Estrogenic G protein-coupled receptor 30 signaling is involved in regulation of endometrial carcinoma by promoting proliferation, invasion potential, and interleukin-6 secretion via the MEK/ERK mitogen-activated protein kinase pathway

Abstract

The regulatory mechanism of endometrial carcinoma and the signal transduction pathways involved in hormone action are poorly defined. It has become apparent that the G protein-coupled receptor (GPR) 30 mediates the non-genomic signaling of 17beta-estradiol (E2). Here we show that GPR30 is highly expressed in endometrial cancer tissues and cancer cell lines and positively regulates cell proliferation and invasion. GPR30 expression was detected in 50 human endometrial carcinomas. The transcription level of GPR30 was significantly higher in the tissue of endometrial carcinoma than in normal endometrium (P < 0.05). Immunohistochemical assays revealed that the positive expression rate of GPR30 protein in endometrial carcinoma tissue (35/50, 70%) was statistically higher than in normal endometrium tissue (8/30, 26.67%) (chi2 = 14.16, P = 0.0002). GPR30 overexpression was correlated with high-grade endometrial carcinoma. GPR30 expression was also found in two human endometrial cancer cell lines: RL95-2 (estrogen receptor positive) and KLE (estrogen receptor negative). The roles of GPR30 in proliferative and invasive responses to E2 and G1, a non-steroidal GPR30-specific agonist, in RL95-2 and KLE cell lines were then explored. We showed that E2 and G1 could initiate the MAPK/ERK mitogen-activated protein kinase pathway in both cell lines. What's more, E2 and G1 promoted KLE and RL95-2 proliferation and stimulated matrix metalloproteinase production and activity via the GPR30-mediated MEK/ERK mitogen-activated protein kinase pathway, as well as increased interleukin-6 secretion. These findings suggest that GPR30-mediated non-genomic signaling could play an important role in endometrial cancer.

Figure 1

G protein‐coupled receptor (GPR) 30 transcription and overexpression in endometrial carcinoma. (a) Normal endometrium tissue: samples 1–6, endometrium of proliferative phase; samples 7–10, endometrium of secretory phase. (b) Endometrial carcinoma: samples 11–20, 10 carcinoma samples. Note: the marker contains the fragments (from top to foot of gel) 2000, 1000, 750, 500, 250, and 100 bp. (c) The y‐axis shows the ratio of optical density of GPR30 to β‐actin (mean ± SD) from (b). *P < 0.05. (d) The relative mRNA levels of GPR30 in normal endometrium and endometrial cancer by real‐time polymerase chain reaction. Ten normal endometrial tissues and 10 endometrial carcinoma tissues were used. (e) Immunochemistry for GPR30 in normal endometrium tissue and endometrial carcinoma. Normal endometrium, endometrial carcinoma, isotype control detected by rabbit IgG, and GPR30 expression detected by rabbit anti‐human GPR30 antibody are shown. Magnification, ×400. (f) Western blotting analysis for GPR30 protein levels in normal endometrium tissue and endometrial carcinoma. (g) showed the densitometric analysis. *P < 0.05. Fifty endometrial carcinoma samples and 17 normal endometrium samples were used in the reverse transcription–polymerase chain reaction, immunochemistry, and western blotting. The results were highly reproducible and these pictures are representative. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.

Figure 2

G protein‐coupled receptor (GPR) 30 expressed in estrogen receptor (ER)‐negative endometrial carcinoma. a and b, c and d, e and f, g and h, I and j are from the same endometrial carcinoma, respectively. (a–f) Endometrioid carcinoma, (g,h) adenosquamous endometrial carcinoma, (i,j) undifferentiated endometrial carcinoma. (a,c) ER protein expression was observed in the nuclei of tumor cells. (b,f,h,j) Carcinoma cells showed GPR30 staining in the cytoplasm and membrane. (d) GPR30 expression was not found in this carcinoma. (e,g) No of ERα expression. (i) Weak staining for ERα in the nuclei of tumor cells.

Figure 3

G protein‐coupled receptor (GPR) 30 expression in endometrial cancer cell lines. RL95‐2 cells were estrogen receptor (ER) positive whereas KLE cells were ER negative. (a–c) ERα mRNA and protein were highly expressed in RL95‐2 but hardly expressed in KLE. (c) ERα protein expression was observed in the nuclei of RL95‐2, but GPR30 immunostaining was found in the cytoplasm and membranes of RL95‐2 and KLE. Rabbit IgG and mouse IgG were used as isotype controls; only one isotype control is displayed here. (a,c,e) Magnification, ×400; (b,d,f) ×200.

Figure 4

G protein‐coupled receptor (GPR) 30 signaling initiated the mitogen‐activated protein kinase (MAPK) pathway in endometrial cancer cell lines. (a–f) Enzyme‐linked immunosorbent assays (ELISA) for MAPK status in KLE and RL95‐2 cell lines. RL95‐2 and KLE were seeded in 10‐cm dishes at 1 × 10⁶/mL, cultured, and starved. Then the cells were incubated with 10⁻⁸ M G1 or 10⁻⁸ M 17β‐estradiol (E2) for 30 min. Next the cells were lysed with cell lysis buffer (400 µL per 10‐cm dish). The supernatants were collected after centrifugation at 10 000g at 4°C for 10 min, and were used for the sandwich ELISA, reading absorbance at 450 nm according to the manufacturers’ protocols. Status of the MAPK pathway activated by G1 and E2 in (a) KLE and (b) RL95‐2 cells. The ratios of p‐mitogen‐activated protein kinase (MEK)/MEK in (c) KLE and (e) RL95‐2 cells, and the ratios of p‐SAPK/JNK/SAPK/JNK in (d) KLE and (f) RL95‐2. (g–h) Western blotting for MAPK status in KLE and RL95‐2 cell lines. The bar graphs show the ratio histograms for the western blotting densitometry. *P < 0.05, ΔP > 0.05. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; JNK, c‐Jun NH2‐terminal kinase; SPAK, Stress‐activated protein kinase.

Figure 5

G protein‐coupled receptor (GPR) 30 via the MEK/ERK mitogen‐activated protein kinase (MAPK) pathway stimulates cell proliferation of endometrial cancer cell lines. KLE and RL95‐2 cells were seeded in 24‐well plates at 1 × 10⁶ cells/mL and cultured for 24 h with Dulbecco's modified Eagle's medium (DMEM)/F12 containing 10% fetal bovine serum. The cells were then starved for another 24 h by replacing the media with phenol red‐free and serum‐free DMEM/F12, followed by addition of different doses of 17β‐estradiol (E2) (10⁻¹¹ M–10⁻⁷ M) or G1 (10⁻¹¹ M–10⁻⁷ M), 10⁻⁸ M E2 or 10⁻⁸ M G1 in the presence of pertussis toxin (PTX) (200 ng/mL) or U0126 (30 µM). Cells were further cultured for 48 h before evaluation of cell proliferation. Vehicle: 0.1% dimethyl sulfoxide/phenol red‐free and serum‐free DMEM/F12. (a) *P < 0.05 versus vehicle; #P < 0.05 versus 10⁻¹¹ M G1; &P < 0.05 versus 10⁻⁷ M G1. (b) *P < 0.05 versus vehicle; #P < 0.05 versus 10⁻¹¹ M and 10⁻⁷ M E2. (c,d) *P < 0.05 versus vehicle; #P < 0.05 versus the others. (g–j) Negative control: cells transfected with HuSH 29‐mer non‐effective against enhanced green fluorescent protein (pRS). RNAi: cells transfected with HuSH 29‐mer short hairpin RNA against GPR30 in pGFP‐V‐RS vector. *P < 0.05; ΔP > 0.05; &P < 0.05 versus cells with the same treament in the negative control.

Figure 6

G protein‐coupled receptor (GPR) 30 signaling via the MEK/ERK mitogen‐activated protein kinase (MAPK) pathway increased matrix metalloproteinase (MMP) production and activity in endometrial cancer cells. (a,b) Enzyme‐linked immunosorbent assay (ELISA) data for MMP‐2 and MMP‐9. Data are expressed as mean ± SD. *P < 0.05 compared with vehicle; #P < 0.05 compared with vehicle and 17β‐estradiol (E2); ΔP < 0.05 compared with vehicle and G1. (c,d) Western blotting showing MMM‐9 and MMP‐2 protein expression. (e,f) Gelatin zymography showing MMP‐9 and MMP‐2 activity. The y‐axis displays normalization of MMP‐9 and MMP‐2 band intensity to the vehicle group. Data are expressed as mean ± SD. *P < 0.05 compared with vehicle; #P < 0.05 compared with vehicle and E2; ΔP < 0.05 compared with vehicle and G1.

Figure 7

G protein‐coupled receptor (GPR) 30 signaling increased interleukin (IL)‐6 production by endometrial cancer cells. RL95‐2 and KLE were seeded in 24‐well plates at 1 × 10⁶ cells/mL, cultured, and starved. Then the cells were incubated with 10⁻⁸ M G1 or 10⁻⁸ M 17β‐estradiol (E2) plus U0126 or pertussis toxin (PTX) for 48 h. The supernatants were harvested, centrifuged at 10 000g for 5 min, and the sediments discarded. The supernatants were stored at –80°C until enzyme‐linked immunosorbent assay. (a) IL‐6 secretion in KLE cells. *P < 0.05 versus vehicle; #P < 0.05 versus E2 + U0126 and E2 + PTX; ΔP < 0.05 versus G1 + U0126 and G1 + PTX. (b) IL‐6 secretion in KLE cells. *P < 0.05 versus vehicle; &P < 0.05 versus G1; P < 0.05 versus E2 + U0126 and E2 + PTX; ΔP < 0.05 versus G1 + U0126 and G1 + PTX.