IDENTIFYING COLLAGEN-I-INDUCED MRNAS IN OSTEOBLASTS

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Chronic diseases of bone mass represent a growing health care problem which demands improved diagnosis and treatment. The importance of extracellular matrix (ECM) components such as type I collagen to the physiology of bone is illustrated by the collagen mutations found in individuals with osteogenesis imperfecta and post menopausal osteoporosis. Yet, ECM-mediated regulation of osseous physiology is not well defined. ECM-ostoblast interactions may be central to the pathophysiology of diseases of bone mass. The goal of the present proposal is to identify end-responses (induced protein expression) of osteoblast -type 1 collagen interactions. This goal will be accomplished using a clonal osteoblast (OB) cell culture model and subtracted cDNA probe screening of a cDNA library to identify cDNAs (representing collagen I-induced proteins) uniquely present in collagen I-stimulated OB. Therefore, the first specific aim of this proposal will be to create cDNA libraries which represent the spectrum of proteins expressed by a) resting OB and b) collagen I-stimulated OB. The second specific aim, to identify the cDNAs that encode proteins whose expression is specific to collagen I-stimulated OB, will require both cDNA libraries to a) synthesize a subtracted probe by the Phenol Reassociation Hybridization Technique (pERT) and b)to screen the collagen I-stimulated OB cDNA library. Finally, the third aim will be to assure that the identified proteins are the result of OB interaction with a collagen I matrix. This will be accomplished by Northern blot comparison of unstimulated and collagen I-stimulated OB expression of transcripts identified as cDNAs in Specific Aim 2. A recent review of bone remodeling suggested that improvements in the clinical management of bone mass will require the identification of the molecular components of bone remodelling. The proposed identification of proteins expressed by ECM-stimulated OB represents the initiation of a research program that seeks to define molecular determinants of osteoblast physiology and bone mass.