Seu-Mei Wang

Mechanism of oxidative stress‐induced intracellular acidosis in rat cerebellar astrocytes and C6 glioma cells

1 Following ischaemic reperfusion, large amounts of superoxide anion (.O2−), hydroxyl radical (.OH) and H2O2 are produced, resulting in brain oedema and changes in cerebral vascular permeability. We have found that H2O2 (100 μm) induces a significant intracellular acidosis in both cultured rat cerebellar astrocytes (0.37 ± 0.04 pH units) and C6 glioma cells (0.33 ± 0.07 pH units). 2 Two membrane‐crossing ferrous iron chelators, phenanthroline and deferoxamine, almost completely inhibited H2O2‐induced intracellular acidosis, while the non‐membrane‐crossing iron chelator apo‐transferrin had no effect. Furthermore, the acidosis was completely inhibited by two potent membrane‐crossing .OH scavengers, N‐(2‐mercaptopropionyl)‐grycine (N‐MPG) and dimethyl thiourea (DMTU). Since .OH can be produced during iron‐catalysed H2O2 breakdown (Fenton reaction), we have shown that a large reduction in pH1 in glial cells can result from the production of intracellular .OH via H2O2 oxidation. 3 We have ruled out the possible involvement of: (i) an increase in intracellular Ca2+ levels; and (ii) inhibition of oxidative phosphorylation. 4 Our results suggest that .OH inhibits glycolysis, leading to ATP hydrolysis and intracellular acidosis. This conclusion is based on the following observations: (i) in glucose‐free medium, or in the presence of iodoacetate or 2‐deoxy‐D‐glucose, H2O2‐induced acidosis is completely suppressed; (ii) H2O2 and iodoacetate both produce an increase in levels of intracellular free Mg2+, an indicator of ATP breakdown; and (iii) direct measurement of intracellular ATP levels and lactate production show 50 and 55% reductions in ATP content and lactate production, respectively, following treatment with 100 μm H2O2. 5 Inhibition of the pH1 regulators (i.e. the Na+–H+ exchange and possibly the Na+–HCO3−–dependent pH1 transporters) resulting from H2O2‐induced intracellular ATP reduction may also be involved in the H2O2‐evoked intracellular acidosis in glial cells.

Decoy receptor 3 (DcR3) induces osteoclast formation from monocyte/macrophage lineage precursor cells

AbstractRecent evidence indicates that the decoy receptor 3 (DcR3) of the TNF receptor superfamily, which initially though prevents cytokine responses of FasL, LIGHT and TL1A by binding and neutralization, can modulate monocyte function through reverse signaling. We show in this work that DcR3 can induce osteoclast formation from human monocytes, murine RAW264.7 macrophages, and bone marrow cells. DcR3-differentiated cells exhibit characteristics unique for osteoclasts, including polynuclear giant morphology, bone resorption, TRAP, CD51/61, and MMP-9 expression. Consistent with the abrogation of osteoclastogenic effect of DcR3 by TNFR-Fc, DcR3 treatment can induce osteoclastogenic cytokine TNF-α release through ERK and p38 MAPK signaling pathways. We conclude that DcR3 via coupling reverse signaling of ERK and p38 MAPK and stimulating TNF-α synthesis is a critical regulator of osteoclast formation. This action of DcR3 might play an important role in significant osteoclastic activity in osteolytic bone metastases.

Mechanism of Hydrogen Peroxide and Hydroxyl Free Radical–Induced Intracellular Acidification in Cultured Rat Cardiac Myoblasts

After a transient ischemic attack of the cardiac vascular system, reactive oxygen-derived free radicals, including the superoxide (O2-.) and hydroxyl (.OH) radicals can be easily produced during reperfusion. These free radicals have been suggested to be responsible for reperfusion-induced cardiac stunning and reperfusion-induced arrhythmia. Hydrogen peroxide (H2O2) is often used as an experimental source of oxygen-derived free radicals. Using freshly dissociated single rat cardiac myocytes and the rat cardiac myoblast cell line, H9c2, we have shown, for the first time, that an intriguing pHiota acidification (approximately 0.24 pH unit) is induced by the addition of 100 micromol/L H2O2 and that this dose is without effect on the intracellular free Ca2+ levels or viability of the cells. Using H9c2 as a model cardiac cell, we have shown that it is the intracellular production of .OH, and not O2-. or H2O2, that results in this acidification. We have excluded any involvement of (1) the three known cardiac pHi regulators (the Na+-H+ exchanger, the Cl--HCO3 exchanger, and the Na+-HCO3 co-transporter), (2) a rise in intracellular Ca2+ levels, and (3) inhibition of oxidative phosphorylation. However, we have found that H2O2-induced acidosis is due to inhibition of the glycolytic pathway, with hydrolysis of intracellular ATP and the resultant intracellular acidification. In cardiac muscle and in skinned cardiac muscle fiber, it has been shown that a small intracellular acidification may severely inhibit contractility. Therefore, the sustained pHi decrease caused by hydroxyl radicals may contribute, in some part, to the well-documented impairment of cardiac mechanical function (ie, reperfusion cardiac stunning) seen during reperfusion ischemia.

Macrophage activation increases the invasive properties of hepatoma cells by destabilization of the adherens junction.

Tumor‐associated macrophages play an important role in tumor progression, but whether they exert a tumor‐progressive effect remains controversial. Here, we demonstrated that activated macrophage‐conditioned medium (AMCM) obtained from RAW macrophages (RAW/AMCM) induced epithelial‐mesenchymal transition (EMT) and stimulated the migratory and invasive activities of HepG2 cells, whereas control conditioned media had no effect. Epithelial‐cadherin (E‐cadherin) and β‐catenin staining patterns were altered at the adherens junctions by RAW/AMCM treatment, with an approximately 50% decrease in E‐cadherin and β‐catenin in the cell membrane. Importantly, levels of β‐catenin‐associated E‐cadherin were also decreased. Following RAW/AMCM treatment, enhanced activation of c‐Src was seen prior to increased tyrosine phosphorylation of β‐catenin, and this led to the destabilization of adherens junctions. Pretreatment of HepG2 cells with the Src kinase inhibitor, PP2, completely abolished the effects of RAW/AMCM on the EMT, migration, invasion, and expression and association of E‐cadherin and β‐catenin. AMCMs obtained from human THP‐1 monocytes and mouse peritoneal macrophages also caused disassembly of the adherens junctions and migration of HepG2 cells. Furthermore, inhibition of the epidermal growth factor receptor (EGFR) with gefitinib partially prevented the downregulation of E‐cadherin and β‐catenin at the adherens junctions and migration behavior induced by RAW/AMCM. Our results suggest that activated macrophages have a tumor‐progressive effect on HepG2 cells which involves the c‐Src‐ and EGFR‐dependent signaling cascades.

Effects of a water-soluble extract of Cordyceps sinensis on steroidogenesis and capsular morphology of lipid droplets in cultured rat adrenocortical cells

Cordyceps sinensiscontains a factor that stimulates corticosteroid production in the animal model. However, it is not known whether this drug acts directly on the adrenal glands or indirectly via the hypothalamus‐pituitary axis. In the present study, we used primary rat adrenal cell cultures to investigate the pharmacological function of a water‐soluble extract of Cordyceps sinensis(CS) and thesignaling pathway involved. Radioimmunoassay of corticosterone indicated that the amount of corticosterone produced by adrenal cells is increased in a positively dose‐dependent manner by CS, reaching a maximun at 25 μg/ml. This stimulating effect was seen 1 h after CS treatment and was maintained for up to 24 h. Concomitantly, the lipid droplets in these cells became small and fewer in number. Immunostaining with a monoclonal antibody, A2, a specific marker for the lipid droplet capsule, demonstrated that detachment of the capsule from the lipid droplet occurs in response to CS application and that the period required for decapsulation is inversely related to the concentration of CS applied. The mechanism of CS‐induced steroidogenesis is apparently different from that for ACTH, since intracellular cAMP levels were not increased in CS‐treated cells. However, combined application with calphostin C, a PKC inhibitor, completely blocked the effect of CS on steroidogenesis, suggesting that activation of PKC may be responsible for the CS‐induced steroidogenesis. J. Cell. Biochem. 69:483–489, 1998.

Studies on the function of Rho A protein in cardiac myofibrillogenesis

The aim of this study was to provide morphological evidence for the presence of rho A protein in developing cardiomyocytes and to investigate its possible role in myofibrillogenesis. Immunostaining with a monoclonal anti‐rho antibody gave a diffuse pattern in the cytosol of cultured cardiomyocytes. Introduction of C3 exoenzyme into the cells by electroporation was used to inactivate rho A protein by ADP‐ribosylation. An immunostaining with anti‐vinculin, anti‐talin, and anti‐integrin antibodies showed the focal adhesions in electroporation control cardiomyocytes to be evenly distributed in the ventral sarcolemma; the costameric structure was also detected using these antibodies. In contrast, in C3 exoenzyme treated cells, focal adhesions were disassembled and costamere were absent; in addition, β‐actin‐positive, non‐striated fibrils were lost and assembly of M‐protein, titin, and α‐actinin into myofibrils was poor, as shown by diffuse and filamentous staining pattern. C3 exoenzyme treatment had a less marked effect on mature cardiomyocytes than on immature cells; in this case, cells became distorted and few myofibrils were seen. The intensity of anti‐phosphotyrosine antibody staining of the focal adhesion was also decreased or diffuse in C3 exoenzyme‐treated cardiomyocytes, suggesting dephosphorylation of focal adhesion components. We therefore conclude that small G protein rho A plays an important role in myofibril assembly in cardiomyocytes. J. Cell. Biochem. 66:43–53, 1997.

Lipopolysaccharide-induced inhibition of connexin43 gap junction communication in astrocytes is mediated by downregulation of caveolin-3.

Astrocytes play a crucial role in maintaining the homeostasis of the brain. Changes to gap junctional intercellular communication (GJIC) in astrocytes and excessive inflammation may trigger brain damage and neurodegenerative diseases. In this study, we investigated the effect of lipopolysaccharide (LPS) on connexin43 (Cx43) gap junctions in rat primary astrocytes. Following LPS treatment, dose- and time-dependent inhibition of Cx43 expression was seen. Moreover, LPS induced a reduction in Cx43 immunoreactivity at cell-cell contacts and significantly inhibited GJIC, as revealed by the fluorescent dye scrape loading assay. Toll-like receptor 4 (TLR4) protein expression was increased 2-3-fold following LPS treatment. To study the pathways underlying these LPS-induced effects, we examined downstream effectors of TLR4 signaling and found that LPS induced a significant increase in phosphorylated extracellular signal-regulated kinase (pERK) levels up to 6 h, followed by signal attenuation and downregulation of caveolin-3 expression. Interestingly, LPS treatment also induced a dramatic increase in inducible nitric oxide synthase (iNOS) levels at 6 h, which were sustained up to 18-24 h. The LPS-induced downregulation of Cx43 and caveolin-3 was prevented by co-treatment of astrocytes with the iNOS cofactor inhibitor 1400W, but not the ERK inhibitor PD98059. Specific knockdown of caveolin-3 using siRNA had a significant inhibitory effect on GJIC and resulted in a downregulation of Cx43. Our results suggest that long-term LPS treatment of astrocytes leads to inhibition of Cx43 gap junction communication by the activation of iNOS and downregulation of caveolin-3 via a TLR4-mediated signaling pathway.

Mechanisms for the magnolol-induced cell death of CGTH W-2 thyroid carcinoma cells.

Magnolol, a substance purified from the bark of Magnolia officialis, inhibits cell proliferation and induces apoptosis in a variety of cancer cells. The aim of this study was to study the effects of magnolol on CGTH W‐2 thyroid carcinoma cells. After 24 h treatment with 80 µM magnolol in serum‐containing medium, about 50% of the cells exhibited apoptotic features and 20% necrotic features. Cytochrome‐c staining was diffused in the cytoplasm of the apoptotic cells, but restricted to the mitochondria in control cells. Western blot analyses showed an increase in levels of activated caspases (caspase‐3 and ‐7) and of cleaved poly (ADP‐ribose) polymerase (PARP) by magnolol. Concomitantly, immunostaining for apoptosis inducing factor (AIF) showed a time‐dependent translocation from the mitochondria to the nucleus. Inhibition of either PARP or caspase activity blocked magnolol‐induced apoptosis, supporting the involvement of the caspases and PARP. In addition, magnolol activated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and inactivated Akt by decreasing levels of phosphorylated PTEN and phosphorylated Akt. These data suggest that magnolol promoted apoptosis probably by alleviating the inhibitory effect of Akt on caspase 9. Furthermore, inhibition of PARP activity, but not of caspase activity, completely prevented magnolol‐induced necrosis, suggesting the notion that it might be caused by depletion of intracellular ATP levels due to PARP activation. These results show that magnolol initiates apoptosis via the cytochrome‐c/caspase 3/PARP/AIF and PTEN/Akt/caspase 9/PARP pathways and necrosis via PARP activation. J. Cell. Biochem. 101: 1011–1022, 2007.

Effects of PPARγ agonists on cell survival and focal adhesions in a Chinese thyroid carcinoma cell line

Peroxisome proliferator‐activated receptor γ (PPARγ) agonists cause cell death in several types of cancer cells. The aim of this study was to examine the effects of two PPARγ agonists, ciglitazone and 15‐deoxy‐Δ 12,14‐prostaglandin J2 (15dPGJ2), on the survival of thyroid carcinoma CGTH W‐2 cells. Both ciglitazone and 15dPGJ2 decreased cell viability in a time‐ and dose‐dependent manner. Cell death was mainly due to apoptosis, with a minor contribution from necrosis. Increased levels of active caspase 3, cleaved poly (ADP‐ribose) polymerase (PARP), and cytosolic cytochrome‐c were noted. In addition, ciglitazone and 15dPGJ2 induced detachment of CGTH W‐2 cells from the culture substratum. Both the protein levels and immunostaining signals of focal adhesion (FA) proteins, including vinculin, integrin β1, focal adhesion kinase (FAK), and paxillin were decreased after PPARγ agonist treatment. Meanwhile, reduced phosphorylation of FAK and paxillin was noted. Furthermore, PPARγ agonists induced expression of protein tyrosine phosphatase‐PEST (PTP‐PEST), and of phosphatase and tensin homologue deleted on chromosome ten (PTEN). The upregulation of these phosphatases might contribute to the dephosphorylation of FAK and paxillin, since pre‐treatment with orthovanadate prevented PPARγ agonist‐induced dephosphorylation of FAK and paxillin. Perturbation of CGTH W‐2 cells with anti‐integrin β1 antibodies induced FA disruption and apoptosis in the same cells, thus the downregulation of integrin β1 by PPARγ agonists resulted in FA disassembly and might induce apoptosis via anoikis. Our results suggested the presence of crosstalk between apoptosis and integrin‐FA signaling. Moreover, upregulation and activation of PTEN was correlated with reduced phosphorylation of Akt, and this consequence disfavored cell survival. In conclusion, PPARγ agonists induced apoptosis of thyroid carcinoma cells via the cytochrome‐c caspase 3 and PTEN‐Akt pathways, and induced necrosis via the PARP pathway. J. Cell. Biochem. 98: 1021–1035, 2006.

Possible involvement of protein kinase C in the induction of adipose differentiation-related protein by Sterol ester in RAW 264.7 macrophages.

The accumulation of lipid droplets in macrophages contributes to the formation of foam cells, an early event in atherosclerosis. It is, therefore, important to elucidate the mechanisms by which lipid droplets accumulate and are utilized. Sterol ester (SE)‐laden RAW 264.7 macrophages accumulated lipid droplets in a time‐dependent manner up to 16 h, which was enhanced by cotreatment with 0.1 μM phorbol 12‐myristate 13‐acetate (PMA). Inhibition of protein kinase C (PKC) activity by cotreatment with 0.3 μM calphostin C CAL for 16 h resulted in coalescence of small lipid droplets into large ones and increased accumulation of lipid droplets, although to a lesser extent than after PMA cotreatment. Immunostaining for adipose differentiation‐related protein (ADRP) revealed a fluorescent rim at the surface of each medium to large lipid droplet. ADRP appearance correlated with lipid droplet accumulation and was regulated by PMA in a time‐dependent manner. Induction of ADRP expression by PMA or CAL required SE, since ADRP levels in PMA‐ or CAL‐treated non‐SE‐laden macrophages were comparable to those in untreated cells. Removal of SE from the incubation medium resulted in the concomitant dissolution of lipid droplets and down‐regulation of ADRP. In conclusion, the above results suggest that ADRP may be an important protein in the regulation of lipid droplet metabolism in lipid‐laden macrophages and that this regulation may be mediated by PKC activity. J. Cell. Biochem. 83: 187–199, 2001.