Project description:Transcriptomic analysis of the mature ER+ population in the mammary gland revealed enrichment for estrogen-responsive genes in the RSK2-KO compared to the wild type.
Project description:Macrophage cholesterol homeostasis is crucial for health and disease and has been linked to the lipid-peroxidizing enzyme arachidonate 15-lipoxygenase type B (ALOX15B), albeit molecular mechanisms remain obscure. We performed global transcriptome and immunofluorescence analysis in ALOX15B-silenced primary human macrophages and observed a reduction of nuclear sterol regulatory element-binding protein (SREBP) 2, the master transcription factor of cellular cholesterol biosynthesis. Consequently, SREBP2-target gene expression was reduced as were the sterol biosynthetic intermediates desmosterol and lathosterol as well as 25- and 27-hydroxycholesterol. Mechanistically, suppression of ALOX15B reduced lipid peroxidation in primary human macrophages and thereby attenuated activation of mitogen-activated protein kinase ERK1/2, which lowered SREBP2 abundance and activity. Low nuclear SREBP2 rendered both, ALOX15B-silenced and ERK1/2-inhibited macrophages refractory to SREBP2 activation upon blocking the NPC intracellular cholesterol transporter 1. These studies suggest a regulatory mechanism controlling macrophage cholesterol homeostasis based on ALOX15B-mediated lipid peroxidation and concomitant ERK1/2 activation.
Project description:Objective: Homeostasis of intestinal stem cells (ISCs) is maintained by external niche signals and complicated intrinsic signaling networks. Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in stem cell self-renewal and differentiation, but the functions and underlying molecular mechanisms are not fully understood. Design: We performed studies with mice deletion of Erk1/2 genes in intestinal epithelial cells at embryonic stages or a control mice. Intestinal tissues were collected and analyzed by histology, gene expression profiling, immunohistochemistry, immunfluorescence, western blotting and organoid culture. Organoids were treated with specific inhibitors for Ras or Akt activity. Mice were treated by intraperitoneal injection of rapamycin to inhibit mTOR signaling. Results: We found that deletion of Erk1/2 genes resulted in an unexpected increase in cell proliferation and migration, expansion of ISCs and formation of polyp-like structures, leading to postnatal death. Deficiency of epithelial Erk1/2 strongly impaired secretory cell differentiation and enterocyte maturation with aberrant Wnt signaling activation. Erk1/2 depletion resulted in loss of feedback regulation leading to Ras/Raf cascade activation. Ras then transactivated Akt activity to stimulate mTOR and phosphorylate β-catenin on Ser552. Suppression of Ras or Akt activity through specific inhibitors significantly repressed Akt downstream signaling and restored cell proliferation and differentiation phenotypes ex vivo. Moreover, inhibition of mTOR signaling by rapamycin partially rescued Erk1/2 depletion-induced intestinal defects and significantly prolonged mutant mouse life span. Conclusions Our study demonstrates an unexplored critical role of ERK/MAPK pathway negative feedback regulation of Akt/mTOR in intestine development and homeostasis, suggesting a complicated regulatory network in ISC function.
Project description:The analysis examined the effects of a global ERK1 and/or tamoxifen-inducible, hepatocyte-specific ERK2 knockout on the liver transcriptome. Transcriptomes from the livers with a ERK1/2 double knockout were compared to the livers of mice with an ERK1 or ERK2 knockout.
Project description:Angiogenesis is a complex process orchestrated by both growth factors and cell adhesion to the extracellular matrix and is initiated by focal degradation of the vascular basement membrane with subsequent migration and proliferation of endothelial cells (EC). The Ras/Raf/MEK/ERK pathway is critical for EC function during angiogensis. Although in vitro studies implicate ERK1 and ERK2 in EC survival, their precise role in EC function in vivo remains poorly defined. Cre/loxP technology was used to inactivate Erk1 and Erk2 in EC during murine development, resulting in embryonic lethality due to a drastic reduction in angiogenesis. The angiogenic defect was linked to diminished EC proliferation and migration, but not to increased cell apoptosis. Expression of key cell cycle regulators was diminished in the double knockout cells. In addition, both Paxillin and Focal Adhesion Kinase were expressed at lower levels and failed to correctly localize to the cell membrane in EC lacking Erk1 and Erk2, leading to defects in the organization of the cytoskeleton and in cell motility. The results demonstrate that ERK1 and ERK2 coordinate cell proliferation and migration during angiogenesis. Lentivirus infected cells to generate ERK1/2 WT and ERK1/2 DKO endothelial cells were cultured, RNA was extracted and Affymetrix gene expression arrays were performed.
Project description:We assessed the effect of RNAi-mediated MAP kinase cascade signaling blockade in primary human keratinocytes. Two sets of siRNA targeting different regions of the Erk1/2 genes were used, enabling identification of off-target siRNA effects. Primary human keratinocytes were electroporated with scrambled control siRNA, a pair of siRNA oligomers against Erk1 and Erk2 (set A), or an independent pair of siRNA oligomers against Erk1 and Erk2 (set B). Four days after electroporation, RNA was harvested. Three biological replicates were performed for each of the three siRNA groups.
Project description:A surge of luteinizing hormone (LH) from the pituitary gland triggers ovulation, oocyte maturation, and luteinization for successful reproduction in mammals. Since the signaling molecules RAS and ERK1/2 are activated by a LH surge in granulosa cells of preovulatory follicles, we disrupted Erk1/2 in mouse granulosa cells and provide in vivo evidence that these kinases are necessary for LH-induced oocyte resumption of meiosis, ovulation, and luteinization. In addition, biochemical analyses and selected disruption of the Cebpb gene in granulosa cells demonstrate that C/EBP is a critical downstream mediator of ERK1/2 activation. These mouse models provide in vivo systems in which to define the context specific and molecular mechanisms by which granulosa cells respond to LH and these mechanisms are relevant to the regulation of human fertility and infertility. Immature wild type or ERK1/2 conditonal knock-out mice were injected with 5IU equine chorionic gonadotropin (eCG)-48h followed by 5 IU hCG injection. The ovarian granulosa cells were collected at hCG 0h, 2.5h, or 4h and the gene expression pforiles were compared by microarray method.
Project description:The Casparian strip constitutes a physical diffusion barrier formed by the polar deposition of lignin in the root endodermis. The polar deposition of lignin is thought to be mediated by the scaffolding activity of membrane bound Casparian Strip domain proteins (CASPs) and the dirigent domain-containing protein Enhanced Suberin1 (ESB1). Here, we show that the endodermis-specific receptor-like kinase (ERK1), is part of this machinery, playing an essential role in the localization of CASP proteins and in the deposition of lignin, which ultimately are required for the formation of afunctional Casparian strip. ERK1 is localized to the cytoplasm and nucleus of the endodermis, and is part of a signalling pathway that implicates the circadian clock regulator Time for Coffee (TIC). In addition, we found that loss of ERK1 and TIC disrupts the Casparian strip organisation and alters composition of the root microbiome.