Abstract 5688: HMGB1 is a Novel Substrate of Dypeptidyl Peptidase IV

Abstract

Introduction: High Mobility Group Box 1 (HMGB1) is a cytokine with a key role in tissue regeneration and angiogenesis. HMGB1 levels are reduced in diabetic skin and topical application of HMGB1 to skin wounds of diabetic mice enhanced vessel density and accelerated wound healing, suggesting that diabetes may affect endogenous HMGB1 functions. CD26 is a protease which selectively removes the N-terminal X-Pro or X-Ala containing motifs from a number of chemokines modulating their activity. High levels of CD26 have been detected in plasma of type II diabetic patients and its inhibition has proved effective in the treatment of diabetes. Since HMGB1 contains potential CD26 cleavage sites we determined whether HMGB1 may be a substrates for CD26 and, eventually, whether CD26-mediated cleavage may alter its biologic activity. Methods and results: Recombinant HMGB1 (5 uM) was incubated overnight with CD26 (2.2 uM). RP-HPLC analysis on Aquapore C18 column yielded a peptide with a molecular weigh lower than 2000 kDa. The CD26-mediated deletion of the N-terminal region of HMGB1 was confirmed by western blot analysis: time-dependent disappearance of the molecular weigh band corresponding to HMGB1 was detected with an anti-HMGB1 antibody which recognizes its first 16 N-terminal amino-acids of HMGB1. It was previously found that HMGB1 promoted endothelial cell migration through the activation of c-Jun N-terminal kinase (JNK) and extracellular regulated kinase (ERK) signaling pathways. To evaluate whether CD26 altered HMGB1 functions, endothelial cells were induced to migrate in presence of HMGB1 and CD26. HMGB1-mediated chemotactic activity was reduced by CD26 in a dose-dependent manner. Similarly, CD26 abolished HMGB1-induced JNK and ERK activation. Since diabetes is associated with the increased CD26 activity, both CD26 activity and HMGB1 levels were measured in serum of diabetic patients. Although HMGB1 serum levels did not significantly differ between diabetic and normoglycemic patients, increased N-terminal deleted HMGB1 was detected in serum from diabetic patients. Conclusions: CD26 cleaves HMGB1 and, via this mechanism, it inhibits its functions. These findings suggest that CD26 may account for the inhibition of HMGB1 effects found in diabetes.