Osteoblast-Restricted Disruption of the Growth Hormone Receptor in Mice Results in Sexually Dimorphic Skeletal Phenotypes

Growth hormone (GH) exerts profound anabolic actions during postnatal skeletal development, in part, through stimulating the production of insulin-like growth factor-1 (IGF-1) in liver and skeletal tissues. To examine the requirement for the GH receptor (GHR) in osteoblast function in bone, we used Cre-LoxP methods to disrupt the GHR from osteoblasts, both in vitro and in vivo. Disruption of GHR from primary calvarial osteoblasts in vitro abolished GH-induced signaling, as assessed by JAK2/STAT5 phosphorylation, and abrogated GH-induced proliferative and anti-apoptotic actions. Osteoblasts lacking GHR exhibited reduced IGF-1-induced Erk and Akt phosphorylation and attenuated IGF-1-induced proliferation and anti-apoptotic action. In addition, differentiation was modestly impaired in osteoblasts lacking GHR, as demonstrated by reduced alkaline phosphatase staining and calcium deposition. In order to determine the requirement for the GHR in bone in vivo, we generated mice lacking the GHR specifically in osteoblasts (ΔGHR), which were born at the expected Mendelian frequency, had a normal life span and were of normal size. Three week-old, female ΔGHR mice had significantly reduced osteoblast numbers, consistent with the in vitro data. By six weeks of age however, female ΔGHR mice demonstrated a marked increase in osteoblasts, although mineralization was impaired; a phenotype similar to that observed previously in mice lacking IGF-1R specifically in osteoblasts. The most striking phenotype occurred in male mice however, where disruption of the GHR from osteoblasts resulted in a feminization of bone geometry in 16 week-old mice, as observed by μCT. These results demonstrate that the GHR is required for normal postnatal bone development in both sexes. GH appears to serve a primary function in modulating local IGF-1 action. However, the changes in bone geometry observed in male ΔGHR mice suggest that, in addition to facilitating IGF-1 action, GH may function to a greater extent than previously appreciated in establishing the sexual dimorphism of the skeleton

Retinoic Acid Increases Proliferation of Human Osteoclast Progenitors and Inhibits RANKL-Stimulated Osteoclast Differentiation by Suppressing RANK

It has been shown that high vitamin A intake is associated with bone fragility and fractures in both animals and humans. However, the mechanism by which vitamin A affects bones is unclear. In the present study, the direct effects of retinoic acid (RA) on human and murine osteoclastogenesis were evaluated using cultured peripheral blood CD14+ monocytes and RAW264.7 cells. Both the activity of the osteoclast marker tartrate resistant acid phosphatase (TRAP) in culture supernatant and the expression of the genes involved in osteoclast differentiation together with bone resorption were measured. To our knowledge, this is the first time that the effects of RA on human osteoclast progenitors and mature osteoclasts have been studied in vitro. RA stimulated proliferation of osteoclast progenitors both from humans and mice. In contrast, RA inhibited differentiation of the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis of human and murine osteoclast progenitors via retinoic acid receptors (RARs). We also show that the mRNA levels of receptor activator of nuclear factor κB (RANK), the key initiating factor and osteoclast associated receptor for RANKL, were potently suppressed by RA in osteoclast progenitors. More importantly, RA abolished the RANK protein in osteoclast progenitors. This inhibition could be partially reversed by a RAR pan-antagonist. Furthermore, RA treatment suppressed the expression of the transcription factor nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and increased the expression of interferon regulatory factor-8 (IRF-8) in osteoclast progenitors via RARs. Also, RA demonstrated differential effects depending on the material supporting the cell culture. RA did not affect TRAP activity in the culture supernatant in the bone slice culture system, but inhibited the release of TRAP activity if cells were cultured on plastic. In conclusion, our results suggest that retinoic acid increases proliferation of human osteoclast progenitors and that it inhibits RANK-stimulated osteoclast differentiation by suppressing RANK

Inhibition of the Progesterone Nuclear Receptor during the Bone Linear Growth Phase Increases Peak Bone Mass in Female Mice

Augmentation of the peak bone mass (PBM) may be one of the most effective interventions to reduce the risk of developing osteoporosis later in life; however treatments to augment PBM are currently limited. Our study evaluated whether a greater PBM could be achieved either in the progesterone nuclear receptor knockout mice (PRKO) or by using a nuclear progesterone receptor (nPR) antagonist, RU486 in mice. Compared to their wild type (WT) littermates the female PRKO mice developed significantly higher cancellous and cortical mass in the distal femurs, and this was associated with increased bone formation. The high bone mass phenotype was partially reproduced by administering RU486 in female WT mice from 1–3 months of age. Our results suggest that the inhibition of the nPR during the rapid bone growth period (1–3 months) increases osteogenesis, which results in acquisition of higher bone mass. Our findings suggest a crucial role for progesterone signaling in bone acquisition and inhibition of the nPR as a novel approach to augment bone mass, which may have the potential to reduce the burden of osteoporosis

Short time administration of antirheumatic drugs - Methotrexate as a strong inhibitor of osteoblast's proliferation in vitro

<p>Abstract</p> <p>Introduction</p> <p>Due to increasing use of disease modifying antirheumatic drugs (DMARDs) as first line therapy in rheumatic diseases, dental and maxillofacial practitioner should be aware of drug related adverse events. Especially effects on bone-metabolism and its cells are discussed controversially. Therefore we investigate the in vitro effect of short time administration of low dose methotrexate (MTX) on osteoblasts as essential part of bone remodelling cells.</p> <p>Methods</p> <p>Primary bovine osteoblasts (OBs) were incubated with various concentrations of MTX, related to tissue concentrations, over a period of fourteen days by using a previously established standard protocol. The effect on cell proliferation as well as mitochondrial activity was assessed by using 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT) assay, imaging and counting of living cells. Additionally, immunostaining of extracellular matrix proteins was used to survey osteogenic differentiation.</p> <p>Results</p> <p>All methods indicate a strong inhibition of osteoblast`s proliferation by short time administration of low dose MTX within therapeutically relevant concentrations of 1 to 1000nM, without affecting cell differentiation of middle-stage differentiated OBs in general. More over a significant decrease of cell numbers and mitochondrial activity was found at these MTX concentrations. The most sensitive method seems to be the MTT-assay. MTX-concentration of 0,01nM and concentrations below had no inhibitory effects anymore.</p> <p>Conclusion</p> <p>Even low dose methotrexate acts as a potent inhibitor of osteoblast’s proliferation and mitochondrial metabolism in vitro, without affecting main differentiation of pre-differentiated osteoblasts. These results suggest possible negative effects of DMARDs concerning bone healing and for example osseointegration of dental implants. Especially the specifics of the jaw bone with its high vascularisation and physiological high tissue metabolism, suggests possible negative effects of DMARD therapy concerning oral and cranio-maxillofacial bone surgery as could be seen in a similar way in bisphosphonate related osteonecrosis of the jaw.</p