Pleiotrophin (PTN) is expressed in vascularized human atherosclerotic plaques: IFN-{gamma}/JAK/STAT1 signaling is critical for the expression of PTN in macrophages

Abstract

Neovascularization is critical to destabilization of atheroma. We previously reported that the angiogenic growth factor pleiotrophin (PTN) coaxes monocytes to assume the phenotype of functional endothelial cells in vitro and in vivo. In this study we show that PTN expression is colocalized with capillaries of human atherosclerotic plaques. Among the various reagents that are critical to the pathogenesis of atherosclerosis, interferon (IFN)-gamma was found to markedly induce PTN mRNA expression in a dose-dependent manner in macrophages. Mechanistic studies revealed that the Janus kinase inhibitors, WHI-P154 and ATA, efficiently blocked STAT1 phosphorylation in a concentration- and time-dependent manner. Notably, the level of phosphorylated STAT1 was found to correlate directly with the PTN mRNA levels. In addition, STAT1/STAT3/p44/42 signaling molecules were found to be phosphorylated by IFN-gamma in macrophages, and they were translocated into the nucleus. Further, PTN promoter analysis showed that a gamma-activated sequence (GAS) located at -2086 to -2078 bp is essential for IFN-gamma-regulated promoter activity. Moreover, electrophoretic mobility shift, supershift, and chromatin immunoprecipitation analyses revealed that both STAT1 and STAT3 bind to the GAS at the chromatin level in the IFN-gamma stimulated cells. Finally, to test whether the combined effect of STAT1/STAT3/p44/42 signaling is required for the expression of PTN in macrophages, gene knockdowns of these transcription factors were performed using siRNA. Cells lacking STAT1, but not STAT3 or p42, have markedly reduced PTN mRNA levels. These data suggest that PTN expression in the human plaques may be in part regulated by IFN-gamma and that PTN is involved in the adaptive immunity.-Li, F., Tian, F., Wang, L., Williamson, I. K., Sharifi, B. G., Shah, P. K. Pleiotrophin (PTN) is expressed in vascularized human atherosclerotic plaques: IFN-gamma/JAK/STAT1 signaling is critical for the expression of PTN in macrophages.

Figure 1.

Representative photomicrographs of PTN expression in advanced human atherosclerotic plaques. A) Advanced human plaques were stained with Movat using standard protocol. B) Brown PTN staining (immunocytochemistry) is associated with microvessels in the intima and at the plaque base (×10). C) Higher magnification (×40) of the intimal area, showing expression of PTN associated with the microvessels and with the inflammatory cells in the plaque. D) Immunostaining using an anti-CD68 antibody (for macrophages; ×20). E) In situ hybridization using an antisense PTN riboprobe (×40).

Figure 2.

Up-regulation of PTN by IFN-γ in THP-1 cells and mouse peritoneal macrophages. A) THP-1 cells were treated with IFN-γ, LPS, M-CSF, and TNF-α for 48 h, and PTN mRNA expression was quantified by real-time PCR. B) Western blotting analysis of PTN protein secreted in the culture medium of IFN-γ-treated THP-1 cells (50 ng/ml for 48 h). C) Real-time PCR analysis of PTN mRNA induction in mouse peritoneal macrophages by IFN-γ (10 ng/ml). Values were normalized against GAPDH mRNA. D) Time course of IFN-γ induction of PTN mRNA expression by THP-1 cells. E) Dose-dependent IFN-γ induction of PTN mRNA expression by THP-1 cells. Cells were treated for 3 h. In all experiments, untreated cells were used as a control.

Figure 3.

IFN-γ up-regulates PTN expression by activating JAK/STAT signal pathway. A, B) THP-1 cells preincubated with the indicated concentrations of JAK inhibitors, WHI-P154 (A) or ATA (B), for 1 h, followed by IFN-γ treatment for 10 min in the presence of each inhibitor. Cell lysates were probed with anti-STAT1 (Tyr 701) antibody by Western blotting. Untreated THP-1 cells and those preincubated with increasing concentrations of the inhibitors for 1 h were further treated with IFN-γ for 2 h. C, D) PTN mRNA levels were quantified by real-time RT-PCR and normalized against GAPDH mRNA. E) THP-1 cells were treated with IFN-γ (50 ng/ml) for the indicated times, and phosphorylation levels of STAT1, STAT3, STAT6, and p44/42 were determined by Western blot.

Figure 4.

Immunofluorescence analysis of nuclear translocation of activated STAT1, STAT3, and p44/42. Immunofluorescence staining of THP-1 cells was performed using a mouse monoclonal anti-STAT1 (A), a rabbit anti-STAT3 (B), and a mouse monoclonal anti-p44/42 MAPK (C). Activated STAT1, STAT3, and p44/42 translocated to the nuclei on IFN-γ stimulation. Red fluorescence shows STAT1 and p44/42 localization (A, C); pink in the merged images shows colocalization of activated STAT1, p44/42 in the nuclei (A, C); green fluorescence shows STAT3 localization (B); blue shows DAPI nuclear stain (all panels).

Figure 5.

Human PTN promoter contains an IFN-γ activated element that interacts with STAT1. A) Schematic representation of human PTN promoter luciferase reporter constructs used in the transient transfection of THP-1 cells, showing position of the IFN-γ activated element GAS. PTN promoter luciferase constructs were cotransfected with an internal control vector, pRL-β-actin. Results are means ± sd of 3 independent experiments performed in triplicates. Asterisk indicates statistically significant difference in luciferase activity. B) EMSA assay showing the binding of THP-1 nuclear extracts to wtGAS following 5-, 10-, 20-, 30-, and 60-min IFN-γ treatments. C) Specificity of binding; 50 (+)- and 100 (++)-fold molar excess of unlabeled probe wtGAS (lane 4 and 5), but not mutant probe mtGAS (lanes 6 and 7), inhibited formation of the nuclear extract/probe complex in the presence of biotin-labeled wtGAS (50 fmol) D) Supershift assay with 1.0 and 2.0 μg (lanes 2 and3, respectively) of an anti-STAT1 monoclonal antibody; unrelated monoclonal antibody had no effect on the nuclear extract/probe complex (lane 4).

Figure 6.

PCR analysis of chromatin immunoprecipitation. Chromatin from IFN-γ treated THP-1 cells was immunoprecipited with antibodies against STAT1, STAT3, p44/42, and normal mouse IgG. After elution of protein-DNA complexes from protein G magnetic beads and reversal of the cross-link, purified DNA was used for PCR analysis and quantification using a densitometer. A) Schematic diagram of PTN promoter. Arrows indicate locations of the primer pairs that cover the GAS motif. B, C) Fold enrichment of PCR products from each antibody, as quantified by densitometery from independent experiments. Data are expressed as the ratio of each antibody signal to IgG signal, calculated by normalization against input chromatin. Anti-STAT1 and anti-STAT3 antibodies markedly enriched PTN promoter DNA with both primer pairs [8.4- and 7.3-fold with primer pair 1 (B); 35- and 29.5-fold with primer pair 2 (C)], but substantially less was detected in the anti-p44/42 and normal IgG control.

Figure 7.

Analysis of siRNA knockdown of STAT1, STAT3, and p42 gene at protein levels and their effects on IFN-γ induced up-regulation of PTN mRNA expression. A) THP-1 cells were transfected with STAT1, STAT3, and p42 siRNAs. At 24 h posttransfection, cells were treated with IFN-γ (50 ng/ml for 3 h), and Western blotting confirmed the knockdown of the expression level of each protein. B) Densitometry of STAT1, STAT3 and p42 levels, normalized against GAPDH. C) Real-time PCR analysis of the effect of STAT1, STAT3, and p42 knockdown on IFN-γ induced PTN mRNA expression.