Abstract
Type I collagen, the major organic component of bone matrix, undergoes a series of post-translational modifications that occur with aging, such as the non-enzymatic glycation. This spontaneous reaction leads to the formation of advanced glycation end products (AGEs), which accumulate in bone tissue and affect its structural and mechanical properties. We have investigated the role of matrix AGEs on bone resorption mediated by mature osteoclasts and the effects of exogenous AGEs on osteoclastogenesis. Using in vitro resorption assays performed on control- and AGE-modified bone and ivory slices, we showed that the resorption process was markedly inhibited when mature osteoclasts were seeded on slices containing matrix pentosidine, a well characterized AGE. More specifically, the total area resorbed per slice, and the area degraded per resorption lacuna created by osteoclasts, were significantly decreased in AGE-containing slices. This inhibition of bone resorption was confirmed by a marked reduction of the release of type I collagen fragments generated by the collagenolytic enzymes secreted by osteoclasts in the culture medium of AGE-modified mineralized matrices. This effect is likely to result from decreased solubility of collagen molecules in the presence of AGEs, as documented by the reduction of pepsin-mediated digestion of AGE-containing collagen. We found that AGE-modified BSA totally inhibited osteoclastogenesis in vitro, most likely by impairing the commitment of osteoclast progenitors into pre-osteoclastic cells. Although the mechanisms remain unknown, AGEs might interfere with osteoclastic differentiation and activity through their interaction with specific cell-surface receptors, because we showed that both osteoclast progenitors and mature osteoclasts expressed different AGEs receptors, including receptor for AGEs (RAGEs). These results suggest that AGEs decreased osteoclast-induced bone resorption, by altering not only the structural integrity of bone matrix proteins but also the osteoclastic differentiation process. We suggest that AGEs may play a role in the alterations of bone remodeling associated with aging and diabetes.
Highlights
Non-enzymatic glycation is a common post-translational modification of proteins induced by the spontaneous condensation of reducing sugars and metabolic intermediates with free amino groups in lysine or arginine residues
The mechanisms remain unknown, advanced glycation end products (AGEs) might interfere with osteoclastic differentiation and activity through their interaction with specific cell-surface receptors, because we showed that both osteoclast progenitors and mature osteoclasts expressed different AGEs receptors, including receptor for AGEs (RAGEs)
Schiff bases and Amadori products undergo a complex series of rearrangements, oxidations, and/or dehydrations along different chemical pathways to produce a class of irreversible adducts to proteins, the advanced glycation end products (AGEs
Summary
In Vitro AGE-Matrix and AGE-BSA Formation—Elephant ivory slices (6-mm diameter, kindly donated by the Centre de conservation et d’etude des collections, Lyon, France) and bone cortical slices (3-mm diameter) from 3-month-old calves were used in these experiments. Murine Osteoclast Differentiation—Bone marrow cells of 6to 8-week-old male NMRI mice were seeded at 5000 cells/mm and cultured for 8 days in differentiation medium: ␣MEM containing 10% FCS (HyClone Laboratories, Perbio Science, Cheshire, UK), 2 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, 30 ng/ml recombinant mouse macrophage-colony stimulating factor, and 30 ng/ml soluble recombinant RANK-L mixed with 2.5 g/ml mouse anti-polyhistidine antibody, according to the manufacturer’s recommendations (R&D Systems Europe, France). Osteoclastogenesis experiments were performed as described previously [37]; 100 cells/mm were seeded on a 96-well tissue culture plate with the differentiation medium, in the presence or absence of 30 ng/ml soluble recombinant RANK-L (mixed with 2.5 g/ml mouse anti-polyhistidine antibody) and BSA derivatives. Comparison between experimental conditions for bone-resorption activity and osteoclastic differentiation assays were assessed using the non-parametric MannWhitney U test
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