Expression and localisation of proteins with a proposed role in mechanosensing in cells of the osteoblast lineage
The main aim of this study was to examine several of the candidate proteins proposed to act as the primary mechanosensory and compare the expression and localisation of these proteins in osteoblasts and osteocytes from the mouse. First, a protocol was developed to isolate primary osteocytes from mouse calvaria, to allow direct comparison with primary mouse osteocytes. Primary osteocytes were then used to examine differences in the expression and localisation of potential mechanosensory molecules. The expression and localisation of focal adhesion proteins, examined via immunohistochemistry, was found to vary between osteoblasts and osteocytes, with osteoblasts having larger, and more numerous focal adhesions. Osteocyte focal adhesions were located mainly at the ends of processes, and not on the main cell body. Additionally, the actin cytoskeleton was markedly different in osteoblasts compared to osteocytes. In osteocytes F-actin is restricted mainly to the processes, whereas in osteoblast F-actin is found throughout the cell body. The main integrin subunits on osteoblastic cells were found to be beta 1 and &agr;5, likely to be associated as &agr;5beta 1, a receptor binding fibronectin. Surprisingly, however, no collagen-binding integrin subunits could be detected. eNOS and caveolin were detected in both osteoblasts and osteocytes, but did not co-localise with each other, or with integrins. No marked differences were found in the expression and localisation of caveolin and eNOS between osteoblasts and osteocytes.