Phospho-eNOS Ser-114 in human mesenchymal stem cells: Constitutive phosphorylation, nuclear localization and upregulation during mitosis

Klinz, Franz‐Josef; Schmidt, Annette; Schinköthe, Timo; Arnhold, Stefan; Desai, Biren; Popken, F.; Brixius, Klara; Schwinger, Robert H. G.; Mehlhorn, Uwe; Staib, Peter; Addicks, Klaus; Bloch, Wilhelm

doi:10.1016/j.ejcb.2005.06.003

Cited by 26 publications

(15 citation statements)

References 26 publications

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“…Samples were obtained following procedures approved by the local ethics committee. MSCs were prepared as previously described 21, 22. The aspirates were diluted in Ca 2+ ‐ and Mg 2+ ‐free PBS, filtered and generated from the interphase fraction of Ficoll gradient‐separated bone marrow (BM) cells (Ficoll‐Paque™ Plus, Amersham Pharmacia Biotech, Uppsala, Sweden).…”

Section: Methodsmentioning

confidence: 99%

Human mesenchymal stem cells: Influence of oxygen pressure on proliferation and chondrogenic differentiation in fibrin glue in vitro

Baumgartner

Arnhold

Brixius³

et al. 2009

J Biomedical Materials Res

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Tissue engineering using biomaterials is a promising solution for cartilage replacement. The purpose of this study was to investigate whether the fibrin sealant Tissucol(R) provides a suitable scaffold for re-implanting stem cells during chondrogenic replacement therapy. Pluripotent stem cells were isolated from adult human bone marrow (hMSCs), cultured and characterized by FACS (CD105+/CD106+, CD45-/CD14-/CD34-). A large-holed porous hMSC-containing fibrin matrix was built that allowed hMSCs to survive throughout the period of culture (42 days) in either proliferation or chondrogenic differentiation medium under normoxic (21% O2) or hypoxic (3% O2) conditions. Morphology (as determined by electron microscopy) and proliferation (Ki67 staining) of the embedded hMSCs did not markedly vary under normoxic and hypoxic culture even after 42 days in culture. The stem cell marker Oct-4 was expressed during the whole culture period. Under chondrogenic differentiation conditions, especially under hypoxic conditions, we observed rounded chondrocyte-like cell types and a chondral phenotype assessed by mRNA expression of collagen II and Alcian blue staining. hMSCs seeded into large-holed porous preparations of Tissucol survive, proliferate and keep their stem cell character. Furthermore, culturing the cells in a corresponding medium induces chondrogenic differentiation, which could be remarkably and significantly enhanced under hypoxic conditions.

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Section: Methodsmentioning

confidence: 99%

Human mesenchymal stem cells: Influence of oxygen pressure on proliferation and chondrogenic differentiation in fibrin glue in vitro

Baumgartner

Arnhold

Brixius³

et al. 2009

J Biomedical Materials Res

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“…Recently it has been shown that activated eNOS can translocate into the nucleus (17)(18)(19)(20) where it binds ER␤ (10). Formation of an eNOS/ER␤ combinatorial complex determines localized remodeling of chromatin, leading to transcriptional activation of previously identified prognostic genes (e.g.…”

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confidence: 99%

Silencing of GSTP1, a Prostate Cancer Prognostic Gene, by the Estrogen Receptor-β and Endothelial Nitric Oxide Synthase Complex

Aiello

Nanni³

et al. 2011

Molecular Endocrinology

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We recently identified in prostate tumors (PCa) a transcriptional prognostic signature comprising a significant number of genes differentially regulated in patients with worse clinical outcome. Induction of up-regulated genes was due to chromatin remodeling by a combinatorial complex between estrogen receptor (ER)-␤ and endothelial nitric oxide synthase (eNOS). Here we show that this complex can also repress transcription of prognostic genes that are down-regulated in PCa, such as the glutathione transferase gene GSTP1. Silencing of GSTP1 is a common early event in prostate carcinogenesis, frequently caused by promoter hypermethylation. We validated loss of glutathione transferase (GST) P1-1 expression in vivo, in tissue microarrays from a retrospective cohort of patients, and correlated it with decreased disease-specific survival. Furthermore, we show that in PCa cultured cells ER␤/eNOS causes GSTP1 repression by being recruited at estrogen responsive elements in the gene promoter with consequential remodeling of local chromatin. Treatment with ER␤ antagonist or its natural ligand 5␣-androstane-3␤,17␤-diol, eNOS inhibitors or ER␤ small interference RNA abrogated the binding and reversed GSTP1 silencing, demonstrating the direct involvement of the complex. In vitro, GSTP1 silencing by ER␤/eNOS was specific for cells from patients with worse clinical outcome where it appeared the sole mechanism regulating GSTP1 expression because no promoter hypermethylation was present. However, in vivo chromatin immunoprecipitation assays on fresh PCa tissues demonstrated that silencing by ER␤/eNOS can coexist with promoter hypermethylation. Our findings reveal that the ER␤/eNOS complex can exert transcriptional repression and suggest that this may represent an epigenetic event favoring inactivation of the GSTP1 locus by methylation. Moreover, abrogation of ER␤/eNOS function by 3␤-adiol emphasizes the significance of circulating or locally produced sex steroid hormones or their metabolites in PCa biology with relevant clinical/therapeutic implications.

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“…Consistent with this observation is a recent study showing a dynamic eNOS translocation from peripheral to nuclear regions in porcine cerebral endothelial cells upon stimulation with lysophsphatidic acid; furthermore, nuclear localization of soluble guanylyl cyclase, the NO downstream effector, was also demonstrated in rat liver specimens by immunogold labeling (Gobeil et al, 2006). In another study, proliferating mesenchymal stem cells derived from human bone marrow exhibit cytosolic and pronounced nuclear localization of eNOS; examination of its phosphorylation states revealed that eNOS phosphorylated at Ser‐114 is heavily enriched in the nucleus, whereas eNOS phosphorylated at Ser‐1177 is localized at filamentous structures in the cytosol (Klinz et al, 2005). In endothelial cells, eNOS has been shown to translocate from the cell membrane to the nuclear fraction within 1–2 min of bradykinin treatment (Wang et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

Protective effects of lipopolysaccharide preconditioning against nitric oxide neurotoxicity

Huang

Yang

Chen

et al. 2007

J of Neuroscience Research

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We have characterized lipopolysaccharide (LPS) preconditioning-induced neuroprotective mechanisms against nitric oxide (NO) toxicity. Pretreatment of rat cortical cultures with LPS attenuated neurotoxicity of NO donors, including sodium nitroprusside (SNP) and diethylamine NONOate (NONOate). A transiently increased expression of endothelial nitric oxide synthase (eNOS) accompanied by an increase in NO production was observed during LPS preconditioning. Application of NOS inhibitors including L-N(5)-(1-iminoethyl)-ornithine (L-NIO) and L-nitroarginine methylester (L-NAME) abolished LPS-dependent protection against SNP toxicity. The LPS effect was also blocked by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). Consistently, application of 8-bromo-cyclic GMP (8-Br-cGMP), a slowly degradable cGMP analogue capable of PKG activation, was neuroprotective. LPS preconditioning resulted in a heightened neuronal expression of Bcl-2 protein that was abolished by L-NAME and KT5823, the respective inhibitors of NOS and PKG. Together, our results reveal the signaling cascade of "LPS --> eNOS --> NO --> cGMP/PKG --> Bcl-2" that might have contributed to the LPS protective effects in cortical neurons.

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Phospho-eNOS Ser-114 in human mesenchymal stem cells: Constitutive phosphorylation, nuclear localization and upregulation during mitosis

Cited by 26 publications

References 26 publications

Human mesenchymal stem cells: Influence of oxygen pressure on proliferation and chondrogenic differentiation in fibrin glue in vitro

Human mesenchymal stem cells: Influence of oxygen pressure on proliferation and chondrogenic differentiation in fibrin glue in vitro

Silencing of GSTP1, a Prostate Cancer Prognostic Gene, by the Estrogen Receptor-β and Endothelial Nitric Oxide Synthase Complex

Protective effects of lipopolysaccharide preconditioning against nitric oxide neurotoxicity

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