Granulocyte-macrophage Colony-stimulating Factor mRNA Stability Research Articles

Contrasting effects of hyperoxia on GM-CSF gene transcription in alveolar epithelial cells and T cells.

Granulocyte/macrophage colony-stimulating factor (GM-CSF) is critically important for normal pulmonary innate immunity and for functional maturation of alveolar macrophages. Alveolar epithelial cells (AEC) are a major source of GM-CSF in the lung and express this growth factor constitutively, whereas most other cells, including T cells, express GM-CSF following inflammatory stimulation. AEC expression of GM-CSF is suppressed by oxidative stress, at least in part through induction of microRNA leading to increased mRNA turnover. In this report, we compare and contrast the effect of hyperoxia on transcriptional aspects of gene regulation of GM-CSF in lung epithelia and T cells of human and mouse origin. Similar to primary murine AEC, human H820 cells that express multiple characteristics of normal alveolar epithelial cells express GM-CSF constitutively, with decreased expression and increased mRNA turnover following exposure to hyperoxia. In contrast, hyperoxia induces augmented GM-CSF expression in human and murine activated T cells, in association with enhanced GM-CSF mRNA stability. Alveolar epithelial cells demonstrate constitutive transcription, with the proximal promoter in an open configuration in normoxia, without change in hyperoxia. Conversely, in both human and murine T cells, hyperoxia increased GM-CSF gene transcription. The proximal promoter was in a closed configuration in unstimulated T cells but became accessible upon activation and still more accessible in activated T cells exposed to hyperoxia. These fundamental differences in molecular regulation of GM-CSF expression highlight the distinctive niche of alveolar epithelial cell expression of GM-CSF and offer insights into the biology of GM-CSF in the setting of acute lung injury.

The Peptidyl-Prolyl Isomerase Pin1 Regulates Granulocyte-Macrophage Colony-Stimulating Factor mRNA Stability in T Lymphocytes

Cytokine production is associated with both the normal and pathologic inflammatory response to injury. Previous studies have shown that the immunosuppressants cyclosporin A or FK506, which interact with the peptidyl-propyl isomerases cyclophilin A and FK506-binding protein (FKBP12), respectively, block cytokine expression. A third member of the peptidyl-propyl isomerase family, Pin1 is expressed by immune and other cells. Pin1 has been implicated in cell cycle progression, is overexpressed in human tumors, and may rescue neurons from tau-associated degeneration. However, the role of Pin1 in the immune system remains largely unknown. In this study, we analyze the role of Pin1 in GM-CSF expression by human PBMC and CD4+ lymphocytes. We show that Pin1 isomerase activity is necessary for activation-dependent, GM-CSF mRNA stabilization, accumulation, and protein secretion, but not non-AU-rich elements containing cytokine mRNAs, including TGF-beta and IL-4. Mechanistically, Pin1 mediated the association of the AU-rich element-binding protein, AUF1, with GM-CSF mRNA, which determined the rate of decay by the exosome.

Pin1 in a mRNA Stability Complex

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that promotes eosinophil survival, maturation, and function. Factors, such as hyaluronic acid, that stimulate production of this cytokine are increased in asthmatic lung, as is the abundance of eosinophils. Eosinophils from asthmatic patients also exhibit elevated GM-CSF. Shen et al. show that Pin1, a peptidyl-prolyl isomerase specific for bonds between phosphorylated Ser-Pro or phosphorylated Thr-Pro, is part of a ribonucleoprotein complex that regulates the stability of GM-CSF mRNA. Pharmacological inhibition of Pin1 with juglone prevented increased mRNA abundance in and secretion of GM-CSF from isolated eosinophils in response to hyaluronic acid. The increase in GM-CSF mRNA was due to increased stability, not increased transcription. Pin1 coimmunoprecipitated with all four subunits of the AU-rich binding protein AUF1 (p45, p42, p40, and p37), of which p45 and p40 each have a potential Pin1 isomerization site. Hyaluronic acid triggered the dissociation of AUF1 from GM-CSF mRNA and the association of hnRNP C with GM-CSF mRNA, but only under conditions in which Pin1 was active. Hyaluronic acid stimulated the dephosphorylation of Pin1, which increased the activity of Pin1, and stimulated the phosphorylation of AUF1. In resting cells, the p37 subunit of AUF1 and Pin1 immunoprecipitated with the exosome-associated protein PM-Scl175, and hyaluronic acid decreased the association of p37 with the exosome. (The exosome is a complex associated with AU-rich mRNA decay.) Juglone blocked the prolonged survival observed for eosinophils isolated from allergen-challenged allergic donor patients, verifying the importance of Pin1 in vivo. The model for Pin1 in mRNA stability is as follows: In resting cells, Pin1 is inactive and the AUF1 complex recruits the exosome to decrease GM-CSF mRNA stability (see Anderson). Upon stimulation, Pin1 becomes dephosphorylated and AUF1 subunits are phosphorylated, leading to the release of the AUF1 complex from the mRNA and the binding of the hnRNP C, which promotes mRNA stability and allows increased production of the GM-CSF protein. Z.-J. Shen, S. Esnault, J. S. Malter, The peptidyl-prolyl isomerase Pin1 regulates the stability of granulocyte-macrophage colony-stimulating factor mRNA in activated eosinophils. Nat. Immunol. 6 , 1280-1287 (2005). [PubMed] P. Anderson, Pin1: A proline isomerase that makes you wheeze? Nat. Immunol. 6 , 1211-1212 (2005). [Online Journal]

Stimulus‐dependent glucocorticoid‐resistance of GM‐CSF production in human cultured airway smooth muscle

For a subpopulation of asthmatics, symptoms persist even with high doses of glucocorticoids. Glucocorticoids reduce the levels of the proinflammatory and fibrogenic cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by human cultured airway smooth muscle (ASM). We have contrasted the effects of a synthetic glucocorticoid, dexamethasone, on thrombin- and IL-1alpha-stimulated GM-CSF production in human ASM cells. Although IL-1alpha stimulated three-fold higher levels of GM-CSF mRNA and protein compared to thrombin, dexamethasone concentration-dependently reduced IL-1alpha-stimulated GM-CSF more potently and to a greater extent than the response to thrombin. This pattern of glucocorticoid regulation was also observed at the GM-CSF mRNA level and was reproduced with other glucocorticoids such as fluticasone propionate. IL-1alpha and thrombin stimulated NF-kappa B-dependent luciferase expression equally. Dexamethasone treatment reduced luciferase expression stimulated by both IL-1alpha and thrombin. The GM-CSF mRNA half life was markedly prolonged by IL-1alpha compared to thrombin. This IL-1alpha-induced GM-CSF mRNA stability was prevented by either dexamethasone or the p38(MAPK) inhibitor, SB203580, neither of which influenced GM-CSF mRNA stability in thrombin-treated cells. Dexamethasone inhibited p38(MAPK) phosphorylation in IL-1alpha-stimulated ASM, whereas thrombin does not stimulate p38(MAPK) phosphorylation. These data suggest that the mechanism underlying the greater potency and efficacy of glucocorticoids in reducing GM-CSF synthesis stimulated by IL-1alpha depends on inhibition of the involvement of p38(MAPK)-induced increases in GM-CSF message stability.

17beta-estradiol enhances the production of granulocyte-macrophage colony-stimulating factor in human keratinocytes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is effective for impaired wound repair. Estrogen is known to enhance wound repair. We examined if 17beta-estradiol (E2) may in vitro enhance GM-CSF production in human keratinocytes. E2 and membrane-impermeable bovine serum albumin-conjugated E2 increased GM-CSF secretion, mRNA stability, and promoter activity. The element homologous to activator protein-1 (AP-1) on the promoter was responsible for the activation. E2 enhanced transcriptional activity and DNA binding of AP-1. E2 transiently generated c-Fos protein, and shifted AP-1 composition from c-Jun homodimers to c-Fos/c-Jun heterodimers in keratinocytes. E2-induced enhancement of GM-CSF secretion, mRNA stability, and promoter activity were not suppressed by estrogen receptor antagonist ICI 182,780, however, suppressed by conventional protein kinase C inhibitor Gö6976 and PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK). Gö6976 and PD98059 suppressed E2-induced c-Fos expression and enhancement of DNA-binding and transcriptional activity at AP-1. E2 induced membrane translocation of protein kinase Calpha, which was suppressed by phosphatidylinositol (PI)-specific phospholipase C (PLC) inhibitor U73122. E2 stimulated the phosphorylation of extracellular signal-regulated kinase (ERK), which was suppressed by PD98059, Gö6976, and U73122. E2 transiently generated inositol 1,4,5-triphosphate in keratinocytes, which was suppressed by U73122 and guanine nucleotide-binding protein inhibitor. These results suggest that E2 may enhance GM-CSF production via guanine nucleotide-binding protein-coupled membrane receptors and signaling cascade of PI-specific PLC/protein kinase Calpha/MEK/ERK.

Histamine Enhances the Production of Granulocyte-Macrophage Colony-Stimulating Factor via Protein Kinase Cα and Extracellular Signal-Regulated Kinase in Human Keratinocytes

The production of granulocyte-macrophage colony-stimulating factor (GM-CSF) in keratinocytes is related to the chronicity of atopic dermatitis. Mast cell-derived histamine contributes to the cross-talk between mast cells and keratinocytes. We examined the effects of histamine on GM-CSF production in human keratinocytes. Histamine increased GM-CSF secretion, mRNA stability and promoter activity. Activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) elements on the promoter were responsible for the activation by histamine. Histamine enhanced transcriptional activity and DNA binding of AP-1 and NF-kappaB. Histamine shifted AP-1 composition form c-Jun homodimers to c-Fos/c-Jun heterodimers, and transiently expressed c-Fos protein. Histamine rapidly induced the phosphorylation and degradation of inhibitory kappaB. Histamine induced membrane translocation of protein kinase Calpha. Histamine-induced GM-CSF production was completely abolished by H1 antagonist pyrilamine and conventional protein kinase C inhibitor Gö6976, and partially suppressed by PD98059 which inhibits the activation of extracellular signal-regulated kinase. Gö6976 and PD98059 suppressed histamine-induced c-Fos expression and AP-1 activation. Gö6976 and PD98059 suppressed histamine-induced enhancement of NF-kappaB transcriptional activity. Histamine-induced phosphorylation and degradation of inhibitory kappaB was suppressed by Gö6976, but not by PD98059. These results suggest that histamine may enhance GM-CSF production at transcriptional and posttranscriptional levels via H1 receptor, protein kinase Calpha and extracellular signal-regulated kinase.

Extracellular signal-regulated kinase mediates granulocyte-macrophage colony-stimulating factor messenger RNA stabilization in tumor necrosis factor-α plus fibronectin-activated peripheral blood eosinophils

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is critical for promoting the long-term survival of lung- or airway-based eosinophils. Previously, we have shown that fibronectin and tumor necrosis factor alpha induced autocrine production of GM-CSF that markedly enhanced eosinophil survival. Cytokine release was preceded by and dependent on messenger RNA (mRNA) stabilization. Here, we show that mitogen-activated protein kinase (MAPK) activation is responsible for GM-CSF mRNA stabilization in peripheral blood eosinophils (pbeos). Activation of extracellular signal-regulated kinase (ERK) but not p38 correlated with GM-CSF mRNA stability. Although ERK inhibition completely prevented GM-CSF mRNA stabilization, p38 inhibition had a partial effect. To establish which MAPK was crucial, we transduced pbeos with dominant-active TatMEK1(E) or TatMKK3b(E) proteins that selectively phosphorylate ERK or p38, respectively. These studies showed that ERK but not p38 was sufficient for GM-CSF mRNA stabilization. These data are in contradistinction to the c-Jun NH(2)-terminal kinase-mediated regulation of interleukin 2 and 3 mRNAs and suggest unique regulatory features for GM-CSF mRNA in eosinophils.

Roles of tumor necrosis factor-α receptor subtypes in the pathogenesis of the tristetraprolin-deficiency syndrome

AbstractTristetraprolin (TTP) is a member of the CCCH tandem zinc-finger class of proteins. It can bind to and destabilize mRNAs encoding tumor necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Conversely, mice deficient in TTP develop a complex syndrome characterized by cachexia, myeloid hyperplasia, and joint and skin inflammation. Studies using anti–TNF-α neutralizing antibodies demonstrated that this syndrome, at least in part, is a consequence of the excess production of TNF-α in the absence of TTP. To evaluate the role played by each TNF-α receptor in the pathogenesis of this syndrome, mice were generated that were deficient in TTP and either or both of the known TNF-α receptors (TNFRs), type 1 (TNFR1) and type 2 (TNFR2). Mice deficient in TTP and TNFR1, or in TTP and both receptors, were protected from developing the TNF-α–induced cachexia and inflammation. In contrast, mice deficient in TNFR2 were more severely affected than mice deficient in TTP alone, suggesting that TNFR2 might play a protective role in the development of the syndrome. In cultured cells derived from these mice, apparent cooperation between the TNFRs was required to achieve normal TNF-α–induced expression of TTP, TNF-α, and GM-CSF mRNAs. Finally, the results showed that TNFR1 plays an important role in mediating TNF-α–induced changes in TNF-α and GM-CSF mRNA stability.

Nitric oxide inhibits rhinovirus-induced granulocyte macrophage colony-stimulating factor production in bronchial epithelial cells.

Infection of asthmatics with human rhinovirus (HRV) enhances airway eosinophilia and airways hyperreactivity. The current studies were performed to further characterize HRV-induced generation by human bronchial epithelial cells of granulocyte macrophage colony-stimulating factor (GM-CSF), a cytokine that could contribute to airway eosinophilia by increasing the survival and activation of eosinophils, and to determine the effects of the antiviral agent nitric oxide (NO) on HRV-induced GM-CSF production. Maximal levels of messenger RNA (mRNA) for GM-CSF were seen 1 h after HRV infection. Expression was sustained through 24 h and declined by 48 h. GM-CSF protein was detected in cell supernatants by 2 h after infection and reached maximal concentrations by 24 h, with the most rapid rate of production occurring from 2 to 7 h. The NO donor 3-(2-hydroxy-2-nitroso-1-propyl-hydrazino)-1-propanamine (NONOate) inhibited HRV-induced GM-CSF protein production in a time- and dose-dependent fashion. NONOate also inhibited HRV-induced GM-CSF mRNA levels at both times (1 and 4 h) examined. NONOate increased GM-CSF mRNA stability, suggesting that reduced mRNA levels were due to inhibition of transcription. The transcription factor nuclear factor-kappa B was rapidly induced by HRV infection, but was not inhibited by NONOate, implying a role for other transcription factors. Thus, NO may play an important anti-inflammatory role in virally induced exacerbations of diseases such as asthma.

Tristetraprolin is a regulator of granulocyte-macrophage colony-stimulating factor mRNA stability

Tristetraprolin (TTP) deficient mice exhibit a complex syndrome characterized by myeloid hyperplasia with extramedullary hematopoiesis, inflammation of skin and joints and cachexia. Macrophages derived from TTP-deficient mice accumulated twice as much TNFα mRNA and secreted 5-7 times more protein than control cells, and TNFα mRNA in these cells had an increased half-life (85 min in TTP-deficient cells versus 39 min in control cells). In transfection experiments, TTP can destabilize mRNAs containing the AU-rich elements from the 3′ untranslated region of both TNFα and GM-CSF mRNAs. To determine whether TTP was acting as a regulator of GM-CSF mRNA stability, we used primary cultures of bone marrow stromal cells (BMSC). In the absence of TTP, BMSC secreted 5-6 times more GM-CSF than control cells, and the steady-state levels of GM-CSF mRNA were also increased, due to the increased half-life of the message. Furthermore, we found that in the absence of TTP, the typical pattern of GM-CSF mRNA (two equally represented species of 0.8 and 1 kb) was changed, so that there was a clear predominance of the 1 kb species; this was found to be the fully polyadenylated form, whereas the 0.8 kb form appeared to be deadenylated. Thus, TTP-deficiency results in the increased accumulation of the fully polyadenylated form of GM-CSF mRNA. These date implicate TTP as a normal physiological regulator of GM-CSF mRNA stability and adenylation, and GM-CSF secretion. They also describe a novel biochemical target for the development of drugs to combat neutropenic conditions.

Developmental Regulation of RNA Transcript Destabilization by A + U-rich Elements is AUF1-dependent

The developmental immaturity of neonatal phagocytic function is associated with decreased accumulation and half-life (t((1)/(2))) of granulocyte/macrophage colony-stimulating factor (GM-CSF) mRNA in mononuclear cells (MNC) from the neonatal umbilical cord compared with adult peripheral blood. The in vivo t((1)/(2)) of GM-CSF mRNA is 3-fold shorter in neonatal (30 min) than in adult (100 min) MNC. Turnover of mRNA containing a 3'-untranslated region (3'-UTR) A + U-rich element (ARE), which regulates GM-CSF mRNA stability, is accelerated in vitro by protein fractions enriched for AUF1, an ARE-specific binding factor. The data reported here demonstrate that the ARE significantly accelerates in vitro decay of the GM-CSF 3'-UTR in the presence of either neonatal or adult MNC protein. Decay intermediates of the GM-CSF 3'-UTR are generated that are truncated at either end of the ARE. Furthermore, the t((1)/(2)) of the ARE-containing 3'-UTR is 4-fold shorter in the presence of neonatal (19 min) than adult (79 min) MNC protein, reconstituting developmental regulation in a cell-free system. Finally, accelerated ARE-dependent decay of the GM-CSF 3'-UTR in vitro by neonatal MNC protein is significantly attenuated by immunodepletion of AUF1, providing new evidence that this accelerated turnover is ARE- and AUF1-dependent.

Modulation of granulocyte-macrophage colony-stimulating factor mRNA stability in vitro by the adenosine-uridine binding factor.

In vitro decay of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA was examined on polysomes prepared from normal human peripheral blood mononuclear cells stimulated with phorbol ester (TPA) and phytohemagglutinin for 14 h. GM-CSF mRNA decayed with a half-life of 90 min while 18 S rRNA was stable. RNA gel mobility assay performed on crude cytosolic lysate (S20) with radiolabeled AUUUA containing RNA identified a 42-kDa RNA-protein complex on SDS-polyacrylamide gel electrophoresis. The binding specificity was identical to that of the previously described adenosine-uridine binding factor (AUBF) (Malter, J. S. (1989) Science 246, 664-666). Further fractionation of the S20 cytosol through a sucrose gradient showed > 90% of AUBF activity associated with polysomes and < 10% with the S130 fraction. Solution phase RNAs containing AUUUA reiterations specifically competed for polysome-bound AUBF and accelerated the decay of GM-CSF mRNA (t1/2 = 17 min). We linked biotinylated AUUUA RNA to streptavidin magnetic beads and removed > 95% of polysome-associated AUBF. A decay system thus depleted of AUBF activity also showed accelerated decay of GM-CSF mRNA (t1/2 = 20 min). These data show that AUBF is preferentially located on polysomes and that its removal destabilizes GM-CSF mRNA. Therefore, AUBF likely prevents GM-CSF mRNA decay by binding to the AUUUA instability determinants in the 3'-untranslated region.