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Induction of CD36 by all-trans retinoic acid: retinoic acid receptor signaling in the pathogenesis of atherosclerosis ¹

DIRK M. WUTTGE, ANNA ROMERT^*, ULF ERIKSSON^*, HANS TÖRMÄ, GÖRAN K. HANSSON and ALLAN SIRSJÖ²

Center for Molecular Medicine and Department of Medicine, Karolinska Institutet;
^* Ludwig Institute for Cancer Research, Stockholm Branch, Stockholm; and
Department of Medical Sciences/Section of Dermatology, University Hospital, Uppsala, Sweden

²Correspondence: Cardiovascular Research Unit, Center for Molecular Medicine, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail: allan.sirsjo{at}cmm.ki.se

SPECIFIC AIM

CD36 has recently attracted increasing attention regarding the involvement of the membrane receptor in the pathogenesis of atherosclerosis. Both the observation that retinoids and CD36 are involved in lipid metabolism and the notion that all-trans retinoic acid (atRA) induces CD36 in malignant astrocytoma (U-251MG) cells led us to examine whether retinoid signaling through retinoic acid receptor (RAR) could be important in regulating CD36 in the context of atherosclerosis.

PRINCIPAL FINDINGS

1. RAR-mediated expression of CD36 in THP-1 cells and increases uptake of oxidized low density lipoprotein (oxLDL)
Treatment of the human monocytic cell line THP-1 with atRA increased CD36 mRNA levels after 12 h as analyzed by quantitative RT-PCR and increased the CD36 protein expression analyzed by FACS after 2 days. The increased CD36 expression was mirrored by enhanced uptake of oxLDL. The scavenger receptors A-1 and -2 were not induced by atRA. The addition of 1 µM of the selective RAR antagonist CD3106 together with 10 nM atRA for 4 days abolished the atRA-induced CD36 expression, whereas CD3106 had no significant effect on the expression of CD36 induced by oxLDL or by BRL49653, an agonist at the proliferation activation receptor (PPAR-). The specific pan-RAR CD367 agonist also increases the CD36 levels in THP-1 cells after 4 days.

2. Synergistic effects of oxLDL and atRA on CD36 expression in THP-1 and primary monocytic cells
To examine whether signaling via RAR would synergize with PPAR- to up-regulate the receptor, we treated the cells with a combination of the pan-RAR agonist CD367 and PPAR- ligand BRL49563. Both the specific pan-RAR agonist CD367 and PPAR- ligand BRL49563 increased surface expression of CD36 to the same degree, whereas a combination of the two agonists doubled the induction (Fig. 1 ). Furthermore, incubation of THP-1 cells with the combination of 50 µg/ml oxLDL with 10 nM atRA resulted in a dramatic increase in CD36 expression compared with either compound alone. In primary monocytes derived from peripheral blood, addition of atRA at 1 µM enhanced the oxLDL-induced CD36 expression whereas atRA alone only slightly increased the CD36 expression.

View larger version (15K): [in this window] [in a new window]   Figure 1. RAR binding mediates the induction of CD36 by atRA and synergizes with the PPAR- signaling. THP-1 cells were incubated for 4 days with vehicle, the RAR agonist CD367 at 10 nM, the PPAR- agonist BRL49653 (5 µM), atRA (1 µM), native LDL (nLDL, 50 µg/ml) or oxLDL (50 µg/ml), alone or in combination. The CD36 expression of the THP-1 cells was analyzed by FACS. Mean +/- SE of 3 different experiments.

3. Identification of RAR and its ligands in atherosclerotic lesions
Since CD36 is expressed in the atherosclerotic lesion and our results indicated that CD36 expression is enhanced by RAR binding, we wondered whether RARs are present in the lesion. We analyzed the expression of RAR-, -ß, and - in human atherosclerotic plaques by using RT-PCR and immunohistochemistry (Fig. 2a ). Figure 2b c d e f g shows human atherosclerotic lesions stained with polyclonal antibodies to RAR- and - in an immunohistochemical assay. Both RAR- and - were present in the lesions. The nuclear receptors were particularly abundant in macrophage-rich areas, where nuclear RAR- and - were found in foam cells. We could not detect any specific staining for RAR-ß (not shown). Also, the mRNA levels of RAR- and - were elevated compared with RAR-ß.

View larger version (58K): [in this window] [in a new window]   Figure 2. RAR and its ligands are present in atherosclerotic lesions. A) The mRNA expression of RAR-, RAR-ß, and RAR- in human atherosclerotic lesions. mRNA was isolated from atherosclerotic lesions of 6 donors and quantified by real-time RT-PCR. Data show the ratio of RAR normalized for the housekeeping gene, ß2-microglobulin. Mean +/- SE of 6 patients. B–G) Identification of RAR- and RAR- protein in human atherosclerotic lesions. The distribution of macrophages and smooth muscle cells is indicated by staining for smooth muscle -actin (B) and CD68 (C), both at 20x magnification. The nuclear staining of cells expressing RAR- (D, 20x; F, 40x) and RAR- (E, 20x; G, 40x) is highlighted by arrowheads. H) Identification of RAR ligands in human atherosclerotic lesions. Atherosclerotic plaque specimens from 4 patients were cultured in medium for 24 h. The conditioned media were added to JEG-3 cells that had been transfected with a RAR-GAL4/luciferase reporter construct and incubated for 24 h. The degree of induced luciferase activity normalized as luciferase/ß-galactosidase (relative activity units) is depicted in the graph. Media without plaque tissue was used as control (n=3).

To further elucidate retinoid signaling in atherosclerosis, the presence of RAR ligands in human atherosclerotic lesions was analyzed using a RAR-GAL4-based reporter assay (Fig. 2h ). When conditioned media from 24 h incubations of human atherosclerotic lesions were added to the transfected reporter cells, a 10-fold increase in RAR signaling was observed.

CONCLUSIONS

The present study shows that 1) atRA stimulates CD36 expression; 2) this stimulation occurs through RAR binding; and 3) binding of RAR greatly enhances CD36 induction mediated by PPAR- in human THP-1 cells and monocyte/macrophages. We further provide evidence suggesting that retinoid signaling is involved in atherosclerosis by demonstrating both the presence of 4) retinoic acid receptors- and - and 5) retinoid ligands in human atherosclerotic plaques.

By using agonists and antagonists for RARs, we could identify RAR as the primary target for atRA-mediated CD36 induction. Previous studies have demonstrated that CD36 could be induced by oxLDL through a PPAR-/RXR-dependent signaling path. atRA enhanced CD36 expression synergistically with ligands for PPAR-, suggesting the collaboration of the two different signaling paths (RAR/RXR and PPAR-/RXR) (Fig. 3 ). Stimulation of macrophages with oxLDL and atRA resulted in an enhanced CD36 expression that was not expected from the experiments with the synthetic ligands themselves. No evidence was obtained for any direct effects of atRA on oxLDL uptake, nor was atRA internalized via oxLDL. Instead, we speculate that the induction of CD36 by atRA and the increased CD36 expression on the surface early in the time course might increase the active transport of oxLDL into the cell. This would lead to increased intracellular levels of ligands for the PPAR-/RXR nuclear factor heterodimer.

View larger version (29K): [in this window] [in a new window]   Figure 3. Schematic diagram of the induction path of CD36. atRA induces CD36 expression after binding to the heterodimer RAR/RXR, whereas oxLDL induces CD36 expression via the heterodimer PPAR-/RXR signaling path. The synergistic effect of both signaling paths leads to an increased expression of CD36 that promotes foam cell formation and development of atherosclerosis. RAR, retinoic acid receptor; RXR, retinoic X receptor; PPAR-, peroxisome proliferator-activated receptor .

The RAR signaling may have consequences for inflammatory activity in the atherosclerotic plaque. AtRA suppresses tumor necrosis factor (TNF) expression and the expression of adhesion molecules, and thus may prevent the development and activation of lesions. On the other hand, atRA has been shown to induce the monocyte chemoattractant protein 1 (MCP-1) in HL-60 and NB4 cell lines, whereas 9-cis-RA was demonstrated to induce MCP-1 in THP-1 cells. Several lines of evidence indicate that MCP-1 may play an important role in the attraction of monocytes to lesions. atRA also inhibits smooth muscle proliferation and reduces restenosis after injury, possibly due to a down-regulation of angiotensin II receptors. Retinoids may also down-regulate interferon (IFN) production by inhibiting the CD28 pathway in T cells; this leads to polarization of adaptive immunity toward Th2, a feature found in atherosclerotic animals with severe hypercholesterolemia. Together, the actions of retinoids would be predicted to increase foam cell formation and lesion growth (through CD36 and MCP-1 up-regulation) but reduce local inflammation (by inhibiting TNF- and IFN. Further experiments will be needed to determine whether retinoid treatment does affect plaque growth and stability.

Vitamin A and its derivatives have been the subject of investigation in atherosclerosis due to their possible roles as lipophilic antioxidants preventing the modification of LDL. However, no significant association between vitamin A levels in plasma and the risk of vascular events due to the atherosclerotic diseases has been observed. Since the plasma levels of vitamin A and its derivatives are tightly controlled, it is difficult to extrapolate from the plasma level of vitamin A to the availability of the retinoids in the local environment of the atherosclerotic lesion. We used another approach; by using the sensitive RAR-GAL-4 reporter assay, we could detect RAR ligands in atherosclerotic lesions. These ligands can be formed by the action of retinol dehydrogenases, which oxidize retinol to generate retinoic acid. We recently observed that proinflammatory cytokines known to be present in the plaque induce retinol dehydrogenase 5 in vascular smooth muscle cells and that this is associated with increased formation of RAR ligands (A Sirsjö et al, unpublished observations). In addition to being synthesized locally, it is possible that atRA from the blood may accumulate in lipid-rich areas of the plaque by hydrophobic interactions. The present finding that atRA increases uptake of oxidized LDL and recent observations that it regulates smooth muscle proliferation indicate that it is a pluripotent factor in the pathogenesis of atherosclerosis, the antioxidant activity of which may play a minor role.

The results of this study unveil a novel mechanism for the regulation of CD36 expression through the RAR receptor family and suggest that this pathway promotes foam cell formation during atherogenesis. Selective blocking of RAR signaling may offer new opportunities for treatment of atherosclerosis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0488fje ; to cite this article, use FASEB J. (March 20, 2001) 10.1096/fj.00-0488fje

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