Promyelocytic leukemia protein (PML) regulates endothelial cell network formation and migration in response to tumor necrosis factor α (TNFα) and interferon α (IFNα)

Abstract

Promyelocytic leukemia protein (PML) is a tumor suppressor that is highly expressed in vascular endothelium and inflamed tissues, yet its role in inflammation-associated cytokine-regulated angiogenesis and underlying mechanism remains largely unclear. We show that tumor necrosis factor α (TNFα) and interferon α (IFNα) stimulate PML expression while suppressing EC network formation and migration, two key events during angiogenesis. By a knockdown approach, we demonstrate that PML is indispensable for TNFα- and IFNα-mediated inhibition of EC network formation. We further demonstrate that signal transducer and activator of transcription 1 (STAT1) binds PML promoter and that is an important regulator of PML expression. Knockdown of STAT1 reduces endogenous PML and blocks TNFα- and IFNα-induced PML accumulation and relieves TNFα- and IFNα-mediated inhibition of EC network formation. Our data also indicate that PML regulates EC migration, in part, by modulating expression of downstream genes, such as negatively regulating integrin β1 (ITGB1). In addition, knockdown of STAT1 or PML alleviates TNFα- and IFNα-mediated inhibition of ITGB1 expression. Antibody blockade demonstrates that ITGB1 is functionally important for PML- and STAT1-regulated EC migration. Taken together, our data provide novel mechanistic insights that PML functions as a negative regulator in EC network formation and migration.

FIGURE 1.

Effects PML knockdown and TNFα on network formation in HUVECs and HMVECs. In vitro network formation in HUVECs (A–D) and HMVECs (E) following PML knockdown. HUVECs or HMVECs were transiently transfected with non-targeting or PML siRNAs for 72 h before the assays. Representative fields of HUVEC-formed networks (A, C) and branch points (B, D, and E) were quantified as described in “Experimental Procedures.” Mean ± S.D. (n = 12). An aliquot of cells was plated for Western blotting (A and E) to examine PML expression levels. Endogenous PML shows a major band around 120 kDa in ECs. C–D, dose-dependent effects of TNFα treatment on HUVEC network formation. Representative fields (C) and the quantitation, mean ± S.D. (n = 6) (D). Statistics: unpaired two-tail t-tests (**, p < 0.001; ***, p < 0.0001).

FIGURE 2.

Effects of TNFα, IFNα, and IFNγ on PML protein levels and network formation in HUVECs and HMVECs. HUVECs (A–C) and HMVECs (D–E) were pre-treated with vehicle control, TNFα (20 ng/ml), IFNα (10³ units/ml), or IFNγ (10³ units/ml) for 16 h followed by in vitro EC network formation assays and quantification of branch points, mean ± S.D. (n = 12). An aliquot of HUVECs (C) or HMVECs (E) was used for Western blotting analysis with α-PML and α-β-actin antibodies. Statistics: unpaired two-tail t-tests (***, p < 0.0001).

FIGURE 3.

PML is essential for TNFα-, IFNα-, and IFNγ-mediated inhibition of EC network formation. A and B, HUVECs were transiently transfected with siControl or siPML for 72 h, and treated with TNFα (20 ng/ml), IFNα (10³ units/ml), or IFNγ (10³ units/ml) for 16 h. The cells were subsequently trypsinized and counted. Equal numbers of cells were plated on the matrix gel for in vitro EC network formation assays. Images shown were taken 20 h after seeding the cells on the extracellular matrix gel (A). Quantitation of branch points is shown as mean ± S.D. (n = 12) (B). C, an aliquot of HUVECs from A and B was plated for Western blotting to examine PML expression. D, similar to B, in vitro EC network formation assays in HMVECs. Statistics: unpaired two-tail t-tests (***, p < 0.0001).

FIGURE 4.

STAT1 participates in the TNFα- and IFNα-induced PML expression and inhibition of EC network formation in HUVECs. A, effects of STAT1 knockdown on PML mRNA levels analyzed by qRT-PCR. B, effects of TNFα and IFNα on endogenous STAT1 and PML protein levels in HUVECs assayed by Western blotting. C, effects of STAT1 knockdown by two siRNAs on PML protein levels following TNFα and IFNα treatment assayed by Western blotting. B and C, Western blotting bands were quantified by Image J as described in “Experimental Procedures.” ND, not detectable. D, effects of STAT1 knockdown on PML NBs examined by immunofluorescence microscopy (DAPI, blue; STAT1, green; PML, red). The microscopic parameters were set identically for all images taken. E, association of STAT1 with the PML promoter following TNFα and IFNα treatment assayed by chromatin immunoprecipitation followed by quantification with qPCR. HUVECs treated with TNFα (20 ng/ml) or IFNα (10³ units/ml) for 16 h followed by ChIP assays as described in “Experimental Procedures.” Putative STAT binding sites were predicted with Genomatix. TNFα and IFNα increase acetylation of histone H3 flanking STAT1 binding sites at the PML promoter. F, effects of knockdown of STAT1 on TNFα- and IFNα-mediated inhibition of EC network formation in HUVECs. Quantitation of branch points after in vitro EC network formation assays was as described in Fig. 1, mean ± S.D. (n = 8). Statistics: unpaired two-tail t-tests (**, p < 0.001; ***, p < 0.0001; ns, not significant).

FIGURE 5.

PML, TNFα, and IFNα inhibit integrin β1 (ITGB1) expression. A, heatmap of a panel of significantly altered genes (>2-fold, p < 0.01) involved in cell migration and angiogenesis identified by microarray gene expression analyses in HUVECs. siCTRL (control siRNA), two PML siRNAs (siPML-1 and siPML-2). Row z-score uses a green-black-red color scheme to represent relatively low-median-high expression levels. Official gene symbols are labeled beside each row. B, effects of knockdown of PML on ITGB1 mRNA examined by qRT-PCR. Fold change is calculated as siPML/siControl. C, effects of knockdown of PML on ITGB1 protein level as assayed by Western blotting in HUVECs. D and E, effects of TNFα and IFNs treatment on ITGB1 accumulation in HUVECs and HMVECs at the mRNA level quantified by qRT-PCR (D) and at the protein level examined by Western blotting (E).

FIGURE 6.

PML and STAT1 are required for TNFα and IFNα to inhibit integrin β1 (ITGB1) expression. A and B, effects of knockdown of STAT1 (A) or PML (B) on TNFα- and IFNα-mediated reduction of ITGB1 expression assayed by Western blotting. C, effects of ITGB1 blocking antibody on HUVEC wound-healing migration following TNFα or IFNα treatment. The width of the initial wound was set as 100%, mean ± S.D. (n = 16). closed means wound width percentage is 0%. D, effects of ITGB1 blocking antibody on HUVEC wound-healing migration following knockdown of STAT1 or PML. siCTRL (control siRNA), two STAT1 siRNAs (siSTAT1–1 and siSTAT1–2), and two PML siRNAs (siPML-1 and siPML-2). An aliquot of the cells was used to determine STAT1 and PML knockdown efficiency as assayed by Western blotting (top panel), and the wound width percentages were quantified (bottom panel). Unpaired two-tail t test (**, p < 0.01; ***, p < 0.001; #, p < 0.00001).

FIGURE 7.

PML regulates transwell migration of endothelial cells. PML was knocked down by a mixture of two siRNAs (siPML-1 and siPML-2) followed by transwell migration assays. A, migration toward 10% fetal bovine serum, FBS. Assays were performed in the presence of TNFα (20 ng/ml) or IFNα (10³ units/ml) following 72 h of transfection of control siRNA (siCTRL) or PML siRNA (siPML). An aliquot of cells was harvested and Western blotting was performed to examine PML knockdown efficiency (left panel). Migration assays were quantified as described in “Experimental Procedures.” B, transwell migration toward the ECM. Experiments were conducted as described in A except that ECM was used to replace FBS. Unpaired two-tail t test was used to determine the statistical significance (**, p < 0.01; ***, p < 0.001; #, p < 0.00001).