Glioma cell proliferation controlled by ERK activity-dependent surface expression of PDGFRA - PubMed (original) (raw)

. 2014 Jan 29;9(1):e87281.

doi: 10.1371/journal.pone.0087281. eCollection 2014.

Duo Zuo 2, Cheng Luan 3, Min Liu 1, Manli Na 1, Liang Ran 2, Yingyu Sun 2, Annette Persson 4, Elisabet Englund 4, Leif G Salford 1, Erik Renström 3, Xiaolong Fan 2, Enming Zhang 3

Affiliations

Glioma cell proliferation controlled by ERK activity-dependent surface expression of PDGFRA

Dongfeng Chen et al. PLoS One. 2014.

Abstract

Increased PDGFRA signaling is an essential pathogenic factor in many subtypes of gliomas. In this context the cell surface expression of PDGFRA is an important determinant of ligand sensing in the glioma microenvironment. However, the regulation of spatial distribution of PDGFRA in glioma cells remains poorly characterized. Here, we report that cell surface PDGFRA expression in gliomas is negatively regulated by an ERK-dependent mechanism, resulting in reduced proliferation of glioma cells. Glioma tumor tissues and their corresponding cell lines were isolated from 14 patients and analyzed by single-cell imaging and flow cytometry. In both cell lines and their corresponding tumor samples, glioma cell proliferation correlated with the extent of surface expression of PDGFRA. High levels of surface PDGFRA also correlated to high tubulin expression in glioma tumor tissue in vivo. In glioma cell lines, surface PDGFRA declined following treatment with inhibitors of tubulin, actin and dynamin. Screening of a panel of small molecule compounds identified the MEK inhibitor U0126 as a potent inhibitor of surface PDGFRA expression. Importantly, U0126 inhibited surface expression in a reversible, dose- and time-dependent manner, without affecting general PDGFRA expression. Treatment with U0126 resulted in reduced co-localization between PDGFRA and intracellular trafficking molecules e.g. clathrin, RAB11 and early endosomal antigen-1, in parallel with enhanced co-localization between PDGFRA and the Golgi cisternae maker, Giantin, suggesting a deviation of PDGFRA from the endosomal trafficking and recycling compartment, to the Golgi network. Furthermore, U0126 treatment in glioma cells induced an initial inhibition of ERK1/2 phosphorylation, followed by up-regulated ERK1/2 phosphorylation concomitant with diminished surface expression of PDGFRA. Finally, down-regulation of surface PDGFRA expression by U0126 is concordant with reduced glioma cell proliferation. These findings suggest that manipulation of spatial expression of PDGFRA can potentially be used to combat gliomas.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Correlation between PDGFRA surface expression and cell proliferation in human primary glioma cells.

A. PDGFRA surface intensity was determined by FACS in glioma cell lines from 8 glioblastomas and 6 grade II astrocytomas. The glioma cell lines were divided into three subgroups according to the level of PDGFRA surface expression. The data represent mean±SD of the percentages of cells with surface PDGFRA expression from three independent FACS experiments. B, the same as A, but EGFR surface expression was assessed. C. The PDGFRA surface expression was detected using TIRFM. The representative images indicate the surface expression of PDGFRA clusters in a single glioma cell. D. The averages of PDGFRA clusters assessed in TIRFM were summarized in the three groups of cell lines. *p<0.05, ***p<0.001 (ANOVA, F-test, n = 14). E. Glioma cell proliferation profiles during 5 days _in vitro_ culture were plotted against the extent of PDGFRA surface expression. *p<0.05, **p<0.01 (ANOVA, F-test, n = 14). F, Same as E, but the proliferation profiles were plotted against the extent of EGFR surface expression. G and H, more BrdU positive cells were detected in glioma cells with high PDGFRA surface expression. **p<0.01 (Student’s _t_-test, n = 8, glioma cell lines from #1, #2, #3 and #5 were in the >70% group and #11, #12, #13 and #14 in the <30% group.).

Figure 2

Figure 2. High-density surface expression of PDGFRA in glioma samples is associated with high cell proliferation rates in vivo.

A. Left panel: representative confocal images of PDGFRA immunohistochemistry staining in glioma samples corresponding to the cell lines with low surface PDGFRA expression as in Figure 1A. Right panel: intensity profile along the white spot line in the middle image, zoom-in from box in left picture. B. Same as A, but sections from glioma samples corresponding to the cell lines with high surface PDGFRA. C. Depiction of the ratio between the mean PDGFRA fluorescence intensity of cell surface (Isurface) to that of the cytosol (Icytosol). The surface area of PDGFRA fluorescence was defined by the staining of cadherin, a plasma membrane marker. D. Average ratios of PDGFRA expression in glioma tissues with high (#2, #3 and #5) or low PDGFRA surface expression (#12, #13 and #14). For each patient’s tissue, 14 cells were chose for the ratio quantitative analysis, n = 6, ***p<0.001 (ANOVA, F-test). E. Mean fluorescence intensity of PDGFRA in the whole cells was detected in tissues with low and high surface expression of PDGFRA. n.s.: no significance by Student’s _t_-test. 19 cells in each of tissues as in D were used for analysis. F. Representative confocal images of Ki-67 immunostaining in the indicated glioma samples. G. Average percentages of Ki67 positive cells under the conditions as in E. n = 3. **p<0.01 (ANOVA, F-test).

Figure 3

Figure 3. PDGFRA surface expression is controlled by cytoskeleton system.

A. Representative confocal images of tubulin immunostaining in the glioma tissue sections with high (n = 3, tissue #2, #3 and #5) or low (n = 3, tissue #12, #13 and #14) surface PDGFRA expression. B. Representative FACS histograms depict the effects of pharmacological inhibitors VBT (500 nM), LB (500 nM) and Dyn (40 µM) on surface PDGFRA expression in cell lines #1, #2 and #3. C. Time dependent decreases of surface PDGFRA expression measured by PDGFRA intensity (MFI, left) and the percentage of PDGFRA-positive cells (right) after treatment of VBT (n = 3, the same cell lines were used as in B. p<0.001, Student _t_-test). D. Increases of surface PDGFRA expression by intensity (left) and percentage (right) after treatment of LB. E. No significance change in the percentage of surface PDGFRA-positive cells but decrease in the surface PDGFRA intensity after dynasore treatment. (n = 3, the same cell lines were used as in B, p<0.001 for MFI, _t_-test). F. Increase of CD71 surface expression after dynasore treatment (n = 3, the same cell lines were used as in B, p<0.001, _t_-test).

Figure 4

Figure 4. PDGFRA surface expression is down-regulated by MEK inhibitor U0126.

A. The inhibitors were screened based on their capacity to down-regulate surface expression of PDGFRA in glioma cells. B. MEK inhibitor U0126 decreased PDGFRA surface expression detected by FACS in cell line #2. C. Representative confocal images showed PDGFRA expression with or without U0126 treatment in cell line #2. D. Average ratios of surface and cytosol PDGFRA expression in a glioma cell line with or without U0126 treatment in cell line #2. 18 cells were used for analysis in each group, ***p<0.001, _t_-test. E. The effect of U0126 on PDGFRA surface expression detected by FACS in 9 cell lines (cell lines #1–7, #9 and #14, p<0.001, _t_-test). F. Dose-dependent effects of U0126 on surface expression of PDGFRA in glioma cells. Data showed as average ± SD. G. Reversible time-dependent effect of U0126 on the PDGFRA expression from the three indicated cell lines. H and I. No significant effect of U0126 on PDGFRA expression at the mRNA and protein levels as analyzed in cell line #2. ΔCt was calculated by the mean Ct value of PDGFRA subtracted with the mean Ct value of GAPDH. The data are presented by average ± SEM of 5 repeats in 3 passages cells. The western blots are representative of three independent replicates. These experiments were performed with the cell line #2.

Figure 5

Figure 5. U0126 induced down-regulation of PDGFRA surface expression is concordant with a positive feedback of ERK activity.

A. Represent immunoblots of ERK activities at different time points following U0126 treatment. The blot is representative from 3 independent experiments. B. Represent immunoblots of ERK activities after 0.5 or 18-free A549 cells. All the experiments were performed in the cell lines #1, #2 and #3.

Figure 6

Figure 6. PDGFRA was deviated from intracellular trafficking system after treatment with U0126 for 18

A. Immunostaining images of PDGFRA and caveolin in cell line #2 following U0126 treatment. The large inserted picture is the zoom-in from the small box. Arrows indicate the colocalized clusters. B. Average PDGFRA co-localization to caveolin detected under the same conditions as in A (12 cells in each treatment, **p<0.01, _t_-test). C. Immunostaining of PDGFRA and clathrin in the cell line #2 with or without U0126 treatment. Arrows indicate the colocalized clusters. D. Average PDGFRA co-localization to clathrin detected under the same conditions as in C (12 cells in each treatment, **p<0.01, _t_-test). E. FACS data show PDGFRA surface expression after treatment of U0126 in combination with VBT in the glioma cell lines #2, #3 and #5 (n = 3, p<0.001, _t_-test). F. No effect of LB on U0126 induced reduction of PDGFRA surface expression in the same cell lines as in E (n = 3, p>0.05, _t_-test). G. Counteraction of U0126 effects on PDGFRA surface expression by dynasore treatment in the same cell lines as in F (n = 3, p<0.001, _t_-test).

Figure 7

Figure 7. U0126 treatment reduces the dwelling of PDGFRA in recycling system.

A. Representative confocal images of PDGFRA and Rab11 staining in glioma tissues of sample #2. B. The same condition as in A, but EEA-1 was detected. C. Immunostaining of PDGFRA and Rab11 with or without U0126 treatment in cell line #2. Arrows indicate the colocalized clusters. D. Average of PDGFRA co-localization to Rab11 detected under conditions as in C (18 cells in each condition, ***p<0.001, _t_-test). E. Immunostaining of PDGFRA and EEA-1 before and after U0126 treatment in cell line #2. Arrows indicate the colocalized clusters. F. Average of PDGFRA co-localization to EEA-1 under the same conditions as in E (28 cells in each condition, **p<0.01, _t_-test). G. Same as in E but Giantin was detected. The three separated images (right), the top, middle and bottom indicating Giantin, PDGFRA and respectively, are the zoom-in from box in left picture. H. Average of PDGFRA co-localization to Giantin under the same conditions as in G (42 cells in each condition. **p<0.01, _t_-test).

Figure 8

Figure 8. Down-regulation of glioma cell proliferation following U0126 treatment.

A. FACS analysis showing BrdU positive glioma cells with low or high surface PDGFRA expression with or without U0126 treatment. B. Statistical analysis of BrdU positive cells under the same conditions as in A. The data were derived from three independent experiments (***p<0.001 ANOVA, F-test). C. Representative histograms showing the inhibition of PDGF stimulated glioma cell growth by U0126 treatment in cell line #2. D. Average percentages of BrdU positive cells analyzed under conditions as in C. n = 3 (cell lines #2, #3 and #5), ***p<0.001 (F-test).

Figure 9

Figure 9. A carton illustrates association of surface PDGFRA with cell proliferation via ERK signaling pathway in glioma cells.

Even in the absence of acute ligand stimulation, PDGFRA is constitutively, but slowly trafficking in glioma cells. In this trafficking process, PDGFRA is transported in vesicles as cargo along cytoskeleton highway. The extent of endocytosis and recycling of PDGFRA is sensed by the ERK activity. High ERK activity could lead to changes resulting in enhanced endocytosis but diminished recycling, with PDGFRA enriched in Golgi apparatus. As a consequence, surface PDGFRA expression is down-regulated. This process would dampen the ligand stimulation.

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