The role of mitogen-activated protein kinases and sterol receptor coactivator-1 in TGF-β-regulated expression of genes implicated in macrophage cholesterol uptake

Abstract

The anti-atherogenic cytokine TGF-β inhibits macrophage foam cell formation by suppressing the expression of key genes implicated in the uptake of modified lipoproteins. We have previously shown a critical role for p38 MAPK and JNK in the TGF-β-mediated regulation of apolipoprotein E expression in human monocytes. However, the roles of these two MAPK pathways in the control of expression of key genes involved in the uptake of modified lipoproteins in human macrophages is poorly understood and formed the focus of this study. TGF-β activated both p38 MAPK and JNK, and knockdown of p38 MAPK or c-Jun, a key downstream target of JNK action, demonstrated their requirement in the TGF-β-inhibited expression of several key genes implicated in macrophage lipoprotein uptake. The potential role of c-Jun and specific co-activators in the action of TGF-β was investigated further by studies on the lipoprotein lipase gene. c-Jun did not directly interact with the minimal promoter region containing the TGF-β response elements and a combination of transient transfection and knock down assays revealed an important role for SRC-1. These studies provide novel insights into the mechanisms underlying the TGF-β-mediated inhibition of macrophage gene expression associated with the control of cholesterol homeostasis.

Figure 1. siRNA-mediated knockdown of p38 MAPK or c-Jun in THP-1 macrophages.

THP-1 monocytes were transfected with the indicated siRNA, differentiated into macrophage using PMA for 24 h, and then incubated with vehicle (−, empty bars) or TGF-β (30 ng/ml) (+, filled bars) for 24 h as described in Materials and Methods. Equal amounts of protein extracts were subjected to western blot analysis using antisera against p38 MAPK, c-Jun or β-actin. The image shows the signal from the immunoreactive p38 MAPK (43 kDa), c-Jun (39 kDa) or β-actin (42 kDa). Protein expression of p38 MAPK or c-Jun was normalized to β-actin and is shown as the fold change relative to GAPDH siRNA-transfected cells (arbitrarily assigned as 1). The data represent mean ± SD of three independent experiments. Statistical analysis was performed using the two-tailed unpaired Student’s t-test, *p < 0.05, **p < 0.01.

Figure 2. p38 MAPK and c-Jun are involved in the TGF-β-regulated expression of key cholesterol uptake genes in human macrophages.

Knockdown of GAPDH or p38 MAPK or c-Jun expression in THP-1 monocytes, differentiation of monocytes into macrophages and incubation with vehicle (−, empty bars) or TGF-β (30 ng/ml) (+, filled bars) for 24 h was carried out as in Fig. 1. Total RNA was subjected to RT-qPCR using primers against CD36, SR-B1, SR-A1, LPL or RPL13A. The mRNA expression levels were determined using the comparative C_t method and normalized to RPL13A with the value from vehicle treated cells arbitrarily assigned as 1. The data represent mean ± SD of three independent experiments. Statistical analysis was performed using the two-tailed unpaired Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. The TGF-β-mediated inhibition of minimal LPL promoter activity in human macrophages is attenuated by transfection of plasmids specifying for DN forms of JNK, SEK-1 and c-Jun.

(A) Schematic representation of the regulatory region of the LPL gene identified in a previous study. The −101 to +187 region linked to the luciferase reporter gene (Luc) is shown. The −31 to +187 sequence contains the TGF-β response elements (TGF-β RE) with three conserved Sp1/Sp3 binding sites required for the response (a single site at position +44 and a dual site at position +62/+65). The +9/+49 and +46/+90 sequences used for EMSA (Fig. 4) are also shown. (B) U937 cells were transfected with the minimal LPL promoter-luciferase construct (−101/+187 in the pGL2 Basic-luciferase vector) and DN JNK, DNSEK-1, DN c-Jun or pcDNA3 control vector. The cells were then differentiated with PMA (1 μM) for 12 h and then treated with vehicle (−, empty bars) or TGF-β (30 ng/ml) for further 12 h (+, filled bars). The luciferase activity was normalized to the protein concentration and is expressed as Relative Luciferase Activity. In each case, the value in cells treated with vehicle has been arbitrarily assigned as 100%. The data represent mean ± SD from three independent experiments. Statistical analysis was performed using the two-tailed unpaired Student’s t-test, *p < 0.05.

Figure 4. AP-1 does not interact with the TGF-β response element in the regulatory region of the LPL gene.

EMSA were carried out using radiolabelled +9/+49 or +46/+90 sequence and whole cell extracts from THP-1 macrophages that were either untreated (0 h) or incubated with 30 ng/ml of TGF-β for 24 h (24 h). +Indicates competition with a 400-fold molar excess of unlabelled complementary oligonucleotide (self), Sp1/Sp3 binding site (Sp1/Sp3) or AP-1 binding site (AP-1). The DNA-protein interactions with the +9/+49 and the +46/+90 sequences have been previously characterized. The region of the autoradiogram containing the Sp1/Sp3 DNA protein complexes (indicated by a vertical line) is shown. The data are representative of three independent experiments.

Figure 5. The TGF-β-mediated inhibition of minimal LPL promoter activity is attenuated by transfection of SRC-1 expression plasmid but not that for p300/CBP.

U937 cells were transfected with the minimal LPL promoter-luciferase construct (−101/+187 in the pGL2 Basic-luciferase vector) and pcDNA3 control vector (pcDNA3) or p300/CBP expression plasmid (p300) (1.5 μg) (A) or SRC-1 expression plasmid (SRC-1) (1.5 and 3.0 μg as indicated) (B). The cells were then differentiated with PMA (1 μM) for 12 h and then either treated with vehicle (−, empty bars) or TGF-β (30 ng/ml) for further 12 h (+, filled bars). The luciferase activity was normalized to the protein concentration and is expressed as Relative Luciferase Activity with the value in cells transfected with the control pcDNA3 plasmid and treated with vehicle arbitrarily assigned as 100%. The data represent mean ± SD from three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6. The TGF-β-mediated inhibition of activation by multiple Sp1/Sp3 sites is attenuated by transfection of SRC-1 expression plasmid.

U937 cells were transfected with the an artificial promoter containing four copies of Sp1/Sp3 site at +62/+65 [Sp1 (62/65)x4] (A) or +44 [Sp1(44)x4 (B)] from the TGF-β response element in the regulatory region of the LPL gene (Fig. 3A) linked to the minimal SV40 promoter, and pcDNA3 control vector (pcDNA3) or SRC-1 expression plasmid (1.5 and 3 μg as indicated). The cells were then differentiated with PMA (1 μM) for 12 h and then incubated with vehicle (−, empty bars) or TGF-β (30 ng/ml) for further 12 h (+, filled bars). The luciferase activity was normalized to the protein concentration and is expressed as Relative Luciferase Activity with the value in cells transfected with the control pcDNA3 plasmid and treated with vehicle arbitrarily assigned as 100%. The data represent mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukeys post-hoc test, *p < 0.05, **p < 0.01.

Figure 7. Knockdown of SRC-1 affects LPL gene expression.

Knockdown of SRC-1 expression in THP-1 monocytes, differentiation of monocytes into macrophages and incubation with vehicle (−, empty bars) or TGF-β (30 ng/ml) (+, filled bars) for 24 h was carried out as in Fig. 1. (A) Equal amounts of protein extracts were subjected to western blot analysis using antisera against SRC-1 or β-actin. The image shows the signal from the immunoreactive SRC-1 (160 kDa) or β-actin (42 kDa). The histogram below the image shows protein expression of SRC-1 normalized to β-actin and is expressed as a fold change relative to GAPDH siRNA-transfected cells (arbitrarily assigned as 1). The data represent mean ± SD of three independent experiments. Statistical analysis was performed using the Student’s t-test, *p < 0.05. (B) RT-qPCR on total RNA was carried out using primers against LPL or RPL13A. The mRNA expression levels were determined using the comparative C_t method and normalized to RPL13A. The value in cells transfected with GAPDH siRNA and treated with vehicle was arbitrarily assigned as 1. The data represent mean ± SD of five independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test, ***p < 0.001.