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

The estrogen sex steroid 17␤-estradiol rapidly inhibits secretagogue-stimulated cAMP-dependent Cl ؊ secretion in the female rat distal colonic crypt by the inhibition of basolateral K ؉ channels. In Ussing chamber studies, both the anti-secretory response and inhibition of basolateral K ؉ current was shown to be attenuated by pretreatment with rottlerin, a PKC␦-specific inhibitor. In whole cell patch-clamp analysis, 17␤-estradiol inhibited a chromanol 293B-sensitive KCNQ1 channel current in isolated female rat distal colonic crypts. Estrogen had no effect on KCNQ1 channel currents in colonic crypts isolated from male rats. Female distal colonic crypts expressed a significantly higher amount of PKC␦ in comparison to male tissue. PKC␦ and PKA were activated at 5 min in response to 17␤-estradiol in female distal colonic crypts only. Both PKC␦-and PKA-associated with the KCNQ1 channel in response to 17␤-estradiol in female distal colonic crypts, and no associations were observed in crypts from males. PKA activation, association with KCNQ1, and phosphorylation of the channel were regulated by PKC␦ as the responses were blocked by pretreatment with rottlerin. Taken together, our experiments have identified the molecular targets underlying the anti-secretory response to estrogen involving the inhibition of KCNQ1 channel activity via PKC␦-and PKA-dependent signaling pathways. This is a novel gender-specific mechanism of regulation of an ion channel by estrogen. The anti-secretory response described in this study provides molecular insights whereby estrogen causes fluid retention effects in the female during periods of high circulating plasma estrogen levels.

Estrogen induces signal transduction through estrogen receptor α (ERα), which localizes to both the plasma membrane and nucleus. Using wild-type mice, ERα knockout (ERKO) mice, or transgenic mice expressing only the ligand-binding domain of ERα exclusively at the plasma membrane (MOER), we compared the transcriptional profiles of liver tissue extracts after mice were injected with the ERα agonist propyl-pyrazole-triol (PPT). The expression of many lipid synthesis-related genes was comparably decreased in livers from MOER or wild-type mice but was not suppressed in ERKO mice, indicating that only membrane-localized ERα was necessary for their suppression. Cholesterol, triglyceride, and fatty acid content was decreased only in livers from wild-type and MOER mice exposed to PPT, but not in the livers from the ERKO mice, validating the membrane-driven signaling pathway on a physiological level. PPT-triggered activation of ERα at the membrane induced adenosine monophosphate-activated protein kinase to phosphorylate sterol regulatory element-binding factor 1 (Srebf1), preventing its association with and therefore its proteolytic cleavage by site-1 protease. Consequently, Srebf1 was sequestered in the cytoplasm, preventing the expression of cholesterol synthesis-associated genes. Thus, we showed that inhibition of gene expression mediated by membrane-localized ERα caused a metabolic phenotype that did not require nuclear ERα.

Liver X receptors (LXRs) are determinants of hepatic stellate cell (HSC) activation and liver fibrosis. Freshly isolated HSCs from Lxrαβ−/− mice have increased lipid droplet (LD) size but the functional consequences of this are unknown. Our aim was to determine whether LXRs link cholesterol to retinoid storage in HSCs and how this impacts activation. Primary HSCs from Lxrαβ−/− and wild-type (WT) mice were profiled by gene array during in vitro activation. Lipid content was quantified by HPLC and mass spectroscopy. Primary HSCs were treated with nuclear receptor ligands, transfected with siRNA and plasmid constructs, and analyzed by immunocytochemistry. Lxrαβ−/− HSCs have increased cholesterol and retinyl esters (CEs & REs). The retinoid increase drives intrinsic retinoic acid receptor (RAR) signaling and activation occurs more rapidly in Lxrαβ−/− HSCs. We identify Rab18 as a novel retinoic acid responsive, lipid droplet associated protein that helps mediate stellate cell activation. Rab18 mRNA, protein, and membrane insertion increase during activation. Both Rab18 GTPase activity and isoprenylation are required for stellate cell lipid droplet loss and induction of activation markers. These phenomena are accelerated in the Lxrαβ−/− HSCs, where there is greater retinoic acid flux. Conversely, Rab18 knockdown retards lipid droplet loss in culture and blocks activation, just like the functional mutants. Rab18 is also induced with acute liver injury in vivo. Conclusion Retinoid and cholesterol metabolism are linked in stellate cells by the LD associated protein, Rab18. Retinoid overload helps explain the pro-fibrotic phenotype of Lxrαβ−/− mice and we establish a pivotal role for Rab18 GTPase activity and membrane insertion in wild-type stellate cell activation. Interference with Rab18 may have significant therapeutic benefit in ameliorating liver fibrosis.

Key points• Oestrogen (E 2 ) exposure leads to a decrease in both Cl − secretion and KCNQ1 current. This inhibition is maintained by a rapid and sustained retrieval of the channel from the plasma membrane.• The E 2 -stimulated internalization of KCNQ1 occurs via a dynamin-and clathrin-dependent mechanism.• KCNQ1 is recycled back to the cell membrane via Rab4 and Rab11 rather than being degraded.• The signalling pathway activated by E 2 and leading to KCNQ1 internalization involves a signalling cascade, in which the activation of protein kinase Cδ induces the phosphorylation of AMP-dependent kinase. Oestrogen stimulated an increase in the association of KCNQ1 with the ubiquitin ligase Nedd4.2.• The findings provide evidence for a hormone-stimulated regulation of KCNQ1 surface density in colonic epithelium. Moreover, this study complements the understanding of the mechanisms for E 2 -induced inhibition of KCNQ1 previously described, and provides new insights on hormonal regulation of ion channel retrieval from the plasma membrane.Abstract The cAMP-regulated potassium channel KCNQ1:KCNE3 plays an essential role in transepithelial Cl − secretion. Recycling of K + across the basolateral membrane provides the driving force necessary to maintain apical Cl − secretion. The steroid hormone oestrogen (17β-oestradiol; E 2 ), produces a female-specific antisecretory response in rat distal colon through the inhibition of the KCNQ1:KCNE3 channel. It has previously been shown that rapid inhibition of the channel conductance results from E 2 -induced uncoupling of the KCNE3 regulatory subunit from the KCNQ1 channel pore complex. The purpose of this study was to determine the mechanism required for sustained inhibition of the channel function. We found that E 2 plays a role in regulation of KCNQ1 cell membrane abundance by endocytosis. Ussing chamber experiments have shown that E 2 inhibits both Cl − secretion and KCNQ1 current in a colonic cell line, HT29cl.19A, when cultured as a confluent epithelium. Following E 2 treatment, KCNQ1 was retrieved from the plasma membrane by a clathrin-mediated endocytosis, which involved the association between KCNQ1 and the clathrin adaptor, AP-2. Following endocytosis, KCNQ1 was accumulated in early endosomes. Following E 2 -induced endocytosis, rather than being degraded, KCNQ1 was recycled by a biphasic mechanism involving Rab4 and Rab11. Protein kinase Cδ and AMP-dependent kinase were rapidly phosphorylated in response to E 2 on their activating phosphorylation sites, Ser643 and Thr172, respectively (as previously shown). Both kinases are necessary for the E 2 -induced endocytosis, because E 2 failed to induce KCNQ1 internalization following pretreatment with specific inhibitors of both protein kinase Cδ and AMP-dependent kinase. The ubiquitin ligase Nedd4.2 binds KCNQ1 in response to E 2 to induce channel internalization. This study has provided the first demonstration of hormonal regulation of KCNQ1 trafficking. In conclusion, we propose that internalization of KCNQ1 is a key event in th...