Advanced glycation end products increase transglutaminase activity in primary porcine tenocytes

Abstract

Objectives: Tendon abnormalities, such as increased stiffness, thickness, and excess calcification, occur commonly in patients with diabetes mellitus and cause considerable disability. These changes are frequently attributed to increased cross-linking of extracellular matrix components by advanced glycation end-products (AGEs). However, cellular effects of AGEs, such as increased activity of the cross-linking transglutaminase (Tgase) enzymes, could also contribute to altered tissue biomechanics and calcification in diabetic tendons. We determined the effect of AGE-modified protein on tenocyte Tgase activity.

Research design and methods: Primary porcine tenocytes were exposed to N- carboxymethyl-lysine (CML)-modified type I collagen in high or normal glucose media. Protein and mRNA levels of the Tgase enzymes and Tgase activity levels were measured, as were markers of apoptosis. We also determined the effect of antioxidants on CML-collagen mediated Tgase activity.

Results: Carboxymethyl-lysine-collagen increased Tgase activity in tenocytes 2.3- to 5.6-fold over unmodified collagen controls in both normal and high glucose media, without altering enzyme protein levels. Anti-oxidant treatment reduced the effect of CML-collagen on Tgase activity. Deoxyribonucleic acid laddering and annexin V protein levels were not altered by CML-collagen exposure.

Conclusions: Carboxymethyl-lysine-collagen increased Tgase activity in tenocytes, likely posttranslationally. Increased levels of Tgase-mediated cross-links may contribute to the excess calcification and biomechanical pathology seen in diabetic tendons.

FIGURE 1.

The effect of CML-collagen on Tgase activity in tenocytes. Tenocytes were incubated in media containing normal (5.5 mM, grey bars) or high (25 mM, black bars) glucose. Various doses of CML-collagen or 400 μg/mL control collagen (control) were added to the media and the cells were incubated for 72 hours. Transglutaminase activity was quantified in the cell layer using a standard radiometric assay corrected for cell protein. Values represent means ± SD (n = 12). Carboxymethyl-lysine-collagen increased Tgase activity over control values at all concentrations tested (*P < 0.001).

FIGURE 2.

The effect of insulin on Tgase activity in tenocytes. Tenocytes were incubated in media containing normal (5.5 mM, grey bars) or high (25 mM, black bars) glucose with or without insulin. After 72 hours of incubation, Tgase activity was measured in the celllayer using a standard radiometric assay corrected for cell protein. Values represent means ± SD (n = 6). Insulin at 100 ng/mL increased Tgase activity in high glucose media (*P < 0.01)

FIGURE 3.

The effects of CML-collagen on Tgase mRNA and protein levels. Tenocytes maintained in media with either normal (5.5 mM, grey bars) or high (25 mM, black bars) glucose were treated with various doses of CML-collagen or 400 μg/mL control collagen. After 72 hours of incubation, mRNA and protein fractions were isolated from the cell layer. Specific primers for type II transglutaminase (panel A) or factor XIIIA (panel C) were used in RT-PCR to estimate mRNA levels for these enzymes. Results are expressed as a ratio of the band of interest to that of 18S. Western blots were also performed using specific antibodies for type II transglutaminase (panel B) and factor XIIIA (panel D). Blots were stripped and re-probed with antitubulin antibodies. Lane 1, low glucose collagen control; lane 2, low glucose 40 μg/mL CML-collagen; lane 3, low glucose 160 μg/mL CML-collagen; lane 4, low glucose 400 μg/mL CML-collagen; lane 5, high glucose collagen control; lane 6, high glucose 40 μg/mL CML-collagen; lane 7, high glucose 160 μg/mL CML-collagen; lane 8, high glucose 400 μg/mL CML-collagen. No significant differences in protein levels were noted on densitometry.

FIGURE 4.

The effects of the antioxidants on Tgase activity in tenocytes. Tenocytes in high glucose media were exposed to 400 μg/mL control collagen (black bars) or 160 μg/mL CML-collagen (grey bars) with or without the antioxidants 500 μM uric acid, 1 mM sodium selenite, or 10 mM N-acetyl cysteine. Transglutaminase activity was measured after 72 hours. All of the antioxidants significantly decreased CML-mediated stimulation of Tgase activity (n = 12; *P ≤ 0.008).

FIGURE 5.

The effect of hydrogen peroxide on Tgase activity. Transglutaminase reaction mixture containing N,N’-dimethylcasein and (³H) putrescine was incubated with or without 1 mM H₂0₂ and/or 20 U/mL catalase for 30 minutes. Forty mU of purified guinea pig liver type II Tgase was added and the reaction was allowed to proceed for 30 minutes. Quantities of ³H putrescine incorporated into N,N’-dimethylcasein were quantified by liquid scintigraphy (n = 6; *P < 0.05).