Galectin-9 protein expression in endothelial cells is positively regulated by histone deacetylase 3

Abstract

Galectin-9 expression in endothelial cells can be induced in response to inflammation. However, the mechanism of its expression remains unclear. In this study, we found that interferon-γ (IFN-γ) induced galectin-9 expression in human endothelial cells in a time-dependent manner, which coincided with the activation of histone deacetylase (HDAC). When endothelial cells were treated with the HDAC3 inhibitor, apicidin, or shRNA-HDAC3 knockdown, IFN-γ-induced galectin-9 expression was abolished. Overexpression of HDAC3 induced the interaction between phosphoinositol 3-kinase (PI3K) and IFN response factor 3 (IRF3), leading to IRF3 phosphorylation, nuclear translocation, and galectin-9 expression. HDAC3 functioned as a scaffold protein for PI3K/IRF3 interaction. In addition to galectin-9 expression, IFN-γ also induced galectin-9 location onto plasma membrane, which was HDAC3-independent. Importantly, HDAC3 was essential for the constitutive transcription of PI3K and IRF3, which might be responsible for the basal level of galectin-9 expression. The phosphorylation of IRF3 was essential for galectin-9 expression. This study provides new evidence that HDAC3 regulates galectin-9 expression in endothelial cells via interaction with PI3K-IRF3 signal pathway.

FIGURE 1.

IFN-γ induces galectin-9 expression in HUVECs at mRNA and protein levels. A and B, HUVECs were treated with 10 ng/ml IFN-γ for the time indicated, followed by quantitative RT-PCR analysis of galectin-9 mRNA (A) and Western blot analysis of galectin-9 protein (B, lower panel represents the relative galectin-9 protein level to GAPDH). *, p < 0.05; **, p < 0.01. C, HUVECs were treated with 10 ng/ml IFN-γ for 16 h, followed by immunofluorescent staining with galectin-9 antibody (green). DAPI was included to counterstain the nucleus (blue). PBS was used as vehicle control. IgG was included as primary antibody control of the staining. Scale bar, 5 μm. Note that the fluorescence intensity in IFN-γ-treated cells is significantly higher than untreated cells. Data presented are representative of mean ± S.E. (error bars) of three independent experiments.

FIGURE 2.

HDAC3 activity is essential for galectin-9 expression. A, IFN-γ increases HDAC activity. HUVECs were treated with 10 ng/ml IFN-γ for the time indicated, followed by HDAC activity assay. Arbitrary unit was defined as A_{420 nm}/μg of protein with untreated set as 1.0. *, p < 0.05. B–D, apicidin decreased the basal level and IFN-γ-induced galectin-9 expression. HUVECs were pretreated with 200 nmol/liter apicidin for 1 h, then treated with 10 ng/ml IFN-γ for 16 h in the presence of apicidin, followed by immunofluorescent staining (B: green, galectin-9; blue, DAPI; Scale bar, 5 μm), Western blot assay (C) and quantitative RT-PCR analysis (D) are shown. *, p < 0.05; ***, p < 0.0001. Data presented are representative of mean ± S.E. (error bars) of three independent experiments. DMSO, dimethyl sulfoxide.

FIGURE 3.

Knockdown of HDAC3 decreases basal level and IFN-γ-induced galectin-9 expression. A–C, knockdown of HDAC3 decreased basal level of galectin-9 expression. HUVECs were infected with nontarget (NTsh) or HDAC3 (HD3sh) shRNA lentiviruses at 1 × 10⁷ units/1 × 10⁶ cells for 72 h, followed by Western blotting (A), routine RT-PCR(B), and quantitative RT-PCR (C) assays. An arbitrary unit was defined as the ratio of target gene mRNA to 18 S RNA with that of NTsh set as 1.0. **, p < 0.01; ***, p < 0.0001. D and E, knockdown of HDAC3 abolishes IFN-γ-induced galectin-9 expression. HUVECs were infected with NTsh or HD3sh RNA at 1 × 10⁷ units/1 × 10⁶ cells for 48 h, then treated with 10 ng/ml IFN-γ for 16 h, followed by membrane fraction isolation and RNA extraction. HDAC3 and galectin-9 protein levels were detected in total cell lysate and membrane fractions by Western blotting (D), and mRNA was detected with quantitative RT-PCR (E). *, p < 0.05; ***, p < 0.0001. Data presented are representative of mean ± S.E. (error bars) of three independent experiments.

FIGURE 4.

Overexpression of HDAC3 increases galectin-9 expression. A–C, HUVECs were transfected with pShuttle2-FLAG-HDAC3 plasmid (pHDAC3, 2 μg/2 × 10⁶cells) via electroporation, followed by Western blotting (A), routine RT-PCR (B), and real time RT-PCR (C) assays 48 h after transfection. pShuttle2-FLAG vector was included as mock control. **, p < 0.01; ***, p < 0.0001. D–F, HUVECs were infected with Ad-FLAG-HDAC3 virus at the m.o.i. indicated, followed by Western blotting (D), routine RT-PCR (E), and real time RT-PCR (F) assays 48 h after infection. Ad-null virus was included as negative control and to compensate the m.o.i. *, p < 0.05; **, p < 0.01. -Fold of induction was defined as the ratio of target gene mRNA to 18 S RNA with that of control group set as 1.0. Data presented are representative of mean ± S.E. (error bars) of three independent experiments.

FIGURE 5.

HDAC3 is essential for IFN-γ-induced PI3K-IRF3 activation and galectin-9 expression. A and B, PI3K is crucial for IFN-γ-induced galectin-9 expression and IRF3 activation. HUVECs were pretreated with PI3K inhibitor LY294002 (5 μmol/liter) for 1 h, then treated with 10 ng/ml IFN-γ for 16 h in the presence of LY294002, followed by immunofluorescent staining (A: green, galectin-9; blue, DAPI; Scale bar, 5 μm) and Western blot assay (B). The same amount of dimethyl sulfoxide was included as vehicle control. p-IRF3 indicates phosphorylation of IRF3 at Ser-386. C–E, HDAC3 is crucial for basal level expression of PI3K and IRF3 and IFN-γ-induced IRF3 activation. HUVECs were infected with NTsh or HD3sh RNA for 48 h, then treated with 10 ng/ml IFN-γ for 16 h, followed by Western blot analysis (C, representative image; D, quantitative analysis) and quantitative RT-PCR assays (E). *, p < 0.05; **, p < 0.01. F, IRF3 phosphorylation is essential for galectin-9 expression. HUVECs were transfected with pcDNA3 (vector), pcDNA3-IRF3 (WT), or pcDNA3-IRF3S386A (S386A) plasmids for 48 h, followed by Western blot analysis of IRF3 phosphorylation and galectin-9 expression. Data presented are representative or average ± S.E. (error bars) of three independent experiments.

FIGURE 6.

IFN-γ enhances the HDAC3-p85α-IRF3 complex formation. A, overexpression of HDAC3 induced IRF3 nuclear translocation. HUVECs were infected with 10 m.o.i. Ad-HDAC3 virus for 48 h, followed by cellular fraction assay. Ad-null virus was included as control. C.E, cytosolic extract; N.E, nuclear extract. B, HDAC3 knockdown ablated IFN-γ-induced IRF3 nuclear translocation. HUVECs were infected with NTsh or HD3sh for 72 h, and then treated with 10 ng/ml IFN-γ for 4 h, followed by cellular fraction assay. Anti-tubulin and histone H4 antibodies were included to identify cytosol and nuclear fractions. C, IFN-γ induced the formation of the HDAC3-p85α-IRF3 complex. HUVECs were infected with Ad-FLAG-HDAC3 virus at 10 m.o.i. and 48 h later treated with 10 ng/ml IFN-γ for 4 h, followed by immunoprecipitation assay with anti-FLAG antibody. 50 μg of lysate was included as input. D, IFN-γ enhanced endogenous HDAC3 association with p85α-IRF3. HUVECs were treated with 10 ng/ml IFN-γ for 4 h, followed by immunoprecipitation assay with anti-HDAC3 antibody. E, HDAC3 activity is unnecessary for HDAC3-p85α-IRF3 complex formation. HUVECs were treated with 10 ng/ml IFN-γ for 4 h in the presence or absence of apicidin, followed by immunoprecipitation with anti-IRF3 antibody. Dimethyl sulfoxide was included as vehicle control.

FIGURE 7.

Schematic illustration of IFN-γ-mediated galectin-9 expression in HUVECs. In HUVECs, HDAC3 is involved in the constitutive expression of IRF3 and the p85α subunit of PI3K. Under IFN-γ treatment, HDAC3, PI3K, and IRF3 form a complex, leading to the phosphorylation of IRF3 at serine 386, which in turn activates galectin-9 expression.