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J Biol Chem, Vol. 275, Issue 2, 1241-1246, January 14, 2000

Transforming Growth Factor-1 (TGF-1) and TGF-2 Decrease Expression of CD36, the Type B Scavenger Receptor, through Mitogen-activated Protein Kinase Phosphorylation of Peroxisome Proliferator-activated Receptor-^*

Jihong Han, David P. Hajjar, James M. Tauras, Jianwei Feng, Antonio M. Gotto Jr., and Andrew C. Nicholson

From the Department of Pathology and Center of Vascular Biology, Weill Medical College of Cornell University, New York, New York 10021

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CD36, the macrophage type B scavenger receptor, binds and internalizes oxidized low density lipoprotein, a key event in the development of macrophage foam cells within atherosclerotic lesions. Expression of CD36 in monocyte/macrophages is dependent on differentiation status and exposure to soluble mediators. In this study, we investigated the effect of transforming growth factor-1 (TGF-1) and TGF-2 on the expression of CD36 in macrophages. Treatment of phorbol ester-differentiated THP-1 macrophages with TGF-1 or TGF-2 significantly decreased expression of CD36 mRNA and surface protein. TGF-1/TGF-2 also inhibited CD36 mRNA expression induced by oxidized low density lipoprotein and 15-deoxy^12,14 prostaglandin J₂, a peroxisome proliferator-activated receptor (PPAR)- ligand, suggesting that the TGF-1/TGF-2 down-regulated CD36 expression by inactivating PPAR--mediated signaling. TGF-1/TGF-2 increased phosphorylation of both mitogen-activated protein (MAP) kinase and PPAR-, whereas MAP kinase inhibitors reversed suppression of CD36 and inhibited PPAR- phosphorylation induced by TGF-1/TGF-2. Finally, MAP kinase inhibitors alone increased expression of CD36 mRNA and surface protein but had no effect on PPAR- protein levels. Our data demonstrate for the first time that TGF-1 and TGF-2 decrease expression of CD36 by a mechanism involving phosphorylation of MAP kinase, subsequent MAP kinase phosphorylation of PPAR-, and a decrease in CD36 gene transcription by phosphorylated PPAR-.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Macrophage scavenger receptors play a significant role in atherosclerotic foam cell development because of their ability to bind and internalize OxLDL¹ (1-4). Two major classes of human scavenger receptors, designated type A and type B, have been identified (class C scavenger receptors are macrophage-specific scavenger receptors from Drosophila (5)). In addition, two other macrophage receptors, MARCO (macrophage receptor with a collagenous structure) and CD68 (macrosialin), may also contribute to the uptake of modified lipoproteins (6, 7). CD36 is a member of a class of cell surface glycoproteins designated as type B scavenger receptors, which also includes SR-BI, a high density lipoprotein receptor (8). CD36 is expressed by monocyte/macrophages (9), platelets, (10) microvascular endothelial cells (11), and adipose tissue (12). Like the type A scavenger receptors (13), CD36 recognizes a broad variety of ligands, including OxLDL (14, 15), anionic phospholipids (16), apoptotic cells (17), thrombospondin (18), collagen (19), and Plasmodium falciparum-infected erythrocytes (20).

Unlike the low density lipoprotein receptor, scavenger receptors are not subject to negative regulation by high levels of intracellular cholesterol. We have shown that OxLDL can stimulate its own uptake by induction of CD36 gene expression (21). The mechanism(s) by which OxLDL up-regulates CD36 involves activation of the transcription factor, peroxisome proliferator-activated receptor (PPAR)- (22, 23). PPAR- is a member of a nuclear hormone superfamily that can heterodimerize with the retinoid X receptor and act as a transcriptional regulator of genes encoding proteins involved in adipogenesis and lipid metabolism (24).

Phorbol esters (phorbol 12-myristate 13-acetate (PMA) in particular), macrophage-colony-stimulating factor and interleukin-4 have also been shown to increase monocyte/macrophage expression of CD36 (25), whereas expression of CD36 is down-regulated in response to cholesterol efflux (26), lipopolysaccharide (25), dexamethasone (25), and interferon  (27). With the exception of OxLDL, which activates PPAR- leading to CD36 gene transcription, the mechanism(s) by which this diverse collection of factors modulates CD36 expression remains undefined.

Transforming growth factor-1 (TGF-1) and TGF-2 are multifunctional mediators that regulate cellular growth, migration, adhesion, extracellular matrix formation, and apoptosis (28). Smads, a novel family of signaling proteins, are the primary downstream signaling mediators activated following TGF- receptor ligation (29). Heteromeric Smad complexes translocate into the nucleus and act as transcription factors for TGF--responsive genes (30). However, in addition to Smad proteins, MAP kinases have also been implicated in mediating downstream TGF- signaling (31-34).

We evaluated the signaling mechanisms involved in the inhibition of CD36 by TGF-1 and TGF-2. We demonstrate that TGF-1 and TGF-2 decrease expression of CD36 by a mechanism involving phosphorylation of MAP kinase, subsequent MAP kinase phosphorylation of PPAR-, and a decrease in CD36 gene transcription by phosphorylated PPAR-.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Lines and Reagents-- THP-1 cells, a human monocytic cell line, were obtained from ATCC (Manassas, VA). They were cultured in RPMI 1640 medium containing 10% fetal calf serum, 50 µg/ml each of penicillin and streptomycin, and 2 mM glutamine. Cells were adjusted to a density of 2.5 × 10⁵ cells/cm² in 100-mm dishes and treated with 200 nM PMA to induce the differentiation of THP-1 monocytes into macrophages. After 8 h of treatment, PMA was removed and cells were washed twice with phosphate-buffered saline (PBS). Incubation was continued overnight in complete medium before initiation of experiments.

TGF-1 and TGF-2 were purchased from R & D Systems (Minneapolis, MN). PMA and 15d-PGJ₂ were obtained from Calbiochem (San Diego, CA).

Isolation of Total RNA, Purification of Poly(A⁺) RNA, and Northern Blotting-- Cells were lysed in RNAzol^TM B (Tel-Test, Inc., Friendswood, TX), chloroform was extracted, and total cellular RNA was precipitated in isopropanol. After washing with 80 and 100% ethanol, the dried pellet of total RNA was dissolved in distilled water and quantified by UV spectroscopy. Poly(A⁺) RNA was purified from approximately 100 µg of total RNA using the Poly(A)Ttract® mRNA isolation system III (Promega, Madison, WI).

Poly(A⁺) RNA was loaded on 1% formaldehyde agarose gels. Following electrophoresis RNA was transferred to a Zeta-probe® GT genomic tested blotting membrane (Bio-Rad) in 10× SSC by capillary force overnight. The blot was UV cross-linked for 2 min and then prehybridized with Hybrisol^TM (Oncor, Inc., Gaithersburg, MD) for 30 min before the addition of ³²P-randomly primed probes for CD36 or GAPDH. After overnight hybridization, membranes were washed twice for 20 min each time with 2× SSC and 0.2% SDS, and twice for 20 min each time with 0.2× SSC and 0.2% SDS at 55 °C. The blot was autoradiographed by exposure to a x-ray film (X-Omat^TM AR, Eastman Kodak Co.). Semiquantitative analysis of autoradiograms was assessed by densitometric scanning using a UMAX (Santa Clara, CA) UC630 flatbed scanner attached to a Macintosh IIci (Apple Computer, Inc., Cupertino, CA) running National Institutes of Health Image software (Bethesda, MD). The probe for CD36 was generated by reverse transcription-polymerase chain reaction. The sequences of 5'- and 3'-oligonucleotides used were ATGGGCTGTGACCGGAACT (285-304) and ACAGACCAACTGTGGTAG (871-889), respectively.

Determination of CD36 Cell Surface Expression-- After treatment, cells were suspended by the addition of trypsin and washed three times with PBS. Approximately 2 × 10⁶ cell were suspended in 300 µl of PBS containing 5% mouse serum and incubated for 30 min at room temperature while shaking. Cells then were incubated with 10 µl of mouse anti-human CD36 conjugated to fluorescine isothiocyanate isomer 1 (Chemicon International Inc., CA). After incubation for 2 h with antibody at room temperature, cells were washed three times with PBS. After suspension in PBS, the cells analyzed by flow cytometry assay with a Coulter FACScan. In addition, photographs were taken of adherent cells on glass slides using a Nikon Labphot2 fluorescent microscope.

Western Analysis of Phospho-MAP kinase and PPAR--- Macrophages were washed twice with cold PBS and then scraped and lysed in ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxychlorate, 1 mM phenylmethylsulfonyl fluoride, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 50 µg/ml aprotinin, and 50 µg/ml leupeptin). The lysate was microcentrifuged for 15 min at 4 °C, and the supernatant was transferred to a new test tube. After determination of protein content by method of Lowry, samples were loaded on an SDS-polyacrylamide electrophoresis gel and transferred onto nylon-enhanced nitrocellulose membrane after electrophoresis. The membrane was blocked with a solution of 0.1% Tween 20/PBS (PBS-T) containing 5% fat-free milk for 2 h. It was next incubated with rabbit polyclonal anti-phospho-p44/42 MAP kinase (New England Bio-Labs) or PPAR- (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature, followed by washing three times for 10 min each with PBS-T buffer. The blot was reblocked with PBS-T containing 5% milk followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG for another 1 h at room temperature. After washing three times for 10 min each with PBS-T, the membrane was incubated for 1 min in a mixture of equal volumes of Western blot chemiluminescence reagents 1 and 2. The membrane was then exposed to film before development.

In Vitro Phosphorylation of PPAR--- PMA-differentiated THP-1 macrophages in 60-mm dishes were incubated with [³²P]H₃PO₄ (0.2 mCi/ml). Following treatment, cells were washed three times with PBS and then lysed in 200 µl of lysis buffer. Supernatants were collected after centrifugation. Protein lysates (50 µg) from each sample were incubated with rabbit polyclonal anti-human PPAR- (1:150) for 1 h at 4 °C. Protein A-agarose (10 µl) was added, and incubation was continued overnight at 4 °C. After washing three times with cold PBS, the slurry was added to loading buffer and boiled for 5 min before loading on a 12% SDS-polyacrylamide electrophoresis gel. After electrophoresis, the gel was dried and exposed to film.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TGF-1 and TGF-2 Decrease CD36 mRNA Expression-- To induce monocyte to macrophage differentiation, THP-1 cells were treated with PMA (200 nM). After several hours of treatment, more than 95% cells became adherent, exhibited spreading, and could not be removed by washing. PMA was removed by washing with PBS, and the cells were incubated overnight in complete medium. To investigate the effect of TGF-1 and TGF-2 on expression of CD36 mRNA, PMA-differentiated THP-1 cells were treated with various concentration of TGF-1 or TGF-2 for 15 h. TGF-1 significantly decreased CD36 mRNA expression at a broad range of concentration in a non-dose-dependent manner (0.5-10.0 ng/ml) with maximum inhibition at 1.0 ng/ml (Fig. 1). In contrast, TGF-2 decreased CD36 mRNA expression only at concentrations 2 ng/ml and in a concentration-dependent manner. A time course (Fig. 2) showed that both TGF-1 and TGF-2 (3 ng/ml) decreased CD36 mRNA expression by 5 h, although the rate of decrease was slightly faster with TGF-2.

View larger version (47K): [in this window] [in a new window]   Fig. 1.   Effect of TGF-1 and TGF-2 on expression of CD36 mRNA. PMA-differentiated THP-1 macrophages were cultured complete RPMI 1640 medium and treated with various concentrations of TGF- 1 or TGF-2 as indicated for 15 h. Total RNA was extracted and used to isolate poly(A+) RNA as described under "Experimental Procedures." The Northern blot was hybridized with a 32P-labeled human CD36 probe and rehybridized with 32P-labeled GAPDH. The data are representative of three separate experiments.

View larger version (52K): [in this window] [in a new window]   Fig. 2.   Time course of CD36 mRNA expression in response to TGF-1 and TGF-2. PMA-differentiated THP-1 macrophages were treated with TGF- or TGF-2 (4 ng/ml) in complete medium for the indicated times. Expression of CD36 mRNA was analyzed as described in Fig. 1.

TGF-1 and TGF-2 Decrease Surface Expression of CD36 Protein-- To determine whether the decrease in CD36 mRNA in response to TGF-1 and TGF-2 was associated with decreased cell surface expression of CD36 protein, we evaluated the CD36 surface expression by fluorescence-activated cell sorting (FACS) and immunohistochemistry using anti-CD36-fluorescein isothiocyanate-conjugated antibody. CD36 surface expression in THP-1 cells was minimal prior to PMA differentiation (Fig. 3, THP) but increased significantly following PMA treatment (Fig. 3, THP/PMA). Consistent with the decrease of CD36 mRNA expression in the Northern assay, treatment of PMA-differentiated THP-1 cells with either TGF-1 (Fig. 3, THP/PMA + TGF1) or TGF-2 (Fig. 3, THP/PMA + TGF2) markedly reduced CD36 surface expression.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Effect of TGF-1 and TGF-2 on CD36 surface protein expression. PMA-differentiated THP-1 macrophages were treated with TGF-1 or TGF-2 (3 ng/ml) for 15 h. After removal with trypsin, the cells (2 × 106) were analyzed for expression of CD36 by FACS and immunohistochemistry as described under "Experimental Procedures." Control cells included both untreated THP-1 monocytes and PMA-differentiated THP-1 macrophages. The data are representative of three separate experiments.

TGF-1 and TGF-2 Inhibit the Increase in CD36 Expression Induced by PPAR- Ligands-- To evaluate the mechanism(s) by which TGF-1 and TGF-2 decreased CD36 expression, PMA-differentiated THP-1 cells were treated with OxLDL or 15d-PGJ₂ in the absence and presence of TGF-1 or TGF-2. Both OxLDL and 15d-PGJ₂ activate PPAR- and increase transcription of PPAR--responsive genes (22, 23). As expected, both OxLDL and 15d-PGJ₂ increased CD36 mRNA expression (Fig. 4). This induction was significantly suppressed by both TGF-1 and TGF-2 (Fig. 4), suggesting that TGF-1 and TGF-2 abrogated PPAR--mediated transcriptional activation of CD36.

View larger version (54K): [in this window] [in a new window]   Fig. 4.   Effect of TGF-1 and TGF-2 on OxLDL- and 15d-PGJ2-induced expression of macrophage CD36 mRNA. PMA-differentiated THP-1 macrophages were treated with OxLDL (100 µg/ml), OxLDL plus TGF-1 or TGF-2 (4 ng/ml), 15d-PGJ2 (3 µM), or 15d-PGJ2 plus TGF-1 or TGF-2 for 15 h. The Northern assay procedure is described under "Experimental Procedures." The data are representative of three separate experiments.

TGF-1 and TGF-2 Phosphorylate the p44 and p42 Isoforms of MAP Kinase-- Inhibition of induction of CD36 mRNA expression in response to PPAR- activation implied that TGF-1 and TGF-2 might be blocking PPAR- transcriptional activity. Because PPAR- contains consensus MAP kinase phosphorylation sequences (35) and because MAP kinase-mediated phosphorylation of PPAR- had been shown to inhibit PPAR- transcriptional activity (35), we evaluated the effect of TGF-1 or TGF-2 on MAP kinase activity and phosphorylation. When macrophages were treated with TGF-1 or TGF-2, both the p44 and p42 isoforms of MAP kinase were rapidly and transiently phosphorylated, with maximum (>2-fold) induction of phosphorylation observed a few minutes after the addition of TGF-1 or TGF-2 (Fig. 5).

View larger version (27K): [in this window] [in a new window]   Fig. 5.   TGF-1 and TGF-2 induce the phosphorylation of p44/42 MAP kinase. PMA-differentiated THP-1 macrophages were treated with TGF- 1 or TGF-2 (4 ng/ml) for the indicated times. Protein lysates (20 µg) from each sample were used to analyze phospho-p44/p42 MAP kinase as described under "Experimental Procedures." The data are representative of three separate experiments.

PPAR- Is Phosphorylated in Response to TGF-1 or TGF-2-- We next evaluated the phosphorylation status of PPAR- in response to TGF-1 and TGF-2. PMA-differentiated THP-1 macrophages were treated with TGF-1 and TGF-2 (4 ng/ml) for 10 h prior to analysis for both PPAR- and phospho-PPAR-. Western blot analysis demonstrated that phospho-PPAR- was undetectable in control macrophages, but treatment with TGF-1 or TGF-2 induced expression of phospho-PPAR- (Fig. 6A). In addition, PMA-differentiated THP-1 macrophages were incubated with [³²P]H₃PO₄ followed by treatment with TGF-1, TGF-2 (4 ng/ml), or the MAP kinase inhibitors PD98059 (10 µM) and UO126 (5 µM) for 10 h. Phosphorylated PPAR- was increased 1.6-fold in TGF-1-treated cells and 1.9-fold in TGF-2-treated cells relative to untreated cells (Fig. 6B). In contrast, two MAP kinase inhibitors, PD98059 and UO126, significantly blocked PPAR- phosphorylation (Fig. 6B). TGF-1, TGF-2 and MAP kinase inhibitors had no effect on PPAR- protein levels (Fig. 6, A and C).

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Western analysis of in vitro phosphorylation of PPAR-. A, induction of phospho-PPAR- by TGF-1 and TGF-2. PMA-differentiated THP-1 macrophages were treated with TGF-1 or TGF-2 (4 ng/ml) for 10 h. Cells were lysed and analyzed for both PPAR- and phospho-PPAR- as described under "Experimental Procedures." B, regulation of phospho-PPAR- by MAP kinase activity. PMA-differentiated THP-1 macrophages were incubated with 0.2 mCi/ml [32P]H3PO4 followed by treatment with TGF-1 or TGF-2 (4 ng/ml) or with MAP kinase inhibitors PD98059 (10 µM) and UO126 (5 µM), as indicated, for 10 h. After cell lysis, protein (50 µg) from each sample was subjected to electrophoresis and 32P-containing phospho-PPAR- was detected as described under "Experimental Procedures." C, effect of MAP kinase inhibitors on PPAR- expression. PMA-differentiated macrophages were treated with different concentrations of MAP kinase inhibitors as indicated for 15 h. After lysis and centrifugation, protein (20 µg) was used to analyze PPAR- protein by Western blotting as described under "Experimental Procedures." The data in each panel are representative of three separate experiments.

MAP Kinase Inhibitors Induce Expression of CD36 and Reverse the Suppression of CD36 Expression by TGF-1 and TGF-2-- TGF- and MAP kinase inhibitors produced opposite effects on PPAR- phosphorylation, implying that MAP kinase inhibitors might directly induce expression of CD36 in macrophages. To test this, macrophages were treated with two MAP kinase inhibitors, PD98059 and UO126. Both MAP kinase inhibitors significantly increased CD36 mRNA (Fig. 7A). FACS analysis demonstrated that, consistent with the increase of CD36 mRNA expression by MAP kinase inhibitors, surface protein of CD36 was also increased (Fig. 7B).

View larger version (49K): [in this window] [in a new window]   Fig. 7.   Effect of MAP kinase inhibitors on CD36 expression. A, PMA-differentiated THP-1 macrophages were treated with various concentrations of the MAP kinase inhibitors PD98059 and UO126 as indicated, for 12 h. Total RNA was extracted, and poly(A+) RNA was isolated to analyze CD36 and GAPDH mRNA as described under "Experimental Procedures." B, PMA-differentiated THP-1 macrophages were treated with the MAP kinase inhibitors PD98059 (10 µM) or UO126 (5 µM) for 15 h. After washing, cells were removed by trypsin and analyzed for CD36 surface protein expression by FACS. The data in both panels are representative of three separate experiments.

Finally, we evaluated the effects of MAP kinase inhibitors on CD36 expression in the presence of TGF-1 and TGF-2. Macrophage treatment with TGF-1 or TGF-2 markedly decreased expression of CD36 mRNA (Fig. 8). MAP kinase inhibitors increased expression of CD36 mRNA and also abrogated the inhibition of CD36 mRNA in response to TGF-1 or TGF-2 (Fig. 8).

View larger version (40K): [in this window] [in a new window]   Fig. 8.   Effect of MAP kinase inhibitors on the inhibition of CD36 expression by TGF-1 and TGF-2. PMA-differentiated THP-1 macrophages were treated with TGF-1 or TGF-2 (4 ng/ml), PD98059 (10 µM), UO126 (5 µM), or combinations of TGF-1 or TGF-2 and MAP kinase inhibitors as indicated for 15 h. Total RNA was extracted, and poly(A+) RNA was isolated to analyze CD36 and GAPDH mRNA as described under "Experimental Procedures." The data are representative of three separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our data demonstrate that TGF-1 and TGF-2 inhibit expression of CD36 by inducing phosphorylation of the p44 and p42 isoforms of MAP kinase, which in turn, results in MAP kinase-mediated phosphorylation of PPAR-. Phosphorylation of PPAR- results in decreased CD36 gene transcription. MAP kinase inhibitors alone increase expression of CD36 by dephosphorylating and activating PPAR-. These data illustrate the complexity of regulation of PPAR--mediated gene expression and demonstrate how multiple signal transduction pathways are utilized to control the transcriptional activities of PPAR- and CD36 gene expression.

PPARs become transcriptionally active when bound to ligand (24). The three PPAR isoforms (, , and /) differ in their C-terminal ligand binding domains. PPARs bind to and are activated by such diverse agents as hypolipidemic drugs (fibrates), long chain fatty acids, arachidonic and linoleic acid metabolites (36), and the thiazolidinedione class of antidiabetic drugs (37).

Growth factors, such as epidermal growth factor and platelet-derived growth factor, have been shown to phosphorylate PPAR- via the MAP kinase signaling pathway and to decrease PPAR- transcriptional activity (38). The NH₂-terminal domain of PPAR- contains a consensus MAP kinase site in a region conserved between PPAR-1 and PPAR-2 isoforms (35). PPAR- proteins migrate on immunoblots as closely spaced doublets, a pattern suggestive of phosphorylation (39, 40). A putative MAP kinase site is phosphorylated by extracellular signal-regulated kinase 2 and Jun NH₂-terminal kinase (35). Phosphorylation significantly inhibits both ligand-independent and ligand-dependent transcriptional activation by PPAR-. (35). This repression is mediated by MAP kinase phosphorylation of Ser-82 on PPAR-1 (38). Mutation of the phosphorylated residue (Ser-82) prevents PPAR-1 phosphorylation as well as growth factor-mediated repression of PPAR--dependent transcription. This phosphorylation-mediated transcriptional repression results from altering the ability of PPAR- to become transcriptionally activated by ligand and is not due to a reduced capacity of the PPAR·retinoid X receptor complex to heterodimerize or recognize its DNA binding site (38).

Three MAP kinase pathways have been identified in mammalian cells. Extracellular signal-regulated kinases 1 and 2 are activated by growth factor stimulation via a Ras-dependent signal transduction cascade (41), whereas Jun NH₂-terminal kinase and p38 kinase are increased by exposure of cells to environmental stress or to cytokines (42, 43). Activated MAP kinases have been shown, in turn, to regulate the activity of specific transcription factors including Elk-1, ATF-2, and c-Jun by phosphorylation of serine or threonine residues (44). The activity of several other nuclear hormone receptors is also regulated by phosphorylation. Phosphorylation of the human 1 thyroid receptor enhances the DNA binding capacity of the protein and increases ligand-mediated transcription (45). Phosphorylation of the retinoic acid receptor and retinoid X receptor modulates heterodimerization of the receptors and consequently increases DNA binding activity (46). In addition, the MAP kinase-dependent phosphorylation of Ser-118 on the estrogen receptor increases transcriptional activation by the AF1 domain (47). Although in general, phosphorylation of nuclear receptors enhances their transcriptional activity, MAP kinase phosphorylation of PPAR- negatively regulates its function.

Although Smads are the primary downstream signaling mediators activated following TGF- receptor ligation (29, 30), MAP kinases have also been demonstrated to modulate downstream TGF--mediated signaling events (31-34). Our data implicate MAP kinase-mediated phosphorylation of PPAR- in inhibiting expression of CD36 in response to TGF- and clearly demonstrate that MAP kinase inhibitors up-regulate expression of CD36. However, we cannot completely rule out the possibility that other downstream signaling events initiated by TGF- activation of MAP kinase can also negatively regulate CD36 expression.

In conclusion, we show for the first time that TGF-, a growth factor expressed within atherosclerotic lesions, induces phosphorylation of PPAR-, inhibits its transcriptional activity, and down-regulates expression of the type B scavenger receptor, CD36. Both TGF- and PPAR- are expressed by monocyte/macrophages (22, 48), and PPAR- has been localized in macrophage-derived foam cells within atherosclerotic lesions (22), where its pattern of expression is correlated with the presence of oxidation-derived epitopes (49). These data may have relevance to both atherosclerotic foam cell formation mediated by CD36 as well as expression of other PPAR--responsive inflammatory mediators expressed within vascular lesions.

	FOOTNOTES

^* This work was supported by a Charles H. Revson and Norman and Rosita Winston Foundation Postdoctoral Fellowship (to J. H.), National Institutes of Health SCOR Grant in Molecular Medicine and Atherosclerosis P50-HL56987 (to A. C. N. and D. P. H.), and the Abercrombie Foundation (to A. M. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pathology, A-626, Cornell University Medical College, 1300 York Ave., New York, NY 10021. Tel.: 212-746-6470; Fax: 212-746-8789; E-mail: nicholso@mail.med.cornell.edu.

	ABBREVIATIONS

The abbreviations used are: OxLDL, oxidized low density lipoprotein; 15d-PGJ₂, 15-deoxy^12,14 prostaglandin J₂; FACS, fluorescence-activated cell sorting; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAP, mitogen-activated protein; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; PPAR, peroxisome proliferator-activated receptor; TGF, transforming growth factor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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