Glucocorticoid upregulation of the annexin-A1 receptor in leukocytes

We tested here whether glucocorticoids modulated myeloid cell expression of a specific G-coupled receptor, termed formyl-peptide receptor like-1 (FPRL-1), recently shown to mediate the anti-inflammatory actions of annexin-A1. Real-time PCR and flow cytometry demonstrated rapid up-regulation of mRNA followed by the protein in HL-60 cells incubated with dexamethasone, with peaks at 2 and 24h, respectively. This effect was not restricted to dexamethasone, since reproduced by glucocorticoids. In addition, it was not restricted to the cell line, since replicated with human peripheral blood monocytes. Glucocorticoid ability to upregulate cell surface expression of FPRL-1 was specific, since no effects upon the related receptor FPR or the integrin CD11b could be detected. In view of the wide range of endogenous ligands known to interact with FPRL-1, including the anti-inflammatory protein annexin-A1, we speculate that the novel effect here described may impact on the clinical immunosuppressive and anti-inflammatory properties of glucocorticoids.     

Inflammation and Cancer: Role of Annexin A1 and FPR2/ALX in Proliferation and Metastasis in Human Laryngeal Squamous Cell Carcinoma

The anti-inflammatory protein annexin A1 (ANXA1) has been associated with cancer progression and metastasis, suggesting its role in regulating tumor cell proliferation. We investigated the mechanism of ANXA1 interaction with formylated peptide receptor 2 (FPR2/ALX) in control, peritumoral and tumor larynx tissue samples from 20 patients, to quantitate the neutrophils and mast cells, and to evaluate the protein expression and co-localization of ANXA1/FPR2 in these inflammatory cells and laryngeal squamous cells by immunocytochemistry. In addition, we performed in vitro experiments to further investigate the functional role of ANXA1/FPR2 in the proliferation and metastasis of Hep-2 cells, a cell line from larynx epidermoid carcinoma, after treatment with ANXA1_2–26 (annexin A1 N-terminal-derived peptide), Boc2 (antagonist of FPR) and/or dexamethasone. Under these treatments, the level of Hep-2 cell proliferation, pro-inflammatory cytokines, ANXA1/FPR2 co-localization, and the prostaglandin signalling were analyzed using ELISA, immunocytochemistry and real-time PCR. An influx of neutrophils and degranulated mast cells was detected in tumor samples. In these inflammatory cells of peritumoral and tumor samples, ANXA1/FPR2 expression was markedly exacerbated, however, in laryngeal carcinoma cells, this expression was down-regulated. ANXA1_2–26 treatment reduced the proliferation of the Hep-2 cells, an effect that was blocked by Boc2, and up-regulated ANXA1/FPR2 expression. ANXA1_2–26 treatment also reduced the levels of pro-inflammatory cytokines and affected the expression of metalloproteinases and EP receptors, which are involved in the prostaglandin signalling. Overall, this study identified potential roles for the molecular mechanism of the ANXA1/FPR2 interaction in laryngeal cancer, including its relationship with the prostaglandin pathway, providing promising starting points for future research. ANXA1 may contribute to the regulation of tumor growth and metastasis through paracrine mechanisms that are mediated by FPR2/ALX. These data may lead to new biological targets for therapeutic intervention in human laryngeal cancer.     

An annexin 1 N-terminal peptide activates leukocytes by triggering different members of the formyl peptide receptor family

The human N-formyl peptide receptor (FPR) is a key modulator of chemotaxis directing granulocytes toward sites of bacterial infections. FPR is the founding member of a subfamily of G protein-coupled receptors thought to function in inflammatory processes. The other two members, FPR-like (FPRL)1 and FPRL2, have a greatly reduced affinity for bacterial peptides or do not bind them at all, with FPRL2 being considered an orphan receptor so far. In this study we show that a peptide derived from the N-terminal domain of the anti-inflammatory protein annexin 1 (lipocortin 1) can activate all three FPR family members at similar concentrations. The annexin 1 peptide initiates chemotactic responses in human monocytes that express all three FPR family members and also desensitizes the cells toward subsequent stimulation with bacterial peptide agonists. Experiments using HEK 293 cells stably expressing a single FPR family member reveal that all three receptors can be activated and desensitized by the N-terminal annexin 1 peptide. These observations identify the annexin 1 peptide as the first endogenous ligand of FPRL2 and indicate that annexin 1 participates in regulating leukocyte emigration into inflamed tissue by activating and desensitizing different receptors of the FPR family.     

Dexamethasone decreases phospholipase C beta1 isozyme expression in human vascular smooth muscle cells

The molecular characterization of the human PLC beta1 gene was just reported by Peruzzi et al. [Biochim. Biophys. Acta 1582 (2002) 46]. This prompted us to investigate the effects of dexamethasone on PLC beta1 expression in two types of human vascular smooth muscle cells--coronary artery smooth muscle cells (hCASMC) and aortic smooth muscle cells (hAoSMC), since glucocorticoids are known to affect the signaling pathways of Gprotein coupled receptors. Semi-quantitative RT-PCR was used to analyze mRNA expression and Western-blot for protein expression. Dexamethasone treatment in the two types of cells studied decreased (mRNA and protein) PLC beta1 isozyme expression. A rapid (2 h) fall in mRNA occurred in hCASMC after treatment, and hCASMC were more sensitive to dexamethasone (1 nM versus 100 nM) than hAoSMC. The major reduction (80%) was observed after 48 h of exposure in both VSMC. Treatment with mifeprisone, an antagonist of glucocorticoid receptors, blunted the dexamethasone effect on PLC beta1 mRNA and showed that this effect was mediated by glucocorticoids receptors.     

Differential effects of adiponectin in osteoblast-like cells

Abstract

The skeleton should maintain an adequate volume, vigour and strength to carry out the role for which it is designed: to hold the whole soft tissue mass that shapes the body and to protect the vital organs. To fulfil this task a satisfactory food intake is required and regulators that are released in the feeding and fasting states, among other signals indicate how much soft mass needs to be built up. Those signals include the secretion of adipocytokines which could represent a relevant link between soft mass (adipose tissue) and skeleton. We studied the presence of adiponectin receptors (AdipoR1, AdipoR2) and its direct effects in osteosarcoma cell line Saos-2. The results indicated that adiponectin receptors were present in the osteoblastic cells with a higher expression of AdipoR1. Human recombinant globular adiponectin was able to increase viability levels and decrease cytotoxicity rates in cell cultures. Also, adiponectin significantly inhibited alkaline phosphatase activity in supernatants. Osteoprotegerin mRNA expression was significantly reduced after 72?h treatment. The FOS induction was studied and the results exhibited a significant increase caused by adiponectin. In conclusion, all these observations suggest that adiponectin influences bone metabolism decreasing the levels of bone formation. Regulators of adiponectin or its receptors could be circulating to modulate the activities of this peptide.     

Functional activation of the formyl peptide receptor by a new endogenous ligand in human lung A549 cells

The formyl peptide receptor (FPR), a heptahelical G protein-coupled receptor on phagocytic leukocytes, can be triggered by bacterially derived oligopeptides of the prototype fMLP. Although FPR expression and activation have been associated with cells of myeloid origin and bacterial inflammation, the receptor has recently been identified in nonmyeloid cells, thus suggesting additional physiological functions and the existence of an endogenous agonist. In this study, we demonstrate the presence and functional activation of the FPR in the human lung cell line A549, which represents an extrahepatic model for the regulation of acute-phase proteins. Activation of the FPR in A549 cells cannot only be triggered by fMLP, but also by an agonistic peptide of the recently identified endogenous FPR ligand, annexin 1. In addition to inducing changes in the F-actin content, annexin 1-mediated triggering of the FPR results in an increased expression of acute-phase proteins. Hence, activation of nonmyeloid FPR by its endogenous ligand annexin 1 could participate in the regulation of acute-phase responses, e.g., during inflammation and/or wound healing.     

Regulation of annexin I in rheumatoid synovial cells by glucocorticoids and interleukin-1

The glucocorticoid (GC)-induced antiinflammatory molecule annexin I is expressed in leukocytes and has antiinflammatory effects in animal models of arthritis, but the expression of annexin I in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) is unknown. We report the constitutive and dexamethasone (DEX)-inducible expression of annexin I in RA FLS. DEX increased FLS annexin I protein translocation and mRNA expression. Interleukin (IL)-1beta also induced annexin I translocation and mRNA but also increased intracellular protein. DEX and IL-1 had additive effects on annexin I mRNA, but DEX inhibited the inducing effect of IL-1beta on cell surface annexin I. These results indicate that glucocorticoids and IL-1beta upregulate the synthesis and translocation of annexin I in RA FLS, but interdependent signalling pathways are involved.     

Spatial and temporal profiles for anti-inflammatory gene expression in leukocytes during a resolving model of peritonitis

The recent appreciation of the role played by endogenous counterregulatory mechanisms in controlling the outcome of the host inflammatory response requires specific analysis of their spatial and temporal profiles. In this study, we have focused on the glucocorticoid-regulated anti-inflammatory mediator annexin 1. Induction of peritonitis in wild-type mice rapidly (4 h) produced the expected signs of inflammation, including marked activation of resident cells (e.g., mast cells), migration of blood-borne leukocytes, mirrored by blood neutrophilia. These changes subsided after 48-96 h. In annexin 1(null) mice, the peritonitis response was exaggerated ( approximately 40% at 4 h), with increased granulocyte migration and cytokine production. In blood leukocytes, annexin 1 gene expression was activated at 4, but not 24, h postzymosan, whereas protein levels were increased at both time points. Locally, endothelial and mast cell annexin 1 gene expression was not detectable in basal conditions, whereas it was switched on during the inflammatory response. The significance of annexin 1 system plasticity in the anti-inflammatory properties of dexamethasone was assessed. Clear induction of annexin 1 gene in response to dexamethasone treatment was evident in the circulating and migrated leukocytes, and in connective tissue mast cells; this was associated with the steroid failure to inhibit leukocyte trafficking, cytokine synthesis, and mast cell degranulation in the annexin 1(null) mouse. In conclusion, understanding how inflammation is brought under control will help clarify the complex interplay between pro- and anti-inflammatory pathways operating during the host response to injury and infection.     

F2L, a peptide derived from heme-binding protein, chemoattracts mouse neutrophils by specifically activating Fpr2, the low-affinity N-formylpeptide receptor

F2L (formylpeptide receptor (FPR)-like (FPRL)-2 ligand), a highly conserved acetylated peptide derived from the amino-terminal cleavage of heme-binding protein, is a potent chemoattractant for human monocytes and dendritic cells, and inhibits LPS-induced human dendritic cell maturation. We recently reported that F2L is able to activate the human receptors FPRL-1 and FPRL2, two members of the FPR family, with highest selectivity and affinity for FPRL2. To facilitate delineation of mechanisms of F2L action in vivo, we have now attempted to define its mouse receptors. This is complicated by the nonequivalence of the human and mouse FPR gene families (three vs at least eight members, respectively). When cell lines were transfected with plasmids encoding the eight mouse receptors, only the one expressing the receptor Fpr2 responded to F2L (EC(50) approximately 400 nM for both human and mouse F2L in both calcium flux and cAMP inhibition assays). This value is similar to F2L potency at human FPRL1. Consistent with this, mouse neutrophils, which like macrophages and dendritic cells express Fpr2, responded to human and mouse F2L in both calcium flux and chemotaxis assays with EC(50) values similar to those found for Fpr2-expressing cell lines ( approximately 500 nM). Moreover, neutrophils from mice genetically deficient in Fpr2 failed to respond to F2L. Thus, Fpr2 is a mouse receptor for F2L, and can be targeted for the study of F2L action in mouse models.     

Induction of an interleukin-1 receptor (IL-1R) on monocytic cells. Evidence that the receptor is not encoded by a T cell-type IL-1R mRNA

Primary human monocytes and the human monocytic cell line THP-1 were induced to express receptors for interleukin-1 alpha (IL-1 alpha) and IL-1 beta. Treatment of primary monocytes with dexamethasone resulted in a 10-fold increase in receptor number over untreated cells, to approximately 2,000 receptors/cell. Treatment of THP-1 cells with phorbol ester followed by prostaglandin E2 and dexamethasone resulted in the expression of approximately 30,000 receptors/cell. Competitive binding assays on THP-1 cells showed that both IL-1 alpha and IL-1 beta bind to the same receptor. The monocyte IL-1R is significantly smaller (63 kDa) than the T cell IL-1R (80 kDa) and is immunologically distinct. However, induction of monocytes and monocytic cell lines leads to the appearance of an abundant mRNA of approximately 5,000 bases which hybridizes to a cDNA probe from the T cell-type IL-1R. Sequence data obtained from a cDNA clone of this mRNA indicate that the message is identical to the T cell IL-1R mRNA throughout the coding region. A smaller mRNA, also homologous to the T cell IL-1R mRNA, accumulated in induced THP-1 cells and has a shorter 3'-untranslated region than the larger. Data are presented which suggest that neither form of this message encodes the 63-kDa IL-1R, but rather that this protein is the product of a separate nonhomologous mRNA.     

RAMPs and CRLR expressions in osteoblastic cells after dexamethasone treatment

Adrenomedullin (ADM) is a potent stimulator of osteoblastic activity and promotes bone growth in vivo. ADM receptors are formed by heterodimerization of the CRLR and a RAMP2 or RAMP3 molecule. Since glucocorticoid responsive elements were recently identified in the human CRLR promoter and that glucocorticoids exert a major action in bones, we investigated the acute effect of dexamethasone (Dex) treatment on ADM receptor components in osteoblastic cell types: the MC3T3-E1 cells and calvaria-derived osteoblastic cells. Changes in expression of CRLR and RAMPs molecules were evaluated at mRNA levels using RT-PCR and at protein levels by Western blot analysis. We found that Dex increased expression of RAMP1 and RAMP2 mRNA in a time-dependent but dose-independent manner, while RAMP3 was unchanged. In contrast, Dex decreased the CRLR mRNA expression and these changes were reflected at protein levels. We suggest that Dex, in osteoblastic cells, altered ADM receptor by inhibition of CRLR expression and consequently could impair the ADM anabolic effect on bone. Our findings could explain in part, the detrimental side effects observed at bone level during glucocorticoid therapy.     

Arrestins block G protein-coupled receptor-mediated apoptosis

G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) activate numerous cellular signals through the combined actions of G proteins, GPCR kinases, and arrestins. Although arrestins have traditionally been thought of as mediating GPCR desensitization, they have now been shown to play important roles in the internalization, trafficking, and signaling of many GPCRs. We demonstrate that in cells devoid of arrestins, the stimulation of numerous GPCRs including the N-formyl peptide receptor (FPR) initiates rapid cell rounding, annexin V positivity, and caspase activation followed by cell death. The apoptotic response is initiated by G protein signaling and involves activation of phosphoinositide 3-kinase, mitogen-activated protein kinases, and c-Src resulting in cytochrome c release from mitochondria and ultimately caspase 9 and caspase 3 activation. Reconstitution with either arrestin-2 or arrestin-3 is completely sufficient to prevent FPR-mediated apoptosis. Surprisingly, a non-desensitizing and non-internalizing mutant of the FPR is unable to initiate apoptosis, indicating that receptor phosphorylation and internalization, but not solely chronic activation due to a lack of desensitization, are critical determinants for the induction of apoptosis by the FPR. We further demonstrate that this response is not unique to the FPR with numerous additional GPCRs, including the V2 vasopressin, angiotensin II (type 1A), and CXCR2 receptors, capable of initiating apoptosis upon stimulation, whereas GPCRs such as the beta(2)-adrenergic receptor and CXCR4 are not capable of initiating apoptotic signaling. These data demonstrate for the first time that arrestins play a critical and completely unexpected role in the suppression GPCR-mediated apoptosis, which we show is a common consequence of GPCR-mediated cellular activation in the absence of arrestins.     

Glucocorticoids Enhance Histamine-Evoked Catecholamine Secretion from Bovine Chromaffin Cells

Abstract: The synthetic glucocorticoid dexamethasone enhanced histamine-evoked catecholamine secretion from cultured bovine chromaffin cells. Dexamethasone enhanced the effects of histamine on both adrenergic (epinephrine-rich) and noradrenergic (norepinephrine-rich) chromaffin cells but had a more dramatic effect on noradrenergic cells. Histamine-evoked secretion in noradrenergic cells appeared to become rapidly inactivated, whereas the rate of secretion in adrenergic cells was nearly constant for up to 2 h; dexamethasone treatment attenuated the inactivation seen in noradrenergic cells. The effect of dexamethasone appeared after a lag of several hours and was maximal by 24 h. The EC₅₀ for dexamethasone was ∼1 n M . The effect of dexamethasone was mimicked by the glucocorticoid agonist RU 28362 and was blocked by the antagonist RU 38486, indicating that the effects of these steroids were mediated by the glucocorticoid or type II corticosteroid receptor. Histamine-evoked catecholamine secretion in both dexamethasone-treated and untreated cells was blocked by the H₁ histamine receptor antagonist mepyramine but was not affected by the H₂ antagonist cimetidine; thus, dexamethasone appeared to enhance an H₁ receptor-mediated process. In the absence of glucocorticoids, H₁ receptor mRNA levels were higher in adrenergic than in noradrenergic cells. Dexamethasone increased H₁ receptor mRNA levels in both cell types. The increased expression of H₁ receptors presumably contributes to the enhancement of histamine-evoked catecholamine secretion by glucocorticoids. Glucocorticoids may play a physiological role in modulating the responsiveness of chromaffin cells to histamine and other stimuli.