Regulation of phospholipase D from human hepatocarcinoma cell line by purine nucleotides and protein kinase A

The regulation of phosphatidylcholine-specific phospholipase D by purine nucleotides and protein kinase A were studied in vitro using an enzyme preparation partially purified from the membranous fraction of 7721 hepatocarcinoma cells. It was found that the enzyme activity was elevated by low concentrations of some purine nucleotides, but the activating effects were decreased when the concentrations of the nucleotides were higher. The optimal concentrations of GTP, GTP[S] , GDP and ATP for maximal activation were 0.1mM, 5M,1 mM and 1 mM respectively. The activation caused by 1mM ADP was lower. The enzyme was not activated by 1mM AMP, but significant activation was observed by the addition of 1mM cAMP. The latter was mediated by protein kinase A, as a specific inhibitor of protein kinase A ablished the activation. There were synergic effects between ATP and GTP, ATP and PIP₂, but not between ATP and GTP[S] , or PIP₂ and GTP[S]. The activating effects of GTP and ATP were abolished by neomycin, a PIP₂ scavenger. These results suggest that phospholipase D is regulated by GTP-binding protein and the presence of PIP₂ is required for the activation induced by GTP. Protein kinase A may be another protein kinase in addition to protein kinase C and protein tyrosine kinase which regulate the activity of phospholipase D, when the intracellular concentration of cAMP is increased.     

Forskolin up-regulates metastasis-related phenotypes and molecules via protein kinase B,but not PI-3K,in H7721 human hepato-carcinoma cell line

Forskolin (FSK) is known as an up-regulator of intracellular cAMP and inhibitor of cancer growth and metastasis. The effects of FSK on the metastasis potential and its mechanisms were studied using a human hepatocarcinoma cell line, H7721. It was found that FSK stimulated cell growth, increased cAMP in the cells, and enhanced the metastasis-related phenotypes, including adhesion to laminin (Ln) and human umbilical vein epithelial cells (HUVEC), chemotactic migration and invasion. These effects were supposed to result from the increase of the SLe^x expression induced by FSK, since only the monoclonal antibody of SLe^x showed a significant attenuation of the enhanced metastasis-associated phenotypes. Using H7721 cells transfected with the sense or antisense cDNA of protein kinase B (PKB) and some inhibitors of signal transduction, it was discovered that FSK up-regulated the expression of SLe^x via PKB, but it was independent of phosphotidylinositide-3-kinase (PI-3K). A subtype of atypical protein kinase C (-PKC) might also participate in the up-regulation of SLe^x expression by FSK, and cAMP/PKA pathway is a negative regulator of SLe^x expression on H7721 cells. It can be concluded that FSK shows a metastasis-promoting effect ex vivo.     

Down-regulation of N-acetylglucosaminyltransferase V by tumorigenesis- or metastasis-suppressor gene and its relation to metastatic potential of human hepatocarcinoma cells

The effects of transfection of the metastasis suppressor gene nm23-H1 and cell-cycle related tumor-suppressor gene p16 on the activity of N-acetylglucosaminyltransferase V (GnT-V) and their relations to cancer metastatic potential were investigated. After transfection of nm23-H1 into 7721 human hepatocarcinoma cells and A549 human lung cancer cells, the activities of GnT-V were decreased by 28%-42% in the cells. In contrast, when p16 was transfected into these two cell lines, the decrease of GnT-V activity was only observed in A549 cells. This was probably to be due to the obvious expression of p16 gene in parental 7721 cells and the deletion of p16 in A549 cells. The decrease of GnT-V mRNA was only observed in nm23-H1-transfected cells, but not in p16-transfected A549 cells, suggesting that these two genes regulated GnT-V via different mechanisms. Horseradish peroxidase (HRP)-lectin staining showed that the 7721 cells transfected with nm23-H1 or the A549 cells transfected with p16 displayed a decreased intensity with HRP-leucoagglutinating phytohemagglutinin and increased intensity with HRP-concanavalin A, indicating the decline of beta1,6 N-acetylglucosamine branching structure on the asparagine-linked glycans of cell-surface and intracellular glycoproteins. The nm23-H1 transfected 7721 cells also displayed some changes in metastasis-related phenotypes, including the increase in cell adhesion to fibronectin (Fn), the decline in cell adhesion to laminin (Ln), and the decreased cell migration and invasion through matrigel. Transfection of antisense GnT-V cDNA into 7721 cells resulted in a decrease of GnT-V activity, an increase of cell adhesion to Fn or Ln, and a decrease in cell migration and invasion through matrigel. These phenotypes bore similarity to those of the 7721 cells transfected with nm23-H1. Our findings indicate that the down-regulation of GnT-V by nm23-H1 contributes to the alterations in metastasis-related phenotypes, and is an important molecular mechanism of metastasis suppression mediated by nm23-H1.     

Reactive oxygen species stimulated human hepatoma cell proliferation via cross-talk between PI3-K/PKB and JNK signaling pathways

Reactive oxygen species (ROS) are important for intracellular signaling mechanisms regulating many cellular processes. Manganese superoxide dismutase (MnSOD) may regulate cell growth by changing the level of intracellular ROS. In our study, we investigated the effect of ROS on 7721 human hepatoma cell proliferation. Treatment with H2O2 (1-10 microM) or transfection with antisense MnSOD cDNA constructs significantly increased the cell proliferation. Recently, the mitogen-activated protein kinases (MAPK) and the protein kinase B (PKB) were proposed to be involved in cell growth. Accordingly, we assessed the ability of ROS to activate MAPK and PKB. PKB and extracellular signal-regulated kinase (ERK) were both rapidly and transiently activated by 10 microM H2O2, but the activities of p38 MAPK and JNK were not changed. ROS-induced PKB activation was abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002, suggesting that PI3-K is an upstream mediator of PKB activation in 7721 cells. Transfection with sense PKB cDNA promoted c-fos and c-jun expression in 7721 cells, suggesting that ROS may regulate c-fos and c-jun expression via the PKB pathway. Furthermore we found that exogenous H2O2 could stimulate the proliferation of PKB-AS7721 cells transfected with antisense PKB cDNA, which was partly dependent on JNK activation, suggesting that H2O2 stimulated hepatoma cell proliferation via cross-talk between the PI3-K/PKB and the JNK signaling pathways. However, insulin could stimulate 7721 cell proliferation, which is independent of cross-talk between PI3-K/PKB and JNK pathways. In addition, H2O2 did not induce the cross-talk between the PI3-K/PKB and the JNK pathways in normal liver cells. Taken together, we found that ROS regulate hepatoma cell growth via specific signaling pathways (cross-talk between PI3-K/PKB and JNK pathway) which may provide a novel clue to elucidate the mechanism of hepatoma carcinogenesis.     

Increased susceptibility to apoptosis of human hepatocarcinoma cells transfected with antisense N-acetylglucosaminyltransferase V cDNA.

The antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V, EC 2. 4.1.155) was constructed as pcDNA3/GnT-V-AS plasmid and transfected into 7721 cells, a human hepatocarcinoma cell line. The transfection was confirmed with Northern blot. By using HPLC and HRP-lectin staining, it was found that the cells transfected with pcDNA3/GnT-V-AS (GnT-V-AS/7721) expressed less GnT-V activity and beta-1,6-GlcNAc branching in the cell glycoproteins compared with the cells mock-transfected with the vector pcDNA3 (pcDNA3/7721). The growth rate of GnT-V-AS/7721 was decreased in serum-containing medium, while the cell death was accelerated in serum-free medium. The GnT-V-AS/7721 cells were more susceptible to the apoptosis induced by ATRA than the mock-transfected cells. This was evidenced by the obvious appearance of a hypoploid sub-G(1) fraction in the DNA histogram using FCM analysis, the more condensed new moon-type nuclei under morphological observation, and the more intensive TUNEL reaction for assaying the fragmented DNA. At the same time as GnT-V down-regulation by GnT-V-AS, an increase of another N-aceylglusaminyltransferase, GnT-III (EC 2.4.1.144), was observed, and the biological significance of this finding was discussed.     

Native group-III metabotropic glutamate receptors are coupled to the mitogen-activated protein kinase/phosphatidylinositol-3-kinase pathways

We used cultured cerebellar granule cells to examine whether native group-III metabotropic glutamate (mGlu) receptors are coupled to the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI-3-K) pathways. Cultured granule cells responded to the group-III mGlu receptor agonist, L-2-amino-4-phosphonobutanoate (l-AP4), with an increased phosphorylation and activity of MAPKs (ERK-1 and -2) and an increased phosphorylation of the PI-3-K target, protein kinase B (PKB/AKT). These effects were attenuated by the group-III antagonists, alpha-methyl-serine-O -phosphate (MSOP) and (R,S )-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), or by pretreatment of the cultures with pertussis toxin. l-AP4 also induced the nuclear translocation of beta-catenin, a downstream effector of the PI-3-K pathway. To assess the functional relevance of these mechanisms we examined the ability of l-AP4 to protect granule cells against apoptosis by trophic deprivation, induced by lowering extracellular K(+) from 25 to 10 mm. Neuroprotection by l-AP4 was attenuated by MSOP and abrogated by the compounds PD98059 and UO126, which inhibit the MAPK pathway, or by the compound LY294002, which inhibits the PI-3-K pathway. Taken together, these results show for the first time that native group-III mGlu receptors are coupled to MAPK and PI-3-K, and that activation of both pathways is necessary for neuroprotection mediated by this particular class of receptors.     

O-GlcNAcylation-Inducing Treatments Inhibit Estrogen Receptor α Expression and Confer Resistance to 4-OH-Tamoxifen in Human Breast Cancer-Derived MCF-7 Cells

O-GlcNAcylation (addition of N-acetyl-glucosamine on serine or threonine residues) is a post-translational modification that regulates stability, activity or localization of cytosolic and nuclear proteins. O-linked N-acetylgluocosmaine transferase (OGT) uses UDP-GlcNAc, produced in the hexosamine biosynthetic pathway to O-GlcNacylate proteins. Removal of O-GlcNAc from proteins is catalyzed by the β-N-Acetylglucosaminidase (OGA). Recent evidences suggest that O-GlcNAcylation may affect the growth of cancer cells. However, the consequences of O-GlcNAcylation on anti-cancer therapy have not been evaluated. In this work, we studied the effects of O-GlcNAcylation on tamoxifen-induced cell death in the breast cancer-derived MCF-7 cells. Treatments that increase O-GlcNAcylation (PUGNAc and/or glucosoamine) protected MCF-7 cells from death induced by tamoxifen. In contrast, inhibition of OGT expression by siRNA potentiated the effect of tamoxifen on cell death. Since the PI-3 kinase/Akt pathway is a major regulator of cell survival, we used BRET to evaluate the effect of PUGNAc+glucosamine on PIP₃ production. We observed that these treatments stimulated PIP₃ production in MCF-7 cells. This effect was associated with an increase in Akt phosphorylation. However, the PI-3 kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, which abolished the effect of PUGNAc+glucosamine on Akt phosphorylation, did not impair the protective effects of PUGNAc+glucosamine against tamoxifen-induced cell death. These results suggest that the protective effects of O-GlcNAcylation are independent of the PI-3 kinase/Akt pathway. As tamoxifen sensitivity depends on the estrogen receptor (ERα) expression level, we evaluated the effect of PUGNAc+glucosamine on the expression of this receptor. We observed that O-GlcNAcylation-inducing treatment significantly reduced the expression of ERα mRNA and protein, suggesting a potential mechanism for the decreased tamoxifen sensitivity induced by these treatments. Therefore, our results suggest that inhibition of O-GlcNAcylation may constitute an interesting approach to improve the sensitivity of breast cancer to anti-estrogen therapy.     

Transmembrane signaling through phospholipase C in human cortical membranes

Stimulation of phosphoinositide-specific phospholipase C (PLC) by carbachol, dopamine and serotonin was measured by supplying exogenous [³H]phosphatidylinositol 4,5-bisphosphate to membranes prepared from human cortex dissected and frozen at autopsy. Subjects with Alzheimer's disease, Parkinson's disease or schizophrenia were compared to age-matched controls with no known neurological disorders. Stimulation of PLC by the neurotransmitters was dependent on the presence of GTPS. Carbachol elicited the greatest stimulations of PLC followed by serotonin and then dopamine. The maximal stimulations of PLC evoked by a neurotransmitter were similar for the various categories of subjects except in Parkinson's patients, where dopamine failed to stimulate PLC beyond the activity attained with carbachol. In the presence of carbachol, the sensitivity of PLC to GTPS was significantly increased in Alzheimer's membranes, but not in age-matched controls or Parkinson's. Overall, the experiments demonstrate the feasibility for using the exogenous substrate assay to study the functionality of the phosphoinositide transmembrane signaling system in human brain.Abbreviations PLC phospholipase C - GTPS guanosine 3-O-thiotriphosphate - Gpp(NH)p 5-guanylyl-imidodiphosphate - DA dopamine - CCh earbachol - 5-HT serotonin - PIP₂ phosphatidylinositol 4,5-bisphosphate - PIP phosphatidylinositol 4-bisphosphate     

N—乙酰氨基葡萄糖转移酶—V过表达对人肝癌细胞迁移及粘附分子表达的影响

The purpose of this paper is to study the effect of N-acetylglucosaminyltransferase V (GnT-V) overexpression on the migration of 7721 cells and its mechanism. The abilities of migration of both 7721 cells transfected with GnT-V cDNA and 7721 cells transfected with pcDNA3 was detected, the expressions of integrin and E-cadherin which are important adhesion molecules on surface membrane and closely related to the abilities of invasion and metastasis. Cell migration abilities were measured by the agarose drop explant method. Flow cytometric analysis (FACS) was applied to determine the relative amounts of integrin alpha 5 and beta 1 subunits on the cell surface while RTPCR was carried out to determine the expression of their mRNA. The expression of E-cadherin was examined by the immunocytochemical ABC method. Western blot analysis was carried out to examine the expression of beta-catenin. GnT-V overexpression enhanced evidently the migration ability of 7721 cells and increased the amount of integrin alpha 5 subunit to 2.9 times of that of control while the amount of beta 1 subunits was not significantly changed. Besides, the expressions of E-cadherin and beta-catenin were enhanced at different levels in GnT-V/7721 cells compared with mocked. The results suggested that the overexpression of GnT-V related to the production of N-linked sugar chains could promote the expressions of integrin, E-cadherin and beta-catenin on 7721 cells so that the migration ability of tumor cells was enhanced.     

Osteopontin enhances the expression and activity of MMP-2 via the SDF-1/CXCR4 axis in hepatocellular carcinoma cell lines

Background and Aims

Osteopontin, SDF-1α, and MMP-2 are important secreted molecules involved in the pathophysiology of human hepatocellular carcinoma (HCC). This study investigates the effect of the SDF-1α/CXCR4 axis on expression and activity of MMP-2 induced by osteopontin.

Methods

The expression of CXCR4, SDF-1α, MMP-2 and their associated cellular signaling cascades, involving Akt and MAP Kinases, were determined by Western blotting. The activities of MMP-2 and MMP-9 were assayed by gel zymography. The role of the osteopontin receptors integrin α_vβ₃ and CD44v6 was evaluated using neutralizing antibodies. We also established CXCR4-deficient SMMC7721 cell lines by transfection with miRNA-CXCR4 plasmids and determined cell invasion activity in a transwell assay.

Results

In comparison with untreated cells, recombinant human osteopontin (rhOPN) up-regulated CXCR4, SDF-1α, and MMP-2 expression about 5-, 4-, and 6-fold on the protein levels through binding to integrin α_vβ₃ and CD44v6 in hepatocellular carcinoma cells (SMMC7721 and HepG2). Inhibition of the SDF-1α/CXCR4 axis down-regulated the rhOPN-induced MMP-2 expression and activity. rhOPN also activated Akt, p38 and JNK. Down-regulation of CXCR4 decreased the rhOPN-induced invasion in SMMC7721 cells.

Conclusion

These results indicate that rhOPN up-regulates MMP-2 through the SDF-1α/CXCR4 axis, mediated by binding to integrin α_vβ₃ and CD44v6 and activating the PI-3K/Akt and JNK pathways in HepG2 and SMMC7721 cells. Therefore, the osteopontin-SDF-1α/CXCR4-MMP-2 system may be a new therapeutic target for treating HCC progression.     

Role of Phosphatidylinositol 4,5-Bisphosphate in Ras/Rac-Induced Disruption of the Cortactin-Actomyosin II Complex and Malignant Transformation

Oncogenic Ras mutants such as v-Ha-Ras cause a rapid rearrangement of actin cytoskeleton during malignant transformation of fibroblasts or epithelial cells. Both PI-3 kinase and Rac are required for Ras-induced malignant transformation and membrane ruffling. However, the signal transduction pathway(s) downstream of Rac that leads to membrane ruffling and other cytoskeletal change(s) as well as the exact biochemical nature of the cytoskeletal change remain unknown. Cortactin/EMS1 is the first identified molecule that is dissociated in a Rac–phosphatidylinositol 4,5-biphosphate (PIP₂)-dependent manner from the actin-myosin II complex during Ras-induced malignant transformation; either the PIP₂ binder HS1 or the Rac blocker SCH51344 restores the ability of EMS1 to bind the complex and suppresses the oncogenicity of Ras. Furthermore, while PIP₂ inhibits the actin-EMS1 interaction, HS1 reverses the PIP₂ effect. Thus, we propose that PIP₂, an end-product of the oncogenic Ras/PI-3 kinase/Rac pathway, serves as a second messenger in the Ras/Rac-induced disruption of the actin cytoskeleton and discuss the anticancer drug potential of PIP₂-binding molecules.     

Characterization of AT2 Receptor Expression in NIH 3T3 Fibroblasts

1. A high expression of angiotensin II receptors and of angiotensin-converting enzyme (ACE) activity was detected in confluent NIH 3T3 fibroblasts.2. Characterization with selective ligands, dithiothreitol, and GTPS, indicated that only the AT₂ subtype was expressed.3. AT₂ receptors and ACE expression were strictly dependent on the cell density and growth phase of the cells, with AT₂ receptors being expressed earlier than ACE. In contrast, high expression of AT₂ receptors irrespective of their growth state was observed in NIH 3T3 cells lacking contact inhibition upon neoplastic transformation with ras.4. Our results imply a possible relation of AT₂ receptors to cell growth and cell–cell contact.     

Effect of p58GTA on β-1,4-galactosyltransferase 1 activity and cell-cycle in human hepatocarcinoma cells

-1,4-galactosyltransferase 1 (1,4-GT 1) is the key enzyme transferring galactose to the terminal N-acetylglucosamine (GlcNAc) forming Gal14GlcNAc structure in the Golgi apparatus. In addition, it also serves as a cell adhesion molecule by recognizing and binding to terminal GlcNAc of glycoconjugates on the adjacent cell surface and matrix through a subpopulation of the enzyme distributed on the cell surface. Transient expression of the p58^GTA protein kinase, which belongs to the p34cdc2-related supergene family, could enhance 1,4-GT 1 total activity in COS cells. In this study, the p58^GTA interaction with 1,4-GT 1 was confirmed using an in vitro assay with the TNT® Coupled Reticulocyte Lysate System. An expression vector containing p58^GTA was stably transfected into 7721 cells, a human hepatocarcinoma cell line, expression was confirmed by Northern and Western blot analyses. The cells transfected with p58^GTA (p58^GTA/7721) contained 1.9 times higher total 1,4-GT 1 activity and 2.6 times higher cell-surface 1,4-GT 1 activity than the mock transfected cells (pcDNA3/7721). However, Ricinus communis agglutinin-I lectin blot analysis revealed that the enhanced 1,4-GT 1 activity did not increase the Gal14GlcNAc groups on most of the membrane proteins in p58^GTA/7721 cells. By flow cytometry analysis, it was found that the p58^GTA/7721 cells were G2/M phase arrested, compared with the pcDNA3/7721 cells. These results suggest that the p58^GTA stable transfection into human hepatocarcinoma cells could enhance the two 1,4-GT 1 subcellular pool activities independently and change its cell-cycle without modifying the -1,4-linked galactose residues on most membrane proteins.     

PAK4 confers cisplatin resistance in gastric cancer cells via PI3K/Akt- and MEK/ERK-dependent pathways

CDDP [cisplatin or cis-diamminedichloroplatinum(II)] and CDDP-based combination chemotherapy have been confirmed effective against gastric cancer. However, CDDP efficiency is limited because of development of drug resistance. In this study, we found that PAK4 (p21-activated kinase 4) expression and activity were elevated in gastric cancer cells with acquired CDDP resistance (AGS/CDDP and MKN-45/CDDP) compared with their parental cells. Inhibition of PAK4 or knockdown of PAK4 expression by specific siRNA (small interfering RNA)-sensitized CDDP-resistant cells to CDDP and overcome CDDP resistance. Combination treatment of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 [the inhibitor of PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B or PKB) pathway] or PD98509 {the inhibitor of MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] pathway} with PF-3758309 (the PAK4 inhibitor) resulted in increased CDDP efficacy compared with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or PD98509 alone. However, after the concomitant treatment of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and PD98509, PF-3758309 administration exerted no additional enhancement of CDDP cytotoxicity in CDDP-resistant cells. Inhibition of PAK4 by PF-3758309 could significantly suppress MEK/ERK and PI3K/Akt signalling in CDDP-resistant cells. Furthermore, inhibition of PI3K/Akt pathway while not MEK/ERK pathway could inhibit PAK4 activity in these cells. The in vivo results were similar with those of in vitro. In conclusion, these results indicate that PAK4 confers CDDP resistance via the activation of MEK/ERK and PI3K/Akt pathways. PAK4 and PI3K/Akt pathways can reciprocally activate each other. Therefore, PAK4 may be a potential target for overcoming CDDP resistance in gastric cancer.     

Oxidative Stress Decreases Phosphatidylinositol 4,5-Bisphosphate Levels by Deactivating Phosphatidylinositol- 4-phosphate 5-Kinase �� in a Syk-dependent Manner

Phosphatidylinositol 4,5-bisphosphate (PIP₂) has many essential functions and its homeostasis is highly regulated. We previously found that hypertonic stress increases PIP₂ by selectively activating the β isoform of the type I phosphatidylinositol phosphate 5-kinase (PIP5Kβ) through Ser/Thr dephosphorylation and promoting its translocation to the plasma membrane. Here we report that hydrogen peroxide (H₂O₂) also induces PIP5Kβ Ser/Thr dephosphorylation, but it has the opposite effect on PIP₂ homeostasis, PIP5Kβ function, and the actin cytoskeleton. Brief H₂O₂ treatments decrease cellular PIP₂ in a PIP5Kβ-dependent manner. PIP5Kβ is tyrosine phosphorylated, dissociates from the plasma membrane, and has decreased lipid kinase activity. In contrast, the other two PIP5K isoforms are not inhibited by H₂O₂. We identified spleen tyrosine kinase (Syk), which is activated by oxidants, as a candidate PIP5Kβ kinase in this pathway, and mapped the oxidant-sensitive tyrosine phosphorylation site to residue 105. The PIP5KβY105E phosphomimetic is catalytically inactive and cytosolic, whereas the Y105F non-phosphorylatable mutant has higher intrinsic lipid kinase activity and is much more membrane associated than wild type PIP5Kβ. These results suggest that during oxidative stress, as modeled by H₂O₂ treatment, Syk-dependent tyrosine phosphorylation of PIP5Kβ is the dominant post-translational modification that is responsible for the decrease in cellular PIP₂.Oxygen-derived free radicals are by-products of metabolic reactions in eukaryotic cells. Reactive oxygen species (ROS)⁴ act as endogenous signaling molecules (1). However, excessive ROS production leads to deleterious effects on cellular homeostasis by inducing DNA damage, lipid/protein oxidation, and ultimately apoptosis or necrosis. Acute and chronic oxidative stress have been implicated in the pathophysiology of shock and sepsis associated with traumatic injuries such as massive thermal burn (2–4), Alzheimer disease, diabetes mellitus, and atherosclerosis (5–7).Phosphatidylinositol 4,5-bisphosphate (PIP₂) has emerged as an integral component of the stress response. This is concordant with its essential role in the regulation of the actin cytoskeleton, endocytosis, exocytosis, plasma membrane (PM) scaffolding, and ion channels/transporter (8). PIP₂ is also essential for InsP₃-mediated Ca²⁺ generation, protein kinase C activation, and PIP₃ generation (9, 10). PIP₂ synthesis is depressed in the heart sarcolemma during oxidative stress, suggesting that PIP₂ depletion may contribute to cardiac dysfunctions (11). Recently, Divecha and colleagues (12) reported that prolonged (many hours) treatment of HeLa cells with hydrogen peroxide (H₂O₂) induces apoptosis by depleting PIP₂. Apoptosis can be attenuated by overexpression of a type I phosphatidylinositol-4-phosphate 5-kinase (PIP5Kβ). We found using isoform-specific PIP5K knockdown by RNA interference (RNAi) that PIP5Kβ synthesizes a large fraction of the ambient PIP₂ pool in HeLa cells (13). Hypertonicity is another type of stress that increases PIP₂ and may be protective against cell injury (14, 15) by activating PIP5Kβ through Ser/Thr dephosphorylation (16). This effect is specific for PIP5Kβ, because depletion of the other two PIP5K isoforms (α and γ) individually does not substantially abrogate the hypertonicity induced PIP₂ increase.In the present study, we used H₂O₂ to model oxidative stress in tissue culture cells, and examined the effect on PIP₂ homeostasis and PIP5Kβ function. We found that a brief H₂O₂ treatment decreases cellular PIP₂ and inactivates PIP5Kβ through tyrosine phosphorylation. We identified spleen tyrosine kinase (Syk) as a candidate kinase in this pathway. Syk is a member of the Syk/Zap-70 nonreceptor tyrosine kinase family that is abundant in hematopoietic cells (17) but is also found in nonhematopoietic lineages (18), including HeLa and COS cells (19, 20).     

MPP+-induced cytotoxicity in neuroblastoma cells: Antagonism and reversal by guanosine

Guanosine exerts neuroprotective effects in the central nervous system. Apoptosis, a morphological form of programmed cell death, is implicated in the pathophysiology of Parkinson’s disease (PD). MPP⁺, a dopaminergic neurotoxin, produces in vivo and in vitro cellular changes characteristic of PD, such as cytotoxicity, resulting in apoptosis. Undifferentiated human SH-SY5Y neuroblastoma cells had been used as an in vitro model of Parkinson’s disease. We investigated if extracellular guanosine affected MPP⁺-induced cytotoxicity and examined the molecular mechanisms mediating its effects. Exposure of neuroblastoma cells to MPP⁺ (10 μM–5 mM for 24–72 h) induced DNA fragmentation in a time-dependent manner (p < 0.05). Administration of guanosine (100 μM) before, concomitantly with or, importantly, after the addition of MPP⁺ abolished MPP⁺-induced DNA fragmentation. Addition of MPP⁺ (500 μM) to cells increased caspase-3 activity over 72 h (p < 0.05), and this was abolished by pre- or co-treatment with guanosine. Exposure of cells to pertussis toxin prior to MPP⁺ eliminated the anti-apoptotic effect of guanosine, indicating that this effect is dependent on a Gi protein-coupled receptor, most likely the putative guanosine receptor. The protection by guanosine was also abolished by the selective inhibitor of the enzyme PI-3-K/Akt/PKB (LY294002), confirming that this pathway plays a decisive role in this effect of guanosine. Neither MPP⁺ nor guanosine had any significant effect on α-synuclein expression. Thus, guanosine antagonizes and reverses MPP⁺-induced cytotoxicity of neuroblastoma cells via activation of the cell survival pathway, PI-3-K/Akt/PKB. Our results suggest that guanosine may be an effective pharmacological intervention in PD.     

An essential role for the SHIP2-dependent negative feedback loop in neuritogenesis of nerve growth factor-stimulated PC12 cells

The local accumulation of phosphatidylinositol (3,4,5) trisphosphate (PIP₃) and resulting activation of Rac1/Cdc42 play a critical role in nerve growth factor (NGF)–induced neurite outgrowth. To further explore the mechanism, we visualized PIP₃, phosphatidylinositol (3,4) bisphosphate, and Rac1/Cdc42 activities by fluorescence resonance energy transfer (FRET) imaging in NGF-stimulated PC12 cells. Based on the obtained FRET images, and with the help of in silico kinetic reaction model, we predicted that PI-5-phosphatase negatively regulates PIP₃ upon NGF stimulation. In agreement with this model, depletion of Src homology 2 domain–containing inositol polyphosphate 5-phosphatase 2 (SHIP2) markedly potentiated NGF-induced Rac1/Cdc42 activation and PIP₃ accumulation and considerably increased the number and the length of neurites in phosphate and tensin homologue–depleted PC12 cells. Further refinement of the computational model predicted Rac1 regulation of PI3-kinase and SHIP2, which was also validated experimentally. We propose that the SHIP2-mediated negative feedback on PIP₃ coordinately works with the PI3-kinase–mediated positive feedback to form an initial protrusive pattern and, later, to punctuate the PIP₃ accumulation to maintain proper neurite outgrowth.     

Increase in beta1-6 GlcNAc branching caused by N-acetylglucosaminyltransferase V directs integrin beta1 stability in human hepatocellular carcinoma cell line SMMC-7721

In this study, an enzymatic inactive mutant of GnT-V (delta cGnT-V) was constructed and transfected in SMMC 7721 cell line. Integrin beta1 in delta cGnT-V transfectants (delta c-7721) showed attenuation of the number of beta1-6 GlcNAc branching, whereas those in wtGnT-V transfectants (wt-7721) presented a beta1-6 GlcNAc-rich pattern. High integrin beta1 expression was observed in wt-7721 compared with mock cells (7721 cell transfected with the vector pcDNA3), while transfection of delta cGnT-V decreased the integrin beta1 expression, despite of no significant changes on integrin beta1 mRNA level in these cell lines. Pulse-chase experiment showed that Integrin beta1 in delta c-7721 was prone to quick degradation and its half-life was less than 3 h, on the contrary, the alleviating degradation of beta1 subunit was observed in wt-7721 where the beta1 subunit half-life was about 16 h, meanwhile, the degradation rate of beta1 subunit in mock cells was in between, about 10 h. More effective in promoting cell migration toward fibronectin and invasion through Matrigel was observed in wt-7721 while this was almost suppressed in delta c-7721. Our results suggest that the addition of beta1-6 GlcNAc branching caused more fully glycosylated mature form on integrin beta1 and inhibited beta1 protein degradation. Glycosylation caused by GnT-V directs integrin beta1 stability and more delivery to plasma membrane, subsequently promotes Fn-based cell migration and invasion.     

A role for G-proteins in the epidermal growth factor stimulation of phospholipase A2 in rat kidney mesangial cells

We have previously demonstrated phospholipase C (PLC) independent activation of phospholipase A₂(PLA₂) by epidermal growth factor (EGF) in glomerular mesangial cells in culture. In the current study using glass beads to permeabilize [³H]- or [¹⁴C]-arachidonate labelled mesangial cells we demonstrate that guanine nucleotides modulate the EGF-mediated stimulation of arachidonic acid release (75% inhibition with 100 M GDPS and 108% augmentation with 100 M GTPS). GTPS alone stimulated both the release of free arachidonic acid and production of diacylglycerol (DAG), while EGF itself neither stimulated DAG nor augmented the DAG response to GTPS. These findings suggest the intermediacy of a G-protein in PLC-independent stimulation of PLA₂ by a growth factor, and provide a model system for determining the relationship between G-protein intermediacy and the intrinsic tyrosine kinase activity of the growth factor receptor.Abbreviations EGF Epidermal Growth Factor - PLC phospholipase C - PLA₂ phospholipase A₂ - DAG Diacylglycerol - NEFA non-esterified fatty acid - GTPS guanosine-5-0-[3-thio]triphosphate - GDP\S guanosine-5-0-[2-thio]diphosphate