount of extracellular matrix components such as collagen. Hepatic stellate cells used in the present experiments were those cultured for 6 days which are considered to be already activated and produce bFGF that can act on themselves in an autocrine manner (24). Thus, it would be reasonable to assume that MS-430 inhibited not only the action of exogenous bFGF to stimulate proliferation of hepatic stellate cells, but also the activation by endogenous bFGF. Such actions of MS-430 would be important as a therapeutic agent for liver fibrosis as well. This possibility should be investigated in future in relation to cell proliferation and cellular expression of procol– lagen mRNA expression. To clarify the mode of action of MS-430 on hepatic stellate cells, smooth muscle a actin and TGF~ expressions in the cells were evaluated, because smooth muscle a actin is a marker of stellate cell activation in vivo (25), and TGF~ is the strongest stimulator of collagen synthesis by hepatic stellate cells (26). Isolated stellate cells are reported to increase smooth muscle a actin expression with increased syntheses of DNA and collagen during culture on plastic dishes (27). However, as shown in Fig 3, smooth muscle a actin expression was decreased in the cells following stimulation by bFGF, suggesting that the expression is not always a marker of hepatic stellate cell activation in vitro. As shown in Fig 4, bFGF increased TGF~ expression in the cells. Considering that smooth muscle a actin expression was decreased in the cells by addition of TGF~ (28), such a decrease in smooth muscle a actin expression is likely to result from in– creased expression of [email protected] did not affect TGFfJexpression in the cells irrespective of the presence of bFGF. However, it significantly attenuated smooth muscle a actin expression, and decreased collagen synthesis stimulated by bFGF. These results may strongly suggest that MS-430 acted on hepatic stellate cells stimulated by bFGF independently of the action of TGF~. In conclusion, MS-430 may stimulate proliferation of hepatocytes as a comitogen for EGF without affecting albumin synthesis. Also, MS-430 may suppress proliferation and collagen synthesis by hepatic stellate cells, MS–430 merits consideration for assessment in vivo as a therapeutic candidate for liver injury that can stimulate liver regeneration and attenuate liver fibrosis. Acknowledgement We thank Ms. Kiyoko Naiki for technical assistance. References 1. J.G.O’GRADY,B.PORTWN and R.WILLIAMS, ~ea.se oft~, Seventh Edition,L.Schiff and E.R.Schiff(Eds)?1077-1090, Lippincott Company, Philadelphia (1993). m Lwer Dzseases,H.Popper and F.Schaffner (Eds), 2. H.POPPER, D.KEPPLER, E2Q+ZWM 209-235, Grune and Stratton, Oriando (1986). 3. H.TSUBOUCHI, S.HIRONO, E.GOHDA, H.NAKAYAMA,K.TAKAHASHI, O.SAKIKIYAMA, H.MIYAZAKI,J.SUGIHARA, E.TOMITA, Y.MUTO, Y.DAIKUHARA and S.HASHIMOTO, Hematology.9875-881 (1989). 4. T.TOMIYA, S.NAGOSHI and K.FUJIWARA,Hematology.M 1-4 (1992). 5. S.L.FRIEDMAN, N.Engl.J.Med. ~ 1828-1835 (1993). 6. S.L.FRIEDMAN, F.J.ROLL, J.BOYLES and M.BISSELL, Proc.Natl.Acad.Sci.USA. 82 8681-8685 (1985). 7. M.OCHI, M.NODA, K.NAKAMITSU, M.DEIE, Y.IKUTA,Y.MAKIand A.AWAYA, Gen.Phannac. 2659-64 (1995). 8. Y.KOYAMA,A.AWAYA,N.ISHIKAWA,S.FUJITA, I.TOMINO, K.YOKOYAMA, S.ARAKI, M.TAKESUE, K.KATO, M.ISHIGURO, T.KITAHARA,N.KIHARA and A.BABA, Biol.Pharma.Bull. (in press). 9. A.NAKATANI,M.YAMADA, T.IKEUCHI, N.ITOKAZU, A.AWAYAand H.HATANAK& Seikagaku. & 567 (1995) (in Japanese).
282
MS-430on LiverCells
Vol. 61, No. 3, 1997
10. K.HIR4TA, I.OGATA,Y.OHTAand K.FUJIWARA,J.Pathol. L5&157-165 (1989). 11. P.O.SEGLEN, Methods.Cell.Biol. H 29-83 (1976). 12. D.L.KNOOK, A.M.SEFFELAAR and A.M. de LEEUW, Exp.Cell.Res. 139468-471 (1982). 13. G.BALLARDINI, M.FALLANI, G.BIAGINI, F.B.BIANCHI and E.PISI, Virchows.Archiv.B.Cell.Pathol. X 45-49 (1988). 14. J.L.FARBER and E.E.YOUNG, Arch.Biochem.Biophys, 211312-320 (1981). 15. R.T.HINEGARDNER, Anal.Biochem. 39197-201 (1971). 16. P.CHOMCZYNSIU and N.SACCHI, Anal.Bioch. J.Q 156-159 (1987). 17. S.KANAME, S.UCHIDA, E.OGATAand K.KUROKAWA,Kidney.Int.Q 1319-1327 (1992). 18. S.YASUHARA, S.KASHIWAGI,H.ITO and A.AWAYA,Int.J.Clin.Pharm.Res. 15167-174 (1995). 19. M.B.SPORN, A.B.ROBERTS, L.M.WAKEFIELDand B. de CROMBRUGGHE, J.Cell.Biol. lQ5 1039-1045 (1987). 20. G.RAMADORI, Virchows.Archiv.B.Cell.Pathol. &l 147-158 (1991). 21. A.PANDURO, F.SHALABY, F.R.WEINER, L.BIEMPICA, M.A.ZERN and D.A.SHA FRITZ, Biochemistry. 251414-1420 (1986). 22. M.PINZANI, L.GESUALDO, G.M.SABBAH and H.E.ABBOUD, J.Clin.Invest. 8417861793 (1989). 23. B.H.DAVIS, J.Cell.Physiol. 136547-553 (1988). 24. O.HIOKI, M.MINEMURA, Y.SHIMIZU,Y.KASII,H.NISHIMORI, T.TAKAHARA, K.HIGUCHI, Y.YOSHITAKE,K.NISHIKAWAand A.WATANABE,J.Hematology.24 217-224 (1996). 25. D.C.ROC@Y,’J.KBOYLES, G.GABBIANIandS.L.FRIEDMAN, J.Submicrosc.Cytol.Pathol. 24193-203 (1992). 26. T.KIWITEL,T.JANNECL L.tiLLER, P.FELLMERandG.RAMADORI,Hematology.24 352-360 (1996). 27. G.RAMADORI,T.VEIT,S.SCHWOGLER,H.P.DIENES,T.KIW’ITEL,H.RIDER and K.-H. MEYER zum BUSCHENFELDE, Virchows.Archiv.B.Cell.Pathol. 59 349357(1990). 28. B.H.DAVIS, U.R.RAPP and N.O.DAVIDSON,Biochem.J. 27843-47 (1991).
PII S0024-3205(97)00383-4 ~••ŒÀÓ«•ˆ••Œ4M¨• •w•æ
EFFECTS OF NEUROTROPIC PYRIMIDINE HETEROCYCLIC COMPOUND, MS-430, ON CULTURED HEPATIC PARENCHYMALAND STELLATE CELLS Mie Inaol, Satoshi Mouhidal, Hitoshi Ikedal~, Akira Awaya3and Keqji Fujiwaral
Depar@ent of Internal Medicine, Saitalna Medical School) 38 Morohongb, Moroyama-cho, Iruma-gun, Saitama 350-04, ZFirstDepartment of I~ternal Medicine, Faculty of Medicine, University of Tokyo? 7+3-1 Hongo, Bunkyo-ku, Tokyo 113, jInstitute of $iologim] Science, Mitsui Pharmaceuticals?Inc.? 3-12-2 Nihonbashi,Chuo-ku,Tokyo 103, Japan. IThird
~(Receivedia finalformApril 14, 1~
Summary MS-430 is a novel sy@hetic pyrimidine derivative that stimulates regeneration of the nerve as a promo~er for various growth factors such as epidermal growth factor (EGF) and ne~e growth factor, and differentiation of astrocytes. The ef– fects of MS-430 on the liver were tested using hepatocytes and stellate cells in primary culture isolatep from rats. MS-430 enhanced EGF-induced DNA synthesis in hepatocytes w~ile it alone failed to increase the basal DNA synthesis. Albumin mRNA expression in the cells and its amount in the medium were not changed by addition $ifEGF or MS–430 alone or both. Basic fibroblast growth factor (bFGF) increas~d DNA and but not collagen synthesis by hepatic stellate cells. Addition of M!?+430 inhibited DNA synthesis by hepatic stellate cells at either presence or ab$ence of bFGF, and collagen synthesis at the presence of bFGF. However, MS~430 had no effects on basal or bFGF-stimulated TGF~ mRNA expression in the cells. These results suggest that MS–430 stimulated proliferation of hepat~cytes as a comitogen for EGF without affecting albumin synthesis, and suppressed proliferation of activated hepatic stellate cells and their collagen synthesis wit~out affecting TGF~ expression. @ Wordr: hepatoeyte,hepaticst@ate ceu liver regeneration% livertibrosis,MS-W
Liver failure occurs as acu{e and chronic types. Acute liver failure often takes a fatal course with multiple organ failure (1). Since massive or submissive liver necrosis is commonly seen in this disease (2), sufficient li~er regeneration is required for survival. However, impaired liver regeneration seems to exist u~der this state, as hepatocyte growth factor (HGF) in the serum is markedly increased in patientp with acute liver failure (3,4). Thus, the use of agents capable of enhancing the action of growth factors for hepatocytes might be a therapeutic strategy for such liver failure. Chronic liver failure develops as a consequence of progressive liver fibrosis due to repetitive necrosis and inflammation (5). Hepatic stellate cells are known to proliferate and produce extracellular matrix components in this process (6), suggesting that the suppression of these functions of hepatic stel~atecells may be therapeutic for this type of liver failure. CorrespondingAuthor: Kenji~Fujiwara,M.D., Ph.D. ThirdDepartment of InternalMedicine, Saitama Medical School, 38 Morohongo,Moroyama-cho, Iruma–gun,Saitama 350–04, Japan. Tel:81-492-76-1198, Fax:81-492-94-8404
274
MS-430on LiverCells
Vol. 61, No. 3, 1997
MS-430, 2-piperodino–5,6 -dihydro-7-methy l-6-oxo (7H) pyrrolo [2,3–d] pyrimidine maleate is a novel synthetic pyrimidine heterocyclic compound that has a variety of actions on peripheral nerves and neuron cells (7-9). MS-430 can promote regeneration of rat sciatic nerve following dissection (7) and differentiation of astrocytes in culture (8). Also, MS-430 is reported to promote neurite sprouting of PC–12 cells, pheochromocytoma-derived cell lines, when stimulated by nerve growth factor (NGF) or epidermel growth factor (EGF) (9), suggesting that MS430 can act as a growth promoting agent on epithelial cells with the receptors for these growth factors. Hepatic stellate cells are pericytes like astrocytes (10). These observations prompted us to test whether MS-430 can act similarly on hepatocytes and hepatic stellate cells. The present paper reports on the effects of MS-430 on rat hepatocytes and hepatic stellate cells in primary culture. Its possibility as a therapeutic candidate for liver failure is also discussed. Materials and Methods Animals Male Sprague-Dawley rats (Charles River Japan, Atsugi, Japan) were housed in a room at 22 t 20C under nomal laboratory li@ting conditions and maintained on a commercial Pelleted ‘iet and water ad libitum. Rats weighing 190-210 g and 290-310 g were subjected to the isolation of hepatocytes and hepatic stellate cells, respectively. All protocols confirmed to National Research Council criteria for human care of animals, and all in vitro experiments were repeated at least 3 times. Preparation of &lS-430 MS–430 (Fig 1) was synthesized as previously reported (8) by Mr. Ikuo Tomino et al. at Synthetic Chemistry and Bioscience Laboratories, Mitsui Petrochemical Industries, Ltd., Tokyo, Japan.
o COOH If
2-piperidino-5,6-dihydro-7-methyl-6-oxo pyrrolo [2,3-d] pyrimidine maleate
(7H)
Fig 1 The Chemical Structure of MS-430. Isolation of Hepatocytes and Hepatic Stelkzte CelLs Hepatocytes were isolated by Seglen’smethod (11). Briefly, the liver was minced after perfusion through the portal vein with 0.05Y0collagenase (type I: Worthington Biochemical Corp., Freefold, NJ) in Hanks’ balanced salt solution (HBSS: Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) at a flow rate of 30 mL/min for 15 min at 37”C. Hepatocytes were separated from nonparenchymal cells by centrifusion at 28g for 2 min at 4“C. Hepatic stellate cells were isolated by the method of Knook et a/. (12) with partial modifica– tions. After liver perfusion with 0.05% collagenase and 0.1% pronase E (Kaken Pharmaceutical Co. Ltd., Urayasu, Japan) in HBSS at a flow rate of 10 mL/min for 15 and 10 rein, respectively, at 37”C, the liver was minced and incubated with HBSS containing both enzymes at 37°C for 30 min. The resultant cells were collected after filtration through a nylon mesh. The recovered cells were overlaid on double–layered metrizamide (Sigma Chemical Company, St. Louis, MO) solu-
Vol. 61, No. 3, 1997
MS-430on LiverCells
275
tions at concentrations of IYZOand 18%, and centrifuged at 1400g for 17 min at room temperature. Hepatic stellate cells were collected from the top layer of 13% metrizamide solution. The viability rates of the collected hepatocytes and hepatic stellate cells were constantly higher than 90%, as tested by trypan blue exclusion. The purity of hepatocytes immediately after isolation and hepatic stellate cells 6 days after incubation on plastic dishes were 90% or more as– sessed by light microscopic examination and immunostaining of desmin (13), respectively. Experimental Design Experiment I: The isolated hepatocytes were suspended in Williams’ medium E (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) containing 10% fetal calf serum (FCS: Gibco Laborato– ries Ltd., Life Technologies Inc., Grand Island, NY) heat–inactivated at 56°C for 30 rein, 10-6M insulin (Sigma Chemical Company) and 10-5 M dexamethasone (Sipa Chemical Company). They were seeded on plastic dishes at a density of 5.0 x 104cells/cm and incubated at 37°C in an atmosphere of 5% C02 and 95% air for 2 hr. Following removal of non-adherent cells by washing with Hank’s balanced salt solution without Ca2+and Mg2+(HBSS(-); Gibco Laboratory Ltd.), adherent hepatocytes were further cultured in Williams’ medium E containing 10% heatinactivated FCS with addition of Oor 50 ng/mL of EGF (Toyobo Co. Ltd., Osaka, Japan) and MS-430 at various concentrations at 24 hr after plating. At 24 hr after MS-430 addition, the medium was collected for the determination of lactate dehydrogenase (LDH) activity and albumin concentration. The hepatocytes were subjected to Northern blot analysis of albumin mRNA and their DNA contents were determined. In another series of experiments, similarly prepared hepatocytes were subjected to the determination of DNA synthesis and protein synthesis in the cells. The same experiments were repeated 3 times. Experiment II: Isolated hepatic stellate cells were suspended in Dulbecco’s Modified Eagle Medium (DMEM: Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) containing 10% heat-inactivated FCS and seeded on plastic dishes at a density of 1.25 x 105cells/cm2.They were incubated for 6 days similarly as was done in hepatocytes. During this period, non–adherent cells were removed by washing with HBSS(-) every 2 days. On 6th day, Oor 10 ng/mL of basic fibroblast growth factor (bFGF: Toyobo Co. Ltd.) and various concentrations of MS-430 were added to the medium and the cells were further cultured. At 24 hr later, the medium was collected for the determination of LDH activity. The cells were subjected to Western and Northern blot analyses of smooth muscle a actin and transforming growth factor (TGF) ~, respectively and their DNA contents were determined. [n another series of experiments, syntheses of DNA, protein and collagen by the cells were determined at 24 hr after MS-430 addition. The same experiments were repeated 3 times. Evaluation of Cell Killing Cytotoxicity of MS-430 was evaluated as cell killing measured by LDH activity in the medium and DNA contents of adherent cells. LDH activity was determined according to the method of Wioblewski et al. (14), which meas– ures the breakdown of pyruvate (Sigma Chemical Company) to lactate detected as the increase in absorbance at 340 nm following addition of b–NADH (Sigma Chemical Company) to the culture medium. Cell killing was expressed as the percentage of LDH activity in the medium to total LDH activity in non-MS-430-treated cells when the cell membrane of those cells was de– stroyed by addition of 0.5% Triton X (Wako Pure Chemical Industries Ltd., Osaka, Japan). For the determination of DNA contents in adherent cells, the cells in dishes were washed 3 times with saline to remove non-adherent cells. The adherent cells were solubilized in 0.1 N NaOH. An aliquot of the cell solution was collected, and kept at 60”C for 45 minutes with the same volume of 0.4 glmL diaminobenzoic acid (Aldrich Chemical Company Inc., Milwaukee, WI) solution in water. DNA contents in the aliquot were measured with a fluorescence spectrophotometer (F-2000, Hitachi, Tokyo, Japan) as previously reported (15). A4easurementofAlbumin Concentration Albumin concentration in the medium was measured by enzyme-linked irnmunosorbent assay as follows. In each well of a 96-well assay plate (Becton Dickinson Labware, Lincoln Park,
276
MS-430on LiverCells
Vol. 61, No. 3, 1997
NJ), 50~g of polyclonal goat IgG antibody against rat albumin (Cappel Research Products, Durham, NC) was distributed as 500 p#mL solution in phosphate buffered saline (PBS), pH 7.4, kept overnight at 4“C, and removed by suction. After 4 washes with 10% Block Ace (blocking agent derived from skim milk; Snow Brand Milk Products Co., Tokyo, Japan) in PBS, pH 7.4, the wells were filled with 400 ~L of 70% Block Ace in PBS, pH 7.2, and kept overnight at 4°C. After removing the solution, the wells were washed 4 times with 10% Block Ace in PBS, pH 7.2 (10% BA). Then, 100 ~L of assay sample and the same volume of 10% BA were added to each well, kept overnight at 4“C, and removed by suction. After 8 washes with 10YoBA, 5 pg of peroxidase-conjugated polyclonal sheep IgG antibody against rat albumin (Cappel Research Products) was distributed as 50 @mL solution in 10% BA, kept overnight at 4“C, and removed by suction. After 8 washes with 10% BA, the wells were added with 200 pL of 2,2’-azino-di(3-ethylbenzthiazoline sulfonate) solution (Boehlinger Mannheim Biochemical, Germany), and kept at room temperature for 30 min. Then, 100 pL of 0.25 M oxalic acid was added to the solution, and the optical density of the solution was determined at 410 nm with an automated microplate reader (MR 5000; Dynatech Laboratories Inc., Chantilly, VA). Measurement of DNA Synthesis At 2 hr before cell harvest, 2 pCi/mL of [methyl-3 H]–thymidine (New England Nuclear, Boston, MA) was added to the culture medium of hepatocytes and hepatic stellate cells. The cells were washed with cold PBS, immersed in 10’%trichloroacetic acid at 4°C for 10 rein, and solubilized in 1 N NaOH at 37°C for 1 hr. An aliquot of the cell solution was collected, and treated with 100Yotrichloroacetic acid at 4“C. The resultant precipitate was further treated with 10% trichloroacetic acid, and solubilized in 0.1 N NaOH. Then, the radioactivity of the solution was measured by liquid scintillation counter. Measurement of Protein Synthesis At 24 hr before cell harvest, 1 pCi/mL of L-[3,4,5-3H]-leucine (New England Nuclear) was added to the culture medium of hepatocytes and hepatic stellate cells. The cells were washed, immersed and solubilized. Then, an aliquot of cell solution was treated with 100!ZO trichloroacetic acid similarly as was done for the measurement of DNA synthesis. The precipitate was further treated with 107otrichloroacetic acid, incubated at 70”C for 10 min and put on ice for 10 min. The resultant precipitate was solubilized in 0.1 N NaOH, and the radioactivity of the solution was measured. Measurement of Collagen Synthesis At 24 hr before cell harvest, 10 ~Ci/mL of L-[2,3-3H]-proline (New England Nuclear), 0.1 mM of ascorbic acid and 0.5 mM ~-aminopropionitrile were added to the culture medium of hepatic stellate cells. At harvest, the medium was removed and the cells were solubilized in 1 N NaOH. The medium and cell solution were precipitated with 10% trichloroacetic acid. The pellet was digested with purified bacterial collagenase (Form III, Advance Biofactures, Lymbrook, NY) at 37°C for 90 rein, and precipitated with trichloroacetic acid and tannic acid. The radioac– tivity in the supematant collected by centrifugation was measured, and collagen synthesis by the cells was expressed as total radioactivity of the supematant obtained from the medium and cells. Northern Blot Analysis of Albumin and TGFf! Total RNA was extracted from cultured hepatocytes and hepatic stellate cells by homogenization in guanidine thiocyanate and centrifugation through phenollchloroform (16). Ten pg of total RNA was subjected to electrophoresis on 1.62% agarose-formaldehyde gel, blotted to nylon filters (Hybond-N; Amersham International Plc., Little Chalfont, England) by upward capillary transfer, and fixed to filters using UV crosslinker (UVP Inc., Upland, CA). cDNA fragments of rat albumin (a gift from Dr. Yasufumi Kaneda through Japanease Cancer Research Resources Bank-Gene, Tokyo, Japan) or rat TGF@(17) were radiolabeled with [a-32P]dCTP (New England Nuclear) using Megaprime DNA labeling system (Amersham International PIc.). Prehybridized filters were hybridized at 42°C for 16 hr with sZp_labeledcDNA fragments, then washed under high-stringency conditions and exposed to XAR film (Eastman Kodak Company, Rochester, NY). Western Blot Analysis of Smooth Muscle aActin After discarding the medium, hepatic stellate cells were incubated in 0.5% sodium dodecyl
277
MS-430on LiverCells
Vol. 61, No. 3, 1997
sulfate (SDS), 60 mM Tris–HCl, pH 6.8 and 100 mM dithiothreitol, boiled for 5 rein, and destroyed by sonication. Samples containing the same amount of protein were separated by SDS polyacrylamide gel electrophoresis (12% acrylamide) under reducing conditions. The gel was electrophoretically transferred onto a nitrocellulose using Trans–Blot SD Semi–Dry Electrophoretic Transfer Cell (Bio Rad, Richmond, CA), exposed to monoclinal antibody against mouse smooth muscle a actin (BioMakor, Rehovot, Israel), and incubated with alkaline phospha– tase conjugated rabbit antibody against IgG (Boehringer Mannheim Biochemical).Then, the filters were washed and developed in 5–bromo-4–chloro–3 -indolyl phosphate/nitro blue tetrazoli– urn solution. Results The baselines of the results ranged in 3 repeated experiments, but the relations among groups were almost similar in each experiment. The following results are those in one of the 3 experiments. Cytotoxicity of MS-430 for Ilepatocytes and Hepatic Stellate Cells in Primary Culture As shown in TABLE I, si~ificant cell killing by MS-430 assessed from LDH activity in the medium was not observed at 0.1 mM or less in both hepatocytes and hepatic stellate cells irres– pective of the presence of EGF or bFGF. However, MS–430 at 1 mM killed most hepatocytes, but much less hepatic stellate cells. The similar results were obtained by trypan blue exclusion test (data not shown). Also, DNA contents in both adherent hepatocytes and stellate cells were not changed by addition of EGF, bFGF and MS-430 at concentrations of 0.1 mM or less. TABLE I Effects of MS-430 on Cell Killing of Cultured Rat Hepatocytes and Hepatic Stellate Cells MS-430 concentration in the medium (mM)
Hepatocytes
Stellate cells
EGF addition (ng/mL) 50 o
bFGF addition (ng/mL) o 10
. . . Actlwty l~m
[fya o)
0 0.001 0.01 0.1 1.0
35.955.3 34.7 ? 6.0 39.4 ? 5.3 32.4 ? 5.4 68.3 ? 21.2*
29.6 ? 2.0 30.7 ? 2.0 31.916.1 26.0 ? 8.2 89.8 ? 33.0”
12.7 t 15.9 ? 17.6 t 15.9 f 22.2 ?
0 0.001 0.01 0.1 1.0
46.3 ? 0.6 46.9 f 0.4 43.5 t 4.1 45.4 ? 1.3 41.4 ? 3.6”
45.0 t 1.2 48.4 ? 0.8 44.9 ? 3.0 43.4 ? 3.6 34.6 ? 3.2”
33.0 ? 1.3 31.9 ? 2.7 29.8 ? 1.2 29.1 t 1.8 23.3 ? 1.2**
2.8 2.8 2.8 2.8 5.5*
12.2 t 4.lb 17.6 ? 2.4 16.3 ~ 0.0 14.9 f 2.3 21.7 ? 2.3$”
35.3 ? 3.2b 35.2 ? 3.5 34.2 z 1.6 33.924.1 22.3 ~ 1,8”
‘The percent of LDH activity in the medium to that in cell lysate as described in Materials and Methods. bMean ? SD of 3 wells. *pcO.1,**p
Vol. 61, No. 3>1997
MS-430on LiverCells
278
H). Albumin mRNA expression in the cells was also unchanged (Fig 2). When MS–430 was added at 0.01 mM to the medium concomitantly with EGF addition, DNA synthesis by hepatocytes was significantly increased compared to controls (TABLE II.).However, the similar change was not seen in hepatocytes cultured without EGF addition. Cellular protein synthesis and albumin concentration in the medium were not changed by MS-430 at this concentration irrespective of the presence of EGF (TABLE II). The same result was also observed in albumin-mRNA expression (Fig 2). TABLE II Effects of MS-430 on DNA, Protein and Albumin Syntheses in Cultured Hepatocytes
MS-430 concentration in the medium (mM)
DNA synthesis (104dpm/well)
Albumin concentration in the medium (w#mL)
EGF concentration (ng/mL) o
0 0.001 0.01 0.1
Protein synthesis (105dpm/well)
3.75? 4.01~ 3.88? 2.53?
50 0.17 0.42 0.36 0.58
0
24.62? 2.69” 2.48 f 26.85* 1.78$ 2.44? 31.48? 2.53*#2.29? 22.41* 2.53* 2.28 ?
50 0.28 0.56 0.22 0.31
3.93? 3.82? 3.47? 3.39 ?
0.60” 0.30” 0.21’ 0.36”
50
0 1.o5 ? 0.90? 1.21 z 0.83?
0.27 0.08 0.37 0.02
1.092 0.85 z 1.70 ? 0.85 z
0.23a 0.09 0.82 1.12
‘Mean * SD of 3 wells. *p
~EGF
Ong/mLv
EGF 50 rig/m< - 28S
a)
- 18S - 28S
b)
=18S
o 10-310-2
10-1
0
10-3 10-210-1
Doses of MS-430(mM) Fig 2 Albumin mRNA Expression in Hepatocytes in Primary Culture. a) Northern blotting, b) Ethidium bromide staining.
MS-43(Ion LiverCells
Vol. 61, No. 3, 1997
279
Effects of MS-430 on the Function of Hepatt”cStellate Cel&in Pn”maryCulture bFGF at 10 ng/mL significantly increased DNA synthesis, but not protein synthesis and collagen synthesis by hepatic stellate cells compared to controls (TABLE III). MS-430 decreased DNA synthesis by the cells in a dose-related manner irrespective of the presence of bFGF. The similar dose-related attenuation by MS-430 was seen in protein synthesis by the cells cultured without bFGF and in collagen synthesis by the cells cultured with bFGF (TABLE III). As shown in Fig 3, bFGF decreased smooth muscle a actin expression in the cells. This decrease was significantly attenuated by addition of MS-430 at 0.001 to 0.1 mM. In the cells cul– tured without bFGF, however, MS–430 did not change smooth muscle a actin expression. bFGF significantly increased TGF~-mRNA expression in the cells (Fig 4). MS-430 did not change this increase as well as TGF(3-mRNA expression in the absence of bFGF. TABLE HI Effects of MS-430 on DNA, Protein and Collagen Syntheses in Cultured Hepatic Stellate Cells
MS-430 concentration in the medium (mM)
DNA synthesis (105dprnAvell)
Collagen Synthesis (104dprn/well)
bFGF concentration (nglmL) o
0 0.001 0.01 0.1
Protein synthesis (105dprn/well)
1.91t 1.58t 1.39? 0.52?
10 0.26 4.29? 0.02 3.70? 0.07# 3.972 0.23# 1,99*
0
0.69” 1.43 ? 0.49” 1.39? 0.11’ 1.23? 0.17*#1.06 ?
10 0.13 1.62? 0.06 1.54? 0.86 1.49? 0.33#1.39 ?
10
0
0.19 3.52 z 0.12 3.25? 0.08$ 3.20? 0.07” 3.18?
0.29 4.12 z 0.08 4.12? 0.59 3.45 ? 0.11#2.84 ?
0.48a 0.67 0.27 0.23#
Qne way analysis of ANOVA p value
<0.01
<0.01
<0.01
0.25
0.44
<0.05
aMean t SD of 3 wells. *p<0,05vs bFGF at Ong/mL, #p
Vol. 61, No. 3, 1997
MS-43(Ion LiverCells
280
factors for hepatocytes and hepatic stellate cells, respectively, in the present experiments. As a marker of hepatocyte differentiation, albumin synthesis, specific function of mature hepatocytes, was evaluated from its concentration in the medium and cellular mRNA expression. Collagen synthesis and smooth muscle a actin expression were measured in hepatic stellate cells as markers of activation.(5) Also, cellular TGFfl expression was determined, since this cytokine regulates both proliferation and activation of the stellate cells in an autocrine manner (19, 20). DNA, protein and collagen syntheses were expressed per well, since DNA contents of hepatocytes and stellate cells were not affected by EGF, bFGF and MS-430 at concentrations of 0.1 mM or less (TABLE I). As shown in TABLE II, DNA synthesis by cultured hepatocytes was markedly increased by addition of EGF, and this increase was further enhanced by concomitant addition of MS-430 at 0.01 mM. However, the enhancement was not seen in hepatocytes in the absence of EGF, suggesting that MS–430 acted as a comitogen for EGF. The concentration of MS-430 was 10 times lower than that reported to be effective for stimulating astrocytes and PC 12 cells (8,9). MS-430 at this concentration did not affect protein synthesis and albumin synthesis by the hepatocytes. It is well known that cell proliferation and differentiation progress inversely (21). From the therapeutic viewpoint, growth factors that stimulate hepatocyte proliferation might decrease the function of mature hepatocytes. In this point, MS-430 may be a promising agent as a comitogen for hepatocytes.
o
10-310-210-1 0
10-310-210-1
Doses of MS-430 (mM) Fig 3 Smooth Muscle a Actin Expression in Hepatic Stellate Cells in Primary Culture.
~ bFGF Orig/m+
bFGF 10 ng/mL=
28s
a)
18S 28S
b)
18S
o
10”310-2
10-1
0 10-3 10-210-1
Doses of MS-430 (mM) Fig 4 TGFfl Expression in Hepatic Stellate Cells in Primary Culture. a) Northern blotting, b) Ethidium bromide staining.
Vol. 61, No. 3, 1997
MS-430on LiverCells
’281
bFGF significantly increased DNA synthesis by hepatic stellate cells without affecting protein synthesis and collagen synthesis, as shown in TABLE III. MS-430 reduced DNA synthesis by the cells in a dose-related manner irrespective of the presence of bFGF. Also, MS-430 suppressed collagen synthesis by the cells in the presence of bFGF. Hepatic stellate cells are known to be activated by platelet derived growth factor, bFGF (22) and TGFf3(23) to produce a large amount of extracellular matrix components such as collagen. Hepatic stellate cells used in the present experiments were those cultured for 6 days which are considered to be already activated and produce bFGF that can act on themselves in an autocrine manner (24). Thus, it would be reasonable to assume that MS-430 inhibited not only the action of exogenous bFGF to stimulate proliferation of hepatic stellate cells, but also the activation by endogenous bFGF. Such actions of MS-430 would be important as a therapeutic agent for liver fibrosis as well. This possibility should be investigated in future in relation to cell proliferation and cellular expression of procol– lagen mRNA expression. To clarify the mode of action of MS-430 on hepatic stellate cells, smooth muscle a actin and TGF~ expressions in the cells were evaluated, because smooth muscle a actin is a marker of stellate cell activation in vivo (25), and TGF~ is the strongest stimulator of collagen synthesis by hepatic stellate cells (26). Isolated stellate cells are reported to increase smooth muscle a actin expression with increased syntheses of DNA and collagen during culture on plastic dishes (27). However, as shown in Fig 3, smooth muscle a actin expression was decreased in the cells following stimulation by bFGF, suggesting that the expression is not always a marker of hepatic stellate cell activation in vitro. As shown in Fig 4, bFGF increased TGF~ expression in the cells. Considering that smooth muscle a actin expression was decreased in the cells by addition of TGF~ (28), such a decrease in smooth muscle a actin expression is likely to result from in– creased expression of [email protected] did not affect TGFfJexpression in the cells irrespective of the presence of bFGF. However, it significantly attenuated smooth muscle a actin expression, and decreased collagen synthesis stimulated by bFGF. These results may strongly suggest that MS-430 acted on hepatic stellate cells stimulated by bFGF independently of the action of TGF~. In conclusion, MS-430 may stimulate proliferation of hepatocytes as a comitogen for EGF without affecting albumin synthesis. Also, MS-430 may suppress proliferation and collagen synthesis by hepatic stellate cells, MS–430 merits consideration for assessment in vivo as a therapeutic candidate for liver injury that can stimulate liver regeneration and attenuate liver fibrosis. Acknowledgement We thank Ms. Kiyoko Naiki for technical assistance. References 1. J.G.O’GRADY,B.PORTWN and R.WILLIAMS, ~ea.se oft~, Seventh Edition,L.Schiff and E.R.Schiff(Eds)?1077-1090, Lippincott Company, Philadelphia (1993). m Lwer Dzseases,H.Popper and F.Schaffner (Eds), 2. H.POPPER, D.KEPPLER, E2Q+ZWM 209-235, Grune and Stratton, Oriando (1986). 3. H.TSUBOUCHI, S.HIRONO, E.GOHDA, H.NAKAYAMA,K.TAKAHASHI, O.SAKIKIYAMA, H.MIYAZAKI,J.SUGIHARA, E.TOMITA, Y.MUTO, Y.DAIKUHARA and S.HASHIMOTO, Hematology.9875-881 (1989). 4. T.TOMIYA, S.NAGOSHI and K.FUJIWARA,Hematology.M 1-4 (1992). 5. S.L.FRIEDMAN, N.Engl.J.Med. ~ 1828-1835 (1993). 6. S.L.FRIEDMAN, F.J.ROLL, J.BOYLES and M.BISSELL, Proc.Natl.Acad.Sci.USA. 82 8681-8685 (1985). 7. M.OCHI, M.NODA, K.NAKAMITSU, M.DEIE, Y.IKUTA,Y.MAKIand A.AWAYA, Gen.Phannac. 2659-64 (1995). 8. Y.KOYAMA,A.AWAYA,N.ISHIKAWA,S.FUJITA, I.TOMINO, K.YOKOYAMA, S.ARAKI, M.TAKESUE, K.KATO, M.ISHIGURO, T.KITAHARA,N.KIHARA and A.BABA, Biol.Pharma.Bull. (in press). 9. A.NAKATANI,M.YAMADA, T.IKEUCHI, N.ITOKAZU, A.AWAYAand H.HATANAK& Seikagaku. & 567 (1995) (in Japanese).
282
MS-430on LiverCells
Vol. 61, No. 3, 1997
10. K.HIR4TA, I.OGATA,Y.OHTAand K.FUJIWARA,J.Pathol. L5&157-165 (1989). 11. P.O.SEGLEN, Methods.Cell.Biol. H 29-83 (1976). 12. D.L.KNOOK, A.M.SEFFELAAR and A.M. de LEEUW, Exp.Cell.Res. 139468-471 (1982). 13. G.BALLARDINI, M.FALLANI, G.BIAGINI, F.B.BIANCHI and E.PISI, Virchows.Archiv.B.Cell.Pathol. X 45-49 (1988). 14. J.L.FARBER and E.E.YOUNG, Arch.Biochem.Biophys, 211312-320 (1981). 15. R.T.HINEGARDNER, Anal.Biochem. 39197-201 (1971). 16. P.CHOMCZYNSIU and N.SACCHI, Anal.Bioch. J.Q 156-159 (1987). 17. S.KANAME, S.UCHIDA, E.OGATAand K.KUROKAWA,Kidney.Int.Q 1319-1327 (1992). 18. S.YASUHARA, S.KASHIWAGI,H.ITO and A.AWAYA,Int.J.Clin.Pharm.Res. 15167-174 (1995). 19. M.B.SPORN, A.B.ROBERTS, L.M.WAKEFIELDand B. de CROMBRUGGHE, J.Cell.Biol. lQ5 1039-1045 (1987). 20. G.RAMADORI, Virchows.Archiv.B.Cell.Pathol. &l 147-158 (1991). 21. A.PANDURO, F.SHALABY, F.R.WEINER, L.BIEMPICA, M.A.ZERN and D.A.SHA FRITZ, Biochemistry. 251414-1420 (1986). 22. M.PINZANI, L.GESUALDO, G.M.SABBAH and H.E.ABBOUD, J.Clin.Invest. 8417861793 (1989). 23. B.H.DAVIS, J.Cell.Physiol. 136547-553 (1988). 24. O.HIOKI, M.MINEMURA, Y.SHIMIZU,Y.KASII,H.NISHIMORI, T.TAKAHARA, K.HIGUCHI, Y.YOSHITAKE,K.NISHIKAWAand A.WATANABE,J.Hematology.24 217-224 (1996). 25. D.C.ROC@Y,’J.KBOYLES, G.GABBIANIandS.L.FRIEDMAN, J.Submicrosc.Cytol.Pathol. 24193-203 (1992). 26. T.KIWITEL,T.JANNECL L.tiLLER, P.FELLMERandG.RAMADORI,Hematology.24 352-360 (1996). 27. G.RAMADORI,T.VEIT,S.SCHWOGLER,H.P.DIENES,T.KIW’ITEL,H.RIDER and K.-H. MEYER zum BUSCHENFELDE, Virchows.Archiv.B.Cell.Pathol. 59 349357(1990). 28. B.H.DAVIS, U.R.RAPP and N.O.DAVIDSON,Biochem.J. 27843-47 (1991).