Acquisition of Secretion of Transforming Growth Factor-β1 Leads to Autonomous Suppression of Scavenger Receptor Activity in a Monocyte-Macrophage Cell Line, THP-1*

Macrophage cells derived from the human monocytic leukemia cell line, THP-1, accumulate esterified cholesterol when cultivated in the presence of acetylated low density lipoprotein (Ac-LDL) through scavenger receptors (ScR). In the present study, we isolated a subtype of THP-1 cells that failed to accumulate esterified cholesterol when cultivated in the presence of Ac-LDL. The cells had negligible amounts of cell association and degradation of Ac-LDL compared with the parent THP-1 cells. The subtype THP-1 cells did not express ScR mRNA as well as that of lipoprotein lipase. In contrast, the expression of apolipoprotein E mRNA was greater than that found in parent THP-1 cells. The culture medium of subtype THP-1 cells treated with 12-O-tetradecanoylphorbol-13-acetate inhibited the uptake of Ac-LDL and the expression of ScR in parent THP-1 cells. After a 48-h incubation in the culture medium containing 12-O-tetradecanoylphorbol-13-acetate, the culture medium of differentiated subtype THP-1 cells contained 6.9 ng/ml transforming growth factor (TGF)-β1, while that of parent THP-1 cells secreted below detection level, which was less than 3 ng/ml. This inhibitory effect of the conditioned medium on the expression of ScR in parent THP-1 cells was abolished by pretreatment of the culture medium with anti-TGF-β1 antibodies. Parent THP-1 cells expressed as much TGF-β1 mRNA as sTHP-1 cells after stimulation of differentiation. Although the precursor forms of TGF-β1 that were synthesized in both parent and subtype THP-1 cells were of similar size and were expressed at similar levels, latent TGF-β1-binding protein, which is necessary for the secretion of TGF-β1, could only be co-immunoprecipitated with anti-TGF-β1 antibody from subtype THP-1 cells. This suggests that subtype THP-1 cells secrete TGF-β1 into the medium by forming a functional complex with the latent TGF-β1-binding protein. We conclude that subtype THP-1 cells could not take up Ac-LDL because ScR was inhibited (leading to a loss of function) caused by the secreted TGF-β1.


Macrophage cells derived from the human monocytic leukemia cell line, THP-1, accumulate esterified cholesterol when cultivated in the presence of acetylated low density lipoprotein (Ac-LDL) through scavenger receptors (ScR). In the present study, we isolated a subtype of THP-1 cells that failed to accumulate esterified cholesterol when cultivated in the presence of Ac-LDL. The cells had negligible amounts of cell association and degradation of Ac-LDL compared with the parent THP-1 cells. The subtype THP-1 cells did not express ScR mRNA as well as that of lipoprotein lipase. In contrast, the expression of apolipoprotein E mRNA was greater than that found in parent THP-1 cells. The culture medium of subtype THP-1 cells treated with 12-O-tetradecanoylphorbol-13-acetate inhibited the uptake of Ac-LDL and the expression of ScR in parent THP-1 cells. After a 48-h incubation in the culture medium containing 12-O-
tetradecanoylphorbol-13-acetate, the culture medium of differentiated subtype THP-1 cells contained 6.9 ng/ml transforming growth factor (TGF)-␤1, while that of parent THP-1 cells secreted below detection level, which was less than 3 ng/ml. This inhibitory effect of the conditioned medium on the expression of ScR in parent THP-1 cells was abolished by pretreatment of the culture medium with anti-TGF-␤1 antibodies. Parent THP-1 cells expressed as much TGF-␤1 mRNA as sTHP-1 cells after stimulation of differentiation. Although the precursor forms of TGF-␤1 that were synthesized in both parent and subtype THP-1 cells were of similar size and were expressed at similar levels, latent TGF-␤1binding protein, which is necessary for the secretion of TGF-␤1, could only be co-immunoprecipitated with anti-TGF-␤1 antibody from subtype THP-1 cells. This suggests that subtype THP-1 cells secrete TGF-␤1 into the medium by forming a functional complex with the latent TGF-␤1-binding protein. We conclude that subtype THP-1 cells could not take up Ac-LDL because ScR was inhibited (leading to a loss of function) caused by the secreted TGF-␤1.
Foam cells carrying lipid droplets are often found in the early stage of atheromatous lesions (1,2). Some of the foam cells are reported to originate from blood monocytes (3,4). When monocytes differentiate into macrophages, they change shape, reduce the number of the microvilli on the cell surface, and generate podocytes. At the terminal stage of the differentiation, low density lipoprotein (LDL) 1 receptors are down-regulated, and acetylated LDL (Ac-LDL) receptors (scavenger receptors; ScR) are induced to express (5,6). In addition, macrophages begin to synthesize and secrete apolipoprotein E (apoE) and lipoprotein lipase (LPL) (7,8), which results in a dramatic change in their lipid metabolism. Macrophages accumulate lipids including cholesterol and become foam cells (9). Since the macrophages in atheromatous lesions express ScR, it is expected that ScR plays an important role in the development of atherosclerotic lesions (10,11). The mechanism of the regulation of ScR activity, however, has not been fully understood.
The induced expression of apoE, LPL, and ScR in macrophages is regulated by a number of factors. The expression of ApoE is stimulated by cholesterol loading (19) and transforming growth factor-␤1 (TGF-␤1) (20), while LPL expression is inhibited by oxysterols (21), TGF-␤ (22), and lipopolysaccharides (23). Bottalico et al. (24) reported that TGF-␤1 inhibits ScR activity in THP-1 cells. TGF-␤ informs a family that inhibits the growth of most epithelial cells, and it has potent effects on the accumulation of extracellular matrix components (25). TGF-␤ is known to induce the expression of natriuretic peptide type C (CNP) in vascular endothelial cells (26), and the secretion of CNP inhibits the proliferation of smooth muscle cells (27). Thus, TGF-␤ may play an important role in vascular remodeling.
In the present study, we have isolated a subtype of THP-1 cells (sTHP-1) that could not accumulate esterified cholesterol by Ac-LDL loading. The cells did not express ScR activity even after they were induced to differentiate. In the differentiated sTHP-1 cells, transcripts of ScR and LPL were below the detection level, and that of apoE was significantly increased compared with that of parent THP-1 cells. The conditioned medium of the differentiated sTHP-1 cells contained sufficient amounts of active TGF-␤1 to suppress the expression of ScR in parent THP-1 cells. sTHP-1 cells synthesized a 190-kDa latent TGF-␤1-binding protein (LTBP), which was not detectable in parent cells. We conclude that differentiated sTHP-1 cells have acquired an ability to secrete large amounts of active form of TGF-␤1, which may due to its ability to express and synthesize LTBP. sTHP-1 cells may prove to be a useful model for understanding the secretion and activation mechanisms of TGF-␤1 and their roles in vascular systems.
Oil Red-O Staining-The differentiated cells that were incubated with 10 g/ml Ac-LDL treatment were fixed in 4% formaldehyde. Then the cells were rinsed with propyleneglycol, and the lipids were stained with 10% oil red-O in propyleneglycol followed by staining of the nuclei with Mayer's hematoxylin (34).
Measurement of Lipids-THP-1 cells were treated with TPA for 4 days, causing differentiation into macrophages, and were subsequently incubated with or without 25-500 g/ml Ac-LDL. The cells were washed and lysed in 1 ml of 1 N NaOH. Lipids were extracted three times with chloroform and methanol (2:1). Total cholesterol and unesterified cholesterol were determined by the enzymatic method (35). The esterified cholesterol content was calculated by subtracting the amount of unesterified cholesterol from that of total cholesterol. The protein concentration was determined by the method of Lowry et al. (36).
Determination of Cell-associated and Degraded Ac-LDL-The binding activity of ScR was measured after incubating the cells with 125 I-Ac-LDL or DiI-Ac-LDL at 37°C. For the measurement of cell-associated and degraded 125 I-Ac-LDL, the cells were incubated with 10 g/ml 125 I-Ac-LDL for 5 h at 37°C. At the end of the incubation, trichloroacetic acid-soluble radioactivity in the medium was determined. This represented the degradation product of 125 I-labeled Ac-LDL. The cells were washed and lysed in 1 ml of 1 N NaOH, and the radioactivities were determined (37). To determine the uptake of DiI-Ac-LDL, the cells were incubated in the presence of 5 g/ml DiI-Ac-LDL for 4 h at 37°C, and they were washed, dissolved, and analyzed for fluorescent intensity by spectrofluorometer (Hitachi, Tokyo) with excitation at 520 nm and emission at 570 nm (38,39). The specific activities for the associated and degraded Ac-LDL were calculated by subtracting the values in the presence of a 100-fold excess amount of unlabeled lipoproteins.
Preparation of Conditioned Medium-After the cells were cultivated in the presence of TPA for 24 -48 h, the culture medium was centrifuged at 1300 ϫ g at 4°C for 15 min. The supernatant fraction was filtered with a 0.22-m pore size filter.
Treatment of Conditioned Medium with Anti-TGF-␤1 Antiserum-To the conditioned culture medium, 100 l of anti-TGF-␤1 antiserum or control nonimmune serum diluted with 300 l of Dulbecco's phosphatebuffered saline was added with 100 l of protein G-Sepharose 4FF (Pharmacia, Uppsala, Sweden). The mixtures were rotated at 4°C overnight. The Sepharose beads, prebound to either anti-TGF-␤1 antibodies (King Brewing Co. Ltd.) or control IgG were added to 2 ml of conditioned medium from sTHP-1 cells that had been treated with TPA for 48 h. The mixtures were incubated at 37°C for 2 h, and the supernatant fractions were recovered by centrifugation at 420 ϫ g for 10 min.
Northern Blot Analysis-Total RNA was isolated from THP-1 cells using the acid guanidium thiocyanate-phenol-chloroform method, described by Chomczynski and Sacchi (43). Total RNA (20 g) was electrophoresed in 0.9% agarose gels containing 2.2 M formaldehyde and then transferred onto nylon membranes (Gene Screen, NEN Life Science Products). Then the membrane was hybridized at 42°C overnight with the indicated probes, which were labeled with [ 32 P]dCTP (Amersham, Buckinghamshire, United Kingdom) by the multipriming method (44). The membranes were washed and exposed to a Fuji Imaging Plate (Fuji Photo Film, Tokyo, Japan). The radioactive bands were determined by a Fuji BAS 2000 image analyzer (Fuji Photo Film).
Immunoprecipitation of TGF-␤1-TGF-␤1 and its complex form were radiolabeled and immunoprecipitated as described previously by Miyazono et al. (45). Briefly, the two types of THP-1 cells, incubated in the medium containing TPA for 3 days, were incubated in cysteine-and methionine-free RPMI 1640 supplemented with 50 ng/ml TPA, 0.1% bovine serum albumin, L-[ 35 S]methionine (200 Ci/ml), and L-[ 35 S]cysteine (200 Ci/ml) at 37°C for 2 h (pulse labeling). Then the culture medium was replaced with the RPMI 1640 supplemented with 50 ng/ml TPA, 0.1% bovine serum albumin, 75 mg/liter L-methionine, and 250 mg/liter L-cysteine and further incubated at 37°C for 4 h (chase). The cells were then lysed and pretreated twice with control mouse IgG1 and protein G-Sepharose. Then 1 ml of the supernatant fractions was immunoprecipitated with 2 g of mouse anti-human TGF-␤1 (Bio-Source International, Camarillo, CA) at 4°C for 2 h. The antigenantibody complex was recovered with 80 l of protein G-Sepharose. The matrix was washed twice with detergent buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.0, 1% sodium deoxycholate, 1% Triton X-100) and once with high salt buffer (500 mM NaCl, 0.2% Triton X-100, 20 mM Tris-HCl, pH 7.0) and eluted with Laemmli's buffer. Samples were electrophoresed in a 8 -16% polyacrylamide gradient gel. The gel was dried and exposed to Fuji Imaging Plate. The visualized data was analyzed using a BAS 2000 image analyzer.

RESULTS
Lipid Accumulation in Parent THP-1 and sTHP-1 Cells-THP-1 cells can be induced to differentiate into macrophagelike cells by TPA treatment. THP-1 cells, thus treated, efficiently accumulate esterified cholesterol into vacuoles when cultivated in the presence of Ac-LDL (14,18). During the course of maintenance of THP-1 cells, we isolated by chance a subtype of THP-1 cells, which, when fully differentiated, failed to accumulate esterified cholesterol, even cultivated in the presence of Ac-LDL. As shown in Fig. 1, these sTHP-1 cells (panel B) differentiated into macrophage-like cells. Like parent THP-1 cells (panel A), they generated pseudopodia, but sTHP-1 cells could not accumulate lipids, even cultivated in the presence of Ac-LDL (10 g/ml). Under identical conditions, parent THP-1 cells accumulated and formed oil red-O-positive vacuoles (Fig.  1A). These vacuoles were rarely found in sTHP-1 cells (Fig. 1B). The accumulation of esterified cholesterol in both cells was determined after incubation with 25-500 g/ml Ac-LDL. In parent THP-1 cells, esterified cholesterol inside the cells significantly increased in a dose-dependent manner. In contrast, when sTHP-1 cells were treated with less than 100 g/ml of Ac-LDL, almost no esterified cholesterol was accumulated (Fig.  2). Parent THP-1 cells accumulated about 2.5-fold more esterified cholesterol than sTHP-1 cells even in the presence of extraordinary high concentration of Ac-LDL (500 g/ml) (Fig.  2). During the proliferative stage, the doubling time of sTHP-1 cells was identical to that of parent THP-1 cells (data not shown).
Determination of ScR Activity-To determine whether the difference in accumulation of esterified cholesterol between the two cell types was directly due to the function of ScR, the binding and degradation activities of 125 I-labeled Ac-LDL were measured (Fig. 3, A and B). In parent THP-1 cells, the binding and degradation activities of labeled Ac-LDL increased after the addition of TPA. However, the cell-associated and degraded Ac-LDL were almost undetectable in sTHP-1 cells even after 5-day treatment with TPA. These data suggest that sTHP-1 cells had some defects in ScR activity.
Expression of ScR, LPL, and ApoE mRNAs-To examine if the defect of ScR activity in sTHP-1 cells was at the transcriptional level, the expression of ScR mRNA was determined. In differentiated parent THP-1 cells, the ScR mRNA was detected after one-day treatment with TPA, and increased thereafter (Fig. 4A). On the other hand, in sTHP-1 cells, ScR mRNA was not detected even after 5-day cultivation. The expression of mRNAs of LPL and apoE, which is reported to be induced after differentiation into macrophages, was also determined. While parent THP-1 cells increased the mRNA level of LPL by the TPA treatment, sTHP-1 cells showed little expression even after a 5-day incubation with TPA (Fig. 4B). After the TPA treatment, the induction of apoE mRNA was faster and greater in sTHP-1 cells than in parent THP-1 cells (Fig. 4C). The induction of apoE expression supports the theory that sTHP-1 cells were differentiated into macrophages by the TPA treatment.
Effect of sTHP-1-conditioned Medium (sT-CM) on the ScR Activity in Parent THP-1 Cells-In sTHP-1 cells, the induction of not only ScR mRNA but also LPL mRNA was suppressed; at the same time, however, induction of apoE was not suppressed FIG. 1. Micrographs of differentiated parent THP-1 cells (A) and sTHP-1 cells. On day 0, 3 ϫ 10 4 cells/mm 2 were seeded and cultivated on cover glass with 0.3 ml of DMEM containing 10% (v/v) fetal calf serum and 50 ng/ml TPA (differentiation medium). On day 3, the cells were washed with PBS, and the medium was replaced with fresh differentiation medium containing 10% LPDS instead of fetal calf serum and supplemented with 10 g/ml Ac-LDL. On day 4, the cells were fixed and stained with Mayer's hematoxylin and oil red-O as described under "Experimental Procedures."

FIG. 2. Accumulation of esterified cholesterol in parent THP-1 and sTHP-1 cells cultivated in the presence of Ac-LDL.
On day 0, 1.5 ϫ 10 7 cells were seeded into 10-cm plastic dishes and cultivated in 6 ml of the differentiation medium containing 10% LPDS. On day 4, the medium was replaced with a fresh one supplemented with 0 -500 g/ml Ac-LDL. On day 6, the cells were harvested, and esterified cholesterol was determined. Each point is the mean of two samples.

FIG. 3. Time courses of the Ac-LDL binding (A) and degradation (B) activities of parent THP-1 and sTHP-1 cells after the addition of TPA.
On day 0, 5 ϫ 10 6 cells were seeded into 6-cm plastic dishes and cultivated in 3 ml of the differentiation medium. After cultivation for the indicated time periods, the cells were assayed for their ability to bind and degrade 125 I-labeled Ac-LDL. Each point is the mean of three samples.

FIG. 4. Northern blot analyses of ScR (A), LPL (B), and ApoE (C) in parent THP-1 and sTHP-1 cells.
On day 0, 1.5 ϫ 10 7 cells were seeded into 10-cm plastic dishes and cultivated in the differentiation medium for the indicated time periods. Total RNA (20 g) was electrophoresed in 0.9% agarose in the presence of 2.2 M formaldehyde, transferred onto nylon membrane, and hybridized with 32 P-labeled probes. Each membrane was rehybridized with human GAPDH probe as an internal standard. by the TPA treatment. Thus, we expected that the phenotype of sTHP-1 cells that lack in ScR activity might be epigenetic. We first tested if the culture medium obtained from sTHP-1 cells treated with TPA (sT-CM) might have some effects on the expression of ScR activity in parent THP-1 cells. Expectedly, the sT-CM obtained after 24 and 48 h of cultivation in the presence of TPA inhibited the ScR activity in parent THP-1 cells 63 and 94%, respectively (Fig. 5). The sT-CM without TPA treatment showed almost no effect on ScR activity in parent THP-1 cells. This inhibitory effect was not directly on the binding of Ac-LDL to ScR, because the measurement of ScR binding activity was not affected with the sT-CM (data not shown). The sT-CM suppressed the transcript level of ScR mRNA in differentiated parent THP-1 cells to 31.8% of that of the control level. The suppression of the ScR activity in parent THP-1 cells was at least at the transcriptional level (Fig. 6A). Similarly, the sT-CM suppressed LPL mRNA expression in differentiated parent THP-1cells (Fig. 6B). These results suggest that the component(s) that was secreted into the medium from the differentiated sTHP-1 cells, was responsible for the observed suppression in parent THP-1 cells.
Measurement of Cytokines in the Conditioned Medium-To identify which component(s) in the sT-CM was responsible for the suppression of the ScR and LPL expression, the concentration of three cytokines, TGF-␤1, TNF-␣, and IFN-␥, which were reported to affect the expression of ScR and LPL, were determined (20, 22, 24, 46 -49). While the culture media from un-differentiated sTHP-1 cells and differentiated and undifferentiated parent cells contained below the detection level of TGF-␤1, those of differentiated sTHP-1 cells obtained after incubation with TPA for 24 and 48 h contained 3.9 and 6.7 ng/ml TGF-␤1, respectively (Table I). Because there is no difference in concentration of TNF-␣ or IFN-␥ in the culture media from parent THP-1 and sTHP-1, TGF-␤1 was the candidate for the factor in the sT-CM that inhibits the expression of ScR and LPL mRNAs in parent THP-1 cells.
Neutralizing Effect of Anti-TGF-␤1 Antibodies on the Inhibitory Effect of sT-CM on ScR Expression-The sT-CM was pretreated with anti-TGF-␤1 antibody bound to protein G-Sepharose. The treated sT-CM, which contained TGF-␤1 below the detection level, lost the inhibitory effect on that ScR activity in differentiated parent THP-1 cells, and the ScR activity was at a level similar to that of the cells treated without sT-CM (Fig.  7). The sT-CM treated with control antibodies suppressed ScR activity to the same extent as that without the antibody treatment. The data suggest that TGF-␤1 significantly secreted from the differentiated sTHP-1 cells is a major factor that suppresses the expression of ScR activity in parent THP-1 cells. Thus, it was strongly suggested that the negligible level of ScR expression in sTHP-1 cells was due to the increased level of TGF-␤1 produced by own cells.
Expression of TGF-␤1 mRNA in sTHP-1 and the Parent THP-1 Cells-TGF-␤1 mRNA expression was determined in parent THP-1 and sTHP-1 cells. Before the TPA treatment, neither type of cells expressed TGF-␤1 mRNA. After TPA treatment, both types of cells started to express TGF-␤1 mRNA (Fig.  8). The expression profiles of TGF-␤1 mRNA in the two cell types were similar.

TABLE I
The concentration of TGF-␤1, TNF-␣, and IFN-␥ in the conditioned medium obtained from the parent THP-1 cells and sTHP-1 with or without TPA treatment The conditioned medium was obtained from the cells with TPA (50 ng/ml) treatment for 24 -48 h (TPA(ϩ)) or without TPA treatment for 48 h (TPA(Ϫ)). Each medium was subjected to measurement of the concentration of each cytokine using an enzyme-linked immunosorbent assay. TGF-␤1 is known to be synthesized as a precursor protein (about 48 kDa) that attaches to LTBP (about 195 kDa) to form the functional secretory complex (45). To know which of these steps is responsible for the overproduction of TGF-␤1 in sTHP-1 cells, all cell proteins were radiolabeled, and then TGF-␤1 was immunoprecipitated with specific antibody. As shown in Fig. 9, four radiolabeled bands, 190, 70, 48, and 45 kDa were observed in sTHP-1 cells (lane 2). The 190-kDa protein band, thought to be LTBP (45), was seen in sTHP-1 cells but not in parent THP-1 cells (compare lanes 1 and 2).

DISCUSSION
ScR are able to bind and internalize modified lipoproteins, such as acetylated and oxidized LDL. Different from LDL receptor, the ScR activity is not regulated by the intracellular concentration of cholesterol. Therefore, the continuous uptake of modified LDL leads to great accumulation of cholesterol into the cells. It has been reported that nonmacrophage cells, transfected with the cDNA encoding ScR, accumulate cholesterol and become foam cells (50). ScR are expected to play an important role in the foam cell formation.
THP-1 cells possess many of the characteristics of monocytemacrophages, especially in the lipid metabolism. They begin to express ScR and secrete greater amounts of apoE and LPL (14 -18). For these reasons, THP-1 cells have been used as a model for foam cell formation (51)(52)(53). In the present study, we identified a subtype of THP-1, sTHP-1 cells, which demonstrated negligible amounts of ScR activity, even after the differentiation. Consequently, the cells failed to become foam cells by cultivation with Ac-LDL at a relatively low concentration. The lack of ScR activity in sTHP-1 cells was due to the suppression of the expression of ScR mRNA. The lack of ScR activity in sTHP-1 cells may not be due to dedifferentiation of the cells, because 1) the doubling time of sTHP-1 cells was similar to those of parent cells, and 2) apoE mRNA, which is a differentiation marker for macrophages, was induced to express in sTHP-1 cells with TPA treatment. A loss of regulation in the expression of these genes was indicated to be the cause for acquired new phenotypes of sTHP-1 cells. As expected, the sT-CM inhibited the ScR activity of parent THP-1 cells. The inhibitory effect of the conditioned medium on the induction of ScR activity depended on the cultivation time of the differentiated sTHP-1 cells. These results supported the hypothesis that factor(s) that caused the suppression of ScR mRNA in the parent THP-1 cells had been secreted from the differentiated sTHP-1 cells.
Macrophages have been reported to produce and secrete a large variety of cytokines, which are known to modulate the macrophage functions and cell marker expression (54). We considered the possibility that there was a factor that could change the expression of ScR and LPL in sTHP-1 cells. First, we measured the concentrations of cytokines in the culture medium of sTHP-1 cells. TGF-␤1, TNF-␣, and IFN-␥ are known to suppress the expression of both ScR and LPL mRNAs (22,24,48,55,56,58). The concentrations of TGF-␤1 in culture media of sTHP-1 cells (sT-CM) and parent THP-1 cells were quite different. The sT-CM contained significantly higher concentrations of TGF-␤1 compared with that of parent cells. TGF-␤1 in the sT-CM increased in a time-dependent manner, which was parallel to the ability to inhibit the ScR activity (see Fig. 5). The treatment of sT-CM with anti-TGF-␤1 antibodies abolished the inhibitory effect of the sT-CM on ScR activity of differentiated THP-1 cells. The results clearly indicate that the component that inhibited the ScR activity was TGF-␤1, suggesting that sTHP-1 cells had acquired the ability to produce TGF-␤1 in the presence of TPA.
TGF-␤1 is reported to decrease the expression of ScR mRNA in THP-1 cells (24) and LPL mRNA in preadipocytes (22) and to increase apoE mRNA in macrophages (20). The present results agree with the findings in previous reports and show that the TGF-␤1 secreted from sTHP-1 cells suppressed the transcript levels of ScR and LPL mRNAs and increased the level of ApoE mRNA. Due to the acquisition of an ability to secrete TGF-␤1, sTHP-1 cells lost their ability to accumulate cholesterol.
Bottalico et al. (24) reported that 24 pM TGF-␤1 has an ability to inhibit 70% of the uptake activity of Ac-LDL through FIG. 8. Northern blot analysis of TGF-␤1 in parent THP-1 and sTHP-1 cells. On day 0, 1.5 ϫ 10 7 cells were seeded into 10-cm plastic dishes and cultivated in the differentiation medium for indicated time periods. Total RNA (20 g) was electrophoresed in 0.9% agarose in the presence of 2.2 M formaldehyde, transferred onto nylon membrane, and hybridized with 32 P-labeled probes. The membrane was rehybridized with human GAPDH probe as an internal standard.
ScR in the differentiated THP-1 cells. The concentration of TGF-␤1 required to acquire maximal suppression of ScR activity in THP-1 cells was 10 times higher in the present study than that reported by Bottalico et al. (24). In the present study, the concentration of TGF-␤1 in the sT-CM after 24 and 48 h of treatment with TPA was 3.9 ng/ml, which is about 160 pM, and 6.7 ng/ml, which is about 270 pM, respectively. These concentrations of TGF-␤1 inhibited 63 and 94% of the uptake activities of Ac-LDL by parent THP-1 cells, respectively (Fig. 5). TGF-␤1 is known to be synthesized and secreted as latent complex from the producer cells. The latent TGF-␤1 complex in human and rat platelets is composed of three different components: mature TGF-␤1, the TGF-␤1 latency-associated peptide, and the LTBP (57,59,60). The latent TGF-␤1 is biologically inactive, and the activation of the latent complexes is an important step for the regulation of TGF-␤1 activity. The Nterminal remnant of the TGF-␤1 precursor is sufficient for TGF-␤1 latency. TGF-␤1 in this latent complex can be activated in vitro by treatment with acid, urea, or heat (59). It is reasonable to speculate that most of the TGF-␤1 in the culture medium of sTHP-1 cells might be in the latent form. Since TGF-␤1 concentration was measured by enzyme-linked immunosorbent assay in acidic conditions, both latent and active forms of TGF-␤1 were included in the measurements. Therefore, a higher concentration of TGF-␤1 was necessary for the maximal inhibition of the ScR activity in our assay system.
In our study, TGF-␤1 was not detected in the culture medium of parent THP-1 cells, even after stimulation of TPA. The parent THP-1 cells showed almost as much TGF-␤1 mRNA as did sTHP-1 cells (Fig. 8). In sTHP-1 cells, considerable amounts of LTBP were immunoprecipitated with anti-TGF-␤1, suggesting that LTBP was forming a functional complex with TGF-␤1 precursor only in sTHP-1 cells. This might explain the high concentration of TGF-␤1 in the culture media of sTHP-1 cells, since LTBP is reported to play a crucial role in the secretion of TGF-␤1 (45). An increased rate of formation of the TGF-␤1-LTBP complex could be explained by either enhanced production of LTBP in sTHP-1 cells or a defect in complex formation in parent THP-1 cells. Since sTHP-1 cells are derived from THP-1 cells, it is reasonable to suppose that a defect(s), if it exists, should occur in sTHP-1 cells rather than in parent THP-1 cells. Therefore, a defect in sTHP-1 cells could possibly be the failure to suppress either the synthesis of LTBP or the formation of a functional secretion complex with LTBP. The latter possibility may be tested when a specific antibody against LTBP becomes available. sTHP-1 cells will be a useful model for investigating the regulation of secretion of TGF-␤1.