title: siRNA-mediated inhibition of Na(+)-K(+)-2Cl- cotransporter (NKCC1) and regulatory volume increase in the chondrocyte cell line C-20/A4.
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.
authors.affiliation: School of Life Sciences, University of Westminter, 115 New Cavendish Street, London W1W 6UW, UK.
authors.forename: Ala
authors.initials: A
authors.lastname: Qusous
authors.forename: Corinne S V
authors.initials: CS
authors.lastname: Geewan
authors.forename: Pamela
authors.initials: P
authors.lastname: Greenwell
authors.forename: Mark J P
authors.initials: MJ
authors.lastname: Kerrigan
mesh_terms: D002460:Cell Line; D048429:Cell Size; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D018613:Microscopy, Confocal; D034622:RNA Interference; D034741:RNA, Small Interfering; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D014162:Transfection
chemical_list: D034741:RNA, Small Interfering; C578877:SLC12A2 protein, human; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2