| Summary: | 碩士 === 國立陽明大學 === 生理學研究所 === 99 === Estrogen promotes not only the development of female reproductive system but also the maintenance of secondary sexual characteristics. Moreover, it can immunosuppress T cell activation and release the inflammatory reactions. In our previous reports, estradiol (E2) does not stimulate intracellular pH changes but rapidly stimulates the increase of intracellular free calcium concentration ([Ca2+]i) and the immunosuppression of phytohemagglutinin (PHA)-induced cytokine secretion and proliferation in human peripheral T cells. Recently, G protein-coupled receptor 30 (GPR30), a novel membrane estrogen receptor (mER), is reported to induce rapid non-genomic responses in various cells. E2 fails to induce lymphocyte apoptosis and thymic atrophy in GPR30 gene knockout mice. To date, it is not clear whether E2 is through GPR30 to stimulate the increase of [Ca2+]i in human T cells and monocytes. Thus, the aim of this study was to investigate whether GPR30 was involved in the increase of [Ca2+]i by E2 and GPR30 agonist, G-1, and the immunosuppression on PHA-induced cell proliferation. Clinically, GPR30 and membrane progesterone receptors (mPRs) possibly play important roles on breast cancer progressions. The peripheral blood mononuclear cells (PBMCs) in breast cancer patients were chosen in order to discuss the relationship between breast cancer, estrogen receptors and progesterone receptors in immune cells. Breast cancer patients, whose tumors do not express nuclear estrogen and progesterone receptors but over express human epidermal growth factor receptor 2 (HER2) [ER-PR-HER2+], were requested to join this study. PBMCs obtained from women with regular menstrual cycle and patients with fibrocystic breast were used as controls. Consequently, the alteration of [Ca2+]i by PHA, E2, G-1 and progesterone (P4), and the mRNA expression of GPR30, membrane progesterone receptors (mPRα, β, γ), progesterone receptor membrane component 1 (PGRMC1) and nuclear estrogen receptor α (ERα) were also examined. RT-PCR and Western blot are used to identify mRNA and protein expression. Fura-2 is applied to detecting [Ca2+]i and cell proliferation is measured by MTT assay. The results demonstrated that GPR30 protein existed in human peripheral T cells. Both E2 and G-1 rapidly induced [Ca2+]i elevation, and then the elevation were blocked by MβCD (methyl-β-cyclodextrin), U73122, 2-APB, SKF96365 and GPR30 antagonist, G-15, but not by pertussis toxin, U73343, staurosporine and ruthenium red. Nevertheless, estrogen receptor selective antagonist ICI182,780 was unable to suppress the calcium elevation caused by E2 but by G-1. When the treatment of G-1 was above 2.5 μM, it could significantly suppress the PHA-stimulated cell proliferation, yet the suppressive effects did not reverse by G-15. There was a trend of suppression of cell proliferation by E2. Taken together, E2 elicited the increase of [Ca2+]i was not mediated by phospholipase Cγ but by phospholipase Cβ via GPR30 protein in T cells. G-1 did exert the immunosuppression on cell proliferation through GPR30 protein. Notably, G-15 dose-dependently antagonized E2-induced calcium signals but failed to reverse immunosuppresive effect on T cell proliferation. Clinical events illustrated that PHA dose-dependently stimulated PBMC proliferation, and the order of extent of proliferation among experimental groups were normal women > fibrocystic breast patients> ER-PR-HER2+ patients. Due to great deviation of samples, if one fold on cell proliferation as a borderline, it could define ER-PR-HER2+ patients as two groups (A and B). For example, the proliferation rate in group A was similar to normal women but above patients with fibrocystic breast. As regards group B, PHA failed to stimulate the proliferation. Furthermore, calcium elevation elicited by PHA, E2 and G-1 had no statistic difference among all groups except P4 that displayed greater calcium signals in ER-PR-HER2+ when compared to normal women and patients with fibrocystic breast. As for the mRNA expressions, mPRα, mPRβ, PGRMC1, and the GPR30 especially in PBMCs from ER-PR-HER2+ were significantly expressed more than normal women and patients with fibrocystic breast; ERα and mPRγ in ER-PR-HER2+ were expressed more than fibrocystic breast; although PGRMC1 in normal women were much higher than patients with fibrocystic breast, mPRβ was the lower. With regards to the group A, expression of the GPR30 particularly were greater than normal women and patients with fibrocystic breast; however, the expressions of ERα, and PGRMC1 in group A were also much more than patients with fibrocystic breast. For B group, only ERα expression was greater than normal women and patients with fibrocystic breast. In comparison of group A and B, the GPR30 expression was less in group B. In summary, owing to the increase of mPRβ expression, P4-induced [Ca2+]i increase in ER-PR-HER2+ patients was significantly elevated, and it might cause the immunosuppression of proliferation of PBMCs in patients with ER-PR-HER2+ cancer and fibrocystic breast. Additionally, increase of GPR30 expression might be the principal cause of the resemblance of proliferative capacity between group A and normal women. And the increase of ERα expression probably led the worst outcome of immmunosuppression on cell proliferation in group B. In conclusion, GPR30 protein might involve in E2-induced [Ca2+]i increase via phospholipase Cβ. GPR30-induced calcium signal might result in the immunosuppression on cell proliferation. Clinical samples of PBMCs displayed the increase of GPR30 expression in ER-PR-HER2+ patients, but the calcium signals by E2 or G-1 remained unchanged. Group A patients exhibited equal proliferative rate in PBMCs to normal women, this might attribute to the over expression of GPR30 in cells. The mPRβ expression as well as P4-induced calcium signal enhances massively in ER-PR-HER2+ patients. Besides, both the proliferative inability in ERα over-expression and the better proliferative rate in GPR30 over-expression patients may down-regulate the immunity in different ways and indirectly contribute to cancer cells becoming progressive and aggressive.
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