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(Circulation Research. 2001;88:637.)
© 2001 American Heart Association, Inc.

Molecular Medicine

Retinoids Inhibit the Actions of Angiotensin II on Vascular Smooth Muscle Cells

Volker Haxsen, Sylvie Adam-Stitah, Eberhard Ritz, Jürgen Wagner

From the Department of Nephrology (V.H., E.R., J.W.), University Hospital, University of Heidelberg, Heidelberg, Germany, and Institut de Génétique et de Biologie Moléculaire et Cellulaire (S.A.-S.), Illkirch-Strasbourg, France.

Correspondence to Dr Jürgen Wagner, Sektion Nephrologie, Medizinische Klinik, Bergheimerstrasse 56a, 69115 Heidelberg, Germany. E-mail juergen_wagner{at}med.uni-heidelberg.de

	Abstract

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Abstract— Retinoids are derivatives of vitamin A and powerful inhibitors of cell proliferation and inflammation. Angiotensin II (Ang II) contributes to vascular lesions by promoting cell growth of vascular smooth muscle cells (VSMCs). Therefore, we examined whether retinoids interfere with the proproliferative actions of Ang II in VSMCs via AT₁ receptor–dependent or activator protein-1 (AP-1)–dependent mechanisms. VSMCs express retinoid receptor proteins, ie, retinoic acid receptor (RAR) and retinoid X receptor (RXR) . Long-term exposure to 1 µmol/L all-trans retinoic acid (RA) dose-dependently inhibited Ang II–induced cell proliferation (P<0.005) as well as DNA and protein synthesis (P<0.001). All-trans RA blocked Ang II stimulation of transforming growth factor-ß₁ mRNA (P<0.005). All-trans RA inhibition of vascular VSMC growth was mediated both via RAR- and RXR-dependent pathways, as shown by receptor-specific synthetic retinoids. Transfection experiments revealed that inhibition of AP-1–dependent gene transcription is one mechanism by which all-trans RA inhibits Ang II action. RAR cotransfection enhanced the anti–AP-1 effects of all-trans RA dose-dependently. AP-1 activity was similarly inhibited by cotransfection with either RAR or RXR. Ang II–induced gene expression of c-fos was abrogated by all-trans RA treatment (P<0.005). In VSMCs, all-trans RA downregulated AT₁ receptor mRNA (P<0.01) and reduced B_max (P<0.001). All-trans RA repressed Ang II–stimulated AT₁ receptor promoter activity. The all-trans RA inhibitory effect was abolished when the AP-1 consensus site on the AT₁ receptor promoter was deleted. Our findings demonstrate that retinoids are potent inhibitors of the actions of Ang II on VSMCs. The findings support the notion that retinoids may interfere with proliferative vascular disease.

Key Words: retinoic acid • angiotensin II • vascular smooth muscle cells • activator protein-1 • proliferation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Apart from causing vasoconstriction, angiotensin II (Ang II) affects the response of vascular smooth muscle cells (VSMCs) to hypertension, arteriosclerosis, or vascular damage of chronic transplant rejection.^{1 2 3} Ang II may act as a comitogen for the proliferation or hypertrophy of VSMCs. It also promotes vascular inflammation and extracellular matrix deposition.^{3 4} Ang II stimulates cell proliferation via the activator protein-1 (AP-1) complex by induction of proto-oncogene expression, such as c-fos and c-jun, which constitute the AP-1.⁵ This type of activation is mediated via the Ang II receptor subtype AT₁ but not AT₂.⁶ Ang II also activates growth and differentiation factors, such as transforming growth factor (TGF)-ß, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and others that in turn modulate the cellular response to Ang II.⁷ The mechanisms by which Ang II stimulates growth and vasoactive factors have not been clarified in detail. AP-1 may again play a role,⁸ although additional Ang II pathways have been described, ie, c-myc, serum response element, and cAMP response element.^{2 9 10}

Retinoids, natural and synthetic derivatives of vitamin A, influence cellular differentiation in organ development.^{11 12} They potently inhibit proliferation of a variety of cell types.^{13 14 15} They act, not exclusively but in part, via inhibition of AP-1–induced activation.¹⁶ Inhibition of AP-1 activity by retinoids is mediated via different mechanisms, which are cell- and context-dependent. These include direct inhibition of c-jun, c-fos downregulation, interruption of c-fos/c-jun dimerization, or inhibition of binding of AP-1 to DNA.^{15 16}

Retinoid signals are transduced by 2 families of receptors, ie, retinoid acid receptors (RARs) and retinoid X receptors (RXRs). RAR and RXR heterodimers bind to polymorphic cis-acting retinoid acid (RA)–responsive and retinoid X–responsive elements.¹⁷ RXRs also form heterodimers with other receptors of the steroid receptor family, such as the peroxisome proliferator–activated receptors and the vitamin D and thyroid hormone receptors, thus enabling crosstalk between different regulatory pathways.¹⁸ Although the vascular system is not considered a traditional target for the retinoids, VSMCs are sensitive to their action.¹³ Expression of RARs by VSMCs has been demonstrated on the RNA level. Functional studies were performed after RA transduction using reporter gene assays.¹³ The antiangiogenetic properties of retinoids have long been known. Retinoids effectively block neointima proliferation induced by balloon arterioplasty injury in the rat.^{14 19 20} The consideration that Ang II has important nonvasoconstrictive effects on VSMCs and that VSMCs constitute a target for retinoids prompted us to examine whether retinoids interfere with the action of Ang II on VSMCs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Culture of VSMCs
Primary rat aortic SMCs were prepared according to Owens et al²¹ and maintained as previously reported in our laboratory.²² Cells were used between passages 3 and 10 out of 3 different isolates. Cells were rendered quiescent after reaching the appropriate degree of confluence by placing them in DMEM/F12 containing 0.5% FCS for 24 hours unless otherwise stated.

Cell Counts
VSMCs (2x10⁴) were plated per well in 24-well dishes, stimulated every 48 hours, and counted in hemocytometers (Neubauer type; Brand).

Measurement of DNA and Protein Synthesis
Cells were plated in 96-well dishes (10⁴ per well) and rendered quiescent for 24 hours. The cells were treated with agonists for another 24 hours and labeled with tritiated thymidine or proline within the final 4 hours of the experiment, as described earlier.²²

Transient Transfection and Luciferase Assay
Confluent VSMCs in 6-well dishes were transfected with 1 µg/well of the reporter gene construct PathDetect AP-1 cis-reporting system (pAP-1-Luc; Stratagene) with FuGENE6 transfection reagent according to the manufacturer’s protocol (Roche Diagnostics). Transfections were normalized against ß-galactosidase activity from a cotransfected (1 µg/well) pSV-ß-galactosidase control vector (Promega). In cotransfection experiments, plasmid cytomegalovirus (pCMV)-R and pCMV-RXR constructs were used (kindly provided by Professor Pierre Chambon, Institut de Génétique et de Biologie Moléculaire et Cellulaire [IGBMC], Illkirch, France). A human AT₁-promoter/exon-1 fragment (AT₁-luc) was amplified via polymerase chain reaction (PCR), sequenced, and cloned into a luciferase vector essentially as in Holzmeister et al.²³ The AP-1 consensus site of AT₁-luc (AP-1) was point-mutated according to Holzmeister et al.²³ After 1-hour pretreatment with 1 µmol/L all-trans RA (Sigma), 1 µmol/L Ang II (Sigma) was added for another 4 (AP-1) or 21 (AT₁) hours, and the luciferase activity was assayed according to the manufacturer’s protocol (Promega). All experiments were repeated 3 to 5 times; assays were performed at least in duplicate or triplicate on 3 different cell isolates.

AT₁ receptor binding was determined in confluent monolayers of VSMCs treated with 1 µmol/L all-trans RA for 24 hours, as described,²⁴ using unlabeled AT₁-specific ligand DuP753 (Losartan) in concentrations ranging from 10 fmol/L to 100 µmol/L and 50 nmol/L of tritiated DuP753 (DuPont LS).

RNA Isolation and Reverse Transcriptase– PCR Analysis
Total RNA was isolated using RNeasy extraction columns (Qiagen). After reverse transcription, quantitative PCR was carried out in triplicates using strand-specific primers for AT₁ receptor (33 cycles), TGF-ß₁ (29 cycles),²² and c-fos (31 cycles).²⁵ For quantification, we used deletion mutants of the respective genes sharing the same primer sequences.²² The results were expressed as ratio of the optical densities of wild-type versus mutant cDNA signal.²²

Immunoblotting and Immunoprecipitation
Whole-cell extracts of VSMCs were prepared from confluent transfected cultures and immunoprecipitated with monoclonal anti-mouse RXR [4RX3A2] or RARß [Ab8ß(F)2] antibodies (IgG1 subclass) covalently linked to protein A beads according to Gaub et al.²⁶ Proteins with or without previous immunoprecipitation were fractionated by SDS-PAGE, electrotransferred onto nitrocellulose filters²⁶ (Schleicher & Schuell), and immunoprobed with polyclonal antibodies raised against RAR [RP(F)], RARß[RPß(F)2], or RXR[RPRX(A)], as described,²⁷ and detected via chemoluminescence.²⁷ The specificity of the reactions was checked by depleting the specific antibodies from the antiserum. COS-1 cell extracts transfected with full-length RAR, RARß, or RXR expression vectors served as positive controls, as described.¹¹

Statistics
Results are presented as mean±SEM. Data were evaluated using the Mann-Whitney test. The zero hypothesis was rejected at P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
VSMCs Express RAR and RXR Protein
To examine whether VSMCs contain RAR protein, we analyzed lysates of VSMCs by immunoblotting with polyclonal anti-RAR and anti-RXR antibodies. The presence of RAR protein is documented in Figure 1A. RAR protein was consistently demonstrated by immunoblotting in 10 and 20 µg of lysate. The 51-kDa band migrated at the same position as the RAR protein overexpressed in control COS cell lysates, which had been transiently transfected with a total RAR construct (pCMV-RAR).

View larger version (30K): [in this window] [in a new window]   Figure 1. VSMCs express RAR and RXR protein. A, Immunoblotting indicates expression of RAR protein in 10- and 20-µg cell lysates of VSMCs (lanes 3 and 4). Control cell lysates were of transiently transfected COS cells expressing pCMV-RAR (lane 2). In the presence of nonimmune serum, only background expression could be detected. RARß: no RARß protein could be detected by Western blot despite the presence of RARß mRNA (lanes 3 and 4). RARß (lanes 1 through 4) was detected in control cell lysates of transiently transfected COS cells expressing pCMV-RARß only (lane 2) but not in VSMC extracts. B, Immunoprecipitation (IP) indicates a 54-kDa band for RXR (lane 4, VSMC-IP) but not in VSMC protein extracts (lane 3, VSMC-extr; 20 µg) compared with transiently transfected COS cell lysates (pCMV-RXR). Control studies in which nonimmune serum was substituted for the primary antibody demonstrated only diffuse and faint background staining (data not shown).

RARß could be demonstrated in VSMCs on the mRNA level only (data not shown). The polyclonal anti-RARß antibody failed to detect RARß protein in VSMC lysates. RARß could also not be detected after immunoprecipitation with specific RARß antibodies or after stimulation of VSMCs with 1 µmol/L all-trans RA (data not shown). In contrast, RARß could be demonstrated in transiently transfected RARß-COS control cell lysates.

RXR protein concentration in VSMC lysates was below the detection limit of immunoblotting when a polyclonal anti-RXR antibody was used. Immunoprecipitation yielded a clear signal for RXR in lysates of VSMCs. Controls were lysates of transiently transfected RXR COS cells (Figure 1B).

All-trans RA Inhibits Ang II–Induced Proliferation of VSMCs
Figure 2A shows the effect of 10 nmol/L or 1 µmol/L all-trans RA on VSMCs treated with 1 µmol/L Ang II in the presence of 0.5% FCS over 10 days. Dose-dependently lower rates of cell proliferation were observed in the cultures treated with all-trans RA plus Ang II compared with the cultures treated with Ang II alone.

View larger version (14K): [in this window] [in a new window]   Figure 2. Antimitogenic effect of all-trans RA. A, Cells were seeded at a density of 2x104/well (24-well; n=6), stimulated every other day, and counted on days 4 and 10. All-trans RA (tRA) 1 µmol/L abrogated the Ang II–induced cell growth to control levels, whereas the effect of 10 nmol/L was intermediate. This result suggests dose dependency of the retinoid effect. **P<0.005 for Ang II plus stimulated cultures vs controls; ##P<0.005 for Ang II plus all-trans RA vs Ang II treatment groups. VSMCs treated for 24 hours with Ang II, all-trans RA, or both (both 1 µmol/L) showed a marked decrease in the incorporation (final 4 hours) of tritiated thymidine (B) and proline (C) when all-trans RA (tRA) was added to Ang II. *P<0.05 for Ang II–stimulated group vs controls (n=12); ##P<0.005 for Ang II plus all-trans RA vs Ang II treatment groups.

Figure 2B indicates that all-trans RA completely abrogated the Ang II–induced DNA synthesis, as assessed by thymidine incorporation, but no effect of all-trans RA on basal DNA synthesis was noted. Similarly, protein synthesis in response to 1 µmol/L Ang II was abrogated by 1 µmol/L all-trans RA (Figure 2C), but basal protein synthesis was not affected. In an additional 48-hour experiment, the cumulative proline incorporation into the precipitable fraction was determined in the presence of 1 µmol/L all-trans RA, 1 µmol/L Ang II alone, or both compounds. The results showed significantly less proline incorporation in the presence of all-trans RA (data not shown).

RAR- and RXR-Specific Agonists Block Ang II–Induced Proliferation of VSMCs
To test whether inhibition of cell proliferation by all-trans RA is mediated via RAR- or RXR-dependent pathways, cells were treated with the RAR-specific agonists AM580 and all-trans RA as well with the RXR-specific agonists BMS649 or 9-cis RA (all reagents 1 µmol/L), respectively. In a 7-day experiment (Figure 3A), 1 µmol/L Ang II stimulated VSMC proliferation 3-fold over controls. In incubations without Ang II, retinoids (all-trans RA, AM580, BMS649, and 9-cis RA) induced a small increase in cell number over control, which was not significant. However, Ang II–induced proliferation was significantly inhibited by all retinoids. There was no difference between RAR- and RXR-specific agonists. To exclude loss of receptor specificity at high concentrations (1 µmol/L), we tested AM580 and BMS649 at 3 different concentrations (1 µmol/L, 100 nmol/L, and 1 nmol/L). The inhibitory efficiency was equivalent (Figure 3B).

View larger version (19K): [in this window] [in a new window]   Figure 3. Antiproliferative effects of retinoids are not receptor specific. A, In a 7-day experiment, Ang II (1 µmol/L) markedly increased proliferation of VSMCs, which was significantly reduced in the presence of retinoids (1 µmol/L). No significant differences in the antiproliferation reaction were observed between RAR-specific (all-trans RA [tRA] and AM580) and RXR-specific (9-cis RA and BMS649) agonists. **P<0.005 for Ang II–stimulated group vs controls; ##P<0.005 for Ang II vs retinoid treatment groups. B, Both the RAR-specific agonist AM580 and the RXR-specific agonist BMS649 dose-dependently inhibited Ang II–induced growth in VSMCs. **P<0.005 for Ang II– stimulated group vs controls; ##P<0.005 for Ang II vs retinoid treatment groups.

All-trans RA Inhibits Ang II–Dependent Activation of TGF-ß₁ Expression
After 4 hours of exposure to 1 µmol/L Ang II, the steady-state expression of TGF-ß₁ mRNA was significantly increased compared with controls. All-trans RA alone (1 µmol/L) reduced basal TGF-ß₁ mRNA concentration and completely abrogated the Ang II–induced stimulation of TGF-ß₁ mRNA (Figure 4).

View larger version (11K): [in this window] [in a new window]   Figure 4. All-trans RA suppresses Ang II–induced TGF-ß1 gene expression. VSMCs treated with 1 µmol/L Ang II for 4 hours exhibited increased TGF-ß1 mRNA. Addition of 1 µmol/L all-trans RA (tRA) resulted in subcontrol levels of TGF-ß1 mRNA. TGF-ß1 mRNA was determined by quantitative reverse transcriptase (RT)–PCR using a TGF-ß1–deletion mutant with the same primer sequences as internal standard. Results are expressed as ratio of the optical densities (OD) of the wild-type and the mutant amplicon. **P<0.005 for Ang II–stimulated group vs controls; ##P<0.005 for Ang II plus all-trans RA vs Ang II treatment groups.

Inhibition of Ang II by All-trans RA Is Mediated Via AP-1
Ang II induced a 24-fold stimulation of luciferase activity of the AP-1 plasmid within 4 hours (Figure 5A). Pretreatment (1 hour) with 1 µmol/L all-trans RA reduced Ang II–dependent AP-1 induction by 30%. Costimulation of Ang II–treated cells with 1 µmol/L Losartan abrogated luciferase activity to control levels (data not shown). Dose-dependent effects of all-trans RA on AP-1-induction could not be detected, presumably because the overall effect was small. The anti–AP-1 activity of all-trans RA in response to Ang II, however, was markedly increased after cotransfection of plasmids encoding CMV promoter–driven retinoid receptors RAR and RXR with the AP-1 reporter gene plasmid (Figure 5B). Cotransfection of both retinoid receptors reduced AP-1 luciferase activity by 50%. When different amounts of RAR were cotransfected, a clear dose-dependency could be demonstrated (Figure 5C).

View larger version (14K): [in this window] [in a new window]   Figure 5. All-trans RA inhibits Ang II–stimulated AP-1 activity. A, VSMCs transfected with an AP-1 luciferase reporter gene construct were stimulated with 1 µmol/L Ang II (4 hours) after 1 hour of pretreatment with various concentrations of all-trans RA (tRA). All-trans RA in concentrations from 1 nmol/L to 1 µmol/L led to a small but consistent decrease in relative luciferase activity in response to Ang II stimulation. *P<0.05 for all-trans RA–treated group vs Ang II–stimulated group. B, Plasmids encoding RAR and RXR (0.5 µg) cotransfected with AP-1 luciferase reporter gene plasmid (1 µg) were pretreated with 1 µmol/L all-trans RA (1 hour) and stimulated with 1 µmol/L Ang II for another 4 hours. Combining RAR and RXR cotransfection (0.5 µg each) led to a maximum inhibition of Ang II–induced AP-1 induction of 50%. Results are expressed as percent of the Ang II induction obtained in the absence of ligand. C, VSMCs transfected with an AP-1 luciferase reporter gene and various amounts of an RAR construct were stimulated with 1 µmol/L Ang II (4 hours) after 1 hour of pretreatment with 1 µmol/L all-trans RA (tRA). Results indicate a strong induction of AP-1 activity by Ang II, a small reduction of AP-1 activity by all-trans RA, and a dose-dependent reduction of Ang II–induced AP-1 activity depending on the amount of cotransfected RAR. B and C, Representative experiments from 5 experiments on 2 different cell isolates. Values are the mean of triplicates and are from a representative experiment.

All-trans RA Abrogates the Induction of c-fos Expression by Ang II
Treatment with 1 µmol/L Ang II for both 30 minutes and 1 hour stimulated c-fos RNA expression significantly. Pretreatment with 1 µmol/L all-trans RA completely abrogated the induction of c-fos mRNA by Ang II (data shown after 1-hour Ang II treatment only; Figure 6).

View larger version (11K): [in this window] [in a new window]   Figure 6. All-trans RA abrogates Ang II–induced c-fos gene expression. VSMCs treated with 1 µmol/L Ang II for 1 hour showed an increase of c-fos mRNA. Pretreatment (1 hour) with 1 µmol/L all-trans RA (tRA) abrogated the Ang II–induced rise of c-fos gene expression. c-fos mRNA was determined by quantitative RT-PCR using a c-fos deletion mutant with the same primer sequences as internal standard. Results are expressed as ratio of the optical densities (OD) of the wild-type and the mutant amplicon. **P<0.005 for Ang II–stimulated group vs controls; ##P<0.005 for Ang II plus all-trans RA vs Ang II treatment groups.

All-trans RA Reduces the Basal Expression of the AT₁ Receptor
To examine whether all-trans RA changes AT₁ receptor gene expression and thus alters Ang II sensitivity, we determined steady-state levels of AT₁ receptor mRNA. Reduction in AT₁ receptor gene expression reached statistical significance after 24 hours of incubation with 1 µmol/L all-trans RA (P<0.05, Figure 7A).

View larger version (10K): [in this window] [in a new window]   Figure 7. All-trans RA alters AT1 receptor. A, Treatment with 1 µmol/L all-trans RA (tRA) significantly lowered AT1 receptor steady-state mRNA levels by 50% after 24 hours. AT1 receptor mRNA was determined by quantitative RT-PCR using an AT1 receptor deletion mutant with the same primer sequences as internal standard. Results are expressed as ratio of the optical densities (OD) of the wild-type and the mutant amplicon. **P<0.005 for all-trans RA–stimulated group vs controls. B, Confluent VSMCs treated with 1 µmol/L all-trans RA (tRA) for 24 hours were incubated with 50 nmol/L tritiated DuP753 and various concentrations of unlabeled DuP753 to assess a competitive binding assay. Bmax decreased by 45%, corresponding to a marked decrease in AT1 receptor numbers. C and D, VSMCs were transfected with AT1 promoter luciferase reporter gene constructs with wild-type (AT1-luc) or point-mutated AP-1 (AT1-luc AP-1) consensus sites. After 1 hour of pretreatment with 1 µmol/L all-trans RA, cells were stimulated with 1 µmol/L Ang II for another 21 hours. C, Ang II–induced wild-type AT1-luc luciferase activity is completely abrogated by all-trans RA. D, Ang II–induced AT1-luc AP-1 activity was not inhibited by all-trans RA. C and D, Representative experiments from 5 experiments using 3 cell isolates. Values are the mean of triplicates and are from a representative experiment.

All-trans RA Downregulates AT₁ Receptor
After 24 hours of treatment with 1 µmol/L all-trans RA (Figure 7B), B_max decreased by 45%, from 995±51 to 564±31 cpm. EC₅₀ decreased from 0.31 to 1.1 nmol/L, indicating a negligible rise in affinity of AT₁ receptors to DuP753 when treated with all-trans RA.

Inhibitory Action of All-trans RA on Ang II–Induced AT₁ Receptor Promoter Activity Is Dependent on AP-1
The effects of 1 µmol/L all-trans RA on the Ang II–induced AT₁ receptor promoter activity were assessed in transiently transfected VSMCs. All-trans RA had no significant effect on the low basal promoter activity of the wild-type AT₁ receptor promoter under quiescent conditions. VSMCs were transfected with an AT₁ receptor promoter luciferase reporter gene construct either with (AT₁-luc) or without (AT₁-luc AP-1) a functionally active AP-1 consensus site. All-trans RA abrogated the Ang II–induced (1 µmol/L) increase of luciferase activity in the wild-type (AT₁-luc) AT₁ promoter construct (Figure 7C). In transfected VSMCs carrying the construct without a functional AP-1 site (AT₁-luc AP-1), all-trans RA was no longer able to inhibit the Ang II–dependent stimulation of AT₁ promoter activity (Figure 7D).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The data document that all-trans RA inhibits the effects of Ang II in VSMCs. This inhibition may involve AP-1 repression and downregulation of the AT₁ receptor. VSMCs express both RAR and RXR subtypes on the RNA level¹³ and, as shown here, on the protein level. These observations suggest that VSMCs may respond not only to RAR ligands, such as all-trans RA, but also to RXR ligands, such as 9-cis RA or BMS649. Expression of RXRs allows for crosstalk with other receptors of the steroid superfamily, such as thyroid hormone, vitamin D, and peroxisome proliferator activator receptors. These receptors also act on cardiovascular tissues and cell growth.^{18 28}

The induction of growth in VSMCs by Ang II has been studied intensively.^{29 30} Ang II may evoke hypertrophic or hyperplastic responses, depending on the balance of antiproliferative and proproliferative autocrine factors, ie, TGF-ß, PDGF, FGF, and others.^{30 31 32} Our data confirm that Ang II stimulated c-fos expression^{2 31} time-dependently and AP-1 activity by a factor of about 20 to 30 using a luciferase vector containing AP-1 consensus sites.⁸

Repression of c-fos gene expression has been identified as a mechanism of retinoid inhibition of AP-1 activity.^{15 33} All-trans RA has been shown to inhibit the serum-induced stimulation of c-fos and c-jun by serum in mesangial cells^{15 33 34} and in our VSMCs.

The anti–AP-1 activity of retinoids has been intensively studied and may be cell-type specific.^{16 35 36} All-trans RA reduced Ang II–induced activation of AP-1 modestly but significantly in reporter gene assays. This suggested low natural abundance of the receptors. Cotransfection with an RAR expression vector dose-dependently inhibited Ang II–induced luciferase activity up to 50%, confirming blockade of Ang II–dependent AP-1 activation by all-trans RA.

All-trans RA dose-dependently blocked Ang II–stimulated cell proliferation, as indicated by proliferation markers, such as cell counts or thymidine and proline incorporation. All-trans RA alone generally had no effect on cell proliferation, although, depending on cell preparation and culture conditions, occasionally a minor increase was observed, which was also described by Chen and Gardner.³⁷ In all doses of all-trans RA used in experiments, no sign of toxicity was present; signs of toxicity were only present at 10 µmol/L.

To clarify whether RAR inhibits Ang II–dependent AP-1 activation alone or with RAR pathways, we assessed retinoid receptor–specific ligands and cotransfection of either RAR or RXR expression plasmids. Receptor-specific ligands, such as AM580 (which is specific for RAR) or BMS649 and 9-cis RA (which are specific for RXRs [Figures 3A and 3B]), showed that both receptor ligands were able to block Ang II–induced cell proliferation. RAR-dependent inhibition of VSMC proliferation is well known.^{13 14} Our data support these findings. In our cells, RXR agonists, 9-cis RA (Figure 3A), and BMS649 (Figures 3A and 3B) were as efficient at inhibiting Ang II–induced cell proliferation as RAR agonists. We used a highly specific RXR agonist, BMS649, which cannot isomerize into other retinoids as 9-cis RA. Its K_d for RXR is 4.9 nmol/L, for RXRß is 2.9 nmol/L, and for RXR is 98 nmol/L. In contrast, the K_d of BMS649 for RAR is 42 500 nmol/L, for RARß is 17 500 nmol/L, and for RXR is 14 000 nmol/L, which is about 10 000-fold higher than for RXRs. Furthermore, in COS-1 cell test systems comparable to Brand et al,³⁸ transient transfections of specific retinoid receptor subtype CAT constructs had shown no stimulation of CAT activity of RAR by BMS649 (C. Zusi, unpublished data, 1991). These data are different from those provided by Neuville et al,¹⁴ who found no RXR-dependent growth inhibition in SMCs. These differences may be explained by the use of 10% serum instead of Ang II and specific SMC populations derived from injured vasculature.¹⁴

Besides retinoid receptor–specific ligands, cotransfection with either RAR or RXR caused inhibition of AP-1 activation by Ang II. This is in good agreement with studies where both RAR and RXR expression vectors were able to confer anti–AP-1 activity.³⁶ The data do not allow conclusions on differences in potency and efficacy between RAR- and RXR-specific compounds, because the effects varied slightly between different cell preparations, and differences were subtle at best.

Ang II induction of cell growth is modulated by autocrine factors, particularly TGF-ß₁, which significantly contributes to the hypertrophic response of VSMCs to Ang II.⁷ Our data of a modest but consistent stimulation of TGF-ß₁ gene expression by Ang II are in accordance with the literature, where an increase of 1.5- to 8-fold, depending on the cellular context, was reported.^{22 31 32} All-trans RA completely abrogated this induction and even reduced basal TGF-ß₁ mRNA levels in these cells. The effects of retinoids on TGF-ß are cell-specific: all-trans RA stimulated TGF-ß₁ mRNA in chondrocytes, where basal TGF-ß is low, and inhibited TGF-ß in myocytes, where basal expression is high.³⁹ The TGF-ß₁ promoter contains 3 AP-1 sites, which are able to confer AP-1–mediated stimulation of gene transcription.³⁶ Salbert et al³⁶ demonstrated that both RARs and RXRs inhibit the TGF-ß₁ gene promoter via inhibition of AP-1. Morishita et al⁴⁰ demonstrated that Ang II–stimulated TGF-ß induction in VSMCs is mediated via AP-1 using an anti–AP-1 oligodeoxynucleotide decoy approach. These findings strongly suggest, but do not provide final proof, that abrogation of Ang II–dependent TGF-ß induction by retinoids may be mediated via AP-1.

The proliferative response to Ang II is mediated via the AT₁ receptor involving protein kinase C and induction of proto-oncogenes.⁸ Our data of a 50% reduction of AT₁ receptor binding confirm the findings of Takeda et al,⁴¹ who also found a reduction in B_max but not K_d values. Holzmeister et al²³ previously demonstrated that the AT₁ receptor promoter contains a functional AP-1 binding site. It is thus conceivable that downregulation of this receptor by retinoids might be mediated via AP-1. To test this hypothesis, we point-mutated the AP-1 binding site on the human AT₁ receptor promoter. All-trans RA had no significant effect on the low basal AT₁ receptor promoter activity of the AT₁ receptor under quiescent conditions. This is in contrast to findings of Takeda et al,⁴¹ where at 10% FCS, all-trans RA reduced AT₁ receptor promoter activity, but inhibition was not AP-1 dependent. In our study, all-trans RA did not inhibit serum-induced AP-1 activity in the presence of 5% or 10% FCS (data not shown). Additionally, under serum conditions of >2%, no Ang II effects could be observed. This suggests that the AP-1 dependency of retinoid effects may be overrun under high-serum stimulation and prompted us to examine the effects of retinoids under low-serum conditions.

In contrast to the wild-type AT₁ receptor construct, the inhibition of Ang II–dependent promoter activity by all-trans RA was now abolished. These findings support the notion that the AP-1 binding site of the AT₁ receptor promoter mediates Ang II stimulation, as has previously been shown for tissue plasminogen activator stimulation by Holzmeister et al.²³

We have shown that retinoids inhibit the proproliferative action of Ang II on different levels, first by inhibiting AP-1–dependent gene transcription and second by reducing responsiveness to Ang II via reduced expression of the AT₁ receptor. Although retinoids belong to the steroid superfamily, they clearly differ in this respect from glucocorticoids. The latter sensitize VSMCs to vasopressors and upregulate the AT₁ receptor.^{24 42}

The effects of retinoids on Ang II are an additional example of their influence on the expression action and of vasoactive factors. Inhibition of endothelin-1, PDGF, inducible nitric oxide synthase, and others by retinoids has been reported in myocytes or mesangial cells.^{35 43 44} Retinoids also inhibit neointima formation in response to vascular balloon injury in the rat.^{14 19 20} Retinoids, therefore, are potentially interesting candidate drugs to modulate agonist-induced transcription in the cardiovascular system.

	Acknowledgments

 
We cordially thank Dr Cécile Rochette-Egly, IGBMC, Illkirch, France, for providing the antibodies and Dr Daniel Metzger and Professor Pierre Chambon, IGBMC, Illkirch, France, for providing us with the RAR and RXR expression vectors. We also thank Dr Christopher Zusi, Bristol-Meyers-Squibb, Wallingford, Conn, for kindly providing us with synthetic retinoids.

	Footnotes

 
Original received January 26, 2000; resubmission received September 12, 2000; revised resubmission received February 20, 2001; accepted February 20, 2001.

	References

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