Elsevier

Bone

Volume 40, Issue 2, February 2007, Pages 345-353
Bone

Advanced glycation end products stimulate osteoblast apoptosis via the MAP kinase and cytosolic apoptotic pathways

https://doi.org/10.1016/j.bone.2006.09.011Get rights and content

Abstract

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 end products (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 MC3T3-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 CML-collagen, 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 end products, which accumulate in diabetic and aged individuals, may promote apoptosis of osteoblastic cells and contribute to deficient bone formation.

Introduction

Advanced glycation end products form when glucose reacts with the proteins to form unstable Schiff bases, which then undergo further modification to form Amadori products [44], [50]. Additional rearrangements or modifications may occur giving rise to advanced glycation end products seen in prolonged hyperglycemia or aged individuals. A common modification is generated by oxidative cleavage of Amadori intermediates to form N-epsilon-(carboxymethyl) lysine (CML) structures. Proteins with long half lives such as collagen are particularly susceptible to formation of advanced glycation end products (AGEs) [36].

AGEs accumulate as a normal process of aging and as a result of hyperglycemia, particularly in long-lived molecules such as collagen. AGEs have been shown to be etiologic factors in diabetes-induced nephropathy, retinopathy, neuropathy and diabetes-accelerated atherosclerosis [40], [44], [50]. AGEs are also thought to contribute to many of the complications of aging including disorders such as osteoarthritis, cataract formation and changes observed in myocardial dysfunction [6], [30], [39].

Advanced glycation end products accumulate in bone and may play a functional role in the development of osteoporosis associated with diabetes and aging [53]. In support of this concept, serum levels of AGEs are elevated in individuals with osteoporosis [18]. In addition, advanced glycation end products may lead to damage of articular surfaces and enhance inflammation, thereby contributing to osteoarthritis [39]. AGEs have also been implicated in the most common form of pathologic bone loss, periodontal disease [26].

AGEs may exert a negative effect on bone by interfering with osteoblast differentiation and the production of matrix proteins such as collagen and osteocalcin [13], [33], [38], [54]. It has recently been reported that AGEs stimulate apoptosis of human mesenchymal stem cells which could limit the formation of adipose tissue, cartilage and bone [25]. Other parameters may also be affected including AGE-inhibited proliferation and differentiation [33]. Although not formally linked, it is possible that problems with fracture repair associated with type 1 diabetes [10], [14], [41] are directly or indirectly related to the effects of AGEs on osteoblasts. Since enhanced osteoblast apoptosis may represent an important mechanism through which bone formation is limited [19], [52], we investigated the capacity of glycated collagen to stimulate apoptosis of osteoblastic cells and investigated the apoptotic pathways that were stimulated.

Section snippets

CML-collagen

CML-collagen was prepared by chemical modification of acid soluble bovine skin collagen (Sigma), as previously described [23], [38]. Briefly, 50 μg of collagen was dissolved 1 mM HCl followed by incubation in sodium cyanoborohydride and glyoxylic acid in PBS. Control collagen was prepared at the same time, except that no glyoxylic acid was added. All samples were then incubated at 37°C for 24 h and then dialyzed against distilled water or PBS. CML-collagen was highly reactive on Western blots

Results

To test whether AGEs stimulated apoptosis of bone-lining cells in vivo, CML-collagen and control collagen were injected adjacent to the scalp periosteum and apoptosis of periosteal cells was measured by the TUNEL assay as we have previously described [16]. CML-collagen stimulated a 5-fold increase in apoptosis of these bone-lining cells compared to unmodified collagen (Fig. 1A). The capacity to induce apoptosis in different osteoblastic cell cultures, primary human and rat neonatal calvarial

Discussion

Diabetes and aging are both characterized by diminished bone formation [1], [42]. There are multiple avenues through which this could occur, one of which is reduced numbers of osteoblastic cells or their precursors. For example, premature aging caused by ablation of the klotho gene is associated with higher levels of osteoblast and osteocyte apoptosis [45]. We have recently shown that diabetes causes enhanced apoptosis of osteoblastic cells following a bacterial stimulus [16]. When apoptosis is

Acknowledgments

We would like to thank Alicia Ruff for help in preparing the manuscript. This work was supported by NIH grants P01AR49920 and R01DE14066.

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