Alterations in Macrophage Cellular Proteome Induced by Calcium Oxalate Crystals: The Association of HSP90 and F‑Actin Is Important for Phagosome Formation

The presence of macrophages in renal interstitium is the key feature of progressive renal inflammation in kidney stone disease. However, response of macrophages to calcium oxalate monohydrate (COM) crystals, the major crystalline composition of kidney stone, remained unclear. This study aimed to inv...

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Bibliographic Details
Published in:Journal of proteome research 2013-08, Vol.12 (8), p.3561-3572
Main Authors: Singhto, Nilubon, Sintiprungrat, Kitisak, Thongboonkerd, Visith
Format: Article
Language:English
Subjects:
Actins - genetics
Actins - metabolism
Calcium Oxalate - pharmacology
Cell Line, Tumor
Cell Movement - drug effects
Cytoskeletal Proteins - genetics
Cytoskeletal Proteins - metabolism
Electrophoresis, Gel, Two-Dimensional
Fluorescent Dyes
Gene Expression
Gene Expression Profiling
HSP90 Heat-Shock Proteins - antagonists & inhibitors
HSP90 Heat-Shock Proteins - genetics
HSP90 Heat-Shock Proteins - metabolism
Humans
Macrophages - cytology
Macrophages - drug effects
Macrophages - metabolism
Microscopy, Fluorescence
Phagocytosis - drug effects
Phagosomes - drug effects
Phagosomes - metabolism
Phagosomes - ultrastructure
Protein Interaction Mapping
Proteome - genetics
Proteome - metabolism
rab5 GTP-Binding Proteins - genetics
rab5 GTP-Binding Proteins - metabolism
RNA, Small Interfering - genetics
RNA, Small Interfering - metabolism
Tubulin - genetics
Tubulin - metabolism
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Summary:The presence of macrophages in renal interstitium is the key feature of progressive renal inflammation in kidney stone disease. However, response of macrophages to calcium oxalate monohydrate (COM) crystals, the major crystalline composition of kidney stone, remained unclear. This study aimed to investigate alterations in the cellular proteome of macrophages induced by COM crystals using a proteomics approach. U937-derived macrophages (by phorbol-12-myristate-13-acetate activation) were incubated without or with 100 μg/mL COM crystals for 24 h. Their cellular proteins were resolved by 2-DE (n = 10 gels; 5 were derived from 5 independent cultures in each group) and visualized with Deep Purple fluorescent dye. Spot matching, quantitative intensity analysis, and statistics revealed 18 differentially expressed protein spots, which were successfully identified by Q-TOF MS and MS/MS analyses. The altered levels of α-tubulin, β-actin and ezrin were validated by Western blot analysis. Protein interaction network analysis using STRING software showed that 90 kDa heat shock protein (HSP90) was associated with β-actin and α-tubulin (all these three proteins were increased in the COM-treated macrophages). Multiple immunofluorescence stainings confirmed the associations of HSP90 with filamentous form of actin (F-actin) and α-tubulin. However, only the association between HSP90 and F-actin was found on the phagosome membrane surrounding COM crystal, indicating that the association of HSP90 with F-actin, but not with α-tubulin, is important for phagosome formation. Silencing of HSP90 (siHSP90) reduced expression of cytoskeletal proteins and phagosome marker (Rab5) and successfully diminished COM crystal-induced phagocytosis and migration of macrophages. Our findings enlightened the significant role of these altered proteins, especially HSP90, in enhanced phagocytic activity of the COM-exposed macrophages.
ISSN:1535-3893
1535-3907
DOI:10.1021/pr4004097