Kwon-Soo Ha

Nestin Is Required for the Proper Self-Renewal of Neural Stem Cells†‡§

The intermediate filament protein, nestin, is a widely employed marker of multipotent neural stem cells (NSCs). Recent in vitro studies have implicated nestin in a number of cellular processes, but there is no data yet on its in vivo function. Here, we report the construction and functional characterization of Nestin knockout mice. We found that these mice show embryonic lethality, with neuroepithelium of the developing neural tube exhibiting significantly fewer NSCs and much higher levels of apoptosis. Consistent with this in vivo observation, NSC cultures derived from knockout embryos show dramatically reduced self‐renewal ability that is associated with elevated apoptosis but no overt defects in cell proliferation or differentiation. Unexpectedly, nestin deficiency has no detectable effect on the integrity of the cytoskeleton. Furthermore, the knockout of Vimentin, which abolishes nestins ability to polymerize into intermediate filaments in NSCs, does not lead to any apoptotic phenotype. These data demonstrate that nestin is important for the proper survival and self‐renewal of NSCs, and that this function is surprisingly uncoupled from nestins structural involvement in the cytoskeleton. STEM CELLS 2010;28:2162–2171

The non-provitamin A carotenoid, lutein, inhibits NF-κB-dependent gene expression through redox-based regulation of the phosphatidylinositol 3-kinase/PTEN/Akt and NF-κB-inducing kinase pathways: Role of H2O2 in NF-κB activation

Reactive oxygen species (ROS) have been implicated in the regulation of NF-kappaB activation, which plays an important role in inflammation and cell survival. However, the molecular mechanisms of ROS in NF-kappaB activation remain poorly defined. We found that the non-provitamin A carotenoid, lutein, decreased intracellular H(2)O(2) accumulation by scavenging superoxide and H(2)O(2) and the NF-kappaB-regulated inflammatory genes, iNOS, TNF-alpha, IL-1beta, and cyclooxygenase-2, in lipopolysaccharide (LPS)-stimulated macrophages. Lutein inhibited LPS-induced NF-kappaB activation, which highly correlated with its inhibitory effect on LPS-induced IkappaB kinase (IKK) activation, IkappaB degradation, nuclear translocation of NF-kappaB, and binding of NF-kappaB to the kappaB motif of the iNOS promoter. This compound inhibited LPS- and H(2)O(2)-induced increases in phosphatidylinositol 3-kinase (PI3K) activity, PTEN inactivation, NF-kappaB-inducing kinase (NIK), and Akt phosphorylation, which are all upstream of IKK activation, but did not affect the interaction between Toll-like receptor 4 and MyD88 and the activation of mitogen-activated protein kinases. The NADPH oxidase inhibitor apocynin and gp91(phox) deletion reduced the LPS-induced NF-kappaB signaling pathway as lutein did. Moreover, lutein treatment and gp91(phox) deletion decreased the expressional levels of the inflammatory genes in vivo and protected mice from LPS-induced lethality. Our data suggest that H(2)O(2) modulates IKK-dependent NF-kappaB activation by promoting the redox-sensitive activation of the PI3K/PTEN/Akt and NIK/IKK pathways. These findings further provide new insights into the pathophysiological role of intracellular H(2)O(2) in the NF-kappaB signal pathway and inflammatory process.

Nitric oxide prevents 6-hydroxydopamine-induced apoptosis in PC12 cells through cGMP-dependent PI3 kinase/Akt activation

Nitric oxide (NO) functions not only as an important signaling molecule in the brain by producing cGMP, but also regulates neuronal cell apoptosis. The mechanism by which NO regulates apoptosis is unclear. In this study, we demonstrated that NO, produced either from the NO donor S‐nitroso‐N‐acetyl‐D,L‐penicillamine (SNAP) or by transfection of neuronal NO synthase, suppressed 6‐hydroxydopamine (6‐OHDA)‐induced apoptosis in PC12 cells by inhibiting mitochondrial cytochrome c release, caspase‐3 and ‐9 activation, and DNA fragmentation. This protection was significantly reversed by the soluble guanylyl cyclase inhibitor 1H‐(1,2,4)‐oxadiazole[4,3‐a]quinoxalon‐1‐one, indicating that cGMP is a key mediator in NO‐mediated anti‐apoptosis. Moreover, the membrane‐permeable cGMP analog 8‐Br‐cGMP inhibited 6‐OHDA‐induced apoptosis. These anti‐apoptotic effects of SNAP and 8‐Br‐cGMP were suppressed by cGMP‐dependent protein kinase G (PKG) inhibitor KT5823, indicating that PKG is a downstream signal mediator in the suppression of apoptosis by NO and cGMP. Both SNAP and 8‐Br‐cGMP induced endogenous Akt activation and Bad phosphorylation, resulting in the inhibition of Bad translocation to mitochondria;these effects were inhibited by KT5823 and the phosphatidylinositol 3‐kinase (PI3K) inhibitors LY294002 and Wortmannin. Our data suggest that the NO/cGMP pathway suppresses 6‐OHDA‐induced PC12 cell apoptosis by suppressing the mitochon‐drial apoptosis signal via PKG/PI3K/Akt‐dependent Bad phosphorylation.—Ha, K.‐S., Kim, K. M., Kwon, Y.‐G., Bai, S.‐K., Nam, W.‐D., Yoo, Y.‐M., Kim, P. K. M., Chung, H.‐T., Billiar, T. R., Kim, Y.‐M. Nitric oxide prevents 6‐hydroxydopamine‐induced apoptosis in PC12 cells through cGMP‐dependent PI3 kinase/Akt activation. FASEB J. 17, 1036–1047 (2003)

β-Carotene inhibits inflammatory gene expression in lipopolysaccharide-stimulated macrophages by suppressing redox-based NF-κB activation

β-Carotene has shown antioxidant and antiinflammatory activities; however, its molecular mechanism has not been clearly defined. We examined in vitro and in vivo regulatory function of β-carotene on the production of nitric oxide (NO) and PGE2 as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, TNF-α, and IL-1β. β-Carotene inhibited the expression and production of these inflammatory mediators in both LPSstimulated RAW264.7 cells and primary macrophages in a dose-dependent fashion as well as in LPS-administrated mice. Furthermore, this compound suppressed NF-κB activation and iNOS promoter activity in RAW264.7 cells stimulated with LPS. β-Carotene blocked nuclear translocation of NF-κB p65 subunit, which correlated with its inhibitory effect on IκBα phosphorylation and degradation. This compound directly blocked the intracellular accumulation of reactive oxygen species in RAW264.7 cells stimulated with LPS as both the NADPH oxidase inhibitor diphenylene iodonium and antioxidant pyrrolidine dithiocarbamate did. The inhibition of NADPH oxidase also inhibited NO production, iNOS expression, and iNOS promoter activity. These results suggest that β-carotene possesses anti-inflammatory activity by functioning as a potential inhibitor for redox-based NF-κB activation, probably due to its antioxidant activity.

Hydrogen Peroxide Activates p70S6k Signaling Pathway

We investigated a possible role of reactive oxygen species (ROS) in p70S6k activation, which plays an important role in the progression of cells from G0/G1 to S phase of the cell cycle by translational up-regulation of a family of mRNA transcripts that encode for components of the protein synthetic machinery. Treatment of mouse epidermal cell JB6 with H2O2 generated extracellularly by glucose/glucose oxidase led to the activation of p70S6k and p90Rsk and to phosphorylation of p42MAPK/p44MAPK. The activation of p70S6k and p90Rsk was dose-dependent and transient, maximal activities being in extracts treated for 15 and 30 min, respectively. Further characterization of ROS-induced activation of p70S6kusing specific inhibitors for p70S6k signaling pathway, rapamycin, and wortmannin revealed that ROS acted upstream of the rapamycin-sensitive component FRAP/RAFT and wortmannin-sensitive component phosphatidylinositol 3-kinase, because both inhibitors caused the inhibition of ROS-induced p70S6k activity. In addition, Ca2+ chelation also inhibited ROS-induced activation of p70S6k, indicating that Ca2+ is a mediator of p70S6k activation by ROS. However, down-regulation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive protein kinase C (PKC) by chronic pretreatment with TPA or a specific PKC inhibitor Ro-31-8220 did not block the activation of p70S6kby ROS, indicating that the activation of TPA-responsive PKC was not required for stimulation of p70S6k activity by H2O2 in JB6 cells. Exposure of JB6 cells to platelet-derived growth factor or epidermal growth factor led to a rapid increase in H2O2, phosphorylation, and activation of p70S6k, which were antagonized by the pretreatment of catalase. Taken together, the results suggest that ROS act as a messenger in growth factor-induced p70S6k signaling pathway.

Requirement of hydrogen peroxide generation in TGF-beta 1 signal transduction in human lung fibroblast cells: involvement of hydrogen peroxide and Ca2+ in TGF-beta 1-induced IL-6 expression.

Stimulation of human lung fibroblast cells with TGF-β1 resulted in a transient burst of reactive oxygen species with maximal increase at 5 min after treatment. This reactive oxygen species increase was inhibited by the antioxidant, N-acetyl-l-cysteine (NAC). TGF-β1 treatment stimulated IL-6 gene expression and protein synthesis in human lung fibroblast cells. Antioxidants including NAC, glutathione, and catalase reduced TGF-β1-induced IL-6 gene expression, and direct H2O2 treatment induced IL-6 expression in a dose-dependent manner. NAC also reduced TGF-β1-induced AP-1 binding activity, which is involved in IL-6 gene expression. It has been reported that Ca2+ influx is stimulated by TGF-β1 treatment. EGTA suppressed TGF-β1- or H2O2-induced IL-6 expression, and ionomycin increased IL-6 expression, with simultaneously modulating AP-1 activity in the same pattern. PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase/extracellular signal-related kinase kinase 1, suppressed TGF-β1- or H2O2-induced IL-6 and AP-1 activation. In addition, TGF-β1 or H2O2 increased MAPK activity which was reduced by EGTA and NAC, suggesting that MAPK is involved in TGF-β1-induced IL-6 expression. Taken together, these results indicate that TGF-β1 induces a transient increase of intracellular H2O2 production, which regulates downstream events such as Ca2+ influx, MAPK, and AP-1 activation and IL-6 gene expression.

Mechanisms underlying TGF-β1-induced expression of VEGF and Flk-1 in mouse macrophages and their implications for angiogenesis

TGF‐β induces vascular endothelial growth factor (VEGF), a potent angiogenic factor, at the transcriptional and protein levels in mouse macrophages. VEGF secretion in response to TGF‐β1 is enhanced by hypoxia and by overexpression of Smad3/4 and hypoxia‐inducible factor‐1α/β (HIF‐1α/β). To examine the transcriptional regulation of VEGF by TGF‐β1, we constructed mouse reporters driven by the VEGF promoter. Overexpression of HIF‐1α/β or Smad3/4 caused a slight increase of VEGF promoter activity in the presence of TGF‐β1, whereas cotransfection of HIF‐1α/β and Smad3/4 had a marked effect. Smad2 was without effect on this promoter activity, whereas Smad7 markedly reduced it. Analysis of mutant promoters revealed that the one putative HIF‐1 and two Smad‐binding elements were critical for TGF‐β1‐induced VEGF promoter activity. The relevance of these elements was confirmed by chromatin immunoprecipitation assay. p300, which has histone acetyltransferase activity, augmented transcriptional activity in response to HIF‐1α/β and Smad3/4, and E1A, an inhibitor of p300, inhibited it. TGF‐β1 also increased the expression of fetal liver kinase‐1 (Flk‐1), a major VEGF receptor, and TGF‐β1 and VEGF stimulated pro‐matrix metalloproteinase 9 (MMP‐9) and active‐MMP‐9 expression, respectively. The results from the present study indicate that TGF‐β1 can activate mouse macrophages to express angiogenic mediators such as VEGF, MMP‐9, and Flk‐1.

Transglutaminase 2: a multi-functional protein in multiple subcellular compartments.

Transglutaminase 2 (TG2) is a multifunctional protein that can function as a transglutaminase, G protein, kinase, protein disulfide isomerase, and as an adaptor protein. These multiple biochemical activities of TG2 account for, at least in part, its involvement in a wide variety of cellular processes encompassing differentiation, cell death, inflammation, cell migration, and wound healing. The individual biochemical activities of TG2 are regulated by several cellular factors, including calcium, nucleotides, and redox potential, which vary depending on its subcellular location. Thus, the microenvironments of the subcellular compartments to which TG2 localizes, such as the cytosol, plasma membrane, nucleus, mitochondria, or extracellular space, are important determinants to switch on or off various TG2 biochemical activities. Furthermore, TG2 interacts with a distinct subset of proteins and/or substrates depending on its subcellular location. In this review, the biological functions and molecular interactions of TG2 will be discussed in the context of the unique environments of the subcellular compartments to which TG2 localizes.

Icariin stimulates angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways in human endothelial cells.

We investigated the molecular effect and signal pathway of icariin, a major flavonoid of Epimedium koreanum Nakai, on angiogenesis. Icariin stimulated in vitro endothelial cell proliferation, migration, and tubulogenesis, which are typical phenomena of angiogenesis, as well as increased in vivo angiogenesis. Icariin activated the angiogenic signal modulators, ERK, phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric oxide synthase (eNOS), and increased NO production, without affecting VEGF expression, indicating that icariin may directly stimulate angiogenesis. Icariin-induced ERK activation and angiogenic events were significantly inhibited by the MEK inhibitor PD98059, without affecting Akt and eNOS phosphorylation. The PI3K inhibitor Wortmannin suppressed icariin-mediated angiogenesis and Akt and eNOS activation without affecting ERK phosphorylation. Moreover, the NOS inhibitor NMA partially reduced the angiogenic activity of icariin. These results suggest that icariin stimulated angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways and may be a useful drug for angiogenic therapy.

Two hevein homologs isolated from the seed of Pharbitis nil L. exhibit potent antifungal activity

Two antifungal peptides (Pn-AMP1 and Pn-AMP2) have been purified to homogeneity from seeds of Pharbitis nil. The amino acid sequences of Pn-AMP1 (41 amino acid0 residues) and Pn-AMP2 (40 amino acid residues) were identical except that Pn-AMP1 has an additional serine residue at the carboxyl-terminus. The molecular masses of Pn-AMP1 and Pn-AMP2 were confirmed as 4299.7 and 4213.2 Da, respectively. Both the Pn-AMPs were highly basic (pI 12.02) and had characteristics of cysteine/glycine rich chitin-binding domain. Pn-AMPs exhibited potent antifungal activity against both chitin-containing and non-chitin-containing fungi in the cell wall. Concentrations required for 50% inhibition of fungal growth were ranged from 3 to 26 micrograms/ml for Pn-AMP1 and from 0.6 to 75 micrograms/ml for Pn-AMP2. The Pn-AMPs penetrated very rapidly into fungal hyphae and localized at septum and hyphal tips of fungi, which caused burst of hyphal tips. Burst of hyphae resulted in disruption of the fungal membrane and leakage of the cytoplasmic materials. To our knowledge, Pn-AMPs are the first hevein-like proteins that show similar fungicidal effects as thionins do.