Development of cardiac fibrosis portends the transition and deterioration from hypertrophy to dilation and heart failure. Here we examined how estrogen blocks this important development. Angiotensin II (AngII) and endothelin-1 induce cardiac hypertrophy and fibrosis in humans. and we find that these agents directly stimulate the transition of the cardiac fibroblast to a myofibroblast. AngII and endothelin-1 stimulated TGFβ1 synthesis in the fibroblast, an inducer of fibrosis that signaled via c-jun kinase to Sma- and Mad-related protein 3 phosphorylation and nuclear translocation in myofibroblasts. As a result, mesenchymal proteins fibronectin and vimentin were produced, as were collagens I and III, the major forms found in fibrotic hearts. 17β-Estradiol (E2) or dipropylnitrile, an estrogen receptor (ER)β agonist, comparably blocked all these events, reversed by estrogen receptor (ER)β small interfering RNA. E2 and dipropylnitrile signaling through cAMP and protein kinase A prevented myofibroblast formation and blocked activation of c-jun kinase and important events of fibrosis. In the hearts of ovariectomized female mice, cardiac hypertrophy and fibrosis were induced by AngII infusion and prevented by E2 administration to wild type but not ERβ knockout rodents. Our results establish the cardiac fibroblast as an important target for hypertrophic/fibrosis-inducing peptides the actions of which were mitigated by E2/ERβ acting in these stromal cells.

Estrogen receptors (ERs) ␣ and ␤ exist as nuclear, cytoplasmic, and membrane cellular pools in a wide variety of organs. The relative contributions of each ER␣ pool to in vivo phenotypes resulting from estrogen signaling have not been determined. To address this, we generated a transgenic mouse expressing only a functional E domain of ER␣ at the plasma membrane (MOER). Cells isolated from many organs showed membrane only localized E domain of ER␣ and no other receptor pools. Liver cells from MOER and wild type mice responded to 17-␤-estradiol (E2) with comparable activation of ERK and phosphatidylinositol 3-kinase, not seen in cells from ER␣KO mice. Mating the MOER female mice with proven male wild type breeders produced no pregnancies because the uterus and vagina of the MOER female mice were extremely atrophic. Ovaries of MOER and homozygous Strasbourg ER␣KO mice showed multiple hemorrhagic cysts and no corpus luteum, and the mammary gland development in both MOER and ER␣KO mice was rudimentary. Despite elevated serum E2 levels, serum LH was not suppressed, and prolactin levels were low in MOER mice. MOER and Strasbourg female mice showed plentiful abdominal visceral and other depots of fat and increased body weight compared to wild type mice despite comparable food consumption. These results provide strong evidence that the normal development and adult functions of important organs in female mice requires nuclear ER␣ and is not rescued by membrane ER␣ domain expression alone. Estrogen receptor (ER)3 ␣ exists in many cellular locations, each potentially contributing to sex steroid action (1). Genetic deletion of ER␣ in mice established important roles of this receptor for normal adult female mammary gland and reproductive tract development and function (2-4). In these regards, adult female ER␣ knock-out (KO) mice show atrophy of the uterus and vagina, abnormal ovarian histology, and rudimentary mammary gland development. As a result, the normal adult functions of these organs were markedly compromised, and many of these abnormalities were phenocopied by aromatase knock-out mice (5). Thus, estrogen or its metabolites acting at ER␣ is necessary for these normal developmental functions.Since the original descriptions of both the Chapel Hill (2) and Strasbourg (4) ER␣KO mice, it has become appreciated that these mice represent depletion of all ER␣ cellular pools. For instance, endothelial cells derived from homozygous ER␣/ER␤ combined deletion mice show no evidence of any cellular ER (6). Furthermore, E2 cannot rapidly signal nor stimulate proliferation and survival in these cells. Thus, in ER␣KO mice, it cannot be determined where estrogen acts in the cell to effect normal development and function. This limits understanding of what specific actions occur through discrete ER␣ pools, contributing to the overall effects of this receptor in vivo.To begin to address this issue we generated a mouse that expresses a functional E domain of ER␣ only at the plasma membrane of cells from multiple organs. No cytoplasmic or n...

The incidence of melanoma is increasing rapidly, with advanced lesions generally failing to respond to conventional chemotherapy. Here, we utilized DNA microarray-based gene expression profiling techniques to identify molecular determinants of melanoma progression within a unique panel of isogenic human melanoma cell lines. When a poorly tumorigenic cell line, derived from an early melanoma, was compared with two increasingly aggressive derivative cell lines, the expression of 66 genes was significantly changed. A similar pattern of differential gene expression was found with an independently derived metastatic cell line. We further examined these melanoma progression-associated genes via use of a tailored TaqMan Low Density Array (LDA), representing the majority of genes within our cohort of interest. Considerable concordance was seen between the transcriptomic profiles determined by DNA microarray and TaqMan LDA approaches. A range of novel markers were identified that correlated here with melanoma progression. Most notable was TSPY, a Y chromosome-specific gene that displayed extensive down-regulation in expression between the parental and derivative cell lines. Examination of a putative CpG island within the TSPY gene demonstrated that this region was hypermethylated in the derivative cell lines, as well as metastatic melanomas from male patients. Moreover, treatment of the derivative cell lines with the DNA methyltransferase inhibitor, 2'-deoxy-5-azacytidine (DAC), restored expression of the TSPY gene to levels comparable with that found in the parental cells. Additional DNA microarray studies uncovered a subset of 13 genes from the above-mentioned 66 gene cohort that displayed re-activation of expression following DAC treatment, including TSPY, CYBA and MT2A. DAC suppressed tumor cell growth in vitro. Moreover, systemic treatment of mice with DAC attenuated growth of melanoma xenografts, with consequent re-expression of TSPY mRNA. Overall, our data support the hypothesis that multiple genes are targeted, either directly or indirectly, by DNA hypermethylation during melanoma progression.

F]DFA to show that ribose preferentially accumulates in the liver, suggesting a striking tissue specificity for ribose metabolism. We demonstrate that solute carrier family 2, member 2 (also known as GLUT2), a glucose transporter expressed in the liver, is one ribose transporter, but we do not know if others exist. [ 18 F]DFA accumulation is attenuated in several mouse models of metabolic syndrome, suggesting an association between ribose salvage and glucose and lipid metabolism. These results describe a tool for studying ribose salvage and suggest that plasma ribose is preferentially metabolized in the liver. molecular imaging | sugar metabolism | Slc2a2