Article Figures & Data
Figures
- Fig. 1.
Interaction between γ synuclein (SNCG) and estrogen receptor (ER)-α, heat-shock protein (Hsp)70, and Hsp90 in SNCG-transfected MCF-7 cells. Cells were transiently transfected with pCI-SNCG or the control vector pCI-neo and then selected with G418 as we described previously (16) . A and B, association of SNCG with endogenous ER-α, Hsp70, and Hsp90 in the absence of estradiol (E2). Total cell lysates were isolated from the cells cultured in the E2-free conditioned medium, and equal amounts of protein were subjected to immunoprecipitation (IP) with different antibodies. IP with anti-ER (A), anti-SNCG (B), anti-Hsp70 antibodies (B), and control rabbit (A) or goat IgG (B) followed by Western blotting for ER-α, SNCG, Hsp70, and Hsp90. C and D, association of SNCG with Hsp70 but not ER-α and Hsp90 in the presence of E2. Total cell lysates were isolated from the cells cultured in the conditioned medium containing 10 nm E2, and equal amounts of protein were subjected to IP with different antibodies. C, IP with anti-ER-α, anti-SNCG antibodies, and control goat IgG followed by Western blot for ER-α, SNCG, Hsp70, and Hsp90. D, IP with anti-Hsp70 antibody and control goat IgG followed by Western blotting for SNCG and Hsp70.
- Fig. 2.
Interaction between endogenous γ synuclein (SNCG), estrogen receptor (ER)-α, heat-shock protein (Hsp)70, heat-shock cognate (Hsc)70, and Hsp90 in T47D cells. A, association of endogenous SNCG with ER-α, Hsp70, and Hsp90 in the absence of estradiol (E2). Total cell lysates were isolated from the cells cultured in the E2-free conditioned medium, and equal amounts of protein were subjected to immunoprecipitation (IP) with anti-ER-α, anti-SNCG antibodies, and normal IgG. The immunoprecipitates were analyzed for Hsp70, SNCG, Hsp90, and ER-α after Western blotting using anti-Hsp70, anti-SNCG, anti-Hsp90, and anti-ER-α antibodies. B, association of endogenous SNCG with Hsp70 in the presence of E2. Total cell lysates were isolated from the cells cultured in the conditioned medium containing 10 nm E2, and equal amounts of protein were subjected to IP with anti-SNCG antibody and normal goat IgG. The nontransfected MCF-7 cell lysates were also subjected to IP as a negative control for SNCG IP. The immunoprecipitates were subjected to Western blotting using antibodies against Hsp70, SNCG. C, association of endogenous SNCG with Hsc70. Lysates from T47-D cells were subjected to IP with anti-SNCG antibody and normal goat IgG. The immunoprecipitates were subjected to Western blotting using antibodies against Hsc70, SNCG.
- Fig. 3.
Estrogen-binding capability for estrogen receptor in γ synuclein (SNCG)-transfected MCF-7 cells (MCF-7-SNCG) and control neo-transfected cells (MCF-7-Neo). Cells were transiently transfected with pCI-SNCG or pCI-neo plasmids. The transfected cells were enriched with Neomycin selection for 12 days before the hormone-binding assay. A, Western blot analysis of SNCG expression in pooled SNCG-transfected MCF-7 cells after selection with G418. B, titration of [3H]estradiol (E2) in MCF-7-Neo and MCF-7-SNCG cells. Inset, enlarged view of [3H]E2 titration from 0 to 1 nm of ligand.
- Fig. 4.
Scatchard analysis of the ligand binding by estrogen receptor from MCF-7-Neo cells (A) and MCF-7-SNCG cells (B). Solid line, high-affinity sites; dashed line, low-affinity sites. C, the high-affinity sites of MCF-7-Neo and MCF-7-SNCG cells. Specific binding was determined by subtracting the nonspecific binding from samples incubated with 100-fold excess of nonlabeled estradiol (E2). Each data point is the mean ± SD of triplicate samples.
- Fig. 5.
Effect of heat-shock protein (Hsp)90 inhibitor geldanamycin (GA) on estrogen-binding capability of estrogen receptor (ER) in MCF-7 cells. A, Western blot analysis of ER and γ synuclein (SNCG) levels in pooled MCF-7-Neo and MCF-7-SNCG cells treated with or without GA. The pooled MCF-7-Neo and MCF-7-SNCG cells were cultured in the presence of 0.1 nm estradiol (E2) and treated with or without 0.25 μm GA for 24 h. Cell lysates were normalized and subjected to Western blot analysis using the antibody against ER-α, SNCG, and actin, respectively. B, GA abolished the stimulatory effect of SNCG on estrogen-binding capability in MCF-7 cells. MCF-7-Neo and MCF-7-SNCG cells were treated with or without 0.25 μm GA for 24 h followed by the ligand binding assay with 0.1 nm [3H]E2. The data were presented as the percentage of the nontreated MCF-7-Neo controls, which was taken as 100%. Each data represent the mean ± SD of triplicate cultures.
- Fig. 6.
Effect of γ synuclein (SNCG) overexpression on ER-α and AKT phosphorylation. A, MCF-7 cells were transiently transfected with pCI-SNCG or pCI-neo plasmids. After G418 selection for 12 days, the pooled population of transfected cells were treated with or without 10-9 m estradiol (E2) or 50 ng/ml epidermal growth factor (EGF). Cell lysates were subjected to Western blot analysis using the antibodies against human Ser167-phosphorylated ER-α, ER-α, and actin, respectively. SNCG overexpression didn’t affect E2 and EGF-induced ER-α phosphorylation. B, the pooled population of SNCG-transfected cells and mock-transfected cells were treated with or without 10-9 m E2 or 50 ng/ml EGF for 15 min. Cell lysates were subjected to Western blot analysis using the antibodies against human AKT and Ser473-phosphorylated AKT, respectively. SNCG overexpression didn’t affect E2 and EGF-induced AKT phosphorylation.
- Fig. 7.
Effects on transcriptional activity of ER-β. MCF-7 cells were transiently cotransfected with pSG5-hERβ and pCI-SNCG or the control vector pCI-neo. After selection with G418, the transfected cells were transfected with pERE4-Luc, as well as control reporter pRL-SV40-Luc, cultured in the ligand-free medium for 4 days, treated with or without 1 nm estradiol (E2) for 24 h before harvesting. The promoter activities were determined by measuring the dual luciferase activity. All values were presented as the fold induction over the control luciferase activity in the nontreated SNCG-negative cells, which was taken as 1. The numbers represent means ± SD of three cultures.
- Fig. 8.
Stimulation of MCF-7 tumor growth by γ synuclein (SNCG). Each of the eight estradiol-supplemented nonovariectomized mice in each group received two injections, one on each side, in the mammary fat pads between the first and second nipples. Tumor size was determined by three-dimensional measurements (mm) using a caliper. Only measurable tumors were used to calculate the mean tumor volume for each clone at each time point. Each point represents the mean of tumors ± SE (bars).
- Fig. 9.
A model for γ synuclein (SNCG)-regulated estrogen receptor (ER)-α activation. In the model, 90 refers to heat-shock protein (Hsp)90, 70 refers to Hsp70, H refers to hormone. According the model, SNCG and Hsp70 preassociate to form SNCG-Hsp70 chaperone complex. This complex binds to the unstable hormone-binding domain of the native receptor. The recruitment of Hsp90 to the ER-α would occur once the receptor was bound with the SNCG-Hsp70 complex. At this stage, the receptor undergoes structure changes and assumes a conformation with high affinity for estradiol. Upon the binding to estradiol, the receptor disassociates with SNCG-Hsp70 complex and Hsp90, which leads to receptor dimerization, interaction with coactivators, and transactivation.
Tables
- Table 1
Effects of SNCG expression on tumor incidence and tumor growth of MCF-7 cellsa,b
Experiment group E2 Tumor incidence Tumor volume (mm3) Tumor/Total(%) Day 21 Day 35 MCF-SNCG2 + 14/16 (88) 234 ± 69 1850 ± 280 MCF-SNCG6 + 15/16 (94) 351 ± 78 2521 ± 390 MCF-neo1 + 10/16 (63) 101 ± 39 491 ± 92 MCF-neo2 + 11/16 (69) 123 ± 25 625 ± 130 -
a Cells were injected into the mammary fat pads, and tumor volumes and tumor incidence were determined as described in “Materials and Methods.” Each mouse received two injections. Tumor volumes were measured at 21 and 35 days after cell injection and are expressed as means ± SEs (number of tumors assayed). All the nonovariectomized mice received an estrogen implantation 1 day before the cell injection. There were total 16 injections for 8 mice in each group, and each injection had 5 × 106 cells. Statistical comparisons for SNCG-positive clones relative to SNCG-negative clones indicated P < 0.01 for the mean tumor sizes and P < 0.05 for the tumor incidence. Statistical comparison for primary tumors was analyzed by Student’st test. A χ2 test was used for statistical analysis of tumor incidence.
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b SNCG, γ synuclein; E2, estradiol.
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- Table 2
Stimulation of estrogen-mediated tumorigenesis by SNCGa,b
Group E2 Tumor incidence (%) Tumor volume (mm3) Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 7 MCF-neo1 - 0 0 0 0 0 0 0 MCF-neo1 + 0 0 0 0 20 30 30 44 ± 10 MCF-SNCG6 - 0 0 0 0 0 0 0 MCF-SNCG6 + 40 80 90 90 90 90 90 194 ± 35 -
a Ovariectomized mice were treated with or without E2 pellet. There were total 10 injections for 5 mice in each group, and each injection had 1.5 × 106 cells. Only measurable tumors were used to calculate the mean tumor volume. Statistical comparisons for SNCG-positive clones relative to SNCG-negative clones indicated P < 0.001 for both tumor incidence and mean tumor sizes in the presence of E2.
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b SNCG, γ synuclein; E2, estradiol.
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Volume 64, Issue 13
