Our group published thatnormal thyrocyte infected with B-RafV600E lentivrus increased in mRNA level of thyroid stimulating hormone(TSHR)(Kim et al., 2014).Accordingly, we assumedthatB-RafV600E-expressing HOSEwould showupregulation of estrogen receptor level. Therefore, we analyzedestrogen receptor alpha and beta expression by real-time PCR and immunoblotting (Fig. 5A and 5B). Unlike our expectation, B-RafV600E-expressing HOSEdid not show the upregulation estrogen receptor α and β.
１８ A.
Fig5.Level of estrogen receptor α and β in B-RafV600E-expressing HOSE.
(A) Measurementof mRNA. At 12 days after infection, we performed Real-time PCR. (B) Measurement of protein. On the same day, we performed immunoblotting.
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6. Estrogen regulates translocation of p-Erk1/2 in B-RafV600E-expressing HOSE.
We found that estrogen inhibits B-RafV600E induced senescence in HOSE. But, we wondered that how estrogen inhibitedOIS.Estrogen regulated translocation of p-Erk1/2 (Chen et al., 2005).For these reasons, we analyzed localization of p-Erk1/2 by using confocal microscopy and counted by using image of immunocytochemistry (Fig. 6A). p-Erk1/2 is mainly accumulated in cytosol in B-RafV600E-expressing HOSE. But, p-Erk1/2 located in nuclear in estrogen treated B-RafV600E-expressing HOSE. Next, we focused on which mechanism is involved in translocation of p-Erk1/2. It was established that PEA15 regulated location of Erk1/2 (Lee et al., 2015). Therefore, we measured level of PEA15 and p-PEA15S104 (Fig. 6B) (Chen et al., 2008; Lee et al., 2015). We observed that p-PEA15S104 is increased in estrogen treated B-RafV600E-expressing HOSE. These results suggested that estrogen regulates translocation of p-Erk1/2 via phosphorylation of PEA15.
２０ A.
２１ B.
Fig6. Estrogen mediated translocation of p- Erk1/2.
(A) Localization of p-Erk1/2. We performed confocal microscopy 19 days after infection and 16 days after treatment of E2and counted by using image J. (B) Level of protein.
Immunoblotting of PEA15, p-PEA15S104 and α-Tubulin is performed19 days after infection and 16 days after treatment of E2.
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IV.Discussion
If residue 600 amino acid val of the B-Raf is optionally substituted with glu, B-Raf always has the active form in cell signaling pathway which causes a more than cell growth and can be oncogene (Michaloglou et al., 2008). B-RafV600E has been reported up to 14% in serous carcinoma (SOC) (Grisham et al., 2013) and up to 71% in serous borderline tumor (SBT) (Malpica and Wong, 2016). For patients receiving the post menopause in women and ovarian cancer resection may increase the risk of ovarian cancer by implementing the estrogen replacement therapy (Risch, 1996; Riman et al., 2002; Morch et al., 2009).
In this study, we observed whether estrogen regulates B-RafV600E-induced senescence in human primary ovarian epithelial cells (HOSE). Thus, we overexpressed B-Raf protein by using lentivirus system. B-RafV600E-expressing HOSE was decreased in cell growth and increased in SA-B-GAL and expression of p21 but not expression of p16. Next, to identify estrogen effect, 40nM 17-b-estradiol (E2) was treated in normal HOSE and B-RafV600E-expressing HOSE every 2 day. B-RafV600E-B-RafV600E-expressing HOSE treated E2 increased in cell growth and level of cyclin D1 and decreased in SA-β-gal compared with B-RafV600E-expressing HOSE. Other hand, no significant difference in normal HOSE and normal HOSE treated E2 better than B-Rafv600e-expressing HOSE. After that, we observed estrogen receptor α and β whether overexpression of B-Raf affects to expression of estrogen receptors.
However, we couldn’t found the difference.To know how estrogen inhibits B-RafV600E-induced senescence and how cell growth was reversed, we focused on relation between estrogen and phosphorylation of Erk1/2. Estrogen can translocate phosphorylation of Erk1/2 into nucleus (Chen et al., 2005). As you know, Erk1/2 must translocate in nucleus to proliferate cells .So, we carried out localization of p-Erk1/2 through Immunofluorescence and confocal microscopy. We proved that p-Erk1/2 was accumulated in cytosol in RafV600E-expressing HOSE. Other hand, p-Erk1/2 was translocated in nucleus in B-RafV600E-expressing HOSE treated E2. It was reported that pea-15 regulates p-Erk1/2(Krueger et al., 2005; Lee et al., 2015). In our experiment, to identify whether PEA15
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was involved in translocation of Erk1/2, we observed level of protein including pea-15, p-pea15S104 and p-Erk1/2. As a result, phosphorylation of pea-15 increased in B-RafV600E-expressing HOSE treated E2.
In further study, we will count amount of p-Erk1/2 in nucleus. And then, we will prove interaction between p-Erk1/2 and PEA15 by performing immunoprecipitation.
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