miR-132/212 knockout mice reveal roles for these miRNAs in regulating cortical synaptic transmission and plasticity

miR-132 and miR-212 are two closely related miRNAs encoded in the same intron of a small non-coding gene, which have been suggested to play roles in both immune and neuronal function. We describe here the generation and initial characterisation of a miR-132/212 double knockout mouse. These mice were viable and fertile with no overt adverse phenotype. Analysis of innate immune responses, including TLR-induced cytokine production and IFNβ induction in response to viral infection of primary fibroblasts did not reveal any phenotype in the knockouts. In contrast, the loss of miR-132 and miR-212, while not overtly affecting neuronal morphology, did affect synaptic function. In both hippocampal and neocortical slices miR-132/212 knockout reduced basal synaptic transmission, without affecting paired-pulse facilitation. Hippocampal long-term potentiation (LTP) induced by tetanic stimulation was not affected by miR-132/212 deletion, whilst theta burst LTP was enhanced. In contrast, neocortical theta burst-induced LTP was inhibited by loss of miR-132/212. Together these results indicate that miR-132 and/or miR-212 play a significant role in synaptic function, possibly by regulating the number of postsynaptic AMPA receptors under basal conditions and during activity-dependent synaptic plasticity

High nuclear MSK1 is associated with longer survival in breast cancer patients

Purpose: Mitogen- and stress- activated kinases (MSKs) are important substrates of the mitogen-activated protein kinase (MAPK)-activated protein kinase family. MSK1 and MSK2 are both nuclear serine/threonine protein kinases, with MSK1 being suggested to potentially play a role in breast cancer cell proliferation, cell cycle progression, cell migration, invasion and tumour growth. The aim of the current study was to assess MSK1 protein expression in breast cancer tumour specimens, evaluating its prognostic significance. Methods: A large cohort of 1902 early stage invasive breast cancer patients was used to explore the expression of MSK1. Protein expression was examined using standard immunohistochemistry on tissue microarrays. Results: Low MSK1 protein expression was associated with younger age (P=0.004), higher tumour grade (P<0.001), higher Nottingham Prognostic Index scores (P=0.007), negative ER (P<0.001) and PR (P<0.001) status, and with triple-negative (P<0.001) and basal-like (P<0.001) phenotypes. Low MSK1 protein expression was significantly associated with shorter time to distant metastasis (P<0.001), and recurrence (P=0.013) and early death due to breast cancer (P=0.01). This association between high MSK1 expression and improved breast cancer-specific survival was observed in the whole cohort (P=0.009) and in the HER2 negative and non-basal like tumours (P=0.006 and P=0.024, respectively). Multivariate analysis including other prognostic variables indicated that MSK1 is not an independent marker of outcome. Conclusions: High MSK1 is associated with improved breast cancer-specific survival in early stage invasive breast cancer patients, and has additional prognostic value in HER2 negative and non-basal like disease. Although not an independent marker of outcome we believe such findings, and significant associations with well-established negative prognostic factors (age, grade, Nottingham Prognostic Index, hormone receptor status, time to distant metastasi

Accelerated apoptotic death and <i>in vivo</i> turnover of erythrocytes in mice lacking functional mitogen- and stress-activated kinase MSK1/2

The mitogen- and stress-activated kinase MSK1/2 plays a decisive role in apoptosis. In analogy to apoptosis of nucleated cells, suicidal erythrocyte death called eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Here, we explored whether MSK1/2 participates in the regulation of eryptosis. To this end, erythrocytes were isolated from mice lacking functional MSK1/2 (msk−/−) and corresponding wild-type mice (msk+/+). Blood count, hematocrit, hemoglobin concentration and mean erythrocyte volume were similar in both msk−/− and msk+/+ mice, but reticulocyte count was significantly increased in msk−/− mice. Cell membrane PS exposure was similar in untreated msk−/− and msk+/+ erythrocytes, but was enhanced by pathophysiological cell stressors ex vivo such as hyperosmotic shock or energy depletion to significantly higher levels in msk−/− erythrocytes than in msk+/+ erythrocytes. Cell shrinkage following hyperosmotic shock and energy depletion, as well as hemolysis following decrease of extracellular osmolarity was more pronounced in msk−/− erythrocytes. The in vivo clearance of autologously-infused CFSE-labeled erythrocytes from circulating blood was faster in msk−/− mice. The spleens from msk−/− mice contained a significantly greater number of PS-exposing erythrocytes than spleens from msk+/+ mice. The present observations point to accelerated eryptosis and subsequent clearance of erythrocytes leading to enhanced erythrocyte turnover in MSK1/2-deficient mice

The Chromatin Modifier MSK1/2 Suppresses Endocrine Cell Fates during Mouse Pancreatic Development

Type I diabetes is caused by loss of insulin-secreting beta cells. To identify novel, pharmacologically-targetable histone-modifying proteins that enhance beta cell production from pancreatic progenitors, we performed a screen for histone modifications induced by signal transduction pathways at key pancreatic genes. The screen led us to investigate the temporal dynamics of ser-28 phosphorylated histone H3 (H3S28ph) and its upstream kinases, MSK1 and MSK2 (MSK1/2). H3S28ph and MSK1/2 were enriched at the key endocrine and acinar promoters in E12.5 multipotent pancreatic progenitors. Pharmacological inhibition of MSK1/2 in embryonic pancreatic explants promoted the specification of endocrine fates, including the beta-cell lineage, while depleting acinar fates. Germline knockout of both Msk isoforms caused enhancement of alpha cells and a reduction in acinar differentiation, while monoallelic loss of Msk1 promoted beta cell mass. Our screen of chromatin state dynamics can be applied to other developmental contexts to reveal new pathways and approaches to modulate cell fates

Mitogen- and Stress-Activated Kinase 1 (MSK1) Regulates Cigarette Smoke-Induced Histone Modifications on NF-κB-dependent Genes

Cigarette smoke (CS) causes sustained lung inflammation, which is an important event in the pathogenesis of chronic obstructive pulmonary disease (COPD). We have previously reported that IKKα (I kappaB kinase alpha) plays a key role in CS-induced pro-inflammatory gene transcription by chromatin modifications; however, the underlying role of downstream signaling kinase is not known. Mitogen- and stress-activated kinase 1 (MSK1) serves as a specific downstream NF-κB RelA/p65 kinase, mediating transcriptional activation of NF-κB-dependent pro-inflammatory genes. The role of MSK1 in nuclear signaling and chromatin modifications is not known, particularly in response to environmental stimuli. We hypothesized that MSK1 regulates chromatin modifications of pro-inflammatory gene promoters in response to CS. Here, we report that CS extract activates MSK1 in human lung epithelial (H292 and BEAS-2B) cell lines, human primary small airway epithelial cells (SAEC), and in mouse lung, resulting in phosphorylation of nuclear MSK1 (Thr581), phospho-acetylation of RelA/p65 at Ser276 and Lys310 respectively. This event was associated with phospho-acetylation of histone H3 (Ser10/Lys9) and acetylation of histone H4 (Lys12). MSK1 N- and C-terminal kinase-dead mutants, MSK1 siRNA-mediated knock-down in transiently transfected H292 cells, and MSK1 stable knock-down mouse embryonic fibroblasts significantly reduced CS extract-induced MSK1, NF-κB RelA/p65 activation, and posttranslational modifications of histones. CS extract/CS promotes the direct interaction of MSK1 with RelA/p65 and p300 in epithelial cells and in mouse lung. Furthermore, CS-mediated recruitment of MSK1 and its substrates to the promoters of NF-κB-dependent pro-inflammatory genes leads to transcriptional activation, as determined by chromatin immunoprecipitation. Thus, MSK1 is an important downstream kinase involved in CS-induced NF-κB activation and chromatin modifications, which have implications in pathogenesis of COPD

Dimethyl fumarate blocks pro-inflammatory cytokine production via inhibition of TLR induced M1 and K63 ubiquitin chain formation

Dimethyl fumarate (DMF) possesses anti-inflammatory properties and is approved for the treatment of psoriasis and multiple sclerosis. While clinically effective, its molecular target has remained elusive - although it is known to activate anti-oxidant pathways. We find that DMF inhibits pro-inflammatory cytokine production in response to TLR agonists independently of the Nrf2-Keap1 anti-oxidant pathway. Instead we show that DMF can inhibit the E2 conjugating enzymes involved in K63 and M1 polyubiquitin chain formation both in vitro and in cells. The formation of K63 and M1 chains is required to link TLR activation to downstream signaling, and consistent with the block in K63 and/or M1 chain formation, DMF inhibits NFκB and ERK1/2 activation, resulting in a loss of pro-inflammatory cytokine production. Together these results reveal a new molecular target for DMF and show that a clinically approved drug inhibits M1 and K63 chain formation in TLR induced signaling complexes. Selective targeting of E2s may therefore be a viable strategy for autoimmunity