An acute phase protein α1-acid glycoprotein mitigates AKI and its progression to CKD through its anti-inflammatory action.

The molecular mechanism for acute kidney injury (AKI) and its progression to chronic kidney disease (CKD) continues to be unclear. In this study, we investigated the pathophysiological role of the acute phase protein α1-acid glycoprotein (AGP) in AKI and its progression to CKD using AGP KO mice. Plasma AGP levels in WT mice were increased by about 3.5-fold on day 1-2 after renal ischemia-reperfusion (IR), and these values then gradually decreased to the level before renal IR on day 7-14. On day 1 after renal IR, the AGP KO showed higher renal dysfunction, tubular injury and renal inflammation as compared with WT. On day 14, renal function, tubular injury and renal inflammation in WT had recovered, but the recovery was delayed, and renal fibrosis continued to progress in AGP KO. These results obtained from AGP KO were rescued by the administration of human-derived AGP (hAGP) simultaneously with renal IR. In vitro experiments using RAW264.7 cells showed hAGP treatment suppressed the LPS-induced macrophage inflammatory response. These data suggest that endogenously induced AGP in early renal IR functions as a renoprotective molecule via its anti-inflammatory action. Thus, AGP represents a potential target molecule for therapeutic development in AKI and its progression CKD.

Shedding of proangiogenic microvesicles from hypertrophic scar myofibroblasts.

Hypertrophic scars are a common complication of burn injuries and represent a major challenge in terms of prevention and treatment. These scars are characterized by a supraphysiological vascular density and by the presence of pathological myofibroblasts (Hmyos) displaying a low apoptosis propensity. However, the nature of the association between these two hallmarks of hypertrophic scarring remains largely unexplored. Here, we show that Hmyos produce signalling entities known as microvesicles that significantly increase the three cellular processes underlying blood vessel formation: endothelial cell proliferation, migration and assembly into capillary-like structures. The release of microvesicles from Hmyos was dose-dependently induced by the serum protein α-2-macroglobulin. Using flow cytometry, we revealed the presence of the α-2-macroglobulin receptor-low-density lipoprotein receptor-related protein 1-on the surface of Hmyos. The inhibition of the binding of α-2-macroglobulin to its receptor abolished the shedding of proangiogenic microvesicles from Hmyos. These findings suggest that the production of microvesicles by Hmyos contributes to the excessive vascularization of hypertrophic scars. α-2-Macroglobulin modulates the release of these microvesicles through interaction with low-density lipoprotein receptor-related protein 1.

Structural and functional insight into pan-endopeptidase inhibition by α2-macroglobulins.

Peptidases must be exquisitely regulated to prevent erroneous cleavage and one control is provided by protein inhibitors. These are usually specific for particular peptidases or families and sterically block the active-site cleft of target enzymes using lock-and-key mechanisms. In contrast, members of the +1400-residue multi-domain α2-macroglobulin inhibitor family (α2Ms) are directed against a broad spectrum of endopeptidases of disparate specificities and catalytic types, and they inhibit their targets without disturbing their active sites. This is achieved by irreversible trap mechanisms resulting from large conformational rearrangement upon cleavage in a promiscuous bait region through the prey endopeptidase. After decades of research, high-resolution structural details of these mechanisms have begun to emerge for tetrameric and monomeric α2Ms, which use 'Venus-flytrap' and 'snap-trap' mechanisms, respectively. In the former, represented by archetypal human α2M, inhibition is exerted through physical entrapment in a large cage, in which preys are still active against small substrates and inhibitors that can enter the cage through several apertures. In the latter, represented by a bacterial α2M from Escherichia coli, covalent linkage and steric hindrance of the prey inhibit activity, but only against very large substrates.

Activated α2 -Macroglobulin Induces Mesenchymal Cellular Migration Of Raw264.7 Cells Through Low-Density Lipoprotein Receptor-Related Protein 1.

Distinct modes of cell migration contribute to diverse types of cell movements. The mesenchymal mode is characterized by a multistep cycle of membrane protrusion, the formation of focal adhesion, and the stabilization at the leading edge associated with the degradation of extracellular matrix (ECM) components and with regulated extracellular proteolysis. Both α2 -Macroglobulin (α2 M) and its receptor, low density lipoprotein receptor-related protein 1 (LRP1), play important roles in inflammatory processes, by controlling the extracellular activity of several proteases. The binding of the active form of α2 M (α2 M*) to LRP1 can also activate different signaling pathways in macrophages, thus inducing extracellular matrix metalloproteinase-9 (MMP-9) activation and cellular proliferation. In the present study, we investigated whether the α2 M*/LRP1 interaction induces cellular migration of the macrophage-derived cell line, Raw264.7. By using the wound-scratch migration assay and confocal microscopy, we demonstrate that α2 M* induces LRP1-mediated mesenchymal cellular migration. This migration exhibits the production of enlarged cellular protrusions, MT1-MMP distribution to these leading edge protrusions, actin polymerization, focal adhesion formation, and increased intracellular LRP1/ß1-integrin colocalization. Moreover, the presence of calphostin-C blocked the α2 M*-stimulated cellular protrusions, suggesting that the PKC activation is involved in the cellular motility of Raw264.7 cells. These findings could constitute a therapeutic target for inflammatory processes with deleterious consequences for human health, such as rheumatoid arthritis, atherosclerosis and cancer. J. Cell. Biochem. 118: 1810-1818, 2017. © 2016 Wiley Periodicals, Inc.

Identification of a new alpha-2-macroglobulin: Multi-spectroscopic and isothermal titration calorimetry study.

A α2M homologue was isolated from sheep (Ovis aries) blood plasma, using a simple two-step procedure, ammonium sulphate fractionation and gel filtration chromatography. Sheep α2M was found to be a large tetrameric glycoprotein of 630 kDa with monomeric subunit of 133 kDa each. Each subunit of sheep α2M was found to be made up of two fragments of 102 and 31 kDa respectively. The proteinase inhibitor from sheep was found to have Stokes radius of 79Ǻ, which makes it much more compact than its human homologue. It entraps only 1 mol of trypsin per mole of inhibitor, like its caprine counterpart. The use of isothermal titration calorimetry has become gold standard for exploring thermodynamics of binding interactions. In this study, binding interaction of trypsin with alpha-2-macroglobulin is studied using ITC. The thermodynamic signatures--enthalpy change (ΔH), entropy change (ΔS) and Gibb's free energy change (ΔG), along with number of binding sites (N) and affinity constant (K) are explored for α2M-trypsin binding for the first time for any known α2M molecule. The thermodynamics of proteinase-antiproteinase association suggests that trypsin-α2M interaction is enthalpy driven event.

Hen egg white ovomacroglobulin promotes fibroblast migration via mediating cell adhesion and cytoskeleton.

BACKGROUND: Hen egg white ovomacroglobulin (OVM) possesses a variety of bioactivities and could potentially be used as a pharmaceutical agent. It has been reported that OVM is involved in wound healing and cancer pathological processes, and previous results suggest that OVM plays a potential role in cell proliferation and migration; however, this has not yet been proven. In the present study, the effects of OVM on fibroblast proliferation and migration were evaluated. RESULTS: Results of cell counting, cell viability, and cell cycle indicated that proliferation of fibroblasts was not altered by OVM treatment. However, scratch assays showed that OVM could promote the migration of 3 T6 mouse embryonic fibroblasts and human skin fibroblasts (HSF). Also, the adhesion of HSF to the collagen matrix was also enhanced by OVM treatment. RT-qPCR and western blot analysis showed that ß1 -integrin, ß-tubulin, and ß-actin were up-regulated while E-cadherin was down-regulated in OVM-treated HSF cells. The effect of OVM was silenced after forming a complex with trypsin, suggesting that the protease inhibitory ability of OVM is important for its effect on cell migration. CONCLUSION: These results suggested that promotion of OVM on cell migration was achieved by enhancing cell adhesion to extracellular matrix, reducing intercellular aggregation, and strengthening cytoskeleton. The finding of the promotion effect of OVM on cell migration is important for understanding its role in wound healing and cancer pathological processes. © 2015 Society of Chemical Industry.

Alpha-2-macroglobulin loaded microcapsules enhance human leukocyte functions and innate immune response.

Synthetic microstructures can be engineered to deliver bioactive compounds impacting on their pharmacokinetics and pharmacodynamics. Herein, we applied dextran-based layer-by-layer (LbL) microcapsules to deliver alpha-2-macroglobulin (α2MG), a protein with modulatory properties in inflammation. Extending recent observations made with dextran-microcapsules loaded with α2MG in experimental sepsis, we focused on the physical and chemical characteristics of these microstructures and determined their biology on rodent and human cells. We report an efficient encapsulation of α2MG into microcapsules, which enhanced i) human leukocyte recruitment to inflamed endothelium and ii) human macrophage phagocytosis: in both settings microcapsules were more effective than soluble α2MG or empty microcapsules (devoid of active protein). Translation of these findings revealed that intravenous administration of α2MG-microcapsules (but not empty microcapsules) promoted neutrophil migration into peritoneal exudates and augmented macrophage phagocytic functions, the latter response being associated with alteration of bioactive lipid mediators as assessed by mass spectrometry. The present study indicates that microencapsulation can be an effective strategy to harness the complex biology of α2MG with enhancing outcomes on fundamental processes of the innate immune response paving the way to potential future development in the control of sepsis.

Damage-associated molecular patterns generated in osteoarthritis directly excite murine nociceptive neurons through Toll-like receptor 4.

OBJECTIVE: To determine whether selected damage-associated molecular patterns (DAMPs) present in the osteoarthritic (OA) joints of mice excite nociceptors through Toll-like receptor 4 (TLR-4). METHODS: The ability of S100A8 and α2 -macroglobulin to excite nociceptors was determined by measuring the release of monocyte chemoattractant protein 1 (MCP-1) by cultured dorsal root ganglion (DRG) cells as well as by measuring the intracellular calcium concentration ([Ca(2+) ]i ) in cultured DRG neurons from naive mice or from mice that had undergone surgical destabilization of the medial meniscus (DMM) 8 weeks previously. The role of TLR-4 was assessed using TLR-4(-/-) cells or a TLR-4 inhibitor. The [Ca(2+) ]i in neurons within ex vivo intact DRGs was measured in samples from Pirt-GCaMP3 mice. Neuronal expression of the Tlr4 gene was determined by in situ hybridization. DMM surgery was performed in wild-type and TLR-4(-/-) mice; mechanical allodynia was monitored, and joint damage was assessed histologically after 16 weeks. RESULTS: DRG neurons from both naive and DMM mice expressed Tlr4. Both S100A8 and α2 -macroglobulin stimulated release of the proalgesic chemokine MCP-1 in DRG cultures, and the neurons rapidly responded to S100A8 and α2 -macroglobulin with increased [Ca(2+) ]i . Blocking TLR-4 inhibited these effects. Neurons within intact DRGs responded to the TLR-4 agonist lipopolysaccharide. In both of the calcium-imaging assays, it was primarily the nociceptor population of neurons that responded to TLR-4 ligands. TLR-4(-/-) mice were not protected from mechanical allodynia or from joint damage associated with DMM. CONCLUSION: Our experiments suggest a role of TLR-4 signaling in the excitation of nociceptors by selected DAMPs. Further research is needed to delineate the importance of this pathway in relation to OA pain.

Activated α2-macroglobulin binding to human prostate cancer cells triggers insulin-like responses.

Ligation of cell surface GRP78 by activated α2-macroglobulin (α2M*) promotes cell proliferation and suppresses apoptosis. α2M*-treated human prostate cancer cells exhibit a 2-3-fold increase in glucose uptake and lactate secretion, an effect similar to insulin treatment. In both α2M* and insulin-treated cells, the mRNA levels of SREBP1-c, SREBP2, fatty-acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, and Glut-1 were significantly increased together with their protein levels, except for SREBP2. Pretreatment of cells with α2M* antagonist antibody directed against the carboxyl-terminal domain of GRP78 blocks these α2M*-mediated effects, and silencing GRP78 expression by RNAi inhibits up-regulation of ATP citrate lyase and fatty-acid synthase. α2M* induces a 2-3-fold increase in lipogenesis as determined by 6-[(14)C]glucose or 1-[(14)C]acetate incorporation into free cholesterol, cholesterol esters, triglycerides, free fatty acids, and phosphatidylcholine, which is blocked by inhibitors of fatty-acid synthase, PI 3-kinase, mTORC, or an antibody against the carboxyl-terminal domain of GRP78. We also assessed the incorporation of [(14)CH3]choline into phosphatidylcholine and observed similar effects. Lipogenesis is significantly affected by pretreatment of prostate cancer cells with fatostatin A, which blocks sterol regulatory element-binding protein proteolytic cleavage and activation. This study demonstrates that α2M* functions as a growth factor, leading to proliferation of prostate cancer cells by promoting insulin-like responses. An antibody against the carboxyl-terminal domain of GRP78 may have important applications in prostate cancer therapy.

Anti-lymphoproliferative activity of alpha-2-macroglobulin in the plasma of hibernating 13-lined ground squirrels and woodchucks.

Plasma from hibernating (HIB) woodchucks (Marmota monax) or 13-lined ground squirrels (Ictidomys tridecemlineatus) suppressed (3)H-thymidine uptake in mouse spleen cell cultures stimulated with Concanavalin A (ConA); plasma from non-hibernating animals were only slightly inhibitory. Maximum inhibition occurred when HIB plasma was added to the cultures prior to ConA. After HPLC size exclusion chromatography of the HIB ground squirrel plasma, a single fraction (fraction-14) demonstrated inhibitory activity. Assay of fraction-14 from 8 HIB squirrels showed inhibition ranging from 13 to 95%; inhibition was correlated to the time the squirrels were exposed to cold prior to hibernation. Western blot analysis showed the factor to be a large molecular weight protein (>300 kDa), and mass spectrometry identified sequences that were 100% homologous with alpha-2-macroglobulin from humans and other species. These findings indicate a hibernation-related protein that may be responsible for immune system down regulation.

Identification of α2-macroglobulin as a master inhibitor of cartilage-degrading factors that attenuates the progression of posttraumatic osteoarthritis.

OBJECTIVE: To determine if supplemental intraarticular α2-macroglobulin (α2 M) has a chondroprotective effect in a rat model of osteoarthritis (OA). METHODS: Using Western blotting, mass spectrometry, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry, α2 M was identified as a potential therapeutic agent through a comparison of α2 M concentrations in serum, synovial fluid (SF), and cartilage from normal subjects and patients with OA. In cultured chondrocytes, the effects of α2 M on interleukin-1 (IL-1)-induced cartilage catabolic enzymes were evaluated by Luminex assay and ELISA. In vivo effects on cartilage degeneration and matrix metalloproteinase 13 (MMP-13) concentration were evaluated in male rats (n = 120) randomized to 1 of 4 treatments: 1) anterior cruciate ligament transection (ACLT) and saline injections, 2) ACLT and 1 IU/kg injections of α2 M, 3) ACLT and 2 IU/kg injections of α2 M, or 4) sham operation and saline injections. Rats were administered intraarticular injections for 6 weeks. The concentration of MMP-13 in SF lavage fluid was measured using ELISA. OA-related gene expression was quantified by real-time quantitative polymerase chain reaction. The extent of OA progression was graded by histologic examination. RESULTS: In both normal subjects and OA patients, α2 M levels were lower in SF as compared to serum, and in OA patients, MMP-13 levels were higher in SF than in serum. In vitro, α2 M inhibited the induction of MMP-13 by IL-1 in a dose-dependent manner in human chondrocytes. In the rat model of ACLT OA, supplemental intraarticular injection of α2 M reduced the concentration of MMP-13 in SF, had a favorable effect on OA-related gene expression, and attenuated OA progression. CONCLUSION: The plasma protease inhibitor α2 M is not present in sufficient concentrations to inactivate the high concentrations of catabolic factors found in OA SF. Our findings suggest that supplemental intraarticular α2 M provides chondral protection in posttraumatic OA.

Microparticle alpha-2-macroglobulin enhances pro-resolving responses and promotes survival in sepsis.

Incorporation of locally produced signaling molecules into cell-derived vesicles may serve as an endogenous mediator delivery system. We recently reported that levels alpha-2-macroglobulin (A2MG)-containing microparticles are elevated in plasma from patients with sepsis. Herein, we investigated the immunomodulatory actions of A2MG containing microparticles during sepsis. Administration of A2MG-enriched (A2MG-E)-microparticles to mice with microbial sepsis protected against hypothermia, reduced bacterial titers, elevated immunoresolvent lipid mediator levels in inflammatory exudates and reduced systemic inflammation. A2MG-E microparticles also enhanced survival in murine sepsis, an action lost in mice transfected with siRNA for LRP1, a putative A2MG receptor. In vitro, A2MG was functionally transferred onto endothelial cell plasma membranes from microparticles, augmenting neutrophil-endothelial adhesion. A2MG also modulated human leukocyte responses: enhanced bacterial phagocytosis, reactive oxygen species production, cathelicidin release, prevented endotoxin induced CXCR2 downregulation and preserved neutrophil chemotaxis in the presence of LPS. A significant association was also found between elevated plasma levels of A2MG-containing microparticles and survival in human sepsis patients. Taken together, these results identify A2MG enrichment in microparticles as an important host protective mechanism in sepsis.

Effects of sFlt-1 and alpha 2-macroglobulin on vascular endothelial growth factor-induced endothelin-1 upregulation in human microvascular endothelial cells.

INTRODUCTION: Soluble fms-like tyrosine kinase-1 (sFlt-1) is a vascular endothelial growth factor (VEGF) binding protein and potent antagonist of VEGF. Alpha 2 macroglobulin (α2M) is another major binding protein for circulating VEGF, which is present in human plasma at higher concentration (2-4 mg/mL) than sFlt-1. This study investigated the effects of sFlt-1 and α2M on VEGF-induced endothelin-1 (ET-1) upregulation in human microvascular endothelial cell-1 (HMEC-1). METHODS: HMEC-1 was cultured and incubated with varying concentrations of sFlt-1 and α2M in combination with VEGF. ET-1 mRNA expression in the cells was measured by real time RT-PCR and ET-1 protein by western blot analysis. RESULTS: ET-1 expression in HMEC-1 incubated with VEGF significantly increased in time- and dose-dependent manners. Next, HMEC-1 was treated with the sFlt-1 (10-1000 ng/mL) or α2M (10-10000 ng/mL) in the presence of VEGF (10 ng/mL). We found that sFlt-1 induced a significant decrease of ET-1 expression upregulated by VEGF, while α2M did not affect the VEGF-induced ET-1 expression. CONCLUSIONS: sFLT-1 suppressed the VEGF-induced the ET-1 expression of HMEC-1. However, α2M did not show a significant effect on the ET-1 expression that was induced by VEGF. The results suggest that a certain proportion of the bound form α2M-VEGF have a biological action involved in the pathophysiology of preeclampsia.

Activated α2-macroglobulin induces Müller glial cell migration by regulating MT1-MMP activity through LRP1.

In retinal proliferative diseases, Müller glial cells (MGCs) acquire migratory abilities. However, the mechanisms that regulate this migration remain poorly understood. In addition, proliferative disorders associated with enhanced activities of matrix metalloprotease 2 (MMP-2) and MMP-9 also present increased levels of the protease inhibitor α2-macroglobulin (α2M) and its receptor, the low-density lipoprotein receptor-related protein 1 (LRP1). In the present work, we investigated whether the protease activated form of α2M, α2M*, and LRP1 are involved with the MGC migratory process. By performing wound-scratch migration and zymography assays, we demonstrated that α2M* induced cell migration and proMMP-2 activation in the human Müller glial cell line, MIO-M1. This induction was blocked when LRP1 and MT1-MMP were knocked down with siRNA techniques. Using fluorescence microscopy and biochemical procedures, we found that α2M* induced an increase in LRP1 and MT1-MMP accumulation in early endosomes, followed by endocytic recycling and intracellular distribution of MT1-MMP toward cellular protrusions. Moreover, Rab11-dominant negative mutant abrogated MT1-MMP recycling pathway, cell migration, and proMMP-2 activation induced by α2M*. In conclusion, α2M*, through its receptor LRP1, induces cellular migration of Müller glial cells by a mechanism that involves MT1-MMP intracellular traffic to the plasma membrane by a Rab11-dependent recycling pathway.

Receptor-recognized α2-macroglobulin binds to cell surface-associated GRP78 and activates mTORC1 and mTORC2 signaling in prostate cancer cells.

OBJECTIVE: Tetrameric α(2)-macroglobulin (α(2)M), a plasma panproteinase inhibitor, is activated upon interaction with a proteinase, and undergoes a major conformational change exposing a receptor recognition site in each of its subunits. Activated α(2)M (α(2)M*) binds to cancer cell surface GRP78 and triggers proliferative and antiapoptotic signaling. We have studied the role of α(2)M* in the regulation of mTORC1 and TORC2 signaling in the growth of human prostate cancer cells. METHODS: Employing immunoprecipitation techniques and Western blotting as well as kinase assays, activation of the mTORC1 and mTORC2 complexes, as well as down stream targets were studied. RNAi was also employed to silence expression of Raptor, Rictor, or GRP78 in parallel studies. RESULTS: Stimulation of cells with α(2)M* promotes phosphorylation of mTOR, TSC2, S6-Kinase, 4EBP, Akt(T308), and Akt(S473) in a concentration and time-dependent manner. Rheb, Raptor, and Rictor also increased. α(2)M* treatment of cells elevated mTORC1 kinase activity as determined by kinase assays of mTOR or Raptor immunoprecipitates. mTORC1 activity was sensitive to LY294002 and rapamycin or transfection of cells with GRP78 dsRNA. Down regulation of Raptor expression by RNAi significantly reduced α(2)M*-induced S6-Kinase phosphorylation at T389 and kinase activity in Raptor immunoprecipitates. α(2)M*-treated cells demonstrate about a twofold increase in mTORC2 kinase activity as determined by kinase assay of Akt(S473) phosphorylation and levels of p-Akt(S473) in mTOR and Rictor immunoprecipitates. mTORC2 activity was sensitive to LY294002 and transfection of cells with GRP78 dsRNA, but insensitive to rapamycin. Down regulation of Rictor expression by RNAi significantly reduces α(2)M*-induced phosphorylation of Akt(S473) phosphorylation in Rictor immunoprecipitates. CONCLUSION: Binding of α(2)M* to prostate cancer cell surface GRP78 upregulates mTORC1 and mTORC2 activation and promotes protein synthesis in the prostate cancer cells.

Inhibition of Aeromonas sobria serine protease (ASP) by α2-macroglobulin.

ASP is a serine protease secreted by Aeromonas sobria. ASP cleaves various plasma proteins, which is associated with onset of sepsis complications, such as shock and blood coagulation disorder. To investigate a host defense mechanism against this virulence factor, we examined the plasma for ASP inhibitor(s). Human plasma inhibited ASP activity for azocasein, which was almost completely abolished by treating plasma with methylamine, which inactivates α2-macroglobulin (α2-MG). The ASP-inhibitor complex in ASP-added plasma was not detected by immunoblotting using anti-ASP antibody; however, using gel filtration of the plasma ASP activity for an oligopeptide, the ASP substrate was eluted in the void fraction (Mw>200 000), suggesting ASP trapping by α2-MG. Indeed, human α2-MG inhibited ASP azocaseinolytic activity in a dose-dependent manner, rapidly forming a complex with the ASP. Fibrinogen degradation by ASP was completely inhibited in the presence of α2-MG. α1-Protease inhibitor, antithrombin, and α2-plasmin inhibitor neither inhibited ASP activity nor formed a complex with ASP. Surprisingly, ASP degraded these plasma serine protease inhibitors. Thus, α2-MG is the major ASP inhibitor in the human plasma and can limit ASP virulence activities in A. sobria infection sites. However, as shown by fluorescence correlation spectroscopy, slow ASP inhibition by α2-MG in plasma may indicate insufficient ASP control in vivo.

The tail nick augments Aeromonas sobria serine protease (ASP) activity in plasma through retarding inhibition by α2-macroglobulin.

ASP is a serine protease secreted by Aeromonas sobria, a sepsis-causing bacterium, and induces sepsis-mimicking disorders through plasma protein cleavage. The pathogen also secretes nASP that has a nick in the carboxy-terminal region. Compared with single-chain ASP (sASP), nASP had near-equivalent activity for small peptide substrates but was less proteolytic. Surprisingly, nASP cleaved proteins more in plasma and was inhibited by human α(2)-macroglobulin more slowly than sASP. Retarded inhibition by α(2)-macroglobulin allows nASP to keep proteolytic activity for longer in the host and exacerbate disorders at Aeromonas sobria infection sites. nASP may be an evolutional form to augment ASP virulence.

Plasma proteins present in osteoarthritic synovial fluid can stimulate cytokine production via Toll-like receptor 4.

INTRODUCTION: Osteoarthritis (OA) is a degenerative disease characterized by cartilage breakdown in the synovial joints. The presence of low-grade inflammation in OA joints is receiving increasing attention, with synovitis shown to be present even in the early stages of the disease. How the synovial inflammation arises is unclear, but proteins in the synovial fluid of affected joints could conceivably contribute. We therefore surveyed the proteins present in OA synovial fluid and assessed their immunostimulatory properties. METHODS: We used mass spectrometry to survey the proteins present in the synovial fluid of patients with knee OA. We used a multiplex bead-based immunoassay to measure levels of inflammatory cytokines in serum and synovial fluid from patients with knee OA and from patients with rheumatoid arthritis (RA), as well as in sera from healthy individuals. Significant differences in cytokine levels between groups were determined by significance analysis of microarrays, and relations were determined by unsupervised hierarchic clustering. To assess the immunostimulatory properties of a subset of the identified proteins, we tested the proteins' ability to induce the production of inflammatory cytokines by macrophages. For proteins found to be stimulatory, the macrophage stimulation assays were repeated by using Toll-like receptor 4 (TLR4)-deficient macrophages. RESULTS: We identified 108 proteins in OA synovial fluid, including plasma proteins, serine protease inhibitors, proteins indicative of cartilage turnover, and proteins involved in inflammation and immunity. Multiplex cytokine analysis revealed that levels of several inflammatory cytokines were significantly higher in OA sera than in normal sera, and levels of inflammatory cytokines in synovial fluid and serum were, as expected, higher in RA samples than in OA samples. As much as 36% of the proteins identified in OA synovial fluid were plasma proteins. Testing a subset of these plasma proteins in macrophage stimulation assays, we found that Gc-globulin, α1-microglobulin, and α2-macroglobulin can signal via TLR4 to induce macrophage production of inflammatory cytokines implicated in OA. CONCLUSIONS: Our findings suggest that plasma proteins present in OA synovial fluid, whether through exudation from plasma or production by synovial tissues, could contribute to low-grade inflammation in OA by functioning as so-called damage-associated molecular patterns in the synovial joint.

Loss of cell surface TFII-I promotes apoptosis in prostate cancer cells stimulated with activated α2 -macroglobulin.

Receptor-recognized forms of α2 -macroglobulin (α2 M) bind to cell surface-associated GRP78 and initiate pro-proliferative and anti-apoptotic signaling. Ligation of GRP78 with α2 M also upregulates TFII-I, which binds to the GRP78 promoter and enhances GRP78 synthesis. In addition to its transcriptional functions, cytosolic TFII-I regulates agonist-induced Ca(2+) entry. In this study we show that down regulation of TFII-I gene expression by RNAi profoundly impairs its cell surface expression and anti-apoptotic signaling as measured by significant reduction of GRP78, Bcl-2, and cyclin D1 in 1-Ln and DU-145 human prostate cancer cells stimulated with α2 M. In contrast, this treatment significantly increases levels of the pro-apoptotic proteins p53, p27, Bax, and Bak and causes DNA fragmentation. Furthermore, down regulation of TFII-I expression activates agonist-induced Ca(2+) entry. In plasma membrane lysates p-PLCγ1, TRPC3, GRP78, MTJ1, and caveolin co-immunoprecipitate with TFII-I suggesting multimeric complexes of these proteins. Consistent with this hypothesis, down regulating TFII-I, MTJ1, or GRP78 expression by RNAi greatly attenuates cell surface expression of TFII-I. In conclusion, we demonstrate that not only does cell surface GRP78 regulate apoptosis, but it also regulates Ca(2+) homeostasis by controlling cell surface localization of TFII-I.