Promyelocytic leukemia protein controls cell migration in response to hydrogen peroxide and insulin-like growth factor-1

Abstract

Promyelocytic leukemia protein (PML) was originally identified as part of a chromosomal translocation that contributes to the development of acute promyelocytic leukemia (APL). Since its discovery, PML has been found to play diverse roles in different cellular processes. Notably, PML has anti-proliferative and pro-apoptotic activity that supports its role as a tumor suppressor. We have previously shown that the peptidyl-prolyl isomerase Pin1 is able to affect cell proliferation and hydrogen peroxide (H(2)O(2))-mediated cell death through modulation of the steady-state levels of PML. We have extended these studies to show that the interaction between PML and Pin1 is targeted by multiple extracellular signals in the cell. We show that H(2)O(2) up-regulates and IGF-1 down-regulates PML expression in a Pin1-dependent manner. Interestingly, we found that H(2)O(2)- and IGF-1-mediated alteration in PML accumulation regulate MDA-MB-231 cell migration. Furthermore, we show that the control of cell migration by PML, and thus H(2)O(2) and IGF-1, results from PML-dependent decreased expression of integrin beta1 (ITGB1). Knockdown of Pin1 leads to decreased cell migration, lower levels of ITGB1 expression and resistance to IGF-1- and H(2)O(2)-induced changes in cell migration and ITGB1 expression. Taken together, our work identifies PML as a common target for H(2)O(2) and IGF-1 and supports a novel tumor suppressive role for PML in controlling cell migration through the expression of ITGB1.

FIGURE 1.

H₂O₂ treatment results in increased PML protein accumulation. A, MDA-MB-231 cells were treated with H₂O₂ for 60 min followed by immunostaining with anti-PML antibodies. B, MDA-MB-231 cells were treated with H₂O₂ for 2 h followed by immunostaining with anti-PML antibodies. Numbers of PML NBs in each cell were scored. The data are derived from two independent experiments; each includes 12 fields from a total of more than 200 cells. C, MDA-MB-231 cells were treated with H₂O₂ for the times and concentrations indicated. Whole cell lysates were prepared and subjected to SDS-PAGE followed by immunoblotting with anti-PML and anti-α-tubulin antibodies. Tubulin served as a loading control. D, MDA-MB-231 cells were treated with or without 100 μm H₂O₂ for 120 min. RNA was isolated and analyzed by standard RT-PCR and real time RT-PCR to analyze changes in the expression of PML. There was no significant difference between the treated and untreated samples. GAPDH was used as an internal control. Error bars indicate S.D.

FIGURE 2.

H₂O₂ treatment decreases the association between PML and Pin1. A, MDA-MB-231 cells were treated as described in Fig. 1C. Whole cell lysates were prepared and used for pulldown assays with either GST or GST-Pin1 proteins. The pulldown fractions were analyzed by immunoblotting with anti-HA antibodies. 30% input is shown. B, MDA-MB-231 cells were transfected with HA-PML 4 (WT) or HA-PML 4 (4×) expression constructs and treated with or without H₂O₂ as described in Fig. 1C. An expression construct for GFP was also included to normalize for transfection efficiency and loading. Whole cell lysates were analyzed by immunoblotting with anti-HA and anti-GFP antibodies. C, MDA-MB-231-shControl or MDA-MB-231-Pin1 shRNA cells were treated with 100 μm H₂O₂ for the times indicated followed by immunostaining with anti-PML antibodies. The number of PML NBs in each cell was counted. At least 200 cells were counted for each treatment.

FIGURE 3.

IGF-1 treatment reduces PML protein accumulation. A, MDA-MB-231 cells were treated with 100 ng/μl IGF-1 for the times indicated. Whole cell lysates were prepared and analyzed by immunoblotting with anti-PML and anti-α-tubulin antibodies. Tubulin served as a loading control. B, MDA-MB-231 cells were treated with 100 ng/μl IGF-1 for 120 min. Total RNA was isolated and analyzed by standard RT-PCR and real-time RT-PCR to analyze PML expression. GAPDH was used as an internal control. There was no significant change observed between treated and untreated samples. C, MDA-MB-231 cells were treated with IGF-1 as described in B and immunostained with anti-PML antibodies. Error bars indicate S.D.

FIGURE 4.

IGF-1 treatment increases the association of PML with Pin1. A, MDA-MB-231 cells were treated with or without 100 ng/μl IGF-1 for 120 min and whole cell lysates were prepared for pulldown assays with GST or GST-Pin1 proteins. The pulldown fractions were analyzed by immunoblotting with anti-HA antibodies. 30% input is shown. B, MDA-MB-231 cells were transfected as described in Fig. 2B and then treated as indicated with 100 ng/μl IGF-1 for 120 min. Whole cell lysates were analyzed by SDS-PAGE followed by immunoblotting with anti-HA and anti-GFP antibodies. C, whole cell lysates from MDA-MB-231-shLuc or -shPin1 cells were treated with IGF-1 as described in panel B and analyzed by SDS-PAGE followed by immunoblotting with anti-HA and anti-tubulin antibodies as indicated.

FIGURE 5.

H₂O₂ and IGF-1 affect cell migration in a PML-dependent manner, and PML is a negative regulator of integrin β1 expression. A, MDA-MB-231 cells were treated with H₂O₂ for the times and concentrations indicated. H₂O₂ was then removed and wound-healing assays were performed as described under “Experimental Procedures.” The results shown indicate changes in six independent fields and are representative of at least two independent experiments. B, MDA-MB-231 cells were transfected with control oligonucleotides or a combination of two siRNA against PML. 72 h after transfection cells were treated with 100 μm H₂O₂ for 120 min followed by wound-healing assays as described in A. Results for each independent siPML construct are found in supplemental Fig. S2. C, MDA-MB-231 cells were transfected with either vector alone or expression plasmids for HA-PML 4. 48 h after transfection cells were treated with IGF-1 as indicated and wound-healing assays were performed. The results shown indicate changes in six independent fields and are representative of at least two independent experiments. Error bars indicate S.D.

FIGURE 6.

PML is a negative regulator of ITGB1 expression. A, total RNA was isolated from MDA-MB-231 cells transfected with either control oligonucleotide or a combination of two siRNA against PML, and real-time RT-PCR was performed to analyze the expression of PML and ITGB1 mRNA. GAPDH served as an internal control. Results for each independent siPML construct are found in supplemental Fig. S2. B, whole cell lysates from MDA-MB-231 cells transfected with control oligonucleotide or a combination of two siRNA against PML were analyzed by immunoblotting with anti-PML, anti-ITGB1, and anti-α-tubulin antibodies. α-Tubulin served as a loading control. Results for each independent siPML construct are found in supplemental Fig. S2. C–E, MDA-MB-231 cells were transfected with siControl or 2 siRNA against Pin1 (siPin1). Cells were harvested 72 h after transfection for mRNA isolation and qRT-PCR (C), whole cell lysate analyzed by immunoblotting (D), and in vitro wound-healing assays (E). Error bars indicate S.D.

FIGURE 7.

The effects of H₂O₂ on cell migration are mediated through PML control of ITGB1 expression. A, MDA-MB-231 cells transfected with control oligonucleotide or a combination of two siRNAs against PML were subjected to wound-healing assays in the presence of control IgG or anti-ITGB1 antibodies. The results shown indicate changes in six independent fields and are representative of two independent experiments. Results for each independent siPML construct are found in supplemental Fig. S2. B, MDA-MB-231 cells were treated with H₂O₂ as described in Fig. 1C. Cells were harvested and whole cell lysates or RNA prepared. Whole cell lysates were analyzed by immunoblotting with anti-ITGB1 and anti-α-tubulin antibodies (left panel). Tubulin served as a loading control. Total RNA was analyzed by real-time RT-PCR for ITGB1 expression. GAPDH served as an internal control (right panel). C, MDA-MB-231 cells transfected with control oligonucleotide or siRNA against PML were treated with or without 100 μm H₂O₂ for 120 min as indicated. Whole cell lysates were analyzed by immunoblotting with anti-ITGB1 and anti-α-tubulin antibodies. Tubulin served as a loading control. D, MDA-MB-231-shLuc and MDA-MB-231-shPin1 cells were treated with H₂O₂ followed by whole cell lysate preparation and immunoblotting with anti-ITGB1 and anti-α-actin antibodies. E, confluent MDA-MB-231-shLuc and MDA-MB-231-shPin1 cells were treated with H₂O₂ prior to scratch wound-healing assays. Error bars indicate S.D.

FIGURE 8.

The role of Pin1 in IGF-1-induced cell migration. A, MDA-MB-231 cells were treated with IGF-1 as indicated in the presence of either IgG or anti-ITGB1 antibodies followed by wound-healing assays. The results shown indicate changes in six independent fields and are representative of at least two independent experiments. B, MDA-MB-231 cells were analyzed as in Fig. 7B except that cells were treated with 100 ng/μl IGF-1 for 120 min. C, MDA-MB-231 cells transfected with vector alone or expression plasmids for HA-PML4 were treated with or without 100 ng/μl IGF-1 for 120 min. as indicated. Whole cell lysates were analyzed by immunoblotting with anti-ITGB1 and anti-α-tubulin antibodies. Tubulin served as a loading control. D, MDA-MB-231-shLuc and MDA-MB-231-shPin1 cells were treated with or without IGF-1 (100 ng/μl) for 120 min as indicated. Whole cell lysates were analyzed by immunoblotting with anti-ITGB1 and anti-α-actin antibodies. E, confluent MDA-MB-231-shLuc and MDA-MB-231-shPin1 cells were treated with or without IGF-1 followed by scratch wound-healing assays. Error bars indicate S.D.