Mutation of the palmitoylation site of estrogen receptor alpha in vivo reveals tissue-specific roles for membrane versus nuclear actions

Estrogen receptor alpha (ERalpha) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERalpha is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERalpha (C451A-ERalpha) to obtain membrane-specific loss of function of ERalpha. The abrogation of membrane localization of ERalpha in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERalpha mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endothelial NO synthase phosphorylation were abrogated in C451A-ERalpha mice. A complementary mutant mouse lacking the transactivation function AF-2 of ERalpha (ERalpha-AF2(0)) provided selective loss of function of nuclear ERalpha actions. In ERalpha-AF2(0), the acceleration of endothelial repair in response to estrogen-dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERalpha-AF2(0), whereas in C451A-ERalpha it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissue-specific roles

Sub-femto-g free fall for space-based gravitational wave observatories: LISA pathfinder results

We report the first results of the LISA Pathfinder in-flight experiment. The results demonstrate that two free-falling reference test masses, such as those needed for a space-based gravitational wave observatory like LISA, can be put in free fall with a relative acceleration noise with a square root of the power spectral density of 5.2 ± 0.1 fm s−2/√Hz or (0.54 ± 0.01) × 10−15 g/√Hz, with g the standard gravity, for frequencies between 0.7 and 20 mHz. This value is lower than the LISA Pathfinder requirement by more than a factor 5 and within a factor 1.25 of the requirement for the LISA mission, and is compatible with Brownian noise from viscous damping due to the residual gas surrounding the test masses. Above 60 mHz the acceleration noise is dominated by interferometer displacement readout noise at a level of (34.8 ± 0.3) fm/√Hz, about 2 orders of magnitude better than requirements. At f ≤ 0.5 mHz we observe a low-frequency tail that stays below 12 fm s−2/√Hz down to 0.1 mHz. This performance would allow for a space-based gravitational wave observatory with a sensitivity close to what was originally foreseen for LISA

Supplementary Material for: Enhanced Liver Regeneration After Partial Hepatectomy in Sterol Regulatory Element-Binding Protein (SREBP)-1c-Null Mice is Associated with Increased Hepatocellular Cholesterol Availability

<b><i>Background/Aims:</i></b> Transient lipid accumulation within hepatocytes preceding the peak proliferative process is a characteristic feature of liver regeneration. However, molecular mediators responsible for this lipid accumulation and their functions are not well defined. Sterol regulatory element-binding proteins-1c (SREBP-1c) are critical transcriptional factors that regulate lipid homeostasis in the liver. We hypothesized that SREBP-1c deficiency induced alterations of lipid metabolism may influence hepatocyte proliferation and liver regeneration. <b><i>Methods:</i></b> 2/3 partial hepatectomy (PH) was performed in wild type C57BL/6J (WT) and <i>Srebp-1c-/-</i> mice. The lipid contents in serum and liver were measured by enzymatic colorimetric methods. Hepatic lipid droplets were detected by Oil Red O staining and immunohistological staining. Hepatic expression of genes involved in lipid metabolism and cellular proliferation was determined by real-time PCR and/or immunoblot. Hepatocyte proliferation and liver regeneration were assessed by BrdU staining and the weight of remanent liver lobes in Srebp-1c-/- mice, respectively. <b><i>Results:</i></b> <i>Srebp-1c-/-</i> mice displayed reduced triglyceride and fatty acids but increased cholesterol in the liver before PH. In response to PH, hepatocellular DNA synthesis was elevated and cell cycle progression was prolonged in <i>Srebp-1c-/-</i> mice, which was associated with enhanced liver regeneration. However, <i>Srebp-1c-/-</i> mice had comparable triglyceride and fatty acid contents and expressions of related genes compared with WT mice during the liver regeneration. In contrast, SREBP-1c-deficiency-induced alteration of cholesterol metabolism was retained during the liver regeneration after PH. <i>Srebp-1c-/-</i> mice exhibited higher cholesterol contents and enhanced expression of SREBP-2 and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR) in the liver than WT mice after PH. Moreover, downregulation of genes involved in cholesterol elimination was observed after PH in <i>Srebp-1c-/-</i> mice. <b><i>Conclusion:</i></b> SREBP-1c deficiency in mice did not interfere with triglyceride and fatty acid metabolism but was associated with significant changes in cholesterol profiles during liver regeneration after PH. These results suggest that increased hepatocellular cholesterol storage and cholesterol availability with the enhanced liver regeneration are identified in <i>Srebp-1c-/-</i> mice. This study also shows that providing requisite cholesterol levels to proliferating hepatocytes and keeping appropriate cholesterol metabolism are required for normal liver regeneration