Data describing expression of formyl peptide receptor 2 in human articular chondrocytes.
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ABSTRACT:
SUBMITTER: Strid Holmertz A
PROVIDER: S-EPMC7326719 | biostudies-literature | 2020 Aug
REPOSITORIES: biostudies-literature
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Project description:Formyl peptide receptor 1 (FPR1) mediates bacterial and mitochondrial N-formyl peptides-induced neutrophil activation. Therefore, FPR1 is an important therapeutic target for drugs to treat septic or sterile inflammatory diseases. Honokiol, a major bioactive compound of Magnoliaceae plants, possesses several anti-inflammatory activities. Here, we show that honokiol exhibits an inhibitory effect on FPR1 binding in human neutrophils. Honokiol inhibited superoxide anion generation, reactive oxygen species formation, and elastase release in bacterial or mitochondrial N-formyl peptides (FPR1 agonists)-activated human neutrophils. Adhesion of FPR1-induced human neutrophils to cerebral endothelial cells was also reduced by honokiol. The receptor-binding results revealed that honokiol repressed FPR1-specific ligand N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein binding to FPR1 in human neutrophils, neutrophil-like THP-1 cells, and hFPR1-transfected HEK293 cells. However, honokiol did not inhibit FPR2-specific ligand binding to FPR2 in human neutrophils. Furthermore, honokiol inhibited FPR1 agonist-induced calcium mobilization as well as phosphorylation of p38 MAPK, ERK, and JNK in human neutrophils. In conclusion, our data demonstrate that honokiol may have therapeutic potential for treating FPR1-mediated inflammatory diseases.
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Project description:Interleukin-1 receptor antagonist (IL-1Ra) is a natural IL-1 inhibitor possessing anti-inflammatory properties. IL-1Ra is produced as different isoforms, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, icIL-1Ra2 and icIL-1Ra3), derived from the same gene. We examined the production of IL-1Ra species by cultured human articular chondrocytes in response to various cytokines. The levels of IL-1Ra were undetectable in culture supernatants of untreated cells, but were significantly increased by IL-1beta. Cell lysates contained very low levels of IL-1Ra, even in response to IL-1beta, suggesting that chondrocytes produce predominantly sIL-1Ra. IL-6, which had no effect on its own, enhanced the effect of IL-1beta, while dexamethasone prevented the response. We observed by RT-PCR that IL-1beta and IL-6 induced primarily the production of sIL-1Ra mRNA. Furthermore, IL-1beta alone or combined with IL-6 increased the levels of nascent unspliced sIL-1Ra mRNA, suggesting that sIL-1Ra expression is regulated at the transcriptional level. Reporter gene assays in immortalized chondrocytes, C-20/A4, consistently showed increased sIL-1Ra promoter activity in response to IL-1beta and IL-6. In conclusion, human articular chondrocytes produce sIL-1Ra in response to IL-1beta and IL-6. The production of sIL-1Ra by chondrocytes may have a protective effect against articular inflammatory and catabolic responses.
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Project description:Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Schönberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human articular and costal (teenage and fetal origin) cartilages. Notably, we found 14 ion channel genes commonly expressed between articular and both types of costal cartilage chondrocytes. There were several other ion channel genes expressed only in articular (6 genes) or costal chondrocytes (5 genes). Significant differences in expression of BEST1 and KCNJ2 (Kir2.1) were observed between fetal and teenage costal cartilage. Interestingly, the large Ca(2+) activated potassium channel (BKα, or KCNMA1) was very highly expressed in all chondrocytes examined. Expression of the gap junction genes for Panx1, GJA1 (Cx43) and GJC1 (Cx45) was also observed in chondrocytes from all cartilage samples. Together, this data highlights similarities between chondrocyte membrane channel gene expressions in cells derived from different anatomical sites, and may imply that common electrophysiological signaling pathways underlie cellular control. The high expression of a range of mechanically and metabolically sensitive membrane channels suggest that chondrocyte mechanotransduction may be more complex than previously thought.
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Project description:Background and purposeIn osteoarthritis (OA), bradykinin (BK) is known to contribute to pain and synovitis, but not to cartilage degradation. Here, we investigated effects of BK and its antagonists on chondrocytes, cells involved in cartilage homeostasis.Experimental approachBK receptor density and affinities of BK, its analogues and antagonists were measured in cultured human and rat chondrocytes by radioligand binding. Effects of BK were assessed by accumulation of inositol phosphates (IP) and release of interleukin (IL)-6 and IL-8.Key resultsDensity of [³H]-BK binding sites was higher (13-30-fold) and BK evoked a greater (48-fold) IP production, in human than in rat chondrocytes. The BK B? receptor antagonists MEN16132 and icatibant displayed similar binding affinity. MEN16132 was 40-fold more potent than icatibant in the IP assay. In human chondrocytes, BK increased release (over 24 h) of IL-6 and IL-8, effects blocked by MEN16132 but not by the B? receptor antagonist Lys-[Leu?][desArg?]BK. BK-induced release of IL-6, but not of IL-8, was partially inhibited by indomethacin (10 µM) and nordihydroguaiaretic acid (10 µM). Antagonists for the prostanoid EP receptors (AH6809 10 µM; L-798,196, 200 nM; L-161,982, 1 µM) were ineffective. Dexamethasone (100 nM) partially inhibited release of both IL-6 and IL-8. Inhibitors of intracellular downstream signalling pathways (SB203580 10 µM; PD98059, 30 µM; SP600125, 30 µM; BAY-117085, 5 µM) indicated the involvement of p38 MAPK and the activation of NF-?B.Conclusion and implicationsBK mediated inflammatory changes and cartilage degradation and B? receptor blockade would, therefore, be a potential treatment for OA.
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Project description:Virulence of emerging community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and other highly pathogenic S. aureus strains depends on their production of phenol-soluble modulin (PSM) peptide toxins, which combine the capacities to attract and lyse neutrophils. The molecular basis of PSM-stimulated neutrophil recruitment has remained unclear. Here, we demonstrate that the human formyl peptide receptor 2 (FPR2/ALX), which has previously been implicated in control of endogenous inflammatory processes, senses PSMs at nanomolar concentrations and initiates proinflammatory neutrophil responses to CA-MRSA. Specific blocking of FPR2/ALX or deletion of PSM genes in CA-MRSA severely diminished neutrophil detection of CA-MRSA. Furthermore, a specific inhibitor of FPR2/ALX and of its functional mouse counterpart blocked PSM-mediated leukocyte infiltration in vivo in a mouse model. Thus, the innate immune system uses a distinct FPR2/ALX-dependent mechanism to specifically sense bacterial peptide toxins and detect highly virulent bacterial pathogens. FPR2/ALX represents an attractive target for new anti-infective or anti-inflammatory strategies.
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Project description:ObjectiveDifferentiated articular chondrocytes express a functional bisoform of the leptin receptor (LRb); however, leptin-LRb signaling in these cells is poorly understood. We hypothesized that leptin-LRb signaling in articular chondrocytes functions to modulate canonical Wnt signaling events by altering the expression of Frizzled (FZD) receptors.MethodsHuman chondrocyte cell lines and primary articular chondrocytes were grown in serum containing growth media for 24h, followed by a media change to Dulbecco's modified Eagle's medium (DMEM) containing 1% Nutridoma-SP to obtain a serum-deficient environment for 24h before treatment. Treatments included recombinant human leptin (10-100nM), recombinant human IL-6 (0.3-3nM), or recombinant human erythropoietin (Epo) (10mU/ml). Cells were harvested 30min-48h after treatment and whole cell lysates were analyzed using immunoblots or luciferase assays.ResultsTreatment of cells with leptin resulted in activation of Janus kinase 2 (JAK2) and subsequent phosphorylation of specific tyrosine residues on LRb, followed by dose- and time-dependent increases in the expression of Frizzled-1 (FZD1) and Frizzled-7 (FZD7). Leptin-mediated increases in the expression of FZD1 were blocked by pre-treatment with the protein synthesis inhibitor cycloheximide or the JAK2 inhibitor AG490. Experiments using a series of hybrid Epo extracellular domain-leptin intracellular domain receptors (ELR) harboring mutations of specific tyrosine residues in the cytoplasmic tail showed that increases in the expression of FZD1 were dependent on LRb-mediated phosphorylation of STAT3, but not ERK1/2 or STAT5. Leptin pre-treatment of chondrocytes prior to Wnt3a stimulation resulted in an increased magnitude of canonical Wnt signaling.ConclusionThese experiments show that leptin-LRb signaling in articular chondrocytes modulates expression of canonical Wnt signaling receptors and suggests that direct cross-talk between these pathways is important in determining chondrocyte homeostasis.
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Project description:Formyl peptide receptors (FPRs) are G protein-coupled chemoattractant receptors expressed mainly in phagocytic leukocytes. High expression of FPRs has also been detected in several cancers but the functions of FPR1 in tumor invasion and metastasis is poorly understood. In this study, we investigated the expression of FPRs in primary human colorectal cancer (CRC) and analyzed the association of FPRs expression with clinicopathological parameters. The levels of FPRs mRNA, especially those of FPR1, were significantly higher in colorectal tumors than in distant normal tissues and adjacent non-tumor tissues. FPR1 mRNA expression was also associated with tumor serosal infiltration. FPR1 protein expression was both in the colorectal epitheliums and tumor infiltrating neutrophils/macrophages. Furthermore, the functions of FPR1 in tumor invasion and tissue repair were investigated using the CRC cell lines SW480 and HT29. Higher cell surface expression of FPR1 is associated with significantly increased migration in SW480 cells compared with HT29 cells that have less FPR1 membrane expression. Finally, genetic deletion of fpr1 increased the survival rate of the resulting knockout mice compared with wild type littermates in a mouse model of colitis-associated colorectal cancer. Our data demonstrate that FPR1 may play an important role in tumor cell invasion in CRC patients.
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Project description:ObjectivesMidkine, a heparin-binding growth factor, promotes population growth, survival and migration of several cell types, but its effect on articular chondrocytes remains unknown. The aim of this study was to investigate its role on proliferation of articular chondrocytes in vitro and in vivo.Materials and methodsBromodeoxyuridine incorporation and MTT assays were performed to examine the proliferative effect of recombinant human midkine (rhMK) on primary articular chondrocytes. Activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) was analysed using western blot analysis. Systemic and local delivery of rhMK into mice and rats was preformed to investigate the proliferative effect of rhMK in vivo, respectively. Histological evaluation, including measurement of articular cartilage thickness, cell density, matrix staining and immunostaining of proliferating cell nuclear antigen was carried out.ResultsrhMK promoted proliferation of articular chondrocytes cultured in a monolayer, which was mediated by activation of ERK and PI3K. The proliferative role of rhMK was not coupled to dedifferentiation of culture-expanded cells. Consistent with its action in vitro, rhMK stimulated proliferation of articular chondrocytes in vivo when it was administered subcutaneously and intra-articularly in mice and rats, respectively.ConclusionOur results demonstrate that rhMK stimulates proliferation of primary articular chondrocytes in vitro and in vivo. The results of this study warrant further examination of rhMK for treatment of animal models of articular cartilage defects.
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Project description:G-protein coupled receptors (GPCRs) participate in a wide range of vital regulations of our physiological actions. They are also of pharmaceutical importance and have become many therapeutic targets for a number of disorders and diseases. Purified GPCR-based approaches including structural study and novel biophysical and biochemical function analyses are increasingly being used in GPCR-directed drug discovery. Before these approaches become routine, however, several hurdles need to be overcome; they include overexpression, solubilization, and purification of large quantities of functional and stable receptors on a regular basis. Here we report milligram production of a human formyl peptide receptor 3 (FPR3). FPR3 comprises a functionally distinct GPCR subfamily that is involved in leukocyte chemotaxis and activation. The bioengineered FPR3 was overexpressed in stable tetracycline-inducible mammalian cell lines (HEK293S). After a systematic detergent screening, fos-choline-14 (FC-14) was selected for subsequent solubilization and purification processes. A two-step purification method, immunoaffinity using anti-rho-tag monoclonal antibody 1D4 and gel filtration, was used to purify the receptors to near homogeneity. Immunofluorescence analysis showed that expressed FPR3 was predominantly displayed on cellular membrane. Secondary structural analysis using circular dichroism showed that the purified FPR3 receptor was correctly folded with >50% α-helix, which is similar to other known GPCR secondary structures. Our method can readily produce milligram quantities of human FPR3, which would facilitate in developing human FPR as therapeutic drug targets.
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Project description:The expression of six cell-surface proteoglycans (syndecan, fibroglycan, amphiglycan, glypican, betaglycan and CD44) was studied at the mRNA level. Analysis was performed by Northern blotting using total RNA preparations from freshly isolated articular chondrocytes obtained from both juveniles and adults. Similar results were obtained for both age groups. By far the most abundant message was that for amphiglycan, CD44 message was next in relative abundance, and the messages for fibroglycan, glypican and betaglycan were all expressed at low levels. Syndecan message could not be detected by this technique. This pattern of expression was different to that observed in cultured skin fibroblasts, where the messages for amphiglycan, CD44, fibroglycan and glypican were all expressed at a similar level. In contrast with the fibroblasts, where the amphiglycan message exhibits no size polymorphism, the chondrocyte amphiglycan message is present in three polymorphic forms, due to the use of alternative polyadenylation signals. When the newly isolated chondrocytes are maintained in monolayer culture for several passages, the amphiglycan message heterogeneity reverts to that characteristic of the fibroblasts. Thus human articular chondrocytes are characterized by both their high level of amphiglycan message expression and their use of alternative polyadenylation signals.
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