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NLRP3 promotes autophagy of urate crystals phagocytized by human osteoblasts.

Allaeys I, Marceau F, Poubelle PE - Arthritis Res. Ther. (2013)

Bottom Line: 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.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.

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ABSTRACT

Introduction: 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.

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Effects of MSU on OB viability and proliferation. (A) Confluent OBs were incubated with vehicle (control) or with MSU from 0.3 to 1 mg/106 cells for 24, 48, or 72 hours. Cells were washed with PBS and removed with Accutase. Propidium iodide (PI) exclusion was analyzed with cytofluorometry. (B) Cells were plated 3 days with vehicle or with MSU in 96 wells and then analyzed with the CellTiter 96 AQueous One proliferation assay. Data are represented as a ratio of MSU-stimulated to vehicle-stimulated cells. Results are given as mean ± SEM of values from four (A) or three (B) different donors. Statistical analysis: one-way ANOVA followed by Bonferroni multiple-comparison test: ns, nonsignificant; means without a common letter differ: P < 0.05.
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Figure 2: Effects of MSU on OB viability and proliferation. (A) Confluent OBs were incubated with vehicle (control) or with MSU from 0.3 to 1 mg/106 cells for 24, 48, or 72 hours. Cells were washed with PBS and removed with Accutase. Propidium iodide (PI) exclusion was analyzed with cytofluorometry. (B) Cells were plated 3 days with vehicle or with MSU in 96 wells and then analyzed with the CellTiter 96 AQueous One proliferation assay. Data are represented as a ratio of MSU-stimulated to vehicle-stimulated cells. Results are given as mean ± SEM of values from four (A) or three (B) different donors. Statistical analysis: one-way ANOVA followed by Bonferroni multiple-comparison test: ns, nonsignificant; means without a common letter differ: P < 0.05.

Mentions: Because MSU can modulate cellular apoptosis and proliferation [59,60], the impact of MSU on OB survival and proliferation was evaluated before studying specialized OB functions. MSU at concentrations up to 1 mg/106 cells for 72 hours of culture did not modify the incorporation of propidium iodide (PI) by OBs, and an average of 80% PI-negative OBs was routinely obtained in control conditions, as well as in the presence of MSU (Figure 2A). In contrast, the proliferation rate of MSU-treated OBs dose-dependently decreased from 0.1 to 1 mg MSU/106 cells (Figure 2B). The significant threshold reduction was observed at 0.3 mg MSU, with a plateau of reduction attained at 0.8 mg MSU. The respective proliferation rates were reduced from 30% to 55% of the OB proliferation rate in control conditions. Thus, although MSU microcrystals at the concentrations tested did not modify the viability of OBs, they significantly decreased the proliferation of OBs and could, in parallel, affect other functions.


NLRP3 promotes autophagy of urate crystals phagocytized by human osteoblasts.

Allaeys I, Marceau F, Poubelle PE - Arthritis Res. Ther. (2013)

Effects of MSU on OB viability and proliferation. (A) Confluent OBs were incubated with vehicle (control) or with MSU from 0.3 to 1 mg/106 cells for 24, 48, or 72 hours. Cells were washed with PBS and removed with Accutase. Propidium iodide (PI) exclusion was analyzed with cytofluorometry. (B) Cells were plated 3 days with vehicle or with MSU in 96 wells and then analyzed with the CellTiter 96 AQueous One proliferation assay. Data are represented as a ratio of MSU-stimulated to vehicle-stimulated cells. Results are given as mean ± SEM of values from four (A) or three (B) different donors. Statistical analysis: one-way ANOVA followed by Bonferroni multiple-comparison test: ns, nonsignificant; means without a common letter differ: P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061723&req=5

Figure 2: Effects of MSU on OB viability and proliferation. (A) Confluent OBs were incubated with vehicle (control) or with MSU from 0.3 to 1 mg/106 cells for 24, 48, or 72 hours. Cells were washed with PBS and removed with Accutase. Propidium iodide (PI) exclusion was analyzed with cytofluorometry. (B) Cells were plated 3 days with vehicle or with MSU in 96 wells and then analyzed with the CellTiter 96 AQueous One proliferation assay. Data are represented as a ratio of MSU-stimulated to vehicle-stimulated cells. Results are given as mean ± SEM of values from four (A) or three (B) different donors. Statistical analysis: one-way ANOVA followed by Bonferroni multiple-comparison test: ns, nonsignificant; means without a common letter differ: P < 0.05.
Mentions: Because MSU can modulate cellular apoptosis and proliferation [59,60], the impact of MSU on OB survival and proliferation was evaluated before studying specialized OB functions. MSU at concentrations up to 1 mg/106 cells for 72 hours of culture did not modify the incorporation of propidium iodide (PI) by OBs, and an average of 80% PI-negative OBs was routinely obtained in control conditions, as well as in the presence of MSU (Figure 2A). In contrast, the proliferation rate of MSU-treated OBs dose-dependently decreased from 0.1 to 1 mg MSU/106 cells (Figure 2B). The significant threshold reduction was observed at 0.3 mg MSU, with a plateau of reduction attained at 0.8 mg MSU. The respective proliferation rates were reduced from 30% to 55% of the OB proliferation rate in control conditions. Thus, although MSU microcrystals at the concentrations tested did not modify the viability of OBs, they significantly decreased the proliferation of OBs and could, in parallel, affect other functions.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: 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.

Show MeSH
Related in: MedlinePlus