Raloxifene Increases Proliferation and Up-regulates Telomerase Activity in Human Umbilical Vein Endothelial Cells

Abstract

Vascular endothelial senescence is involved in human atherosclerosis. Telomerase activity is known to be critical in cellular senescence and its level is modulated by regulation of telomerase catalytic subunit (telomerase reverse transcriptase (TERT)) at both the transcriptional and post-transcriptional levels. Since the cardioprotective effect of estrogen itself has not been ruled out, we examined that of raloxifene, which has been classified as a selective estrogen receptor modulator, on the proliferation and telomerase activity of human umbilical vein endothelial cells (HUVECs). Raloxifene, like estrogen, clearly induced the telomerase activity and human TERT (hTERT) expression via estrogen receptor (ER) α and ERβ. Treatment with raloxifene for 5 days significantly induced cell growth, and either cotreatment with a telomerase inhibitor, 3′-azido-3′-deoxythymidine, or transfection with hTERT-specific small interfering RNA significantly attenuated the raloxifene-induced cell growth. Raloxifene also induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, significantly attenuated the raloxifene-induced telomerase activity. In addition, raloxifene induced both the phosphorylation of hTERT and IκB. Moreover, cotreatment with an IκBα phosphorylation inhibitor, BAY-11–7082, or a specific NFκB nuclear translocation inhibitor, SN50, significantly attenuated the raloxifene-induced telomerase activity and the association of NFκB with hTERT. These results show that raloxifene induced the up-regulation of telomerase activity not only by the transcriptional regulation of hTERT but also by post-translational regulation of the phosphorylation of Akt and hTERT and the association of hTERT with NFκB in HUVECs. Thus, the up-regulation of telomerase activity in vascular endothelial cells might be one mechanism contributing to the potential atheroprotective effect of raloxifene. Vascular endothelial senescence is involved in human atherosclerosis. Telomerase activity is known to be critical in cellular senescence and its level is modulated by regulation of telomerase catalytic subunit (telomerase reverse transcriptase (TERT)) at both the transcriptional and post-transcriptional levels. Since the cardioprotective effect of estrogen itself has not been ruled out, we examined that of raloxifene, which has been classified as a selective estrogen receptor modulator, on the proliferation and telomerase activity of human umbilical vein endothelial cells (HUVECs). Raloxifene, like estrogen, clearly induced the telomerase activity and human TERT (hTERT) expression via estrogen receptor (ER) α and ERβ. Treatment with raloxifene for 5 days significantly induced cell growth, and either cotreatment with a telomerase inhibitor, 3′-azido-3′-deoxythymidine, or transfection with hTERT-specific small interfering RNA significantly attenuated the raloxifene-induced cell growth. Raloxifene also induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, significantly attenuated the raloxifene-induced telomerase activity. In addition, raloxifene induced both the phosphorylation of hTERT and IκB. Moreover, cotreatment with an IκBα phosphorylation inhibitor, BAY-11–7082, or a specific NFκB nuclear translocation inhibitor, SN50, significantly attenuated the raloxifene-induced telomerase activity and the association of NFκB with hTERT. These results show that raloxifene induced the up-regulation of telomerase activity not only by the transcriptional regulation of hTERT but also by post-translational regulation of the phosphorylation of Akt and hTERT and the association of hTERT with NFκB in HUVECs. Thus, the up-regulation of telomerase activity in vascular endothelial cells might be one mechanism contributing to the potential atheroprotective effect of raloxifene. The risk of cardiovascular disease steeply increases after menopause. Many epidemiological and basic studies have shown that estrogen has the significant function in the vasculature of preventing the primary development of cardiovascular disease in women (1Stampfer M.J. Colditz G.A. Willett W.C. Manson J.E. Rosner B. Speizer F.E. Hennekens C.H. N. Engl. J. Med. 1991; 325: 756-762Crossref PubMed Scopus (1987) Google Scholar, 2Mendelsohn M.E. Karas R.H. N. Engl. J. Med. 1999; 340: 1801-1811Crossref PubMed Scopus (2509) Google Scholar). In the Women's Health Initiative, a large prospective randomized controlled study, although women on the conjugated equine estrogen-medroxyprogesterone acetate arm had an increase in the relative risk of cardiovascular events and breast cancer (3Writing Group for the Women's Health Initiative InvestigatorsJ. Am. Med. Assoc. 2002; 288: 321-333Crossref PubMed Scopus (14017) Google Scholar), the more recent reports indicated that on women on the conjugated equine estrogenonly treatment arm experienced a significant increase in the risk of stroke compared with women treated with placebo but did not show an increase in cardiovascular disease (4Women's Health Initiative Steering CommitteeJ. Am. Med. Assoc. 2004; 291: 1701-1712Crossref PubMed Scopus (4166) Google Scholar). Thus, the cardioprotective effect of estrogen itself has not been ruled out by the results of the Women's Health Initiative study. Recently, we reported that medroxyprogesterone acetate attenuates the induction of both endothelial nitric-oxide synthase (eNOS) 2The abbreviations used are: eNOS, endothelial nitric-oxide synthase; HUVEC, human umbilical vein endothelial cell; ER, estrogen receptor; ERK, extracellular signal-regulated kinase; TERT, telomerase reverse transcriptase; siRNA, small interfering RNA; TRAP, telomeric repeat amplification protocol; CSS, charcoal-stripped serum; RT, reverse transcription; E2, estradiol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PI3K, phosphatidylinositol 3-kinase; KO, knock-out. activity and NO production by estrogen in human umbilical vein endothelial cells (HUVECs) (5Oishi A. Ohmichi M. Takahashi K. Takahashi T. Mori-Abe A. Kawagoe J. Otsu R. Mochizuki Y. Inaba N. Kurachi H. Biochem. Biophys. Res. Commun. 2004; 324: 193-198Crossref PubMed Scopus (23) Google Scholar). Thus, it is possible that progestin has adverse effects on the cardiovascular system. Therefore, there is a need for an ideal estrogen analog that does not require the addition of progestin to protect the uterine endometrium. Raloxifene is a nonsteroidal benzothiophene that has been classified as a selective estrogen receptor modulator based on the fact that it exerts estrogen-agonistic effects on bone (6Delmas P.D. Bjarnason N.H. Mitlak B.H. Ravoux A.C. Shah A.S. Huster W.J. Draper M. Christiansen C. N. Engl. J. Med. 1997; 337: 1641-1647Crossref PubMed Scopus (1600) Google Scholar) and lipid metabolism (7Walsh B.W. Kuller L.H. Wild R.A. Paul S. Farmer M. Lawrence J.B. Shah A.S. J. Am. Med. Assoc. 1998; 279: 1445-1451Crossref PubMed Scopus (672) Google Scholar) and estrogen-antagonistic effects on the uterine endometrium (6Delmas P.D. Bjarnason N.H. Mitlak B.H. Ravoux A.C. Shah A.S. Huster W.J. Draper M. Christiansen C. N. Engl. J. Med. 1997; 337: 1641-1647Crossref PubMed Scopus (1600) Google Scholar) and breast tissue (8Cummings S.R. Eckert S. Krueger K.A. Grady D. Powles T.J. Cauley J.A. Norton L. Nickelsen T. Bjarnason N.H. Morrow M. Lippman M.E. Black D. Glusman J.E. Costa A. Jordan V.C. J. Am. Med. Assoc. 1999; 281: 2189-2197Crossref PubMed Scopus (1845) Google Scholar). However, its ability to protect against cardiovascular disease has yet to be proven. We demonstrated previously that the effects (and the mechanisms thereof) of raloxifene on vascular endothelial and smooth muscle cells were similar to those of estrogen. For instance, in vascular endothelial cells, raloxifene induces eNOS phosphorylation, as estrogen does. Its effect is mediated by estrogen receptor (ER) α via a transcription-independent mechanism, a so-called nongenomic mechanism, and is differentially mediated by an Akt- and ERK-dependent cascade (9Hisamoto K. Ohmichi M. Kanda Y. Adachi K. Nishio Y. Hayakawa J. Mabuchi S. Takahashi K. Tasaka K. Miyamoto Y. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 47642-47649Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 10Hisamoto K. Ohmichi M. Kurachi H. Hayakawa J. Kanda Y. Nishio Y. Adachi K. Tasaka K. Miyoshi E. Fujiwara N. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 3459-3467Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). In vascular smooth muscle cells, raloxifene exerts an antiproliferative effect on cells treated with platelet-derived growth factor mediated by ERα, as estrogen does, in part via a transcription-dependent mechanism, a so-called genomic mechanism (11Takahashi K. Ohmichi M. Yoshida M. Hisamoto K. Mabuchi S. Arimoto-Ishida E. Mori A. Tsutsumi S. Tasaka K. Murata Y. Kurachi H. J. Endocrinol. 2003; 178: 319-329Crossref PubMed Scopus (54) Google Scholar), and in part via a nongenomic mechanism (12Mori-Abe A. Tsutsumi S. Takahashi K. Toya M. Yoshida M. Du B. Kawagoe J. Nakahara K. Takahashi T. Ohmichi M. Kurachi H. J. Endocrinol. 2003; 178: 417-426Crossref PubMed Scopus (54) Google Scholar). The incidence of atherosclerosis increases with age. Aging is associated with endothelial dysfunction. On a cellular level, aging leads to an irreversible state of cell cycle arrest known as senescence (13Goldstein S. Science. 1990; 249: 1129-1133Crossref PubMed Scopus (600) Google Scholar). It is generally believed that an important factor in regulating cellular life span is the telomere length (14Buys C.H. N. Engl. J. Med. 2000; 342: 1282-1283Crossref PubMed Scopus (59) Google Scholar). Telomerase, an RNA-dependent DNA polymerase, catalyzes the addition of telomeric repeat sequences to chromosome ends and delays the development of senescence (14Buys C.H. N. Engl. J. Med. 2000; 342: 1282-1283Crossref PubMed Scopus (59) Google Scholar). It was reported that telomerase inactivation precedes the aging of vascular endothelial cells (15Vasa M. Breitschopf K. Zeiher A.M. Dimmeler S. Circ. Res. 2000; PubMed Scopus Google Scholar) and cells with in human T. H. Yoshida T. Y. Yoshida H. 2002; PubMed Scopus Google Scholar). In addition, the expression of human telomerase reverse transcriptase the catalytic subunit of in endothelial cells was reported to to the induction of J. E. A.M. Y. A. A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, endothelial cell senescence induced by telomere to We reported that raloxifene induces eNOS phosphorylation via the Akt cascade (9Hisamoto K. Ohmichi M. Kanda Y. Adachi K. Nishio Y. Hayakawa J. Mabuchi S. Takahashi K. Tasaka K. Miyamoto Y. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 47642-47649Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). NO was reported to telomerase and endothelial cell senescence (15Vasa M. Breitschopf K. Zeiher A.M. Dimmeler S. Circ. Res. 2000; PubMed Scopus Google Scholar). In addition, it was reported that the in hTERT to a for phosphorylation by Akt and that Akt human telomerase activity phosphorylation of hTERT T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). These to raloxifene telomerase activity and to to the mechanism In the we show that raloxifene telomerase activity not only by transcriptional regulation of hTERT but also by post-translational regulation of the phosphorylation of hTERT and the association of hTERT with and were and were and were siRNA, siRNA, and were was The cell proliferation was The telomeric repeat amplification was of were The umbilical vein was with and for to the of endothelial The endothelial cells were in and in endothelial cell growth for cell or endothelial cells were in and as by the were in cell or were in expression or estrogen and endothelial cells not were on cells that had previously of human by growth factor were days after to a and or charcoal-stripped but growth factor and for cells were by and at in cell of the to of were for telomerase activity by a telomeric repeat amplification which is based on the by Res. 1997; PubMed Scopus Google Scholar). Telomerase activity was the of the of the telomeric repeat at to that of the in were to the an of cells in Telomerase activity is which a of the to and to the in the by of The and of as a of telomerase activity have been by Norton Scopus Google Scholar). RNA was by in were in a at a to that hTERT or was with of of were in a of the were to the and by of of and were and the was to the cell The cell was on a for The was to and the was for were in with or raloxifene in the or of The was the The of cells was by the at days and 5 in and for The cell and the cells in for as reported previously Y. E. S. Biol. 2003; PubMed Scopus Google Scholar). However, were with similar were growth of cells was by in for by with with or raloxifene for The of cells was by the at of the after addition of for as previously J. Ohmichi M. Kurachi H. Kanda Y. Hisamoto K. Nishio Y. Adachi K. Tasaka K. T. Murata Y. Res. 2000; Google Scholar). of cellular RNA was The expression of hTERT and glyceraldehyde-3-phosphate was by amplification as previously S. M. T. K. Nishio Y. A. M. Res. 1999; Google Scholar). hTERT were the and was of RNA a RNA with of RNA was by to The the and of was the of the were by of in of and and of DNA and specific at cycle of at for at for and at for were by in a The of was by with glyceraldehyde-3-phosphate as previously S. M. T. K. Nishio Y. A. M. Res. 1999; Google Scholar). were via after the were and raloxifene. were treated with or raloxifene by for were by an of after the treatment were and were and with a RNA was and we and to the of TERT The used in were as reverse glyceraldehyde-3-phosphate and reverse The of was in cells were in for and treated with were with and in and The were at at for and the of the were the of were by and to was in in were with specific primary For of hTERT or of the association of hTERT with NFκB cell were The were with and for with were with and by with or in the were with or by the system. was by and was The as the Raloxifene via the of Telomerase and hTERT examined raloxifene the proliferation of HUVECs. The of cells was significantly by with raloxifene We treated with raloxifene in the of an of reverse transcriptase S. Y. Y. M. Y. J. T. A. B. J. 1998; PubMed Google Scholar, C. Res. PubMed Scopus Google Scholar, 1997; Google Scholar, M. T. T. N. D. Res. 2002; Google Scholar), to telomerase activity was involved in the induction of endothelial cell proliferation by raloxifene. did not the cell significantly the induction of proliferation by raloxifene to raloxifene or estrogen induced the telomerase were treated with raloxifene or estrogen for and to to the telomerase activity. Treatment with either raloxifene or estrogen significantly the telomerase activity Moreover, we used hTERT-specific (hTERT) to the transcriptional of hTERT was involved in the induction of endothelial cell proliferation by raloxifene. We that the expression of hTERT in with hTERT was significantly that in with transfection with had effect on the induction of cell proliferation by raloxifene, transfection with hTERT significantly the induction of cell proliferation by raloxifene Raloxifene Telomerase via and We examined the is involved in the raloxifene-induced telomerase were treated with raloxifene in the or of and to to the telomerase activity. Treatment with clearly attenuated the raloxifene-induced telomerase that the is involved in the induction of telomerase activity by raloxifene we examined which of is involved in the raloxifene-induced telomerase both and We that the expression of and in with and were significantly that in with Moreover, transfection with either or significantly the induced activity S. Ohmichi M. A. Y. Hisamoto K. Arimoto-Ishida E. Nishio Y. Takahashi K. Tasaka K. Murata Y. 2004; PubMed Scopus Google Scholar), that the estrogen not telomerase activity was attenuated by transfection with or These that both and involved in the induction of telomerase activity by raloxifene. Raloxifene hTERT in and in were to or not the raloxifene-induced increase in the telomerase activity was to an increase in the expression of hTERT Treatment of with raloxifene or estrogen for the of hTERT Moreover, raloxifene as as estrogen the expression of in of These results that the up-regulation of telomerase activity by raloxifene and estrogen at in to an increase in the expression of hTERT Raloxifene Telomerase via the activity also be by post-translational of the It has been reported that the in hTERT to a for phosphorylation by Akt and that Akt human telomerase activity the phosphorylation of hTERT T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, we examined raloxifene induces the phosphorylation of were treated with raloxifene for and the cell were by and to with or raloxifene did not the of Akt it induced phosphorylation of Akt and with a effect at we examined raloxifene induces the phosphorylation of hTERT at the Akt phosphorylation For cells were treated with raloxifene for the indicated and the cell were with and to with raloxifene did not the expression of the increase by raloxifene of the phosphorylation of hTERT at the Akt phosphorylation a at and and the cascade is involved in the and telomerase were treated with raloxifene or estrogen in the or of and to to the telomerase activity. clearly attenuated both the and telomerase that the cascade is involved in the induction of telomerase activity by raloxifene and estrogen. Raloxifene Telomerase by the of NFκB with has been reported that and telomerase activity via in a human cell M. T. T. N. D. Res. 2002; Google Scholar), and NFκB was reported to be a post-translational of telomerase that by the of hTERT M. T. T. N. D. Res. 2003; Google Scholar). NFκB is its association with an which NFκB in the of by its NFκB to to the with hTERT J.A. 1999; PubMed Scopus Google Scholar, J.A. S.R. 1999; PubMed Scopus Google Scholar, R. J. T. M.E. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). We examined or not raloxifene induces the phosphorylation of IκB. For cells were treated with raloxifene and by with or raloxifene did not the expression of Akt it the level of with a effect at and Moreover, we examined raloxifene induces the association of NFκB with hTERT. were treated with raloxifene and used to cell that were with and to with Raloxifene did not the expression of but the association of hTERT with NFκB was by raloxifene with a effect at and with an IκBα phosphorylation R. J. T. M.E. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) attenuated the raloxifene-induced association of hTERT with NFκB with a specific NFκB nuclear translocation or also attenuated the induction of telomerase activity by raloxifene that association NFκB and hTERT was involved in the raloxifene-induced telomerase the of the effects of LY294002, SN50, and on the we examined the effects of LY294002, SN50, and on the raloxifene-induced which we reported previously (9Hisamoto K. Ohmichi M. Kanda Y. Adachi K. Nishio Y. Hayakawa J. Mabuchi S. Takahashi K. Tasaka K. Miyamoto Y. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 47642-47649Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). For cells were treated with raloxifene LY294002, SN50, or and to with with LY294002, SN50, or had effect on the raloxifene-induced of the in was that telomerase activity was induced by raloxifene and the telomerase was involved in the raloxifene-induced cell proliferation of HUVECs. The was that raloxifene induced the up-regulation of telomerase activity by mechanisms in by the of hTERT hTERT phosphorylation the cascade and the association of hTERT with NFκB We have reported that the increase of the phosphorylation of Akt induced by raloxifene is for the raloxifene-induced of NO in endothelial cells (9Hisamoto K. Ohmichi M. Kanda Y. Adachi K. Nishio Y. Hayakawa J. Mabuchi S. Takahashi K. Tasaka K. Miyamoto Y. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 47642-47649Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Thus, the phosphorylation of Akt is involved in both NO and cell proliferation in vascular endothelial cells, Akt have an important in the of vascular endothelial It that raloxifene-induced hTERT phosphorylation raloxifene-induced expression of hTERT as we previously J. Ohmichi M. Takahashi T. C. Mabuchi S. Takahashi K. H. Mori-Abe A. M. Du B. T. A. S. M. Kurachi H. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, B. Ohmichi M. Takahashi K. Kawagoe J. C. H. Mori-Abe A. M. T. A. M. N. Takahashi T. Kurachi H. J. Endocrinol. 2004; PubMed Scopus Google Scholar, A. Ohmichi M. Kawagoe J. S. Mabuchi S. Takahashi T. C. Arimoto-Ishida E. Nishio Y. M. Kurachi H. Tasaka K. Murata Y. 2004; PubMed Scopus Google Scholar). Since telomerase activity is not only by transcriptional regulation of hTERT but also by post-transcriptional regulation via phosphorylation of the of raloxifene-induced telomerase activity might be that of raloxifene-induced expression of hTERT transcriptional and post-translational regulation in or at the in and be to We previously reported that raloxifene the up-regulation of telomerase activity in human breast cancer cells J. Ohmichi M. Takahashi T. C. Mabuchi S. Takahashi K. H. Mori-Abe A. M. Du B. T. A. S. M. Kurachi H. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), raloxifene, like estrogen, induced the telomerase activity in cells B. Ohmichi M. Takahashi K. Kawagoe J. C. H. Mori-Abe A. M. T. A. M. N. Takahashi T. Kurachi H. J. Endocrinol. 2004; PubMed Scopus Google Scholar). Raloxifene also induced the telomerase activity in like estrogen and Thus, raloxifene an effect in human breast cancer cells, it an effect in and HUVECs. We previously demonstrated that the hTERT an and expression of the hTERT that is for the transcriptional by estrogen A. Ohmichi M. Kawagoe J. S. Mabuchi S. Takahashi T. C. Arimoto-Ishida E. Nishio Y. M. Kurachi H. Tasaka K. Murata Y. 2004; PubMed Scopus Google Scholar), that hTERT is a of estrogen. Recently, were reported to in the tissue of selective estrogen receptor B.W. 2004; PubMed Scopus Google Scholar). involved in the activity of raloxifene in breast cancer However, the mechanism by which raloxifene exerts its effect in vascular endothelial cells We the of in the raloxifene-induced telomerase activity in vascular endothelial Raloxifene the cell proliferation by of up-regulation of telomerase activity via post-translational regulation of phosphorylation of hTERT in human breast cancer cells J. Ohmichi M. Takahashi T. C. Mabuchi S. Takahashi K. H. Mori-Abe A. M. Du B. T. A. S. M. Kurachi H. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Raloxifene also the platelet-derived growth cell proliferation in vascular smooth muscle cells, in part of the expression of by of mediated by via a transcription-dependent mechanism, a so-called genomic mechanism (11Takahashi K. Ohmichi M. Yoshida M. Hisamoto K. Mabuchi S. Arimoto-Ishida E. Mori A. Tsutsumi S. Tasaka K. Murata Y. Kurachi H. J. Endocrinol. 2003; 178: 319-329Crossref PubMed Scopus (54) Google Scholar), and in part by a cascade is mediated by via a nongenomic mechanism (12Mori-Abe A. Tsutsumi S. Takahashi K. Toya M. Yoshida M. Du B. Kawagoe J. Nakahara K. Takahashi T. Ohmichi M. Kurachi H. J. Endocrinol. 2003; 178: 417-426Crossref PubMed Scopus (54) Google Scholar). In raloxifene, like estrogen B.W. 2004; PubMed Scopus Google Scholar, L. M. J. 1999; Full Text PDF PubMed Scopus Google Scholar, J. S. H. K. Y. Res. 2002; PubMed Scopus Google Scholar), induced cell proliferation in vascular endothelial cells by the up-regulation of telomerase activity via post-translational regulation of phosphorylation of hTERT. Thus, the tissue of selective estrogen receptor also as reported previously for the It has been reported that estrogen receptor be in and and for the regulation of NO in vascular endothelial cells R.A. Am. J. 2004; Scopus Google Scholar). However, of in the induction of cell proliferation by selective estrogen receptor via the nongenomic mechanism and in as reported previously M.J. 2002; Scopus Google Scholar). is involved in the raloxifene-induced telomerase We have reported that estrogen K. Ohmichi M. Kurachi H. Hayakawa J. Kanda Y. Nishio Y. Adachi K. Tasaka K. Miyoshi E. Fujiwara N. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 3459-3467Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar) and raloxifene (9Hisamoto K. Ohmichi M. Kanda Y. Adachi K. Nishio Y. Hayakawa J. Mabuchi S. Takahashi K. Tasaka K. Miyamoto Y. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 47642-47649Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) induced the of eNOS via to but not in vascular endothelial Moreover, we have shown that estrogen and raloxifene induced telomerase activity in cells with the B. Ohmichi M. Takahashi K. Kawagoe J. C. H. Mori-Abe A. M. T. A. M. N. Takahashi T. Kurachi H. J. Endocrinol. 2004; PubMed Scopus Google Scholar) and estrogen induced the of Akt via ERα, but not via in human cancer cells S. Ohmichi M. A. Nishio Y. Arimoto-Ishida E. N. Tasaka K. Murata Y. 2004; PubMed Scopus Google Scholar). However, estrogen treatment the vascular in the and M.J. 2002; Scopus Google Scholar, Karas R.H. M. M.E. J. PubMed Scopus Google Scholar, R.H. J.B. M. M. J.A. M.E. S. A. 1999; PubMed Scopus Google Scholar) but does not the in R.H. H. M.J. C. J.A. M.E. Circ. Res. 2001; PubMed Scopus Google Scholar), that and in vascular that raloxifene induced telomerase activity in via to and estrogen and raloxifene NO production (9Hisamoto K. Ohmichi M. Kanda Y. Adachi K. Nishio Y. Hayakawa J. Mabuchi S. Takahashi K. Tasaka K. Miyamoto Y. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 47642-47649Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 10Hisamoto K. Ohmichi M. Kurachi H. Hayakawa J. Kanda Y. Nishio Y. Adachi K. Tasaka K. Miyoshi E. Fujiwara N. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 3459-3467Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar) and telomerase activity via the Akt cascade in vascular endothelial cells, that both the vascular endothelial In the addition of progestin is to protect the uterine endometrium. We reported that medroxyprogesterone acetate attenuates the induction of both eNOS activity and NO production by estrogen in (5Oishi A. Ohmichi M. Takahashi K. Takahashi T. Mori-Abe A. Kawagoe J. Otsu R. Mochizuki Y. Inaba N. Kurachi H. Biochem. Biophys. Res. Commun. 2004; 324: 193-198Crossref PubMed Scopus (23) Google Scholar). Since raloxifene exerts estrogen-antagonistic effects on the uterine endometrium (6Delmas P.D. Bjarnason N.H. Mitlak B.H. Ravoux A.C. Shah A.S. Huster W.J. Draper M. Christiansen C. N. Engl. J. Med. 1997; 337: 1641-1647Crossref PubMed Scopus (1600) Google Scholar), the addition of progestin is not In addition, although estrogen induces the of and raloxifene has adverse effects on the of and N. Engl. J. Med. 2003; PubMed Scopus Google Scholar). The of raloxifene was a randomized to raloxifene the risk of in women with E. Grady D. A. K. J. Am. Med. Assoc. 2002; PubMed Scopus Google Scholar). The development of cardiovascular disease was a of the In raloxifene had effect on the incidence of cardiovascular events in the in a of women at risk for cardiovascular those to raloxifene compared with placebo experienced significantly cardiovascular there is a that the effects of raloxifene be more in endothelial cells more to atherosclerosis and more to the aging and be to the for the which is a randomized to raloxifene the incidence of cardiovascular disease in is Circ. Res. 1999; PubMed Scopus Google Scholar), and we the