We have previously shown that diabetes significantly enhances apoptosis of osteoblastic cells in vivo and that the enhanced apoptosis contributes to diabetes impaired new bone formation. A potential mechanism is enhanced apoptosis stimulated by advanced glycation endproducts (AGEs). To investigate this further, an advanced glycation product, carboxymethyl lysine modified collagen (CML-collagen) was injected in vivo and stimulated a 5 fold increase in calvarial periosteal cell apoptosis compared to unmodified collagen. It also induced apoptosis in primary cultures of human or neonatal rat osteoblastic cells or MC-3T3-E1 cells in vitro. Moreover, the apoptotic effect was largely mediated through RAGE receptor. CML-collagen increased p38 and JNK activity 3.2 and 4.4 fold, respectively. Inhibition of p38 and JNK reduced CML-collagen stimulated apoptosis by 45% and 59% and by 90% when used together (P<0.05). The predominant apoptotic pathway induced by CML-collagen involved caspase-8 activation of caspase-3 and was independent of NF-κB activation. When osteoblastic cells were exposed to a long-term low dose incubation with CMLcollagen there was a higher degree of apoptosis compared to short term incubation. In more differentiated osteoblastic cultures apoptosis was enhanced even further. These results indicate that advanced glycation endproducts, which accumulate in diabetic and aged individuals may promote apoptosis of osteoblastic cells and contribute to deficient bone formation.

Advanced glycation end products (AGEs) are elevated in aged and diabetic individuals and are associated with pathological changes associated with both. Previously we demonstrated that the AGE N(epsilon)-(carboxymethyl)lysine (CML)-collagen induced fibroblast apoptosis through the cytoplasmic and mitochondrial pathways and the global induction of proapoptotic genes. In the present study we investigated upstream mechanisms of CML-collagen-induced apoptosis. CML-collagen induced activation of the proapoptotic transcription factor FOXO1 compared with unmodified collagen. When FOXO1 was silenced, CML-collagen-stimulated apoptosis was reduced by approximately 75% compared with fibroblasts incubated with nonsilencing small interfering RNA, demonstrating the functional significance of FOXO1 activation (P < 0.05). CML-collagen but not control collagen also induced a 3.3-fold increase in p38 and a 5.6-fold increase in JNK(1/2) activity (P < 0.05). With the use of specific inhibitors, activation of p38 and JNK was shown to play an important role in CML-collagen-induced activation of FOXO1 and caspase-3. Moreover, inhibition of p38 and JNK reduced CML-collagen-stimulated apoptosis by 48 and 57%, respectively, and by 89% when used together (P < 0.05). In contrast, inhibition of the phosphatidylinositol 3-kinase/Akt pathway enhanced FOXO1 activation. p38 and JNK stimulation by CML-collagen was almost entirely blocked when formation of ROS was inhibited and was partially reduced by NO and ceramide inhibitors. These inhibitors also reduced apoptosis to a similar extent. Together these data support a model in which AGE-induced apoptosis involves the formation of ROS, NO, and ceramide and leads to p38 and JNK MAP kinase activation, which in turn induces FOXO1 and caspase-3.