BAG3 Protein Is Involved in Endothelial Cell Response to Phenethyl Isothiocyanate

Abstract

Phenethyl isothiocyanate (PEITC), a cruciferous vegetable-derived compound, is a versatile cancer chemopreventive agent that displays the ability to inhibit tumor growth during initiation, promotion, and progression phases in several animal models of carcinogenesis. In this report, we dissect the cellular events induced by noncytotoxic concentrations of PEITC in human umbilical vein endothelial cells (HUVECs). In the early phase, PEITC treatment elicited cells' morphological changes that comprise reduction in cell volume and modification of actin organization concomitantly with a rapid activation of the PI3K/Akt pathway. Downstream to PI3K, PEITC also induces the activity of Rac1 and activation of c-Jun N-terminal kinase (JNK), well-known regulators of actin cytoskeleton dynamics. Interestingly, PEITC modifications of the actin cytoskeleton were abrogated by pretreatment with JNK inhibitor, SP600125. JNK signaling led also to the activation of the c-Jun transcription factor, which is involved in the upregulation of several genes; among them is the BAG3 protein. This protein, a member of the BAG family of heat shock protein (Hsp) 70 cochaperones, is able to sustain survival in different tumor cell lines and neoangiogenesis by directly regulating the endothelial cell cycle. Furthermore, BAG3 is involved in maintaining actin folding. Our findings indicate that BAG3 protein expression is induced in endothelial cells upon exposure to a noncytotoxic concentration of PEITC and its expression is requested for the recovery of normal cell size and morphology after the stressful stimuli. This assigns an additional role for BAG3 protein in the endothelial cells after a stress event.

Figure 1

PEITC induces actin cytoskeleton alterations and activation of the PI3K/Akt pathway in HUVECs. (a) HUVECs from 2 different donors were plated at 5 ×10³/cm² and then treated with PEITC at indicated concentrations for 24 and 48 h. Then, cells were subjected to the MTT assay, and results are shown in a bar graph as % of viable cells in respect to controls (untreated or 0.01% DMSO-treated cells). (b) HUVECs from 2 different donors were treated as described above. After 24 h, a cell cycle assay was performed by using PI on permeabilized cells. Results are expressed as % of cells in each cell cycle phase. (c) Phase-contrast images and confocal analysis of HUVECs treated with (A, D) DMSO (control) at a final concentration (0.01%), with (B) PEITC (10 μM) for 15 min, and with (C, E) PEITC (10 μM) for 120 min. (B, C, E) Images of PEITC-treated cells showing blebbing formation in the outer cytoskeletal domain. (d) HUVECs were seeded as described above and treated with PEITC; at the indicated time points, cells were harvested and subjected to cell lysis. Protein contents were analyzed by Western blot to analyze the levels of phospho-FAK protein. Tubulin was used as a loading control. (e) HUVECs were treated with 0.01% DMSO (C) and with 10 μM PEITC for 15, 30, 60, and 120 min. Total protein extracts were analyzed by Western blot using anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-PI3K, and anti-PI3K to test PI3K/Akt activation levels. The anti-GAPDH antibody was used as an internal loading control. Each lane was loaded with 20 μg protein. The bar graph depicts densitometric analysis of the data (expressed as phospho-PI3K/PI3K and phospho-Akt/Akt ratios) corresponding to the left panel. Results were obtained from at least two independent experiments and are expressed as the mean ± SEM. ^∗P < 0.05, ^#P < 0.01, and ^§P < 0.001, statistically significant differences, compared to DMSO-treated cells (C), were calculated by Student's t-test for unpaired data.

Figure 2

PEITC induces Rac1 activity via PI3K, and Rac1 in turn activates JNK. (a) HUVECs were treated with 0.01% DMSO (C) or treated with 10 μM of PEITC (P) for 2 h and subjected to Western blot analysis of total proteins to measure the effect of PEITC on Rac1-GTPase activity and Rac1 total protein levels and on JNK activation. The left and middle panels display columns representing densitometric analysis of Rac1 activity and of Rac1 total protein levels (expressed as Rac1/GAPDH ratios). Representative blot analysis for Rac1 after a pull-down assay and for total Rac1 protein. (b) HUVECs were treated as described above. Total cell protein content was subjected to Western blot analysis to measure phospho-JNK levels. The left panel displays columns representing densitometric analysis of the data (expressed as phospho-JNK/JNK ratios) corresponding to the right panel. The anti-GAPDH antibody was used as an internal loading control. (c) The levels of GTP-bound Rac1, total Rac1, and phospho-JNK in lysates of pretreated or not with 10 μM of LY2940021 (1 h) were analyzed in HUVECs in the presence or absence of 10 μM PEITC stimulation for 2 h. The lower bar graphs depict densitometric analysis of Rac1 activity, Rac1 protein levels, and phospho-JNK levels. (d) The levels of GTP-bound Rac1, phospho-Akt, and phospho-JNK were analyzed in lysates of pretreated or not with 100 μM of NSC23766 HUVECs for 4 h, in the presence/absence of 10 μM PEITC stimulation for 2 h. The lower bar graphs depict densitometric analysis of Rac1 activity, phospho-JNK, and phospho-Akt levels. Results were obtained from at least two independent experiments and are expressed as the mean ± SEM. ^∗P < 0.05, ^#P < 0.01, and ^§P < 0.001, statistically significant differences, compared to DMSO-treated cells (C), were calculated by Student's t-test for unpaired data.

Figure 3

JNK activity is involved in actin remodeling processes during PEITC treatment. (a) The upper panel displays phase-contrast images and confocal analysis of HUVECs treated with (A, E) 0.01% DMSO (control), (B, F) 10 μM of SP600125 (JNK inhibitor) for 1 h (C, G), and 10 μM of PEITC for 2 h and (D, H) pretreated with 10 μM of SP600125 1 h before PEITC administration. Scale bars: d = 15 μm and h = 5 μm. (b) The levels of GTP-bound Rac1, phospho-JNK, and phospho-c-Jun in lysates of HUVECs, pretreated or not with 10 μM of SP600125 for 1 h, were analyzed in the presence/absence of 10 μM PEITC stimulation for 2 h. The left bar graphs depict densitometric analysis of Rac1 activity, phospho-JNK, and phospho-c-JUN levels. Results were obtained from at least two independent experiments and are expressed as the mean ± SEM. ^∗P < 0.05, ^#P < 0.01, ^§P < 0.001, statistically significant differences, compared to DMSO-treated cells (C), were calculated by Student's t-test for unpaired data.

Figure 4

PEITC treatment induces BAG3 expression and its delocalization in HUVECs. (a) Immunofluorescence analysis of HUVECs in control conditions and after PEITC treatment. Cells were stained with the BAG3 antibody (A–E), phalloidin (B–F) that allows for the visualization of F-actin, and Hoechst (C–G), and D–H are merged images. The merged image shows overlapping localization of BAG3 and F-actin. (b) HUVECs were treated with 0.01% DMSO (control) and with 10 μM PEITC for 16 h. Total protein extracts were analyzed by Western blot using the anti-BAG3 antibody and anti-calregulin antibody, as an internal loading control. The lower panel displays columns representing densitometric analysis of the data (expressed as the BAG3/calregulin ratio) corresponding to the upper panel (n = 2). ^∗P < 0.05, statistically significant differences, were calculated by Student's t-test for unpaired data. (c) Analysis of bag3 mRNA levels by qRT-PCR. Fold induction of bag3 mRNA levels (y-axis) in HUVEC controls and PEITC-treated cells is expressed relative to β-actin mRNA levels. Data are the mean values ± SD from two independent experiments performed in triplicate. ^∗P < 0.05 and ^#P < 0.01, statistically significant differences, compared to DMSO-treated cells (C), were calculated by one-way ANOVA with Dunnett's post hoc test using SigmaPlot12.0 software. (d) Phase-contrast images of HUVECs treated with (A) DMSO (control) at a final concentration (0.01%), with (B) PEITC (10 μM) for 120 min, and with (C) PEITC (10 μM) for 16 h. (D, E, F, G) HUVECs were transfected for 48 h with bag3 siRNA (100 nM) or with a NT siRNA (100 nM) and treated with (D, F) DMSO (control) at a final concentration (0.01%) and with (E, G) PEITC (10 μM) for 16 h. (e) HUVECs were transfected as described above. Total cell protein content was subjected to Western blot analysis to measure BAG3 levels and anti-calregulin antibody, as an internal loading control.

Figure 5

Schematic representation of the proposed model for PI3K/Rac1/JNK/c-Jun pathway activation and involvement of BAG3 as a target of the PEITC effect.