okadaic-acid and Pituitary-Neoplasms

The human (hPRL) PRL gene proximal promoter (-164/+15) is the target for numerous signal transduction pathways involving protein kinases. The inhibitor of Ser/Thr-protein phosphatases okadaic acid (OA) was shown to induce this promoter in rat pituitary GH3B6 through a synergism between increased amounts of the ubiquitous factor AP-1 and the pituitary-specific factor Pit-1. Here we show that this activation results mainly from transcriptional stimulation of the c-fos promoter leading to increased AP-1 activity. We report the surprising absence of the hPRL and c-fos promoter stimulation by OA in GH3 cells, closely related to GH3B6 cells, and we use this discrepancy to dissect the precise mechanism of action. c-fos gene activation involves the mitogen-activated kinase (MAPK)-ternary complex factor (TCF) pathway and can be obtained by expressing active V12ras in both cell lines. We show that OA acts by inhibiting protein phosphatase PP1, thereby protecting MAPK kinase (MEK)1/2 and/or a MEK1/2-kinase from dephosphorylation. PP1 inhibition of MEK activation by V12ras does not occur in GH3 cells, indicating that a distinct, PP1-sensitive phosphorylation site is used in GH3B6 cells to activate the TCF pathway in GH3B6 cells. Finally, we show that the synergistic OA activation of the hPRL promoter by Pit-1 and AP-1 is independent of the Pit-1 transactivation domain and is mediated by the general coactivator (CRE-binding protein)-binding protein (CBP)/p300.

Topics: Animals; DNA-Binding Proteins; E1A-Associated p300 Protein; Enzyme Inhibitors; Flavonoids; Humans; Isoenzymes; MAP Kinase Kinase Kinase 1; Mitogen-Activated Protein Kinases; Nuclear Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Pituitary Neoplasms; Prolactin; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-fos; ras Proteins; Rats; Signal Transduction; Trans-Activators; Transcription Factor AP-1; Transcription Factor Pit-1; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured

1. The role of non-calcineurin protein phosphatases in the cyclic AMP signal transduction pathway was examined in mouse pituitary corticotroph tumour (AtT20) cells. 2. Blockers of protein phosphatases, calyculin A and okadaic acid, were applied in AtT20 cells depleted of rapidly mobilizable pools of intracellular calcium and activated by various cyclic AMP generating agonists. Inhibitors of cyclic nucleotide phosphodiesterases were present throughout. The accumulation of cyclic AMP was monitored by radioimmunoassay, phosphodiesterase activity in cell homogenates was measured by radiometric assay. 3. Neither calyculin A nor okadaic acid altered basal cyclic AMP levels but cyclic AMP formation induced by 41 amino acid residue corticotrophin releasing-factor (CRF) was strongly inhibited (up to 80%), 1-Norokadaone was inactive. Similar data were also obtained when isoprenaline or pituitary adenylate cyclase activating peptide1-38 were used as agonists. 4. Pertussis toxin did not modify the inhibition of CRF-induced cyclic AMP production by calyculin A. 5. Pretreatment with calyculin A completely prevented the stimulation of cyclic AMP formation by cholera toxin even in the presence of 0.5 mM isobutylmethylxanthine (IBMX) and 0.1 mM rolipram. Cholera toxin mediated ADP-ribosylation of the 45 K and 52 K molecular weight Gs alpha isoforms in membranes from calyculin A-pretreated cells was enhanced to 150-200% when compared with controls. 6. Cholera toxin-induced cyclic AMP was reduced by calyculin A within 10 min when calyculin A was applied after a 90 min pretreatment with cholera toxin. Under these conditions the effect of calyculin A could be blocked by the combination of 0.5 mM IBMX and 0.1 mM rolipram, but not by 0.5 mM IBMX alone. 7. Phosphodiesterase activity in AtT20 cell homogenates showed a significant, 2.7 fold increase after treatment with calyculin A. In control cells phosphodiesterase activity was blocked by 80% in the presence of IBMX (0.5 mM), or IBMX plus rolipram (0.1 mM). In calyculin A-treated cells phosphodiesterase activity was also strongly inhibited by IBMX, but because of the stimulating effect of calyculin A, the activity remaining was still 55% of that found in control homogenates. This activity was reduced to 5% of control by using IBMX and rolipram in combination. Assay of phosphodiesterase in Ca2+ free conditions showed that calyculin A markedly increases the activity of rolipram sensitive (type 4) phosphodiesterase. 8. Taken togeth

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Adenylate Cyclase Toxin; Adrenocorticotropic Hormone; Animals; Cholera Toxin; Colforsin; Cyclic AMP; Enzyme Activation; Enzyme Inhibitors; GTP-Binding Proteins; Marine Toxins; Mice; Okadaic Acid; Oxazoles; Pertussis Toxin; Phosphoprotein Phosphatases; Pituitary Neoplasms; Signal Transduction; Tumor Cells, Cultured; Virulence Factors, Bordetella

Pituitary GH3 cells die by apoptosis when treated with okadaic acid, a specific inhibitor of ser/thr phosphatases. Incubations starting at concentrations of 5 and 12.5 nM followed by stepwise rises resulted in two populations (the S1 and S2 sublines) that proliferated at initially lethal 30 nM. Cells were partially resistant to higher concentrations of okadaic acid and its derivative methyl okadaate. Toxicity of the structurally distinct inhibitors cantharidic acid and calyculin A was differently affected in the two resistant lines. The enhanced expression of the P-glycoprotein was one mechanism of resistance in S1 and S2. Resistance was reversed completely (S1) or partially (S2) by the addition of verapamil. In addition, phosphatase activity, presumably PP2A, was increased in S2. Therefore, pharmacokinetic and pharmacodynamic mechanisms can protect pituitary GH3 cells from apoptotic cell death by okadaic acid.

Topics: Animals; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; DNA Fragmentation; Drug Resistance, Multiple; Okadaic Acid; Pituitary Neoplasms; Rats; Tumor Cells, Cultured

Natriuretic peptides inhibit the release and action of many hormones through cyclic guanosine monophosphate (cGMP), but the mechanism of cGMP action is unclear. In frog ventricular muscle and guinea-pig hippocampal neurons, cGMP inhibits voltage-activated Ca2+ currents by stimulating phosphodiesterase activity and reducing intracellular cyclic AMP; however, this mechanism is not involved in the action of cGMP on other channels or on Ca2+ channels in other cells. Natriuretic peptide receptors in the rat pituitary also stimulate guanylyl cyclase activity but inhibit secretion by increasing membrane conductance to potassium. In an electrophysiological study on rat pituitary tumour cells, we identified the large-conductance, calcium- and voltage-activated potassium channels (BK) as the primary target of another inhibitory neuropeptide, somatostatin. Here we report that atrial natriuretic peptide also stimulates BK channel activity in GH4C1 cells through protein dephosphorylation. Unlike somatostatin, however, the effect of atrial natriuretic peptide on BK channel activity is preceded by a rapid and potent stimulation of cGMP production and requires cGMP-dependent protein kinase activity. Protein phosphatase activation by cGMP-dependent kinase could explain the inhibitory effects of natriuretic peptides on electrical excitability and the antagonism of cGMP and cAMP in many systems.

Topics: Animals; Atrial Natriuretic Factor; Charybdotoxin; Cyclic AMP; Cyclic GMP; Ethers, Cyclic; Membrane Potentials; Okadaic Acid; Phosphoprotein Phosphatases; Pituitary Neoplasms; Potassium Channels; Protein Kinases; Scorpion Venoms; Tetraethylammonium; Tetraethylammonium Compounds; Thionucleotides; Tumor Cells, Cultured

The properties of the calcium/calmodulin-dependent protein phosphatase calcineurin and its potential role in stimulus-secretion coupling were examined in AtT20 mouse pituitary corticotrope tumor cells. Protein phosphatase activity was assayed by measuring the liberation of 32P from 32P-casein, adrenocorticotropin secretion was measured by radioimmunoassay. About 60% of the total phosphatase activity was inhibited by 500 nM okadaic acid, suggesting the presence of protein phosphatases 1 and/or 2A. A further 25-30% reduction of phosphatase activity was achieved by chelating free calcium. Addition of the EF-hand protein blocker trifluoperazine or a calcineurin autoinhibitory peptide fragment markedly reduced okadaic acid resistant and calcium-dependent protein phosphatase activity indicating that calcium-dependent 32P release is largely due to calcineurin (protein phosphatase 2B). The remaining 10-15% of total activity was Mg2+ dependent and blocked by NaF, hence possibly due to protein phosphatase 2C. Calcineurin activity was inhibited by the immunosuppressants FK506 and cyclosporin A, either when added to the cell lysates or after preincubation of intact cells with the drugs for 30 min at 37 degrees C. When added to lysates, cyclosporin A inhibited calcium/calmodulin-dependent phosphatase more effectively than FK506. However, when tested on intact cells, FK506 proved 10-fold more potent than cyclosporin A. Both immunosuppressive agents enhanced the calcium-dependent release of adrenocorticotropic hormone into the medium, once more, FK506 was 10-fold more potent than cyclosporin A. Taken together, these data suggest that calcineurin is an inhibitory element in the signal transduction pathway controlling exocytotic secretion in pituitary cells that express voltage-operated calcium channels. This is in direct contrast with leukocytes where voltage-operated calcium channels are not found, and calcineurin is an important element for agonist-induced activation.

Topics: Adrenocorticotropic Hormone; Animals; Calcineurin; Calcium; Calmodulin-Binding Proteins; Cyclosporine; Ethers, Cyclic; Kinetics; Magnesium; Mice; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorus Radioisotopes; Pituitary Neoplasms; Tacrolimus; Trifluoperazine; Tumor Cells, Cultured