FHL2 protein is a novel co-repressor of nuclear receptor Nur77

Abstract

The three members of the NR4A orphan nuclear receptor subfamily Nur77, Nurr1, and NOR-1, regulate a variety of biological functions including vascular disease and metabolism. In this study, we identified Four and a half LIM domains protein-2 (FHL2) as a novel interacting protein of NR4A nuclear receptors by yeast two-hybrid screen and co-immunoprecipitation studies. Each of the four LIM domains of FHL2 can bind Nur77, and both the amino-terminal domain and the DNA binding domain of Nur77 are involved in the interaction between FHL2 and Nur77. FHL2 represses Nur77 transcriptional activity in a dose-dependent manner, and short hairpin RNA-mediated knockdown of FHL2 results in increased Nur77 transcriptional activity. ChIP experiments on the enolase3 promoter revealed that FHL2 inhibits the association of Nur77 with DNA. FHL2 is highly expressed in human endothelial and smooth muscle cells, but not in monocytes or macrophages. To substantiate functional involvement of FHL2 in smooth muscle cell physiology, we demonstrated that FHL2 overexpression increases the growth of these cells, whereas FHL2 knockdown results in reduced DNA synthesis. Collectively, these studies suggest that association of FHL2 with Nur77 plays a pivotal role in vascular disease.

FIGURE 1.

FHL2 interacts with Nur77. A, HEK293T cells were transfected with expression vectors encoding HA-tagged FHL2 and Myc-tagged Nur77, as indicated. Whole cell extracts were immunoprecipitated for FHL2 with an anti-HA antibody (IP: FHL2), and the bound protein was analyzed by Western blotting using anti-Myc antibody (WB: Nur77). 10% of the cell lysate was used as input. Data are representative of at least three independent experiments. B, in vitro transcribed/translated Nur77 was incubated with GST-FHL2 fusion protein, and binding of Nur77 to FHL2 was analyzed by Western blotting. GST-DCOH protein was used as a control. 10% of the cell lysate was used as input. Data are representative of at least two independent experiments. C, schematic represents full-length FHL2 protein. D, HEK293T cells were co-transfected with expression vectors encoding Nur77 and FHL2-mutants, as indicated. Whole cell extracts were prepared and immunoprecipitated with anti-HA antibody (IP: LIMs). Immunoprecipitated samples were resolved on 12% SDS-PAGE and analyzed by Western blotting with anti-Myc antibody (WB: Nur77). The input represents 10% of the total cell lysate. Data are representative of at least three independent experiments. Protein molecular mass markers (M) are shown in kilodaltons (kDa).

FIGURE 2.

The amino-terminal domain and DBD of Nur77 interact with FHL2. A, schematic diagrams Nur77 deletion mutants and their ability to interact with FHL2. B, HEK 293T cells were co-transfected with expression vectors encoding FHL2 and Nur77 domains, as indicated. Whole cell extracts were immunoprecipitated using the anti-HA antibody (IP: FHL2). Immunoprecipitated samples were resolved on 12% SDS-PAGE and analyzed by Western blotting (WB) with anti-Myc antibody (WB: Nur77). C, HEK293T cells were transfected with the amino-terminal domain of Nur77 (N-Term) without or with FHL2. Prior to lysis the cells were treated with proteasomal inhibitor MG-132 for 8 h. Whole cell extracts were incubated with anti-Myc antibody (IP: N-Term), subjected to SDS-PAGE, and analyzed by Western blotting with anti-HA antibody (WB: FHL2). The input represents 10% of the total cell lysate in all co-immunoprecipitation experiments. Data are representative of at least three independent cell culture experiments. Protein molecular mass marker is shown in kilodaltons (kDa).

FIGURE 3.

FHL2 is a co-repressor of Nur77. The activity of Nur77 was monitored either with the NBRE-luc or with the NurRE-luc plasmid. A and B, in HEK293T cells FHL2 inhibits the activity of Nur77 on NurRE (A) and the NBRE (B) luciferase reporters. C, FHL2 also inhibits the transcriptional activity of Nur77 in SMCs. SMCs were transduced with lentivirus encoding Nur77 and FHL2, and the cells were subsequently transfected with reporter plasmid. D, effect of distinct FHL2 mutants on Nur77 activity was assayed, showing that each LIM domain gives full inhibition of Nur77 activity, except LIM0, which contains only half an LIM domain. In all luciferase experiments, the transfection efficiency was normalized using Renilla luciferase, which was co-transfected with the reporter plasmids. All experiments were performed in triplicate and repeated at least three times. Values represent mean ± S.D. (error bars). *, p ≤ 0.05.

FIGURE 4.

FHL2 regulates Nur77 transcriptional activity. A and B, human SMCs were transduced with lentiviruses encoding shRNA targeting FHL2 (shFHL2#1 and #2) or a control shRNA (shCon). After 72 h of transduction, semiquantitative RT-PCR for FHL2 expression (A) and Western blot analysis for FHL2 protein (B) were performed. Tubulin was used as a loading control. C, human SMCs were transduced with recombinant lentiviral particles as indicated and incubated for 4 days. Then cells were transfected with NurRE and luciferase activity measured after 36 h. The total amount of DNA was kept constant by adding empty vector. The transfection efficiency was normalized using Renilla luciferase. Values represent mean ± S.D. (error bars). *, p ≤ 0.05. D, human SMCs were transduced with recombinant lentiviral particles as indicated and incubated for 4 days. The expression of enolase3, which is a known target gene of Nur77, was analyzed by real-time RT-PCR. Acidic ribosomal phosphoprotein P0 was used as an internal control for cDNA content of the samples. E, ChIP analyses were performed with enolase3 promoter-specific PCR primers and Nur77-specific antibodies. In the left panel PCR products after agarose gel electrophoresis are shown, and in the right panel are the data from semiquantitative PCR, with the appropriate controls. Data are representative of at least two independent experiments in two different batches of SMCs. Values represent mean ± S.D. *, p ≤ 0.05.

FIGURE 5.

Knockdown of FHL2 decreases SMC proliferation. A, semiquantitative RT-PCR was performed to assess expression of FHL2 mRNA in mouse heart, human SMCs and endothelial cells, in THP-1 monocytes and macrophages, and mouse bone marrow-derived macrophages. Acidic ribosomal phosphoprotein P0 was used as an internal control for cDNA content of the samples. Values represent mean ± S.D. (error bars), and data are representative of at least two independent experiments. B–D, recombinant lentiviral particles encoding Nur77 and FHL2 (B), shFHL2 (C), or shNur77 (D) were transduced in SMCs. After serum stimulation, cells were pulse-labeled with BrdU to measure DNA synthesis. All experiments were performed in quadruplicate and repeated at least four times. Values represent mean ± S.D. E, semiquantitative RT-PCR was performed to assess knockdown of Nur77 in SMCs using shRNA. Values represent mean ± S.D. *, p ≤ 0.05.