NLRP3 promotes autophagy of urate crystals phagocytized by human osteoblasts.
BACKGROUND
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). 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.


METHODS
Normal 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.


RESULTS
Most 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.


CONCLUSIONS
MSU 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.
Isabelle
I
Allaeys
François
F
Marceau
Patrice E
PE
Poubelle
D000368:Aged; D001343:Autophagy / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D015151:Immunoblotting; D008297:Male; D018613:Microscopy, Confocal; D008875:Middle Aged; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D010006:Osteoblasts / Q000378:metabolism*; D010587:Phagocytosis / Q000502:physiology; D034741:RNA, Small Interfering; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D014527:Uric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; D034741:RNA, Small Interfering; D014527:Uric Acid