Glycation of collagen interferes with discoidin domain receptor 2 (DDR2) mediated collagen induction of lysyl oxidase in osteoblasts

Abstract

Diabetes doubles the risk of bone fracture. Poor quality of bone in diabetes is largely linked to diminished bone formation and bone organic phase abnormalities. Studies indicated that in diabetes, glycation of type I collagen, the most abundant protein in bone extracellular matrix, prompts abnormal arrangement of collagen molecules leading to weak bones. In addition, diabetic bone fragility is attributed to reduced levels of lysyl oxidase enzyme-dependent collagen cross-links. However, the mechanism underlying the presence of lower levels of enzymatic collagen cross-links in diabetic bone has not been directly investigated. Thus, we investigated the regulation of lysyl oxidase in bone under diabetic conditions. Our in-vitro model examined collagen and glycated-collagen regulation of lysyl oxidase in primary rodent osteoblasts. Our findings indicate that collagen up-regulates lysyl oxidase in osteoblasts, while glycated-collagen fails to regulate lysyl oxidase. To determine the mechanism of collagen upregulation of lysyl oxidase, we studied two classes of collagen receptors: Discoidin Domain-Receptor 2 (DDR2) and integrins. Inhibitor and knockdown studies indicated that collagen up-regulates lysyl oxidase through DDR2. Furthermore, glycated-collagen-DDR2 binding and activation analyses showed that collagen glycation disrupted collagen-DDR2 binding and activation, pointing to a mechanism for the diminished levels of lysyl oxidase and consequent low lysyl oxidase-derived cross-links in diabetic bone. Our in-vivo studies focused on lysyl oxidase regulation in mouse calvarial bone healing tissues under diabetic conditions. These experiments revealed that diabetes up-regulates lysyl oxidase mRNA and enzyme activity in partially healed bone (day 7). We next performed histological analyses on day 7 healing bone. Data suggest that hematomas, which form in the initial stage of bone healing, do not resolve in diabetic mice. Unresolved hematomas are rich in growth factors, which could explain elevated levels of lysyl oxidase in diabetic healing bone. Taken together, this study provides a novel mechanism by which collagen glycation disrupts the regulation of lysyl oxidase in diabetic bone fragility. Additionally, we found that the excess formation of non-mineralized fibrotic matrix might be one of the mechanisms behind impaired intramembranous bone healing in diabetes.