Retinoids Stimulate Fibrinogen Production Both In Vitro (Hepatocytes) and In Vivo
Induction Requires Activation of the Retinoid X Receptor
Abstract The in vitro effects of retinoids on fibrinogen synthesis were investigated in HepG2 cells and primary human hepatocytes. In vivo effects were studied in the rat. In HepG2 cells, maximal stimulation (twofold) of fibrinogen secretion was obtained when cells were incubated in the presence of 1 μmol/L all-trans retinoic acid (T-RA) for 24 hours. A comparable increase was observed for both de novo fibrinogen synthesis and fibrinogen β chain mRNA level. In primary cultures of human hepatocytes, treatment with 1 μmol/L T-RA for 72 hours also gave a twofold increase in fibrinogen production. Furthermore, rats treated for 6 days with 100 mg · kg−1 · d−1 T-RA presented increased fibrinogen plasma levels (110%). A selective retinoic X receptor (RXR) agonist, 4-[1-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzoic acid (3-methyl TTNEB), as well as 9-cis retinoic acid, a natural RXR ligand, mimicked the effects of T-RA on fibrinogen synthesis in vitro at lower concentrations. In contrast, a selective retinoic A receptor α (RARα) agonist was a poor activator. The ED50 of the different retinoids on fibrinogen secretion by HepG2 cells was 25 nmol/L for T-RA, 4 nmol/L for 9-cis retinoic acid, 11 nmol/L for the synthetic RXR agonist, and >500 nmol/L for the RARα agonist. However, incubation of HepG2 cells with RXR agonist together with RARα agonist resulted in a further increase in fibrinogen production. The secretion of two other acute-phase proteins, α-antichymotrypsin and caeruloplasmin, was also stimulated by retinoids in HepG2 cells but by a different regulatory mechanism. We conclude that activation of RXR by a specific ligand upregulates fibrinogen production by hepatocytes. Elevated levels of fibrinogen in rats treated with T-RA indicate that retinoids may be involved in the physiological regulation of fibrinogen, with a key role for RXR.
- Received April 4, 1995.
- Accepted June 27, 1995.
Human fibrinogen is a plasma glycoprotein that participates in the final phase of the blood coagulation cascade. It is a large protein with a molecular mass of 340 000 D corresponding to two symmetrical half molecules, each consisting of one α, one β, and one γ chain with molecular weights of 66 000, 52 000, and 46 000 D.1 These chains are encoded by three different genes.
Several epidemiological studies have demonstrated that fibrinogen is an independent risk factor for cardiovascular disease.2 3 Thus, it would be interesting to identify hypofibrinogenemic drugs. In this context, it is important to elucidate the mechanisms controlling fibrinogen synthesis by the liver. Fibrinogen synthesis is positively regulated by its degradation products, the fragments D and E, via a monocyte-dependent pathway and/or via a direct effect on hepatocytes.4 5 This constitutive positive-feedback loop could represent the major regulation of fibrinogen synthesis by the liver under physiological conditions. Fibrinogen is also a positive acute-phase protein. In an inflammatory state, fibrinogen synthesis is under the control of two mediators in the liver, glucocorticoids and IL-6. Two sequences, located between −2900 to −1500 bp and −150 to −82 bp upstream of the start site of transcription of the β chain gene, have been characterized and identified as response elements for glucocorticoids and IL-6, respectively.6 The stimulation of fibrinogen synthesis by IL-6 and glucocorticoids has been demonstrated in HepG2 cells, making these cells a useful model in which to study the in vitro modulation of fibrinogen production.7
In the human hepatocarcinoma HepG2, β chain synthesis is the rate-limiting step in fibrinogen production,8 although expression of the three fibrinogen genes is coordinated. Overexpression of any fibrinogen chain by transfection of the corresponding cDNA into HepG2 cells stimulates the expression of the other two genes.9
Vitamin A and its active metabolite T-RA have profound effects on cell growth and differentiation.10 The pleiotropic effects of retinoids are mediated by two known families of nuclear receptors, both belonging to the steroid hormone receptor superfamily. These nuclear receptors function as ligand-dependent transcription factors. The RAR gene family comprises three subtypes, RARα, RARβ, and RARγ, with each gene encoding a variable number of isoforms.11 All members of this family bind T-RA with the same affinity. The second family of receptors, RXR, also consists of three distinct isoforms designated RXR α, β, and γ. Instead of T-RA, they bind its stereoisomer 9-cis RA.12 13 14 15 The classification of these receptors is based on differences in amino acid sequence, responsiveness to different retinoids, and ability to modulate expression of different target genes. Thus, the multiple effects of retinoic acid can be analyzed by using ligands specific for the known receptors. These ligands can be used to distinguish the respective contributions of RXR and RAR to retinoid effects. It has been postulated that RAR requires heterodimerization with RXR for efficient DNA binding and gene regulation, while RXR can also bind to DNA and functions as a homodimer in the presence of 9-cis RA.16 Synthetic retinoids with strong preferences for each of the RAR subtypes have been described.17 However, a selective pharmacological action of these compounds has not been proven. A new synthetic RXR agonist, 3-methyl TTNEB, displays a very strong selectivity for the RXR receptor.18 A biological effect of a specific RXR agonist has not yet been described.
In the present study we demonstrate that T-RA induces a twofold increase in fibrinogen synthesis and secretion by HepG2 cells. This effect can be mimicked by the RXR agonist 3-methyl TTNEB, indicating a positive role of RXR agonist in the production of fibrinogen. Furthermore, we show that T-RA upregulates the production of fibrinogen by primary human hepatocytes, leading to the assumption that fibrinogen synthesis by the liver is under the control of retinoids. The physiological importance of this hypothesis is supported by our in vivo results in the rat, in which T-RA treatment induced a large increase in fibrinogen plasma levels.
T-RA was purchased from Sigma. 9-cis RA and 3-methyl TTNEB were synthesized in our laboratory. AM 580 (RARα agonist) was kindly provided by the Centre International de Recherche en Dermatologie, CIRD-GALDERMA, Nice, France. Cell culture media were obtained from GIBCO. Collagen-coated culture plates were from J. Bibby. Recombinant IL-6 was purchased from Genzyme, and purified human fibrinogen was obtained from Diagnostica Stago. Anti-human fibrinogen monoclonal antibody was from Cederlane. Anti-rat fibrinogen polyclonal antibody as well as horseradish peroxidase (HRPO)–labeled anti-human fibrinogen polyclonal antibody were from Cappel-Organon Teknika. Anti-human fibrinogen polyclonal antibody used in the immunoprecipitation technique was obtained from Dakopatts. Antisera against human α-antichymotrypsin, caeruloplasmin, and albumin were purchased from The Binding Site. [35S]Protein labeling mix (1000 Ci/mmol) and [α-33P]dCTP (1000 Ci/mmol) were obtained from New England Nuclear. The RNA-SR extraction kit was purchased from Biogenesis-Realef. The DNA labeling kit “Ready To Go” was from Pharmacia. Hybond-N nylon membranes and rapid hybridization buffer were from Amersham. The human β chain fibrinogen cDNA, human GAPDH cDNA, and HepG2 cells were purchased from American Type Culture Collection. Male Sprague Dawley rats were obtained from Iffa Credo. All other chemicals were of the best available commercial grade.
HepG2 cells were cultured in BME Eagle’s medium containing 2 mmol/L glutamine, 0.1 mmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, 100 U/L each penicillin and streptomycin, and 10% FCS and were grown at 37°C in a 5% CO2 incubator. When cells were 70% to 90% confluent they were incubated for further studies in RPMI-1640 supplemented with 1% FCS in the presence of drugs. Medium was renewed every 24 hours. All retinoids were dissolved in dimethyl sulfoxide at a stock solution that was 500-fold concentrated and stored at −20°C until used. For each concentration used, the final dimethyl sulfoxide concentration was 0.2%. At the end of the incubation, fibrinogen concentration in the supernatants was quantified by ELISA.
Metabolic Labeling of the Cells
In some experiments, HepG2 cells were labeled for 24 hours in RPMI-1640 without methionine supplemented with 10 μmol/L methionine, 1% FCS, and 10 μCi/mL [35S]protein labeling mix. Radiolabeled secreted fibrinogen and other acute-phase proteins were analyzed by immunoprecipitation of the supernatants.
For fibrinogen neosynthesis experiments, HepG2 cells were starved for 15 minutes in RPMI-1640 without methionine and then pulsed for 10 minutes in the same medium supplemented with 80 μCi/mL [35S]protein labeling mix. Cells were then chased for 15 minutes with RPMI-1640 supplemented with an excess of cold methionine. Cells were lysed in 10 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L EDTA, and 1% Nonidet P40, pH 7.4. Lysates were used for fibrinogen immunoprecipitation.
Primary Human Hepatocytes
Primary human hepatocytes were isolated from liver biopsies by using the collagenase perfusion technique.19 The medium used for the maintenance of the cells was 50% Williams E, 50% nutrient mixture Ham’s F-12, 100 U/L each penicillin and streptomycin, 2 mmol/L glutamine, 7 mmol/L d-glucose, 0.4 mmol/L l-ornithine, 0.6 mmol/L l-arginine, 284 μmol/L l-ascorbic acid, 10 nmol/L glucagon, 333 nmol/L insulin, 1.27 μmol/L transferrin, 0.1 μmol/L dexamethasone, 10 IU/mL heparin, and 2% FCS. Cells were seeded at a density of 300 000 cells/well in 24-well plates precoated with collagen and were allowed to adhere overnight at 37°C in a 5% CO2 incubator. Cells were then washed twice and incubated with the compounds for 72 hours in the labeling medium, which contained 89% minimum essential medium without methionine, 5% nutrient mixture Ham’s F-12, 5% Williams E, 100 U/L each penicillin and streptomycin, 2 mmol/L glutamine, 7 mmol/L d-glucose, 0.4 mmol/L l-ornithine, 0.6 mmol/L l-arginine, 284 μmol/L l-ascorbic acid, 10 nmol/L glucagon, 333 nmol/L insulin, 1.27 μmol/L transferrin, 0.1 μmol/L dexamethasone, 10 IU/mL heparin, 1% FCS, and 20 μCi/mL [35S]protein labeling mix. Radiolabeled secreted fibrinogen was analyzed by immunoprecipitation of the culture media.
Quantification of Fibrinogen in Supernatants by ELISA
Anti-human fibrinogen monoclonal antibody (0.75 μg/well) in PBS (0.040 mol/L phosphate and 0.14 mol/L NaCl, pH 7.4) was adsorbed onto microtiter-plate wells for 1 hour at 37°C and then overnight at 4°C. Plates were washed with 0.002% Tween 80 in PBS (PBS-Tween) and blocked with 1% BSA in PBS for 2 hours at room temperature. Plates were then washed three times. Cell supernatants and purified human fibrinogen were diluted in PBS-Tween supplemented with 0.1% BSA. The diluted samples were added to the wells and incubated for 1 hour at 37°C. After three washes, HRPO–labeled polyclonal antibody against human fibrinogen was diluted in PBS-Tween at a final concentration of 1.6 μg/mL, and 150 μL was added to each well. Plates were incubated for 30 minutes at 37°C. Thereafter, the plates were washed three times and examined by using 1 mg/mL orthophenylene diamine in 0.05 mol/L citrate/0.03% H2O2, pH 5 to 6. After 10 minutes, the reaction was stopped by the addition of 4N H2SO4, and the absorbance at 492 nm was read. A sample concentration was obtained by comparison with a standard curve that was linear from 5 to 110 ng/mL fibrinogen.
Immunoprecipitation of Fibrinogen and Other Acute-Phase Proteins
The radiolabeled proteins were immunoprecipitated with specific rabbit or sheep anti–human plasma protein antibodies isolated with protein A or G Sepharose beads. Beads were washed with Tris-HCL 10 mmol/L, NaCl 500 mmol/L, EDTA 1 mmol/L, and Nonidet P40 1%, pH 7.4. The final pellet was resuspended in sodium dodecyl sulfate sample buffer, heated at 100°C for 5 minutes, and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The radiolabeled bands were quantified on a PhosphorImager (Molecular Dynamics).
Northern and Slot-Blot Analysis of mRNA Levels
Total RNA from 20 to 30×106 cells was extracted by using an RNA-SR kit. For slot-blot analysis, 10 μg total RNA was loaded directly on Hybond-N nylon membranes. For Northern blot analysis, 15 μg total RNA was electrophoresed in a 1% agarose gel by using the formamide/formaldehyde technique20 and transferred to Hybond-N membranes in 20× standard saline citrate buffer. Membranes were baked for 3 hours at 80°C. Linearized plasmids containing β chain fibrinogen cDNA or GAPDH cDNA were radiolabeled with 5′ [α-33P]dCTP by using a random priming method (“Ready To Go” kit). Hybridization was performed for 2 hours at 65°C with an 8-ng/mL probe in the rapid hybridization buffer. Membranes were then washed twice at 65°C for 10 minutes in 2× standard saline citrate and 0.1% sodium dodecyl sulfate and exposed by using PhosphorImager screens.
T-RA was dissolved in 5% ethanol, 0.04 mol/L NaOH, and 0.15 mol/L NaCl. Male Sprague Dawley rats were treated for 6 days with 100 mg · kg−1 · d−1 (1 mL PO) of the T-RA compound. Control animals received vehicle. During treatment the animals were fed a standard chow. Blood samples were obtained by intracardiac puncture and collected in citrate tubes. Plasma was analyzed for its fibrinogen content by using an ELISA.
Quantification of Fibrinogen in Rat Plasma by ELISA
Buffers used in this procedure are the same as the ones described in the human fibrinogen ELISA. Polyclonal antibody to rat fibrinogen (1 μg/well) was adsorbed on microtiter plates for 1 hour at 37°C and overnight at 4°C. Plates were washed with PBS-Tween and blocked with 2% BSA in PBS for 2 hours at 37°C. Rat plasma was diluted 50 000- to 150 000-fold with 0.1% BSA in PBS-Tween. Human purified fibrinogen was used for the standard curve as this ELISA cross-reacts with human fibrinogen. Samples were incubated for 2 hours at 37°C. Plates were then washed three times. HRPO–labeled anti-human fibrinogen polyclonal antibody (1:10 000 dilution in 0.1% BSA–PBS-Tween) was added for 2 hours at 37°C. The reaction was examined by using 2 mg/mL orthophenylene diamine in citrate buffer and stopped by adding 4N H2SO4. The absorbance at 492 nm was read. The standard curve was linear from 5 to 50 ng/mL fibrinogen.
Effects of T-RA on In Vitro and In Vivo Fibrinogen Production
HepG2 cells were incubated for 24 hours with various concentrations of T-RA. As shown in Fig 1⇓, T-RA induced a dose-dependent increase in fibrinogen secretion with an ED50 of 25 nmol/L and a maximal effect at 1 μmol/L. Higher doses of T-RA were cytotoxic. Similar results were obtained by using two other human hepatoma cells (data not shown). To determine if the retinoid effect could also be observed on primary cultures, human hepatocytes were incubated for 72 hours with 1 μmol/L T-RA, and the fibrinogen content of the supernatants was analyzed by immunoprecipitation. T-RA (1 μmol/L) induced a twofold increase in fibrinogen secretion after quantification on a PhosphorImager (Fig 1⇓). IL-6, used as a positive control of induction of fibrinogen production by these cells, gave a fourfold increase. The stimulation of fibrinogen synthesis in primary human hepatocytes was marginal after a 24-hour incubation period with 1 μmol/L T-RA but became significant with longer incubations.
To investigate whether the effects observed with retinoids in human hepatoma cells and primary human hepatocytes were physiologically relevant, we performed an in vivo experiment by using Sprague Dawley rats. Animals were treated with T-RA 100 mg · kg−1 · d−1 PO. T-RA induced a significant increase (110±20%, P=.0001) in plasma levels of fibrinogen after a 6-day treatment (Table⇓). Similar results were obtained in BALB/c mice (data not shown). These experiments show that T-RA also stimulates the hepatic production of fibrinogen in vivo.
To further characterize the in vitro effect of T-RA in HepG2 cells, we performed a kinetic experiment. Cells were treated for various periods with 1 μmol/L T-RA. The effect of T-RA on the intracellular synthesis of the three fibrinogen chains was analyzed by immunoprecipitation after a [35S]methionine pulse chase. The neosynthesis of the α, β, and γ chains was strongly induced by T-RA (Fig 2⇓). A pretreatment of at least 4 hours with T-RA was required to observe an effect. There was a coordinate increase in the neosynthesis of the three chains, but the effect of T-RA on the β chain was significantly higher than the effects observed for the α and γ chains: 74% induction for the β, 54% for the α, and 32% for the γ chain were observed after 24 hours of T-RA treatment. We next examined the effect of T-RA at the β chain mRNA level, because the synthesis of the β chain is the rate-limiting step for fibrinogen production by HepG2 cells.8 T-RA (1 μmol/L) induced a 130% increase in β chain mRNA level after a 24-hour incubation, but the GAPDH mRNA level was not affected (Fig 2⇓). T-RA also induced a 148% increase in α chain mRNA (data not shown). The induction of β chain mRNA was also detectable after 4 hours of treatment with T-RA (data not shown). These results clearly demonstrate an effect of T-RA on fibrinogen synthesis, thus suggesting the existence of a transcriptional or posttranscriptional control by T-RA.
Effects of Various Retinoids on Fibrinogen Production
Most retinoid effects are exerted by receptor-mediated regulation of gene expression. To date, two families of nuclear receptors have been characterized as ligand-inducible transcription factors.11 12 13 14 15 Specific ligands of each RAR17 and RXR18 subtype have been synthesized. The specificity of 3-methyl TTNEB, an RXR agonist, is 100- to 1000-fold higher for RXR subtypes than for RAR subtypes, leading to the conclusion that this compound is a highly selective RXR agonist.18
To investigate which receptor subtype was involved in the fibrinogen induction, HepG2 cells were treated with various retinoids known for their specificity for one of the retinoid receptors. With 9-cis RA, a natural ligand for RXR, a strong increase in fibrinogen production by HepG2 cells was observed (Fig 3⇓). The maximal effect was quite comparable in magnitude to that obtained with T-RA, but the ED50 was about sixfold lower (4 versus 25 nmol/L), suggesting an important role for RXR in the stimulation of fibrinogen production by retinoids. However, as isomerization and metabolization of 9-cis RA and T-RA may occur in HepG2 cells, it was not possible to make firm conclusions on the importance of RXR. We therefore studied the effect of specific RXR and RAR agonists. AM 580 is a specific RARα agonist with a Kd of 6.4 nmol/L,17 and 3-methyl TTNEB is a potent and specific RXR agonist.18 We tested these different agonists on HepG2 cells. The RAR agonist was a very poor activator of fibrinogen production, (Fig 3⇓), giving only a slight increase at very high concentrations. In contrast, 3-methyl TTNEB increased fibrinogen production with an ED50 of 11 nmol/L. Like other retinoids, maximal effect was obtained at 1 μmol/L. These results clearly indicated that the activation of the RXR receptor is preferentially involved in fibrinogen production.
To confirm this preferential effect of RXR agonists, we studied the effects of the various agonists at high concentrations on β chain mRNA level. As expected, when used at a 1-μmol/L concentration, 3-methyl TTNEB, like 9-cis RA, induced a nearly twofold increase (85%) in β chain mRNA level compared with the RAR agonist (30%). In the same experiment the level of GAPDH mRNA was not significantly modified by the retinoids (Fig 4⇓). These results are in accordance with a key role of the RXR receptor(s) in fibrinogen production by human hepatoma cells.
Combination of RXR and RAR Agonists on Fibrinogen Production
To study how RXR receptor activates the fibrinogen β gene (ie, RXR homodimer versus RXR/RAR heterodimer), HepG2 cells were incubated with increasing concentrations of RXR and RAR agonists. At each dose the combined treatment of RXR and RAR agonists gave an additive and sometimes synergistic effect on fibrinogen production (Fig 5A⇓). The shape of the curve obtained with the combination of agonists was similar to that observed for 9-cis RA (Fig 3⇑). This result indicated that the heterodimer RXR/RAR could give a maximal stimulation of fibrinogen production.
To test this assumption, we performed the same type of experiment by using lower concentrations of retinoids (2.5 nmol/L). At this dose, the specificity of each agonist for its receptor is very high, and T-RA preferentially activates RAR but not RXR.21 To obtain a better stimulation of fibrinogen production under these conditions, we performed longer incubations (48 hours). AM 580 and T-RA induced a small increase (25%) in fibrinogen production, while the RXR agonists 9-cis RA and 3-methyl TTNEB were more potent (75%) (Fig 5B⇑). The combination of RAR and RXR agonists gave an induction that was stronger than the predicted additive effect (black arrow). This response characterizes a synergy between the RAR and RXR agonists on the fibrinogen gene.
All these results clearly indicate that activation of the RAR receptor alone is not sufficient to increase fibrinogen production in HepG2 cells, whereas activation of the RXR receptor alone is sufficient. However, simultaneous activation of RXR and RAR leads to a stronger effect than RXR alone, suggesting that although the homodimer RXR/RXR may activate the fibrinogen β gene, the heterodimer RXR/RAR gives a maximal activation of the fibrinogen β gene.
Effects of Retinoids on Other Acute-Phase Proteins
In this article we describe the activation of fibrinogen production by retinoids on HepG2 cells. Because the production of fibrinogen, a positive acute-phase protein, is stimulated by IL-6, we wondered if retinoids modulate the production of other acute-phase proteins that are sensitive to IL-6. By using immunoprecipitation techniques we identified three acute-phase proteins other than fibrinogen that are secreted by HepG2 cells: α-antichymotrypsin, caeruloplasmin, and albumin. The production of α-antichymotrypsin and caeruloplasmin was stimulated by 1 μmol/L T-RA, but albumin production was not significantly affected (Fig 6⇓). However, the induction pattern by retinoids was not similar for α-antichymotrypsin and caeruloplasmin. While the RXR agonist induced a stronger activation on caeruloplasmin production than the RAR agonist, leading to a profile of activation similar to the one observed for fibrinogen, no difference could be observed between the RAR and RXR agonists effects on α-antichymotrypsin production. These results suggest the existence of different mechanisms for the effect of retinoids on the regulation of acute-phase protein production by hepatoma cells.
The cellular mechanism of action of retinoids remains the central problem in retinoid biology. Because retinoids are actually used as therapeutic molecules in dermatology and cancerology, it becomes crucial to understand the different pathways involved in the pleiotropic effects of these compounds. The discovery of the retinoid receptor superfamily opened a new area of investigation and prompted research of specific agonists.22 However, the activation of a gene by a specific agonist has not yet been demonstrated at the cellular level. Retinoids activate a large number of genes, including growth factors and their receptors, hormones, cellular enzymes, and effectors.23 Retinoids also act on hepatocytes and hepatoma cells and regulate the transcription of a variety of genes, eg, the phosphoenolpyruvate carboxykinase gene24 and the alcohol dehydrogenase-3 gene.25 Retinoic acid also stimulates the α-fetoprotein mRNA in the Morris rat hepatoma-derived cell line McA-RH8994 but not in the McA-RH7777 hepatoma line, suggesting that different mechanisms are involved.26 Retinoids have been described as inducers of apoA-I synthesis in primary cultures of hepatocytes obtained from cynomolgus monkeys27 and rats.28 However, in both cases, the effects required high concentrations of T-RA (10 μmol/L) and/or long incubations (72 hours).
We have demonstrated here that all T-RA induces a significant activation of fibrinogen production in the human hepatoma cell line HepG2. This effect was obtained with physiological concentrations of T-RA and was reproduced in primary human hepatocytes. In vivo experiments on rats reinforced the physiological relevance of our data since we observed a large increase in plasma levels of fibrinogen after retinoid treatment. Intracellular kinetic analysis of the neosynthesis of the three fibrinogen chains induced by T-RA on HepG2 cells did not reveal a lag time between the induction of one chain with respect to the others. There was a coordinate increase in the neosynthesis of the three chains, but the effect of T-RA on the β chain was greater than the effects observed for the α and γ chains. These results are in agreement with studies indicating that the synthesis of the three chains of fibrinogen in HepG2 cells is coordinated.8 9 T-RA acts at the mRNA level; we observed that the β chain mRNA steady-state level is also induced in HepG2 cells treated with 1 μmol/L T-RA. Our results and the fact that multiple retinoid effects are mediated by specific nuclear receptors lead to the assumption that T-RA activates the transcription of the fibrinogen genes. However, an increase in the stability of the fibrinogen mRNA cannot be excluded. Additional experiments are needed to determine if the fibrinogen gene response to T-RA is a direct or indirect effect.
HepG2 cells express RARα transcripts,29 and expression of RXRα is abundant in the liver.12 However, no research has considered the expression of RXR receptors in hepatoma cells. The synthesis of the very potent and selective RXR agonist 3-methyl TTNEB18 has provided an ideal pharmacological tool with which to elucidate the biological role of the individual retinoid receptors. We tested this selective RXR agonist and found that it is as potent as T-RA in inducing fibrinogen production by HepG2 cells. 3-Methyl TTNEB is able to stimulate fibrinogen secretion with an ED50 of 11 nmol/L. This value is comparable to the Kd of this compound for its binding to RXRα.18 By using the same cellular model, we demonstrated that a specific RARα agonist marginally activates fibrinogen production, but only at high concentrations. Identical results were obtained for the β chain mRNA levels. This indicates that activation of the RXR receptor is a prerequisite for the increase in fibrinogen production by HepG2 cells, while activation of RAR is not. This result would be the first evidence for a biological role of the RXR receptor on gene activation. The RXR receptor has been described as an auxiliary protein able to form heterodimers with many other nuclear receptors, eg, thyroid hormone receptor, vitamin D3 receptor, and RAR.30 31 Heterodimer formation with RXR seems to be necessary to induce maximal transcriptional activation, so this is considered as the main function of RXR.32 The induction of fibrinogen production by a selective RXR agonist could indicate that the RXR receptor can induce gene expression either through an RXR homodimer or an RXR-activated RXR/RAR heterodimer. Data supporting transactivation through an RXR homodimer have been reported.16
Incubation of HepG2 cells with RAR and RXR agonists simultaneously gave a higher induction than with RXR agonist alone, suggesting that the heterodimer RXR/RAR is involved in the maximal activation of fibrinogen production. This dual function of RXR has been demonstrated with transactivation and gel retardation experiments.32 The results obtained in the present study with natural retinoids such as T-RA and 9-cis RA also reinforce the central role of RXR. At a concentration of 1 μmol/L, both retinoids stimulated fibrinogen production. However, at a concentration of 2.5 nmol/L, T-RA was a poor activator of fibrinogen production, as T-RA activates only RAR, compared with 9-cis RA, which activates both RAR and RXR. These results confirm our hypothesis that activation of RXR is essential for the stimulation of hepatic fibrinogen production by retinoids.
Fibrinogen is not the only acute-phase protein whose synthesis is affected by retinoids. The secretion of two other acute-phase proteins, α-antichymotrypsin and caeruloplasmin, were also activated by retinoids in HepG2 cells. However, these two proteins do not share the same profile of activation with synthetic retinoids. While the RXR agonist is more active than the RAR agonist on caeruloplasmin production, the induction of α-antichymotrypsin, like fibrinogen production, is quite similar for the RAR and RXR agonists. This indicates that retinoids may act through different pathways in the same cells. For instance, the homodimer RXR/RXR may be less efficient in activating some genes (eg, α-antichymotrypsin) than others (eg, fibrinogen and caeruloplasmin). Analysis of the sequence of the promoters of these proteins could help in understanding these differences. A direct-repeat 5 has been reported to be a retinoic acid–responsive element, whereas a direct-repeat 1 is a retinoic X–responsive element. The presence and the nature of the retinoid-responsive elements in the promoter of different acute-phase proteins may explain the results that we describe here.
Retinoids do not activate the production of all acute-phase proteins, as albumin secretion was not affected. Moreover, preliminary results clearly indicated that production of C-reactive protein and α1-microglobulin by HepG2 cells was not significantly altered by retinoids. Interestingly, the secretion of these proteins is not stimulated by IL-6, suggesting a correlation between IL-6 and the retinoid sensitivity of acute-phase proteins. Taken together, these results show that retinoids strongly affect the synthesis of some acute-phase proteins by hepatocytes.
Fibrinogen is a powerful and independent risk factor for cardiovascular diseases.2 3 High levels of plasma fibrinogen can induce a prothrombotic state that favors thrombus formation. Therefore, it is important to understand the mechanisms involved in the regulation of fibrinogen synthesis to identify molecules able to decrease the plasma level of fibrinogen. Our results indicate that retinoids, in particular RXR agonists, upregulate fibrinogen production by hepatocytes both in vitro and in vivo. Our data suggest that retinoid therapy could favor a prothrombotic state. This should be taken into account in future clinical studies in which retinoids are used.
Selected Abbreviations and Acronyms
|BSA||=||bovine serum albumin|
|9-cis RA||=||9-cis retinoic acid|
|ELISA||=||enzyme-linked immunosorbent assay|
|FCS||=||fetal calf serum|
|3-methyl TTNEB||=||4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzoic acid|
|RAR||=||retinoic A receptor|
|RXR||=||retinoic X receptor|
|T-RA||=||all-trans retinoic acid|
We gratefully thank Drs J. Hiernaux and A. Kaptein for their critical reading of the manuscript, R. Guillard for her technical assistance, and Dr A. Bouillot for the synthesis of 9-cis RA and 3-methyl TTNEB. We also thank Drs Shroot and Charpentier from CIRD Galderma, France, for giving us the AM 580.
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