Effects of FGF-2 on E11-mediated osteocytogenesis in skeletal health and disease

Fibroblast growth factor 2 (FGF-2) is known to be released from cartilage upon injury and is able to influence chondrocyte gene expression in vitro. In cartilage, FGF-2 regulates E11/podoplanin expression in murine joints following surgical destabilisation (DMM model of osteoarthritis (OA)), and in cartilage explant injury models. In bone, E11 is critical for the early stages of osteocytogenesis and is responsible for the acquisition of the osteocyte dendritic phenotype. This dendritic phenotype is dysregulated in OA and given the known role of the osteocyte in controlling bone remodelling, this may contribute to the subchondral bone thickening observed in OA. Hence, the aim of this study was to elucidate the nature of FGF-2- mediated E11 expression and osteocytogenesis in skeletal health and disease. This thesis has shown that FGF-2 dose-dependently increased E11 mRNA expression in MC3T3 cells, primary osteoblasts and in primary calvaria organ cultures, which was confirmed by E11 protein western blotting data. The FGF-2 induced changes in E11 expression were accompanied by significant increases in the mRNA expression of the osteocyte markers Phex and Dmp1, and significant decreases in the mRNA expression of the osteoblast markers Col1a1, Postn, Bglap and Alpl expression. This thus supports the hypothesis that FGF-2 drives osteocytogenesis. The acquisition of osteocyte phenotype involves the re-organisation of the cytoskeleton, such as F-actin. This step is important for the transition of cuboidal-shaped osteoblasts to the stellate-shaped osteocyte phenotype. FGF-2 stimulation of MC3T3 cells and primary osteoblasts revealed more numerous and longer dendrites, as visualised by phalloidin staining for F-actin and indicative of the acquisition of the osteocyte phenotype. In contrast, control cells had a typical rounded morphology with fewer and shorter dendrites. Furthermore, immunofluorescence labelling for E11 in control cells revealed uniform distribution throughout the cytoplasm, especially in the perinuclear region. In contrast, FGF-2 treated cells showed a modified distribution where E11 was negligible in the cytoplasm, but concentrated in the dendrites. The use of siRNA knockdown of E11 achieved a 70% reduction of basal E11 mRNA expression. This knockdown also effectively abrogated FGF-2-related changes in E11 expression and dendrite formation as disclosed by mRNA and protein expression, immunofluorescence and F-actin staining with phalloidin. Despite these FGF-2 driven increases in E11 and osteocyte dendrite formation in vitro, immunohistochemical labelling revealed no differences in E11 expression in subchondral, trabecular and cortical osteocytes from naïve Fgf-2 deficient mice in comparison to wild-type mice. Similar results were observed upon sclerostin immunolabelling. FGF-2 stimulation of MC3T3 cells elicited activation of ERK1/2, Akt and p38 MAPK. However, inhibition of the aforementioned pathways failed to reduce FGF-2- mediated E11 expression and as such, the specific signalling pathway responsible remains unclear. Upstream, the expression of Fgfr1 was increased (>10-fold) over 24 h time point, while a reduction was seen in Fgfr2/3 expression over same time point especially in the FGF-2 treated cultures. This suggests that increased E11 expression and the acquisition of the osteocyte phenotype may be speculatively though upregulation of Fgfr1. The expression of E11 and sclerostin in OA pathology in mice, human and dogs were investigated. Initially sequence homology using the Clustal Omega alignment program showed both proteins to be homologous in the domestic animals under study. A comparative study using canine subchondral bone osteocytes revealed increased E11 expression in the OA samples relative to the control. This feature may be related to newly embedded osteocytes during sclerosis. However, E11 and sclerostin were unchanged in both murine (DMM) and human OA subchondral bone osteocytes in comparison to controls. In mice, this may be due to limited OA development; whilst in humans the sample size, age, stage of the disease and sourcing from same diseased joint may be important in the interpretation of the results. The expression of E11 and sclerostin during OA pathology was also investigated in Fgf-2 deficient mice in which OA was induced using the DMM model. There was no difference in E11 expression between the OA and control (sham-operated) samples, suggesting that compensation of E11 expression may be mediated by growth factors from the FGF family. Surprisingly, increased E11 expression was observed in the control Fgf-2 deficient mice, in comparison to the wild-type control mice. This suggests a potential adjustment to loading by the contralateral knee, as this was not observed in naïve mice from both groups. Together, these data show that FGF-2 promotes the osteocyte phenotype, and that this is mediated by increased E11 expression. These results may help explain (1) the altered osteocyte phenotype and (2) increased subchondral bone thickening observed in OA. This knowledge will be of interest in the search for disease modifying therapeutics for skeletal health, including OA and osteoporosis.