Jiahn-Chun Wu

Role of catenins in the development of gap junctions in rat cardiomyocytes

Gap junctions are intercellular communicating channels responsible for the synchronized activity of cardiomyocytes. Recent studies have shown that the membrane‐associated guanylate kinase protein, zonula occludens‐1 (ZO‐1) can bind to catenins in epithelial cells and act as an adapter for the transport of the connexin isotype, Cx43 during gap junction formation. The significance of catenins in the development of gap junctions and whether complexes between catenins and ZO‐1 are formed in cardiomyocytes are not clear. In this study, immunofluorescence and confocal microscopy showed sequential redistribution of α‐catenin, β‐catenin, ZO‐1, and Cx43 to the plasma membrane when rat cardiomyocytes were cultured in low Ca2+ (<5 μM) medium, then shifted to 1.8 mM Ca2+ medium (Ca2+ switch). Diffuse cytoplasmic staining of α‐catenin, β‐catenin, ZO‐1, and Cx43 was seen in the cytoplasm when cardiomyocytes were cultured in low Ca2+ medium. Staining of α‐catenin, β‐catenin, and ZO‐1 was detected at the plasma membrane of cell–cell contact sites 10 min after Ca2+ switch, whereas Cx43 staining was first detected, colocalized with ZO‐1 at the plasma membrane, 30 min after Ca2+ switch. Distinct junctional and extensive cytoplasmic staining of α‐catenin, β‐catenin, ZO‐1, and Cx43 was seen 2 h after Ca2+ switch. Immunoprecipitation of Triton X‐100 cardiomyocyte extracts using anti‐β‐catenin antibodies showed that β‐catenin was associated with α‐catenin, ZO‐1, and Cx43 at 2 h after Ca2+ switch. Intracellular application of antisera against α‐catenin, β‐catenin, or ZO‐1 by electroporation of cardiomyocytes cultured in low Ca2+ medium inhibited the redistribution of Cx43 to the plasma membrane following Ca2+ switch. These results suggest the formation of a catenin–ZO‐1–Cx43 complex in rat cardiomyocytes and that binding of catenins to ZO‐1 is required for Cx43 transport to the plasma membrane during the assembly of gap junctions.

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.

Signal mechanisms of vascular endothelial growth factor and interleukin-8 in ovarian hyperstimulation syndrome: dopamine targets their common pathways

BACKGROUND Ovarian hyperstimulation syndrome (OHSS) is a serious complication of ovarian stimulation with massive ascites, pleural effusion and hemoconcentration. The pathophysiological signal mechanisms of OHSS are still unclear and merit further investigation. METHODS Various angiogenic cytokines of follicular fluid and ascites of patients with risk of OHSS were measured, and examined for inducing endothelial permeability. These include vascular endothelial growth factor (VEGF), interleukin (IL)-6, IL-8, basic fibroblast growth factor, tumor necrosis factor-alpha, IL-1alpha, IL-1beta and platelet-derived growth factor. We explore the molecular signal pathways of major contributing cytokines in granulosa-lutein cells and endothelial cells possibly involved in OHSS. RESULTS Neutralizing antibodies of VEGF or IL-8 significantly decreased follicular fluid- and ascites-induced endothelial permeability. Human chorionic gonadotrophin induced VEGF secretion of granulosa-lutein cells through the Sp1 and CREB dependent pathways. IL-8 activated CXCR1/2 of endothelial cells leading to VEGF receptor (VEGFR)-2 transactivation. Both VEGF and IL-8 of follicular fluid enhanced endothelial permeability via VEGFR-2-mediated Rho/Rock activation, actin polymerization and phosphorylations of VE-cadherin and occludin, resulting in opening of adherens junctions and tight junctions. Dopamine (2 microM) inhibited follicular fluid-induced VEGFR-2 signals and endothelial permeability, without diminishing migration and tube formation. CONCLUSIONS Our results suggest that VEGF and IL-8 secreted from corpora luteae may play major roles in OHSS. Delineation of signal pathways would be helpful for treatment. Dopamine may block VEGF- and IL-8-induced endothelial permeability by inhibiting common VEGFR-2 dependent signals.

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.

OxLDL Upregulates Caveolin―1 Expression in Macrophages: Role for Caveolin-1 in the Adhesion of oxLDL―Treated Macrophages to Endothelium

Caveolin‐1, a principle component of caveolae, is present in several cell types known to play an important role in the development of atherosclerosis. In this study, its distribution and expression were studied in the arterial walls of hypercholesterolemic rabbits and apo‐E‐deficient mice and in oxidized low‐density lipoprotein (oxLDL)‐treated RAW264.7 macrophages. Immunohistochemical studies showed that staining for caveolin‐1 expression was stronger in atherosclerotic lesions in hypercholesterolemic rabbits and apo‐E‐deficient mice compared to normal rabbits and mice and was closely associated with macrophages. OxLDL treatment increased caveolin‐1 protein expression in RAW264.7 macrophages in a time‐ and dose‐dependent manner. The increase in caveolin‐1 expression was dependent on phosphorylation of the mitogen‐activated protein kinases (MAPKs) extracellular signal‐regulated kinase1/2 (ERK1/2), p38, and Jun N‐terminal kinase (JNK) and the transcriptional activation and translocation of nuclear factor‐?B (NF‐κB). OxLDL also induced caveolin‐1 mRNA expression and this effect was not seen in the presence of inhibitors for transcription or de novo protein synthesis. OxLDL increased the adhesion of RAW264.7 macrophages to endothelial cells via an increase in caveolin‐1 expression, and the adhesion was reduced by the use of anti‐caveolin‐1 antibody or caveolin‐1‐specific shRNA. These results show that oxLDL increases caveolin‐1 expression in macrophages through the MAPKs/NF‐κB pathway. The caveolin‐1 levels are closely associated with the adherence of monocytes/macrophages to endothelial cells and their accumulation within the arterial intima after hypercholesterolemia insult, resulting in the progression of atherosclerosis. J. Cell. Biochem. 107: 460–472, 2009.

Early metabolic inhibition-induced intracellular sodium and calcium increase in rat cerebellar granule cells

1 Possible mechanisms responsible for the increases in intracellular calcium ([Ca2+]i) and sodium ([Na+]i) levels seen during metabolic inhibition were investigated by continuous [Ca2+]i and [Na+]i measurement in cultured rat cerebellar granule cells. An initial small mitochondrial Ca2+ release was seen, followed by a large influx of extracellular Ca2+. A large influx of extracellular Na+ was also seen. 2 The large [Ca2+]i increase was not due to opening of voltage‐dependent or voltage‐independent calcium channels, activation of NMDA/non‐NMDA channels, activation of the Na+i‐Ca2+o exchanger, or inability of plasmalemmal Ca2+‐ATPase to extrude, or mitochondria to take up, calcium. 3 The large [Na+]i increase was not due to activation of the TTX‐sensitive Na+ channel, the Na+i‐Ca2+o exchanger, the Na+‐H+ exchanger, or the Na+‐K+‐2Cl− cotransporter, or an inability of Na+‐K+‐ATPase to extrude the intracellular sodium. 4 Phospholipase A2 (PLA2) activation may be involved in the large influx, since both were completely inhibited by PLA2 inhibitors. Moreover, melittin (a PLA2 activator) or lysophosphatidylcholine or arachidonic acid (both PLA2 activation products) caused similar responses. Inhibition of PLA2 activity may help prevent the influx of these ions that may result in serious brain injury and oedema during hypoxia/ischaemia.

Effects of hyperthermia on the cytoskeleton and focal adhesion proteins in a human thyroid carcinoma cell line.

Hyperthermia is reported to act as a sensitizer to chemotherapeutic drugs in the treatment of cancer. Thyroid follicular carcinoma were used to elucidate the effects of hyperthermic treatment (41–43°C) on cell morphology, cytoskeleton, and the focal adhesion complex. The critical temperature that resulted in inhibition of cell proliferation as the cell number in the same area did not increase over a 23 h time course and irreversible changes in cell morphology was 42–43°C. An immunofluorescence study on heat‐treated cells (43°C, 1–5 h) demonstrated that depolymerization of actin filaments, intermediate filaments, and microtubules accounted for the rounding‐up of cells and detachment from the substratum. Characteristic staining patterns for integrin αv, focal adhesion kinase, and vinculin were noted in untreated cells, but the immunoreactive intensities for these proteins became weaker with time of heat treatment. Anti‐phosphotyrosine staining revealed less immunoreactivity in the focal adhesions in treated cells compared with control cells. The disappearance of integrin αv from the cell surface may result in inhibition of integrin‐mediated activation of focal adhesion kinase, which results in dephosphorylation of focal adhesion components and its disassembly. These results indicate that hyperthermia induces disruption of integrin‐mediated actin cytoskeleton assembly and, possibly, of other integrin‐mediated signaling pathways. J. Cell. Biochem. 75:327–337, 1999.

Lysophosphatidic acid induces ovarian cancer cell dispersal by activating Fyn kinase associated with p120‐catenin

Lysophosphatidic acid (LPA), known as the “ovarian cancer activating factor,” is a natural phospholipid involved in important biological functions, such as cell proliferation, wound healing and neurite retraction. LPA causes colony dispersal in various carcinoma cell lines by inducing morphological changes, including membrane ruffling, lamellipodia formation, cell–cell dissociation and single cell migration. However, its effects on cell–cell dissociation and cell–cell adhesion of ovarian cancer cells have not been studied. In our study, we showed that LPA induced sequential events of intercellular junction dispersal and “half‐junction” formation in ovarian cancer SKOV3 cells and that Src‐family kinases were involved in both processes, since the effects were abolished by the selective tyrosine kinase inhibitor PP2. LPA induced rapid and transient activation of Src family kinases, which were recruited to cell–cell junctions by increasing the association with the adherens junction protein p120‐catenin. We identified the Src family kinase, Fyn, as the key component associated with p120‐catenin after LPA stimulation in SKOV3 cells. Our study provides evidence that LPA induces junction dispersal in ovarian cancer SKOV3 cells by activating the Src family kinase Fyn and increasing its association with p120‐catenin at the cell–cell junction.