AGEs induce caspase-mediated apoptosis of rat BMSCs via TNF alpha production and oxidative stress

作者：Weinberg Evgeny; Maymon Tal; Weinreb Miron^*

来源：Journal of Molecular Endocrinology, 2014, 52(1): 67-76.

DOI：10.1530/JME-13-0229

• diabetes
• apoptosis
• bone marrow stromal cells
• osteopenia

Diabetic humans and animals exhibit lower bone mass and healing, resulting from diminished bone formation. We have recently reported that type 1 diabetic rats have fewer bone marrow osteoprogenitor cells, and since the formation of advanced glycation end products (AGEs) in bone increases in diabetes, we explored possible mechanisms involved in AGE-induced apoptosis of rat bone marrow stromal cells (BMSCs). BMSCs isolated from 4-month-old rats were exposed to 10-400 mu g/ml AGE-BSA for 16 h and apoptosis was quantified with PI/annexin V staining and flow cytometry. Signaling mechanisms were evaluated by preincubating the cells with appropriate inhibitors. The formation of reactive oxygen species (ROS) was quantified by flow cytometric analysis of DCFDA fluorescence and the expression of genes by RT-PCR analysis. AGE-BSA at a concentration of 400 mg/ml increased the apoptosis of BMSCs two-to threefold, an effect completely blocked by a pan-caspase inhibitor. BSA or high concentrations of glucose had no effect. AGE-BSA-induced BMSC apoptosis was attenuated by a p38 inhibitor but not by an NF-kappa B inhibitor. Treatment with AGE-BSA induced the expression of several pro-apoptotic ligands and receptors, most notably tumor necrosis factor a (TNF alpha), TRAIL, lymphotoxin alpha, CD40, and TNFR2. Furthermore, AGE-BSA-induced apoptosis was completely blocked by pirfenidone, an inhibitor of TNFa production/secretion. Finally, AGE-BSA increased the production of ROS in BMSCs, and its apoptogenic effect was blocked by the antioxidant N-acetylcysteine (N-acetyl-L-cysteine). Thus, AGE-BSA increases the apoptosis of rat BMSCs via the activation of caspases, involving TNFa production/secretion, p38 MAPK signaling, and oxidative stress. We propose that increased protein glycation, such as that occurring under hyperglycemia, causes the apoptosis of BMSCs, which might significantly contribute to the development of osteopenia in diabetic animals.

• 出版日期2014-2