Function Of Skeletal Matrix Genes

Dental &Craniofacial Research

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Abstract

The Molecular Biology of Bones and Teeth Unit is directed by Dr. Marian F. Young, and during this fiscal year included Drs. Sunil Wadhwa, Yanming Bi, Colette Inkson and Ms. Mildred Embree and Ms. Tina Kilts. The goal of this unit is to study the function of matrix proteins made by cells in skeletal tissues. Biglycan (bgn) is an extracellular proteoglycan that is highly expressed in the matrices of bones and teeth. To understand what role it plays in skeletal tissues, mice were created that were unable to make the protein (knockout/KO). These mice suffer from age dependent osteoporosis with decreased bone mass and bone strength compared to wild-type (wt) mice. The goal of one of our studies was to explore the cell and molecular connections between biglycan and bone function using this unique animal model. Previously we showed that bgn was important for TGF-beta induction of osteogenic bone marrow stromal cells. TGF-beta is part of a superfamily that includes the morphogenic proteins called BMPs. Using bone cells isolated from bgn deficient mice, we showed that the absence of bgn caused less BMP-4 binding, which reduced the sensitivity of osteoblasts to BMP-4 stimulation. The loss of sensitivity resulted in reduced expression of the osteogenic master gene, cbfa1/runx2, which ultimately led to a defect in the differentiation of osteoblasts. The response of bgn deficient osteoblasts to BMP-4 was completely rescued by reintroduction of bgn by viral transfection. Based on these observations, we theorize that bgn can modulate BMP-induced bone cell differentiation. Microarray experiments were next performed to determine global gene expression patterns in normal and bgn deficient osteoblasts treated with or without BMP. Extensive bioinformatics analysis showed several unique sets of gene clusters altered in way that was dependent on bgn, BMP or both. Mimican is a small proteoglycan related to biglycan that was shown to be significanly down-regulated in bgn deficient osteoblasts. We plan to analyze the bones from mimican KO mice to determine if a decrease in mimican could play a role in the osteopenia observed in the bgn KO. We are also examining the skeletal function of several other interesting genes discovered by array analysis including wnt, wisp (wnt inducible secreted protein) and SLPI (serine leukocyte protease inhibitor). These experiments will deepen our understanding of how biglycan affects bone cell signaling, differentiation and function. Several types of analysis showed that when biglycan is absent there is a induction of a related protein called decorin. In order to determine whether decorin (dcn) could compensate for the absence of bgn we created mice deficient in both proteoglycans. The doubly mutant mice had earlier and more severe osteopenia compared to bgn or dcn singly deficient mice. To determine the cellular basis for this skeletal defect, ostegenic stem cells were isolated from the marrow of adult bgn/dcn doubly deficent mice and were shown to have 1) increased proliferation 2) increased TGF-beta signaling and 3) premature apoptosis (programmed cell death). To determine the mechanistic foundation for these observations we examined the localization of TGF-beta in normal and mutant cells and showed that in the absence of both bgn and dcn TGF beta inefficiently binds to the extracellular matrix and is then liberated to stimulate TGF-beta induced downstream signaling pathways. The outcome of this uncontrolled growth factor over-activation is premature cell death of the osteogenic stem cell leading to decreased osteogenesis and ultimately osteopenia. These data have important implications for understanding the role of the osteogenic adult stem cell micro environment in controlling stem cell fate and, ultimately, skeletal form and function.