Raloxifene improves the ovariectomy-induced impairment in endothelium-dependent vasodilation.

OBJECTIVE: To examine the effect of raloxifene on the endothelial dysfunction caused by surgical menopause. DESIGN: Ten premenopausal women who underwent gynecological surgery with ovariectomy were divided into two groups. Five participants used raloxifene (60 mg/d) for 7 days staring 1 week after the surgery, and the other five participants did not use raloxifene. We examined the changes in flow-mediated dilatation (FMD) of the brachial artery using ultrasonography. Vasodilation in response to nitroglycerin was also studied. We also measured the brachial-ankle pulse wave velocity to examine the change in arterial stiffness in these participants before and after surgical menopause. RESULTS: In both the raloxifene and control groups, a significant decrease in FMD was observed 1 week after the surgery. Although no further changes in FMD were observed in the control group at 2 weeks after surgery, FMD was significantly increased in the raloxifene group. No remarkable changes in nitroglycerin or brachial-ankle pulse wave velocity were observed after surgery in either group. CONCLUSIONS: Raloxifene rapidly restored FMD that was impaired after surgical menopause. Therefore, raloxifene may be effective for ameliorating and maintaining endothelial function in premenopausal women who undergo ovariectomy.

Medroxyprogesterone acetate induces cell proliferation through up-regulation of cyclin D1 expression via phosphatidylinositol 3-kinase/Akt/nuclear factor-kappaB cascade in human breast cancer cells.

The mechanism of medroxyprogesterone acetate (MPA)-induced cell proliferation in human breast cancer cells remains elusive. We examined the mechanism by which MPA affects the cyclin D1 expression in progesterone receptor (PR)-positive T47D human breast cancer cells. MPA (10 nM) treatment for 48 h induced proliferation of the cells (1.6-fold induction). MPA induced cyclin D1 expression (3.3-fold induction), and RU486, a selective PR antagonist, blocked the MPA-induced cell proliferation and cyclin D1 expression (23% inhibition). MPA increased both the protein level (2.2-fold induction) and promoter activity (2.7-fold induction) of cyclin D1 in MCF-7 cells transfected with PRB but not with PRA. Although MPA transcriptionally activated cyclin D1 expression, cyclin D1 promoter does not have progesterone-responsive element-related sequence. We further examined the mechanism for the regulation of the cyclin D1 expression. Because the cyclin D1 promoter contains three putative nuclear factor-kappaB (NFkappaB)-binding motifs and NFkappaB is a substrate of Akt, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)/Akt/NFkappaB cascade on the responses of cyclin D1 to MPA. MPA induced the transient phosphorylation of Akt (2.7-fold induction at 5 min), and treatment with PI3K inhibitor (wortmannin) attenuated the MPA-induced up-regulation of cyclin D1 expression (40% inhibition) and cell proliferation (40% inhibition). MPA also induced phosphorylation of inhibitor of NFkappaBalpha (IkappaBalpha) (2.3-fold induction), and treatment with wortmannin attenuated the MPA-induced IkappaBalpha phosphorylation (60% inhibition). Treatment with an IkappaBalpha phosphorylation inhibitor (BAY 11-7085) or a specific NFkappaB nuclear translocation inhibitor (SN-50) attenuated the MPA-induced up-regulation of both cyclin D1 expression (80 and 50% inhibition, respectively) and cell proliferation (55 and 34% inhibition, respectively). Because MPA induced a transient phosphorylation of Akt and the cyclin D1 promoter contains no progesterone-responsive element-related sequence, the MPA-induced cell proliferation through PRB by up-regulation of cyclin D1 expression via the PI3K/Akt/NFkappaB cascade may be a nongenomic mechanism.

Growth factors change nuclear distribution of estrogen receptor-alpha via mitogen-activated protein kinase or phosphatidylinositol 3-kinase cascade in a human breast cancer cell line.

In the present study, to examine the dynamic changes in the localization of nuclear estrogen receptor (ER)alpha induced by growth factors, we used time-lapse confocal microscopy to directly visualized ERalpha fused with green fluorescent protein (GFP-ERalpha) in single living cells treated with epidermal growth factor (EGF) or IGF-I. We observed that 17beta-estradiol (E2) changed the normally diffuse distribution of GFP-ERalpha throughout the nucleoplasm to a hyperspeckled distribution within 10 min. Both EGF and IGF-I also changed the nuclear distribution of GFP-ERalpha, similarly to E2 treatment. However, the time courses of the nuclear redistribution of GFP-ERalpha induced by EGF or IGF-I were different from that induced by E2 treatment. In the EGF-treated cells, the GFP-ERalpha nuclear redistribution was observed at 30 min and reached a maximum at 60 min, whereas in the IGF-I-treated cells, the GFP-ERalpha nuclear redistribution was observed at 60 min and reached a maximum at 90 min. The EGF-induced redistribution of GFP-ERalpha was blocked by pretreatment with a MAPK cascade inhibitor, PD98059, whereas the IGF-I-induced redistribution of GFP-ERalpha was blocked by pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002. Analysis using an activation function-2 domain deletion mutant of GFP-ERalpha showed that the change in the distribution of GFP-ERalpha was not induced by E2, EGF, or IGF-I treatment. These data suggest that MAPK and phosphatidylinositol 3-kinase cascades are involved in the nuclear redistribution of ERalpha by EGF and IGF-I, respectively, and that the activation function-2 domain of ERalpha may be needed for the nuclear redistribution of ERalpha.

Both estrogen and raloxifene protect against beta-amyloid-induced neurotoxicity in estrogen receptor alpha-transfected PC12 cells by activation of telomerase activity via Akt cascade.

Although estrogen is known to protect against beta-amyloid (Abeta)-induced neurotoxicity, the mechanisms responsible for this effect are only beginning to be elucidated. In addition, the effect of raloxifene on Abeta-induced neuro-toxicity remains unknown. Here we investigated whether raloxifene exhibits similar neuro-protective effects to estrogen against Abeta-induced neurotoxicity and the mechanism of the effects of these agents in PC12 cells transfected with the full-length human estrogen receptor (ER) alpha gene (PCER). Raloxifene, like 17beta-estradiol (E2), significantly inhibited Abeta-induced apoptosis in PCER cells, but not in a control line of cells transfected with vector DNA alone (PCCON). Since telomerase activity, the level of which is modulated by regulation of telomerase catalytic subunit (TERT) at both the transcriptional and post-transcriptional levels, is known to be involved in suppressing apoptosis in neurons, we examined the effect of E2 and raloxifene on telomerase activity. Although both E2 and raloxifene induced telomerase activity in PCER cells, but not in PCCON cells, treated with Abeta, they had no effect on the level of TERT expression. These results suggest that neither E2 nor raloxifene affects the telomerase activity at the transcriptional level. We therefore studied the mechanism by which E2 and raloxifene induce the telomerase activity at the post-transcriptional level. Both E2 and raloxifene induced the phosphorylation of Akt, and pre-treatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, attenuated both E2- and raloxifene-induced activation of the telomerase activity. Moreover, both E2 and raloxifene induced both the phosphorylation of TERT at a putative Akt phosphorylation site and the association of nuclear factor kappaB with TERT. Our findings suggest that and raloxifene exert neuroprotective effects by E2 telomerase activation via a post-transcriptional cascade in an experimental model relevant to Alzheimer's disease.

Long-term hormone replacement therapy delays the age related progression of carotid intima-media thickness in healthy postmenopausal women.

OBJECTIVES: Carotid intima-media thickness (IMT) is an appropriate intermediate end point to investigate clinically relevant effects on atherogenesis. The study objective was to clarify whether long-term hormone replacement therapy (HRT) modifies the progress of age-related IMT in healthy postmenopausal Japanese women. METHODS: One hundred and eighty-eight healthy postmenopausal women aged 42-69 years were recruited into the retrospective study. IMT was measured by B-mode real-time ultrasound in the following three groups of patients. One hundred and fifteen women who were prescribed estrogen plus progestin or estrogen alone were classified into two groups according to the HRT treated period: short-term (<2 years of treatment, n = 52) and long-term (> or =2 years, n = 63) HRT groups. The third group consisted an age-matched women (n = 73), who were never treated with HRT (non-HRT group) as a control. RESULTS: Each group was divided into three subgroups according to age: < or =49 years, 50-59 years and 60 years or older. IMT in patients of age > or =60 years in the non-HRT group was 0.607 +/- 0.064 mm and was significantly higher compared with that in the other two age subgroups of non-HRT patients (< or =49 years: [0.495 +/- 0.051 mm; 50-59 years: 0.505 +/- 0.068 mm) (P < 0.05). In the short-term HRT group, IMT of > or =60-year-old-subjects (0.588 +/- 0.074 mm) was also significantly higher compared with that in the other two age subgroups (< or =49 years: 0.480 +/- 0.034 mm; 50-59 years: 0.511 +/- 0.062 mm). However, in the long-term HRT group, IMT was not significantly different among the three age subgroups. There was a significant relationship between IMT and age in non-HRT (r = 0.594, P < 0.0001) and short-term HRT (r = 0.542, P < 0.001) groups, but no significant relationship was observed in the long-term HRT (r = 0.195 , P = 0.1266) group. CONCLUSIONS: In long-term HRT, more than 2 years may delay the age-related increase in IMT in healthy postmenopausal Japanese women.

Raloxifene increases proliferation and up-regulates telomerase activity in human umbilical vein endothelial cells.

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) alpha and ERbeta. 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 IkappaB. Moreover, cotreatment with an IkappaBalpha phosphorylation inhibitor, BAY-11-7082, or a specific NFkappaB nuclear translocation inhibitor, SN50, significantly attenuated the raloxifene-induced telomerase activity and the association of NFkappaB 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 NFkappaB 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.

Impact of menopause on the augmentation of arterial stiffness with aging.

BACKGROUND/AIMS: Aortic stiffness, determined by pulse wave velocity (PWV), is an independent marker of cardiovascular risk. PWV is mainly influenced by age-associated alterations in arterial wall structure and blood pressure. The present study was conducted to assess the impact of menopause on the brachial-ankle PWV (baPWV) in healthy women. METHODS: Fifty premenopausal women aged 22-54 years and 40 postmenopausal women aged 40-73 years were recruited for this study. Subjects with hypertension, diabetes, and hyperlipidemia were strictly excluded. The results of baPWV were analyzed chronologically by 10- or 5-year age intervals. RESULTS: There was no significant difference in baPWV between premenopausal and postmenopausal women in their 40s and 50s. The baPWV of postmenopausal women aged over 60 years was significantly higher than that of postmenopausal women in their 50s. To clarify the age-dependent elevation in baPWV in detail, women their 50s and 60s were divided into subgroups by 5-year age intervals. There was no significant difference in baPWV among the 50-54-, 55-59- and 60-64-year subgroups. baPWV significantly increased in the 65-69- year subgroup (p< 0.05). There was a significant relationship between baPWV and age in premenopausal (r = 0.452, p = 0.001) and postmenopausal (r = 0.581, p < 0.0001) women. The slope of the regression line for baPWV plotted against age was steeper in postmenopausal than in premenopausal women. CONCLUSIONS: This study produces suggestive evidence that menopause amplifies the age-dependent increase in arterial stiffness.

Medroxyprogesterone acetate attenuates estrogen-induced nitric oxide production in human umbilical vein endothelial cells.

We report the novel observation that medroxyprogesterone acetate (MPA) attenuates the induction by 17beta estradiol (E2) of both nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) activity in human umbilical vein endothelial cells. Although MPA had no effect on basal NO production or basal eNOS phosphorylation or activity, it attenuated the E2-induced NO production and eNOS phosphorylation and activity. Moreover, we examined the mechanism by which MPA attenuated the E2-induced NO production and eNOS phosphorylation. MPA attenuated the E2-induced phosphorylation of Akt, a kinase that phosphorylates eNOS. Treatment with pure progesterone receptor (PR) antagonist RU486 completely abolished the inhibitory effect of MPA on E2-induced Akt phosphorylation and eNOS phosphorylation. In addition, the effects of actinomycin D were tested to rule out the influence of genomic events mediated by nuclear PRs. Actinomycin D did not affect the inhibitory effect of MPA on E2-induced Akt phosphorylation. Furthermore, the potential roles of PRA and PRB were evaluated. In COS cells transfected with either PRA or PRB, MPA attenuated E2-induced Akt phosphorylation. These results indicate that MPA attenuated E2-induced NO production via an Akt cascade through PRA or PRB in a non-genomic manner.

Raloxifene inhibits estrogen-induced up-regulation of telomerase activity in a human breast cancer cell line.

The mechanism by which raloxifene acts in the chemoprevention of breast cancer remains unclear. Because telomerase activity is involved in estrogen-induced carcinogenesis, we examined the effect of raloxifene on estrogen-induced up-regulation of telomerase activity in MCF-7 human breast cancer cell line. Raloxifene inhibited the induction of cell growth and telomerase activity by 17beta-estradiol (E2). Raloxifene inhibited the E2-induced expression of the human telomerase catalytic subunit (hTERT), and transient expression assays using luciferase reporter plasmids containing various fragments of the hTERT promoter showed that the estrogen-responsive element appeared to be partially responsible for the action of raloxifene. E2 induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, attenuated the E2-induced increases of the telomerase activity and hTERT promoter activity. Raloxifene inhibited the E2-induced Akt phosphorylation. In addition, raloxifene also inhibited the E2-induced hTERT expression via the PI3K/Akt/NFkappaB cascade. Moreover, raloxifene also inhibited the E2-induced phosphorylation of hTERT, association of NFkappaB with hTERT, and nuclear accumulation of hTERT. These results show that raloxifene inhibited the E2-induced up-regulation of telomerase activity not only by transcriptional regulation of hTERT via an estrogen-responsive element-dependent mechanism and the PI3K/Akt/NFkappaB cascade but also by post-translational regulation via phosphorylation of hTERT and association with NFkappaB.