and by the Morton Gressman Scholar Award (Sir William Dunn. School of ...... Thomas, A. P., Marks, J. S., Coll, K. E., and Williamson, J. R.. (1983) J. Bid. Chem.
THEJ O U R N A L
Vol. 264, No. 16, Issue of June 5, pp. 9583-9591, 1989 Printed in U.S.A.
OF BIOLOGICAL CHEMISTRY
Vasopressin Inhibits Type-ICollagen and Albumin Gene Expressionin Primary Culturesof Adult Rat Hepatocytes* (Received for publication, December 19, 1988)
Mario ChojkierSs, David A. BrennerSll, and Hyam L. Leffertll** From the $Department of Medicine, Veterans Administration Medical Center and the1) Department of Pharmacology and Center for Molecular Genetics, University of California, S a n Diego, California 92161
The mechanisms that regulatecollagen gene expression in hepatic cells are poorly understood. Accelerated Ca2+fluxes are associated with inhibitingcollagen synthesis selectively in human fibroblasts (Flaherty, M., and Chojkier, M. (1986) J. Biol. Chem. 261, 1206012065). In suspension cultures of isolated hepatocytes, the Ca2+agonist vasopressin increases cytosolic levels of free Ca2+(Thomas, A. P., Marks, J. S., Coil, K. E., and Williamson, J. R. (1983) J. Biol. Chem. 258, 5716-5725). However, whether vasopressin’s interactions with plasma membrane VI receptors attenuate hepatic collagen production is unknown. We investigated thisproblem by studying vasopressin’s effects on collagen synthesis and Ca2+efflux in long-term primary culturesof differentiated andproliferation-competent adultrat hepatocytes. Twelve-day-old quiescent cultures were exposed to test substances and labeled with [5-3H]proline.Determinations of radioactivity in collagenase-sensitive andcollagenase-resistant proteins were used to calculate the relative levels of collagen production. Synthetic [8-arg]vasopressin stimulated 46Ca2’efflux within 1 min and inhibited hepatocyte collagen production within 3 h by 50%; overall rates of protein synthesis were not affected significantly. In cultureslabeled with [35S]methionine,vasopressin also decreased the levels of newly synthesized and secreted albumin, but not fibrinogen, detected in specific immunoprecipitates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Northernblot analyses using specific [32P]cDNAprobes revealed 70% decreases in hybridizable levels of collagen al(1) mRNA in hepatocyte cultures treated with eithervasopressin or Ca2+ionophore A23187; hybridizable levels of albumin mRNA also fell -50% following vasopressin treatment. Vasopressin did not affect collagen production in quiescent cultures of mouse Swiss 3T3, human myofibroblast or rat smooth muscle cells; and hepatocyte collagen production was unaffected by treatment with glucagon or dibutyryl CAMP. Thus, accelerated Ca2+fluxes induced by vasopressin are associated with decreased produc-
* This study was supported, in part, by Veterans Administration Grants (to M. C. and D. A. B.), National Institute of Health Grants DK 38652 (to M. C.) DK 39996 (to D. A. B.) DK 28215 (to H. L. L.), and National Science Foundation Grant DCB 86-16740 (to H. L. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I Recipient of a Research Career Development Award from the Veterans’ Administration. ll PEW Scholar in the Biomedical Sciences. ** Supported in part by the John Simon Guggenheim Foundation and by the Morton Gressman Scholar Award (Sir William Dunn School of Pathology, Oxford).
tion of hepatocyte collagen and albumin in primary cultures that simulatequiescent adult rat liver.
Hepatic collagen production and collagen content are markedly increased in patients with liver cirrhosis (1, 2). Thus, considerable effort has been devoted toward identifying hepatocellular and biochemical mechanisms that might normally regulate the synthesis anddegradation of collagens associated with interstitial and basal laminar elements (3). Since it has been difficult to obtain normal human liver tissue systematically for in vitro study, many investigators have resorted to using short-term primary cultures of adult rat hepatocytes hoping to simplify biochemical and morphological studies of the regulation of collagen production in normal hepatic cells (for review, see Ref. 4). However, conflicting results have been obtained (these various systems employ different culture conditions), and it has been concluded that hepatocytes (5-8), hepatic lipocytes (It0 cells) (9-lo), or hepatic endothelial cells (11) are the principal collagen-producing cells in these cultures. Such differences have complicated interpretation of results of regulatory studies, for example those where corticosteroids inhibited collagen production (12, 13). In thisreport, we approached the problem of the regulation of hepatic collagen production by using a long-term primary culture system of well-differentiated and proliferation-competent adult rat hepatocytes (14). Our work is derived from recent observations linking Ca2+ionophore and cholecystokinin-accelerated Ca2+fluxes with selective inhibition of collagen synthesis in cultured human fibroblasts (15), and from reported increases in the levels of cytosolic-free Ca2+ and inositol triphosphate that are induced by vasopressin in suspensions of freshly isolated hepatocytes (16-18). Since little is known about the effects of vasopressin in the liver, the function(s) of the hepatocyte VI vasopressin receptor, or the role of Ca2+fluxes in hepatic cellular physiology (19, 20), we wondered if vasopressin might attenuate hepatic differentiated function. In this study, we demonstrate that vasopressin decreases Type I collagen and albumin synthesis and the mRNA levels for both of these proteins in long-term hepatocyte cultures. EXPERIMENTALPROCEDURES
Materials-Uniformly labeled ~-[“C]proline(273 mCi/mmol), and ~-[2,3-~H]ornithine (20 mCi/mmol) were obtained from Du PontNew England Nuclear. ~-[l-[’~C]Ornithine (55 mCi/mmol), L - [ ~ - ~ H ] proline (22 mCi/mmol), [35S]methionine(1.4 Ci/mmol), 45CaC1,(30 mCi/mg Ca2+), and aqueous counting scintillant fluid were purchased from Amersham Corp. Chromatographically purified bacterial collagenase form I11 was obtained from Advance Biofactures Company (Lynbrook, NY). Synthetic [8-arg]vasopressin and porcine insulin and glucagon were generously provided by N. Ling (Salk Institute, La Jolla) and W. Bromer (Lilly), respectively. A pharmaceutical
9583
Vasopressin Inhibits
9584
CollagenAlbumin and
preparation of synthetic [8-arg]vasopressin was obtained from ParkeDavis. [(u-32P]2r-Deoxycytidine 5”triphosphate was purchased from ICN (Irvine, CA). Deoxyribonucleotides, Klenow fragment of DNA polymerase I, bovine serum albumin, and Dextran sulfate were purchased from Pharmacia LKB Biotechnology Inc. Agarose and restriction endonucleases were purchased from Bethesda Research Laboratories., AG-50W-X8 resin (100-200 mesh) and low melting agarose were from Bio-Rad. CsCl was obtained from IBI (New Haven, CT). Byodine transfer membrane was purchased from Pall (Glen Cove, NY). Guanidine isothiocyanate was obtained from Eastman. L-AScorbic acid, calcium ionophore A23187, Type I bacterial collagenase, dibutyryl cyclic AMP, N-ethylmaleimide and ethidium bromide were obtained from Sigma. Sephadex G-50 prespun columns were obtained from Boehringer Mannheim. Agar, tryptone, and yeast extract were obtained fromDifco (Detroit, MI). Pronase (102p.u.k./mg)was purchased from Behring Diagnostics. Human fetal AF, fibroblasts, rat smooth muscle cells, and themedia used for their invitro culturing were obtained from the Core Cell Culture Facility (University of California, San Diego, CA). Swiss 3T3 fibroblasts and human myofibroblasts from Dupuytren’s dermic nodules, were the gift of S. Mendoza and J. Vanderberg (University of California, San Diego, CA), respectively. Hepatocyte Cultures-Primary adult hepatocyte cultures were generated from livers obtained from Fisher/344 male rats (200 g) as describedpreviously (21,22). Hepatocyteswere plated (lo6cells/2 ml of medium) into untreated 35-mm Falcon plastic tissue culture dishes and cultured at 37 “C under standard conditions without media changes in humidified 10% C02/air incubators. At the time of plating, arginine-free media (DVMEM)’ were supplemented with pretested heat-inactivated 15% dialyzed, heat inactivated FBS,L-ornithine (0.4 mM), and 10 pg each of porcine insulin, inosine, and hydrocortisonesuccinate/ml. Mouse fibroblasts, myofibroblasts, and muscle cells were cultured in standard DVMEM supplemented with 10% FBS. Radiolabelingof Cells-Quiescent well-differentiated 10-13-day-old primary hepatic cell cultures were used for all biochemical measurements (23, 24). The cells were shifted (“zero time”) from their spent media into 1-2 ml of fresh arginine-free DVMEM supplemented with 0.2 mM Na+ ascorbate and 0.1 mM L-proline. Depending upon the experiment, test substances, including 15% dialyzed, heat inactivated FBS, vasopressin, A23187, porcine glucagon, or dibutyryl-cyclic AMP also were added at zero time (see legends to figures). For protein synthesis measurements, the cultures were labeled with [5-3H]proline, [35S]methionine,or a mixture of either [5-3H]proline plus [14C]ornithine, or [“C]proline plus [2,3-3H]ornithine starting a t 15 min until 3-6 hafter the fluid change. Protein synthesisrates were linear during this time. All isotopes were purified prior to use over a Dowex AG50W-X8 ion-exchange column (1 X 45 cm) (25). Swiss 3T3, myofibroblast, and smooth muscle cells were grown to confluence in P-100 culture dishes (Falcon0) a t 37 “C in a humidified 5% CO,, 95% air incubator, and fluid changed into 10 ml of standard DVMEM, supplemented with 10% FBS and 0.1 mM L-proline. Labeling conditions for these cultures were similar to those described above. Measurement of Collagen Production-The production of collagen and noncollagen protein was determined by incubation with purified bacterial collagenase (26) as described previously (15, 27). Radioactivity in collagenase-sensitive and collagenase-insensitive labeled proteins was used to calculate the relative rates of collagen production. The levels of radioactivity in collagenase-sensitive proteins released into culture fluids were used to quantify procollagen “secretion” (15, 27). Collagen Purification-Standard purification procedures were followed (27). Soluble proteins were precipitated with 200mgof [NH,J2S04/ml,pH 8.0, and centrifuged at 23,400 X g for 1 h at 4 ‘C. The precipitates were collected, dialyzed against 0.5 M acetic acid, and incubated with 10 pg of pepsin/ml at 4 “C for 20 h with stirring. The resulting suspensions were centrifuged at 30,000 X g for 1 h a t 4 “C,and the supernatants adjusted to pH 7.5 with 2 N NaOH. The [NH,],SO, precipitation step was repeated twice (for culture samples derived from individual treatments) and pooled precipitates were washed with 70% ethanol and dissolved in abuffer containing 50 mM Tris, pH 7.5, 0.1 M NaCl, 0.3 mM phenylmethylsulfonyl fluoride, and 0.25 mM [-aminocaproic acid. Radioactive collagen from culture fluids or from culturefluids plus cell layers was purified as described above ~
~
~~~
~
~
The abbreviations used are: DVMEM, Dulbecco/Vogt’s modified Eagles medium; FBS, fetal bovine serum; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; COLL, collagen; ALB, albumin; kb, kilobases.
Expression Gene
using 1-2 mg of either purified rat collagen (obtained from 12-dayold primary culture fluids) or calf skin collagen (Behring Diagnostics) as carrier. Purified collagens were fractionated by gel chromatography using a column (0.9 X 58 cm) containing Sephadex G-25, equilibrated first with 30 ml of 50 mM Tris, pH 7.5,30% (w/v) NaCl, and thenwith 15 mlof 50 mM Tris,pH 7.5, 25% (w/v) NaCl. The samples were preequilibrated (50 mM Tris, pH 7.5, 0.87% (w/v) NaCl), applied to the column and eluted (flow rate -1 ml/min) with a step gradientof 20 ml of 20% (w/v), 15% (w/v), 10% (w/v), and 80 mlof 5% (w/v) NaCl(28). The purity of the resulting fractions was assessed by SDSPAGE and by their sensitivity to purified bacterial collagenase (25, 29.30). Determination of Radiolabeled Albumin and Fibrinogen-Twelveday-old primary hepatocyte cultures were incubated with [35S]methionine in fresh methionine-free plating media under standard conditions and culture fluids harvested and centrifuged 3-4 h later (23, 31). In some experiments, albumin was purified from these fluids using an Affi-Gel Blue column as described previously (25). Radiolabeled albumin and fibrinogen secreted into culture fluids were recovered by immunoprecipitation using rabbit anti-rat albumin (Cappel) or rabbit anti-human fibrinogen (DAKO) antibodies, respectively. Immune complexes were precipitated with Protein-A (25, 32), washed, and centrifuged four times, and analyzed by SDS-PAGE and autoradiography. Sodium Dodecyl Sulfate-PolyacrylamideGel Electrophoresis-SDSPAGE and gel staining with Coomassie Brilliant Blue wereperformed according to Laemmli (33) as described previously (27). When necessary, gels were dried under a vacuum and exposed to X-Omat film (Eastman) a t -70 “C. Intensifying screens or flashed film (fluorogram (30)) were used as needed. Hepatocyte Contribution to Hepatic Collagen Production-This calculation was made with a previously validated method used for in vivo studies of hepatic collagen synthesis in intact rats(25,32). After primary rat hepatic cell cultures were incubated with the isotope mixture containing [3H]proline and [14C]ornithine,the relative contribution of hepatocytes to the total collagen production in these cultures was determined by comparing the ratio of [3H]proline/[14C] arginine in hepatic collagen to the same ratio in albumin. We calculated the predicted [3H]proline radioactivity in hepatic collagen produced by hepatocytes, as reported previously (25). Hepatocyte [3H]Pro~oLL
The ratio of proline (+ hydroxyproline)/arginine is approximately 3.5 times greater in hepatic collagen than in albumin (34-36). The relative contribution of hepatocytes and nonparenchymal cells (NPC) to hepatic collagen production was calculated as reported previously (25). Hepatocyte contribution (%) =
Hepatocyte I 3 H 1 P r o ~ ox~ ~ Hepatic [3H]ProCoLL
The NPC collagen production was determined as follows: NPC [3H]ProCoLL = Hepatic [ 3 H ] ] P r o ~ o~L Hepatocyte [3H]Pr~,LL.
Amino Acid Analysis-Low molecular weight fractions of cell culture fluid supernatant (obtained following fractionation with 66% ethanol) were evaporated to dryness, dissolved in distilled water, and chromatographed over Bio-Gel P2. The retained material was eluted and the resulting fractions used for amino acid analysis. Proteins precipitated in 66% ethanol or proteins recovered from purification or immunoprecipitation procedures described above were hydrolyzed in 6 N HCl at 120 ‘C for 3 h and evaporated to dryness. Amino acid analyses were performed as described previously (25,32). Radioactivity was measured in aqueous scintillant using a Mark 111 counter; automatic corrections were applied for spillover ofI4C radioactivity into the 3H channel and for respective counting efficiencies. Determination of 45Cuz+Efflux-Twelve-day-old primary hepato-
Vasopressin Inhibits
Collagen and Albumin Gene Expression
cyte cultures were incubated for 24 h with spent plating media (-1.5 ml/dish) in the presence of 33 pCi of ‘‘CaCI, (15,37). Thefluids were aspirated, the cultures were washed three times with 2 mlof fresh plating media, and shifted (time 0) into 2 ml of similar media without or supplemented with 100 nM [8-arg]vasopressin. Fifty pl of culture fluids were sampled from cultures in both groups at varying times thereafter. Northern Blotting-Primary hepatocyte cultures were generated and incubated, as described above, with or without test substances. Cells were processed as described previously (27), and total cellular RNA isolated by standard procedures (38). For treatment of cell cultures with Pronase, culture fluids were removed and cells washed twice with 50 mM Tris, 0.15 M NaC1, pH 7.5. Pronase (0.25% w/v) was incubated in 0.15 M NaCl at 37 “C for 1 h, and 1 ml was added to each dish. Cells were treated with the Pronase solution for 7 min, and then washed with 50 mM Tris, 0.15 M NaCl, pH 7.5, at 4 “C. The resulting RNA was chromatographed over oligo(dT) columns (30), and the recovered poly(A)+ RNA was used for Northern blotting as described previously (27, 39). Autoradiograms were made with intensifying screens and quantified by ascanning laser densitometer interfaced with an integrator. The plasmid pHF677 (40) containing cDNA encoding the human al(1) collagen gene was provided by F. Ramirez (SUNY Health Science Center, Brooklyn, NY). The plasmid LK 280 containing cDNA encoding the rat /3 actin genewas provided by L. Kedes (Stanford University, Palo Alto, CA). The plasmid malb2 containing cDNA encoding the mouse albumin gene wasprovided by S. Tilghman (Princeton University, Princeton, NJ) (41). Plasmid DNA was purified by alkali lysis and CsCl gradient centrifugation as described previously (39). The cDNA inserts were purified by digestion with the appropriate restriction endonucleases (EcoRI for pHF677, Hind111 for malb2, and PuuII for LK 280), electrophoresis on a low melting point agarose gel, and elution on an anion exchange column (Elutip). The cDNA fragments were radiolabeled with [a-”PIdCTP using the random primer synthesis method (42). The labeled cDNA probes were separated from dCTP by centrifugation through a G-50 spin column. The specific activity of the cDNA probes were approximately 1 X lo9 cpm/pg of DNA. Statistical Analysis-Unless noted, results were expressed as means f S.E. Student’s t test was used to evaluate the difference of the means between groups, acceptingp < 0.05 as significant (43).
9585
TABLEI Effect of vasopressin on collagenproduction in primary cultures of adult rat hevatocvtes Experimental condition”
Collagenb
Nonco’lagen proteinb
Relative collagen production‘
dpm x IO”
nM
%
Experiment 1 Control Vasopressin, 50 Vasopressin, 100
1.4 f 0.2 1.3 f 0.3 0.7 f 0.1
62.9 f 2.7 64.0 f 2.4 75.2 f 7.5
Experiment 2 Control Vasopressin, 10 Vasopressin, 100
9.6 f 0.5 7.7 0.2 4.3 f 0.4
302.3 f 1.7 273.3 & 1.3 198.3 f 2.1
*
* *
2.2 0.3 2.0 0.5 0.5 f 0.1
3.1 2.8 2.2
* 0.1 * 0.2 * 0.1
Experiment 3 Control 5.1 f 0.1 3.1 0.1 159.8 3.0 4.2 f 0.2 158.4 7.7 Vasopressin, 10 2.6 f 0.1 Vasopressin, 100 0.6 f 0.1 90.6 f 5.8 0.6 f 0.1 Twelve-day-old primary rat hepatocyte cultures were incubated for 3 h with or without [8-arg]vasopressin, as described under “Experimental Procedures.” In experiment 1synthetic [8-arglvasopressin was provided byN. Ling (Salk Institute). In experiments 2 and 3 synthetic [8-arglvasopressin was from Parke-Davis, and 56 p M chlorobutanol (vehicle) was added to control plates. * Determined from the 3H radioactivity incorporated into collagenase-sensitive and collagenase-resistant proteinsafter labeling cell cultures with 20FCi of [5-3H]proline for 3 h. Values are means f S.E. of quadruplicate samples. Calculated from the formula (26):
* *
3H collagen d ~ m / ( ~noncollagen H dpm x 5.4
*
+ 3H collagen dpm).
2.1.3.31, (21, 25, 45, 46))inthese cultures. When labeled proline and ornithine are presented to hepatic cells all of the cells incorporate proline into proteins, but only hepatocytes generate proteins containing labeled arginine derived from ornithine via the urea cycle (21, 25, 45, 46). Four h after the RESULTS fluid change, total secreted proteins (panel A ) including albumin (panel B ) , contained -80% of the 14Cradioactivity as Collagen Production in PrimaryHepatocyte Cultures-Collagen production was measured in 12-day-old proliferation- arginine. Most of the 14C radioactivity remaining in the culcompetentcultures composed of hepatocytes (-80%) and ture fluid amino acids (5.3 x lo5dpm) was found in proline (94.8%). Negligible amounts nonparenchymal cells (-20%). Such cultures display a broad and in other related amino acids of 14C radioactivity remaining in the culture fluid as amino spectrum of hepatocyte-specific functions (44). During a 24h interval following fluid change, therelative level of collagen acids were identified as arginine (0.6%) and ornithine(4.6%). cellular uptake of [“C] to total protein production was 2-3% (see control experiments These observations indicated that the ornithine was rapid and nearly quantitative; and, that among in Table I). Within this time frame, about 70%of newly synthesized procollagenwassecreted; the relative level of [ “Clornithine’s metabolites, proline alone was significantly collagen to total protein in the culture fluids was 4-6%. More exported into the culturemedium. Forty-five percent of the 14Cin collagen purified from these than 95% of the preexisting and newly synthesized collagen cultures was found as [“Clarginine (Fig. 3C). The collagen hadtheelectrophoreticcharacteristics of al(1) anda2(I) chains as shown in stained reducing SDS-gels (Fig. lA)fol- [I4C]arginine radioactivity was -70% of the level expected lowing fluorography (Fig. 1B). Gel chromatography of this were collagen produced exclusively by hepatocytes (25). In purified collagen demonstrated (Fig. 2) that the preexisting contrast, when human fibroblast cultures were dual-labeled (major peak of proteins eluting at 90 ml) and newly synthe- under similar conditions there was, as expected (45), a neglisized material, which coeluted with authentic Type-I collagen, gible amount of “C radioactivity present in arginine in hywere also composed solely of al(1)and a2(I) subunits(Fig. 2, drolyzed fibroblastproteins (Fig. 3 0 ) . Since [14C]arginine produced by hepatocytes is unlikely to have been transferred inset). to nonparenchymal cells (47), these experiments suggested, The relative contributions of hepatocytes and nonparenchymal cells to collagen production were estimated in 12-day- based uponprevious analyses (251, that more than two-thirds old cultures using two different approaches. First, a method of the newly synthesized collagen in this in vitro system was wasemployed that involves amino acid analysis of newly produced by hepatocytes. synthesized proteins in cultures dual-labeled with [3H]proline Evidence supporting this conclusion was obtained from a second type of experiment, in which Pronase was used to and [“Clornithine. The results are shown in Fig. 3. The rationale underlying this approach was based upon the selectively destroy cultured hepatocytes (48, 49), in 12-daypresence in hepatocytes and absence in nonparenchymal cells old cultures.Theresultingnonparenchymal cell survivors of argininebiosyntheticcapacity(principally due tothe (-20%of the initial numbers of attached cells) were then hepatocyte-specific function, ornithine transcarbamylase [EC examined for the presence of al(1) and albumin mRNA by
Vasopressin Inhibits
9586
Collagen and Albumin Gene Expression m
kDa
Y ,
4
OR(
1110
J
J
-200
7
n 900
- 66
600
f
B
A
0
FIG. 1. Analysis of preexisting and newly synthesized collagen in primary cultures of adult rat hepatocytes. Purified collagens were obtained by salt fractionation and enzymatic digestion from 12-day-old cultures; the proteins were pretreated with 100 mM dithiothreitol and applied to a 6% SDS-polyacrylamide gel as descrikd under "Experimental Procedures." The gel was subjected to 50 mA constant currentfor 3 h a t 21 'C, fixed, stained with Coomassie Blue ( l a n e A ) and subjected to fluorography ( l a n e B). A , analysis of preexisting collagen obtained from 84 dishes as described under "Experimental Procedures." B, analysis of newly synthesized collagen obtained from media and cell layers (14 dishes) of cultures labeled for 3 h with 50 pCi of [5-3H]proline/ml of media. Calf skin collagen (1.4 mg) was used as carrier.
12000
6000
0
FRACTION No.
Detection of hepatocyte-specific protein synthesis in primary cultures of adult rat hepatocytes by dual ['Hlproline and ["C]ornithine labeling. A , 12-day-old hepatocyte cultures were incubated in 2 ml of DVMEM (15% dialyzed, heat-inactivated FBS, 0.2 mM Na'-ascorbate, 0.4 mM proline, 0.4 mM ornithine) and labeled with 50 pCi of [5-3H]proline and 50 pCi of ["Clornithine for 24h. Proteins in the media were precipitated with 66% ethanol. Hydrolyzates of precipitated proteins (0.2 N sodium citrate, 0.1% Brij 35,pH 2.2) were obtained as described under "Experimental Procedures," applied to a 1 X 45-cm AG-50 column, and eluted (1ml/min) a t 50 "C with 120 ml of 0.7 N sodium citrate, 0.1% Brij 35, pH 8.6, and thenwith 0.2 N NaOH. Four-ml fractions were collected. Elution of authentic proline, ornithine, and arginine standards is indicated (arrows). Recoveries of standards of radioactive amino acids on this column were quantitative. Radioactivity was measured by liquid scintillation spectroscopy. B, newly synthesized albumin secreted into the medium during the 24-h labeling interval was purified from 12day-old hepatocyte cultures by Affi-Gel Blue chromatography as described under "Experimental Procedures." Hydrolyzates were obtained and analyzed as described in A . C, newly synthesized collagen secreted intothe medium during the 24-h labeling interval was purified from 12-day-oldhepatocyte cultures using carrier rat collagen from 12-day-old primary rat hepatocyte cultures as described under "Experimental Procedures." Hydrolyzates were obtained and analyzed as described in A . D,confluent AF, human fibroblasts were incubated in 5 ml of minimum essential medium, (10% FBS, 0.1 mM proline) and labeled for 24 h with 25 pCi [14C]prolineand 50 pCi [2,33H]ornithine. Hydrolyzates of cell layer proteins were obtained and analyzed as described in A. FIG. 3.
.20
r
1loo 80
x)
0
0
20
40
60
80
100
120
(ml)
FIG.2. Subunit structure of highly purified collagen synthesized by primary cultures of adult rat hepatocytes. Preexisting and newly synthesized collagens were obtained and purified from media of 12-day-old cultures as described in Fig. 1 ( p a n e l B ) . Collagens were fractionated by gel chromatography as described under "Experimental Procedures." Conductivity and absorbance were determined for each fraction. The arrow indicates the elution position of collagen Type I (90 ml). Proteins elutingin this peak were dialyzed, precipitated with 66% ethanol, and dissolved in 0.2 N NaCl, 1 mM phenylmethylsulfonyl fluoride, 50 mM Tris, pH 7.5. The resulting material was analyzed by SDS-PAGE and gel staining (inset) as described in Fig. 1.
Northern blots. The cultured cells contained, before Pronase treatment, 5.5- and 2.2-kb species of collagen d(1) and albuminmRNA,respectively(Fig. 4, lane l ) . After Pronase
Vasopressin Inhibits
9587
Gene Expression
CollagenAlbumin and 2000 -
1 ”
1
1500-
X
r
L
W
+ N o
- 5.5 kb
h
k
s
1000-
V
n
500-
0-
- 2.2
I
kb
0
5
15
10
20
25
30
Time (Min.)
FIG.5. Effects of vasopressin on
4 FIG.4. Detection of n l ( 1 ) collagen and albumin mRNA in primary cultures of adult rat hepatocytes before and after pronase treatment. Twelve-day-old cultures and total cellular RNA preparations were generated as described under “Experimental Procedures.” Poly(A)’ RNA was obtained and electrophoresed ona formaldehyde, 1% agarose gel and transferred to a nylon filter by capillary blotting (2pg of RNA loaded/lane). The quality and integrity of RNA following electrophoresis was checked by visual examination of ethidium bromide-stained gels under transillumination. Bound RNA was hybridized to RzP-labeledcDNA plasmids (human collagen al(1)and mouse albumin) as described under “Experimental Procedures.” The filters were exposed to x-ray film at -70 “C with an intensifying screen. Lanes 1-3 show RNA from untreated cells (undiluted, diluted 1:16, diluted 1:64,respectively); RNA from Pronase-treatedcultures,obtained as described under“Experimental Procedures,” is shown in lane 4. treatment, neither collagen mRNA nor albumin mRNA were detected (Fig. 4, lane 4 ) , although quantitative yields (-20% of the total RNA obtained from untreated cultures (10-40 pg RNA/106 cells)) of intact 18 S and 23 S RNA were recovered together with a strong B actin signal, the internal cDNA hybridization control used in the Northern blot studies (not shown). The lack of albumin mRNA signal (Fig. 4, lane 4 ) indicated effective Pronase digestion of hepatocytes, since albumin mRNA was negligibleor not detectable in dilutions of total RNA recovered fromuntreated cultures (%6 and Vm), (Fig. 4, lanes 2 and 3, respectively). Thus, the results from both dual-labelingand RNA blotting indicated that hepatocytes generated the bulk of the Type I collagen under the conditions of long-term culture that were used in these experiments. Effects of Vasopressin on Protein Synthesis and 4sCa2+Efflux-Vasopressin inhibited collagen production in a dosedependent fashion in 12-day-old primary hepatocyte cultures (Table I). Significant inhibitory effects were observed at 10 nM peptide. This response was obtained with either synthetic pharmaceutical preparations, a 99% pure octapeptide, or synthetic [8-arg]vasopressin. The averagemaximal inhibitory effect was about 50% (IDso -80-100nM); only small (1020%) and generally insignificant effects on noncollagen protein production were observed (Table I). Similar results were obtained when the cultures were fluid changed into serumfree medium containing [8-arg]vasopressin(data not shown). Vasopressin alsostimulated initial rates of 4sCa2+ efflux under similar culture conditions (Fig. 5) and in a manner like that reported for vasopressin-stimulated 4sCa2+ fluxes in several nonhepatocyte cell systems (50-52). However, in contrast to
“Ca2+ efflux in primary cultures of adult rat hepatocytes. Twelve-day-old cultures were incubated for 24 h with spent plating media (1.5 ml) in the presence of 33 pCi 4sCaC12(1.5 mM). Media were aspirated,cultures were washed three times with 2 ml of fresh plating media, and thenshifted (time zero) into 2 ml of the same media without or with 100 nM [8arglvasopressin. Fifty rl of culture fluids were sampled from triplicate cultures in both groups a t varying times thereafter. Values shown are mean f S.E.; p < 0.05 up to 10 min.
TABLE I1 Effect of vasopressin oncollngen production in nonheDatic cells in culture Experimental condition” nM
Collagenb
Noncollagen
proteinb
dpm x IO”
Relative collagen
production‘ %
Swiss 3T3 cells Control Vasopressin 25 Vasopressin, 50 Vasopressin, 100
4.0 f 0.2 6.2 f 0.9 4.9 f 0.5 5.5 f 0.5
285.8 f 16.7 245.8 f 88.7 306.4 f 25.8 335.3 f 25.9
1.4 f 0.1 1.6 f 0.1 1.6 f 0.1 1.6 f 0.1
Myofibroblasts Control Vasopressin, 100
6.3 f 1.0 7.1 f 0.4
604.1 f 19.5 545.4 f 7.0
1.0 f 0.2 1.3 f 0.1
Smooth muscle cells Control Vasopressin, 100
2.8 f 0.1 166.8 f 11.0 1.6 f 0.1 1.7 f 0.1 2.2 f 0.1 126.9 f 10.9 “Confluent cells were incubated for 4 h with or without [8-arg] vasopressin. b.e Determined as described in Table I.
primary hepatocyte cultures, Table I1 shows that vasopressin did not attenuate collagen production in confluent cultures of Swiss mouse 3T3 fibroblasts, human myofibroblasts, or rat smooth muscle cells. Vasopressin’sinhibitory effects on cultured hepatocyte protein synthesis were selective,but not entirely specific, as the levels of newly synthesized and secreted albumin were also decreased 50% after 4 h of exposure to 100 nM peptide (Fig. 6 A ) whereas the levels ofnewly synthesized and secreted fibrinogen, another major hepatocyte-specific plasma protein, were unaffected (Fig. 6 B ) . Effects of A23187, Glucagon, and Dibuty&cAMP on Collugen Production-calcium ionophore A23187 (0.6 PM) decreased the production of newly synthesized collagen in primary hepatocyte cultures (Table 111). Similar results were obtained with cultured smooth muscle cells and myofibroblasts (data notshown) and with human fibroblasts (15). The ionophore’s inhibitory effects were detected after 2 h of incubation and persisted for at least 6 h after contact with cells; full attenuation required exposure up to 24 h (not shown). Unlike vasopressin, the ionophore exerted more of a nonspe-
Vasopressin InhibitsCollagen and Albumin Gene Expression
9588
kDo
t-
b
-200
-116
- 93 - 66
a
-45
FIG. 6. Effects of vasopressin on albumin and fibrinogen synthesis in primary cultures ofadult rat hepatocytes. Twelveday-old cultures were incubated with 60 pCi of ["SS]methionine in fresh methionine-free plating media for 3 h. Newly synthesized secreted albumin (panel A ) and fibrinogen a, 8, and y chains (panel B ) were recovered from culture fluids specifically by immunoprecipitation using rabbit anti-rat albumin or rabbit anti-human fibrinogen antibodies, respectively. Immune complexes were precipitated with Protein-A, washed, and centrifuged. Immunoprecipitates were pretreated with 100 mM dithiothreitol and analyzed by SDS-PAGE and the resulting gels subjected to autoradiography with intensifying screens as described under "Experimental Procedures." The specificity of the immunoprecipitation reactions was indicated by the failure to immunoprecipitate similar radiolabeled bands when culture fluids were incubated under similar conditions but with preimmune sera (data notshown).
TABLE I11 Effect of Ca2' ionophore A23187 on collagen production in primary cultures of adult rat hepatocytes Experimental condition*
Relative
2-h Incubation Control A23187
Noncollaen cottagen proteinb
Cottagenb
nrodr~ctinn'
dpm
Albumin
X
1.3 f32.2 0.1 0.4 f 0.1
10"
%
f3.9 7.1 17.6 f 1.4 2.4
& 0.8 & 0.4
6-h Incubation 56.4 f 3.4 3.5 & 0.4 2.2 f 0.3 Control 0.7 f 0.1 38.7 f 6.4 1.9 f 0.1 A23187 a Twelve-day-old primary rat hepatocyte cultures were incubated with or without A23187 (0.6 p ~ )as, described under "Experimental Procedures." Control cultures received ionophore diluent (17 mM ethanol, final concentration in the medium). *.' Determined as described in Table I.
TABLE IV Effect of glucagon and CAMP on collagen production in primary cultures of adult rat hepatocytes
FIG.7. Effects ofvasopressin on al(1)collagen and albumin steady-state mRNA levels in primary cultures of adult rat hepatocytes. Twelve-day-old cultures were fluid changed and incubated in the absence (-) or presence (+) of 100 nM [8-arg]vasopressin for 3 h asdescribed under "Experimental Procedures." Poly(A)' RNA was isolated, electrophoresed, and hybridized to ["'PJcDNA probes as described in Fig. 4. cific "biphasic" effect, particularly at 2 h, when a significant reduction in noncollagenprotein was observed that was somewhat dissipated by 6 h. In contrast to vasopressin and A23187, neither 10-100 nM glucagon (a CAMP-generating dose range in this system (53)) nor 1 PM dibutyryl CAMPaffected collagen production significantly in 12-day-oldhepatocyte cultures (Table IV). Effects of Vasopressin on Collagen and Albumin mRNA LeueLs-Twelve-day-old hepatocyte cultures were incubated with 100 nM vasopressin; RNA was recovered from the cultures 3-4 h later and analyzed for hybridization to different "P-labeled cDNA probes.(No morphological differenceswere observed in either treated or mock-treated cultures prior to isolation of the RNA which, in both cases, was obtained in similar yield and free of degradation as analyzed onethidium bromide stained gels.) Northern blots revealed (Fig. 7) that vasopressin-treated cells contained levels of 2.2-kb albumin and 5.5-kb collagend ( 1 )mRNA that, in comparison to mocktreated cells, were reducedby 50 and 70%, respectively. Similar attenuation also was seen in the level of a second putative al(1) mRNA speciesof -6 kb (Fig. 7) and, again, for allthree mRNAspeciesin cultures treated with A23187 (data not shown). In contrast, the hybridization of "'P-labeled probes to 2-kb p actin mRNAwas similar in its intensity when analyzed on Northern blots of RNA fromvasopressin-treated and mock-treated cultures (data not shown). DISCUSSION
dpm X lo"
Control Glucagon, 10 nM Glucagon, 100 nM Dibutywl CAMP, 1 pM
2.2 f 0.4 2.5 f 0.3 3.3 f 0.4 2.2 f 0.5
124.3 f 16.0 120.9 f 20.7 139.8 f 8.7 109.3 f 9.0
%
1.7 & 0.1 2.0 f 0.1 2.5 k 0.2 2.0 f 0.3
Twelve-day-old primary rat hepatocyte cultures were incubated for 3 h with no additions or in the presence of glucagon or CAMP,as described under "Experimental Procedures." b.c Determined as described in Table I.
Developmental, proliferative, and functional processes in animal cells are markedly dependent on extracellular matrices in contact with the cell surface (54). Recent reports of proliferative and differentiated properties of short-term proliferation competent primary cultures of adult rat hepatocytes, cultured on plastic substrata coated with differing combinations of matrix components, lend further support to this biological principle (55, 56). As major components of various tissue matrices, collagenous proteins are being scrutinized for their structural properties and cellular attachment sites (57);
Vasopressin Inhibits
CollagenAlbumin and
Expression Gene
9589
the molecular mechanisms that regulate collagen production Similar conclusions were reached from similar studies using short-term primary culturesof adult rat hepatocytes (65,66). (58). In hepatic tissue, also are under intense investigation Taken together, the combined results clearly indicate that where collagen overproduction is associated intimately with liver cirrhosis, controversy prevailsover which hepatic cell(s) hepatocytes synthesize TypeI collagen i n vivo and i n vitro. Other investigators found that contamination with hepatic synthesize and secrete collagen, the chemical type(s) of collagen produced, and the regulatory mechanisms involved. In lipocytes (“1t0” cells) are responsible for the increased collathis report, using well-differentiated long-term proliferation-gen production in primary hepaticcell cultures (from -2.5 to competent primary cultures of adult rat hepatocytes as a -9% relative rates) undersome culture conditions(10). These short-term cultures,however, display sharp differences in the model system, we providebiochemicalevidence fromdual [3H]proline/[’4C]ornithinelabeling studies showing that hep- behavior of the recovered cell populations that eventually atocytes synthesize and secrete Type I collagen. We also have survive during the firstweek postplating. Thisis not surprisfound that vasopressin, an osmoregulatory nonapeptide (59) ing since fundamental differences exist between the two prirapidly reduces the synthesis of both albumin and Type I mary systems with respect to the conditions of hepatocyte collagen together with coordinate reductions of -50 and 70%, isolation, plating, and culturing. In the short-term cultures, cell respectively, in the mRNA levels for both of these proteins. although special measures are used to “purify” the initial suspension recovered following tissue dispersion and to coat These observations suggest that vasopressin regulates hepathe plastic culture dishes with Type I collagen, the plated tocyte gene expression. overgrown by nonSeveral findingsreinforce this conclusion. First, in addition hepatocytes eventually deteriorate and are parenchymal contaminants. Moreover, in our studies, 12-dayto the pharmaceutical preparations employed, the synthetic analog [8-arg]vasopressin (CYFQNCPRG) also reduced col- old hepatocyte cultures produced collagen at rates that were similar (2% of total proteins) to thoseobserved in “nonconlagen and albumin synthesis and the mRNA levels of each of taminated 8-day-old hepatocyte cultures following centrifuthese proteins in these cultures. These results eliminate the gal elutriation and gradient separation (10). In addition, the possibility that nonspecific contaminants in the pharmaceuproportion of [14C]arginine recovered in newly synthesized tical preparations acted as the inducing agents. The i n vitro collagen was that expected if 70% of the collagen were prorequirement of supraphysiological levels of vasopressin (its duced by hepatocytes. We recognize that a potential pitfall of basal plasma levels range between 2-7 PM, its plasma halfthe dual isotopic labeling approach is that a fraction of the life ranges 17-35 min, and -33%of its clearance in rats is [14C]arginine produced by hepatocytes might be transported performed by the liver (60) has not yet been explained, but it t o nonparenchymal cells and incorporated into their proteins. is unlikely to be the consequence of long-term hepatocyte Were this tooccur to any significant extent, itwould clearly culture conditions since nanomolarlevels of [8-arg]vasopres- complicate the interpretation of an hepatocellular origin of sin also were required for the activation of the sodium pump synthesis of an ubiquitous protein-like Type I collagen. Howand 45Ca2+ fluxes in suspension cultures of freshly isolated ever, since the amount of [14C]arginine present in the media adult rat hepatocytes (17,19). Second, although isitarguable after the 4-h incubation period used in the current studies that vasopressin’s affects are indirectvia the peptide’s inter- was only 0.003%of the starting dpm ((3000/1.1 x 10’ dpm actions with nonparenchymal cell subpopulations present in [14C]ornithine)), itwould seem unlikely that any significant hepatocyte cultures, this seems unlikely as vasopressin failed amount of extracellular [14C]arginine (relative to[‘Hlproline) to elicit detectable effects on Type I collagen production in could have entered the precursorpool of protein synthesis in three different kinds of nonhepatic cell cultures, including nonparenchymal cells (where further reductions in arginine’s smooth musclecells,Swiss 3T3fibroblastsand myofibro- specific activity would have occurred) andhave been detected blasts,all of which harborvasopressinreceptors (50-52). in newly synthesized protein (see also Ref. 42). Direct cell-toFurthermore, the Pronase digestion of hepatocytes in primary cell transfer of [I4C]arginine from hepatocytes to nonparencultures eliminated all of the hybridizable albumin and Type chymal cellsalso would seemunlikely inthe absence of I collagen mRNA molecules detected in Northern blots, inelectronmicroscopically detectable intercellular junctionsbedicating thata Pronase-sensitive cell population was respon- tween hepatocytes and nonparenchymalcells in vitro.’ sible for both albumin and most of the collagen production in Further experiments are needed to delineate the mechathese cultures. Third,vasopressin’s ability to inhibit albumin nism(s) and the causal relationships, if any, by which vasosynthesis and attenuate the levels of albumin mRNAreflected pressin lowers the synthesis and mRNA levels of Type I a specific effect on hepatocytes, as the synthesis of albumin collagen andalbuminincultured hepatocytes. By itself, in these cultures and in vivo is solely due to expressionof this CAMP, althoughimplicated earlier in the regulation of collagene in hepatocytes. The failure of vasopressin to inhibit the gen synthesis in certain fibroblast systems (67), is apparently synthesis of fibrinogen, another uniquely hepatocyte-specific not involved, since neither dibutyryl-CAMP nor glucagon (a function,furtherindicatesthat vasopressin’seffects were CAMP-generating peptide in these cultured hepatocytes (53)) selectively pleiotropic. These findings, plus morphological affected collagen and albumin production. observations showing no obvious cellular alterations in vasoThe available evidence suggests involvementof one or more pressin-treated cultures,imply that sucheffects on hepatocyte Ca’+-dependentprocesses,possiblyincluding activation of function did not arise fromgeneralized cytotoxicity. protein kinase C (68): (i) vasopressin accelerates 4sCa2+fluxes The use of the dual-labeling method in these i n vitro studies, in this andin other adult rat hepatocyte systems(16, 17), (ii) to identify the hepatocellular origin of a ubiquitous protein- vasopressin’s effects are reportedly additive with phorbol eslike Type Icollagen, has been validated earlier by i n vivo ters that rapidly activate hepatocyte Na+-K+ [ATPase] and studies using adult rats (25); observations made with long- =Rb+ flux (68),and(iii) calciumionophore A23187 also in vivo findings. inhibits collagen andalbuminsynthesisintheseprimary term cultures are entirely consistent with Moreover, direct evidence fromstudies employing insitu cultures and collagen synthesis in cultured fibroblasts(15). hybridization (61) and immunohistochemistry combined Although the ionophore concentration that was used in the either with lightmicroscopic (62) or withelectronmicroscopic present studies bordered upon a level that suppresses initia(63, 64) analyses of liver tissue sections also indicated previI collagen i n vivo. K. Dempo and H. L. Leffert, unpublished observations. ously that hepatocytes synthesize Type
9590
Vasopressin InhibitsCollagen and Albumin Gene Expression
tion of hepatocyte DNA synthesis in this system (76), its nonselective inhibitory effects on hepatocyte protein synthesis dissipated with time. Lastly, recent work indicates that collagenase (69) and albumin gene expression (70, 71) are regulated by a complex network of nuclear factors that bind specifically to regions of untranslated 5”control sequences in both genes. Whether vasopressin affects some of these transacting factors remains to be determined. It is reasonable to ask if the inhibitory effects of vasopressin observed in thishepatocyte culture system are physiologically relevant in uiuo. While these cultureshave demonstrated their physiological relevance in many instances, particularly when used to probe into the mechanisms controlling liver regeneration (21, 31, 44, 49), thus far there is no evidence that liver collagen and albumin gene expression might be regulated by vasopressin. On the other hand, there is mounting evidence that various stressful conditions clearly alter hepatocyte gene expression. For example, malnutrition (30), inflammation (72), and exposure to tumor necrosis factor (Y (73) inhibit hepatic albumin synthesis and alter the distribution of hepatocyte production of many of the acute-phase proteins (74). Further studies of hepatic vasopressin levels and receptor function in various physiological states andin various animal models, like vasopressin-deficient Brattleboro rats (75), combined with primary culture studies of hepatocytes from such animals, may provide new insightsinto the regulation of hepatic gene expression and protein synthesis in normal and diseased liver. Acknowledgments-We thank Michael Filip, Martina Buck, Hal Skelly, and Linda Veloz for their excellent technical assistance and Kris Beaver for her skillful . Preparation of this manuscriDt. . REFERENCES 1. Seyer, J. M., Hutchenson, E. T., and Kang, A. H. (1977) J. Clin. Znuest. 5 9 , 241-248 2. Rojkind, M., Giambrone, M.-A., and Biempica, L.(1979) Gastroenterology 76, 710-719 3. Chojkier, M., and Brenner, D. A. (1988) Hepatology 8, 176-182 4. Rojkind, M., Gatmaitan, Z., Mackensen, S., Giambrone, M.-A., Ponce, P., and Reid, L. M. (1980) J. Cell B i d . 87, 255-263 5. Hata, R.-I., Ninomiya, Y., Nagai, Y., and Tsukada, Y.(1980) Biochemistry 19, 169-176 6. Diegelmann, R. F., Guzelian, P. S., Gay, R., and Gay, S. (1983) Science 219, 1343-1345 7. Tseng, S. C. G., Smuckler, E. A., and Stern, R. (1983) Hepatology 3,955-963 8 Tsutsumi, M., Takada, A., Takase, S., and Ooshima, A. (1988) Lab. Znuest. 58, 88-92 9. Friedman, S. L., Roll, F. J., Boyles, J., and Bissell, D. M. (1985) Proc. Natl. Acad. Sci. U. S. A . 82,8681-8685 10. Maher, J. J., Bissell, D. M., Friedman, S. L., and Roll, F. J. (1988) J. Clin. Znuest. 8 2 , 450-459 11. Voss, B., Rauterbert, J., Pott, G., Brehmer, U., Allam, S., Lehmann, R., and Bassewitz, D. B. v. (1982) Hepatology 2, 19-28 12. Guzelian, P. S., Lindblad, W. J., and Diegelmann, R. F. (1984) Gastroenterology 86,897-904 13. Weiner, F. R., Czaja, M. J., Jefferson, D. M., Giambrone, M.-A., Tur-Kaspa, R., Reid, L. M., and Zern, M.A. (1987) J. Biol. Chem. 262,6955-6958 14. Leffert, H. L., and Koch, K. S. (1982) Cold Spring Harbor Symp. Cell Proliferation 9 , 597-613 15. Flaherty, M., and Chojkier, M. (1986)J. Biol. Chem. 261,1206012065 16. Thomas, A. P., Marks, J. S., Coll, K. E., and Williamson, J. R. (1983) J. Bid. Chem. 258,5716-5725 17. Rhodes, D., Prpik, V., Exton, J. H., and Blackmore, P. F. (1983) J . B i d . Chem. 258, 2770-2773 18. Thomas, A. P., Alexander, J., and Williamson, J. R. (1984) J . Biol. Chem. 259, 5574-5584 19. Exton, J. H. (1988) Hepatology 8, 152-166 20. Exton, J. H. (1988) FASEB J. 2 , 2670-2676
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