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siRNA-Mediated Inhibition of Na+ –K+–2Cl− Cotransporter (NKCC1) and Regulatory Volume Increase in the Chondrocyte Cell Line C-20/A4

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Abstract

The Na+–K+–2Cl− cotransporter (NKCC1) is an essential membrane transporter and has been linked to the regulation of volume, matrix synthesis and bone growth in chondrocytes; the sole resident cell type of articular cartilage. Despite the integral nature of NKCC1, its regulation is currently poorly understood, and therefore here we describe a NKCC1 knockdown technique that will permit the easier study of this transporter. Small interfering RNA (siRNA), designed to knock down NKCC1, was transfected into the chondrocyte cell line C-20/A4 and the efficacy determined at the message, protein and functional levels. NKCC1 expression was analyzed by reverse-transcriptase polymerase chain reaction, where NKCC1 expression declined to 25.10 ± 1.08% after 12 h of transfection and did not show any rise in the following 36 h. The efficacy of the designed siRNA molecules was confirmed by both Western blot and immunocytochemistry. The effect of the knockdown on regulatory volume increase (RVI, a novel assay for NKCC1 function) was investigated by confocal laser scanning microscopy in response to a 43% hypertonic challenge, whereby control chondrocytes underwent a decrease in volume to 67.38 ± 1.70%, followed by volume restoration to 82.17 ± 2.23 at 20 min (t½ = 22.11 ± 3.23 min). Conversely, upon knockdown, chondrocytes exhibited a slower rate of RVI (t½ = 43.26 ± 5.64 min), thus suggesting that NKCC1 plays an important and yet partial role in RVI in C-20/A4 chondrocytes. Together, these data provide a robust protocol for the study of NKCC1 in chondrocytes and suggest a mechanism for C-20/A4 chondrocyte RVI.

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Acknowledgments

The authors thank Dr. Ian Locke for donating C-20/A4 chondrocytes, Dr. Peter Flatman for the initial sample of T4 antibodies and Dr. Sanjiv Rughooputh for his assistance and feedback. This work was partly funded by the Wellcome Trust and made possible thanks to Mr. Aiman T. Qusous and Ms. Najda Goussous. T4 antibody (contribution of Lytle and Forbush III) was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology (Iowa City, IA 52242).

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  1. School of Life Sciences, University of Westminter, 115 New Cavendish Street, London, W1W 6UW, UK

    Ala Qusous, Corinne S. V. Geewan & Pamela Greenwell

  2. School of Science, University of Greenwich, Chatham Maritime, ME4 4TB, UK

    Mark J. P. Kerrigan

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  1. Ala Qusous
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Correspondence to Mark J. P. Kerrigan.

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Qusous, A., Geewan, C.S.V., Greenwell, P. et al. siRNA-Mediated Inhibition of Na+ –K+–2Cl− Cotransporter (NKCC1) and Regulatory Volume Increase in the Chondrocyte Cell Line C-20/A4. J Membrane Biol 243, 25 (2011). https://doi.org/10.1007/s00232-011-9389-z

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