The fall in circulating oestrogen (E2) after the menopause leads to an increased rate of bone remodelling, excessive osteoclast activity and a greater fracture risk. Until recently hormone replacement therapy (HRT) was prescribed to post-menopausal women to prevent bone loss, however HRT is associated with an elevated incidence of cardiovascular disease, stroke and cancer. These side-effects led to an interest in naturally occurring compounds with oestrogenic action such as phytoestrogens (PEs), which are non-steroidal-plant derived compounds. Human trials and animal studies suggest a beneficial effect of PEs on bone mass, although their ability to modify osteoclast formation in response to key inflammatory cytokines has not been examined. The aim of the following studies was to determine the effect of physiologically relevant concentrations of genistein, coumestrol and daidzein on TNF-α-induced osteoclast formation, osteoblasts differentiation and T cell activity. Genistein (10-7 M), daidzein (10-5 M), and coumestrol (10-7 M) significantly reduced TNF-α-induced TRAP positive osteoclast formation and bone resorption, which was prevented by the E2 antagonist ICI 182,780. The suppressive action on osteoclast formation was associated with a significant reduction in TNF-α-induced c-fos and NFATc1 mRNA expression and NFATc1 nuclear translocation. Constitutive c-fos expression prevented the inhibitory action of PEs on osteoclast differentiation, resorption and NFATc1 expression. The effect of PEs, in the presence or absence of the anabolic nutritional factor zinc, on osteoblasts differentiation and bone nodule formation was examined in-vitro. Coumestrol (10-5 to 10-7 M), daidzein (10-5 to 10-6 M) and genistein (10-5 M) enhanced bone nodule formation and ALP activity in human osteoblasts, and this effect was significantly augmented in the presence of zinc (10-5 M). Furthermore, PEs and zinc increased Runx2 mRNA expression and Zn2+ augmented the inhibitory effect of PEs on RANKL/OPG ratio. This suggests that in addition to the direct inhibitory effect on osteoclast formation PEs also in-directly reduce the osteoblastsic stimulus for osteoclast formation and promote bone formation. E2 deficiency is thought to promote osteoclastogenesis by modifying Thelper1 (Th1) cell proliferation and inflammatory cytokine production in particular TNF-α. I therefore examined the effect of PEs on T cell proliferation and inflammatory cytokine production. All PEs prevented the augmentative effect of con A stimulated T cells on osteoclast formation in co-culture. However the mechanism of action varied, genistein reduced con A stimulated TNF-α, IL-1β and RANKL expression with little effect on viability, coumestrol decreased cell viability and TNF-α expression whereas the inhibitory effect of daidzein was mediated via suppression of viable T cell number. This study provides novel evidence that PEs have multiple effects on bone cell activity, directly inhibiting TNF-α-induced osteoclast formation, reducing the osteoblasts and T cell derived stimulus for osteoclast formation and augmenting osteoblasts differentiation and bone formation. Thus, PEs have a potential role in the treatment of post-menopausal osteoporosis and inflammatory skeletal disorders and that the beneficial effect noted in previous studies is mediated through multiple mechanisms.

INTRODUCTION: In this study, we identified the association of Vitamin D with orthodontic tooth movement and the impact of Vitamin D 1,25OHD and 25OHD forms on osteoblast function. MATERIALS AND METHODS: This study is comprised of two parts; a clinical and a laboratory part. In part I, saliva samples were collected from orthodontic patients each month for the first six months of orthodontic treatment along with casts at the beginning and the end of the study period. The samples were measured for Vitamin D binding protein (VitDBP) and alkaline phosphatase (ALP) and correlated with clinical tooth movement using absolute change in irregularity index (II). In part II, osteoblasts were collected from the calvaria of 3-5 day old healthy wild-type mice and cultured with differing concentrations of 1,25OHD (1, 10 and 100nmol) and 25OHD (100, 200, 400 nmol). ALP, OPG, and RANKL were measured as outcomes of Vitamin D treatment of osteoblasts. Intracellular signaling in response to Vitamin D was assessed by identifying the phosphorylation of ERK 1/2, p38 and NLK in primary osteoblasts. RESULTS: Measurement of salivary Vitamin D binding protein (VitDBP) showed that both low (<2.75 ng/ml) and high (>6.48 ng/ml) logVitDBP were associated with reduced tooth movement. There was no significant correlation between ALP levels and orthodontic treatment. Significant seasonal changes in VitDBP using a two-season year model were found with lower levels noticed in the summer (Mar-Sept) than in the winter (Oct-Feb) at p<0.05. A decrease in OPG production with higher concentrations of 1,25OHD and 25OHD with a corresponding increase in RANKL levels in primary osteoblast cultures was found. Similar to the clinical findings, ALP levels were not significantly affected by increasing concentrations of both 1,25OHD and 25OHD. The ERK 1/2 showed upregulation in response to treatment with 1,25OHD and downregulation in response to treatment with 25OHD concentrations. Meanwhile, p38 and NLK were affected by 1,25OHD and not by 25OHD. CONCLUSIONS: Clinical outcomes of orthodontic treatment are associated with a range of optimal Vitamin D binding protein (VitDBP) as detected in saliva. Different forms of Vitamin D affect osteoblast response and signaling differently.

Osteoblasts are bone-forming cells and therefore they are responsible of the synthesis of type I collagen, the main component of bone matrix. However, there is an apparent disconnect between the regulation of osteoblast differentiation and bone formation since the synthesis of Type I collagen precedes the expression of Runx2, the earliest determinant of osteoblast differentiation. Recently, genetic experiments in the mouse have revealed the existence of an unexpected cross-regulation between bone and other organs. In particular this body of work has highlighted the importance of osteoblasts as endocrine cells to regulate whole-body glucose homeostasis by secretion of a hormone, osteocalcin. However, the fundamental question of why bone regulates glucose homeostasis remained to be answered. Therefore, in my thesis, considering that bone is a metabolically demanding organ that constantly renews itself, I hypothesized that characterizing the connection between the need of glucose as a main nutrient in osteoblasts and bone development will provide a key to deeper understanding of why bone regulates glucose homeostasis. My work shows here that glucose uptake through GLUT1 in osteoblasts is needed for osteoblast differentiation by suppressing the AMPK-dependent activation by phosphorylation at S148 of Smurf1 that targets Runx2 for degradation. I also uncovered the mechanism of action of Smurf1 in this setting. In a distinct but synergetic way, glucose uptake promotes bone formation by inhibiting a distinct function of AMPK. In turn, Runx2 favors Glut1 expression, and this feedforward regulation between Runx2 and Glut1 determines the onset of osteoblast differentiation during development and the extent of bone formation throughout life. Furthermore, I also identified that Smurf1 not only regulates osteoblast differentiation by targeting Runx2 for degradation but also contributes to whole-body glucose homeostasis by regulating the activation of osteocalcin by targeting the insulin receptor for degradation in vivo. These results identify Smurf1 as a determinant of osteoblast differentiation during development, of bone formation and glucose homeostasis post-natally. Most importantly, we show that these Smurf1 functions required AMPK-phosphorylation site S148 in vivo. Altogether, these results revealed the absolute necessity of glucose as a regulator of Runx2 accumulation during osteoblast differentiation and bone formation in vivo and highlight the fundamental importance of the intricate cross-talk between bone and whole-body glucose metabolism.

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.

Prolonged periods of non-weightbearing activity result in a significant loss of bone mass which increases the risk of fracture with the initiation of mechanical loading. The loss of bone mass is partially driven by declines in bone formation yet the mechanisms responsible for this decline are unclear. To investigate the limitations of osteoblasts during disuse, marrow ablation was superimposed on hindlimb unloaded mice. Marrow ablation is a useful model to study osteoblast functionality as new cancellous bone is rapidly formed throughout the marrow of a long bone while hindlimb unloading is the most common method used to produce skeletal unloading. The specific hypotheses of this study were aimed at determining if changes in osteoblast functionality, differentiation, and/or proliferation were compromised in non-weightbearing bone in response to a bone formation stimulus. Additionally, the influence of having compromised osteoblast functionality at the time of stimulation was assessed in non-weightbearing bones. Key outcome measures used to address these hypotheses included static and dynamic cancellous bone histomorphometry, bone densitometry, and real-time polymerase chain reaction (PCR) analyses of gene expression. The results document similar ablation-induced increases of cancellous bone in both weightbearing and unloaded animals. Similarly, there was no influence of load on ablation-induced increases in cancellous bone forming surface or mineral apposition rate. Unloading did significantly attenuate the ablation-induced increase in bone formation rate, due to reduced levels of total surface mineralization. When osteoblast functionality was compromised prior to marrow ablation, bone formation rate increases were also attenuated in ablated animals due to reduced mineralization. Additionally, increases in forming surface were attenuated as compared to unloaded animals having normal osteoblast function at the time of ablation. Collectively, these data identify mineralization as the limiting step in new bone formation during periods of disuse. The caveat, however, is that when bone formation is stimulated after a period of unloading sufficient to compromise osteoblast functionality, increases in osteoblast recruitment to the bone surface are compromised.

Prolonged periods of non-weightbearing activity result in a significant loss of bone mass which increases the risk of fracture with the initiation of mechanical loading. The loss of bone mass is partially driven by declines in bone formation yet the mechanisms responsible for this decline are unclear. To investigate the limitations of osteoblasts during disuse, marrow ablation was superimposed on hindlimb unloaded mice. Marrow ablation is a useful model to study osteoblast functionality as new cancellous bone is rapidly formed throughout the marrow of a long bone while hindlimb unloading is the most common method used to produce skeletal unloading. The specific hypotheses of this study were aimed at determining if changes in osteoblast functionality, differentiation, and/or proliferation were compromised in non-weightbearing bone in response to a bone formation stimulus. Additionally, the influence of having compromised osteoblast functionality at the time of stimulation was assessed in non-weightbearing bones. Key outcome measures used to address these hypotheses included static and dynamic cancellous bone histomorphometry, bone densitometry, and real-time polymerase chain reaction (PCR) analyses of gene expression. The results document similar ablation-induced increases of cancellous bone in both weightbearing and unloaded animals. Similarly, there was no influence of load on ablation-induced increases in cancellous bone forming surface or mineral apposition rate. Unloading did significantly attenuate the ablation-induced increase in bone formation rate, due to reduced levels of total surface mineralization. When osteoblast functionality was compromised prior to marrow ablation, bone formation rate increases were also attenuated in ablated animals due to reduced mineralization. Additionally, increases in forming surface were attenuated as compared to unloaded animals having normal osteoblast function at the time of ablation. Collectively, these data identify mineralization as the limiting step in new bone formation during periods of disuse. The caveat, however, is that when bone formation is stimulated after a period of unloading sufficient to compromise osteoblast functionality, increases in osteoblast recruitment to the bone surface are compromised.

Thesis (Ph. D.)--School of Dentistry. University of Missouri--Kansas City, 2006. / "A dissertation in oral biology and cell biology and biophysics." Advisor: Lynda F. Bonewald. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Nov. 12, 2007. Includes bibliographical references (leaves 121-139). Online version of the print edition.

Diss. (sammanfattning) Umeå : Umeå universitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Upon amputation, zebrafish innately regenerate lost or damaged bone by precisely positioning injury-induced, lineage-restricted osteoblast progenitors (pObs). While substantial progress has been made in identifying the cellular and molecular mechanisms underlying this fascinating process, the cell-specific function of these pathways is poorly understood. Understanding how molecular signals initiate osteoblast dedifferentiation, balance progenitor renewal and re-differentiation, and control bone shape during regeneration are of paramount importance for developing human therapies. We show that fin amputation induces a Wnt/β-catenin-dependent epithelial to mesenchymal transformation (EMT) of osteoblasts to generate proliferative Runx2+ pObs. Localized Wnt/β-catenin signaling maintains this progenitor population towards the distal tip of the regenerative blastema. As they become proximally displaced, pObs upregulate sp7 and subsequently mature into re-epithelialized Runx2-/sp7+ osteoblasts that extend pre-existing bone. Autocrine Bone Morphogenetic Protein (BMP) signaling promotes osteoblast differentiation by activating sp7 expression and counters Wnt by inducing Dickkopf-related Wnt antagonists. As such, opposing activities of Wnt and BMP coordinate the simultaneous demand for growth and differentiation during bone regeneration. Previous studies have implicated Hedgehog/Smoothened (Hh/Smo) signaling in controlling the re-establishment of stereotypically branched bony rays during fin regeneration. Using a photoconvertible patched2 reporter, we resolve active Hh/Smo output to a narrow distal regenerate zone comprising pObs and neighboring migratory basal epidermal cells. Hh/Smo activity is driven by epidermal Sonic hedgehog a (Shha) rather than Ob-derived Indian hedgehog a (Ihha), which instead uses non-canonical signaling to support bone maturation. Using high-resolution imaging and BMS-833923, a uniquely effective Smo inhibitor, we show that Shha/Smo promotes branching by escorting pObs into split groups that mirror transiently divided clusters of Shha-expressing epidermis. Epidermal cellular protrusions directly contact pObs only where an otherwise occluding basement membrane remains incompletely assembled. These intimate interactions progressively generate physically separated pOb pools that then regenerate independently to collectively re-form a now branched bone. Our studies elucidate a signaling network model that provides a conceptual framework to understand innate bone repair and regeneration mechanisms and rationally design regenerative therapeutics. This dissertation includes previously published co-authored material. / 10000-01-01