Maria J. G. Fernandes

Angiomotin family proteins are novel activators of the LATS2 kinase tumor suppressor

The Hippo pathway kinase LATS2 promotes contact inhibition of growth. How LATS2 is activated in response to changes in cell density is unknown. It is found that tight junction protein AMOTL2 is a novel activator of LATS2, raising the possibility that tight junction assembly promotes LATS2-dependent inhibition of cell proliferation.

Regulation of lysophosphatidic acid receptor expression and function in human synoviocytes: Implications for rheumatoid arthritis?

Lysophosphatidic acid (LPA), via interaction with its G-protein coupled receptors, is involved in various pathological conditions. Extracellular LPA is mainly produced by the enzyme autotaxin (ATX). Using fibroblast-like synoviocytes (FLS) isolated from synovial tissues of patients with rheumatoid arthritis (RA), we studied the expression profile of LPA receptors, LPA-induced cell migration, and interleukin (IL)-8 and IL-6 production. We report that FLS express LPA receptors LPA1-3. Moreover, exogenously applied LPA induces FLS migration and secretion of IL-8/IL-6, whereas the LPA3 agonist l-sn-1-O-oleoyl-2-methyl-glyceryl-3-phosphothionate (2S-OMPT) stimulates cytokine synthesis but not cell motility. The LPA-induced FLS motility and cytokine production are suppressed by LPA1/3 receptor antagonists diacylglycerol pyrophosphate and (S)-phosphoric acid mono-(2-octadec-9-enoylamino-3-[4-(pyridine-2-ylmethoxy)-phenyl]-propyl) ester (VPC32183). Signal transduction through p42/44 mitogen-activated protein kinase (MAPK), p38 MAPK, and Rho kinase is involved in LPA-mediated cytokine secretion, whereas LPA-induced cell motility requires p38 MAPK and Rho kinase but not p42/44 MAPK. Treatment of FLS with tumor necrosis factor-α (TNF-α) increases LPA3 mRNA expression and correlates with enhanced LPA- or OMPT-induced cytokine production. LPA-mediated superproduction of cytokines by TNF-α-primed FLS is abolished by LPA1/3 receptor antagonists. We also report the presence of ATX in synovial fluid of patients with RA. LPA1/3 receptor antagonists and ATX inhibitors reduce the synovial fluid-induced cell motility. Together the data suggest that LPA1 and LPA3 may contribute to the pathogenesis of RA through the modulation of FLS migration and cytokine production. The above results provide novel insights into the relevance of LPA receptors in FLS biology and as potential therapeutic targets for the treatment of RA.

Scaffold-Guided Subchondral Bone Repair: Implication of Neutrophils and Alternatively Activated Arginase-1+ Macrophages

Background: Microfracture and drilling elicit a cartilage repair whose quality depends on subchondral bone repair. Alternatively activated (AA) macrophages express arginase-1, release angiogenic factors, and could be potential mediators of trabecular bone repair. Hypothesis: Chitosan–glycerol phosphate (GP)/blood implants elicit arginase-1+ macrophages in vivo through neutrophil-dependent mechanisms and improve trabecular bone repair of drilled defects compared with drilling alone. Study Design: Controlled laboratory study. Methods: Bilateral trochlear cartilage defects were created in 15 rabbits, microdrilled, and treated or not with chitosan-GP/blood implant to analyze AA macrophages, CD-31+ blood vessels, bone, and cartilage repair after 1, 2, or 8 weeks. Neutrophil and macrophage chemotaxis to rabbit subcutaneous implants of autologous blood and chitosan-GP (±blood) was quantified at 1 or 7 days. In vitro, sera from human chitosan-GP/blood and whole blood clots cultured at 37°C were analyzed by proteomics and neutrophil chemotaxis assays. Results: Chitosan-GP/blood clots and whole blood clots released a similar profile of chemotactic factors (PDGF-BB, IL-8/CXCL8, MCP-1/CCL2, and no IL-1β or IL-6), although chitosan clot sera attracted more neutrophils in vitro. Subcutaneous chitosan-GP (±blood) implants attracted more neutrophils (P < .001) and AA macrophages than whole blood clots in vivo. In repairing subchondral drill holes, chitosan-GP/blood implant attracted more AA macrophages at 1 and 2 weeks and more blood vessels at 2 weeks compared with drilled controls. Treatment elicited a more complete woven bone repair at 8 weeks than controls (P = .0011) with a more uniform, integrated collagen type II+ cartilage repair tissue. Conclusion and Clinical Relevance: AA macrophages may play a role in the regeneration of subchondral bone, and chitosan-GP can attract and transiently accumulate these cells in the repair tissue. The resulting improved subchondral repair could be advantageous toward enhancing integration of a restored chondral surface to the subchondral bone.

Crystal-induced neutrophil activation. VII. Involvement of Syk in the responses to monosodium urate crystals.

The inflammatory response in acute gouty arthritis is in large part a result of the interaction between neutrophils and monosodium urate (MSU) crystals. The tyrosine kinase Syk, which has been largely associated with the phagocytic response by Fc receptors and with spreading mediated by integrins, has been identified as one of the major proteins tyrosine‐phosphorylated in human neutrophils upon stimulation by MSU crystals and is known to be mediated in part by the Fc receptor, CD16. This has led to the present examination of the implication of Syk in the activation pathways used by MSU crystals. The tyrosine‐phosphorylation patterns induced by MSU crystals and by the ligation of CD16 were inhibited by piceatannol, which, conversely, only slightly delayed but did not diminish the peak of tyrosine phosphorylation induced by cross‐linking CD32 or by the addition of fMet‐Leu‐Phe. Moreover, piceatannol inhibited the activity of Syk as monitored by in vitro kinase assays, by its in situ tyrosine phosphorylation, and by its activity toward exogenous substrates after stimulation by MSU crystals. We also measured the impact of piceatannol on the mobilization of calcium, the production of superoxide anions, and the activity of PLD stimulated by MSU crystals. We noted a distinct inhibition of all these responses by piceatannol. Finally, the morphological changes observed in neutrophils as characteristic of MSU crystal internalization were diminished significantly by piceatannol. The results obtained show that Syk plays a critical and central role in the signal‐transduction pathways called upon by MSU crystals subsequent to their interaction with human neutrophils.

Differential expression of adenosine receptors in human neutrophils: up‐regulation by specific Th1 cytokines and lipopolysaccharide

Four types of adenosine receptors have been identified in different tissues and cell types, namely, A1, A2A, A2B, and A3 receptors. We report that A2AR but not A2BR mRNA in freshly isolated polymorphonuclear neutrophils (PMN) is maximally up‐regulated after 4 h stimulation with lipopolysaccharide (LPS) or tumor necrosis factor α (TNF‐α) and to a lesser extent, with interleukin (IL)‐1β. These effects were maintained up to 21 h. Consistent with changes in A2AR mRNA expression, up‐regulation of A2AR protein was also detected after 4 h of LPS or TNF‐α exposure. Up‐regulation of A2AR protein expression was transient and returned to near basal levels after 12 h or 16 h stimulation with TNF‐α or LPS, respectively. Conversely, IL‐1β failed to promote A2AR protein expression. Suppression of thapsigargin‐induced leukotriene synthesis by the selective A2AR agonist CGS‐21680 was found to be more pronounced when PMN were cultured for 4 h with LPS or TNF‐α. In contrast, the up‐regulation of A2AR has no impact on CGS‐21680‐induced inhibition of phospholipase D activation and superoxide production in response to formyl‐Met‐Leu‐Phe. These results demonstrate that the A2AR is up‐regulated by specific T helper cell type 1 cytokines and LPS. Although this could represent a potential feedback mechanism to control inflammation, the effect of A2AR up‐regulation varied depending on the stimulus used to stimulate PMN functional responses after their incubation with proinflammatory mediators.

Signaling through CD16b in human neutrophils involves the Tec family of tyrosine kinases

Tec kinases belong to the second largest family of nonreceptor tyrosine kinases. Although these kinases are expressed in myeloid cells, little is known about their implication in neutrophil function. We recently reported the participation of Tec kinases in the responses of human neutrophils to the bacterial peptide N‐formyl‐l‐methionyl‐l‐leucyl‐l‐phenylalanine via G‐coupled protein receptors. In this study, we extended our investigations of Tec kinases to the signaling of the glycosylphosphatidylinositol‐linked receptor CD16b, which is highly and specifically expressed in neutrophils. The results obtained indicate that Tec is translocated to the plasma membrane, phosphorylated, and activated upon CD16b cross‐linking and that the activation of Tec is inhibited by Src‐specific inhibitors as well as by the phosphatidylinositol‐3 kinase inhibitor, wortmannin. As no specific inhibitor of Tec exists, the role of Tec kinases was further investigated using a‐Cyano‐b‐hydroxy‐b‐methyl‐N‐(2,5‐dibromophenyl)propenamide (LFM‐A13), a compound known to inhibit Bruton’s tyrosine kinase. We show that this compound also inhibits the kinase activity of Tec and provide evidence that the mobilization of intracellular calcium and the tyrosine phosphorylation of phospholipase Cγ2 (PLCγ2) induced upon CD16b engagement are inhibited by LFM‐A13. We also show that Tec kinases are important for CD16b‐dependent degranulation of neutrophils. In summary, we provide direct evidence for the implication of Tec in CD16b signaling and suggest that Tec kinases are involved in the phosphorylation and activation of PLCγ2 and subsequently, in the mobilization of calcium in human neutrophils.

Human angiomotin-like 1 associates with an angiomotin protein complex through its coiled-coil domain and induces the remodeling of the actin cytoskeleton

Angiostatin is a potent inhibitor of angiogenesis. One mechanism through which angiostatin inhibits angiogenesis is by binding to the cell surface protein p80-angiomotin. The p80-angiomotin protein promotes angiogenesis, in part, by conferring a hypermigratory phenotype to endothelial cells. Although p80-angiomotin is extensively characterized, less is known about the related protein angiomotin-like 1. We report that angiomotin-like 1 forms part of a protein complex containing p80-angiomotin. Structure-function studies revealed that angiomotin-like 1 associates with this p80-angiomotin-containing complex via its coiled-coil domain. Since p80-angiomotin plays a role in cell migration, a process that involves the remodeling of the actin cytoskeleton, we then addressed the hypothesis that angiomotin-like 1 may interact with the cytoskeleton. Immunofluorescence studies reveal that angiomotin-like 1 not only co-localizes with filamentous actin but also significantly modifies the architecture of the actin cytoskeleton. Regarding migration, angiomotin-like 1 increases the velocity of migration and decreases the persistence of migration directionality. Together these observations strongly suggest that angiomotin-like 1 is involved in actin-cytoskeleton-based processes, in part, via its interaction with a p80-angiomotin-containing complex and the actin cytoskeleton. These findings have important implications for angiogenesis-driven disease since angiomotin and angiomotin-like 1 are both expressed in capillaries.

Early Events in the Activation of FcγRIIA in Human Neutrophils: Stimulated Insolubilization, Translocation to Detergent-Resistant Domains, and Degradation of FcγRIIA

The signal transduction mechanisms associated with the ligation of FcγRIIA in human neutrophils are as yet only incompletely characterized. In the present study, we have investigated the distribution and fate of FcγRIIA following its cross-linking. The results obtained indicate that cross-linking of FcγRIIA led, within a few seconds, to its translocation into a nonionic detergent-insoluble fraction. This was followed, within a couple of minutes, by a substantial loss of immunoreactive FcγRIIA in the cells. The stimulated degradation of FcγRIIA was blocked by the Src kinase inhibitor PP1 but not by wortmannin, ST-638, piceatannol, or cytochalasin B. Cross-linked FcγRIIA could be solubilized by saponin (in the presence of Nonidet P-40) and by β-octylglucoside. Sucrose gradient analysis of the distribution of FcγRIIA revealed that its cross-linking led to its translocation into the pellets and not the light buoyant density fractions classically associated with lipid rafts. Disruption of cholesterol-containing membrane microdomains with filipin prevented the degradation of FcγRIIA but did not inhibit the stimulation of the pattern of tyrosine phosphorylation or the mobilization of calcium that followed FcγRIIA cross-linking. These data suggest that both cholesterol-rich domains and Src kinases are required for the degradation of the activated FcγRIIA and provide new insights into the early events following FcγRIIA cross-linking.

Specific and overlapping sphingosine-1-phosphate receptor functions in human synoviocytes: impact of TNF-a

Sphingosine-1-phosphate (S1P), via interaction with its G protein-coupled receptors, regulates various physiological and pathological responses. The present study investigated the role of S1P/S1P receptor signaling in several functional responses of human fibroblast-like synoviocytes (FLSs) that may contribute to the pathogenesis of rheumatoid arthritis (RA). We report that FLSs express the S1P(1), S1P(2), and S1P(3) receptors. Moreover, exogenously applied S1P induces FLS 1) migration, 2) secretion of inflammatory cytokines/chemokines, and 3) protection from apoptosis. Using specific S1P receptor agonists/antagonists, we determined that S1P stimulates FLS migration through S1P(1) and S1P(3), induces cytokine/chemokine secretion through S1P(2) and S1P(3), and protects from cell apoptosis via S1P(1). The S1P-mediated cell motility and cytokine/chemokine secretion seem to be regulated by the p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, and Rho kinase signal transduction pathways. Interestingly, treatment of FLSs with tumor necrosis factor-alpha increases S1P(3) expression and correlates with the enhancement of S1P-induced cytokine/chemokine production. Our data suggest that S1P(1), S1P(2), and S1P(3) play essential roles in the pathogenesis of RA by modulating FLS migration, cytokine/chemokine production, and cell survival. Moreover, the cytokine-rich environment of the inflamed synovium may synergize with S1P signaling to exacerbate the clinical manifestations of this autoimmune disease.

CD16b associates with high-density, detergent-resistant membranes in human neutrophils

CD16b is unique in that it is the only Fc receptor linked to the plasma membrane by a GPI (glycosylphosphatidylinositol) anchor. GPI-anchored proteins often preferentially localize to DRMs (detergent-resistant membranes) that are rich in sphingolipids and cholesterol and play an important role in signal transduction. Even though the responses to CD16b engagement have been intensively investigated, the importance of DRM integrity for CD16b signalling has not been characterized in human neutrophils. We provide direct evidence that CD16b constitutively partitions with both low- and high-density DRMs. Moreover, upon CD16b engagement, a significant increase in the amount of the receptor is observed in high-density DRMs. Similarly to CD16b, CD11b also resides in low- and high-density DRMs. In contrast with CD16b, the partitioning of CD11b in DRMs does not change in response to CD16b engagement. We also provide evidence for the implication of Syk in CD16b signalling and its partitioning to DRMs in resting and activated PMNs (polymorphonuclear neutrophils). Additionally, DRM-disrupting agents, such as nystatin and methyl-beta-cyclodextrin, alter cellular responses to CD16b receptor ligation. Notably, a significant increase in the mobilization of intracellular Ca2+ and in tyrosine phosphorylation of intracellular substrates after CD16b engagement is observed. Altogether, the results of this study provide evidence that high-density DRMs play a role in CD16b signalling in human neutrophils.