Article
Role of aquaporin-1 in the pathogenesis of human osteoarthritis
Search Medline for
Authors
Published: | September 1, 2015 |
---|
Outline
Text
Introduction: Therapeutic options for osteoarthritis (OA) are still limited to symptomatic treatment and, finally, end-stage surgical interventions. Screening for potential causative molecular options revealed the transmembrane water channel aquaporin-1 (AQP1) being up-regulated in damaged OA articular cartilage compared to intact zones. Therefore, we analyzed the putative functional role of AQP1 during OA by induction and silencing of its gene expression in human articular chondrocytes.
Methods: Chondrocytes were obtained from knee explants of OA patients undergoing knee replacement surgery. For AQP1 knockdown, cells were nucleofected with siRNA targeting human AQP1. For overexpression, respectively, a plasmid vector carrying human AQP1 gene was used for nucleofection. After verification of successful silencing and induction on the RNA and protein level, the implications for marker gene expression of the chondrocyte metabolism as well as for other relevant molecules were analyzed using real-time quantitative PCR.
Results: AQP1 mRNA was significantly decreased 24 h after AQP1 knockdown in human OA chondrocytes (p≤0.001) compared to cells treated with non-targeting siRNA. This was verified also on the protein level by immunocytochemistry. After 48 h, there was no significant change in the expression of collagen types II (1.49; ≤0.079) (fold change; p-value), I (1.50; ≤0.176) and IX (1.28; ≤0.215), Sox9 (1.22; ≤0.605), ADAMTS-4 (1.43; ≤0.701) and -5 (0.79; ≤0.058), MMP9 (0.88; ≤0.585) and 13 (2.53; ≤0.122), and Lin-2 (1.20; ≤0.549). 72 hours after nucleofection, AQP1 overexpression, in contrast, also did not lead to any significant changes in the expression of collagens type I, MMP9, ADAMTS-4 and -5, Lin-2 and -7, respectively, when compared to control cells. TIMP3, as well, was not influenced. Although the expression of collagens type II (-2.71±1.1) and X (-3.36±0.03) as well as of SOX9 (-1.76±0.8) was more obviously down-regulated, this was also seen without statistical significance.
Conclusion: Neither AQP1 silencing nor over-expression showed significant influence on typical metabolic or homeostatic parameters in cultivated chondrocytes. This supports the hypothesis that water channels indeed are functionally restricted to fluid and cell volume regulation. Using a more homogeneous study population, comparing with healthy donors as well as monitoring a broader spectrum of biological parameters might be helpful to gain additional relevant functional insights.