The Orphan Nuclear Receptor Small Heterodimer Partner as a Novel Coregulator of Nuclear Factor- (cid:1) B in Oxidized Low Density Lipoprotein-treated Macrophage Cell Line RAW 264.7*

Small heterodimer partner (SHP), specifically expressed in liver and a limited number of other tissues, is an unusual orphan nuclear receptor that lacks the conventional DNA binding domain. In this work, we found that SHP expression is abundant in murine macrophage cell line RAW 264.7 but was suppressed by oxidized low density lipoprotein (oxLDL) and its constituent 13-hy-droxyoctadecadienoic acid, a ligand for peroxisome pro-liferator-activated receptor (cid:2) . Furthermore, SHP acted as a transcription coactivator of nuclear factor- (cid:1) B (NF (cid:1) B) and was essential for the previously described NF (cid:1) B transactivation by palmitoyl lysophosphatidylcholine, one of measured using the luciferase assay kit (Promega), and the results were normalized to the LacZ expression. Northern, Western, and Reverse Transcriptase-Polymerase Chain Re- action (RT-PCR) Analyses— Total RNA from cell monolayers was iso-lated using guanidinium isothiocyanate and phenol (22). RNA (20 (cid:4) g) was separated on a 1% agarose-formaldehyde gel and transferred to Zeta-Probe GT membranes (Bio-Rad) in 10 (cid:2) SSC buffer (1.5 M NaCl, 0.1 M NaH 2 PO 4 , 0.01 M EDTA) by capillary action. Blots were hybrid-ized with the 32 P-labeled SHP probe for 16 h at 65 °C in 250 m M Na 2 HPO 4 , pH 7.2 and 7% SDS. After washing twice in 20 m M Na 2 HPO 4 , pH 7.2 and 5% SDS at 65 °C for 45 min and once in 20 m M Na 2 HPO 4 , pH 7.2 and 1% SDS for 45 min at 65 °C, the blots were analyzed by exposure to X-Omat AR film (Eastman Kodak Co.) at (cid:3) 70 °C. The 1.5-kilobase probe for 28 S rRNA (ATCC no. 77235) was used for normalization of expression. Protein extracts from oxLDL-treated RAW264.7 cells or HeLa cells cotransfected with HA-SHP expression vector were prepared with lysis buffer (10 m M HEPES, pH 7.9, 10 m M KCl, 2 m M MgCl 2 , 0.5 m M dithiothreitol, 1 m M phenylmethylsulfonyl fluoride, 5 (cid:4) g/ml aprotinin, 5 (cid:4) g/ml pepstatin A, 5 (cid:4) g/ml leupeptin, and 1% Triton X-100), separated by 12% SDS-polyacrylamide gel electro- phoresis, and analyzed by Western blot using HA-monoclonal and goat anti-murine PPAR (cid:2) polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and an enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech). After detection of PPAR (cid:2) , the mem- brane was stripped and reprobed with anti-actin antibody (Santa Cruz Biotechnology). For RT-PCR analyses, total cDNA synthesized from 2 (cid:4) g of total RNA was amplified for 35 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min. The oligonucleotide primer set (5 (cid:4) -ACT CTG GAT TCA GCT GGT CG-3 (cid:4) and 5 (cid:4) -GTT CAT GCT TGT GAA GGA TG-3 (cid:4) ) was used for amplification of a 250-base pair fragment of SHP. The reaction products from the PCR were examined by 1% agarose gel electrophoresis and normalized by comparison to the RT-PCR product for glyceraldehyde-3-phosphate dehydrogenase mRNA. the indicated reporter constructs along with PPAR (cid:2) expression vector (10 (cid:4) g) and LacZ expres- sion vector and the indicated time periods with either vehicle only (ethanol), nLDL (100 (cid:4) g/ml), oxLDL (100 (cid:4) g/ml), lysoPC (10 (cid:4) M ), or 13-HODE (20 (cid:4) g/ml). Normalized luciferase expressions from three independent experiments performed in triplicate samples are presented relative to the LacZ expressions, the error our results suggest a model in which SHP serves as a transcriptional coactivator molecule of NF (cid:1) B and PPAR (cid:2) . In particular, SHP appears to be essential for the NF (cid:1) B transactivation by oxLDL as indicated by a solid line . In addition, resting macrophage cells, like RAW 264.7 cells, may express a putative repressor molecule of PPAR (cid:2) that is likely subjected to down-regulation by oxLDL and dominates over the stimu-lating activity of SHP on the PPAR (cid:2) transactivation (see the text for further details).

The nuclear receptor superfamily includes receptors for a variety of small hydrophobic ligands such as steroids, T3, and retinoids as well as a large number of related proteins that do not have known ligands, referred to as orphan nuclear receptors (for review, see Ref. 1). The receptor proteins are direct regulators of transcription that function by binding to specific DNA sequences named hormone response elements in promoters of target genes. Nearly all the superfamily members bind as dimers to DNA elements. Although some apparently bind only as homodimers, thyroid hormone receptors, vitamin D receptor, retinoic acid receptors (RARs), 1 the peroxisome proliferator-activated receptors (PPARs), and several orphan nuclear receptors bind their specific response elements with high affinity as heterodimers with the retinoid X receptors. Receptors have dimerization interfaces in both their DNA binding domain and ligand binding domain. The DNA binding domain interfaces are quite different for each receptor, whereas the ligand binding domain interface is primarily based on a conserved motif referred to as the ninth heptad or the I-box (2). This motif is required for both heterodimerization and homodimerization of many nuclear receptors, including retinoid X receptor, thyroid hormone receptor, RAR, hepatocyte nuclear factor 4, and chicken ovalbumin upstream promoter transcription factor (2)(3)(4).
Small heterodimer partner (SHP) is an orphan nuclear receptor specifically expressed in liver and a limited number of other tissues, and its activities are in some ways opposite to those of retinoid X receptor (5). SHP, like the orphan nuclear receptor DAX-1 (for review, see Ref. 6), lacks the conventional DNA binding domain. Both direct biochemical and the yeast two-hybrid results demonstrated that SHP interacts with many members of the receptor superfamily (5). As expected from its lack of a DNA binding domain, addition of SHP inhibited in vitro DNA binding by nuclear receptors with which it interacted, and in mammalian cell cotransfections, SHP repressed their transactivation. In addition, the SHP sequences required for interaction with other superfamily members have recently been localized to the central portion of SHP (7), distinct from the I-box (2)(3)(4).
Macrophages are thought to play critical pathogenic roles in several chronic inflammatory diseases, including atherosclerosis (for review, see Ref. 8). The formation of an atherosclerotic lesion is a complex process involving intracellular lipid accumulation and various signal transduction pathways in vascular cells, such as endothelial cells, smooth muscle cells, and monocytes/macrophages. Each vascular cell is capable of oxidatively modifying low density lipoprotein (LDL) to generate oxidized LDL (oxLDL) (for review, see Ref. 9). Circulating monocytes adhere to activated endothelium, migrate into intima, and subsequently differentiate into macrophages that express various scavenger receptor genes. Accumulation of oxLDL through scavenger receptors mediates the formation of foam cells from macrophages and consequently fatty streaks, the earliest visible lesions of atherosclerosis. The oxidative modification results in a number of important biological activities of LDL, including the induction of a number of genes encoding cytokines and growth factors (10). Among the growth factors and cytokines that are secreted from vascular cells, a regulatory network is thought to exist, and a lesion may result from their collective action.
Cross-talk between transcription factors of distinct families is an important phenomenon in regulating gene transcription and has recently become the subject of intensive investigation. In particular, the transcription factor nuclear factor-B (NFB) has been shown to functionally interact with numerous other transcription factors, including members of the nuclear receptor superfamily (for review, see Ref. 11), resulting in mutually repressed biological activity of these transcription factors. These include glucocorticoid receptor, estrogen receptor, progesterone receptor, and androgen receptor (11). More recently, PPAR␥ and RAR/retinoid X receptor were also added to the list of nuclear receptors functionally interacting with NFB (12)(13)(14)(15). NFB, composed of homo-and heterodimeric complexes of members of the Rel (NFB) family of polypeptides, is important for the inducible expression of a wide variety of cellular and viral genes (for review, see Ref. 16). In vertebrates, this family comprises p50, p65 (RelA), c-Rel, p52, and RelB. These proteins share a 300-amino acid region, known as the Rel homology domain, that binds to DNA and mediates homo-and heterodimerization. This domain also is the target of various IB inhibitors. In the majority of cells, NFB exists in an inactive form in the cytoplasm, bound to the inhibitory IB proteins. Treatment of cells with various inducers results in the degradation of IB proteins. The bound NFB is released and translocates to the nucleus where it activates appropriate target genes.
In this study, we identified SHP as a novel transcription coactivator of NFB. We have further presented the experimental results indicating that the modulation of SHP expression appears to function as a distinct regulatory component of the transcriptional activities of NFB in oxLDL-treated, resting macrophage cells. These results could have an important implication for the differentiation mechanism of resting macrophage cells into foam cells and resulting atherogenesis.
Yeast Two-hybrid Test-For the yeast two-hybrid tests, plasmids encoding LexA fusions and B42 fusions were cotransformed into Saccharomyces cerevisiae EGY48 strain containing the LacZ reporter plasmid SH/18-34 (21). Liquid assays of ␤-galactosidase expression were carried out as described previously (21). Similar results were obtained in more than two similar experiments.
Glutathione S-Transferase (GST) Pull-down Assays-The GST fusions or GST alone was expressed in Escherichia coli, bound to glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech), and incubated with either cell lysates cotransfected with HA-SHP expression vector or labeled proteins expressed by in vitro translation by using the TNT-coupled Transcription-Translation System with conditions as described by the manufacturer (Promega, Madison, WI). Specifically bound proteins were eluted from beads with 40 mM reduced glutathione in 50 mM Tris (pH 8.0) and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography as described previously (21). To introduce serial deletions into p65, the p65 expression vector was digested with the appropriate restriction enzymes before being subjected to the TNT-coupled Transcription-Translation System.
Cell Culture and Transfections-RAW 264.7, HeLa, and CV-1 cells were cultured in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum, 100 g/ml penicillin, and 100 g/ml streptomycin at 37°C in 5% CO 2 (Life Technologies, Inc.). For transfections, cells were grown in six-well plates with medium supplemented with 10% fetal bovine serum for 24 h and transfected with the indicated plasmid(s) by the calcium phosphate coprecipitation method (12). The total amounts of expression vectors were kept constant by adding the CDM8 expression vector to transfections. After 24 h, cells were washed and incubated in serum-free Dulbecco's modified Eagle's medium in the absence or presence of either oxLDL or other reagent(s). Cells were harvested after the indicated time periods, luciferase activity was measured using the luciferase assay kit (Promega), and the results were normalized to the LacZ expression.
Northern, Western, and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analyses-Total RNA from cell monolayers was isolated using guanidinium isothiocyanate and phenol (22). RNA (20 g) was separated on a 1% agarose-formaldehyde gel and transferred to Zeta-Probe GT membranes (Bio-Rad) in 10ϫ SSC buffer (1.5 M NaCl, 0.1 M NaH 2 PO 4 , 0.01 M EDTA) by capillary action. Blots were hybridized with the 32 P-labeled SHP probe for 16 h at 65°C in 250 mM Na 2 HPO 4 , pH 7.2 and 7% SDS. After washing twice in 20 mM Na 2 HPO 4 , pH 7.2 and 5% SDS at 65°C for 45 min and once in 20 mM Na 2 HPO 4 , pH 7.2 and 1% SDS for 45 min at 65°C, the blots were analyzed by exposure to X-Omat AR film (Eastman Kodak Co.) at Ϫ70°C. The 1.5-kilobase probe for 28 S rRNA (ATCC no. 77235) was used for normalization of expression. Protein extracts from oxLDL-treated RAW264.7 cells or HeLa cells cotransfected with HA-SHP expression vector were prepared with lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 2 mM MgCl 2 , 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 g/ml aprotinin, 5 g/ml pepstatin A, 5 g/ml leupeptin, and 1% Triton X-100), separated by 12% SDS-polyacrylamide gel electrophoresis, and analyzed by Western blot using HA-monoclonal and goat anti-murine PPAR␥ polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and an enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech). After detection of PPAR␥, the membrane was stripped and reprobed with anti-actin antibody (Santa Cruz Biotechnology). For RT-PCR analyses, total cDNA synthesized from 2 g of total RNA was amplified for 35 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min. The oligonucleotide primer set (5Ј-ACT CTG GAT TCA GCT GGT CG-3Ј and 5Ј-GTT CAT GCT TGT GAA GGA TG-3Ј) was used for amplification of a 250-base pair fragment of SHP. The reaction products from the PCR were examined by 1% agarose gel electrophoresis and normalized by comparison to the RT-PCR product for glyceraldehyde-3-phosphate dehydrogenase mRNA.

RESULTS AND DISCUSSION
SHP as a Novel Transcription Coactivator of NFB-Many nuclear receptors have been shown to functionally interact with NFB (for review, see Ref. 11). In this work, we examined whether the NFB components p50 and p65 also interact with the unusual orphan nuclear receptor SHP. GST fusion to SHP interacted with radiolabeled p65 but not with luciferase or p50 (Fig. 1A, left). In addition, SHP interacted with SRC-1, a transcription coactivator of nuclear receptors and other transcription factors (for review, see Ref. 23) including NFB (18). Accordingly GST fusion to SHP specifically interacted with the radiolabeled C-terminal subregions of SRC-1 (i.e. SRC-D and SRC-E containing the SRC-1 residues 759 -1141 and 1101-1441, respectively) (Fig. 1A, left). It is noted that these regions are distinct from the region containing the previously shown receptor binding site (i.e. SRC-C consisting of the SRC-1 residues 568 -779) that includes the nuclear receptor-interacting LXXLL motifs (24,25) in which L and X denote leucine and any amino acid, respectively. Similar results were also obtained with the mammalian two-hybrid-based tests (results not shown). The SHP interaction interface was further mapped to the N-terminal 283 residues of p65 (Fig. 1A, right). In addition, HeLa nuclear extracts cotransfected with HA-tagged SHP expression vector retained with GST fusion to p65 but not with GST alone contained SHP as demonstrated by Western analyses with HA-monoclonal antibody (Fig. 1B). Consistent with these results, B42 fusion to the N-terminal domain of p65 (i.e. p65N consisting of the p65 residues 1-283), but not B42 alone or B42 fusion to p50, stimulated the LexA-SHP-mediated transactivation of the LacZ reporter construct controlled by upstream LexA binding sites ( Fig. 2A, bottom). SHP also interacted with SRC-D and SRC-E but not with SRC-C in yeast ( Fig.  2A), whereas RAR interacted with SRC-C as well as the Cterminal region of SRC-1 (i.e. SRC-E) as previously shown (23). Confirming the functionality of these interactions, cotransfected SHP enhanced transactivation by NFB in a dose-dependent manner either alone or in synergy with SRC-1 (Fig.  2B). In contrast, SHP efficiently suppressed transactivation by RAR as previously described (5) and had no significant effect on transactivation directed by Gal4 fusion to VP16 (results not shown). These results clearly demonstrate that SHP directly interacts with p65 and is a positive transcriptional coregulator of NFB. This positive regulatory role is in sharp contrast to the previously reported inhibitory role of SHP on a variety of receptor-dependent signaling pathways (5).
Down-regulation of SHP by oxLDL in RAW 264.7-SHP was previously demonstrated to be specifically expressed in liver and a limited number of tissues (26). During our recent search for additional tissues or cell lines that may express SHP, we found that SHP mRNA is abundant in the mouse macrophage cell line RAW 264.7. Surprisingly this expression was significantly repressed by oxLDL, but not by native LDL (nLDL), in a time-dependent manner (Fig. 3A). However, the repression was not observed in the presence of the endocytosis blocker cytochalasin B (12) (Fig. 3B), suggesting that the inhibitory effect of oxLDL likely involves endocytosis of oxLDL. We have previously shown that 13-HODE, a constituent of oxLDL and a ligand for PPAR␥, functions in an endocytosis-dependent man- FIG. 1. SHP interacts with the NFB component p65. A, the full-length p50 and p65, luciferase (Luc.), and various SRC-1 fragments (18) as well as a series of C-terminally truncated p65 generated by digesting with the appropriate restriction enzymes were labeled with [ 35 S]methionine by in vitro translation and incubated with glutathione beads containing GST alone and GST-SHP as indicated. Beads were washed, and specifically bound material was eluted with reduced glutathione and resolved by SDS-polyacrylamide gel electrophoresis. Approximately 20% of the total reaction mixture was loaded as input. B, HeLa nuclear extracts cotransfected with vector alone or HA-SHP expression vector were incubated with glutathione beads containing GST alone and GST-p65 as indicated. Specifically bound material was eluted with reduced glutathione, resolved by SDS-polyacrylamide gel electrophoresis, and probed with HA-monoclonal antibody. Approximately 20% of the total reaction mixture was loaded as input.

FIG. 2. SHP as a novel coactivator of NFB. A, the indicated B42
and LexA plasmids were transformed into a yeast strain containing an appropriate LacZ reporter gene as described previously (21). The data are representative of at least two similar experiments, and the error bars are as indicated. B, HeLa cells were transfected with LacZ expression vector, expression vectors for p65, SHP, and SRC-1, and a reporter gene, B-LUC, as indicated. Normalized luciferase expressions from triplicate samples were calculated relative to the LacZ expressions, and the results were expressed as -fold activation (n-fold) over the value obtained with the reporter alone. The data are representative of three similar experiments, and the error bars are as indicated.

FIG. 3. Suppression of the SHP mRNA level by oxLDL.
Total RNA from RAW 264.7 cells treated with nLDL (100 g/ml) or oxLDL (100 g/ml) for the indicated time periods was extracted and subjected to Northern blot analysis using SHP as a probe (A). Similarly RAW 264.7 cells were treated for 4 h with nLDL (100 g/ml) or oxLDL (100 g/ml) in the absence or presence of cytochalasin B, an inhibitor of endocytosis (B), or for 4 h with nLDL (100 g/ml), oxLDL (100 g/ml), 13-HODE (20 g/ml), troglitazone (15 M), lysoPC (10 M), or 7-ketocholesterol (25 M) (C). RAW 264.7 cells were treated for 20 min with actinomycin D (5 g/ml) and incubated with nLDL (100 g/ml) or oxLDL (100 g/ml) for the indicated time periods (D). Intensity of each band was measured and plotted against time; the band intensity from untreated cells was used as a standard (100%). A cDNA encoding 28 S rRNA was used as a control for loading equivalence of RNA. ner (12). Thus, we examined if this inhibitory effect of oxLDL stemmed from its constituent 13-HODE. As shown in Fig. 3C, 13-HODE was indeed responsible for the inhibitory effect of oxLDL. Troglitazone, a synthetic PPAR␥ ligand, also showed similar effects. In contrast, lysoPC, another constituent of ox-LDL that transactivates NFB (12,27), was without any effect. However, the oxLDL-mediated repressive effects were not recapitulated with a luciferase reporter construct directed by the previously described native SHP promoter (26) in cotransfections of CV1 and RAW 264.7 cells (results not shown). Notably 13-HODE, in addition to its role as a direct ligand for PPAR␥, may have a multitude of other effects as recently demonstrated for another PPAR␥ ligand 15-deoxy-⌬ 12,14 -prostaglandin J 2 in inhibiting the NFB transactivation (14). As shown in Fig. 3D, SHP mRNA from oxLDL-treated cells degraded much faster than nLDL-treated cells in the presence of the transcription blocker actinomycin D. Under this condition, the half-life of SHP mRNA in the presence of oxLDL was ϳ1 h relative to 2 h with nLDL. Interestingly cycloheximide, a protein synthesis blocker, had no effect on the inhibitory action of oxLDL (results not shown), suggesting that new protein synthesis is not required for the repression. Overall these results strongly suggest that oxLDL suppresses SHP expression in resting macrophage cells likely through modulating its mRNA stability.
SHP as a Modulator of the NFB Activities in oxLDL-treated RAW 264.7-SHP has previously been shown to down-regulate transactivation by nuclear receptors with which it interacts (5, 7, 28, 29). As shown in Fig. 2, however, SHP is apparently a transcriptional coactivator of NFB. Notably SHP also interacted with PPAR␥ in the yeast two-hybrid tests (results not shown). Thus, we examined the effects of cotransfected SHP on either oxLDL-mediated transactivation of NFB or PPAR␥ in CV1 cells. Surprisingly oxLDL did not readily enhance the NFB transactivation unless SHP was coexpressed (Fig. 4A), suggesting the essentiality of SHP in the NFB transactivation by oxLDL. Furthermore, SHP was an activator of both the basal and ligand-induced levels of PPAR␥ transactivation in CV-1 cells (Fig. 4B) as opposed to its previously described inhibitory effects with other nuclear receptors (5,7,28,29). However, it is not currently known if SHP could still be an activator of the PPAR␥ transactivation in RAW 264.7 cells. Consistent with an idea that SHP alone may not effectively stimulate the PPAR␥ transactivation in RAW 264.7 cells, the PPAR␥ transactivation level was relatively low during the early phase of oxLDL treatment in which SHP expression was still abundant (Fig. 5). From these results, a putative repressor molecule of PPAR␥, which is dominant over the stimulatory effect of SHP and suppressible by oxLDL, was predicted to exist in the resting RAW 264.7 cells. This issue is currently under intense investigation. Interestingly treatment of RAW 264.7 cells with oxLDL did not lead to significant changes in PPAR␥ expression (Fig. 4C) in contrast to the previously described results with THP-1 monocytic leukemia cells (30). In addition, the previously described 13-HODE/PPAR␥-mediated repression of the NFB transactivation (12) became impaired with the increasing amount of SHP expression vector (Fig. 4D). The 13-HODE-dependent repression was readily observed only in the presence of 10 ng of cotransfected PPAR␥. Under this condition, however, the -fold repression progressively de- Western blot (W) analyses of PPAR␥ in RAW264.7 cells were executed as described previously (21). Total RNA or cell lysate was prepared from cells treated with oxLDL (100 g/ml) for the time periods as indicated. The 250-base pair PCR fragment of PPAR␥ as well as the antibodydirected PPAR␥ band are as shown. Equivalent loading of RNA and protein was verified by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR product and actin protein, respectively.

FIG. 5. Time-dependent transactivation of NFB and PPAR␥.
A, RAW264.7 cells were transfected with the indicated reporter constructs along with PPAR␥ expression vector (10 g) and LacZ expression vector and treated for the indicated time periods with either vehicle only (ethanol), nLDL (100 g/ml), oxLDL (100 g/ml), lysoPC (10 M), or 13-HODE (20 g/ml). Normalized luciferase expressions from three independent experiments performed in triplicate samples are presented relative to the LacZ expressions, and the error bars are as indicated. B, our results suggest a model in which SHP serves as a transcriptional coactivator molecule of NFB and PPAR␥. In particular, SHP appears to be essential for the NFB transactivation by oxLDL as indicated by a solid line. In addition, resting macrophage cells, like RAW 264.7 cells, may express a putative repressor molecule of PPAR␥ that is likely subjected to down-regulation by oxLDL and dominates over the stimulating activity of SHP on the PPAR␥ transactivation (see the text for further details). creased from ϳ3 to 2 and 0 as the amount of SHP expression vector increased from 0 to 100 and 200 ng, respectively (Fig.  4D). Considering all of these results together, the NFB transactivation was expected to gradually diminish in oxLDL-treated RAW 264.7 cells as the SHP expression becomes repressed (Fig. 3). Indeed stimulation of the NFB transactivation was readily observed with oxLDL or lysoPC for the initial 4 -8 h after treatment, whereas lysoPC was much less effective and oxLDL and 13-HODE were rather repressive with the NFB transactivation for the 12-24 h post-treatment (Fig. 5). In particular, the latter results likely result from the previously described PPAR␥-mediated repression of the NFB transactivation (12), which does not seem to operate when the SHP level is relatively high (Fig. 4D). Currently it is not clear why the PPAR␥ transcriptional activities were much higher for the 12-24 h post-treatment generating a biphasic transition from the NFB to PPAR␥ transactivation during oxLDL treatment, although the putative, oxLDL-suppressible repressor may initially exist in the resting RAW 264.7 cells as mentioned already. Overall these results suggest that the oxLDL-mediated downregulation of SHP, coupled with the PPAR␥-mediated repressive actions (12)(13)(14), may result in efficient shut-down of the NFB transcriptional activities in oxLDL-treated RAW 264.7 cells.
LysoPC has been shown to induce the expression of cell adhesion molecules and growth factors and to induce macrophage proliferation (31). Because NFB is usually involved with proliferation, this lysoPC-mediated proliferation of macrophages may involve its action with NFB (27). Interestingly our preliminary results 2 indicated that the SHP protein level also drops in RAW 264.7 cells upon oxLDL treatment with a time dependence similar to its mRNA expression profile (Fig.  3), and human foam cells from atherogenic lesion did not express SHP at all. These results imply that, although the role of oxLDL-PPAR␥ in regulating macrophage differentiation is still unclear, an efficient shut-down of the NFB transactivation may play a pivotal role in these processes. Thus, our results suggest the possibility of new therapeutic interventions in atherogenesis by targeting SHP for regulated expression or modulating transcriptional activities by identifying its putative ligands. More detailed studies with various monocytic cells will provide further details as to the action of SHP in these processes. Finally it is important to note that oxLDL treatment may also affect a variety of nuclear receptor-dependent signaling pathways in macrophages based on the previously defined modulatory role of SHP with receptors (5).
In summary, we identified SHP as a novel transcription coactivator of NFB and have further presented the experimental results indicating that targeted expression of SHP appears to function as a distinct regulatory component of the transcriptional activities of NFB in oxLDL-treated, resting macrophage cells.