Transforming Growth Factor-β1 (TGF-β1) and TGF-β2 Decrease Expression of CD36, the Type B Scavenger Receptor, through Mitogen-activated Protein Kinase Phosphorylation of Peroxisome Proliferator-activated Receptor-γ*

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-β1 (TGF-β1) and TGF-β2 on the expression of CD36 in macrophages. Treatment of phorbol ester-differentiated THP-1 macrophages with TGF-β1 or TGF-β2 significantly decreased expression of CD36 mRNA and surface protein. TGF-β1/TGF-β2 also inhibited CD36 mRNA expression induced by oxidized low density lipoprotein and 15-deoxyΔ12,14 prostaglandin J2, a peroxisome proliferator-activated receptor (PPAR)-γ ligand, suggesting that the TGF-β1/TGF-β2 down-regulated CD36 expression by inactivating PPAR-γ-mediated signaling. TGF-β1/TGF-β2 increased phosphorylation of both mitogen-activated protein (MAP) kinase and PPAR-γ, whereas MAP kinase inhibitors reversed suppression of CD36 and inhibited PPAR-γ phosphorylation induced by TGF-β1/TGF-β2. Finally, MAP kinase inhibitors alone increased expression of CD36 mRNA and surface protein but had no effect on PPAR-γ protein levels. Our data demonstrate for the first time that TGF-β1 and TGF-β2 decrease expression of CD36 by a mechanism involving phosphorylation of MAP kinase, subsequent MAP kinase phosphorylation of PPAR-γ, and a decrease in CD36 gene transcription by phosphorylated PPAR-γ.

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).
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
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 g/ml each of penicillin and streptomycin, and 2 mM glutamine. Cells were adjusted to a density of 2.5 ϫ 10 5 cells/cm 2 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.
Isolation of Total RNA, Purification of Poly(A ϩ ) 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 ϩ ) RNA was purified from approximately 100 g of total RNA using the Poly(A)Ttract® mRNA isolation system III (Promega, Madison, WI).
Poly(A ϩ ) 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 10ϫ 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 32 P-randomly primed probes for CD36 or GAPDH. After overnight hybridization, membranes were washed twice for 20 min each time with 2ϫ SSC and 0.2% SDS, and twice for 20 min each time with 0.2ϫ 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 5Ј-and 3Ј-oligonucleotides used were ATGGGCTGTGACCGGAACT (285-304) and ACAGACCAACTGTGG-TAG (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 ϫ 10 6 cell were suspended in 300 l of PBS containing 5% mouse serum and incubated for 30 min at room temperature while shaking. Cells then were incubated with 10 l 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 g/ml aprotinin, and 50 g/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 [ 32 P]H 3 PO 4 (0.2 mCi/ml). Following treatment, cells were washed three times with PBS and then lysed in 200 l of lysis buffer. Supernatants were collected after centrifugation. Protein lysates (50 g) from each sample were incubated with rabbit polyclonal anti-human PPAR-␥ (1:150) for 1 h at 4°C. Protein A-agarose (10 l) 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
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 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.
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 phosphoryla- tion. 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 (Ͼ2-fold) induction of phosphorylation observed a few minutes after the addition of TGF-␤1 or TGF-␤2 (Fig. 5).

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).
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). DISCUSSION 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 NH 2 -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 NH 2 -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␥⅐retinoid 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 NH 2 -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)(32)(33)(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.