Estrogen and insulin-like-growth factor 1 (IGF-1) are potent mitogenic stimuli that share important properties in the control of cellular proliferation. However, the coupling between the signaling cascades of estrogen receptors ␣ and  and the IGF-1 receptor (IGF-1R) is poorly understood. Therefore, we selectively transfected estrogen receptor ␣ or  in COS7 and HEK293 cells, which contain IGF-1R. In presence of estrogen receptor ␣ but not , 17-estradiol (E2) rapidly induces phosphorylation of the IGF-1R and the extracellular signal-regulated kinases 1/2. Furthermore, upon stimulation with E2, estrogen receptor ␣ but not  bound rapidly to the IGF-1R in COS7 as well as L6 cells, which express all investigated receptors endogenously. Control experiments in the IGF-1R-deficient fibroblast cell line R ؊ showed that after stimulation with E2 only estrogen receptor ␣ bound to the transfected IGF-1R. Overexpression of dominant negative mitogen-activated protein kinases kinase inhibited this effect. Finally, estrogen receptor ␣ but not  is required to induce the activation of the estrogen receptor-responsive reporter ERE-LUC in IGF-1-stimulated cells. Taken together, these data demonstrate that ligand bound estrogen receptor ␣ is required for rapid activation of the IGF-1R signaling cascade.Estrogen as well as insulin-like growth factor 1 (IGF-1) 1 are potent mitogens that are involved in a large array of processes that control proliferation and differentiation in mammalian cells (1, 2). Both mitogens act through receptor-mediated signaling pathways. The cross-talk between these two signaling pathways is currently under investigation (3-6). Estrogen is a steroid hormone that binds to members of the nuclear receptor superfamily (7), whereas IGF-1 as a peptide-growth factor binds to a transmembrane tyrosine kinase receptor, which signals via a series of phosphorylation events (2).Two different estrogen receptors, ER␣ and ER, which are encoded by genes located on different chromosomes, have been identified so far (8,9). Sequence analysis demonstrates a high degree of homology between ER␣ and ER in the DNA-binding domain and the ligand-binding domain. However, there are significant differences in regions that would be expected to influence transcriptional activity. The ability of estrogen receptors to activate target gene transcription has been attributed to two regions: the N-terminal activation function 1 (AF-1) and the ligand-dependent AF-2, which is localized in the C-terminal hormone-binding domain (10, 11). AF-1 and AF-2 can activate transcription independently and synergistically, and they act in a promoter-and cell-specific manner (12, 13). Phosphorylation of a serine residue at position 118 is required for full action of the AF-1 (14). Both AF-1 and AF-2 are required to enhance transcription of target genes through AP-1 sites (15). Interestingly, ER␣ and ER act differently at AP-1 sites (16), which may be due to differences in their AF domains (17). ER␣ and ER can form homo-and heterodimers (18), and thus t...
Background-The AT 1 receptor has been implicated in the pathogenesis of hypertension and atherosclerosis. Estrogen deficiency is also associated with cardiovascular diseases. Therefore, we examined the AT 1 receptor gene expression in ovariectomized rats with and without estrogen replacement therapy and the influence of estrogen on AT 1 receptor expression in cultured vascular smooth muscle cells. Methods and Results-Rat aortic tissue was examined 5 weeks after ovariectomy. In one group, estrogen (1.7 mg estradiol) was administered during the 5-week period. Functional experiments assessed angiotensin II-induced contraction of aortic rings. AT 1 receptor mRNA levels were measured by quantitative polymerase chain reaction and Northern blotting. AT 1 receptor density was assessed by radioligand binding assays. These techniques were also applied in cultured vascular smooth muscle cells. The efficacy of angiotensin II on vasoconstriction was significantly increased in aortas from ovariectomized rats. As assessed by radioligand binding assays, AT 1 receptor density was increased to 160% without changes in receptor affinity during estrogen deficiency. AT 1 receptor mRNA levels were consistently increased to 187% in ovariectomized rats compared with sham-operated animals. Estrogen substitution therapy in ovariectomized rats reversed this AT 1 receptor overexpression. To explore the underlying mechanisms, the direct influence of estradiol on AT 1 receptor expression was investigated in VSMCs. Estradiol (1 mol/L) led to a time-dependent downregulation of AT 1 receptor mRNA, with a maximum of 33.3% at 12 hours. There was a correlative decrease in AT 1 receptor density. Conclusions-This novel observation of estrogen-induced downregulation of AT 1 receptor expression could explain the association of estrogen deficiency with hypertension and atherosclerosis, because activation of the AT 1 receptor plays a key role in the regulation of blood pressure, fluid homeostasis, and vascular cell growth. (Circulation. 1998;97:2197-2201.)Key Words: angiotensin Ⅲ hypertension Ⅲ hormones Ⅲ genes Ⅲ muscle, smooth Ⅲ atherosclerosis T he low incidence of vascular diseases in premenopausal women and the rapid increase of the risk of cardiovascular events after menopause as well as the beneficial effects of estrogen replacement therapy on cardiac and vascular morbidity have suggested a important role of estrogens in the pathogenesis of atherosclerosis. [1][2][3] In addition to its effects on classic cardiovascular risk factors, eg, in the sense of a decrease of cholesterol plasma levels, 4,5 estrogen has been recognized to directly influence vascular as well as myocardial cells. Indeed, VSMCs, myocytes, and cardiac fibroblasts have been shown to contain functional estrogen receptors. [6][7][8] Moreover, there is increasing evidence that estrogen interferes with the RAS. The production of angiotensinogen is enhanced, whereas ACE levels are decreased, by estrogens. According to a recent report, plasma renin levels are also reduced during estroge...
Gender-based differences found in cardiovascular diseases raise the possibility that estrogen may have direct effects on cardiac tissue. Therefore we investigated whether cardiac myocytes and fibroblasts express functional estrogen receptors. Immunofluorescence demonstrated estrogen receptor protein expression in both female and male rat cardiac myocytes and fibroblasts. Nuclear translocation of the estrogen receptor protein was observed after stimulation of cardiomyocytes with 17ß-estradiol (E 2 ). Cells transfected with an estrogen-responsive reporter plasmid showed that treatment with E 2 induced a significant increase in reporter activity. Furthermore, E 2 induced a significant increase in expression of the estrogen receptors a and ß, progesterone receptor and connexin 43 in cardiac myocytes. Cardiac myocytes and fibroblasts contain functional estrogen receptors and estrogen regulates expression of specific cardiac genes. These data suggest that gender-based differences in cardiac diseases may in part be due to direct effects of estrogen on the heart.
Background and AimsDeoxynivalenol (DON) is a Fusarium derived mycotoxin, often occurring on cereals used for human and animal nutrition. The intestine, as prominent barrier for nutritional toxins, has to handle the mycotoxin from the mucosa protected luminal side (apical exposure), as well as already absorbed toxin, reaching the cells from basolateral side via the blood stream. In the present study, the impact of the direction of DON exposure on epithelial cell behaviour and intestinal barrier integrity was elucidated.MethodsA non-transformed intestinal porcine epithelial cell line (IPEC-J2), cultured in membrane inserts, serving as a polarised in vitro model to determine the effects of deoxynivalenol (DON) on cellular viability and tight junction integrity.ResultsApplication of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3. Epithelial cell number decreased and nuclei size was enlarged after 72 h incubation of 4000 ng/mL DON from basolateral. Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL.ConclusionsSeverity of impact of the mycotoxin deoxynivalenol on the intestinal epithelial barrier is dependent on route of application. The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity.
Saturated very long chain fatty acids (VLCFAs; > or =C22:0) accumulate in X-linked adrenoleukodystrophy (X-ALD, OMIM 300100), a severe hereditary neurodegenerative disease, due to peroxisomal impairment. Previous studies analysed the development of X-ALD in humans and gene knockout animal models. However, the toxic effect of VLCFA leading to severe symptoms with progressive and multifocal demyelination, adrenal insufficiency and inflammation still remains unclear. To understand the toxic effects of VLCFA in the brain, here we exposed neural cells to VLCFA and analysed the cellular consequences. We found that oligodendrocytes and astrocytes challenged with docosanoic- (C22:0), tetracosanoic- (C24:0) and hexacosanoic acids (C24:0) die within 24 h. VLCFA-induced depolarization of mitochondria in situ and increased intracellular Ca2+ level in all three brain cell types provides indications about the mechanism of toxicity of VLCFA. Interestingly, VLCFAs affect to the largest degree the myelin-producing oligodendrocytes. In isolated mitochondria, VLCFAs exert a detrimental effect by affecting the inner mitochondrial membrane and promoting the permeability transition. In conclusion, we suggest that there is a potent toxic activity of VLCFA due to dramatic cell physiological effects with mitochondrial dysfunction and Ca2+ deregulation. This provides the first evidence for mitochondrial-based cell death mechanisms in neurodegenerative disease with peroxisomal defects and subsequent VLCFA accumulation.
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