8-bromocyclic-gmp and Liver-Neoplasms

βig-h3 is a TGF-β (transforming growth factor β)-induced ECM (extracellular matrix) protein that induces the secretion of MMPs (matrix metalloproteinases). However, the mechanism of induction is yet to be established. In this study, siRNAs (small interfering RNAs) targeted against βig-h3 were transfected into SMMC-7721 cells [a HCC (human hepatocellular carcinoma) cell line] to knockdown the expression of βig-h3. We found that NiCl2, a potent blocker of extracellular Ca2+ entry, reduced βig-h3-induced secretion of MMP-2 and -9. Further investigation suggested that reduction in the levels of βig-h3 decreased the secretion of MMP-2 and -9 that was enhanced by an increase in the concentration of extracellular Ca2+. SNAP (S-nitroso-N-acetylpenicillamine), a NO (nitric oxide) donor, and 8-Br-cGMP (8-bromo-cGMP) inhibited thapsigargin-induced Ca2+ entry and MMP secretion in the invasive potential of human SMMC-7721 cells. Further, the inhibitory effects of 8-Br-cGMP and SNAP could be significantly enhanced by down-regulating βig-h3. βig-h3 attenuates the negative regulation of NO/cGMP-sensitive store-operated Ca2+ entry. Our findings suggest that the expression of βig-h3 might play an important role in the regulation of store-operated Ca2+ entry to increase the invasive potential of HCC cells.

Topics: Calcium; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclic GMP; Extracellular Matrix Proteins; Fluorescent Antibody Technique; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Nickel; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; S-Nitroso-N-Acetylpenicillamine; Thapsigargin; Transfection; Transforming Growth Factor beta

This study aimed to investigate cGMP-regulated store-operated Ca(2+)entry in human 7721 hepatoma cells. [Ca(2+)](i)was measured using Fura2/AM. After incubation of the cells with 4 microm thapsigargin, Ca(2+)entry was evoked by application of 1 mMm Ca(2+)to extracellular solution and was blocked by 3 m m Ni(2+), indicating the presence of store-operated Ca(2+)entry in human 7721 hepatoma cell line. Application of 8-Br-cGMP reduced the [Ca(2+)](i)in hepatoma 7721 cells by 80%. These data demonstrated for the first time that store-operated Ca(2+)entry pathway is present in human hepatoma cells, which is regulated by cGMP.

Topics: Calcium; Calcium Channels; Calcium-Transporting ATPases; Carcinoma, Hepatocellular; Cyclic GMP; Enzyme Inhibitors; Humans; Liver Neoplasms; Nickel; Thapsigargin; Tumor Cells, Cultured

The present study examined the effect of hepatoma-associated antigen HAb18G (homologous to CD147) expression on the NO/cGMP-regulated Ca(2+) mobilization and metastatic process of human hepatoma cells. HAb18G/CD147 cDNA was transfected into human 7721 hepatoma cells to obtain a cell line stably expressing HAb18G/CD147, T7721, as demonstrated by Northern blot and immunocytochemical studies. 8-Bromo-cGMP (cGMP) inhibited the thapsigargin-induced Ca(2+) entry in a concentration-dependent manner in 7721 cells. The cGMP-induced inhibition was abolished by an inhibitor of protein kinase G, KT5823 (1 microm). However, expression of HAb18G/CD147 in T7721 cells decreased the inhibitory response to cGMP. A similar concentration-dependent inhibitory effect on the Ca(2+) entry was observed in 7721 cells in response to a NO donor, (+/-)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca(2+) entry was significantly reduced in HAb18G/CD147-expressing T7721 cells, indicating a role for HAb18G/CD147 in NO/cGMP-regulated Ca(2+) entry. Experiments investigating metastatic potentials demonstrated that HAb18G/CD147-expressing T7721 cells attached to the Matrigel-coated culture plates and invaded through Matrigel-coated permeable filters at the rate significantly greater than that observed in 7721 cells. Both the attachment and invasion rates could be suppressed by SNAP, and the inhibitory effect of SNAP could be reversed by NO inhibitor, N(G)-nitro-l-arginine methyl ester. The sensitivity of the attachment and invasion rates to cGMP was significantly reduced in T7721 cells as compared with 7721 cells when cells were pretreated with thapsigargin. The difference in the sensitivity between the two cells could be abolished by a Ca(2+) channel blocker, Ni(2+) (3 mm). These results suggest that HAb18G/CD147 enhances metastatic potentials in human hepatoma cells by disrupting the regulation of store-operated Ca(2+) entry by NO/cGMP.

Topics: Alkaloids; Antigens, CD; Antigens, Neoplasm; Antigens, Surface; Avian Proteins; Basigin; Blood Proteins; Blotting, Northern; Calcium; Carbazoles; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line; Cell Movement; Collagen; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; DNA, Complementary; Dose-Response Relationship, Drug; Drug Combinations; Enzyme Inhibitors; Humans; Immunohistochemistry; Indoles; Laminin; Liver Neoplasms; Membrane Glycoproteins; Neoplasm Metastasis; NG-Nitroarginine Methyl Ester; Nickel; Nitric Oxide; Penicillamine; Proteoglycans; Signal Transduction; Thapsigargin; Time Factors; Transfection; Tumor Cells, Cultured

Nitric oxide (NO) is believed to play an important, but as yet undefined, role in regulating hypoxia inducible gene expression. Recently, we have reported evidence suggesting that the human insulin-like growth factor-binding protein-1 (IGFBP-1) gene is directly regulated by hypoxia through the hypoxia-inducible factor-1 pathway. The goal of the current study was to investigate NO regulation of hypoxic induction of IGFBP-1 gene expression using HepG2 cells, a model system of hepatic gene expression. We report that a NO generator, sodium nitroprusside, significantly diminishes hypoxic activation of IGFBP-1 protein and messenger ribonucleic acid expression. Furthermore, these effects are independent of guanylate cyclase/ cGMP signaling, as two different inhibitors, LY 83583, a specific inhibitor of guanylate cyclase, and KT 5823, a protein kinase G inhibitor, had no effect on IGFBP-1 induction by hypoxia. Hypoxic induction of a reporter gene containing four tandemly ligated hypoxia response elements was completely blocked by sodium nitroprusside, but not by 8-bromo-cGMP, an analog ofcGMP. These results suggest that NO blocks hypoxic induction of IGFBP-1 by a guanylate cyclase/ cGMP-independent pathway, possibly at the level of oxygen sensing. The impaired hypoxia regulation of IGFBP-1 by nitric oxide may play a key role in the hyperinduction of IGFBP-1 observed in pathophysiological conditions such as fetal hypoxia and preeclampsia where dysregulation of NO has been observed.

Topics: Alkaloids; Aminoquinolines; Base Sequence; Binding Sites; Carbazoles; Carcinoma, Hepatocellular; Cell Hypoxia; Conserved Sequence; Cyclic GMP; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation; Guanylate Cyclase; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Indoles; Insulin-Like Growth Factor Binding Protein 1; Kinetics; Liver; Liver Neoplasms; Luciferases; Molecular Sequence Data; Nitric Oxide; Nitroprusside; Nuclear Proteins; Protein Biosynthesis; Recombinant Fusion Proteins; Signal Transduction; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Expression of the asialoglycoprotein receptor by the human hepatocellular carcinoma cell line HuH-7 in response to intracellular cGMP concentrations was previously shown to be regulated at the translational level. In a cell-free system, initiation of asialoglycoprotein receptor mRNA translation was dependent on the presence of the 7-methylguanylate cap site and was independent of 8-bromo-cGMP levels in which the cells were grown prior to RNA isolation. Stable transfection of COS-7 cells with deletion constructs of the asialoglycoprotein receptor H2b subunit localized the cGMP-responsive cis-acting element to the mRNA 5'-untranslated region (UTR). Addition of biotin (an activator of guanylate cyclase) induced the expression of beta-galactosidase present as a chimeric plasmid containing the H2b 187-nucleotide 5'-UTR. An RNA gel retardation assay identified a 37-nucleotide cognate sequence within this 187-nucleotide region. Titration of the 5'-UTR with a cytosolic fraction isolated from HuH-7 grown in the presence or absence of 8-bromo-cGMP or biotin provided direct evidence for an RNA-binding protein responsive to intracellular levels of cGMP. Based on these findings, it seems reasonable to propose that reduction of intracellular levels of cGMP by biotin deprivation results in a negative trans-acting factor associating with the 5'-UTR of asialoglycoprotein receptor mRNAs, thereby inhibiting translation.

Topics: Animals; Asialoglycoprotein Receptor; Asialoglycoproteins; Biotin; Carcinoma, Hepatocellular; COS Cells; Cyclic GMP; Guanosine; Humans; Liver Neoplasms; Protein Biosynthesis; Receptors, Cell Surface; Restriction Mapping; RNA, Messenger; Transfection; Tumor Cells, Cultured

Many inflammatory diseases are associated with a hypoproliferative anaemia. Patients with this anaemia often present with serum erythropoietin (EPO) concentrations that are too low for the degree of their anaemia. Proinflammatory cytokines, in addition to their inhibitory effects on proliferation of erythroid progenitors, could contribute to the pathogenesis of this anaemia by reducing EPO production. Because several cytokines stimulate nitric oxide (NO) synthase we propose that nitric oxide might mediate the suppression of EPO production during inflammation. In order to test this hypothesis we investigated the effects of NO donors on 24-h hypoxia-induced EPO production in the hepatocellular carcinoma cell line HepG2. Following application of the NO donors sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP), EPO production was dose-dependently reduced: compared to the untreated control EPO production was lowered by 89% with SNP (1000 microM), by 66% with SIN-1 (1000 microM), and by 72% with SNAP (500 microM). In contrast, 8-bromo-cGMP did not inhibit EPO formation. Since pyrogallol (300 microM) and H2O2 (250 microM) showed a comparable suppression of EPO synthesis, we propose that NO might affect EPO production either by a similar direct influence on the cellular redox state or via increasing the cellular content of reactive oxygen species.

Topics: Carcinoma, Hepatocellular; Cyclic GMP; Cytokines; Erythropoietin; Humans; Hydrogen Peroxide; Liver Neoplasms; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Penicillamine; Reactive Oxygen Species; S-Nitroso-N-Acetylpenicillamine; Tumor Cells, Cultured

The effects of 8-bromoguanosine 3':5'-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (gj) in rat Cx43-transfected cells by 24.0 +/- 3.7% (mean +/- SEM, n = 5), whereas gj was not affected in human Cx43-transfected cells by the same treatment. The relaxation of gj in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (gammaj) of about 20, 40-45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the gammaj distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [32P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [32P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased gj, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.

Topics: Amino Acids; Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Line; Cyclic GMP; DNA, Complementary; Gap Junctions; GAP-43 Protein; Humans; Ion Channels; Liver Neoplasms; Membrane Glycoproteins; Membrane Potentials; Nerve Tissue Proteins; Patch-Clamp Techniques; Phosphorylation; Precipitin Tests; Protein Kinases; Rats; Transfection

Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Base Sequence; Carcinoma, Hepatocellular; Cell Communication; Connexin 26; Connexins; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; DNA Primers; Enzyme Inhibitors; Gap Junctions; Gene Expression; Homeostasis; Humans; Liver Neoplasms; Membrane Potentials; Molecular Sequence Data; Phosphorylation; Polymerase Chain Reaction; Protein Kinase C; Recombinant Proteins; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured

Post-transcriptional regulation of the asialoglyco-protein receptor (ASGR) in the HepG2 cell line can be mediated by the presence of biotin in the culture medium. To determine if the induction by biotin of intracellular cGMP affects ASGR expression, HepG2 were grown in biotin-depleted medium with the cell-permeant 8-bromo-cGMP (8-Br-cGMP). Both cell-surface and total ASGR binding of iodinated asialoorosomucoid (125I-ASOR) was increased from 30 to 95% of control levels by the addition of increasing concentrations of 8-Br-cGMP. The rate of ASGR-mediated endocytosis of 125I-ASOR also increased with increasing concentrations of 8-Br-cGMP. Estimates of the steady state levels of ASGR by transblot analysis utilizing both antisera to affinity-purified ASGR and to isoform-specific antibodies prepared against synthetic peptides confirmed that the increase in 125I-ASOR binding was due to an increase in ASGR expression. Metabolic labeling of biotin-deprived HepG2 with [35S] cysteine and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitants revealed an increase of radiolabeled ASGR within 30 min of the addition of 8-Br-cGMP. Induction of cGMP by atrial natriuretic factor also increased the metabolic labeling of ASGR. ASGR expression in a second hepatocellular carcinoma cell line, HuH-7, responded in a similar fashion to the addition of 8-Br-cGMP. In contrast to 8-Br-cGMP, exposure to 8-bromo-cAMP results in a reduction of ASGR expression even in the presence of biotin-containing medium. The antagonistic roles of cGMP and cAMP suggest a balance between cyclic nucleotides is required for the maintenance of differentiated functions by the hepatocyte.

Topics: Amino Acid Sequence; Asialoglycoprotein Receptor; Asialoglycoproteins; Atrial Natriuretic Factor; Carcinoma, Hepatocellular; Cell Line; Cyclic GMP; Endocytosis; Humans; Kinetics; Liver Neoplasms; Molecular Sequence Data; Oligopeptides; Receptors, Immunologic; Second Messenger Systems