Glucosamine affects intracellular signalling through inhibition of mitogen-activated protein kinase phosphorylation in human chondrocytes

Abstract

The aim of this study was to determine the effects of glucosamine on matrix metalloprotease (MMP) production, on mitogen-activated protein kinase (MAPK) phosphorylation, and on activator protein (AP)-1 transcription factor activation in human chondrocytes. The human immortalized cell line lbpva55 and healthy human chondrocytes (obtained from healthy donors) were subjected to challenge with 10 ng/ml IL-1beta after pretreatment with 2.5 or 10 mmol/l glucosamine. MMP mRNA expression levels were evaluated using quantitative real-time PCR, and MMP protein production levels were evaluated in the culture supernatant using ELISA. MAPK phosphorylation was evaluated using Western blotting. AP-1 transcription factor activation was evaluated by measuring AP-1 DNA-binding activity. After IL-1beta stimulation, levels of MMP-1, MMP-3 and MMP-13 production were markedly increased. Treatment with 2.5 and 10 mmol/l glucosamine reduced expression of these metalloproteases. MMP expression is regulated by transcription factors such as the AP-1 complex, which is activated by phosphorylated MAPKs. IL-1beta stimulated phosphorylation of c-jun amino-terminal kinase, p38 MAPK and extracellular signal-regulated kinase-1/2. Glucosamine inhibited c-jun amino-terminal kinase and p38 phosphorylation, and consequently c-jun binding activity. These findings demonstrate, for the first time, that glucosamine inhibits IL-1beta-stimulated MMP production in human chondrocytes by affecting MAPK phosphorylation.

Figure 1

Effect of glucosamine on MMP-1 and MMP-13 expression in lbpva55 cells stimulated with 10 ng/ml IL-1β. Cells were pretreated for 2 hours with 2.5 or 10 mmol/l glucosamine (G2.5 and G10, respectively), and then stimulated with IL-1β for 22 hours. mRNA was extracted and analyzed by quantitative real-time PCR, and cell supernatant was analyzed by ELISA. Shown are (a) matrix metalloprotease (MMP)-1 and (b) MMP-13 mRNA levels, and (c) MMP-1 and (d) MMP-13 protein amounts. Quantitative real-time PCR results are expressed in relative arbitrary units (AU), and ELISA results are expressed in ng/ml or pg/ml. Results are expressed as mean ± standard error, obtained in three different experiments. *P ≤ 0.05. CTL, control.

Figure 2

Effect of glucosamine on MMP-3 expression in lbpva55 cell line stimulated with 10 ng/ml IL-1β. Cells were pretreated for 2 hours with 2.5 or 10 mmol/l glucosamine (G2.5 and G10, respectively), and then stimulated with IL-1β for 22 hours. mRNA was extracted and analyzed by quantitative real-time PCR, and cell supernatant was analyzed by ELISA. Shown are (a) matrix metalloprotease (MMP)-3 mRNA and (b) MMP-3 protein levels. Quantitative real-time PCR results are expressed in relative arbitrary units (AU) and ELISA results are expressed in ng/ml. Results are expressed as mean ± standard error, obtained in three different experiments. *P ≤ 0.05. CTL, control.

Figure 3

Effect of glucosamine on MMP-1 and MMP-13 expression in HPCs stimulated with 10 ng/ml IL-1β. Cells were pretreated for 2 hours with 2.5 and 10 mmol/l glucosamine (G2.5 and G10, respectively), and then stimulated with IL-1β for 22 hours. mRNA was extracted and analyzed by quantitative real-time PCR, and cell supernatant was analyzed by ELISA. Shown are (a) matrix metalloprotease (MMP)-1 and (b) MMP-13 mRNA levels, and (c) MMP-1 and (d) MMP-13 protein amounts. Quantitative real-time PCR results are expressed in relative arbitrary units (AU) and ELISA results are expressed in ng/ml or pg/ml. Results are expressed as mean ± standard error, obtained in six different experiments. *P ≤ 0.05. CTL, control; HPC, human primary chondrocyte.

Figure 4

Effect of glucosamine on MMP-3 expression in HCPs stimulated with 10 ng/ml IL-1β. Cells were pretreated for 2 hours with 2.5 or 10 mmol/l glucosamine (G2.5 and G10, respectively), and then stimulated with IL-1β for 22 hours. mRNA was extracted and analyzed by quantitative real-time PCR, and cell supernatant was analyzed by ELISA. Shown are (a) matrix metalloprotease (MMP)-3 mRNA level and (b) MMP-3 protein level. Quantitative real-time PCR results are expressed in relative arbitrary units (AU) and ELISA results are expressed in ng/ml. Results are expressed as mean ± standard error, obtained in six different experiments. *P ≤ 0.05. CTL, control; HPC, human primary chondrocyte.

Figure 5

Effect of glucosamine MAPK phosphorylation in lbpva55 cells stimulated with 10 ng/ml IL-1β. Cells were pretreated for 2 hours with 2.5 or 10 mmol/l glocosamine (G2.5 and G10, respectively), and then stimulated with IL-1β for 15 minutes. Whole cell extract was prepared as described in Materials and methods. Proteins were resolved on SDS-PAGE, electrotransferred, and immunoblotted. Antibodies to (a) phosphorylated (p)-c-jun amino-terminal kinase (JNK) and total JNK, (b) p-p38 and total p38, and (c) p-extracellular signal-regulated kinase (ERK)1/2 and total ERK1/2 were used to visualize mitogen-activated protein kinase (MAPK) phosphorylation. Representative data from three independent experiments are shown. CTL, control.

Figure 6

Effect of glucosamine MAPK phosphorylation in HPCs stimulated with 10 ng/ml IL-1β. Cells were pretreated for 2 hours with 2.5 or 10 mmol/l glucosamine (G2.5 and G10, respectively), and then stimulated with IL-1β for 15 minutes. Whole cell extract was prepared as described in Materials and methods. Proteins were resolved on SDS-PAGE, electrotransferred and immunoblotted. Antibodies to (a) phosphorylated (p)-c-jun amino-terminal kinase (JNK) and total JNK, (b) p-p38 and total p38, and (c) p-extracellular signal-regulated kinase (ERK)1/2 and total ERK1/2 were used to visualize mitogen-activated protein kinase (MAPK) phosphorylation. Representative data from six independent experiments are shown. CTL, control; HPC, human primary chondrocyte.

Figure 7

Effect of glucosamine on c-jun DNA-binding activity in HPCs stimulated with 10 ng/ml IL-1β. Cells were pretreated with 2.5 and 10 mmol/l glucosamine (G2.5 and G10, respectively) for 2 hours and then stimulated with IL-1β for 15 minutes. Nuclear extract was prepared as described in Materials and methods. Results are expressed as optical density (OD) measured at 450 nm and represent the mean ± standard error of data obtained in six different experiments. *P ≤ 0.05. CTL, control; HPC, human primary chondrocyte.