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Hyperosmolarity regulates SOX9 mRNA posttranscriptionally in human articular chondrocytes.

Tew SR, Peffers MJ, McKay TR, Lowe ET, Khan WS, Hardingham TE, Clegg PD - Am. J. Physiol., Cell Physiol. (2009)

Bottom Line: The effect was strongest and most reproducible when actin stress fibers were disrupted by the Rho effector kinase inhibitor Y27632, or by culturing the cells within alginate beads.Freshly isolated chondrocytes, used within 24-48 h of isolation, did not contain actin stress fibers and upregulated SOX9 mRNA in response to hyperosmolarity in the presence and absence of Y27632.The results showed that the osmotic environment regulated both SOX9 and COL2A1 mRNA posttranscriptionally, but in fresh cells resulted in increased SOX9, but decreased COL2A1.

View Article: PubMed Central - PubMed

Affiliation: Veterinary Clinical Sciences, University of Liverpool Veterinary Teaching Hospital, Leahurst, Neston, Cheshire, United Kingdom. tew@liverpool.ac.uk

ABSTRACT
The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. We previously showed that activation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA (Tew SR and Hardingham TE. J Biol Chem 281: 39471-39479, 2006). In this study we investigated whether regulation of p38 MAPK caused by changes in osmotic pressure could control SOX9 mRNA levels expression by a similar mechanism. Primary human articular chondrocytes isolated from osteoarthritic cartilage at passage 2-4 showed significantly raised SOX9 mRNA levels when exposed to hyperosmotic conditions for 5 h. The effect was strongest and most reproducible when actin stress fibers were disrupted by the Rho effector kinase inhibitor Y27632, or by culturing the cells within alginate beads. Freshly isolated chondrocytes, used within 24-48 h of isolation, did not contain actin stress fibers and upregulated SOX9 mRNA in response to hyperosmolarity in the presence and absence of Y27632. In these freshly isolated chondrocytes, hyperosmolarity led to an increase in the half-life of SOX9 mRNA, which was sensitive to the p38 MAPK inhibitor SB202190. SOX9 protein levels were increased by hyperosmotic culture over 24 h, and, in passaged chondrocytes, the activity of a COL2A1 enhancer driven luciferase assay was upregulated. However, in freshly isolated chondrocytes, COL2A1 mRNA levels were reduced by hyperosmotic conditions and the half-life was decreased. The results showed that the osmotic environment regulated both SOX9 and COL2A1 mRNA posttranscriptionally, but in fresh cells resulted in increased SOX9, but decreased COL2A1.

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Effect of hyperosmolarity on SOX9 mRNA levels in passaged human articular chondrocytes (HAC). A: real-time PCR analysis of SOX9 mRNA levels in passaged HAC cultured under different osmotic conditions (given in mosM on x-axis) in the presence and absence of Y27632 (10 μM) for 5 h. Data presented were normalized using GAPDH expression and represent means and SDs from experiments conducted on cells from between 4 and 6 different donors. *Mixed-effects linear regression-interaction between hyperosmotic conditions and Y27632 (P < 0.05). B: real-time PCR analysis of SOX9 mRNA levels in passaged HAC cultured in alginate beads for 3 days in standard growth media before rinsing 3 times in serum-free DMEM and then cultured for 5 h in serum-free DMEM adjusted to 270, 380, or 550 mosM. Data are presented as means and SDs of expression values in HAC from 3 donors. *P < 0.05 vs. both 270 and 380 mosM, one-way ANOVA and Bonferroni post hoc test.
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Figure 1: Effect of hyperosmolarity on SOX9 mRNA levels in passaged human articular chondrocytes (HAC). A: real-time PCR analysis of SOX9 mRNA levels in passaged HAC cultured under different osmotic conditions (given in mosM on x-axis) in the presence and absence of Y27632 (10 μM) for 5 h. Data presented were normalized using GAPDH expression and represent means and SDs from experiments conducted on cells from between 4 and 6 different donors. *Mixed-effects linear regression-interaction between hyperosmotic conditions and Y27632 (P < 0.05). B: real-time PCR analysis of SOX9 mRNA levels in passaged HAC cultured in alginate beads for 3 days in standard growth media before rinsing 3 times in serum-free DMEM and then cultured for 5 h in serum-free DMEM adjusted to 270, 380, or 550 mosM. Data are presented as means and SDs of expression values in HAC from 3 donors. *P < 0.05 vs. both 270 and 380 mosM, one-way ANOVA and Bonferroni post hoc test.

Mentions: Initially, HAC which had been expanded in monolayer culture to passage 2-3 were used to determine the effect of medium osmolarity on SOX9 mRNA expression (Fig. 1A). Culture media with a normal osmolarity (380 mosM) was used as a control condition, with 270 mosM and 550 mosM media providing hypo- and hyperosmotic stimulation, respectively. In monolayer cultures after 5 h there was no significant difference between SOX9 mRNA levels at any osmolarity examined. However, when the chondrocytes were cultured with the ROCK inhibitor Y27632, SOX9 mRNA was upregulated by 2.4-fold in the 550 mosM cultures compared with 380 mosM cultures. Mixed-effects linear regression of the results showed that there was a specific interaction between 550 mosM conditions and the presence of Y27632 leading to the higher expression of SOX9 mRNA (P < 0.05). The effect of further modification of the cytoskeleton was investigated by culturing the chondrocytes in alginate beads, which leads to rounded cell morphology and a cortical actin distribution with no stress fibers (34, 43) (Fig. 1B). The passaged HAC were grown in alginate for 3 days before this experiment, and during this time, SOX9 mRNA levels increased markedly due to cellular redifferentiation caused by the rounded cell shape (34). A 5-h exposure to hyperosmotic culture was able to induce a further, significant (P < 0.01) 3.2-fold increase in the expression of SOX9 mRNA than was observed in 380 mosM cultures, and this was without a requirement for Y27632 treatment.


Hyperosmolarity regulates SOX9 mRNA posttranscriptionally in human articular chondrocytes.

Tew SR, Peffers MJ, McKay TR, Lowe ET, Khan WS, Hardingham TE, Clegg PD - Am. J. Physiol., Cell Physiol. (2009)

Effect of hyperosmolarity on SOX9 mRNA levels in passaged human articular chondrocytes (HAC). A: real-time PCR analysis of SOX9 mRNA levels in passaged HAC cultured under different osmotic conditions (given in mosM on x-axis) in the presence and absence of Y27632 (10 μM) for 5 h. Data presented were normalized using GAPDH expression and represent means and SDs from experiments conducted on cells from between 4 and 6 different donors. *Mixed-effects linear regression-interaction between hyperosmotic conditions and Y27632 (P < 0.05). B: real-time PCR analysis of SOX9 mRNA levels in passaged HAC cultured in alginate beads for 3 days in standard growth media before rinsing 3 times in serum-free DMEM and then cultured for 5 h in serum-free DMEM adjusted to 270, 380, or 550 mosM. Data are presented as means and SDs of expression values in HAC from 3 donors. *P < 0.05 vs. both 270 and 380 mosM, one-way ANOVA and Bonferroni post hoc test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2770738&req=5

Figure 1: Effect of hyperosmolarity on SOX9 mRNA levels in passaged human articular chondrocytes (HAC). A: real-time PCR analysis of SOX9 mRNA levels in passaged HAC cultured under different osmotic conditions (given in mosM on x-axis) in the presence and absence of Y27632 (10 μM) for 5 h. Data presented were normalized using GAPDH expression and represent means and SDs from experiments conducted on cells from between 4 and 6 different donors. *Mixed-effects linear regression-interaction between hyperosmotic conditions and Y27632 (P < 0.05). B: real-time PCR analysis of SOX9 mRNA levels in passaged HAC cultured in alginate beads for 3 days in standard growth media before rinsing 3 times in serum-free DMEM and then cultured for 5 h in serum-free DMEM adjusted to 270, 380, or 550 mosM. Data are presented as means and SDs of expression values in HAC from 3 donors. *P < 0.05 vs. both 270 and 380 mosM, one-way ANOVA and Bonferroni post hoc test.
Mentions: Initially, HAC which had been expanded in monolayer culture to passage 2-3 were used to determine the effect of medium osmolarity on SOX9 mRNA expression (Fig. 1A). Culture media with a normal osmolarity (380 mosM) was used as a control condition, with 270 mosM and 550 mosM media providing hypo- and hyperosmotic stimulation, respectively. In monolayer cultures after 5 h there was no significant difference between SOX9 mRNA levels at any osmolarity examined. However, when the chondrocytes were cultured with the ROCK inhibitor Y27632, SOX9 mRNA was upregulated by 2.4-fold in the 550 mosM cultures compared with 380 mosM cultures. Mixed-effects linear regression of the results showed that there was a specific interaction between 550 mosM conditions and the presence of Y27632 leading to the higher expression of SOX9 mRNA (P < 0.05). The effect of further modification of the cytoskeleton was investigated by culturing the chondrocytes in alginate beads, which leads to rounded cell morphology and a cortical actin distribution with no stress fibers (34, 43) (Fig. 1B). The passaged HAC were grown in alginate for 3 days before this experiment, and during this time, SOX9 mRNA levels increased markedly due to cellular redifferentiation caused by the rounded cell shape (34). A 5-h exposure to hyperosmotic culture was able to induce a further, significant (P < 0.01) 3.2-fold increase in the expression of SOX9 mRNA than was observed in 380 mosM cultures, and this was without a requirement for Y27632 treatment.

Bottom Line: The effect was strongest and most reproducible when actin stress fibers were disrupted by the Rho effector kinase inhibitor Y27632, or by culturing the cells within alginate beads.Freshly isolated chondrocytes, used within 24-48 h of isolation, did not contain actin stress fibers and upregulated SOX9 mRNA in response to hyperosmolarity in the presence and absence of Y27632.The results showed that the osmotic environment regulated both SOX9 and COL2A1 mRNA posttranscriptionally, but in fresh cells resulted in increased SOX9, but decreased COL2A1.

View Article: PubMed Central - PubMed

Affiliation: Veterinary Clinical Sciences, University of Liverpool Veterinary Teaching Hospital, Leahurst, Neston, Cheshire, United Kingdom. tew@liverpool.ac.uk

ABSTRACT
The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. We previously showed that activation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA (Tew SR and Hardingham TE. J Biol Chem 281: 39471-39479, 2006). In this study we investigated whether regulation of p38 MAPK caused by changes in osmotic pressure could control SOX9 mRNA levels expression by a similar mechanism. Primary human articular chondrocytes isolated from osteoarthritic cartilage at passage 2-4 showed significantly raised SOX9 mRNA levels when exposed to hyperosmotic conditions for 5 h. The effect was strongest and most reproducible when actin stress fibers were disrupted by the Rho effector kinase inhibitor Y27632, or by culturing the cells within alginate beads. Freshly isolated chondrocytes, used within 24-48 h of isolation, did not contain actin stress fibers and upregulated SOX9 mRNA in response to hyperosmolarity in the presence and absence of Y27632. In these freshly isolated chondrocytes, hyperosmolarity led to an increase in the half-life of SOX9 mRNA, which was sensitive to the p38 MAPK inhibitor SB202190. SOX9 protein levels were increased by hyperosmotic culture over 24 h, and, in passaged chondrocytes, the activity of a COL2A1 enhancer driven luciferase assay was upregulated. However, in freshly isolated chondrocytes, COL2A1 mRNA levels were reduced by hyperosmotic conditions and the half-life was decreased. The results showed that the osmotic environment regulated both SOX9 and COL2A1 mRNA posttranscriptionally, but in fresh cells resulted in increased SOX9, but decreased COL2A1.

Show MeSH
Related in: MedlinePlus