CELLULAR BASIS OF CRANIOFACIAL BONE DISORDERS

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Normal bone development and remodeling require complex humoral, cell- cell, and cell-matrix interactions. The integration of humoral and local signals is necessary to permit proper development, maintain mineral homeostasis, insure mechanical strength, and support haematopoeisis. There is now good evidence that osteoblasts can orchestrate osteoclast development and modulate osteoclast activity via paracrine signals. Information is just beginning to emerge that osteoclasts themselves synthesize and release factors that may influence bone remodeling. We therefore hypothesize that osteoclast-derived signals may function as autocrine effectors of osteoclast development and activity and therefore influence normal and pathological osteoclastogenesis and osteoclast-mediated bone resorption. We have shown that osteoclasts produce small proinflammatory molecules known as chemokines, such as IL-8 and GRO alpha and that such molecules can act to modulate osteoclast precursor recruitment, development, and activity. Therefore, to further investigate and define osteoclast autocrine regulation we propose the following specific aims: 1) Identify a profile of chemokines produced by human and mouse OC and determine which exhibit autocrine effects on OC function (bone resorption, motility, free radical production, TRAP, cathepsin K, and carbonic anhydrase II expression). The regulation of this subset of chemokines by select known modulators of OC function will be analyzed. Included here will be studies employing selective chemokine and chemokine receptor antagonists, chemokine neutralizing antibodies, and mouse OC-like cells formed in vitro from bone marrow obtained from IL-8 receptor knockout mice. 2) Identify and characterize the profile of chemokine receptors expressed on OC as a function of OC physiology, and pathophysiology. As part of this aim we will begin to elucidate the intracellular signal transduction pathways involved in chemokine modulation of OC activity. 3) Examine the potential role of chemokines in OC precursor recruitment and differentiation in vitro, in vivo, and in ovo. 4. Examine spatial and temporal aspects of chemokine and chemokine receptor expression in normal and pathological bone tissue by in situ hybridization and immunohistochemistry. Included here are mouse IL-8 receptor knockout studies. All of the above studies will use a combination of in vivo and in vitro approaches, and model systems including the mouse calvarial injection model for histomorphometric studies, human tissue sections for in situ hybridization and immunohistochemical analysis, isolated human and avian Ocs, human Oc-like cells, the mouse Oc-like cell developmental model, and cells obtained from an IL-8 receptor knockout mouse. Oc- chemokine production, mRNA steady state levels and regulation will be assessed by RT-PCP, RNAse protection assay, chemokine ELISA, and in situ hybridization techniques. Osteoclast development and activity will be evaluated based on functional, biochemical and molecular markers of the osteoclast phenotype, including bone resorption, osteoclast antigen expression tartarate-resistant acid phosphatase activity, and calcitonin receptor levels. Such studies are anticipated to reveal new aspects of normal bone remodeling mechanisms such as tooth eruption and have potential to lend insight into skeletal pathologies such as periodontal disease, implant loosening, osteoarthritis, other inflammatory skeletal disorders, and osteoporosis.