Tamoxifen elicits atheroprotection through estrogen receptor α AF-1 but does not accelerate reendothelialization.

Based on both experimental and clinical data, tamoxifen has been proposed to have cardiovascular benefits, although the mechanism(s) contributing to that protective effect are still poorly understood. In vitro experiments demonstrated that tamoxifen elicits its transcriptional effect through estrogen receptor (ER) α, but other targets can participate in its actions. However, although tamoxifen selectively activates the activating function (AF)-1 of ERα, we recently showed that this ERα subfunction is dispensable for the atheroprotective action of 17ß-estradiol (E2), the main ligand of ERs. The goal of the present work is to determine to which extent ERα and its AF-1 mediate the vasculoprotective action of tamoxifen. Our data confirm that tamoxifen exerts an atheroprotective action on low density lipoprotein receptor (LDL-r(-/-)) female mice, but, in contrast to E2, it fails to accelerate reendothelialization after carotid electric injury. Tamoxifen and E2 elicit differences in gene expression profiles in the mouse aorta. Finally, the atheroprotective action of tamoxifen is abrogated in ERα(-/-)LDL-r(-/-) mice and in LDL-r(-/-) mice selectively deficient in ERαAF-1 (ERαAF-1(0/0)LDL-r(-/-)). Our results demonstrate, for the first time to our knowledge, that tamoxifen mediates its actions in vivo through the selective activation of ERαAF-1, which is sufficient to prevent atheroma, but not to accelerate endothelial healing.

Activation function 2 (AF2) of estrogen receptor-alpha is required for the atheroprotective action of estradiol but not to accelerate endothelial healing.

17ß-Estradiol (E2) regulates estrogen receptor-α (ERα) target gene transcription through the two independent activation functions (AFs), AF1 and AF2, located in the N-terminal and ligand binding domain of ERα, respectively. We previously reported that ERα is required for the E2 atheroprotective action as well as for its accelerative action on endothelial healing, but its AF1 function is dispensable. Here, we investigated the role of ERαAF2 in these two major beneficial actions of E2 by electively targeting ERαAF2 (named ERαAF2(0)). Our results prove four points. (i) Compared with WT ERα, the ability of ERαAF2(0) to stimulate the C3 complement or the estrogen response element-thymidine kinase promoter in two cell lines was dramatically decreased, confirming the importance of AF2 in the E2-induced transcriptional activity of ERα. (ii) The uterotrophic action of E2 was totally absent in ERαAF2(0) mice, showing the crucial role of ERαAF2 in E2-induced uterus hyperplasia. (iii) ERαAF2 was dispensable for the accelerative action of E2 on endothelial healing, underlining the functionality of ERαAF2(0) in vivo. (iv) Finally, the atheroprotective effect of E2 was abrogated in ERαAF2(0) LDL-r(-/-) mice. Thus, whereas ERαAF1 and ERαAF2 are both required for the uterotrophic action of E2, we show that only ERαAF2 is necessary for its atheroprotective effect.

A natural protective function of invariant NKT cells in a mouse model of innate-cell-driven lung inflammation.

Activation of invariant natural killer T (iNKT) cells by treatment with their α-galactosyl ceramide ligand provides therapeutic benefits in several immune inflammatory settings. Given the artificial nature of this stimulation, the natural regulatory functions of iNKT remain uncertain. Addressing this issue in a mouse model of innate-cell-driven lung inflammation induced by the cytokine/alarmin IL-33 that targets iNKT cells, we found that eosinophil and neutrophil recruitment was markedly increased in treated iNKT cell-deficient (Jα18 KO) mice, as was the local production of eotaxin and keratinocyte chemoattractant chemokines. By contrast, lung inflammation decreased after adoptive transfer of iNKT cells, which restored the WT inflammatory response in Jα18 KO mice. Finally, we established that this natural anti-inflammatory function of iNKT cells depends on their IFN-γ production and on endogenous IL-12. Our study provides the first evidence of a protective role of iNKT cells during lung inflammation that does not require pharmacological TCR engagement.

Prevention of skin flap necrosis by estradiol involves reperfusion of a protected vascular network.

Although 17beta-estradiol (E2) is protective in experimental models of myocardial and brain ischemia, its effect on skin ischemia remains unknown. Here, we assessed the protective effect of E2 in a mouse model of skin ischemia, mimicking the surgery of skin flaps. Whereas necrosis appeared in the half portion of the skin flap within 1 week after surgery in ovariectomized mice, it was reduced up to 10-fold when mice were pretreated with E2, at least 3 days before the surgery. The beneficial effect of E2 appeared to be attributable to an increase in skin survival, revealed by measuring viability of ex vivo explants and enhancement of the antiapoptotic Bcl-2 protein expression in vivo. This protective effect on the skin contributed to the protection of the vascular network and facilitated reperfusion, which was found to be accelerated in ovariectomized E2-treated mice, whereas hemorrhages were observed in untreated mice. E2 also increased expression of fibroblast growth factor-2 isoforms in the skin and circulating vascular endothelial growth factor in the serum. Finally, this protective effect of E2 was abolished in estrogen receptor-deficient mice (ERalpha(-/-)) but maintained in chimeric mice reconstituted with ERalpha-deficient bone marrow, indicating dispensable action of E2 in bone marrow-derived cells. This protective effect of E2 was mimicked by treatment with tamoxifen, a selective estrogen receptor modulator. In conclusion, we have demonstrated for the first time that E2 exerts a major preventive effect of skin flap necrosis through a prevention of ischemic-induced skin lesions, including those of the vascular network, which contributes to accelerate the reperfusion of the skin flap.

Endothelial estrogen receptor-alpha plays a crucial role in the atheroprotective action of 17beta-estradiol in low-density lipoprotein receptor-deficient mice.

BACKGROUND: The prevention of early atheroma by estrogens has been clearly demonstrated in all animal models and appears to be mediated through a direct action on the arterial wall rather than through an effect on the lipoprotein profile. The goal of the present study was to evaluate which cellular target is crucial in this beneficial action of estradiol. METHODS AND RESULTS: We first confirmed the key role of estrogen receptor-alpha (ERalpha) in the atheroprotective effect of estradiol, because this action was completely abolished in mice deficient in both the low-density lipoprotein receptor (LDLr) and ERalpha. Second, using chimeric mice with an ERalpha deficiency in the hematopoietic lineage, we showed the persistence of the protective action of estradiol, which suggests the involvement of extrahematopoietic ERalpha. Third, we showed that loxP-flanked ERalpha mice (ERalpha(flox/flox)) bred with Tie2-Cre(+) mice on an LDLr(-/-) background had complete inactivation of ERalpha in most hematopoietic and all endothelial cells. Remarkably, in this mouse model, the atheroprotective effect of estradiol was completely abolished. Fourth, the atheroprotective effect of estradiol remained abolished in Tie2-Cre(+) ERalpha(flox/flox) LDLr(-/-) mice transplanted with either Tie2-Cre(+) ERalpha(flox/flox) or ERalpha(-/-) bone marrow, whereas it was present in analogous chimeric Tie2-Cre(-) ERalpha(flox/flox) LDLr(-/-) receivers expressing endothelial ERalpha. CONCLUSIONS: We demonstrate directly and for the first time that endothelial ERalpha represents a key target of the atheroprotective effect of estradiol, whereas hematopoietic ERalpha is dispensable. Selective estrogen receptor modulators that mimic the endothelial action of estradiol should now be considered in atheroprotection.

The AF-1 activation function of estrogen receptor α is necessary and sufficient for uterine epithelial cell proliferation in vivo.

Estrogen receptor-α (ERα) regulates gene transcription through the 2 activation functions (AFs) AF-1 and AF-2. The crucial role of ERαAF-2 was previously demonstrated for endometrial proliferative action of 17ß-estradiol (E2). Here, we investigated the role of ERαAF-1 in the regulation of gene transcription and cell proliferation in the uterus. We show that acute treatment with E2 or tamoxifen, which selectively activates ERαAF-1, similarly regulate the expression of a uterine set of estrogen-dependent genes as well as epithelial cell proliferation in the uterus of wild-type mice. These effects were abrogated in mice lacking ERαAF-1 (ERαAF-1(0)). Four weeks of E2 treatment led to uterine hypertrophy and sustained luminal epithelial and stromal cell proliferation in wild-type mice, but not in ERαAF-1(0) mice. However, ERαAF-1(0) mice still presented a moderate uterine hypertrophy essentially due to a stromal edema, potentially due to the persistence of Vegf-a induction. Epithelial apoptosis is largely decreased in these ERαAF-1(0) uteri, and response to progesterone is also altered. Finally, E2-induced proliferation of an ERα-positive epithelial cancer cell line was also inhibited by overexpression of an inducible ERα isoform lacking AF-1. Altogether, these data highlight the crucial role of ERαAF-1 in the E2-induced proliferative response in vitro and in vivo. Because ERαAF-1 was previously reported to be dispensable for several E2 extrareproductive protective effects, an optimal ERα modulation could be obtained using molecules activating ERα with a minimal ERαAF-1 action.

Lysosomal serine protease CLN2 regulates tumor necrosis factor-alpha-mediated apoptosis in a Bid-dependent manner.

Apoptosis is a highly organized, energy-dependent program by which multicellular organisms eliminate damaged, superfluous, and potentially harmful cells. Although caspases are the most prominent group of proteases involved in the apoptotic process, the role of lysosomes has only recently been unmasked. This study investigated the role of the lysosomal serine protease CLN2 in apoptosis. We report that cells isolated from patients affected with late infantile neuronal ceroid lipofuscinosis (LINCL) having a deficient activity of CLN2 are resistant to the toxic effect of death ligands such as tumor necrosis factor (TNF), CD95 ligand, or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not to receptor-independent stress agents. CLN2-deficient cells exhibited a defect in TNF-induced Bid cleavage, release of cytochrome c, and caspase-9 and -3 activation. Moreover, extracts from CLN2-overexpressing cells or a CLN2 recombinant protein were able to catalyze the in vitro cleavage of Bid. Noteworthy, correction of the lysosomal enzyme defect of LINCL fibroblasts using a medium enriched in CLN2 protein enabled restoration of TNF-induced Bid and caspase-3 processing and toxicity. Conversely, transfection of CLN2-corrected cells with small interfering RNA targeting Bid abrogated TNF-induced cell death. Altogether, our study demonstrates that genetic deletion of the lysosomal serine protease CLN2 and the subsequent loss of its catalytic function confer resistance to TNF in non-neuronal somatic cells, indicating that CLN2 plays a yet unsuspected role in TNF-induced cell death.