Chulhee Choi

Differential Regulation and Function of Fas Expression on Glial Cells

Fas/Apo-1 is a member of the TNF receptor superfamily that signals apoptotic cell death in susceptible target cells. Fas or Fas ligand (FasL)-deficient mice are relatively resistant to the induction of experimental allergic encephalomyelitis, implying the involvement of Fas/FasL in this disease process. We have examined the regulation and function of Fas expression in glial cells (astrocytes and microglia). Fas is constitutively expressed by primary murine microglia at a low level and significantly up-regulated by TNF-α or IFN-γ stimulation. Primary astrocytes express high constitutive levels of Fas, which are not further affected by cytokine treatment. In microglia, Fas expression is regulated at the level of mRNA expression; TNF-α and IFN-γ induced Fas mRNA by ∼20-fold. STAT-1α and NF-κB activation are involved in IFN-γ- or TNF-α-mediated Fas up-regulation in microglia, respectively. The cytokine TGF-β inhibits basal expression of Fas as well as cytokine-mediated Fas expression by microglia. Upon incubation of microglial cells with FasL-expressing cells, ∼20% of cells underwent Fas-mediated cell death, which increased to ∼60% when cells were pretreated with either TNF-α or IFN-γ. TGF-β treatment inhibited Fas-mediated cell death of TNF-α- or IFN-γ-stimulated microglial cells. In contrast, astrocytes are resistant to Fas-mediated cell death, however, ligation of Fas induces expression of the chemokines macrophage inflammatory protein-1β (MIP-1β), MIP-1α, and MIP-2. These data demonstrate that Fas transmits different signals in the two glial cell populations: a cytotoxic signal in microglia and an inflammatory signal in the astrocyte.

ICAM-1-Induced Expression of Proinflammatory Cytokines in Astrocytes: Involvement of Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase Pathways

ICAM-1 is a transmembrane glycoprotein of the Ig superfamily involved in cell adhesion. ICAM-1 is aberrantly expressed by astrocytes in CNS pathologies such as multiple sclerosis, experimental allergic encephalomyelitis, and Alzheimer’s disease, suggesting a possible role for ICAM-1 in these disorders. ICAM-1 has been shown to be important for leukocyte diapedesis through brain microvessels and subsequent binding to astrocytes. However, other functional roles for ICAM-1 expression on astrocytes have not been well elucidated. Therefore, we investigated the intracellular signals generated upon ICAM-1 engagement on astrocytes. ICAM-1 ligation by a mAb to rat ICAM-1 induced mRNA expression of proinflammatory cytokines such as IL-1α, IL-1β, IL-6, and TNF-α. Examination of cytokine protein production revealed that ICAM-1 ligation results in IL-6 secretion by astrocytes, whereas IL-1β and IL-1α protein is expressed intracellularly in astrocytes. The involvement of mitogen-activated protein kinases (MAPKs) in ICAM-1-mediated cytokine expression in astrocytes was tested, as the MAPK extracellular signal-regulated kinase (ERK) was previously shown to be activated upon ICAM-1 engagement. Our results indicate that ERK1/ERK2, as well as p38 MAPK, are activated upon ligation of ICAM-1. Studies using pharmacological inhibitors demonstrate that both p38 MAPK and ERK1/2 are involved in ICAM-1-induced IL-6 expression, whereas only ERK1/2 is important for IL-1α and IL-1β expression. Our data support the role of ICAM-1 on astrocytes as an inflammatory mediator in the CNS and also uncover a novel signal transduction pathway through p38 MAPK upon ICAM-1 ligation.

CXC Chemokine Receptor 4 Expression and Function in Human Astroglioma Cells

Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-γ-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-α, IL-1β, and IFN-γ. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-α and IL-1β. The peak of CXCR4 expression in response to TNF-α and IL-1β was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-α and IL-1β (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1α (SDF-1α), resulted in an elevation in intracellular Ca2+ concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1α treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-γ-inducible protein-10. SDF-1α-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation.

Tumor necrosis factor-related apoptosis-inducing ligand induces caspase-dependent interleukin-8 expression and apoptosis in human astroglioma cells.

ABSTRACT Among the tumor necrosis factor (TNF) family of cytokines, FasL and TNF-related apoptosis-inducing ligand (TRAIL) are known to induce cell death via caspase activation. Recently, other biological functions of these death ligands have been postulated in vitro and in vivo. It was previously shown that Fas ligation induces chemokine expression in human glioma cells. In this study, we investigated whether the TRAIL-DR5 system transduces signals similar to those induced by other TNF family ligands and receptors. To address this issue, two human glioma cell lines, CRT-MG and U87-MG, were used, and an agonistic antibody against DR5 (TRA-8) and human recombinant TRAIL were used to ligate DR5. We demonstrate that DR5 ligation by either TRAIL or TRA-8 induces two functional outcomes, apoptosis and expression of the chemokine interleukin-8 (IL-8); the nonspecific caspase inhibitor Boc-D-Fmk blocks both TRAIL-mediated cell death and IL-8 production; the caspase 3-specific inhibitor z-DEVD-Fmk suppresses TRAIL-mediated apoptosis but not IL-8 induction; caspase 1- and 8-specific inhibitors block both TRAIL-mediated cell death and IL-8 production; and DR5 ligation by TRAIL mediates AP-1 and NF-κB activation, which can be inhibited by caspase 1- and 8-specific inhibitors. These findings collectively indicate that DR5 ligation on human glioma cells leads to apoptosis and that the activation of AP-1 and NF-κB leads to the induction of IL-8 expression; these responses are dependent on caspase activation. Therefore, the TRAIL-DR5 system has a role not only as an inducer of apoptotic cell death but also as a tranducer for proinflammatory and angiogenic signals in human brain tumors.

Ginsenosides compound K and Rh2 inhibit tumor necrosis factor-α-induced activation of the NF-κB and JNK pathways in human astroglial cells

Ginsenosides, the main component of Panax ginseng, have been known for the anti-inflammatory and anti-proliferative activities. In this study, we investigated the molecular mechanisms responsible for the anti-inflammatory effects of ginsenosides on activated astroglial cells. Among 13 different ginsenosides, intestinal bacterial metabolites Rh(2) and compound K (C-K) showed a significant inhibitory effect on tumor necrosis factor-alpha (TNF-alpha)-induced expression of intercellular adhesion molecule-1 in human astroglial cells. Pretreatment with C-K or Rh(2) suppressed TNF-alpha-induced phosphorylation of IkappaBalpha kinase and the subsequent phosphorylation and degradation of IkappaBalpha. Additionally, the same treatment inhibited TNF-alpha-induced phosphorylation of MKK4 and the subsequent activation of the JNK-AP-1 pathway. The inhibitory effect of ginsenosides on TNF-alpha-induced activation of the NF-kappaB and JNK pathways was not observed in human monocytic U937 cells. These results collectively indicate that ginsenoside metabolites C-K and Rh(2) exert anti-inflammatory effects by the inhibition of both NF-kappaB and JNK pathways in a cell-specific manner.

Mitochondrial Network Determines Intracellular ROS Dynamics and Sensitivity to Oxidative Stress through Switching Inter-Mitochondrial Messengers

Oxidative stresses caused by reactive oxygen species (ROS) can induce rapid depolarization of inner mitochondrial membrane potential and subsequent impairment of oxidative phosphorylation. Damaged mitochondria produce more ROS, especially the superoxide anion (O2 −) and hydrogen peroxide (H2O2), which potentiate mitochondria-driven ROS propagation, so-called ROS-induced ROS release (RIRR), via activation of an inter-mitochondria signaling network. Therefore, loss of function in only a fraction of mitochondria might eventually affect cell viability through this positive feedback loop. Since ROS are very short-lived molecules in the biological milieu, mitochondrial network dynamics, such as density, number, and spatial distribution, can affect mitochondria-driven ROS propagation. To address this issue, we developed a mathematical model using an agent-based modeling approach, and tested the effect of mitochondrial network dynamics on RIRR for mitochondria under various conditions. Simulation results show that the intracellular ROS signaling pattern, such as ROS propagation speed and oxidative stress vulnerability, are critically affected by mitochondrial network dynamics. Mitochondrial network dynamics of mitochondrial distribution, density, activity, and size can mediate inter-mitochondrial signaling under certain conditions and determine the identity of the ROS signaling pattern. We further elucidated the potential mechanism of these actions, i.e., conversion of major messenger molecules involved in ROS signaling. If the average distance between neighboring mitochondria is large or mitochondrial distribution becomes randomized, messenger molecule of the ROS signaling network can be switched from O2 − to H2O2. In this case, mitochondria-driven ROS propagation is efficiently blocked by introduction of excess cytosolic glutathione peroxidase 1, while introduction of cytosolic superoxide dismutase has no effect. Together, these results suggest that mitochondrial network dynamics is a major determinant for cellular responses to RIRR through changing the key messenger molecules.

Oxidative Stress-Induced Necrotic Cell Death via Mitochondira-Dependent Burst of Reactive Oxygen Species

Oxidative stress is deeply involved in various brain diseases, including neurodegenerative diseases, stroke, and ischemia/reperfusion injury. Mitochondria are thought to be the target and source of oxidative stress. We investigated the role of mitochondria in oxidative stress-induced necrotic neuronal cell death in a neuroblastoma cell line and a mouse model of middle cerebral artery occlusion. The exogenous administration of hydrogen peroxide was used to study the role of oxidative stress on neuronal cell survival and mitochondrial function in vitro. Hydrogen peroxide induced non-apoptotic neuronal cell death in a c-Jun N-terminal kinase- and poly(ADP-ribosyl) polymerase-dependent manner. Unexpectedly, hydrogen peroxide treatment induced transient hyperpolarization of the mitochondrial membrane potential and a subsequent delayed burst of endogenous reactive oxygen species (ROS). The inhibition of mitochondrial hyperpolarization by diphenylene iodonium or rotenone, potent inhibitors of mitochondrial respiratory chain complex I, resulted in reduced ROS production and subsequent neuronal cell death in vitro and in vivo. The inhibition of mitochondrial hyperpolarization can protect neuronal cells from oxidative stress-induced necrotic cell death, suggesting a novel method of therapeutic intervention in oxidative stress-induced neurological disease.

Efficient differentiation of human pluripotent stem cells into functional CD34+ progenitor cells by combined modulation of the MEK/ERK and BMP4 signaling pathways.

Differentiation of human pluripotent stem cells (hPSCs) into functional cell types is a crucial step in cell therapy. In the present study, we demonstrate that functional CD34(+) progenitor cells can be efficiently produced from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) by combined modulation of 2 signaling pathways. A higher proportion of CD34(+) cells (∼ 20%) could be derived from hPSCs by inhibition of mitogen-activated protein kinase (MAPK) extracellular signal-regulated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling and activation of bone morphogenic protein-4 (BMP4) signaling. hPSC-derived CD34(+) progenitor cells further developed to endothelial and smooth muscle cells with functionality. Moreover, they contributed directly to neovasculogenesis in ischemic mouse hind limbs, thereby resulting in improved blood perfusion and limb salvage. Our results suggest that combined modulation of signaling pathways may be an efficient means of differentiating hPSCs into functional CD34(+) progenitor cells.

Microfluidic Self-Sorting of Mammalian Cells to Achieve Cell Cycle Synchrony by Hydrophoresis

Cell cycle studies for examining regulatory mechanisms and progression invariably require synchronization of cell cultures at a specific phase of the cell cycle. Current implementations to produce synchronous cell populations, however, tend to perturb normal cellular progression and metabolism and typically require complex, time-consuming preparations. Thus, it is challenging for the development of a simple, noninvasive, and effective means for cell cycle synchronization. We demonstrate the use of hydrophoretic size separation to sort cells in target phases of the cell cycle entirely based on a hydrodynamic principle. With this method, we found that there is a linear relationship between a cells size and its position distribution in the hydrophoretic device. We also demonstrate the robustness of the hydrophoretic method for practical applications by sorting cells in the G(0)/G(1) and G(2)/M phases out of the original, asynchronous cells with a high level of synchrony of 95.5% and 85.2%, respectively. These results show that the hydrophoretic size separation can be used in order to collect cells at the same phase of the cell cycle in a gentle, noninvasive way.

Peroxiredoxin II is an essential antioxidant enzyme that prevents the oxidative inactivation of VEGF receptor-2 in vascular endothelial cells.

Cellular antioxidant enzymes play crucial roles in aerobic organisms by eliminating detrimental oxidants and maintaining the intracellular redox homeostasis. Therefore, the function of antioxidant enzymes is inextricably linked to the redox-dependent activities of multiple proteins and signaling pathways. Here, we report that the VEGFR2 RTK has an oxidation-sensitive cysteine residue whose reduced state is preserved specifically by peroxiredoxin II (PrxII) in vascular endothelial cells. In the absence of PrxII, the cellular H(2)O(2) level is markedly increased and the VEGFR2 becomes inactive, no longer responding to VEGF stimulation. Such VEGFR2 inactivation is due to the formation of intramolecular disulfide linkage between Cys1199 and Cys1206 in the C-terminal tail. Interestingly, the PrxII-mediated VEGFR2 protection is achieved by association of two proteins in the caveolae. Furthermore, PrxII deficiency suppresses tumor angiogenesis in vivo. This study thus demonstrates a physiological function of PrxII as the residential antioxidant safeguard specific to the redox-sensitive VEGFR2.