Abstract
IntroductionMonosodium urate (MSU) microcrystals present in bone tissues of chronic gout can be ingested by nonprofessional phagocytes like osteoblasts (OBs) that express NLRP3 (nucleotide-binding domain and leucine-rich repeat region containing family of receptor protein 3). MSU is known to activate NLRP3 inflammasomes in professional phagocytes. We have identified a new role for NLRP3 coupled to autophagy in MSU-stimulated human OBs.MethodsNormal human OBs cultured in vitro were investigated for their capacity for phagocytosis of MSU microcrystals by using confocal microscopy. Subsequent mineralization and matrix metalloproteinase activity were evaluated, whereas regulatory events of phagocytosis were deciphered by using signaling inhibitors, phosphokinase arrays, and small interfering RNAs. Statistics were carried out by using paired or unpaired t tests, and the one-way ANOVA, followed by multiple comparison test.ResultsMost of the OBs internalized MSU in vacuoles. This process depends on signaling via PI3K, protein kinase C (PKC), and spleen tyrosine kinase (Syk), but is independent of Src kinases. Simultaneously, MSU decreases phosphorylation of the protein kinases TOR (target of rapamycin) and p70S6K. MSU activates the cleavage of microtubule-associated protein light chain 3 (LC3)-I into LC3-II, and MSU microcrystals are coated with GFP-tagged LC3. However, MSU-stimulated autophagy in OBs absolutely requires the phagocytosis process. We find that MSU upregulates NLRP3, which positively controls the formation of MSU-autophagosomes in OBs. MSU does not increase death and late apoptosis of OBs, but reduces their proliferation in parallel to decreasing their competence for mineralization and to increasing their matrix metalloproteinase activity.ConclusionsMSU microcrystals, found locally encrusted in the bone matrix of chronic gout, activate phagocytosis and NLRP3-dependent autophagy in OBs, but remain intact in permanent autophagosomes while deregulating OB functions.
Highlights
Monosodium urate (MSU) microcrystals present in bone tissues of chronic gout can be ingested by nonprofessional phagocytes like osteoblasts (OBs) that express NLRP3
The addition of MSU to OBs transfected with green fluorescent protein (GFP)-tagged light chain 3 (LC3) showed a rapid increase of labeled vacuoles in their cytosol, as well as MSU coated with GFPtagged LC3 (Figure 5D). These results indicate that MSU in human OBs induced endogenous LC3 conversion and stimulated the process of autophagy while they were progressively engulfed in OBs
No modification of IκB phosphorylation was detected in OBs activated by MSU, whereas TNF-α addition to OBs was typically associated with changes of IκB phosphorylation (Figure 6C). These results indicate that OBs respond to MSU (a) by a primary nonconventional phagocytosis followed by (b) a secondary autophagy, (c) by activating NLRP3 protein without concomitant IL-1β generation, and (d) by no signal through the NF-κB pathway
Summary
Monosodium urate (MSU) microcrystals present in bone tissues of chronic gout can be ingested by nonprofessional phagocytes like osteoblasts (OBs) that express NLRP3 (nucleotide-binding domain and leucine-rich repeat region containing family of receptor protein 3). We have identified a new role for NLRP3 coupled to autophagy in MSU-stimulated human OBs. Uric acid is an obligatory physiologic breakdown product of purine metabolism. Uric acid is an obligatory physiologic breakdown product of purine metabolism This compound is soluble in the cytosol of cells and in plasma. The same crystals were shown nonspecifically to bind to dendritic cell-surface lipids with activation of immunoreceptor tyrosine-based motifs and subsequent recruitment of spleen tyrosine kinase (Syk) and PI3K activation [13]. These different pathways of cell activation by MSU are followed by phagocytosis of the solid particles
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