R J Flower

Glucocorticoids act within minutes to inhibit recruitment of signalling factors to activated EGF receptors through a receptor-dependent, transcription-independent mechanism.

Recruitment to activated tyrosine kinase growth factor receptors of Grb2 and p21ras leads to downstream activation of the kinases Raf, MAPK/Erk kinase (Mek) and, subsequently, extracellular signal‐regulated kinase (Erk). Activated Erk phosphorylates specific serine residues within cytosolic phospholipase A2 (PLA2), promoting enzyme translocation to membranes and facilitating liberation of arachidonic acid (AA). In the A549 human adenocarcinoma cell line dexamethasone inhibited epidermal growth factor (EGF)‐stimulated cytosolic PLA2 (cPLA2) activation and AA release by blocking the recruitment of Grb2 to the activated EGF receptor (EGF‐R) through a glucocorticoid receptor (GR)‐dependent (RU486‐sensitive), transcription‐independent (actinomycin‐insensitive), mechanism. The dexamethasone‐induced block of Grb2 recruitment was parallelled by changes in phosphorylation status and subcellular localization of lipocortin 1 (LC1) and an increase in the amount of the tyrosine phosphoprotein co‐localized with EGF‐R. Like dexamethasone, peptides containing E‐Q‐E‐Y‐V from the N‐terminal domain of LC1 also blocked ligand‐induced association of Grb2, p21ras and Raf. Our results point to an unsuspected rapid effect of glucocorticoids, mediated by occupation of GR but not by changes in gene transcription, which is brought about by competition between LC1 and Grb2 leading to a failure of recruitment off signalling factors to EGF‐R

Different glucocorticoids vary in their genomic and non‐genomic mechanism of action in A549 cells

We have examined the effects of 12 glucocorticoids as inhibitors of A549 cell growth. Other than cortisone and prednisone, all the glucocorticoids inhibited cell growth and this was strongly correlated (r=0.91) with inhibition of prostaglandin (PG)E2 formation. The molecular mechanism by which the active steroids prevented PGE2 synthesis was examined and three groups were identified. Group A drugs did not inhibit arachidonic acid release but inhibited the induction of COX2. Group B drugs were not able to inhibit the induction of COX2 but inhibited arachidonic acid release through suppression of cPLA2 activation. Group C drugs were apparently able to bring about both effects. The inhibitory actions of all steroids was dependent upon glucocorticoid receptor occupation since RU486 reversed their effects. However, group A acted through the NF‐κB pathway to inhibit COX2 as the response was blocked by the inhibitor geldanamycin which prevents dissociation of GR and the effect was blocked by APDC, the NF‐κB inhibitor. On the other hand, the group B drugs were not inhibited by NF‐κB inhibitors or geldanamycin but their effect was abolished by the src inhibitor PP2. Group C drugs depended on both pathways. In terms of PGE2 generation, there is clear evidence of two entirely separate mechanisms of glucocorticoid action, one of which correlates with NF‐κB mediated genomic actions whilst the other, depends upon rapid effects on a cell signalling system which does not require dissociation of GR. The implications for these findings are discussed.

Role of lipocortin-1 in the anti-hyperalgesic actions of dexamethasone

The effect of dexamethasone, lipocorton‐12–26 and an antiserum to lipocortin‐12–26 (LCPS1) upon the hyperalgesic activities in rats of carrageenin, bradykinin, tumour necrosis factor α (TNFα), interleukin‐12, interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), prostaglandin Eβ (PGE2) and dopamine were investigated in a model of mechanical hyperalgesia. Hyperalgesic responses to intraplantar (i.pl.) injections of carrageenin (100 μg), bradykinin (500 ng), TNFα (2.5 pg), IL‐1β (0.5 pg), and IL‐6 (1.0 ng), but not responses to IL‐8 (0.1 ng), PGE2 (100 ng) and dopamine (10 μg), were inhibited by pretreatment with dexamethasone (0.5 mg kg−1, subcutaneously, s.c., or 0.04–5.0 μg/paw). Inhibition of hyperalgesic responses to injections (i.pl.) of bradykinin (500 ng) and IL‐1β (0.5 pg) by dexamethasone (0.5 mg kg−1, s.c.) was reversed by LCPS1 (0.5 ml kg−1, injected s.c., 24 h and 1 h before hyperalgesic substances) and hyperalgesic responses to injections (i.pl.) of bradykinin (500 ng), TNFα (2.5 pg) and IL‐1β (0.5 pg), but not responses to PGE2 (100 ng), were inhibited by pretreatment with lipocortin‐12–26 (100 μg/paw). Also, lipocortin‐12–26 (30 and 100 μg ml−1) and dexamethasone (10 μg ml−1) inhibited TNFα release by cells of the J774 (murine macrophage‐like) cell‐line stimulated with LPS (3 μg ml−1), and LCPS1 partially reversed the inhibition by dexamethasone. These data are consistent with an important role for endogenous lipocortin‐12–26 in mediating the anti‐hyperalgesic effect of dexamethasone, with inhibiton of TNFα production by lipocortin‐12–26 contributing, in part, to this role. Although arachidonic acid by itself was not hyperalgesic, the hyperalgesic response to IL‐1β (0.25 pg, i.pl.) was potentiated by arachidonic acid (50 μg) and the potentiated response was inhibited by dexamethasone (50 μg, i.pl.) and lipocortin‐12–26 (100 μg, i.pl.). Also, lipocortin‐12–26 (30 and 100 μg ml−1) inhibited/abolished PGE2 release by J774 cells stimulated with LPS (3 μg ml−1). These data suggest that, in inflammatory hyperalgesia, inhibition of the induction of cyclo‐oxygenase 2 (COX‐2), rather than phospholipase A2, by dexamethasone and lipocortin‐12–26 accounts for the anti‐hyperalgesic effects of these agents. The above data support the notion that induction of lipocortin by dexamethasone plays a major role in the inhibition by dexamethasone of inflammatory hyperalgesia evoked by carrageenin, bradykinin and the cytokines TNFα, IL‐1β and IL‐6, and provides additional evidence that the biological activity of lipocortin resides within the peptide lipocortin‐12–26. Further, the data suggest that inhibition of lipocortin‐12–26 of eicosanoid production by COX‐2 also contributes to the anti‐hyperalgesic effect of lipocortin‐1.

Role of inducible nitric oxide synthase in the regulation of neutrophil migration in zymosan-induced inflammation.

In the present study, by comparing the responses in wild‐type mice and mice lacking the inducible (or type 2) nitric oxide synthase (iNOS), we investigated the role played by iNOS in the regulation of polymorphonuclear granulocyte (PMN) accumulation and chemokine production in the mouse peritoneal cavity in response to administration of zymosan (0·2 mg). Zymosan injection induced the production of nitric oxide, and triggered a time‐dependent PMN immigration into the peritoneal cavity. This response was associated with increases in the level of the chemokines macrophage inflammatory protein (MIP)‐1α, MIP‐2, monocyte chemo‐attractant protein (MCP)‐1 and cytokine‐induced neutrophil chemo‐attractant (KC), as measured in the peritoneal cavities. Injection of zymosan also induced a time‐dependent increase in the production of the anti‐inflammatory cytokine interleukin‐10 (IL‐10) in the peritoneal cavity. When comparing the response between wild‐type and iNOS knockout (KO) mice, we observed that the low‐level PMN accumulation measured at 1 hr was slightly but significantly increased in the absence of functional iNOS. On the other hand, the delayed response (2–4 hr after zymosan) of PMN accumulation was suppressed in the iNOS KO mice. The early enhancement of PMN infiltration in the iNOS‐deficient mice was associated with increased peritoneal levels of MIP‐2, KC and IL‐10 proteins. The delayed suppression of PMN infiltration was associated with reduced MIP‐2 and IL‐10 levels in the peritoneal cavity. The lack of iNOS did not affect the release of MIP‐1α and MCP‐1 at any of the time‐points studied. The current data demonstrate that iNOS regulates the production of certain CXC (but not CC) proinflammatory chemokines, the production of IL‐10 and exerts a biphasic regulatory effect on PMN accumulation in zymosan‐induced acute inflammation.

Post-translational modification plays an essential role in the translocation of annexin A1 from the cytoplasm to the cell surface

Annexin A1 (ANXA1) has an important role in cell‐cell communication in the host defense and neuroendocrine systems. In both systems, its actions are exerted extracellularly via membrane‐bound receptors on adjacent sites after translocation of the protein from the cytoplasm to the cell surface of adjacent cells. This study used molecular, microscopic, and pharmacological approaches to explore the mechanisms underlying the cellular exportation of ANXA1 in TtT/GF (pituitary folliculo‐stellate) cells. LPS caused serine‐phosphorylation of ANXA1 (ANXA1‐S27‐PO4) and translocation of the phosphorylated protein to the cell membrane. The fundamental requirement of phosphorylation for membrane translocation was confirmed by immunofluorescence microscopy on cells transfected with wild‐type or mutated (S27/A) ANXA1 constructs tagged with enhanced green fluorescence protein. The trafficking of ANXA1‐S27‐PO4 to the cell surface was dependent on PI3‐kinase and MAP‐kinase. It also required HMG‐coenzyme A and myristoylation. The effects of HMG‐coenzyme A blockade were overcome by mevalonic acid (the product of HMG‐coen‐zyme A) and farnesyl‐pyrophosphate but not by geranyl‐geranylpyrophosphate or cholesterol. Together, these results suggest that serine‐27 phosphorylation is essential for the translocation of ANXA1 across the cell membrane and also identify a role for isoprenyl lipids. Such lipids could target consensus sequences in ANXA1. Alternatively, they may target other proteins in the signal transduction cascade (e.g., transporters).—Solito, E., Christian, H. C., Festa, M., Mulla, A., Tierney, T., Flower, R. J., Buckingham, J. C. Post‐transla‐tional modification plays an essential role in the translocation of annexin A1 from the cytoplasm to the cell surface. FASEB J. 20, E677–E687 (2006)

Anti-inflammatory actions of an N-terminal peptide from human lipocortin 1

An acetylated polypeptide corresponding to residues 2–26 of human lipocortin 1 was synthesized and the anti‐inflammatory activity assessed in three models of acute inflammation in rat and mouse. In the carrageenin rat paw oedema test, the peptide produced a maximal inhibition of approximately 41% at the 3 h time point with a 10 μg dose. When rat paw oedema was induced by the injection of venom phospholipase A2, the peptide produced a significant inhibition (31%) at the top dose of 20 μg per paw. In the mouse air‐pouch model, systemic treatment with the peptide produced a dramatic reduction in cytokine‐induced leukocyte migration with an ID50 of approximately 40 μg per mouse. The N‐terminal peptide 2–26 shares the actions of lipocortin 1 in these acute models of inflammation.

Tamoxifen inhibits growth of oestrogen receptor-negative A549 cells

The non-steroidal anti-oestrogen tamoxifen inhibits proliferation of the A549 human lung adenocarcinoma cell line (EC50 congruent to 10 nM) yet there was no evidence of oestrogen receptor expression as determined by ligand binding assay and northern blotting. 17-beta-Oestradiol had no effect on A549 cell proliferation (1 pM-1 microM) and moreover a 100-fold excess failed to reverse the effect of 10 nM tamoxifen as did a 100-fold excess of the steroidal anti-oestrogens ICI 164384 and ICI 182780. However, 4-hydroxytamoxifen which had no significant effect on A549 cell growth (1 pM-1 microM) completely antagonized the effect of 10 nM tamoxifen when used at a 100-fold excess. In the presence of oleic acid and stearic acid (10 microM) the growth inhibitory effect of tamoxifen in A549 cells was greatly enhanced, unlike effects mediated by the anti-oestrogen binding protein described in other cells where these fatty acids had no effect. These results indicate the presence of a unique and highly sensitive mechanism in A549 cells whereby concentrations of tamoxifen relevant to classical receptor binding can inhibit cell growth in the absence of the oestrogen receptor.

Lipocortin 1 and the control of cPLA2 activity in A549 cells. Glucocorticoids block EGF stimulation of cPLA2 phosphorylation.

Epidermal growth factor (EGF) rapidly stimulates the release of arachidonic acid in A549 cells by a mechanism that is sensitive to pertussis toxin [1]. We show that EGF treatment of A549 cells stimulates phosphorylation of cytosolic phospholipase A2 (cPLA2) through a mechanism that is similarly inhibited by pertussis toxin. The level of cPLA2 expression is, apparently, not changed during this period. Pretreatment of cells with dexamethasone (10-100 nM) for 3 hr prevents this activation of cPLA2 by EFG, without changing the level of cPLA21 expression. The effect of dexamethasone is reversed in the presence of the neutralizing antilipocortin Mab 1A but not by the nonneutralizing antilipocortin 1 control Mab 1B. This strongly suggests that lipocortin 1 mediates the effect of dexamethasone by inhibiting activation of cPLA2. This concept is supported by the fact that a peptide Lc13-25 (10-200 micrograms/mL), derived from the N-terminus of lipocortin 1, also inhibits activation of cPLA2 by EGF in these cells.

Attenuation of glucocorticoid functions in an Anx-A1-/- cell line.

The Ca(2+)- and phospholipid-binding protein Anx-A1 (annexin 1; lipocortin 1) has been described both as an inhibitor of phospholipase A(2) (PLA(2)) activity and as a mediator of glucocorticoid-regulated cell growth and eicosanoid generation. Here we show that, when compared with Anx-A1(+/+) cells, lung fibroblast cell lines derived from the Anx-A1(-/-) mouse exhibit an altered morphology characterized by a spindle-shaped appearance and an accumulation of intracellular organelles. Unlike their wild-type counterparts, Anx-A1(-/-) cells also overexpress cyclo-oxygenase 2 (COX 2), cytosolic PLA(2) and secretory PLA(2) and in response to fetal calf serum, exhibit an exaggerated release of eicosanoids, which is insensitive to dexamethasone (10(-8)- 10(-6) M) inhibition. Proliferation and serum-induced progression of Anx-A1(+/+) cells from G(0)/G(1) into S phase, and the associated expression of extracellular signal-regulated kinase 2 (ERK2), cyclin-dependent kinase 4 (cdk4) and COX 2, is strongly inhibited by dexamethasone, whereas Anx-A1(-/-) cells are refractory to the drug. Loss of the response to dexamethasone in Anx-A1(-/-) cells occurs against a background of no apparent change in glucocorticoid receptor expression or sensitivity to non-steroidal anti-inflammatory drugs. Taken together, these observations suggest strongly that Anx-A1 functions as an inhibitor of signal-transduction pathways that lead to cell proliferation and may help to explain how glucocorticoids regulate these processes.

Dexamethasone inhibition of leucocyte adhesion to rat mesenteric postcapillary venules: role of intercellular adhesion molecule 1 and KC

BACKGROUND A previous study showed that the glucocorticoid dexamethasone, at doses of 100 μg/kg and above, inhibited leucocyte adhesion to rat mesenteric postcapillary venules activated with interleukin 1β (IL-1β), as assessed by videomicroscopy. AIMS To identify whether the adhesion molecule, intercellular adhesion molecule 1 (ICAM-1), or the chemokine KC could be targeted by the steroid to mediate its antiadhesive effect. METHODS Rat mesenteries were treated with IL-1β (20 ng intraperitoneally) and the extent of leucocyte adhesion measured at two and four hours using intravital microscopy. Rats were treated with dexamethasone, and passively immunised against ICAM-1 or KC. Endogenous expression of these two mediators was validated by immunohistochemistry, ELISA, and the injection of specific radiolabelled antibodies. RESULTS Dexamethasone greatly reduced IL-1β induced leucocyte adhesion, endothelial expression of ICAM-1 in the postcapillary venule, and release of the mast cell derived chemokine KC. Injection of specific antibodies to the latter mediators was also extremely effective in downregulating (>80%) IL-1β induced leucocyte adhesion. CONCLUSIONS Induction by IL-1β of endogenous ICAM-1 and KC contributes to leucocyte adhesion to inflamed mesenteric vessels. Without excluding other possible mediators, these data clearly show that dexamethasone interferes with ICAM-1 expression and KC release from mast cells, resulting in suppression of leucocyte accumulation in the bowel wall, which is a prominent feature of several gastrointestinal pathologies.