High glucose concentration up-regulates the expression of matrix metalloproteinase-9 and -13 in tendon cells

Abstract

Background: Diabetes mellitus is associated with tendinopathy or tendon injuries. However, the mechanism underlying diabetic tendinopathy is unclear. The purpose of this study was to examine the effects of high glucose concentrations on the activity and expression of matrix metalloproteinases, type I collagen, and type III collagen in tendon cells.

Methods: Tendon cells from rat Achilles tendons were treated with 6 mM, 12 mM, and 25 mM glucose, and then cell proliferation was evaluated by the 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Messenger RNA (mRNA) expression of MMP-2, MMP-8, MMP-9, and MMP-13 and type I and type III collagen was assessed by quantitative real-time polymerase chain reaction (PCR). The enzymatic activity of MMP-2 and MMP-9 was measured by gelatin zymography.

Results: The MTT assay results showed that the glucose concentration did not affect tendon cell proliferation. The results of the real-time PCR assay revealed that the mRNA expression of MMP-9 and MMP-13 was up-regulated by treatment with 25 mM glucose, whereas the mRNA expression of type I and III collagen was not affected. Gelatin zymography showed that 25 mM glucose increased the enzymatic activity of MMP-9.

Conclusions: High glucose concentration up-regulates the expression of MMP-9 and MMP-13 in tendon cells, which may account for the molecular mechanisms underlying diabetic tendinopathy.

Figure 1

MTT assay for the effect of glucose on tendon cells. A high glucose concentration did not affect the proliferation of tendon cells (p > 0.05 by Kruskal-Wallis test, n = 3; X-axis, glucose concentration; Y-axis, percentage of optic density (OD) value).

Figure 2

Real-time PCR assay to assess the effect of a high glucose concentration on the mRNA expression of MMP-2, MMP 8, MMP-9, and MMP-13 (*p < 0.05 by Kruskal-Wallis test, n = 3; X-axis, glucose concentration; Y-axis, relative fold change).

Figure 3

Real-time PCR assay to assess the effect of a high glucose concentration on the mRNA expression of type I and III collagen. A high glucose concentration had no effect on the mRNA expression of type I and III collagen (p > 0.05 by Kruskal-Wallis test, n = 3; X-axis, glucose concentration; Y-axis, fold change relative to expression in 6 mM glucose).

Figure 4

Gelatin zymography for the effect of glucose on tendon cells. (A) Gelatin zymography showed that MMP-2 and MMP-9 had enzymatic activities at a molecular size of 72 kDa and 95 kDa, respectively. (B) Densitometric analysis of the MMP-2 and MMP-9 zymogram revealed that the enzymatic activity of MMP-9 in tendon cells increased after glucose treatment (*p < 0.05, n = 3).