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. 2013:2013:679763.
doi: 10.1155/2013/679763. Epub 2013 Mar 19.

Dysfunction of collagen synthesis and secretion in chondrocytes induced by wisp3 mutation

Min Wang  1 , Xiao-Fei Man, Ya-Qing Liu, Er-Yuan Liao, Zhi-Feng Shen, Xiang-Hang Luo, Li-Juan Guo, Xian-Ping Wu, Hou-De Zhou
Affiliations

Dysfunction of collagen synthesis and secretion in chondrocytes induced by wisp3 mutation

Min Wang et al. Int J Endocrinol. 2013.

Abstract

Wisp3 gene mutation was shown to cause spondyloepiphyseal dysplasia tarda with progressive arthropathy (SRDT-PA), but the underlying mechanism is not clear. To clarify this mechanism, we constructed the wild and mutated Wisp3 expression vectors and transfected into human chondrocytes lines C-20/A4; Wisp3 proteins subcellular localization, cell proliferation, cell apoptosis, and Wisp3-mediated gene expression were determined, and dynamic secretion of collagen in transfected chondrocytes was analyzed by (14)C-proline incorporation experiment. Mutated Wisp3 protein increased proliferation activity, decreased apoptosis of C-20/A4 cells, and aggregated abnormally in cytoplasm. Expression of collagen II was also downregulated in C-20/A4 cells transfected with mutated Wisp3. Wild type Wisp3 transfection increased intracellular collagen content and extracellular collagen secretion, but the mutated Wisp3 lost this function, and the peak phase of collagen secretion was delayed in mutated Wisp3 transfected cells. Thus abnormal protein distribution, cell proliferation, collagen synthesis, and secretion in Wisp3 mutated chondrocytes might contribute to the pathogenesis of SEDT-PA.

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Figures

Figure 1
Figure 1
Localization of wild and mutated Wisp3 protein in C20/A4 cells by confocal microscope. Recombined plasmids WT-Wisp3/pEGFP-C2, MUT1000T/C/pEGFP-C2, and MUT840delT/pEGFP-C2 were transfected transiently into human chondrocyte cell line C20/A4, and pEGFP-C2 vector was used as a control. The cells were observed using a confocal laser scanning microscope after 48 hours of transfection at magnification 1000x. (a) EGFP; (b) WT-Wisp3; (c) MUT1000T/C; (d) MUT840delT; Green fluorescence indicate the Wisp3 EGFP fusion protein. Blue fluorescence shows cell nuclei dye by DAPI. Note the distribution of WT-Wisp3 in cytoplasm and cell membrane uniformly. In contrast, the majority of MUT1000T/C and MUT840delT were aggregated to speckles or agglomerates in cytoplasm.
Figure 2
Figure 2
Cell viability and cycle analysis in C20/A4 cells transfected with Wisp3. Cells stably transfected with MUT1000T/C/pcDNA3.1(+), MUT840delT/pcDNA3.1(+) or empty vector, and cell viability (a) were determined by MTT and cell cycle was evaluated using flow cytometry. (b) Empty vector; (c) MUT1000T/C; (d) MUT840delT. *P < 0.01 compared with C20/A4 cells transfected with empty vector.
Figure 3
Figure 3
Cell apoptosis analysis in C20/A4 cells transfected with mutant Wisp3 Cells stably transfected with MUT1000T/C/pcDNA3.1(+), MUT840delT/pcDNA3.1(+), or empty vector; cell apoptosis was evaluated by using FAC flow cytometry (a–c) and acridine orange/ethidium bromide staining (d–f) (apoptotic cells stained with yellow, condensed, or fragmented nuclei) analysis. (a) and (d) empty vector apoptosis rate is 27.1%; (b and e) MUT1000T/C apoptosis rate is 9.2%; (c and f) MUT840delT apoptosis rate is 7.8%. Magnification is 200 x.
Figure 4
Figure 4
mRNA expression of cartilage-specific genes in C20/A4 cells transfected with wild and mutant Wisp3. Cells stably transfected with WT-Wisp3/pcDNA3.1(+), MUT1000T/C/pcDNA3.1(+), MUT840delT/pcDNA3.1(+), or empty vector. mRNA expression of cartilage-specific genes in C20/A4 cells was determined by RT-PCR. (a)–(g) represent Wisp3, COL2A1, COL1A1, SOX9, fibronectin, MMP-1, and β-actin separately.
Figure 5
Figure 5
Wisp3 and COL2A1 protein expression in C20/A4 cells transfected with wild and mutant Wisp3 analyzed by western blot.
Figure 6
Figure 6
Change of intracellular collagen content and extracellular collagen secretion in mutant chondrocytes analyzed by 14C-proline incorporation assay. (a) Time course of 14C-proline labeled collagen (detected by radioactivity) secreted to the supernatant of the cultured chondrocytes. (b) Time course of 14C-proline labeled collagen content in cultured chondrocytes. (c) Ratio of secreted collagen to intracellular collagen.
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