Complications of diabetes result in part from elevated serum glucose levels. This leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGE's). AGE's play a significant role in many complications of diabetes. Osteopenia and low mineral density and weak bones is a complication of Type 1 diabetes and is known as """"""""diabetic bone disease"""""""". Surprisingly little is known regarding the role of AGE's in modulating bone healing, bone formation, and osteoblast function. For example, the presence and activity of the receptor for advanced glycation end products (RAGE) has not been reported in osteoblasts. The principal hypothesis of the proposed research is that AGE's inhibit bone healing and formation by binding and activating RAGE in osteoblasts. We propose that this results in NFKB activation and transcriptional repression and down-regulation of key osteoblast growth factors and extracellular matrix genes.
Aim 1 will measure in vivo the expression of selected growth factors and extracellular matrix products (BMP-1, BMP- 2, BMP-4, and type I collagen) in healing calvaria defects made in diabetic and non-diabetic mice. Diabetes will be induced by the multiple low dose streptozotocin protocol in Balb/c mice; selected studies will be performed in the nonchemically-induced murine diabetic model (NOD strain). The degree of inhibition of bone healing in diabetic animals and expression patterns of RAGE will be determined by quantitative histomorphometric and quantitative immunohistochemical measurements. Studies will directly determine the role of AGE's in diminished diabetic bone formation by local application of AGE's to calvaria defects in non-diabetic mice. The degree to which this mimics diabetic bone will be determined by measuring inhibition of healing and regulation of the same growth factors and extracellular matrix products.
Aim 2 will determine in vitro in primary rat osteoblast cell cultures that AGE's inhibit production of osteoblast growth factors and type I collagen via RAGE activated NF-KB. RAGE function blocking antibody studies will identify the AGE/RAGE-dependent NF-KB activation mechanism in the regulation of the specified osteoblast genes. The role of NF-KB activation in down-regulating target osteoblast genes will be directly determined by transfection with the super-repressor 32A/36A IKB-alpha, a potent and specific inhibitor of NF-KB activation. These studies will identify a new mechanism that contributes to diabetic bone disease; and should lead to the identification of new therapeutic treatment targets for this increasingly prevalent clinical condition.
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