Negative regulation of CXCR4-mediated chemotaxis by the lipid phosphatase activity of tumor suppressor PTEN

Abstract

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a multifunctional tumor suppressor, has been shown to play a regulatory role in cell migration. Dictyostelium discoideum cells lacking PTEN exhibited impaired migration toward chemoattractant gradients. In the present study, we investigated the involvement of PTEN in chemotaxis of mammalian cells by examining PTEN-null Jurkat T cells. We observed that, in contrast to observations made in D discoideum, PTEN-null Jurkat T cells exhibited potent chemotactic responses to the chemokine stromal cell-derived factor 1alpha (SDF-1alpha), indicating that PTEN was not requisite for CXC chemokine receptor 4 (CXCR4)-mediated chemotaxis of Jurkat cells. Conversely, reconstitution of PTEN in Jurkat cells by using a tetracycline (Tet-on)-inducible expression system down-regulated CXCR4-mediated chemotaxis. Furthermore, we established the lipid phosphatase activity of PTEN as essential for its inhibitory effect on chemotaxis. In addition, using PTEN-expressing T-cell lines and primary T cells, we demonstrated that down-regulation of PTEN expression with vector-based small interfering RNAs (siRNAs) enhanced CXCR4-mediated chemotaxis. Based on these results, we conclude that PTEN expression negatively regulates chemotaxis of lymphoid mammalian cells via its lipid phosphatase activity. Our findings may account for the reported increase in metastatic activity of PTEN-null tumor cells.

Figure 1.

PTEN-null Jurkat T cells exhibited potent chemotactic response to SDF-1α. (A) Jurkat cells were PTEN deficient. Lanes 1 to 6 contained lysates of wild-type Jurkat cells obtained from the laboratories of Drs Ji-Ming Wang, William L. Farrar, and Daniel W. McVicar, as well as our lab (all at NCI-Frederick, Frederick, MD), respectively, and the Tet-on and Tet-off Jurkat T-cell lines purchased from Clontech (Palo Alto, CA). Lanes 7 to 9 showed the lysates of HEK293 cells and PBMCs, used as positive controls. Cultured cells were lysed and electrophoresed on an SDS–polyacrylamide gel electrophoresis (PAGE) gel, transferred to nitrocellulose membranes, and probed with anti-PTEN or anti-ERK1/2 as a loading control. (B) Jurkat cells express CXCR4. The surface expression of chemokine receptor CXCR4 in Jurkat cells was measured by flow cytometry. Cells were stained with anti-hCXCR4 (solid line) or isotype-matched human IgG2a as a negative control (dotted line). (C) Jurkat cells exhibit dose-dependent chemotactic responses to SDF-1α. Chemotaxis was performed using 48-well chemotaxis chambers as described in “Materials and methods.” Different concentrations (0-1000 ng/mL) of SDF-1α were used for chemotaxis. (D) SDF-1α–induced chemotaxis of Jurkat cells was PTX sensitive. Jurkat cells were pretreated with different doses of PTX for 30 minutes at room temperature (RT) and chemotaxis assay was carried out as described in “Materials and methods”. All the results in this figure were representative of at least 3 independent experiments. (C-D) Data shown are means ± SD.

Figure 2.

Reconstitution of PTEN in Jurkat cells down-regulated CXCR4-mediated chemotaxis. (A) Doxycycline (dox) induced PTEN expression in PIJ-17 but not in Col-18 or Tet-on Jurkat cells. PIJ-17, Con-18, and Tet-on Jurkat cells were treated with 1 μg/mL dox for 48 hours and the expression of PTEN (top panel) and Ser473-phosphorylated AKT (middle panel) was measured by Western blotting. The samples were also blotted with anti-ERK1/2 as loading control (bottom panel). (B) Expression of PTEN down-regulated SDF-1α–mediated chemotaxis. PIJ-17 and Con-18 Jurkat cells were cultured with or without the presence of dox (1 μg/mL) for 48 hours and chemotaxis was carried out as in Figure 1. The results were representative of at least 5 independent experiments. Data shown are means ± SD. (C-D) Time course of PTEN expression under dox induction and its effects on chemotaxis. PIJ-17 Jurkat cells were treated with 1 μg/mL dox for the indicated periods of time, and the levels of PTEN and Ser473-phosphorylated AKT were detected by Western blotting (C). The same cells were also assayed for chemotactic responses (D). Data shown are means ± SD. The results were representative of 3 independent experiments. (E) PTEN expression had no effects on CXCR4 surface expression. PIJ-17 Jurkat T cells were cultured with or without dox (1 μg/mL) for 48 hours and CXCR4 expression were detected by flow cytometry. The results were representative of 2 independent experiments. The histograms show cell-surface staining with anti-hCXCR4 (solid line) or isotype-matched human IgG2a (dashed line).

Figure 3.

The lipid phosphatase activity was essential for PTEN's role as a negative regulator of chemotaxis. Tet-on Jurkat cells were transiently transfected with Tet-inducible vectors expressing PTEN/wt, PTEN/G129E, PTEN/G129R, or a mock vector. After 6 hours, dox (1 μg/mL) was added to the culture medium. Cells were cultured for an additional 48 hours and then harvested for Western blotting (A) and chemotaxis (B). The results were representative of 3 independent experiments. Data shown are means ± SD.

Figure 4.

PI3K inhibitors reproduced the inhibitory effect of PTEN on CXCR4-mediated chemotaxis. PIJ-17 Jurkat cells were cultured with or without dox (1 μg/mL) and were then pretreated with wortmannin (A) or LY294002 (B) for 1 hour at RT at the indicated concentrations. The cells were then subjected to chemotaxis assays with SDF-1α (100 ng/mL). The basal migration in the absence of SDF-1α was indicated as medium only. The results were representative of 4 independent experiments. Data shown are means ± SD.

Figure 5.

Restoration of PTEN expression in Jurkat cells down-regulated IGF1-mediated chemotaxis. (A) The chemotactic responses of cultured PIJ-17 Jurkat cells (without dox) to different chemoattractants were tested. The concentrations used were determined previously as optimal for chemotaxis of different T lymphocyte subsets: RANTES (50 ng/mL), MDC (100 ng/mL), eotaxin3 (100 ng/mL), LARC (1000 ng/mL), IP-10 (100 ng/mL), I-309 (10 ng/mL), MIP-3β (100 ng/mL), IGF-1 (10 ng/mL), SDF-1α (100 ng/mL). The results were representative of 3 independent experiments. (B) PIJ-17 Jurkat cells were cultured with or without dox (1 μg/mL) for 48 hours and chemotaxis assays were performed as in panel A. The results were representative of 3 independent experiments. Data shown are means ± SD.

Figure 6.

Inhibition of PTEN expression by vector-based siRNA enhanced SDF-1α–induced chemotaxis of T cells. (A) Western blotting confirmed down-regulation of PTEN expression in siRNA/PTEN-transfected cells. The nontransfected cells and the cells transfected with siRNA/PTEN constructs or mock vectors (stable transfection for 2D6 and 2B4 T-cell lines and transient transfection for primary T cells) were lysed and PTEN protein expression detected by Western blotting as described in “Materials and methods”. The samples were also blotted with anti-ERK1/2 as loading control (bottom panel). The results were representative of 3 independent experiments for T-cell lines and 2 independent experiments for primary T cells. (B-D) CXCR4-mediated chemotaxis of 2D6 T cells (B), 2B4 T cells (C), and primary CD4 T cells (D) was enhanced by inhibition of PTEN expression. The chemotaxis assays were performed as described in “Materials and methods.” The results were representative of 3 independent experiments for T-cell lines and 2 independent experiments for primary T cells. Data shown are means ± SD.