Korean J Physiol Pharmacol.  2017 Sep;21(5):531-546. 10.4196/kjpp.2017.21.5.531.

Time-dependent proteomic and genomic alterations in Toll-like receptor-4-activated human chondrocytes: increased expression of lamin A/C and annexins

• Ha SH
• Kim HK
• Anh NT
• Kim N
• Ko KS
• Rhee BD
• Han J

• KMID: 2388698
• DOI: http://doi.org/10.4196/kjpp.2017.21.5.531

Abstract

Activation of Toll-like receptor-4 (TLR-4) in articular chondrocytes increases the catabolic compartment and leads to matrix degradation during the development of osteoarthritis. In this study, we determined the proteomic and genomic alterations in human chondrocytes during lipopolysaccharide (LPS)-induced inflammation to elucidate the underlying mechanisms and consequences of TLR-4 activation. Human chondrocytes were cultured with LPS for 12, 24, and 36 h to induce TLR-4 activation. The TLR-4-induced inflammatory response was confirmed by real-time PCR analysis of increased interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) expression levels. In TLR-4-activated chondrocytes, proteomic changes were determined by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-mass spectroscopy analysis, and genomic changes were determined by microarray and gene ontology analyses. Proteomics analysis identified 26 proteins with significantly altered expression levels; these proteins were related to the cytoskeleton and oxidative stress responses. Gene ontology analysis indicated that LPS treatment altered specific functional pathways including "˜chemotaxis', "˜hematopoietic organ development', "˜positive regulation of cell proliferation', and "˜regulation of cytokine biosynthetic process'. Nine of the 26 identified proteins displayed the same increased expression patterns in both proteomics and genomics analyses. Western blot analysis confirmed the LPS-induced increases in expression levels of lamin A/C and annexins 4/5/6. In conclusion, this study identified the time-dependent genomic, proteomic, and functional pathway alterations that occur in chondrocytes during LPS-induced TLR-4 activation. These results provide valuable new insights into the underlying mechanisms that control the development and progression of osteoarthritis.

Keyword

Annexin 4/5/6; Chondrocyte; Lamin A/C; Omics; Toll-like receptor-4

MeSH Terms

Annexins*
Blotting, Western
Chondrocytes*
Cytoskeleton
Electrophoresis
Gene Ontology
Genomics
Humans*
Inflammation
Interleukin-1beta
Interleukin-6
Osteoarthritis
Oxidative Stress
Proteomics
Real-Time Polymerase Chain Reaction
Spectrum Analysis
Tumor Necrosis Factor-alpha
Annexins
Interleukin-1beta
Interleukin-6
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 LPS-induced changes in chondrocyte morphology and cytokine expression(A) Morphology of cultured human chondrocytes. Images of untreated control (CON), and after LPS treatment for 12, 24, and 36 h to activate TLR-4 (×200 magnification). Scale bar=100 µm. (B) Relative changes in mRNA expression of selected cytokines including TNF-α, IL-6, and IL-1β. Internal standard was GAPDH.

• 

  Fig. 2 Two-dimensional gel electrophoresis patterns of proteins in control and LPS-treated chondrocytes.(A)Proteins (200 µg each) isolated from of untreated control (Con) chondrocytes and after LPS treatment for 12, 24, and 36 h to activate TLR-4. Proteins were separated in the 20×24 cm gel with pH ranging from 4 to 10. (B) Twenty-seven significantly changed protein spots were selected for MALDI-TOF MS analysis to identify proteins (C). Histogram of time-dependent protein expression levels in untreated control (CON) chondrocytes and after LPS treatment for 12, 24, and 36 h to activate TLR-4 (*p<0.05).

• 

  Fig. 3 Protein-protein interaction (PPI) analysis of identified proteins.(A) The functional cluster network was constructed using 13 experimentally identified proteins. PPI and time-dependent protein expression changes were mapped in the network. (B~D) LPS-treated time-dependent comparative protein expression changes were visualized using the Cytoscape program after LPS treatment for 12 h (B), 24 h (C), and 36 h (D). Color bar expresses the fold-change ratio (LPS treatment/Control). Molecular and Biological Function: : energy metabolism antioxidant cytoskeletal and structure.

• 

  Fig. 4 Microarray results and biological pathway analysis in LPS-treated TLR-4-activated chondrocytes.(A) Venn diagram of up-regulated and down-regulated gene numbers in TLR-4 treated chondrocytes at 12, 24 and 36 h (B) Top 10 upregulated biological pathways in chondrocytes treated with LPS. (C) Top 10 downregulated biological pathways in chondrocytes treated with LPS.

• 

  Fig. 5 Cytokine-cytokine receptor interaction pathway map in KEGG presented with relative gene expression of 12, 24 and 36 h vs. Con.Red: up-regulated genes, Blue: down-regulated genes, Green: unchanged genes.

• 

  Fig. 6 Confirmation of lamin A/C and annexin expression by immunoblotting.(A) Representative immunoblot of lamin A/C expression in LPS-treated chondrocytes. (B) Histogram of untreated control (CON) chondrocytes and after LPS treatment for 12, 24, and 36 h to activate TLR-4 (*p<0.05). (C) Representative immunoblot of annexin 4/5/6 expression in LPS-treated chondrocytes. (D) Histogram of untreated control (CON) chondrocytes and after LPS treatment for 12, 24, and 36 h to activate TLR-4 (*p<0.05) (n=3).

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