Bone matrix-osteoblast feedback mechanisms

Collagen, the main component of connective tissues, including bone extracellular matrix (ECM), comprises a family of structurally related proteins, with a defined super-molecular organization. The basic element of collagen is the fiber, which undergoes several intra- and extracellular modifications, before arranged in stable fibrils. One major process of collagen / matrix maturation is collagen cross-link formation. This process is tissue-specific, and involves a variety of cellular and matrix derived signals. Inhibition of lysyl oxidase, one enzymatic key player in cross-link formation, leads to improper collagen cross-linking and altered fibrillogenesis. In vitro and in vivo experiments confirmed that these aberrations in matrix formation are accompanied by loss of bone mineral, reduced bone strength and altered mineralization. Based on this knowledge, we assume that the temporal and spatial sequence of how matrix, specifically collagen, is laid down does influence the rate of matrix deposition, as well as its maturation. Therefore, an alteration in the matrix properties should generate signals that affect the function and activity of matrix producing cells. The aim of this project is to identify and characterize feedback mechanisms that signal from the matrix to the matrix-producing cells. To accomplish this goal we first want to compare the in vitro secreted matrix of matrix- producing cell lines like MC3T3-E1 cells, by determining their pattern of cross-link formation, fiber properties and expression of cell differentiation markers. These data will tell us something about the qualities of different in vitro secreted matrices, and if their properties are comparable to the in vivo situation. By the use of Attenuated Total Reflection FTIR we also want to study the kinetics of matrix production in vitro. Second, we want to monitor the effects of different lathyrogens, inhibitors of lysyl oxidase, on matrix deposition in cell-culture with the methods described before. Based on these experiments we will be able to produce normal and altered matrix after lathyrogen treatment. We then plan to seed fresh cells onto these matrices and compare the expression levels of markers for osteoblastic activity and proliferation of cells grown on the different types of matrices. Any alteration in marker expression should reflect a feedback-signaling cascade from the matrix to the cell. Employing expression microarrays we then plan to identify genes involved in these potential feedback mechanisms, and their specific expression levels, in more detail. Having the genes of these potential signaling cascades in hand should make it possible to influence matrix maturation and adaptation in any tissue.

Collagen, the main component of connective tissues, including bone extracellular matrix (ECM), comprises a family of structurally related proteins, with a defined super-molecular organization. The basic element of collagen is the fiber, which undergoes several intra- and extracellular modifications, before arranged in stable fibrils. One major process of collagen / matrix maturation is collagen cross-link formation. This process is tissue-specific, and involves a variety of cellular and matrix derived signals. Inhibition of lysyl oxidase (Lox), one enzymatic key player in cross-link formation, leads to improper collagen cross-linking and altered fibrillogenesis. In vitro and in vivo experiments confirmed that these aberrations in matrix formation are accompanied by loss of bone mineral, reduced bone strength and altered mineralization. Based on this knowledge, we performed experiments that showed that the temporal and spatial sequence of how matrix, specifically collagen, is laid down does influence the rate of matrix deposition, as well as its maturation. Therefore, an alteration in the matrix properties should generate signals that affect the function and activity of matrix producing cells, since experiments showed that osteoblastic cells cultured on a disrupted matrix alter their behaviour at the mRNA expression level. Moreover, the way the initial matrix is disturbed has a direct influence on how the cells react. A specialized form of infrared spectroscopy, namely FTIR-ATR (attenuated total reflection), was applied so as to enable the culturing of cells in a flow though apparatus that allows the real-time monitoring and quantitative and qualitative analysis of the extracellular matrix produced under dynamic rather than static conditions, a situation much closer to the in vivo one. One way to disturb the matrix is through the introduction of homocysteine (Hcys) in the culture medium. Since it has recently been implicated as a major contributor to fracture risk in humans, attention was focused on its effect on cells of osteoblastic lineage, specifically MC3T3-E1. Interestingly, the experiments performed showed the breadth of the Hcys effect on cellular gene expression at the mRNA level. In addition to the genes commonly associated with osteoblastic function such as the runt related transcription factor 2 (Runx2) and Lox , genes like interleukin 6 (IL-6), DNA methyl-transferases (Dnmt`s) and serum amyloid A3 (Saa3) were also affected, offering a glimpse into potential underlying mechanisms for a plethora of metabolic and musculoskeletal diseases. In summary, we showed that Hcys influences osteoblastic proliferation, differentiation, and function at several levels. For this reason, Hcys is a major risk factor for development of the fragility and fracture disease of bone (osteoporosis).