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The inhibition of Bax activation-induced apoptosis by RasGRP2 via R-Ras-PI3K-Akt signaling pathway in the endothelial cells.

ABSTRACT: Apoptosis of endothelial cells is a very important event in various diseases and angiogenesis. We recently reported that ras guanyl nucleotide releasing protein 2 (RasGRP2), which is a guanine nucleotide exchange factor, was expressed in the human umbilical vein endothelial cells (HUVECs) and that Rap1 activation by its overexpression inhibited apoptosis by suppressing tumor necrosis factor-? induced-reactive oxygen species (ROS) production. However, other signaling pathways and roles of RasGRP2 not mediated via Rap1 are not well understood. Therefore, we compared the Mock (M) and the RasGRP2-stable overexpression (R) immortalized HUVECs using BAM7 and anisomycin, which are apoptosis inducers. BAM7 and anisomycin induced apoptosis without causing ROS production, and such apoptosis was significantly increased in M cells, but not in R cells. RasGRP2 suppressed BAM7- and anisomycin-induced apoptosis, but not via the Rap1 pathway as observed using Rap1 knockdown. Furthermore, RasGRP2 activated not only Rap1 but also R-Ras, and suppressed apoptosis by activating R-Ras-phosphoinositide 3-kinase (PI3K)-Akt signaling pathway. The phosphorylation of Akt by RasGRP2 inhibited Bax translocation by promoting translocation of hexokinase-2 (HK-2) from cytoplasm to mitochondria. Taken together, it was suggested that RasGRP2 suppresses the Bax activation-induced apoptosis by promoting HK-2 translocation to mitochondria via R-Ras-PI3K-Akt signaling pathway.

SUBMITTER: Takino JI

PROVIDER: S-EPMC6854084 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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The inhibition of Bax activation-induced apoptosis by RasGRP2 via R-Ras-PI3K-Akt signaling pathway in the endothelial cells.

Takino Jun-Ichi JI Sato Takuma T Nagamine Kentaro K Hori Takamitsu T

Scientific reports 20191113 1

Apoptosis of endothelial cells is a very important event in various diseases and angiogenesis. We recently reported that ras guanyl nucleotide releasing protein 2 (RasGRP2), which is a guanine nucleotide exchange factor, was expressed in the human umbilical vein endothelial cells (HUVECs) and that Rap1 activation by its overexpression inhibited apoptosis by suppressing tumor necrosis factor-α induced-reactive oxygen species (ROS) production. However, other signaling pathways and roles of RasGRP2 ...[more]

PMID: 31723205

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Project description:BackgroundMandibular distraction osteogenesis (MDO) is a kind of endogenous tissue engineering technology that lengthens the jaw and opens airway so that a patient can breathe safely and comfortably on his or her own. Endothelial progenitor cells (EPCs) are crucial for MDO-related angiogenesis. Moreover, emerging evidence suggests that heat shock protein 20 (Hsp20) modulates angiogenesis under hypoxic conditions. However, the specific role of Hsp20 in EPCs, in the context of MDO, is not yet known. The aim of this study was to explore the expression of Hsp20 during MDO and the effects of Hsp20 on EPCs under hypoxia.MethodsMandibular distraction osteogenesis and mandibular bone defect (MBD) canine model were established. The expression of CD34, CD133, HIF-1α, and Hsp20 in callus was detected by immunofluorescence on day 14 after surgery. Canine bone marrow EPCs were cultured, with or without optimal cobalt chloride (CoCl2) concentration. Hypoxic effects, caused by CoCl2, were evaluated by means of the cell cycle, cell apoptosis, transwell cell migration, and tube formation assays. The Hsp20/KDR/PI3K/Akt expression levels were evaluated via immunofluorescence, RT-qPCR, and western blot. Next, EPCs were incorporated with either Hsp20-overexpression or Hsp20-siRNA lentivirus. The resulting effects were evaluated as described above.ResultsCD34, CD133, HIF-1α, and Hsp20 were displayed more positive in the callus of MDO compared with MBD. In addition, hypoxic conditions, generated by 0.1 mM CoCl2, in canine EPCs, accelerated cell proliferation, migration, tube formation, and Hsp20 expression. Hsp20 overexpression in EPCs significantly stimulated cell proliferation, migration, and tube formation, whereas Hsp20 inhibition produced the opposite effect. Additionally, the molecular mechanism was partly dependent on the KDR/PI3K/Akt pathway.ConclusionIn summary, herein, we present a novel mechanism of Hsp20-mediated regulation of canine EPCs via Akt activation in a hypoxic microenvironment.

Project description:BACKGROUND:Neuroinflammation is an important host defense response to secondary brain injury after intracerebral hemorrhage (ICH). Triggering receptor expressed on myeloid cells 2 (TREM2) confers strong neuroprotective effects by attenuating neuroinflammation in experimental ischemic stroke. Recent studies suggest that apolipoprotein E (apoE) is a novel, high-affinity ligand of TREM2. This study aimed to investigate the effects of TREM2 activation on neuroinflammation and neuronal apoptosis in a mouse model of ICH. METHODS:Adult male CD1 mice (n = 216) were subjected to intrastriatal injection of bacterial collagenase. The TREM2 ligand, apoE-mimetic peptide COG1410 was administered intranasally at 1?h after ICH induction. To elucidate the underlying mechanism, TREM2 small interfering RNA (siRNA) and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 were administered intracerebroventricularly prior to COG1410 treatment. Neurobehavioral tests, brain water content, immunofluorescence, western blotting, and Fluoro-Jade C- and terminal deoxynucleotidyl transferase dUTP nick end labeling staining were performed. RESULTS:Endogenous TREM2 expression was increased and peaked at 24?h after ICH. TREM2 was expressed on microglia, astrocytes, and neurons. COG1410 improved both short-term and long-term neurological functions, reduced brain edema, inhibited microglia/macrophage activation and neutrophil infiltration, and suppressed neuronal apoptotic cell death in perihematomal areas after ICH. Knockdown of endogenous TREM2 by TREM2 siRNA aggravated neurological deficits and decreased the expression of TREM2 in naïve and ICH mice. COG1410 was associated with upregulation of TREM2, PI3K, phosphorylated-Akt, and Bcl-2 and downregulation of TNF-?, IL-1?, and Bax after ICH. The neuroprotective effects of COG1410 were abolished by both TREM2 siRNA and PI3K inhibitor LY294002. CONCLUSIONS:Our finding demonstrated that TREM2 activation improved neurological functions and attenuated neuroinflammation and neuronal apoptosis after ICH, which was, at least in part, mediated by activation of PI3K/Akt signaling pathway. Therefore, activation of TREM2 may be a potential therapeutic strategy for the management of ICH patients.

Project description:ObjectivePolycythemia vera (PV) is characterized by erythrocytosis associated with the presence of the activating JAK2(V617F) mutation in a variable proportion of hematopoietic cells. JAK2(V617F) is detected in other myeloproliferative neoplasms, does not appear to be the PV-initiating event, and its specific role in deregulated erythropoiesis in PV is incompletely understood. We investigated the pathogenesis of PV to characterize abnormal proliferation and apoptosis responses and aberrant oncogenic pathway activation in primary PV erythroid precursors.Materials and methodsPeripheral blood CD34(+) cells isolated from PV patients and healthy controls were grown in liquid culture to expand a population of primary erythroblasts for experiments designed to analyze cellular proliferation, apoptosis, JAK2(V617F) mutation status, cytokine-dependent protein phosphorylation and gene expression profiling using Affymetrix microarrays.ResultsThe survival and proliferation of PV erythroblasts were growth factor-dependent under strict serum-free conditions requiring both erythropoietin (EPO) and stem cell factor. PV erythroblasts exhibited EPO hypersensitivity and enhanced cellular proliferation associated with increased EPO-mediated extracellular signal-regulated kinases 1 and 2 phosphorylation. EPO-induced AKT phosphorylation was observed in PV but not normal erythroblasts, an effect associated with apoptosis resistance in PV erythroblasts. Analysis of gene expression and oncogenic pathway activation signatures revealed increased RAS (p<0.01) and phosphoinositide-3 kinase (p<0.05) pathway activation in PV erythroblasts.ConclusionDeregulated erythropoiesis in PV involves EPO hypersensitivity and apoptosis resistance of erythroid precursor cells associated with abnormally increased activation of RAS-ERK and phosphoinositide-3 kinase-AKT pathways. These data suggest that investigation of the mechanisms of abnormal RAS and phosphoinositide-3 kinase pathway activation in erythroblasts may contribute to our understanding of the molecular pathogenesis of PV.

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The inhibition of Bax activation-induced apoptosis by RasGRP2 via R-Ras-PI3K-Akt signaling pathway in the endothelial cells.

Publications

The inhibition of Bax activation-induced apoptosis by RasGRP2 via R-Ras-PI3K-Akt signaling pathway in the endothelial cells.

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