Interleukin-1ß-induced matrix metalloproteinase-3 via ERK1/2 pathway to promote mesenchymal stem cell migration.

Human umbilical cord Wharton's jelly derived mesenchymal stem cells (hUCMSCs), a source of cell therapy, have received a great deal of attention due to their homing or migrating ability in response to signals emanating from damaged sites. It has been found that IL-1ß possesses the ability to induce the expression of matrix metalloproteinase-3 (MMP-3) in bone marrow MSCs. MMP-3 is involved in cell migration in various types of cells, including glioblastoma, vascular smooth muscle, and adult neural progenitor cells. In this study, we proposed that IL-1ß influences hUCMSCs migration involving MMP-3. The expression level of MMP-3 in IL-1ß-induced hUCMSCs was verified using cDNA microarray analysis, quantitative real-time PCR, ELISA and Western blot. Wound-healing and trans-well assay were used to investigate the cell migration and invasion ability of IL-1ß-treated hUCMSCs. In addition, we pre-treated hUCMSCs with interleukin-1 receptor antagonist, MMP-3 inhibitors (ALX-260-165, UK 356618), or transfected with MMP-3 siRNA to confirm the role of MMP3 in IL-1ß-induced cell migration. Our results showed that IL-1ß induced MMP-3 expression is related to the migration of hUCMSCs. Moreover, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) inhibitor U0126, p38 inhibitor SB205380, JNK inhibitor SP600125 and Akt inhibitor GSK 690693 decreased IL-1ß-induced MMP-3 mRNA and protein expression. The migration and invasion ability analyses showed that these inhibitors attenuated the IL-1ß-induced migration and invasion ability of hUCMSCs. In conclusion, we have found that IL-1ß induces the expression of MMP-3 through ERK1/2, JNK, p38 MAPK and Akt signaling pathways to enhance the migration of hUCMSCs. These results provide further understanding of the mechanisms in IL-1ß-induced hUCMSCs migration to injury sites.

Interleukin-1ß Enhances Umbilical Cord Mesenchymal Stem Cell Adhesion Ability on Human Umbilical Vein Endothelial Cells via LFA-1/ICAM-1 Interaction.

The migration of administered mesenchymal stem cells (MSCs) to sites of injury via the bloodstream has been demonstrated. However, the underlying mechanisms of umbilical cord MSC adhesion to endothelial cells during transendothelial migration are still unclear. In this study, our data showed that IL-1ß induced LFA-1 expression on MSCs and ICAM-1 expression on HUVECs. We then pretreated MSCs with protein synthesis inhibitor cycloheximide. The results showed that IL-1ß induced LFA-1 expression on the surface of MSCs via the protein synthesis pathway. Through the p38 MAPK signaling pathway inhibitor SB 203580, we found that IL-1ß induces the expression of LFA-1 through p38 MAPK signaling and enhances ICAM-1 expression in HUVECs. In addition, IL-1ß-induced MSC adhesion to HUVECs was found to be inhibited by IL-1RA and the LFA-1 inhibitor lovastatin. These results indicate that IL-1ß promotes the cell adhesion of MSCs to HUVECs through LFA-1/ICAM-1 interaction. We address the evidence that the cell adhesion mechanism of IL-1ß promotes MSC adhesion to HUVECs. The implications of these findings could enhance the therapeutic potential of MSCs.

HYS-32-Induced Microtubule Catastrophes in Rat Astrocytes Involves the PI3K-GSK3beta Signaling Pathway.

HYS-32 is a novel derivative of combretastatin-A4 (CA-4) previously shown to induce microtubule coiling in rat primary astrocytes. In this study, we further investigated the signaling mechanism and EB1, a microtubule-associated end binding protein, involved in HYS-32-induced microtubule catastrophes. Confocal microscopy with double immunofluorescence staining revealed that EB1 accumulates at the growing microtubule plus ends, where they exhibit a bright comet-like staining pattern in control astrocytes. HYS-32 induced microtubule catastrophes in both a dose- and time-dependent manner and dramatically increased the distances between microtubule tips and the cell border. Treatment of HYS-32 (5 µM) eliminated EB1 localization at the microtubule plus ends and resulted in an extensive redistribution of EB1 to the microtubule lattice without affecting the ß-tubulin or EB1 protein expression. Time-lapse experiments with immunoprecipitation further displayed that the association between EB-1 and ß-tubulin was significantly decreased following a short-term treatment (2 h), but gradually increased in a prolonged treatment (6-24 h) with HYS-32. Further, HYS-32 treatment induced GSK3ß phosphorylation at Y216 and S9, where the ratio of GSK3ß-pY216 to GSK3ß-pS9 was first elevated followed by a decrease over time. Co-treatment of astrocytes with HYS-32 and GSK3ß inhibitor SB415286 attenuated the HYS-32-induced microtubule catastrophes and partially prevented EB1 dissociation from the plus end of microtubules. Furthermore, co-treatment with PI3K inhibitor LY294002 inhibited HYS-32-induced GSK3ß-pS9 and partially restored EB1 distribution from the microtubule lattice to plus ends. Together these findings suggest that HYS-32 induces microtubule catastrophes by preventing EB1 from targeting to microtubule plus ends through the GSK3ß signaling pathway.

Lipopolysaccharide induces degradation of connexin43 in rat astrocytes via the ubiquitin-proteasome proteolytic pathway.

The astrocytic syncytium plays a critical role in maintaining the homeostasis of the brain through the regulation of gap junction intercellular communication (GJIC). Changes to GJIC in response to inflammatory stimuli in astrocytes may have serious effects on the brain. We have previously shown that lipopolysaccharide (LPS) reduces connexin43 (Cx43) expression and GJIC in cultured rat astrocytes via a toll-like receptor 4-mediated signaling pathway. In the present study, treatment of astrocytes with LPS resulted in a significant increase in levels of the phosphorylated forms of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) -1, -2, and -3 for up to 18 h. An increase in nuclear transcription factor NF-κB levels was also observed after 8 h of LPS treatment and was sustained for up to 18 h. The LPS-induced decrease in Cx43 protein levels and inhibition of GJIC were blocked by the SAPK/JNK inhibitor SP600125, but not by the NF-κB inhibitor BAY11-7082. Following blockade of de novo protein synthesis by cycloheximide, LPS accelerated Cx43 degradation. Moreover, the LPS-induced downregulation of Cx43 was blocked following inhibition of 26S proteasome activity using the reversible proteasome inhibitor MG132 or the irreversible proteasome inhibitor lactacystin. Immunoprecipitation analyses revealed an increased association of Cx43 with both ubiquitin and E3 ubiquitin ligase Nedd4 in astrocytes after LPS stimulation for 6 h and this effect was prevented by SP600125. Taken together, these results suggest that LPS stimulation leads to downregulation of Cx43 expression and GJIC in rat astrocytes by activation of SAPK/JNK and the ubiquitin-proteasome proteolytic pathway.

HYS-32, a novel analogue of combretastatin A-4, enhances connexin43 expression and gap junction intercellular communication in rat astrocytes.

HYS-32 [4-(3,4-dimethoxyphenyl)-3-(naphthalen-2-yl)-2(5H)-furanone] is a new analogue of the anti-tumor compound combretastatin A-4 containing a cis-stilbene moiety. In this study, we investigated its effects on Cx43 gap junction intercellular communication (GJIC) and the signaling pathway involved in rat primary astrocytes. Western blot analyses showed that HYS-32 dose- and time-dependently upregulated Cx43 expression. A confocal microscopic study and scrape-loading/dye transfer analyses demonstrated that HYS-32 (5µM) induced microtubule coiling, accumulation of Cx43 in gap junction plaques, and increased GJIC in astrocytes. The HYS-32-induced microtubule coiling and Cx43 accumulation in gap junction plaques was reversed when HYS-32 was removed. Treatment of astrocytes with cycloheximide resulted in time-dependent degradation of by co-treatment with HYS-32 by increasing the half-life of Cx43. Co-treatment with HYS-32 also prevented the LPS-induced downregulation of Cx43 and inhibition of GJIC in astrocytes. HYS-32 induced activation of PKC, ERK, and JNK, and co-treatment with the PKC inhibitor Go6976 or the ERK inhibitor PD98059, but not the JNK inhibitor SP600125, prevented the HYS-32-induced increase in Cx43 expression and GJIC. Go6976 suppressed the HYS-32-induced PKC phosphorylation and increase in phospho-ERK levels, while PD98059 did not prevent the HYS-32-induced increase in phospho-PKC levels, suggesting that PKC is an upstream effector of ERK. In conclusion, our results show that HYS-32 increases the half-life of Cx43 and enhances Cx43 expression and GJIC in astrocytes via a PKC-ERK signaling cascade. These novel biological effects of HYS-32 on astrocyte gap junctions support its potential for therapeutic use as a protective agent for the central nervous system.

Antofine-induced connexin43 gap junction disassembly in rat astrocytes involves protein kinase Cß.

Antofine, a phenanthroindolizidine alkaloid derived from Cryptocaryachinensis and Ficusseptica in the Asclepiadaceae milkweed family, is cytotoxic for various cancer cell lines. In this study, we demonstrated that treatment of rat primary astrocytes with antofine induced dose-dependent inhibition of gap junction intercellular communication (GJIC), as assessed by scrape-loading 6-carboxyfluorescein dye transfer. Levels of Cx43 protein were also decreased in a dose- and time-dependent manner following antofine treatment. Double-labeling immunofluorescence microscopy showed that antofine (10ng/ml) induced endocytosis of surface gap junctions into the cytoplasm, where Cx43 was co-localized with the early endosome marker EEA1. Inhibition of lysosomes or proteasomes by co-treatment with antofine and their respective specific inhibitors, NH4Cl or MG132, partially inhibited the antofine-induced decrease in Cx43 protein levels, but did not inhibit the antofine-induced inhibition of GJIC. After 30min of treatment, antofine induced a rapid increase in the intracellular Ca(2+) concentration and activation of protein kinase C (PKC)α/ßII, which was maintained for at least 6h. Co-treatment of astrocytes with antofine and the intracellular Ca(2+) chelator BAPTA-AM prevented downregulation of Cx43 and inhibition of GJIC. Moreover, co-treatment with antofine and a specific PKCß inhibitor prevented endocytosis of gap junctions, downregulation of Cx43, and inhibition of GJIC. Taken together, these findings indicate that antofine induces Cx43 gap junction disassembly by the PKCß signaling pathway. Inhibition of GJIC by antofine may undermine the neuroprotective effect of astrocytes in CNS.

Lysophosphatidic acid modulates the association of PTP1B with N-cadherin/catenin complex in SKOV3 ovarian cancer cells.

LPA (lysophosphatidic acid) is a natural phospholipid that plays important roles in promoting cancer cell proliferation, invasion and metastases. We previously reported that LPA induces ovarian cancer cell dispersal and disruption of AJ (adherens junction) through the activation of SFK (Src family kinases). In this study, we have investigated the regulatory mechanisms during the early phase of LPA-induced cell dispersal. An in vitro model of the ovarian cancer cell line SKOV3 for cell dispersal was used. LPA induces rapid AJ disruption by increasing the internalization of N-cadherin-ß-catenin. By using immunoprecipitations, LPA was shown to induce increased tyrosine phosphorylation of ß-catenin and alter the balance of ß-catenin-bound SFK and PTP1B (phosphotyrosine phosphatase 1B). The altered balance of tyrosine kinase/phosphatase correlated with a concomitant disintegration of the ß-catenin-α-catenin, but not the ß-catenin-N-cadherin complex. This disintegration of ß-catenin from α-catenin and the cell dispersal caused by LPA can be rescued by blocking SFK activity with the chemical inhibitor, PP2. More importantly, PP2 also restores the level of PTP1B bound to ß-catenin. We propose that LPA signalling alters AJ stability by changing the dynamics of tyrosine kinase/phosphatase bound to AJ proteins. This work provides further understanding of the early signalling events regulating ovarian cancer cell dispersal and AJ disruption induced by LPA.

Pigment epithelium-derived factor reduces the PDGF-induced migration and proliferation of human aortic smooth muscle cells through PPARγ activation.

Our previous study demonstrated that pigment epithelium-derived factor (PEDF) plays an important role in the proliferation and migration of human aortic smooth muscle cells (HASMCs). In the present study, we examined whether PEDF inhibited platelet-derived growth factor (PDGF)-stimulated HASMC migration and proliferation. PEDF dose-dependently reduced PDGF-induced HASMC migration and proliferation in vitro and also arrested cell cycle progression in the G0/G1 phase, and this was associated with decreased expression of cyclin D1, cyclin E, CDK2, CDK4, and p21(Cip1) and increased expression of the cyclin-dependent kinase inhibitor p27(Kip1). The antiproliferative and antimigratory effects of PEDF were partially blocked by the PPARγ antagonist GW9662, but not by the PPARα antagonist MK886. In in vivo studies, the femoral artery of C57BL/6 mice was endothelial-denuded and the mice injected intravenously with PEDF or vehicle. After 2 weeks, both the neointima/media area ratio and cell proliferation (proliferating cell nuclear antigen-positive cells) in the neointima were significantly reduced and again these effects were partially reversed by GW9662 pretreatment. Our data show that PEDF increases PPARγ activation, preventing entry of HASMCs into the cell cycle in vitro and reducing the neointimal area and cell proliferation in the neointima in vivo. Thus, PEDF may represent a safe and effective novel target for the prevention and treatment of vascular proliferative diseases.

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.

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.

The interaction of estrogen receptor alpha and caveolin-3 regulates connexin43 phosphorylation in metabolic inhibition-treated rat cardiomyocytes.

Caveolin-3, the major caveolin isoform in cardiomyocytes, plays an important role in the rapid signaling pathways initiated by stimulation of the membrane-associated molecules. To examine the role of caveolin-3 in regulating estrogen receptor alpha in cardiomyocytes, we investigate whether the membrane estrogen receptor alpha associates with caveolin-3 and whether this association is linked to the 17beta-estradiol-mediated signals. In control cardiomyocytes, following discontinuous sucrose gradient centrifugation, caveolin-3 was found predominantly in the lipid raft buoyant fractions, whereas it was distributed to both the buoyant and non-lipid raft heavy fractions following metabolic inhibition treatment. Confocal microscopy showed that estrogen receptor alpha co-localized with caveolin-3 on the plasma membrane of neonatal and adult rat cardiomyocytes. This membrane labeling of estrogen receptor alpha was not seen following treatment with the cholesterol-depleting agent methyl-beta-cyclodextrin (5mM), whereas metabolic inhibition had little effect on the membrane distribution of estrogen receptor alpha. Metabolic inhibition induced tyrosine phosphorylation of caveolin-3 and decreased its association with estrogen receptor alpha, both effects being mediated via a Src activation mechanism, since they were inhibited by the selective tyrosine kinase inhibitor PP2. Metabolic inhibition also induced tyrosine phosphorylation of connexin43 and increased its association with c-Src, both effects being prevented by 17beta-estradiol (200 nM). The effect of 17beta-estradiol on metabolic inhibition-induced tyrosine phosphorylation of connexin43 was inhibited by the specific estrogen receptor antagonist ICI182780. These data identify cardiac caveolin-3 as juxtamembrane scaffolding for estrogen receptor alpha docking at caveolae, which provide a unique compartment for conveying 17beta-estradiol-elicited, rapid signaling to regulate connexin43 phosphorylation during ischemia.

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-kappaB). 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-kappaB 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.

Protein kinase C-Fyn kinase cascade mediates the oleic acid-induced disassembly of neonatal rat cardiomyocyte adherens junctions.

Oleic acid (OA) affects assembly of gap junctions in neonatal cardiomyocytes. Adherens junction (AJ) regulates the stability of gap junction integrity; however, the effect of OA on AJ remains largely unexplored. The distribution of N-cadherin and catenins at cell-cell junction was decreased by OA. OA induced activation of protein kinase C(PKC)-alpha and -epsilon and Src family kinase, and all three kinases were involved in the oleic acid-induced disassembly of the adherens junction, since it was blocked by pretreatment with Gö6976 (a PKCalpha inhibitor), epsilonV1-2 (a PKCepsilon inhibitor), or PP2 (a Src family kinase inhibitor). Src family kinase appeared to be the downstream of PKC-alpha and -epsilon, as blockade of either PKC-alpha or -epsilon activity prevented the OA-induced activation of Src family kinase. Immunoprecipitation analyses showed that OA activated Fyn and Fer. OA promoted the association of p120 catenin/beta-catenin with Fyn and Fer and caused increased tyrosine phosphorylation of p120 catenin and beta-catenin, resulting in decreased binding of the former to N-cadherin and of the latter to alpha-catenin. Pretreatment with PP2 abrogated this OA-induced tyrosine phosphorylation of p120 catenin and beta-catenin and restored the association of N-cadherin with p120 catenin and that of beta-catenin with alpha-catenin. In conclusion, these results show that OA activates the PKC-Fyn signaling pathway, leading to the disassembly of the AJ. Therefore, inhibitors of PKC-alpha/-epsilon and Src family kinase are potential candidates as cardioprotection agents against OA-induced heart injury during ischemia-reperfusion.

Effects of 18-glycyrrhetinic acid on serine 368 phosphorylation of connexin43 in rat neonatal cardiomyocytes.

18Beta-glycyrrhetinic acid (18beta-GA) regulates serine/threonine dephosphorylation of connexin43 (Cx43). Phospho-specific antibodies were used here to determine the effect of 18beta-GA on serine 368-phosphorylated Cx43 (pSer368Cx43) in cultured rat neonatal cardiomyocytes by immunofluorescence microscopy and immunoblot analyses. 18beta-GA caused a time-dependent increase in pSer368Cx43 levels and induced gap junction disassembly, shown by a change in pSer368Cx43 immunostaining from large aggregates to dispersed punctates at cell-cell contact areas. 18beta-GA also induced a time-dependent increase in the levels of serine 729-phosphorylated PKCepsilon, the active form of PKCepsilon. The 18beta-GA-induced increase in pSer368Cx43 levels and changes in pSer368Cx43 staining pattern were abolished by the PKC inhibitor, chelerythrine. Furthermore, 18beta-GA increased the co-immunoprecipitation of Cx43 with PKCepsilon. However, the 18beta-GA-induced increase in pSer368Cx43 levels and increased association of Cx43 with PKCepsilon were inhibited by co-treatment with the protein phosphatase type 1 and type 2A inhibitor, calyculin A. We conclude that 18beta-GA induces Ser368 phosphorylation of Cx43 via PKCepsilon.

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.

An acidic extracellular pH induces Src kinase-dependent loss of beta-catenin from the adherens junction.

Little attention has been paid to the role of adherens junctions (AJs) in acidic extracellular pH (pHe)-induced cell invasion. Incubation of HepG2 cells in acidic medium (pH 6.6) induced cell dispersion from tight cell clusters, and this change was accompanied by downregulation of beta-catenin at cell junctions and a rapid activation of c-Src. Pretreatment with PP2 prevented the acidic pH-induced downregulation of beta-catenin at AJ and in the membrane fractions. The acidic pHe-induced c-Src activation increased tyrosine phosphorylation of beta-catenin and decreased the amount of beta-catenin-associated E-cadherin. The depletion of membrane-bound beta-catenin coincided with enhanced cell migration and invasion, and this acidic pHe-increased cell migration and invasion was prevented by PP2. In conclusion, this study characterizes a novel signaling pathway responsible for acidic microenvironment-promoted migration and invasive behaviors of cancer cells.

Mechanism of oleic acid-induced myofibril disassembly in rat cardiomyocytes.

This study investigated the mechanism of oleic acid (OA)-induced disassembly of myofibrils in cardiomyocytes. OA treatment disrupted myofibrils, as revealed by the disorganization of several sarcomeric proteins. Since focal adhesions (FAs) are implicated in myofibril assembly, we examined structural changes in FAs after OA treatment. Immunofluorescence studies with antibodies against FA proteins (vinculin, integrin beta1D, and paxillin) showed that FAs and costameres disintegrated or disappeared after OA treatment and that the changes in FA proteins occurred prior to myofibril disassembly. The effects of OA on FAs and myofibrils were reversed after removal of OA. OA decreased expression of integrin beta1D, paxillin, vinculin, and actin, and induced tyrosine dephosphorylation of FA kinase (FAK) and paxillin. These effects were blocked by pretreatment with sodium orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor. This inhibitor also prevented OA-induced myofibril disassembly, indicating the involvement of PTP in myofibril disassembly. Furthermore, OA increased protein levels of PTP-PEST. The upregulation of this phosphatase correlated with the tyrosine dephosphorylation of paxillin and FAK, which are targets for PTP-PEST. In addition, OA decreased RhoA activity and the phosphorylation of cofilin, a downstream target of RhoA. Cofilin dephosphorylation increased its actin-severing activity and led to the depolymerization of F-actin, which might provide another potential mechanism for OA-induced myofibril disassembly.

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 microM 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.

18beta-glycyrrhetinic acid promotes src interaction with connexin43 in rat cardiomyocytes.

The mechanism by which 18beta-glycyrrhetinic acid regulates gap junction intercellular communication (GJIC) remains poorly understood. In this study, treatment of cultured rat neonatal cardiomyocytes with 18beta-glycyrrhetinic acid resulted in dose-dependent inhibition of GJIC as assessed by fluorescent dye transfer analysis. 18beta-Glycyrrhetinic acid induced time-dependent serine/threonine dephosphorylation and redistribution of connexin43 (Cx43) in cardiomyocytes and the induced Cx43 dephosphorylation was prevented by the protein phosphatase inhibitor, calyculin A. However, functional analyses showed that the inhibitory effect of 18beta-glycyrrhetinic acid on dye spreading among cardiomyocytes was not blocked by calyculin A, but was blocked by the Src-selective tyrosine kinase inhibitor, PP2. 18beta-Glycyrrhetinic acid also induced an increase in the levels of phosphorylated Src, and this effect was prevented by PP2. Immunoprecipitation using anti-Cx43 and anti-p-Src antibodies showed that 18beta-glycyrrhetinic acid increased the association between p-Src and Cx43 and induced tyrosine phosphorylation of Cx43. We conclude that the inhibitory effect of 18beta-glycyrrhetinic acid on GJIC in cardiomyocytes involves Src-mediated tyrosine phosphorylation of Cx43.

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

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists cause cell death in several types of cancer cells. The aim of this study was to examine the effects of two PPARgamma agonists, ciglitazone and 15-deoxy-delta(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 beta1, focal adhesion kinase (FAK), and paxillin were decreased after PPARgamma agonist treatment. Meanwhile, reduced phosphorylation of FAK and paxillin was noted. Furthermore, PPARgamma 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 PPARgamma agonist-induced dephosphorylation of FAK and paxillin. Perturbation of CGTH W-2 cells with anti-integrin beta1 antibodies induced FA disruption and apoptosis in the same cells, thus the downregulation of integrin beta1 by PPARgamma 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, PPARgamma agonists induced apoptosis of thyroid carcinoma cells via the cytochrome-c caspase 3 and PTEN-Akt pathways, and induced necrosis via the PARP pathway.