The effects of REV5901 on intracellular calcium signalling in freshly isolated bovine articular chondrocytes.

REV5901 is an inhibitor of regulatory volume decrease (RVD) a mechanotransduction pathway regulating cell volume in response to hypotonicity, with protective properties upon chondrocyte trauma impact in situ. As the mechanism of action of REV5901 is unknown and changes in intracellular calcium ([Ca2+]i) have been linked to REV5901-loading, we investigated the effects of REV5901 on a known calcium signalling pathway. Upon REV5901 loading, there was significant increase in [Ca2+]i reaching 37.97 ± 5.67%, above basal levels which was reduced to 27.86 ± 3.15% in the presence of 2 mmol/l EGTA. In the presence of U73122 or neomycin there was a decrease in calcium with inhibition factors (I.F.) of 0.39 ± 0.09 and 0.37 ± 0.08, respectively, whereas rottlerin abolished the REV5901-induced [Ca2+]i rise. The role of calcium channels in contributing to the REV5901-induced calcium rise was investigated whereby the calcium rise was inhibited in the absence of extracellular sodium and by the addition of Gd3+ and Ruthenium red. These data show a phospholipase Cß3-dependent release of calcium from intracellular stores as well as a sodium calcium exchanger-mediated influx in response to REV5901 loading, suggesting a potential role for calcium signalling in mediating the action of REV5901 in chondrocytes.

Novel methods for the quantification of changes in actin organization in chondrocytes using fluorescent imaging and linear profiling.

We present three novel reproducible methodologies for the quantification of changes in actin organization from microscope images. Striation and integrative analysis were devised for the investigation of trans-cellular filaments and F-actin localization, respectively, in response to physiological or mechanical actin-modulatory conditions. Additionally, the Parker-Qusous (PQ) formula was developed as a measure of total quantity of F-actin, independent of cell volume changes, whereby fluorescence intensity was divided by the cube root of cell volume, squared. Values obtained were quantified in Mauricean Units (Mu; pixel/µm(3)). Upon isolation, there was a 49% decrease in total F-actin fluorescence from 1.91 ± 0.16 pixel/µm(3) (Mu) to 0.95 ± 0.55 Mu, whereas upon culture, an apparent increase in total fluorescence was deemed insignificant due to an increase in average cell volume, with a rise, however, in striation units (StU) from 1 ± 1 to 5 ± 1 StU/cell, and a decrease in percentage cortical fluorescence to 30.45% ± 1.52% (P = 7.8 × 10(-5)). Freshly isolated chondrocytes exhibited a decrease in total F-actin fluorescence to 0.61 ± 0.05 Mu and 0.32 ± 0.02 Mu, 10 min posthypertonic and hypotonic challenges, respectively. Regulatory volume decrease was inhibited in the presence of REV5901 with maintenance of actin levels at 1.15 Mu. Following mechanical impact in situ, there was a reduction in total F-actin fluorescence to 0.95 ± 0.08 Mu and 0.74 ± 0.06 Mu under isotonic and hypotonic conditions, respectively, but not under hypertonic conditions. We report simple methodologies for quantification of changes in actin organization, which will further our understanding of the role of actin in various cellular stress responses. These techniques can be applied to better quantify changes in localization of various proteins using fluorescent labeling.

The phenotypic characterization of A13/BACii, a novel bovine chondrocytic cell line with differentiation potential.

In cartilage research bovine articular cartilage is used as an alternative to human tissue. However, animal material is subject to availability and primary cultures undergo senescence, limiting their use. Here we report the immortalization of primary bovine chondrocytes, which could be used as a surrogate for freshly isolated chondrocytes. Chondrocytes were isolated from cartilage explants and immortalized using 1.0 µg/ml benzo[alpha]pyrene. For 3-dimensional culture, chondrocytes were resuspended in 0.5% low-melt agarose at high density (HD) and cultured for 24 h prior to determining changes in expression profile and morphology. A13/BACii chondrocytes acquired a 'flat' irregular morphology and a foetal-like cell volume (1,509.59 ± 182.04 µm(3)). The human cell line C-20/A4 showed a statistically similar volume and length to A13/BACii. Two-dimensional-cultured A13/BACii expressed elevated levels of type I collagen (col1), reduced levels of type II collagen (col2) compared to freshly isolated chondrocytes and an overall col2 to col1 expression ratio (col2:col1) of 0.11 ± 0.01. Upon 3-dimensional encapsulation, there was a significant rise in col2 expression in both A13/BACii and C-20/A4, suggesting a capacity for redifferentiation in both cell lines with a return of col2:col1 values of A13/BACii to values previously observed in primary chondrocytes. A13/BACii chondrocytes expressed aggrecan, matrix metalloproteinase (MMP)-3, MMP-9 and MMP-13, further supporting indications of the differentiated phenotype. Here we report the creation of a novel chondrocytic cell line and demonstrate its strong potential for redifferentiation upon HD 3-dimensional encapsulation, providing an alternative to conventional dedifferentiated cell lines and primary culture.

Quantification of Changes in Morphology, Mechanotransduction, and Gene Expression in Bovine Articular Chondrocytes in Response to 2-Dimensional Culture Indicates the Existence of a Novel Phenotype.

OBJECTIVE: Matrix-induced autologous chondrocyte implantation (ACI) offers a potential solution for cartilage repair but is currently hindered by loss of the chondrocyte differentiated phenotype. To further our understanding of the mechanism of dedifferentiation, changes in the phenotype in relation to mechanotransduction were recorded in response to monolayer culture. METHODS: Bovine cartilage explants were excised and chondrocytes cultured for 9 days (P1), 14 days (P2), and 21 (P3) days. Changes in morphology and regulatory volume increase (RVI; a mechanotransduction response) were determined by the expression of key genes by RT-PCR and confocal microscopy, respectively. RESULTS: A loss of a differentiated phenotype was observed in P1 with a reduction in sphericity and an overall increase in cell volume from 474.7 ± 32.1 µm(3) to 725.2 ± 35.6 µm(3). Furthermore, the effect of 2-dimensional (2-D) culture-induced dedifferentiation on mechanotransduction was investigated, whereby RVI and Gd(3+)-sensitive REV5901-induced calcium rise were only observed in 2-D cultured chondrocytes. A significant up-regulation of types I and II collagens and Sox9 was observed in P1 chondrocytes and no further significant change in type I collagen but a return to baseline levels of type II collagen and Sox9 upon further culture. CONCLUSION: These data indicated the presence of an intermediate, mesodifferentiated phenotype and highlight the importance of mechanotransduction as a marker of the chondrocytic cell type.

siRNA-mediated inhibition of Na(+)-K(+)-2Cl- cotransporter (NKCC1) and regulatory volume increase in the chondrocyte cell line C-20/A4.

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.

Stimulation of regulatory volume increase (RVI) in avian articular chondrocytes by gadolinium chloride.

Chondrocytes, the resident cell-type of articular cartilage, are responsible for the regulation of the extracellular matrix (ECM) in response to their physico-chemical environment. Due to the nature of cartilage loading, chondrocytes are exposed to constant changes in extracellular osmolality with a gradual increase throughout the day. As an increase in osmolality attenuates matrix synthesis, we have studied cell volume regulation (regulatory volume increase (RVI)) after hypertonic challenge and the regulation of RVI by the actin cytoskeleton. Using freshly isolated avian articular chondrocytes, changes in actin organisation were studied by confocal laser scanning microscopy following a 43% increase in extracellular osmolality. Using calcein-loading chondrocytes, the capacity for RVI was determined and the rate of volume recovery (t1/2) mathematically extrapolated. Following an increase in extracellular osmolality there was a significant increase (p < 0.05) in cortical actin, inhibited by the removal of extracellular calcium EGTA or by the addition of 100 micromol.L-1 gadolinium chloride. Most cells exhibited slow RVI (t1/2 = 55.5 +/- 5.5 min), whereby inhibition of actin polymerisation by gadolinium chloride or the removal of extracellular calcium significantly increased the rate of volume recovery via a bumetanide-sensitive pathway (t1/2 of 29.6 +/- 6.5 min and 13.8 +/- 3.1 min, respectively). These data suggest the Na+-K+-2Cl(-) (NKCC) co-transporter regulated by the actin cytoskeleton is involved in avian chondrocyte RVI.

Regulatory volume increase (RVI) by in situ and isolated bovine articular chondrocytes.

Metabolism of the matrix by chondrocytes is sensitive to alterations in cell volume that occur, for example, during static loading and osteoarthritis. The ability of chondrocytes to respond to changes in volume could be important, and this study was aimed at testing the hypothesis that chondrocytes can regulate their volume following cell shrinking by regulatory volume increase (RVI). We used single cell fluorescence imaging of in situ bovine articular chondrocytes, cells freshly isolated into 280 or 380 mOsm, or 2-D cultured chondrocytes loaded with calcein or fura-2, to investigate RVI and changes to [Ca2+]i during shrinkage. Following a 42% hyperosmotic challenge, chondrocytes rapidly shrunk, however, only approximately 6% of the in situ or freshly isolated chondrocytes demonstrated RVI. This contrasted with 2D-cultured chondrocytes where approximately 54% of the cells exhibited RVI. The rate of RVI was the same for all preparations. During the 'post-RVD/RVI protocol', approximately 60% of the in situ and freshly isolated chondrocytes demonstrated RVD, but only approximately 5% showed RVI. There was no relationship between [Ca2+]i and RVI either during hyperosmotic challenge, or during RVD suggesting that changes to [Ca2+]i were not required for RVI. Depolymerisation of the actin cytoskeleton by latrunculin, increased RVI by freshly isolated chondrocytes, in a bumetanide-sensitive manner. The results showed that in situ and freshly isolated articular chondrocytes have only limited RVI capacity. However, RVI was stimulated by treating freshly isolated chondrocytes with latrunculin B and following 2D culture of chondrocytes, suggesting that cytoskeletal integrity plays a role in regulating RVI activity which appears to be mediated principally by the Na+ - K+ -2Cl- cotransporter.