Advanced glycation end products increase transglutaminase activity in primary porcine tenocytes.
OBJECTIVE
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.


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.
Rosenthal
Ann K
AK
Department of Medicine, Division of Rheumatology, Medical College of Wisconsin, Milwaukee, WI, USA. akrose@mcw.edu
Gohr
Claudia M
CM
Mitton
Elizabeth
E
Monnier
Vincent
V
Burner
Todd
T
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D013264:Stifle / Q000378:metabolism; D013552:Swine; D013710:Tendons / Q000187:drug effects / Q000201:enzymology*; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D011503:Transglutaminases / Q000235:genetics / Q000378:metabolism*
D000975:Antioxidants; D017127:Glycation End Products, Advanced; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013844:Thiazoles; C048496:N(6)-carboxymethyllysine; D011503:Transglutaminases; C022616:thiazolyl blue; D008239:Lysine