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G Proteins Are Involved in the Suppression of Collagen α1(I) Gene Expression in Cultured Rat Hepatic Stellate Cells
Cell. Signal. Vol. 10, No. 3, pp. 173–183, 1998 Copyright 1998 Elsevier Science Inc.
ISSN 0898-6568/98 $19.00 PII S0898-6568(97)00036-3
G Proteins Are Involved in the Suppression of Collagen a1(I) Gene Expression in Cultured Rat Hepatic Stellate Cells Jose´ A. Solı´s-Herruzo,* Inmaculada Herna´ndez, Paz De la Torre, Inmaculada Garcı´a, Jose A. Sa´nchez, Inmaculada Ferna´ndez, Gregorio Castellano and Teresa Mun˜oz-Yagu¨e Gastroenterologı´a, Centro de Investigacio´n, Hospital Universitario “Doce de Octubre,” Universidad Complutense, Madrid, Spain
ABSTRACT. We analyse the role of the G proteins in regulating collagen gene expression by measuring collagen a1(I) mRNA levels in cultured hepatic stellate cells in basal conditions and after stimulating or inhibiting the major intracellular signalling pathways. Stimulation of Gs protein and adenylyl cyclase or the addition of 8Br-cAMP to the cells led to a decrease in collagen a1(I) mRNA levels, while blocking protein kinase A abolished this effect. Blocking Gi protein, phospholipase A2 and C, calcium channels and calmodulin resulted in a significant increase in collagen mRNA levels. PKC stimulation led to a marked decrease in these levels. These results suggest that collagen gene expression is inhibited by a number of intracellular pathways. A Gs and a pertussis toxin-sensitive G protein seem to initiate cellular response. Transcription factors, acting in these pathways, must be identified. However, it seems that they do not need to be synthesised. cell signal 10;3:173–183, 1998. 1998 Elsevier Science Inc. KEY WORDS. Collagen, Fibrogenesis, Gene expression, Hepatic stellate cells, Signal transduction pathways
INTRODUCTION Hepatic stellate cells (HSC) (fat-storing cells, Ito cells, hepatic lipocytes) are sinusoidal cells, localised within the space of Disse of the liver, that have been identified as the principal cellular source of extracellular matrix in chronic active liver disease [1]. In normal liver, these cells are recognised because they also express desmin and contain prominent intracellular droplets storing vitamin A [2, 3]. In cirrhotic liver, large numbers of transitional cells with characteristics of both HSC and myofibroblastlike cells are present [4]. Little is known about mechanisms and intracellular pathways involved in modulating collagen gene expression by cultured HSC in basal conditions. It is well known that the surface of cells is a target of chemicals, cytokines, hormones, growth factors and physical signals that activate membrane-bound receptors. These activated receptors promote a cascade of signals that passes through a set of coupling proteins, called G proteins, to intracellular effector enzymes or ion channels. Changes in effector activity cause changes in second messenger levels (cyclic adenosine monophosphate (cAMP), inositol 1,4,5-triphosphate (IP3), eicosa*Author to whom correspondence should be addressed at: Hospital “12 de Octubre.” Servicio de Aparato Digestivo, C/Andalucia, Km 4.5, 28041Madrid, Spain. E-Mail: [email protected] This study was supported in part by Grants 90/145, 90/148 and 95/609 from the “Fondo de Investigaciones Sanitarias,” and PB-92/316 and PB94/ 001 from DGICYT, Spain. Received 26 July 1996; and accepted 18 December 1996.
noids, diacylglycerol (DAG), ceramide) or in ionic composition (Ca21, K1, Na1), that ultimately lead to a cellular response, including the modulation of gene expression [5, 6, 7, 8]. G proteins are heterotrimers that are made up of a and bg subunits and couple many kinds of cell-surface receptors. Upon binding of an agonist, the receptor becomes activated and the a-subunit of G proteins undergoes a conformational change, allowing GTP to bind in place of GDP. Once GTP is bound, the a subunit dissociates from the receptor and from bg subunit and interacts with effectors. The function of a G protein depends on its subunit structure. There are four major families of G proteins. The Gs family, which activates adenylyl cyclase to produce cAMP. The Gi family, composed of: (1) the Gi protein, which inhibits adenylyl cyclase and voltage-sensitive Ca21 channels and activates atrial K1 and renal Na1 channels and a phosphoinositidespecific phospholipase C (PLC), (2) Go protein, which inhibits the Ca21 channels in neurons, and (3) Gt protein (transducin), which activates cGMP phosphodiesterase of the retinal rods. The Gq family, which activates PLC-b and, finally, the G12 and G13 family, whose function is not well known. While cholera toxin (CTX) activates Gs protein indefinitely, pertussis toxin (PTX) inhibits Gi, Go and Gt proteins [5, 6, 7, 8]. The role played by the G proteins in the control of collagen gene expression is not known. However, preliminary studies suggest that these proteins may be involved in the
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suppression of this gene [9]. The aim of this study was to analyse the role played by the G proteins in regulating collagen gene expression. We used a line of HSC obtained by Greenwel et al. [10] from a cirrhotic liver of carbon tetrachloride-treated rat. This cell line displays many features of activated HSC, and has proved to be very useful in studying factors that regulate extracellular matrix production.
MATERIALS AND METHODS The HSC line CFSC was used in these experiments. These cells were a gift from Dr. M. Rojkind and P. Greenwel (Albert Einstein College of Medicine, NY) and were derived from a carbon tetrachloride cirrhotic rat liver after spontaneous immortalization in culture [10]. The cells have been maintained in culture for over 100 passages and have maintained the same phenotype with regard to cell proliferation and extracellular matrix production since passage 30. This cell line expresses a phenotype similar to that of early passage of fat-storing cells, and has morphologic features of myofibroblasts [10]. Confluent CFSC were cultured at 378C, in an atmosphere of 5% CO2, 95% air in cell culture flasks using 10 ml minimum essential medium Eagle with Hanks’ BSS (HMEM) containing 10% foetal calf serum (FCS) (Flow, Irvine, UK). Cells were plated at a density of 5 3 106/80 cm2 flask and studies were performed after 4–6 days: Intracellular signal-transduction pathways involved in the regulation of collagen gene expression were explored by measuring the steady-state levels of collagen a1(I) mRNA and the activity of chloramphenicol acetyl transferase (CAT) in CFSC after their treatment with inhibitors or, eventually, activators of these pathways. The role played by G proteins was studied by incubating these cells with 0.05–0.4 mg/ml CTX (Sigma Chemical Co. Alcobendas, Madrid, Spain) or with 1–10 ng/ml PTX (Sigma). Adenylyl cyclase/cAMP/protein kinase A (PKA) pathway was explored by using 100 mM forskolin (Sigma), 1 mM 8Br-cAMP (Sigma) or 50 mM N-(2-methylamino) ethyl)-5 isoquinolinesulfonamide dihydrochloride (H-8). The role of PLC was examined by treating cells with 500 mM neomycin. The role played by intracellular calcium was studied by blocking calcium channels with 100 mM verapamil (Sigma) and by increasing intracellular calcium levels with 10 mM calcium ionophore A23187 (Sigma). Calmodulin was inhibited with 10 mM calmidazolium (Sigma), 10 mM trifluoperazine dimaleate (TFP) (Calbiochem, San Diego, CA) or 10 mM N-(6 aminohexyl)-5-chloro 1 naphthalenesulfonamide (W-7) (Sigma). Protein kinase C (PKC) was inhibited with 50 mM 1-(5-isoquinolinesulfonyl)-2-methylpiper dihydrochloride (H-7) (Sigma), or 0.5 nM staurosporine (Sigma), and stimulated with 10 nM phorbol 12-myrisrate 13-acetate (PMA) (Sigma) for 24 h, or 63 mM oleyl acetyl glycerol (OAG) (Sigma). The role played by newly synthesised proteins was studied by blocking protein synthesis with 0.1 mM cycloheximide (Sigma). This concentration of cycloheximide blocked protein synthesis by 96% in these CFSC but did not affect the
FIGURE 1. Cholera toxin induces a time- and dose-dependent
decrease in collagen a1(I) mRNA levels. (A) Dose-response effect of cholera toxin on the levels of collagen a1(I) mRNA. Confluent CFSC were incubated for 24 h in HMEM and 10% FCS in the absence or presence of increasing concentrations of cholera toxin (CTX). (B) Time course of the effect of cholera toxin on collagen a1(I) mRNA levels. Cells were treated with 0.4 mg/ml cholera toxin for 1–24 h. Ten mg of total RNA were electrophoresed on a formaldehyde, 1% agarose gel and transferred to a nylon filter by capillary blotting. The quality and integrity of RNA following electrophoresis was checked by visual examination of ethidium bromide stained gels under transillumination. Bound RNA was hybridised to 32P-labelled cDNA probe [rat collagen a1(I)] as described under Methods. The filters were exposed to X-ray film at 2708C with an intensifying screen. The autoradiograms were quantitated by scanning laser densitometry. The level of collagen a1(I) mRNA in each sample was normalised to the level of a-tubulin mRNA. Coll.a1(I): collagen a1(I) mRNA; a-Tubulin: a tubulin mRNA; Coll.a1(I)/a-Tub.: Collagen a1(I)/a-Tubulin mRNA ratio.
steady state level of collagen a1(I) mRNA. All these experiments were repeated at least 3 times. Preparation of Complementary DNA (cDNA) Probes Rat cDNA for the alpha 1 chain of type I collagen (1.6 kilobase Pst1 fragment) was generously provided by Dr. D. Rowe (Farmington, CO) [11]. The cDNA fragments were radiolabelled with [a-32P]-dCTP (specific activity 5 3000 Ci/mmol) using a primer extension kit (Multiprime DNA labelling system from Amersham International, England, UK) to a specific activity of 1 3 109 cpm per mg of DNA. Northern Blotting RNA was isolated from confluent cells in culture by the phenol/chloroform extraction method of White and Ban-
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TABLE 1. Effect of blockade or, eventually, activation of intracellular signal transduction pathways on the steady-state level of collagen a1(I) mRNA
a1(I) collagen mRNA a-tubulin mRNA ratio (% of control) Pathway
Agent
Experiment 1
Experiment 2
Experiment 3
Means 6 SD
100.0%
100.0%
100.0%
100.0%
Control
Culture medium without additive
Gs protein/ Adenylyl cyclase PKA
Cholera toxin, 0.4 mg/ml Forskolin, 100 mM 8 Br-cAMP, 1 mM H-8, 50 mM 1 Cholera toxin, 0.4 mg/ml
34.5 51.8 69.7
22.7 52.3 62.5
26.1 49.6 65.8
27.8 6 6.0 51.2 6 1.4 66.0 6 3.6
105.0
97.3
107.7
102.5 6 4.5
Gi-Proteins
Pertussis toxin, 1 ng/ml 5 ng/ml 10 ng/ml
153.6 184.4 205.5
129.0 167.4 198.8
140.8 178.2 186.3
141.1 6 12 176.4 6 8.6 196.8 6 9.7
PLC
Neomycin, 500 mM
205.9
221.0
177.0
201.3 6 22.3
Calcium
Verapamil, 100 mM A 23187, 10 mM
216.1 79.6
164.8 39.4
198.0 53.7
193.0 6 26 57.5 6 20
Calmodulin
TFP, 10 mM W-7, 10 mM Calmidazolium, 10 mM
204.1 212.4 141.1
202.0 232.0 129.0
236.0 136.0 147.0
180.7 6 38.7 226.8 6 12.6 139.0 6 9.1
PKC
PMA 10 nM, 24 hours Staurosporine, 0.5 nM
47.4 90.3
58.0 108.0
72.0 92.0
59.1 6 12.3 96.7 6 9.7
PLA2
BPB, 20 mM Quinacrine, 0.5 mM 1.0 mM 2.0 mM
156.1 149.0 181.0 179.0
175.8 122.0 169.0 183.0
153.8 178.9 182.4 238.1
161.9 6 12.1 149.9 6 28 177.4 6 7.2 200.0 6 33
Cycloxigenase
Indomethacin, 100 mM
245.0
186.5
185.2
205.5 6 34
Lipoxigenase
NDGA, 24 mM
112.8
98.3
122.0
111.0 6 12
Protein synthesis
Cycloheximide, 0.1 mM
108.2
102.4
99.6
103.4 6 4.4
Confluent CFSC were cultured in 10 ml HMEM containing 10% FCS. Intracellular signal transduction pathways involved in the regulation of collagen gene expression were explored by measuring the steady-state levels of collagen a1(I) mRNA and a-tubulin mRNA in CFSC after their treatment with inhibitors or activators of these pathways. The level of collagen a1(I) mRNA in each sample was normalised to the level of a-tubulin mRNA.
croft [12]. Ten mg of total RNA were electrophoresed in a 1% agarose 2.2 M formaldehyde gel in 20 mM 3-(N-morpholino) propanesulfonic acid buffer. RNA samples were stained with ethidium bromide and transferred overnight by capillary blotting in 20 3 sodium saline citrate (SSC) to nylon filters (Amersham International, UK). The RNA was UV-crosslinked to the filter. The nylon filters were prehybridised at 658C in 7% sodium dodecyl sulphate (SDS) and 0.5 M buffer phosphate (pH 7.0) and 50 mg/ml of sonicated salmon sperm DNA. The nylon filters were hybridised overnight at 658C in freshly prepared hybridisation solution with addition of 106 cpm/ml of the labelled cDNA probe. The nylon filters were washed at a stringency of 2 3 SSC, 0.1% SDS at room temperature for 30 min and 0.1 3 SSC, 0.1% SDS at 658C for 15 min and exposed to X-ray film at 2708C with an intensifying screen. The autoradiograms were quantitated by scanning laser densitometry DeskTopY Scanner Plus (Pharmacia Biotech SA Barcelona, Spain). The level of collagen a1(I) mRNA in each sample was normalised to the level of a-tubulin mRNA (Clontech Laboratories Inc., Palo Alto, CA).
Chloramphenicol Acetyl Transferase (CAT) Assay CFSC were transfected prior to confluence by electroporation using a Cellject electroporation system (Eurogentec. Liege, Belgium). The transfected plasmid (COLCAT1) included part of the first exon and the promoter region of the mouse alpha 1 type I collagen gene fused to the bacterial CAT gene [13]. This plasmid was generously provided by Dr. D. Brenner (University of North Carolina, Chapel Hill, NC). On the day before transfection, cells were plated at a density of 4 3 104/cm2 in 160-cm2 flasks. Cells were harvested with trypsin and centrifuged at 250 g. The pellet was washed 3 times with PBS and resuspended in culture medium. Cell density of this suspension was 1 3 107/ml. Onehalf ml of cell suspension was transferred to a tube and sonicated salmon sperm DNA (40 mg/ml) and plasmid DNA (18 mg/ml) were added. Electroporation was performed at 08C by delivering two pulses: the first of 700 V at 40 mF and the second of 150 V at 1200 mF. All electroporated cells were collected in the same tube, mixed, plated in 80 cm2 flasks for different experimental conditions and incubated at 378C. Inhibitors or activators of intracellular signal trans-
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International, plc., UK) according to the manufacturer’s instructions. Cells were incubated at 378C with either control medium or medium containing 10 ng/ml PTX, 0.4 mg/ml CTX, 1 mM 8 Br-cAMP or 100 mM forskolin for various periods of times. The cells were subsequently placed on ice and the medium was removed. Cultures were rinsed twice with cold PBS and then scraped in 10 ml of cold 65% ethanol. The cells were then pelleted by cold centrifugation at 2000 3 g for 15 min and the resulting supernatants were dried under vacuum. The extracts were dissolved in 2 ml assay buffer before Elisa analysis. PKA Assay
FIGURE 2. Northern blot hybridisation analysis demonstrating
the effects of cholera toxin (0.4 mg/ml), forskolin (100 mM), 8 Bromo-cyclic adenosine monophosphate (1 mM) (8BrAMP) (A) cholera toxin (0.4 mg/ml), cholera toxin and H-8 (50 mM) and H-8 (B) on the steady-state levels of a1(I) collagen mRNA. Confluent CFSC were incubated for 24 h in HMEM, 10% FCS or in culture medium that contained cholera toxin (CTX), forskolin (Frsk), 8 bromo cyclic AMP (8BrAMP) or H-8. C: control untreated cells; Coll.a1(I): collagen a1(I) mRNA; a-Tubulin: a tubulin mRNA; Coll.a1(I)/a-Tub.: Collagen a1(I)/ a-Tubulin mRNA ratio. The results presented are representative of 2 to 4 separate experiments.
duction pathways were added to the cells 6 h after electroporation, and this treatment was generally maintained for 48 h. Cultured cells were harvested and transferred to a microcentrifuge tube on ice. These cells were lysated by three cycles of freezing in dry ice/ethanol for 5 min and thawed at 378C for 5 min. The particulate was removed by centrifugation at 10,000 3 g for 5 min at 48C. Protein content of the supernatant was assayed by the Bradford method [14], and aliquots containing equal amounts of proteins were analysed for CAT activity, using a modification of the method of Gorman et al. [15]. The substrate and acetylated form of [14C]chloramphenicol (Amersham) were separated by thin layer chromatography on silica plates (Scharlau, Barcelona, Spain) in 95:5 chloroform/methanol and visualised by autoradiography with Kodak X-Omat film at room temperature for 12–24 h. Autoradiograms were quantitated by a scanning laser densitometer. Individual spots were located and excised, and their [14C] content was measured by liquid scintillation spectrophotometry. Measurement of Intracellular cAMP Intracellular concentrations of cAMP were determined using an enzyme immunoassay (Biotrack cAMP, Amersham
Cells were treated with 1 mM 8Br-cAMP with or without pre-treatment with 50 mM H-8. After the indicated times, cells were suspended in 20 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA, 100 mM NaCl and 40 mg/ml leupeptin. Aliquots were taken for measurement of protein content and the rest was sonicated for 30 s. PKA activity was measured using a commercially available kit (Promega Co., Madison, WI) following the manufacturer’s instructions. Briefly, 10 ml of samples were incubated with 5 ml Pep Tag 53 reaction buffer, 5 ml Pep Tag A1 peptide and 5 ml PKA activation solution in a final volume of 25 ml for 30 min at 308C. The reaction was stopped by boiling the samples for 10 min. Afterward, samples were loaded on a 1% agarose gel and electrophoresed for 45 min in 50 mM Tris-HCl, pH 8.0, until phosphorylated and unphosphorylated Pep Tag A1 peptides were completely separated by migration to positive and negative electrodes, respectively. The band of phosphorylated peptide was excised from the gel, boiled to dissolve the agarose and mixed with 75 ml gel solubilization solution, 100 ml glacial acetic acid and 200 ml of distilled water. Absorbency of the samples was read and PKA activity was calculated and expressed in pmol/min/mg protein. The Level of Intracellular Free Ca21 This was determined by flow cytometry as described by Vandenberghe and Ceuppens [16]. CFSC (1 3 106 cells) were suspended in 1 ml HMEM and treated with 5 mg/ml TABLE 2. Effect of cholera toxin, pertussis toxin, forskolin
and 8 Br-cAMP on intracellular cyclic AMP levels cAMP levels (pmol/106 cells)
Control CTX, 0.4 mg/ml PTX, 10 ng/ml Forskolin, 100 mM 8 Br-cAMP, 1 mM
2 hours
24 hours
8.9 6 0.8 377 6 21a ND 144 6 5.6a 36.5 6 6.7b
8.7 6 1.3 259 6 15 a 36 6 2.7a 235 6 21a 78.4 6 16a
CFSC were treated with either 0.4 mg/ml cholera toxin (CTX), 10 ng/ml pertussis toxin (PTX), 100 mM forskolin or 1 mM 8 bromo cyclic AMP (8Br-cAMP) for 2 and 24 h. Cyclic AMP was determined as described in the text. Data are shown as means 6 SD of triplicate determinations. ND 5 not done. a P , 0.001 against control cells. b P , 0.01 compared with control cells.
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of the calcium sensitive Fluo-3M at 378C for 20 min. The cells were washed 33 and resuspended at a concentration of 1 3 106 cells/ml in a hepes-buffered saline, containing 137 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 5 mM glucose, 1 mM CaCl2, 0.5 mM MgCl2, 1 g/l bovine serum albumin, 10 mM hepes, pH 7.4. Before each assay, the cells were incubated for 10 min in a 378C water bath and subsequently analysed on an Epics Elite flow cytometer (Coulter Electronics, Inc., Hialeah, FL). The laser was set at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and operated at 50 mW laser power. Throughout the experiments, temperature was maintained at 378C. Green fluorescence and forward angle light scatter were collected for lineally or logarithmically amplified (4-decades, 1024 channels) signals. The mean fluorescence of events was recorded and expressed as arbitrary units (channel number). After recording basal fluorescence, cells were treated with either 10 mM A23187 or 500 mM neomycin. The effect of neomycin and PTX on cytosolic Ca21 was studied with or without stimulation with 0.6 nM TNFa.
FIGURE 3. Steady-state levels of a1(I) collagen mRNA in pertus-
sis toxin-treated cells. Confluent CFSC were incubated for 24 h in HMEM 10% FCS or in the same culture medium containing pertussis toxin (1, 5 and 10 ng/ml). O: control untreated cells; Coll.a1(I): collagen a1(I) mRNA; a-Tubulin: a tubulin mRNA; Coll.a1(I)/a-Tub.: Collagen a1(I)/a-Tubulin mRNA ratio. Blots are representative of as least three separate experiments.
This was measured as described by Berridge et al. [17]. CFSC were incubated in serum-free HMEM containing 1% bovine serum albumin and prelabeled with 5 mCi/ml myo[3H]inositol for 24 h. The cells were washed 33 with serum-free medium containing 10 mM LiCl to minimise inositol phosphate hydrolysis and further incubated at 378C for 15 min. Cells were then exposed to 0.6 nM TNFa for various times with or without preincubation with 500 mM neomycin for 2 h. Media were then aspirated and ice-cold 10% TCA added. Cell layers were washed with 0.5 ml 10% TCA and the TCA fractions were treated with water-saturated diethylether. After adjusting the pH to 7.0–7.5 with NH4OH, the ether extracts were applied to AG1X8 anion exchange Dowex columns (100–200 mesh, formate form) (BioRad, Richmond, CA). The inositol triphosphate fraction was eluted with 1 M ammonium formate in 0.1 M formic acid and radioactivity was determined by liquid scintillation counting.
cultured in HMEM supplemented with 10%. When required, cells were treated with 10 ng/ml PTX for 24 h or with 20 mM BPB or 2 mM quinacrine for 2 h. After incubation with appropriate stimuli for the indicated times, cells were scraped in the PLA2 buffer (see below) and homogenised by sonication. Aliquots of cell homogenate (up to 100 mg of protein) were incubated for 30 min at 378C in 50 mM phosphate buffer (1 mM MgCl2, 1mM EGTA, pH 7.5) 50 mM phosphatidylcholine substrate (1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3phosphocholine, 25,000 dpm/assay), in a final volume of 0.25 ml. The phospholipid substrate was used in the form of vesicles obtained by sonication in assay buffer until the solution clarified. The reactions were terminated by adding chloroform/methanol (1:2, v/v). The lipids were extracted and separated by thinlayer chromatography. Plates were run with n-hexane/diethylether/acetic acid (70:30:1, by volume) as a solvent system. Authentic arachidonic acid was cochromatographed and visualised by exposing the plates to iodine vapours. The arachidonic acid fraction was scraped from the plate and monitored for radioactivity by liquid-scintillation counting. Counts in arachidonic acid were normalised for the amount of radioactivity recovered in each lane of the plate.
Assay for PLA2 Activity
Measurement of PKC Activity
PLA2 activity was measured as described previously [18]. Cells, plated at a density of 5 3 105 cells/well in 6-well plates, were
PKC activity was measured according to the method of Kitano et al. [19]. Cultured CFSC were washed twice with ice-cold
Accumulation of Inositol Phosphates
TABLE 3.
Protein kinase A activity (pmol/min/ng protein) 5 min Control 8Br-cAMP, 1 mM 8Br-cAMP, 1 mM 1 H8, 50 mM
147 6 10 311 6 20 a b 184 6 19 c
30 min 163 6 8.6 567 6 19 a b 291 6 24 a
60 min 156 6 13 579 6 13 a b 189 6 21
CFSC were treated with 1 mM 8Br-cAMP for 5, 30 or 60 min with or without previous incubation with 50 mM H-8 for 2 h. After these times, PKA activity was measured as described in Material and Methods. Data are the mean 6 SD of one experiment with triplicate determinations. a P , 0.001 as compared with the control cells. b P , 0.001 compared with H-8 pretreated cells. c P , 0.05 as compared with the control cells.
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(100,000 3 g, 1 h) in a Bekman L-80 ultracentrifuge. Aliquots (60 ml) of the cytosolic or membrane fraction were added to 190 ml of mixture containing 20 mM Tris-HCl, pH 7.5, 5 mM magnesium acetate, 0.2 mg/ml histone type IIIs, and 10 mM [g32P]ATP. Reactions were performed in the presence and absence of 1.5 mM CaCl2, 25 m/ml a-phosphatidyl serine and 0.5 mg/ml diolein. Incubations were performed at 308C for 10 min. The reaction was terminated with the addition of 1 ml ice-cold stopping solution containing 10% TCA and 2 mM ATP and followed by the addition of 100 ml of 0.5% bovine serum albumin. After centrifugation at 800 3 g for 20 min, the supernatant was discarded and the pellet was resuspended in 0.1 ml of 0.1 N NaOH and immediately reprecipitated with 1 ml of ice-cold stopping solution. The precipitated protein was collected on a 0.45 mm filter, and washed 5 times with 3 ml of 5% TCA, dried and the radioactivity on the filter was measured. PKC activity reflected the difference in activity measured in the presence of calcium, phosphatidylserine and diolein. Results are expressed as pmol of [32P] incorporated/ minutes/mg protein. Protein concentration was determined by the method of Bradford [20]. FIGURE 4. Effects of neomycin, calcium ionophore A23187, ver-
apamil or calmodulin inhibition on the steady-state levels of a1(I) collagen mRNA in cultured hepatic stellate cells. CFSC were incubated for 24 h in HMEM, 10% FCS lacking (C) or containing 10 mM calcium ionophore A23187 (Ion), 500 mM neomycin (Neo) or 100 mM verapamil (Vera) (A) 10 mM calmidazolium (Calm), 10 mM trifluoperazine (TFP) or 10 mM W-7 (B). Total RNA was isolated, electrophoresed, transferred to nylon filters and hybridised as described in Fig. 1. W-7: N-(6 aminohexyl)-5-chlor-1-naphthalenesulfonamide; Coll.a1(I): collagen a1(I) mRNA; a-Tubulin: a tubulin mRNA; Coll.a1(I)/a-Tub.: Collagen a1(I)/a-Tubulin mRNA ratio. These blots are representative of three separate experiments.
PBS and then scraped off from the flasks in sonication buffer (20 mM Tris-HCl, pH 7.5; 0.5 mM EGTA, 1 mM dithiothreitol, 0.1 mg/ml pepstatine, 1mg/ml leupeptin, 2 mM benzamidine) and centrifuged for 5 min at 1000 3 g at 48C. The cell pellet was resuspended in 1 ml sonication buffer and sonicated for 1 min. After sonication, cytosolic and membrane fractions were separated by centrifugation
Statistical Analysis All results are expressed as mean 6 SD unless otherwise mentioned. The Student’s t-test was used to evaluate the difference of means between groups, accepting P , 0.05 as level of significance [21]. RESULTS Gs Protein We studied the role played by the Gs proteins by incubating CFSC in the presence of CTX. This study demonstrated that treatment of cells with this toxin resulted in a doseand time-dependent decrease in the steady-state levels of collagen a1(I) mRNA (Figs. 1A and 1B) Thus, incubation of cells with 0.4 mg/ml CTX for 24 h reduced the steadystate levels of collagen a1(I) mRNA to 27.8 6 6.0% of the control level (Table 1; Fig. 2A). This toxin alters the conformation of the Gs protein, which results in a prolonged activation of adenylyl cyclase and elevation in cAMP levels
TABLE 4.
Incorporation of [3H]inositol into IP3 (cpm/106 cells) 0 min Control PTX, 10 ng/ml 1 TNFa, 0.6 nM TNFa, 0.6 nM Neomycin, 0.5 mM 1 TNFa, 0.6 nM PTX, 10 ng/ml
3172 1951 3046 2691 1903
6 6 6 6 6
243 134 (*) 154 (NS) 253 189 (NS)
5 min 3066 2693 5970 3169 2146
6 6 6 6 6
283 165 (*) 196a (*) 233 228 (**)
15 min 3138 2280 5054 3257 2073
6 6 6 6 6
241 132 (*) 266a (*) 178 146 (NS)
30 min 3072 2140 3095 2810 1971
6 6 6 6 6
304 179 (**) 208 (NS) 206 183 (NS)
Cells were labelled with myo[3H]inositol for 24 h as described under Material and Methods and treated with 0.6 nM TNFa for the indicated times with or without pre-treatment with 500 mM neomycin for 2 h or with 10 ng/ml pertussis toxin (PTX) for 24 h. Values are means 1 SD from a single experiment which was conducted in triplicate. a P , 0.001 compared with control cells. * 5 P , 0.001 compared with TNFa-treated cells; ** 5 P , 0.05; NS 5 not significant.
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TABLE 5.
TABLE 6.
[Ca21]i (arbitrary units of fluorescence) Additive
Basal
Additive
(%)
TNFa, 0.6 nM
7.2
16.7
232
Neomycin, 500 mM
5.6
4.9
88
Neomycin, 500 mM 1 TNFa, 0.6 nM
6.3
7.9
126
A32187, 10 mM
6.8
19.6
288
PTX, 10 ng/ml 1 TNFa, 0.6 nM
4.6
5.9
128
Intracellular free calcium level in CFSC were determined as described in Material and Methods. “Basal” represents prestimulation fluorescence and “additive” means peak fluorescence values after the addition of test substances. Data are representative of two independent experiments. % 5 Percentage of basal level after additive.
[8]. To document increases in cAMP levels after exposure to cholera toxin, cAMP was measured in cells after 2 and 24 h of treatment with 0.4 mg/ml CTX. Cellular levels of cAMP increased between 30- and 40-fold on exposure to CTX for 2–24 h (Table 2). Treatment of cells with 100 mM forskolin for 24 h, which activates adenylyl cyclase and increases cAMP levels (Table 2), led to a decrease in collagen a1(I) mRNA levels to 51.2 6 1.4% of control levels (Table 1; Fig. 2A). Likewise, when CFSC were exposed to the cAMP-analogue 1 mM 8Br-cAMP, for 24 h, the level of collagen a1(I) mRNA decreased to 66.0 6 3.6% of control levels (Table 1; Fig. 2A). On the other hand, 50 mM H8, which inhibited 8Br-cAMP-activated PKA by about 50% (Table 3), blocked the effect of CTX on collagen a1(I) mRNA (Table 1; Fig. 2B).
PKC activity (pmol[32P] incorporated/min/mg protein)
Control PMA, 60 nM, 1 hour H7, 50 mM 1 PMA, 60 nM Staurosporine, 0.5 nM 1 PMA, 60 nM PMA, 10 nM, 24 hours
Cytosolic
Membrane
141 6 21 41.8 6 12.3
67 6 19.2 537 6 36a
96.3 6 22.4
60 6 18.5
72.7 6 9.3 63.9 6 11.7
25.6 6 13.9 b 265 6 16.1a
Cells were treated with 10 or 60 nM PMA for 24 or 2 h, respectively, with or without pre-incubation with 0.5 nM staurosporine or 50 mM H7 for 2 h. Subsequently, cells were washed and sonicated as described in Material and Methods. Cytosolic and membrane fractions were separated by ultracentrifugation and PKC activity in both fractions was measured as described. Data represent means 6 SD of one experiment performed in triplicate. a P , 0.001. b P , 0.05.
the incorporation of [3H]inositol into IP3 and the increase in intracellular free calcium level induced by TNFa treatment (Tables 4 and 5). Incubation of CFSC for 24 h with 10 mM calcium ionophore A23187, which allows calcium to move into the cytoplasm from the extracellular fluid and reproduces the effects of IP3, decreased collagen a1(I) mRNA to 57.5 6 20% of the control level (Table 1; Fig. 4A). In contrast, 0.1 mM verapamil, which decreases cytosolic free calcium, increased collagen a1(I) mRNA level 2-fold. Likewise, blocking calmodulin with 10 mM calmidazolium, 10 mM TFP or 10 mM W-7 resulted in a marked increase in collagen a1(I) mRNA by 39 6 9% to 126.8 6 12% over the control level (Table 1; Fig. 4B). Blockade of G proteins with 10 ng/ml PTX for 24 h led to an inhibition of the TNFa-induced increase in both cytosolic IP3 and calcium (Tables 4 and 5).
PTX-Sensitive G Proteins The role played by PTX-sensitive G proteins was investigated by incubating CFSC for 24 h with a range of PTX concentrations between 1 ng/ml and 10 ng/ml. This study demonstrated that this toxin induced a dose-related increase in intracellular steady-state levels of collagen a1(I) mRNA of 96.8 6 9.7% over the control level at a concentration of PTX of 10 ng/ml (Table 1; Fig. 3). Likewise, PTXtreatment (10 ng/ml) for 24 h enhanced cAMP levels from about 8.7 6 1.3 to 36 6 2.7 pmol/l06 cells (Table 2). PLC and Intracellular Calcium Because a PTX-sensitive G protein may stimulate PLC and liberate inositol 1,4,5-triphosphate (IP3) into the cytosol, we treated CFSC with neomycin, an inhibitor of this enzyme. Neomycin binds phosphatidylinositol 4,5-biphosphate (PIP2) and inhibits inositol 1,4,5-triphosphate (IP3) release [22]. Treatment of cells with 500 mM neomycin for 24 h led to an increase in this mRNA level by 101.3 6 22.3% over controls (Table 1; Fig. 4A), and blunted both
PKC The diacylglycerol, the second hydrolysis product of the PLC pathway, is a potent activator of PKC, which, in turn, might phosphorylate proteins involved in the modulation of gene expression. Therefore, we examined the role of this protein kinase in the regulation of collagen gene expression by incubating CFSC for 24 h with 0.5 nM staurosporine, a PKC inhibitor (Table 6). Three independent experiments demonstrated that staurosporine did not change significantly the steady-state levels of collagen a1(I) mRNA (Table 1; Fig. 5A). One the other hand, activation of PKC by treating cells with 10 nM PMA for 24 h (Table 6) resulted in a decrease in the intracellular content of this mRNA Fig. 5A). Similar results were obtained, when a chimeric plasmid containing of segments of the collagen 59-flanking region ligated to the chloramphenicol acetyl transferase (CAT) gene (COLCAT1) was transfected into CFSC and CAT activity was assayed. PMA (10 nM, 24 h), as well as the membrane-permeable protein kinase C activator OAG (63 mM),
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FIGURE 5. Effects of protein kinase C stimulation and inhibi-
tion on collagen a1 type I mRNA level and chloramphenicol acetyltransferase (CAT) activity. (A) CFSC were incubated for 24 h in culture medium lacking (C) or containing PMA (10 nM) or staurosporine (0.5 nM). Collagen a1(I) mRNA level was analysed as described in Fig. 1. (B) DNA fragments containing part of the first exon and 3700 bp of the mouse a1(I) collagen promoter region fused to the bacterial chloramphenicol acetyltransferase gene (COLCAT1) were transfected into CFSC by electroporation as described in Methods. Transfected cells were collected in a common pool, mixed, and aliquots of cell suspension were plated in culture flasks for different experimental conditions. Six hours after electroporation, cells were left untreated (control) or treated for 24 h with 50 mM H-7, 0.5 nM staurosporine, 10 nM PMA or 100 mM OAG. Aliquots of cell lysates containing equal amounts of proteins were analysed for CAT activity. The thin-layer plates were exposed to X-ray film for 24 h. Individual spots were localised and excised, and radioactivity in each spot was determined by scintillation counting. The percent of acetylation was calculated. The results presented are representative of three separate experiments. C, control. PMA, phorbol myristate acetate. STAU, staurosporine. OAG, oleyl acetyl glycerol. Coll.a1(I), collagen a1(I) mRNA. a-Tubulin, a-tubulin mRNA. Coll.a1(I)/a-Tub., Collagen a1(I)/a-Tubulin mRNA ratio. Chl, [14C]chloramphenicol. Ac Chl, monoacetylated [14C]chloramphenicol. Ac Chl.(%), percentage of monoacetylated [14C]chloramphenicol.
decreased CAT activity to 31% and 5%, respectively, of control levels, while the protein kinase C inhibitors H7 (50 mM) and staurosporine (0.5 nM) increased CAT activity slightly (18% and 7%, respectively) (Fig. 5B). PLA2 Because some cell receptors are coupled to PLA2 through a G protein [23], we studied the role played by this enzyme in the modulation of collagen gene expression. Cells were in-
FIGURE 6. Effect of blockade of phospholipase A2 pathway on
the steady-state levels of collagen a1(I) mRNA. Culture of confluent CFSC were incubated in control medium (0) or in medium containing 20 mM BPB (A), 0.5–2 mM quinacrine (B), 24 nM NDGA or 100 mM indomethacin (INDO) (C). Total RNA was isolated, electrophoresed, transferred to nylon filters and hybridized as described in Material and Methods and Fig. 1. Coll.a1(I), collagen a1(I) mRNA. a-Tubulin, a tubulin mRNA. Coll.a1(I)/a-Tub., Collagen a1(I)/a-Tubulin mRNA ratio. BPB, bromophenacylbromide. NDGA, nordihydroguaiaretic acid.
cubated with two different inhibitors of PLA2 (Table 7) for 24 h. BPB (20 mM) induced a marked increase in the steady-state level of collagen a1(I) mRNA, which rose by 61.9 6 12% over that in control cells (Fig. 6A). Likewise, increasing concentrations (0.5 mM to 2 mM) of quinacrine resulted in an elevation of collagen a1(I) mRNA content in cells by 50 6 28% to 100 6 33% above the central levels (Fig. 6B). Indomethacin (100 mM), used as cycloxygenase inhibitor, increased the collagen a1(I) mRNA content in the cells by 105.5% 6 34%, and 24 mM NDGA, a lipoxygenase inhibitor, raised these levels slightly by 11 6 12% over the control level (Table 1; Fig. 6C). “De Novo” Synthesis of Proteins Cycloheximide at 0.1 mM, a concentration that blocks protein synthesis by 95% in CFSC, did not affect the steadystate levels of collagen a1(I) mRNA, indicating that de novo synthesis of a protein is not required for the expression of collagen gene at basal level (Table 1).
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TABLE 7.
Phospholipase A2 activity (nmol/min/mg) 0
5
10
30
90 min
Control
0.574 6 0.011
0.569 6 0.013
0.571 6 0.016
0.566 6 0.012
0.572 6 0.011
BPB, 20 mM
0.230 6 0.01
0.206 6 0.013
0.194 6 0.02
0.179 6 0.019
a
0.172 6 0.011a
Quinacrine, 2 mM
0.330 6 0.014a
0.328 6 0.011a
0.320 6 0.016a
0.314 6 0.015a
a
a
a
0.320 6 0.013a
Phospholipase A2 activity was measured in triplicate at 50 mM phosphatidyl choline substrate (1-palmitoyl-2-(1-14C)arachidonylglycero-3-phosphocholine in the presence or absence of 20 mM BPB or 2 mM quinacrine. Data are given as mean values 6 SD. a P , 0.001 as compared with control cells.
DISCUSSION The results of the present study indicate that a Gs protein is involved in the suppression of collagen gene expression. Thus, when CFSC were treated with CTX, an enzyme that activates Gs proteins, there was a 30-fold increase in cellular cAMP level and a marked reduction in the steady-state levels of collagen a1(I) mRNA (Table 1; Fig. 1). To confirm that suppression of collagen a1(I) mRNA induced by CTX treatment was mediated through an activation of adenylyl cyclase and an elevation of cAMP levels and not through other properties of this toxin, cells were treated with forskolin, a diterpene which elevates cAMP levels (18-fold) (Table 2) by activating adenylyl cyclase. This treatment led also to a marked reduction in the steady-state level of collagen a1(I) mRNA (Table 1; Fig. 2A). This effect may be mediated by the cAMP released by activation of adenylyl cyclase, since addition of 8Br-cAMP to the cells resulted in a 34% decrease in collagen a1(I) mRNA level (Table 1; Fig. 2A). As far as we know, no studies have been published on the role played by Gs-protein in the modulation of collagen a1(I) mRNA levels. However, a number of studies have shown that cAMP decreases collagen production (24–26). In Schwann cells treated with either forskolin or cAMP derivatives, collagen types I and III mRNA were significantly suppressed [27]. The mechanism whereby cAMP decreases the expression of the collagen gene is not known. Cyclic AMP generated in response to cell receptor occupancy causes PKA activation and phosphorylation of transcription factors [28–30]. Our results point to the mediation of this enzyme in the cAMP-induced inhibition of collagen gene. When PKA was inhibited by pre-incubating CFSC with H-8, the intracellular steady-state level of collagen a1(I) mRNA did not decrease after treating these cells with CTX (Table 1; Fig. 2B). All members of the Gi family, including Gi, Go and Gt proteins, can be blocked by PTX [7]. This enzyme uncouples the Gi family from receptors and therefore these proteins fail to interact with effecters. Thus, PTX inhibits the actions mediated by this class of G proteins. In order to evaluate the role played by these proteins on the modulation of collagen a1(I) gene expression, cells were treated with increasing concentrations of PTX. Our results provide evidence for an implication of a PTX-sensitive G protein in
the suppression of type collagen a1 gene expression. Indeed, this treatment resulted in a dose-related increase in type collagen a1 mRNA content in these cells (Table 1; Fig. 3), so that 10 ng/ml PTX elevated these levels 2-fold (Table 1). Obviously, this response cannot be ascribed to the inhibitory action of the Gi protein on adenylyl cyclase, since PTX increased cAMP levels (Table 2) and, consequently, should decrease, but not increase, collagen a1(I) mRNA levels. It is unlikely that PTX-induced increase in mRNA levels can be imputed to Gt or Go proteins either, as these proteins are found only in the retina and neurons, respectively [7, 8]. Gi protein, in addition to its action on adenylyl cyclase, may also activate a phosphatidylinositol-specific PLC [5, 31]. Stimulation of this Gi protein in response to cell receptor occupancy leads to the activation of PLC, which cleaves phosphatidylinositol 4,5-biphosphate (PIP2) to yield inositol 1,4,5-triphosphate (IP3) and DAG [32]. These two metabolites act as second messengers of cellular activation. IP3 increases cytosolic free calcium concentration [33] while DAG stimulates PKC. Our results suggest that PLC may be activated by a PTX-sensitive G protein in CFSC. Thus, treatment of CFSC with PTX led to a marked inhibition of the TNFa-induced increase in cytosolic IP3 and Ca21 concentrations (Tables 4 and 5), that may be the results of a reduced PLC activity. On the other hand, blocking PLC by treating CFSC with neomycin, an inhibitor of IP3 formation [22], led to a marked increase in the intracellular levels of collagen a1(I) mRNA (Fig. 4A). These results point to an inhibitory role of the pathway initiated in the PLC. IP3, one of the two metabolites cleaved by PLC from PIP2, promotes the release of Ca21 from intracellular storage sites, increasing the cytosolic free Ca21 concentration [33]. There is very little information about the role played by calcium ion in collagen gene expression. Our results show that this intracellular second messenger plays a role in the modulation of the expression of collagen gene. Preincubation of CFSC with ionophore A23187, which increases intracellular Ca21, reduced the steady-state level of collagen a1(I) mRNA (Fig. 4A). In contrast, verapamil, a calcium channel blocker, increased the level of collagen a1(I) mRNA (Fig. 4A). These results concur with those previously reported by Flaherty and Chojkier [34] and Chojkier et al. [35]. Many of the intracellular actions of calcium, including the expression of some genes, occur by its binding to cal-
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modulin [36]. In favour of the involvement of a calmodulin dependent system in the inhibition of collagen gene expression are our results after blocking calmodulin with three different anticalmodulin drugs (calmidazolium, TFP, W-7). Inhibition of this calcium-binding protein resulted in a “marked” increase in the levels of collagen a1(I) mRNA (Fig. 4B). DAG, a product cleaved from PIP2 by PLC activation, stimulates PKC [32]. Our results show that PMA decreased steady-state levels of collagen a1(I) mRNA (Fig. 5A), as well as CAT activity in CFSC transfected with COLCAT1 (Fig. 5B). In contrast, blocking PKC with H7 or staurosporine did not change significantly CAT activity or collagen a1(I) mRNA levels (Table 1). These results suggest that DAG and PKC are also parts of an inhibitory pathway of collagen gene, which, in basal conditions, seems to be little active. These results are in agreement with those obtained by other authors [34–38], who found that phorbol esters markedly inhibited collagen production in cultured fibroblasts. Likewise, Goldstein et al. [38] reported that this PMA-mediated inhibition in collagen production was associated with a marked reduction in the a1(I) collagen mRNA level and gene transcription. PLA2, one of the enzyme coupled to membrane receptors by G proteins [23], has also been implicated in intracellular signalling mechanisms. This enzyme cleaves arachidonic acid from phospholipids and generates a variety of metabolites including prostaglandins, leucotrienes and lipoxins [39]. These metabolites of arachidonic acid have also been implicated as direct mediators of post-receptor events in a number of cell lines [40]. Our study provides evidence for an implication of PLA2 in modulating the expression of collagen gene. Treatment of CFSC with some PLA2 inhibitors, such as BPB or quinacrine, or with indomethacin, a cycloxigenase inhibitor, led to a marked increase in type I collagen a1 mRNA level in these cells (Table 1; Fig. 6), while the inhibition of lipoxygenase induced no significant change in this mRNA. Thus, these results suggest that PLA2 and prostaglandins are implicated in a pathway involved in the inhibition of collagen gene expression. Activated protein kinases may modify the phosphorylation of a number of proteins which may channel signals from the cell membrane to the transcription apparatus in the nucleus. Transcription of a1(I) collagen gene is regulated by specific interaction of trans-acting transcription factors and cis-acting DNA elements located in the promoter, 59-flanking region and the first intron of the gene. A number of studies have demonstrated that both positive and negative elements are present in these regions and various trans-acting factors have been partially characterised [41]. However, little is known about the trans-acting factors acting as third messengers in each signal-transducing pathway and the cis-regulating elements in the type I collagen a1 gene. Nevertheless, this study shows that no synthesis of a protein is necessary to modulate basal expression of collagen gene, since inhibition of protein synthesis with cycloheximide did not modify cellular levels of collagen a1(I)
mRNA (Table 1). These results concur with those we described in human skin fibroblasts and NIH 3T3 cells [42]. In conclusion, this study shows that collagen gene expression is inhibited by a number of intracellular pathways which transduce signals from the cell membrane to the nucleus. A Gs protein and a PTX-sensitive G protein seems to initiate cellular response to receptor occupancy. cAMP and PKA act as second messengers of the Gs protein activation, while PLC/IP3/calcium/calmodulin, DAG/PKC and PLA2/ prostaglandins seem to be effecters of the PTX-sensitive G protein. It is conceivable that fibrogenic activity of fat-storing cells is hindered and secured by multiple intracellular pathways, diverting the activity of these cells to other functions. Nuclear factors, acting as third messenge is in these pathways, have to be identified. However, it seems that they do not require to be synthesised. It is most likely that they are still present within the cells, but need to be activated. The authors are grateful to Patricia Greenwel and Dr. Marcos Rojkind (Albert Einstein College of Medicine, Bronx, NY) for the gift of the CFSC cell line, Dr David A. Brenner (University of North Carolina, Chapel Hill, NC) for the kind gift of COLCAT1 plasmid and Dr. David Rowe (University of Connecticut Health Centre) for providing us the collagen a1(I) rat probe.
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ISSN 0898-6568/98 $19.00 PII S0898-6568(97)00036-3
G Proteins Are Involved in the Suppression of Collagen a1(I) Gene Expression in Cultured Rat Hepatic Stellate Cells Jose´ A. Solı´s-Herruzo,* Inmaculada Herna´ndez, Paz De la Torre, Inmaculada Garcı´a, Jose A. Sa´nchez, Inmaculada Ferna´ndez, Gregorio Castellano and Teresa Mun˜oz-Yagu¨e Gastroenterologı´a, Centro de Investigacio´n, Hospital Universitario “Doce de Octubre,” Universidad Complutense, Madrid, Spain
ABSTRACT. We analyse the role of the G proteins in regulating collagen gene expression by measuring collagen a1(I) mRNA levels in cultured hepatic stellate cells in basal conditions and after stimulating or inhibiting the major intracellular signalling pathways. Stimulation of Gs protein and adenylyl cyclase or the addition of 8Br-cAMP to the cells led to a decrease in collagen a1(I) mRNA levels, while blocking protein kinase A abolished this effect. Blocking Gi protein, phospholipase A2 and C, calcium channels and calmodulin resulted in a significant increase in collagen mRNA levels. PKC stimulation led to a marked decrease in these levels. These results suggest that collagen gene expression is inhibited by a number of intracellular pathways. A Gs and a pertussis toxin-sensitive G protein seem to initiate cellular response. Transcription factors, acting in these pathways, must be identified. However, it seems that they do not need to be synthesised. cell signal 10;3:173–183, 1998. 1998 Elsevier Science Inc. KEY WORDS. Collagen, Fibrogenesis, Gene expression, Hepatic stellate cells, Signal transduction pathways
INTRODUCTION Hepatic stellate cells (HSC) (fat-storing cells, Ito cells, hepatic lipocytes) are sinusoidal cells, localised within the space of Disse of the liver, that have been identified as the principal cellular source of extracellular matrix in chronic active liver disease [1]. In normal liver, these cells are recognised because they also express desmin and contain prominent intracellular droplets storing vitamin A [2, 3]. In cirrhotic liver, large numbers of transitional cells with characteristics of both HSC and myofibroblastlike cells are present [4]. Little is known about mechanisms and intracellular pathways involved in modulating collagen gene expression by cultured HSC in basal conditions. It is well known that the surface of cells is a target of chemicals, cytokines, hormones, growth factors and physical signals that activate membrane-bound receptors. These activated receptors promote a cascade of signals that passes through a set of coupling proteins, called G proteins, to intracellular effector enzymes or ion channels. Changes in effector activity cause changes in second messenger levels (cyclic adenosine monophosphate (cAMP), inositol 1,4,5-triphosphate (IP3), eicosa*Author to whom correspondence should be addressed at: Hospital “12 de Octubre.” Servicio de Aparato Digestivo, C/Andalucia, Km 4.5, 28041Madrid, Spain. E-Mail: [email protected] This study was supported in part by Grants 90/145, 90/148 and 95/609 from the “Fondo de Investigaciones Sanitarias,” and PB-92/316 and PB94/ 001 from DGICYT, Spain. Received 26 July 1996; and accepted 18 December 1996.
noids, diacylglycerol (DAG), ceramide) or in ionic composition (Ca21, K1, Na1), that ultimately lead to a cellular response, including the modulation of gene expression [5, 6, 7, 8]. G proteins are heterotrimers that are made up of a and bg subunits and couple many kinds of cell-surface receptors. Upon binding of an agonist, the receptor becomes activated and the a-subunit of G proteins undergoes a conformational change, allowing GTP to bind in place of GDP. Once GTP is bound, the a subunit dissociates from the receptor and from bg subunit and interacts with effectors. The function of a G protein depends on its subunit structure. There are four major families of G proteins. The Gs family, which activates adenylyl cyclase to produce cAMP. The Gi family, composed of: (1) the Gi protein, which inhibits adenylyl cyclase and voltage-sensitive Ca21 channels and activates atrial K1 and renal Na1 channels and a phosphoinositidespecific phospholipase C (PLC), (2) Go protein, which inhibits the Ca21 channels in neurons, and (3) Gt protein (transducin), which activates cGMP phosphodiesterase of the retinal rods. The Gq family, which activates PLC-b and, finally, the G12 and G13 family, whose function is not well known. While cholera toxin (CTX) activates Gs protein indefinitely, pertussis toxin (PTX) inhibits Gi, Go and Gt proteins [5, 6, 7, 8]. The role played by the G proteins in the control of collagen gene expression is not known. However, preliminary studies suggest that these proteins may be involved in the
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suppression of this gene [9]. The aim of this study was to analyse the role played by the G proteins in regulating collagen gene expression. We used a line of HSC obtained by Greenwel et al. [10] from a cirrhotic liver of carbon tetrachloride-treated rat. This cell line displays many features of activated HSC, and has proved to be very useful in studying factors that regulate extracellular matrix production.
MATERIALS AND METHODS The HSC line CFSC was used in these experiments. These cells were a gift from Dr. M. Rojkind and P. Greenwel (Albert Einstein College of Medicine, NY) and were derived from a carbon tetrachloride cirrhotic rat liver after spontaneous immortalization in culture [10]. The cells have been maintained in culture for over 100 passages and have maintained the same phenotype with regard to cell proliferation and extracellular matrix production since passage 30. This cell line expresses a phenotype similar to that of early passage of fat-storing cells, and has morphologic features of myofibroblasts [10]. Confluent CFSC were cultured at 378C, in an atmosphere of 5% CO2, 95% air in cell culture flasks using 10 ml minimum essential medium Eagle with Hanks’ BSS (HMEM) containing 10% foetal calf serum (FCS) (Flow, Irvine, UK). Cells were plated at a density of 5 3 106/80 cm2 flask and studies were performed after 4–6 days: Intracellular signal-transduction pathways involved in the regulation of collagen gene expression were explored by measuring the steady-state levels of collagen a1(I) mRNA and the activity of chloramphenicol acetyl transferase (CAT) in CFSC after their treatment with inhibitors or, eventually, activators of these pathways. The role played by G proteins was studied by incubating these cells with 0.05–0.4 mg/ml CTX (Sigma Chemical Co. Alcobendas, Madrid, Spain) or with 1–10 ng/ml PTX (Sigma). Adenylyl cyclase/cAMP/protein kinase A (PKA) pathway was explored by using 100 mM forskolin (Sigma), 1 mM 8Br-cAMP (Sigma) or 50 mM N-(2-methylamino) ethyl)-5 isoquinolinesulfonamide dihydrochloride (H-8). The role of PLC was examined by treating cells with 500 mM neomycin. The role played by intracellular calcium was studied by blocking calcium channels with 100 mM verapamil (Sigma) and by increasing intracellular calcium levels with 10 mM calcium ionophore A23187 (Sigma). Calmodulin was inhibited with 10 mM calmidazolium (Sigma), 10 mM trifluoperazine dimaleate (TFP) (Calbiochem, San Diego, CA) or 10 mM N-(6 aminohexyl)-5-chloro 1 naphthalenesulfonamide (W-7) (Sigma). Protein kinase C (PKC) was inhibited with 50 mM 1-(5-isoquinolinesulfonyl)-2-methylpiper dihydrochloride (H-7) (Sigma), or 0.5 nM staurosporine (Sigma), and stimulated with 10 nM phorbol 12-myrisrate 13-acetate (PMA) (Sigma) for 24 h, or 63 mM oleyl acetyl glycerol (OAG) (Sigma). The role played by newly synthesised proteins was studied by blocking protein synthesis with 0.1 mM cycloheximide (Sigma). This concentration of cycloheximide blocked protein synthesis by 96% in these CFSC but did not affect the
FIGURE 1. Cholera toxin induces a time- and dose-dependent
decrease in collagen a1(I) mRNA levels. (A) Dose-response effect of cholera toxin on the levels of collagen a1(I) mRNA. Confluent CFSC were incubated for 24 h in HMEM and 10% FCS in the absence or presence of increasing concentrations of cholera toxin (CTX). (B) Time course of the effect of cholera toxin on collagen a1(I) mRNA levels. Cells were treated with 0.4 mg/ml cholera toxin for 1–24 h. Ten mg of total RNA were electrophoresed on a formaldehyde, 1% agarose gel and transferred to a nylon filter by capillary blotting. The quality and integrity of RNA following electrophoresis was checked by visual examination of ethidium bromide stained gels under transillumination. Bound RNA was hybridised to 32P-labelled cDNA probe [rat collagen a1(I)] as described under Methods. The filters were exposed to X-ray film at 2708C with an intensifying screen. The autoradiograms were quantitated by scanning laser densitometry. The level of collagen a1(I) mRNA in each sample was normalised to the level of a-tubulin mRNA. Coll.a1(I): collagen a1(I) mRNA; a-Tubulin: a tubulin mRNA; Coll.a1(I)/a-Tub.: Collagen a1(I)/a-Tubulin mRNA ratio.
steady state level of collagen a1(I) mRNA. All these experiments were repeated at least 3 times. Preparation of Complementary DNA (cDNA) Probes Rat cDNA for the alpha 1 chain of type I collagen (1.6 kilobase Pst1 fragment) was generously provided by Dr. D. Rowe (Farmington, CO) [11]. The cDNA fragments were radiolabelled with [a-32P]-dCTP (specific activity 5 3000 Ci/mmol) using a primer extension kit (Multiprime DNA labelling system from Amersham International, England, UK) to a specific activity of 1 3 109 cpm per mg of DNA. Northern Blotting RNA was isolated from confluent cells in culture by the phenol/chloroform extraction method of White and Ban-
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TABLE 1. Effect of blockade or, eventually, activation of intracellular signal transduction pathways on the steady-state level of collagen a1(I) mRNA
a1(I) collagen mRNA a-tubulin mRNA ratio (% of control) Pathway
Agent
Experiment 1
Experiment 2
Experiment 3
Means 6 SD
100.0%
100.0%
100.0%
100.0%
Control
Culture medium without additive
Gs protein/ Adenylyl cyclase PKA
Cholera toxin, 0.4 mg/ml Forskolin, 100 mM 8 Br-cAMP, 1 mM H-8, 50 mM 1 Cholera toxin, 0.4 mg/ml
34.5 51.8 69.7
22.7 52.3 62.5
26.1 49.6 65.8
27.8 6 6.0 51.2 6 1.4 66.0 6 3.6
105.0
97.3
107.7
102.5 6 4.5
Gi-Proteins
Pertussis toxin, 1 ng/ml 5 ng/ml 10 ng/ml
153.6 184.4 205.5
129.0 167.4 198.8
140.8 178.2 186.3
141.1 6 12 176.4 6 8.6 196.8 6 9.7
PLC
Neomycin, 500 mM
205.9
221.0
177.0
201.3 6 22.3
Calcium
Verapamil, 100 mM A 23187, 10 mM
216.1 79.6
164.8 39.4
198.0 53.7
193.0 6 26 57.5 6 20
Calmodulin
TFP, 10 mM W-7, 10 mM Calmidazolium, 10 mM
204.1 212.4 141.1
202.0 232.0 129.0
236.0 136.0 147.0
180.7 6 38.7 226.8 6 12.6 139.0 6 9.1
PKC
PMA 10 nM, 24 hours Staurosporine, 0.5 nM
47.4 90.3
58.0 108.0
72.0 92.0
59.1 6 12.3 96.7 6 9.7
PLA2
BPB, 20 mM Quinacrine, 0.5 mM 1.0 mM 2.0 mM
156.1 149.0 181.0 179.0
175.8 122.0 169.0 183.0
153.8 178.9 182.4 238.1
161.9 6 12.1 149.9 6 28 177.4 6 7.2 200.0 6 33
Cycloxigenase
Indomethacin, 100 mM
245.0
186.5
185.2
205.5 6 34
Lipoxigenase
NDGA, 24 mM
112.8
98.3
122.0
111.0 6 12
Protein synthesis
Cycloheximide, 0.1 mM
108.2
102.4
99.6
103.4 6 4.4
Confluent CFSC were cultured in 10 ml HMEM containing 10% FCS. Intracellular signal transduction pathways involved in the regulation of collagen gene expression were explored by measuring the steady-state levels of collagen a1(I) mRNA and a-tubulin mRNA in CFSC after their treatment with inhibitors or activators of these pathways. The level of collagen a1(I) mRNA in each sample was normalised to the level of a-tubulin mRNA.
croft [12]. Ten mg of total RNA were electrophoresed in a 1% agarose 2.2 M formaldehyde gel in 20 mM 3-(N-morpholino) propanesulfonic acid buffer. RNA samples were stained with ethidium bromide and transferred overnight by capillary blotting in 20 3 sodium saline citrate (SSC) to nylon filters (Amersham International, UK). The RNA was UV-crosslinked to the filter. The nylon filters were prehybridised at 658C in 7% sodium dodecyl sulphate (SDS) and 0.5 M buffer phosphate (pH 7.0) and 50 mg/ml of sonicated salmon sperm DNA. The nylon filters were hybridised overnight at 658C in freshly prepared hybridisation solution with addition of 106 cpm/ml of the labelled cDNA probe. The nylon filters were washed at a stringency of 2 3 SSC, 0.1% SDS at room temperature for 30 min and 0.1 3 SSC, 0.1% SDS at 658C for 15 min and exposed to X-ray film at 2708C with an intensifying screen. The autoradiograms were quantitated by scanning laser densitometry DeskTopY Scanner Plus (Pharmacia Biotech SA Barcelona, Spain). The level of collagen a1(I) mRNA in each sample was normalised to the level of a-tubulin mRNA (Clontech Laboratories Inc., Palo Alto, CA).
Chloramphenicol Acetyl Transferase (CAT) Assay CFSC were transfected prior to confluence by electroporation using a Cellject electroporation system (Eurogentec. Liege, Belgium). The transfected plasmid (COLCAT1) included part of the first exon and the promoter region of the mouse alpha 1 type I collagen gene fused to the bacterial CAT gene [13]. This plasmid was generously provided by Dr. D. Brenner (University of North Carolina, Chapel Hill, NC). On the day before transfection, cells were plated at a density of 4 3 104/cm2 in 160-cm2 flasks. Cells were harvested with trypsin and centrifuged at 250 g. The pellet was washed 3 times with PBS and resuspended in culture medium. Cell density of this suspension was 1 3 107/ml. Onehalf ml of cell suspension was transferred to a tube and sonicated salmon sperm DNA (40 mg/ml) and plasmid DNA (18 mg/ml) were added. Electroporation was performed at 08C by delivering two pulses: the first of 700 V at 40 mF and the second of 150 V at 1200 mF. All electroporated cells were collected in the same tube, mixed, plated in 80 cm2 flasks for different experimental conditions and incubated at 378C. Inhibitors or activators of intracellular signal trans-
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International, plc., UK) according to the manufacturer’s instructions. Cells were incubated at 378C with either control medium or medium containing 10 ng/ml PTX, 0.4 mg/ml CTX, 1 mM 8 Br-cAMP or 100 mM forskolin for various periods of times. The cells were subsequently placed on ice and the medium was removed. Cultures were rinsed twice with cold PBS and then scraped in 10 ml of cold 65% ethanol. The cells were then pelleted by cold centrifugation at 2000 3 g for 15 min and the resulting supernatants were dried under vacuum. The extracts were dissolved in 2 ml assay buffer before Elisa analysis. PKA Assay
FIGURE 2. Northern blot hybridisation analysis demonstrating
the effects of cholera toxin (0.4 mg/ml), forskolin (100 mM), 8 Bromo-cyclic adenosine monophosphate (1 mM) (8BrAMP) (A) cholera toxin (0.4 mg/ml), cholera toxin and H-8 (50 mM) and H-8 (B) on the steady-state levels of a1(I) collagen mRNA. Confluent CFSC were incubated for 24 h in HMEM, 10% FCS or in culture medium that contained cholera toxin (CTX), forskolin (Frsk), 8 bromo cyclic AMP (8BrAMP) or H-8. C: control untreated cells; Coll.a1(I): collagen a1(I) mRNA; a-Tubulin: a tubulin mRNA; Coll.a1(I)/a-Tub.: Collagen a1(I)/ a-Tubulin mRNA ratio. The results presented are representative of 2 to 4 separate experiments.
duction pathways were added to the cells 6 h after electroporation, and this treatment was generally maintained for 48 h. Cultured cells were harvested and transferred to a microcentrifuge tube on ice. These cells were lysated by three cycles of freezing in dry ice/ethanol for 5 min and thawed at 378C for 5 min. The particulate was removed by centrifugation at 10,000 3 g for 5 min at 48C. Protein content of the supernatant was assayed by the Bradford method [14], and aliquots containing equal amounts of proteins were analysed for CAT activity, using a modification of the method of Gorman et al. [15]. The substrate and acetylated form of [14C]chloramphenicol (Amersham) were separated by thin layer chromatography on silica plates (Scharlau, Barcelona, Spain) in 95:5 chloroform/methanol and visualised by autoradiography with Kodak X-Omat film at room temperature for 12–24 h. Autoradiograms were quantitated by a scanning laser densitometer. Individual spots were located and excised, and their [14C] content was measured by liquid scintillation spectrophotometry. Measurement of Intracellular cAMP Intracellular concentrations of cAMP were determined using an enzyme immunoassay (Biotrack cAMP, Amersham
Cells were treated with 1 mM 8Br-cAMP with or without pre-treatment with 50 mM H-8. After the indicated times, cells were suspended in 20 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA, 100 mM NaCl and 40 mg/ml leupeptin. Aliquots were taken for measurement of protein content and the rest was sonicated for 30 s. PKA activity was measured using a commercially available kit (Promega Co., Madison, WI) following the manufacturer’s instructions. Briefly, 10 ml of samples were incubated with 5 ml Pep Tag 53 reaction buffer, 5 ml Pep Tag A1 peptide and 5 ml PKA activation solution in a final volume of 25 ml for 30 min at 308C. The reaction was stopped by boiling the samples for 10 min. Afterward, samples were loaded on a 1% agarose gel and electrophoresed for 45 min in 50 mM Tris-HCl, pH 8.0, until phosphorylated and unphosphorylated Pep Tag A1 peptides were completely separated by migration to positive and negative electrodes, respectively. The band of phosphorylated peptide was excised from the gel, boiled to dissolve the agarose and mixed with 75 ml gel solubilization solution, 100 ml glacial acetic acid and 200 ml of distilled water. Absorbency of the samples was read and PKA activity was calculated and expressed in pmol/min/mg protein. The Level of Intracellular Free Ca21 This was determined by flow cytometry as described by Vandenberghe and Ceuppens [16]. CFSC (1 3 106 cells) were suspended in 1 ml HMEM and treated with 5 mg/ml TABLE 2. Effect of cholera toxin, pertussis toxin, forskolin
and 8 Br-cAMP on intracellular cyclic AMP levels cAMP levels (pmol/106 cells)
Control CTX, 0.4 mg/ml PTX, 10 ng/ml Forskolin, 100 mM 8 Br-cAMP, 1 mM
2 hours
24 hours
8.9 6 0.8 377 6 21a ND 144 6 5.6a 36.5 6 6.7b
8.7 6 1.3 259 6 15 a 36 6 2.7a 235 6 21a 78.4 6 16a
CFSC were treated with either 0.4 mg/ml cholera toxin (CTX), 10 ng/ml pertussis toxin (PTX), 100 mM forskolin or 1 mM 8 bromo cyclic AMP (8Br-cAMP) for 2 and 24 h. Cyclic AMP was determined as described in the text. Data are shown as means 6 SD of triplicate determinations. ND 5 not done. a P , 0.001 against control cells. b P , 0.01 compared with control cells.
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of the calcium sensitive Fluo-3M at 378C for 20 min. The cells were washed 33 and resuspended at a concentration of 1 3 106 cells/ml in a hepes-buffered saline, containing 137 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 5 mM glucose, 1 mM CaCl2, 0.5 mM MgCl2, 1 g/l bovine serum albumin, 10 mM hepes, pH 7.4. Before each assay, the cells were incubated for 10 min in a 378C water bath and subsequently analysed on an Epics Elite flow cytometer (Coulter Electronics, Inc., Hialeah, FL). The laser was set at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and operated at 50 mW laser power. Throughout the experiments, temperature was maintained at 378C. Green fluorescence and forward angle light scatter were collected for lineally or logarithmically amplified (4-decades, 1024 channels) signals. The mean fluorescence of events was recorded and expressed as arbitrary units (channel number). After recording basal fluorescence, cells were treated with either 10 mM A23187 or 500 mM neomycin. The effect of neomycin and PTX on cytosolic Ca21 was studied with or without stimulation with 0.6 nM TNFa.
FIGURE 3. Steady-state levels of a1(I) collagen mRNA in pertus-
sis toxin-treated cells. Confluent CFSC were incubated for 24 h in HMEM 10% FCS or in the same culture medium containing pertussis toxin (1, 5 and 10 ng/ml). O: control untreated cells; Coll.a1(I): collagen a1(I) mRNA; a-Tubulin: a tubulin mRNA; Coll.a1(I)/a-Tub.: Collagen a1(I)/a-Tubulin mRNA ratio. Blots are representative of as least three separate experiments.
This was measured as described by Berridge et al. [17]. CFSC were incubated in serum-free HMEM containing 1% bovine serum albumin and prelabeled with 5 mCi/ml myo[3H]inositol for 24 h. The cells were washed 33 with serum-free medium containing 10 mM LiCl to minimise inositol phosphate hydrolysis and further incubated at 378C for 15 min. Cells were then exposed to 0.6 nM TNFa for various times with or without preincubation with 500 mM neomycin for 2 h. Media were then aspirated and ice-cold 10% TCA added. Cell layers were washed with 0.5 ml 10% TCA and the TCA fractions were treated with water-saturated diethylether. After adjusting the pH to 7.0–7.5 with NH4OH, the ether extracts were applied to AG1X8 anion exchange Dowex columns (100–200 mesh, formate form) (BioRad, Richmond, CA). The inositol triphosphate fraction was eluted with 1 M ammonium formate in 0.1 M formic acid and radioactivity was determined by liquid scintillation counting.
cultured in HMEM supplemented with 10%. When required, cells were treated with 10 ng/ml PTX for 24 h or with 20 mM BPB or 2 mM quinacrine for 2 h. After incubation with appropriate stimuli for the indicated times, cells were scraped in the PLA2 buffer (see below) and homogenised by sonication. Aliquots of cell homogenate (up to 100 mg of protein) were incubated for 30 min at 378C in 50 mM phosphate buffer (1 mM MgCl2, 1mM EGTA, pH 7.5) 50 mM phosphatidylcholine substrate (1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3phosphocholine, 25,000 dpm/assay), in a final volume of 0.25 ml. The phospholipid substrate was used in the form of vesicles obtained by sonication in assay buffer until the solution clarified. The reactions were terminated by adding chloroform/methanol (1:2, v/v). The lipids were extracted and separated by thinlayer chromatography. Plates were run with n-hexane/diethylether/acetic acid (70:30:1, by volume) as a solvent system. Authentic arachidonic acid was cochromatographed and visualised by exposing the plates to iodine vapours. The arachidonic acid fraction was scraped from the plate and monitored for radioactivity by liquid-scintillation counting. Counts in arachidonic acid were normalised for the amount of radioactivity recovered in each lane of the plate.
Assay for PLA2 Activity
Measurement of PKC Activity
PLA2 activity was measured as described previously [18]. Cells, plated at a density of 5 3 105 cells/well in 6-well plates, were
PKC activity was measured according to the method of Kitano et al. [19]. Cultured CFSC were washed twice with ice-cold
Accumulation of Inositol Phosphates
TABLE 3.
Protein kinase A activity (pmol/min/ng protein) 5 min Control 8Br-cAMP, 1 mM 8Br-cAMP, 1 mM 1 H8, 50 mM
147 6 10 311 6 20 a b 184 6 19 c
30 min 163 6 8.6 567 6 19 a b 291 6 24 a
60 min 156 6 13 579 6 13 a b 189 6 21
CFSC were treated with 1 mM 8Br-cAMP for 5, 30 or 60 min with or without previous incubation with 50 mM H-8 for 2 h. After these times, PKA activity was measured as described in Material and Methods. Data are the mean 6 SD of one experiment with triplicate determinations. a P , 0.001 as compared with the control cells. b P , 0.001 compared with H-8 pretreated cells. c P , 0.05 as compared with the control cells.
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(100,000 3 g, 1 h) in a Bekman L-80 ultracentrifuge. Aliquots (60 ml) of the cytosolic or membrane fraction were added to 190 ml of mixture containing 20 mM Tris-HCl, pH 7.5, 5 mM magnesium acetate, 0.2 mg/ml histone type IIIs, and 10 mM [g32P]ATP. Reactions were performed in the presence and absence of 1.5 mM CaCl2, 25 m/ml a-phosphatidyl serine and 0.5 mg/ml diolein. Incubations were performed at 308C for 10 min. The reaction was terminated with the addition of 1 ml ice-cold stopping solution containing 10% TCA and 2 mM ATP and followed by the addition of 100 ml of 0.5% bovine serum albumin. After centrifugation at 800 3 g for 20 min, the supernatant was discarded and the pellet was resuspended in 0.1 ml of 0.1 N NaOH and immediately reprecipitated with 1 ml of ice-cold stopping solution. The precipitated protein was collected on a 0.45 mm filter, and washed 5 times with 3 ml of 5% TCA, dried and the radioactivity on the filter was measured. PKC activity reflected the difference in activity measured in the presence of calcium, phosphatidylserine and diolein. Results are expressed as pmol of [32P] incorporated/ minutes/mg protein. Protein concentration was determined by the method of Bradford [20]. FIGURE 4. Effects of neomycin, calcium ionophore A23187, ver-
apamil or calmodulin inhibition on the steady-state levels of a1(I) collagen mRNA in cultured hepatic stellate cells. CFSC were incubated for 24 h in HMEM, 10% FCS lacking (C) or containing 10 mM calcium ionophore A23187 (Ion), 500 mM neomycin (Neo) or 100 mM verapamil (Vera) (A) 10 mM calmidazolium (Calm), 10 mM trifluoperazine (TFP) or 10 mM W-7 (B). Total RNA was isolated, electrophoresed, transferred to nylon filters and hybridised as described in Fig. 1. W-7: N-(6 aminohexyl)-5-chlor-1-naphthalenesulfonamide; Coll.a1(I): collagen a1(I) mRNA; a-Tubulin: a tubulin mRNA; Coll.a1(I)/a-Tub.: Collagen a1(I)/a-Tubulin mRNA ratio. These blots are representative of three separate experiments.
PBS and then scraped off from the flasks in sonication buffer (20 mM Tris-HCl, pH 7.5; 0.5 mM EGTA, 1 mM dithiothreitol, 0.1 mg/ml pepstatine, 1mg/ml leupeptin, 2 mM benzamidine) and centrifuged for 5 min at 1000 3 g at 48C. The cell pellet was resuspended in 1 ml sonication buffer and sonicated for 1 min. After sonication, cytosolic and membrane fractions were separated by centrifugation
Statistical Analysis All results are expressed as mean 6 SD unless otherwise mentioned. The Student’s t-test was used to evaluate the difference of means between groups, accepting P , 0.05 as level of significance [21]. RESULTS Gs Protein We studied the role played by the Gs proteins by incubating CFSC in the presence of CTX. This study demonstrated that treatment of cells with this toxin resulted in a doseand time-dependent decrease in the steady-state levels of collagen a1(I) mRNA (Figs. 1A and 1B) Thus, incubation of cells with 0.4 mg/ml CTX for 24 h reduced the steadystate levels of collagen a1(I) mRNA to 27.8 6 6.0% of the control level (Table 1; Fig. 2A). This toxin alters the conformation of the Gs protein, which results in a prolonged activation of adenylyl cyclase and elevation in cAMP levels
TABLE 4.
Incorporation of [3H]inositol into IP3 (cpm/106 cells) 0 min Control PTX, 10 ng/ml 1 TNFa, 0.6 nM TNFa, 0.6 nM Neomycin, 0.5 mM 1 TNFa, 0.6 nM PTX, 10 ng/ml
3172 1951 3046 2691 1903
6 6 6 6 6
243 134 (*) 154 (NS) 253 189 (NS)
5 min 3066 2693 5970 3169 2146
6 6 6 6 6
283 165 (*) 196a (*) 233 228 (**)
15 min 3138 2280 5054 3257 2073
6 6 6 6 6
241 132 (*) 266a (*) 178 146 (NS)
30 min 3072 2140 3095 2810 1971
6 6 6 6 6
304 179 (**) 208 (NS) 206 183 (NS)
Cells were labelled with myo[3H]inositol for 24 h as described under Material and Methods and treated with 0.6 nM TNFa for the indicated times with or without pre-treatment with 500 mM neomycin for 2 h or with 10 ng/ml pertussis toxin (PTX) for 24 h. Values are means 1 SD from a single experiment which was conducted in triplicate. a P , 0.001 compared with control cells. * 5 P , 0.001 compared with TNFa-treated cells; ** 5 P , 0.05; NS 5 not significant.
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TABLE 5.
TABLE 6.
[Ca21]i (arbitrary units of fluorescence) Additive
Basal
Additive
(%)
TNFa, 0.6 nM
7.2
16.7
232
Neomycin, 500 mM
5.6
4.9
88
Neomycin, 500 mM 1 TNFa, 0.6 nM
6.3
7.9
126
A32187, 10 mM
6.8
19.6
288
PTX, 10 ng/ml 1 TNFa, 0.6 nM
4.6
5.9
128
Intracellular free calcium level in CFSC were determined as described in Material and Methods. “Basal” represents prestimulation fluorescence and “additive” means peak fluorescence values after the addition of test substances. Data are representative of two independent experiments. % 5 Percentage of basal level after additive.
[8]. To document increases in cAMP levels after exposure to cholera toxin, cAMP was measured in cells after 2 and 24 h of treatment with 0.4 mg/ml CTX. Cellular levels of cAMP increased between 30- and 40-fold on exposure to CTX for 2–24 h (Table 2). Treatment of cells with 100 mM forskolin for 24 h, which activates adenylyl cyclase and increases cAMP levels (Table 2), led to a decrease in collagen a1(I) mRNA levels to 51.2 6 1.4% of control levels (Table 1; Fig. 2A). Likewise, when CFSC were exposed to the cAMP-analogue 1 mM 8Br-cAMP, for 24 h, the level of collagen a1(I) mRNA decreased to 66.0 6 3.6% of control levels (Table 1; Fig. 2A). On the other hand, 50 mM H8, which inhibited 8Br-cAMP-activated PKA by about 50% (Table 3), blocked the effect of CTX on collagen a1(I) mRNA (Table 1; Fig. 2B).
PKC activity (pmol[32P] incorporated/min/mg protein)
Control PMA, 60 nM, 1 hour H7, 50 mM 1 PMA, 60 nM Staurosporine, 0.5 nM 1 PMA, 60 nM PMA, 10 nM, 24 hours
Cytosolic
Membrane
141 6 21 41.8 6 12.3
67 6 19.2 537 6 36a
96.3 6 22.4
60 6 18.5
72.7 6 9.3 63.9 6 11.7
25.6 6 13.9 b 265 6 16.1a
Cells were treated with 10 or 60 nM PMA for 24 or 2 h, respectively, with or without pre-incubation with 0.5 nM staurosporine or 50 mM H7 for 2 h. Subsequently, cells were washed and sonicated as described in Material and Methods. Cytosolic and membrane fractions were separated by ultracentrifugation and PKC activity in both fractions was measured as described. Data represent means 6 SD of one experiment performed in triplicate. a P , 0.001. b P , 0.05.
the incorporation of [3H]inositol into IP3 and the increase in intracellular free calcium level induced by TNFa treatment (Tables 4 and 5). Incubation of CFSC for 24 h with 10 mM calcium ionophore A23187, which allows calcium to move into the cytoplasm from the extracellular fluid and reproduces the effects of IP3, decreased collagen a1(I) mRNA to 57.5 6 20% of the control level (Table 1; Fig. 4A). In contrast, 0.1 mM verapamil, which decreases cytosolic free calcium, increased collagen a1(I) mRNA level 2-fold. Likewise, blocking calmodulin with 10 mM calmidazolium, 10 mM TFP or 10 mM W-7 resulted in a marked increase in collagen a1(I) mRNA by 39 6 9% to 126.8 6 12% over the control level (Table 1; Fig. 4B). Blockade of G proteins with 10 ng/ml PTX for 24 h led to an inhibition of the TNFa-induced increase in both cytosolic IP3 and calcium (Tables 4 and 5).
PTX-Sensitive G Proteins The role played by PTX-sensitive G proteins was investigated by incubating CFSC for 24 h with a range of PTX concentrations between 1 ng/ml and 10 ng/ml. This study demonstrated that this toxin induced a dose-related increase in intracellular steady-state levels of collagen a1(I) mRNA of 96.8 6 9.7% over the control level at a concentration of PTX of 10 ng/ml (Table 1; Fig. 3). Likewise, PTXtreatment (10 ng/ml) for 24 h enhanced cAMP levels from about 8.7 6 1.3 to 36 6 2.7 pmol/l06 cells (Table 2). PLC and Intracellular Calcium Because a PTX-sensitive G protein may stimulate PLC and liberate inositol 1,4,5-triphosphate (IP3) into the cytosol, we treated CFSC with neomycin, an inhibitor of this enzyme. Neomycin binds phosphatidylinositol 4,5-biphosphate (PIP2) and inhibits inositol 1,4,5-triphosphate (IP3) release [22]. Treatment of cells with 500 mM neomycin for 24 h led to an increase in this mRNA level by 101.3 6 22.3% over controls (Table 1; Fig. 4A), and blunted both
PKC The diacylglycerol, the second hydrolysis product of the PLC pathway, is a potent activator of PKC, which, in turn, might phosphorylate proteins involved in the modulation of gene expression. Therefore, we examined the role of this protein kinase in the regulation of collagen gene expression by incubating CFSC for 24 h with 0.5 nM staurosporine, a PKC inhibitor (Table 6). Three independent experiments demonstrated that staurosporine did not change significantly the steady-state levels of collagen a1(I) mRNA (Table 1; Fig. 5A). One the other hand, activation of PKC by treating cells with 10 nM PMA for 24 h (Table 6) resulted in a decrease in the intracellular content of this mRNA Fig. 5A). Similar results were obtained, when a chimeric plasmid containing of segments of the collagen 59-flanking region ligated to the chloramphenicol acetyl transferase (CAT) gene (COLCAT1) was transfected into CFSC and CAT activity was assayed. PMA (10 nM, 24 h), as well as the membrane-permeable protein kinase C activator OAG (63 mM),
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FIGURE 5. Effects of protein kinase C stimulation and inhibi-
tion on collagen a1 type I mRNA level and chloramphenicol acetyltransferase (CAT) activity. (A) CFSC were incubated for 24 h in culture medium lacking (C) or containing PMA (10 nM) or staurosporine (0.5 nM). Collagen a1(I) mRNA level was analysed as described in Fig. 1. (B) DNA fragments containing part of the first exon and 3700 bp of the mouse a1(I) collagen promoter region fused to the bacterial chloramphenicol acetyltransferase gene (COLCAT1) were transfected into CFSC by electroporation as described in Methods. Transfected cells were collected in a common pool, mixed, and aliquots of cell suspension were plated in culture flasks for different experimental conditions. Six hours after electroporation, cells were left untreated (control) or treated for 24 h with 50 mM H-7, 0.5 nM staurosporine, 10 nM PMA or 100 mM OAG. Aliquots of cell lysates containing equal amounts of proteins were analysed for CAT activity. The thin-layer plates were exposed to X-ray film for 24 h. Individual spots were localised and excised, and radioactivity in each spot was determined by scintillation counting. The percent of acetylation was calculated. The results presented are representative of three separate experiments. C, control. PMA, phorbol myristate acetate. STAU, staurosporine. OAG, oleyl acetyl glycerol. Coll.a1(I), collagen a1(I) mRNA. a-Tubulin, a-tubulin mRNA. Coll.a1(I)/a-Tub., Collagen a1(I)/a-Tubulin mRNA ratio. Chl, [14C]chloramphenicol. Ac Chl, monoacetylated [14C]chloramphenicol. Ac Chl.(%), percentage of monoacetylated [14C]chloramphenicol.
decreased CAT activity to 31% and 5%, respectively, of control levels, while the protein kinase C inhibitors H7 (50 mM) and staurosporine (0.5 nM) increased CAT activity slightly (18% and 7%, respectively) (Fig. 5B). PLA2 Because some cell receptors are coupled to PLA2 through a G protein [23], we studied the role played by this enzyme in the modulation of collagen gene expression. Cells were in-
FIGURE 6. Effect of blockade of phospholipase A2 pathway on
the steady-state levels of collagen a1(I) mRNA. Culture of confluent CFSC were incubated in control medium (0) or in medium containing 20 mM BPB (A), 0.5–2 mM quinacrine (B), 24 nM NDGA or 100 mM indomethacin (INDO) (C). Total RNA was isolated, electrophoresed, transferred to nylon filters and hybridized as described in Material and Methods and Fig. 1. Coll.a1(I), collagen a1(I) mRNA. a-Tubulin, a tubulin mRNA. Coll.a1(I)/a-Tub., Collagen a1(I)/a-Tubulin mRNA ratio. BPB, bromophenacylbromide. NDGA, nordihydroguaiaretic acid.
cubated with two different inhibitors of PLA2 (Table 7) for 24 h. BPB (20 mM) induced a marked increase in the steady-state level of collagen a1(I) mRNA, which rose by 61.9 6 12% over that in control cells (Fig. 6A). Likewise, increasing concentrations (0.5 mM to 2 mM) of quinacrine resulted in an elevation of collagen a1(I) mRNA content in cells by 50 6 28% to 100 6 33% above the central levels (Fig. 6B). Indomethacin (100 mM), used as cycloxygenase inhibitor, increased the collagen a1(I) mRNA content in the cells by 105.5% 6 34%, and 24 mM NDGA, a lipoxygenase inhibitor, raised these levels slightly by 11 6 12% over the control level (Table 1; Fig. 6C). “De Novo” Synthesis of Proteins Cycloheximide at 0.1 mM, a concentration that blocks protein synthesis by 95% in CFSC, did not affect the steadystate levels of collagen a1(I) mRNA, indicating that de novo synthesis of a protein is not required for the expression of collagen gene at basal level (Table 1).
G Proteins and Collagen Gene Expression
181
TABLE 7.
Phospholipase A2 activity (nmol/min/mg) 0
5
10
30
90 min
Control
0.574 6 0.011
0.569 6 0.013
0.571 6 0.016
0.566 6 0.012
0.572 6 0.011
BPB, 20 mM
0.230 6 0.01
0.206 6 0.013
0.194 6 0.02
0.179 6 0.019
a
0.172 6 0.011a
Quinacrine, 2 mM
0.330 6 0.014a
0.328 6 0.011a
0.320 6 0.016a
0.314 6 0.015a
a
a
a
0.320 6 0.013a
Phospholipase A2 activity was measured in triplicate at 50 mM phosphatidyl choline substrate (1-palmitoyl-2-(1-14C)arachidonylglycero-3-phosphocholine in the presence or absence of 20 mM BPB or 2 mM quinacrine. Data are given as mean values 6 SD. a P , 0.001 as compared with control cells.
DISCUSSION The results of the present study indicate that a Gs protein is involved in the suppression of collagen gene expression. Thus, when CFSC were treated with CTX, an enzyme that activates Gs proteins, there was a 30-fold increase in cellular cAMP level and a marked reduction in the steady-state levels of collagen a1(I) mRNA (Table 1; Fig. 1). To confirm that suppression of collagen a1(I) mRNA induced by CTX treatment was mediated through an activation of adenylyl cyclase and an elevation of cAMP levels and not through other properties of this toxin, cells were treated with forskolin, a diterpene which elevates cAMP levels (18-fold) (Table 2) by activating adenylyl cyclase. This treatment led also to a marked reduction in the steady-state level of collagen a1(I) mRNA (Table 1; Fig. 2A). This effect may be mediated by the cAMP released by activation of adenylyl cyclase, since addition of 8Br-cAMP to the cells resulted in a 34% decrease in collagen a1(I) mRNA level (Table 1; Fig. 2A). As far as we know, no studies have been published on the role played by Gs-protein in the modulation of collagen a1(I) mRNA levels. However, a number of studies have shown that cAMP decreases collagen production (24–26). In Schwann cells treated with either forskolin or cAMP derivatives, collagen types I and III mRNA were significantly suppressed [27]. The mechanism whereby cAMP decreases the expression of the collagen gene is not known. Cyclic AMP generated in response to cell receptor occupancy causes PKA activation and phosphorylation of transcription factors [28–30]. Our results point to the mediation of this enzyme in the cAMP-induced inhibition of collagen gene. When PKA was inhibited by pre-incubating CFSC with H-8, the intracellular steady-state level of collagen a1(I) mRNA did not decrease after treating these cells with CTX (Table 1; Fig. 2B). All members of the Gi family, including Gi, Go and Gt proteins, can be blocked by PTX [7]. This enzyme uncouples the Gi family from receptors and therefore these proteins fail to interact with effecters. Thus, PTX inhibits the actions mediated by this class of G proteins. In order to evaluate the role played by these proteins on the modulation of collagen a1(I) gene expression, cells were treated with increasing concentrations of PTX. Our results provide evidence for an implication of a PTX-sensitive G protein in
the suppression of type collagen a1 gene expression. Indeed, this treatment resulted in a dose-related increase in type collagen a1 mRNA content in these cells (Table 1; Fig. 3), so that 10 ng/ml PTX elevated these levels 2-fold (Table 1). Obviously, this response cannot be ascribed to the inhibitory action of the Gi protein on adenylyl cyclase, since PTX increased cAMP levels (Table 2) and, consequently, should decrease, but not increase, collagen a1(I) mRNA levels. It is unlikely that PTX-induced increase in mRNA levels can be imputed to Gt or Go proteins either, as these proteins are found only in the retina and neurons, respectively [7, 8]. Gi protein, in addition to its action on adenylyl cyclase, may also activate a phosphatidylinositol-specific PLC [5, 31]. Stimulation of this Gi protein in response to cell receptor occupancy leads to the activation of PLC, which cleaves phosphatidylinositol 4,5-biphosphate (PIP2) to yield inositol 1,4,5-triphosphate (IP3) and DAG [32]. These two metabolites act as second messengers of cellular activation. IP3 increases cytosolic free calcium concentration [33] while DAG stimulates PKC. Our results suggest that PLC may be activated by a PTX-sensitive G protein in CFSC. Thus, treatment of CFSC with PTX led to a marked inhibition of the TNFa-induced increase in cytosolic IP3 and Ca21 concentrations (Tables 4 and 5), that may be the results of a reduced PLC activity. On the other hand, blocking PLC by treating CFSC with neomycin, an inhibitor of IP3 formation [22], led to a marked increase in the intracellular levels of collagen a1(I) mRNA (Fig. 4A). These results point to an inhibitory role of the pathway initiated in the PLC. IP3, one of the two metabolites cleaved by PLC from PIP2, promotes the release of Ca21 from intracellular storage sites, increasing the cytosolic free Ca21 concentration [33]. There is very little information about the role played by calcium ion in collagen gene expression. Our results show that this intracellular second messenger plays a role in the modulation of the expression of collagen gene. Preincubation of CFSC with ionophore A23187, which increases intracellular Ca21, reduced the steady-state level of collagen a1(I) mRNA (Fig. 4A). In contrast, verapamil, a calcium channel blocker, increased the level of collagen a1(I) mRNA (Fig. 4A). These results concur with those previously reported by Flaherty and Chojkier [34] and Chojkier et al. [35]. Many of the intracellular actions of calcium, including the expression of some genes, occur by its binding to cal-
J. A. Solı´s-Herruzo et al.
182
modulin [36]. In favour of the involvement of a calmodulin dependent system in the inhibition of collagen gene expression are our results after blocking calmodulin with three different anticalmodulin drugs (calmidazolium, TFP, W-7). Inhibition of this calcium-binding protein resulted in a “marked” increase in the levels of collagen a1(I) mRNA (Fig. 4B). DAG, a product cleaved from PIP2 by PLC activation, stimulates PKC [32]. Our results show that PMA decreased steady-state levels of collagen a1(I) mRNA (Fig. 5A), as well as CAT activity in CFSC transfected with COLCAT1 (Fig. 5B). In contrast, blocking PKC with H7 or staurosporine did not change significantly CAT activity or collagen a1(I) mRNA levels (Table 1). These results suggest that DAG and PKC are also parts of an inhibitory pathway of collagen gene, which, in basal conditions, seems to be little active. These results are in agreement with those obtained by other authors [34–38], who found that phorbol esters markedly inhibited collagen production in cultured fibroblasts. Likewise, Goldstein et al. [38] reported that this PMA-mediated inhibition in collagen production was associated with a marked reduction in the a1(I) collagen mRNA level and gene transcription. PLA2, one of the enzyme coupled to membrane receptors by G proteins [23], has also been implicated in intracellular signalling mechanisms. This enzyme cleaves arachidonic acid from phospholipids and generates a variety of metabolites including prostaglandins, leucotrienes and lipoxins [39]. These metabolites of arachidonic acid have also been implicated as direct mediators of post-receptor events in a number of cell lines [40]. Our study provides evidence for an implication of PLA2 in modulating the expression of collagen gene. Treatment of CFSC with some PLA2 inhibitors, such as BPB or quinacrine, or with indomethacin, a cycloxigenase inhibitor, led to a marked increase in type I collagen a1 mRNA level in these cells (Table 1; Fig. 6), while the inhibition of lipoxygenase induced no significant change in this mRNA. Thus, these results suggest that PLA2 and prostaglandins are implicated in a pathway involved in the inhibition of collagen gene expression. Activated protein kinases may modify the phosphorylation of a number of proteins which may channel signals from the cell membrane to the transcription apparatus in the nucleus. Transcription of a1(I) collagen gene is regulated by specific interaction of trans-acting transcription factors and cis-acting DNA elements located in the promoter, 59-flanking region and the first intron of the gene. A number of studies have demonstrated that both positive and negative elements are present in these regions and various trans-acting factors have been partially characterised [41]. However, little is known about the trans-acting factors acting as third messengers in each signal-transducing pathway and the cis-regulating elements in the type I collagen a1 gene. Nevertheless, this study shows that no synthesis of a protein is necessary to modulate basal expression of collagen gene, since inhibition of protein synthesis with cycloheximide did not modify cellular levels of collagen a1(I)
mRNA (Table 1). These results concur with those we described in human skin fibroblasts and NIH 3T3 cells [42]. In conclusion, this study shows that collagen gene expression is inhibited by a number of intracellular pathways which transduce signals from the cell membrane to the nucleus. A Gs protein and a PTX-sensitive G protein seems to initiate cellular response to receptor occupancy. cAMP and PKA act as second messengers of the Gs protein activation, while PLC/IP3/calcium/calmodulin, DAG/PKC and PLA2/ prostaglandins seem to be effecters of the PTX-sensitive G protein. It is conceivable that fibrogenic activity of fat-storing cells is hindered and secured by multiple intracellular pathways, diverting the activity of these cells to other functions. Nuclear factors, acting as third messenge is in these pathways, have to be identified. However, it seems that they do not require to be synthesised. It is most likely that they are still present within the cells, but need to be activated. The authors are grateful to Patricia Greenwel and Dr. Marcos Rojkind (Albert Einstein College of Medicine, Bronx, NY) for the gift of the CFSC cell line, Dr David A. Brenner (University of North Carolina, Chapel Hill, NC) for the kind gift of COLCAT1 plasmid and Dr. David Rowe (University of Connecticut Health Centre) for providing us the collagen a1(I) rat probe.
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