Functional Genomic Analysis of the Regulation of Bone Cells by a Bioactive Lipid
Norm Karin, Principal Investigator
Bone mass homeostasis is a function of the activity of osteoblasts (bone-forming cells) and osteoclasts (bone-degrading cells). It is a process regulated by hormones, growth factors, and physical stimuli. The Functional Genomic Analysis of the Regulation of Bone Cells by a Bioactive Lipid project team at Pacific Northwest National Laboratory (PNNL) is creating capabilities that permit the regulation of osteoblast function to be analyzed at the genomic and proteomic levels.
A combination of microarray screening, liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of secreted proteins, and quantitative imaging of fluorescent reporter expression is being applied to a mouse osteoblast cell line responding to the bioactive lipid, lysophosphatidic acid (LPA). Genomic and proteomic data is being evaluated in the context of a complex cell behavior output, LPA-induced osteoblast motility.
We are generating new databases corresponding to LPA-linked transcriptional regulation and LPA-induced changes in osteoblast secretory activity. The latter database has the additional potential of containing candidates for LPA-specific biomarkers of osteoblast functional control. Because the major source of LPA in vivo is platelets and blood clots, the results of this project have particular relevance to bone fracture repair.
Schematic representation of the steps in bone fracture healing.
LPA-induced changes in the osteoblast actin cytoskeleton. Cells were serum starved overnight after which serum-free medium or medium containing 2.5 µM LPA was added. After 24 hours, the cells were fixed and stained with fluorescent phalloidin to reveal the F-actin cytoskeleton. Left: control cells. Right: LPA-treated cells. Arrowhead points to a dendritic process; asterisks indicate bare areas on culture dish.
Effect of LPA on osteoblast migration. MC3T3-E1 cells were serum-starved overnight after which fresh, serum-free medium or medium containing 2.5 µM LPA was added. Time-lapse video images were collected over a 22-hour period. The migration distance of 14 cells was analyzed from each treatment. Screen-capture images are shown from the start (A, C) and end (B, D) of the experiment. A, B: control cells. C, D: LPA-treated cells.
