Interleukin 1 induces early protein phosphorylation and requires only a short exposure for late induced secretion of beta-endorphin in a mouse pituitary cell line.

Abstract

Previous work has shown that prolonged pretreatment of a mouse anterior pituitary cell line, AtT-20 cells, with the cytokine interleukin 1 (IL-1) stimulates /3-endorphin release and potentiates the secretion induced by many secretagogues. Prolonged treatment with IL-1 abolishes the capacity of cytokine to induce the phosphorylation of 20-and 60-kDa proteins.The presence of IL-1 was required initially only for a short time to induce late secretion in AtT-20 cells. These observations indicate that once IL-1 generates an early signal, its presence is no longer necessary for the subsequent secretion of (3-endorphin. by IL-1 (1). These observations suggested that the mechanism by which IL-1 enhanced TPA-induced,-endorphin secretion was mediated by increasing PKC activity. This report shows that IL-1 induces an early phosphorylation of three proteins, independent of PKC and cAMPdependent kinase. Once an early signal is triggered by IL-1, its presence is no longer required for subsequent B-endorphin secretion. MATERIALS AND METHODSCell Culture. AtT-20/D16-16 mouse anterior pituitary tumor cells, obtained from S. Sabol (National Heart, Lung and Blood Institute, Bethesda, MD), were grown in Dulbecco's modified Eagle medium (DMEM) containing 4.5 g of glucose per liter, 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 ,g/ml). The cells were maintained in a humidified atmosphere of 10% CO2 at 37°C. For /-endorphin release experiments the cells were subcultured in 24-well Costar plates at an initial density of 105 cells per well and were used 5-7 days later (80-90% confluency). For phosphorylation experiments, the cells were subcultured in 6-well Costar plates at an initial density of 2 x 105 cells per well and were grown to -75% confluency. CRF and TPA in 100-fold stock solution were diluted to final concentrations of 0.1 ,uM in the culture wells. IL-la final concentration in all experiments was 1 nM.Phosphorylation Experiments. AtT-20 cells were preincubated for 15 min in phosphate-free DMEM and then labeled for 45 min with 100 ,uCi of [32P]orthophosphate per ml (1 Ci = 37 GBq) in phosphate-free DMEM or phosphate-free Krebs-Ringer buffer. Secretagogue solutions were added and, after incubation periods ranging from 5 to 120 min, the cells were dissolved in a buffer containing 50 mM Tris phosphate, 100 mM NaF, 10 mM EDTA, 5 mM EGTA, 4 mg of leupeptin per liter, and 1% (vol/vol) Triton X-100 (pH 7.4). Cytosolic fractions were obtained by centrifugation of cell homogenates (15,000 rpm, 5 min) followed by collection of the supernatants. After precipitation ofproteins with ice-cold 6% trichloroacetic acid (TCA) and a second centrifugation step (15,000 rpm, 5 min), the resulting pellets were homogenized twice in 50% ethanol/50% diethyl ether using a sonicator, which was followed each time by centrifugation (15,000 rpm, 5 min). Pellets were then resuspended in phosphate-buffered saline, aliquots were removed for determination of protein concentration, and the remainder of the samples was lyophilized...

Nur77 induction and activation are necessary for interleukin‐1 stimulation of proopiomelanocortin in AtT‐20 corticotrophs

“…In concordance, several phosphorylation sites, present in the Nur77 protein, may be the target for p38 regulation. IL‐1 treatment of AtT‐20 cells stimulates the phosphorylation of a protein with the same molecular weight than Nur77 [32]. Further studies are required to identify whether p38 modulation is exerted by direct phosphorylation of Nur77 as is the direct phosphorylation of Nur77 by extracellular signal‐regulated kinase (ERK)2 [27].…”

Investigation of guanine-nucleotide-binding protein involvement and regulation of cyclic AMP metabolism in interleukin 1 signal transduction

“…However, in some of these reports changes were marginal, and in other reports no direct effects of ILl on cAMP synthesis were observed in T-cells (Didier et al, 1988), osteosarcoma cells (Rodan et al, 1990), pituitary tumour cells (Fagarasan et al, 1990) or astrocytoma cells (Kasahara et al, 1990). Still further reports have shown that ILI could elevate intracellular cyclic AMP levels, but that this effect was poorly reproducible (Bomsztyk et al, 1990).…”

“…Despite the finding that ILI stimulation induces production of diacylglycerol, evidence for a concomitant ILl-induced activation of protein kinase C is controversial (Abraham et al, 1987;Ostrowski et al, 1988;Munoz et al, 1990 lation has been reported to increase intracellular cAMP acutely in several cell types (Shirakawa et al, 1988;Zhang et al, 1988;Bomsztyk et al, 1990;Munoz et al, 1990) and to stimulate adenylate cyclase activity in membrane preparations (Shirakawa et al, 1988;Chedid et al, 1989). Others, however, have failed to observe cAMP responses to ILl (Didier et al, 1988;Shiroo & Matsushima, 1990;Fagarasan et al, 1990;Kasahara et al, 1990;Rodan et al, 1990), and in T-cells elevation of cAMP concentration by pharmacological agents was found to inhibit the ability of ILI to induce interleukin 2 (IL2) secretion (Rollins et al, 1991).…”

Phosphorylation pattern of liver proteins during the early stages of the acute‐phase response.

“…In the present study we have addressed the problem of the nature of the intracellular signals that are activated by the inflammatory mediators external to the liver cell. Recent 0309-1651/93/040425--08/$08.00/0 reports indicate that IL-l or TNF can induce in their target cells the phosphorylation of specific proteins (Bird and Saklatvala, 1989;Fagarasan et al, 1990): therefore we have analyzed the pattern of liver phosphoproteins during the early stages of acute-phase response induced in the rat by a subcutaneous injection of turpentine.…”