Orthopedic implants containing biodegradable magnesium have been used for fracture repair with considerable efficacy; however, the underlying mechanisms by which these implants improve fracture healing remain elusive. Here we show the formation of abundant new bone at peripheral cortical sites after intramedullary implantation of a pin containing ultrapure magnesium into the intact distal femur in rats. This response was accompanied by substantial increases of neuronal calcitonin gene-related polypeptide-α (CGRP) in both the peripheral cortex of the femur and the ipsilateral dorsal root ganglia (DRG). Surgical removal of the periosteum, capsaicin denervation of sensory nerves or knockdown in vivo of the CGRP-receptor-encoding genes Calcrl or Ramp1 substantially reversed the magnesium-induced osteogenesis that we observed in this model. Overexpression of these genes, however, enhanced magnesium-induced osteogenesis. We further found that an elevation of extracellular magnesium induces magnesium transporter 1 (MAGT1)-dependent and transient receptor potential cation channel, subfamily M, member 7 (TRPM7)-dependent magnesium entry, as well as an increase in intracellular adenosine triphosphate (ATP) and the accumulation of terminal synaptic vesicles in isolated rat DRG neurons. In isolated rat periosteum-derived stem cells, CGRP induces CALCRL-and RAMP1-dependent activation of cAMP-responsive element binding protein 1 (CREB1) and SP7 (also known as osterix), and thus enhances osteogenic differentiation of these stem cells. Furthermore, we have developed an innovative, magnesium-containing intramedullary nail that facilitates femur fracture repair in rats with ovariectomy-induced osteoporosis. Taken together, these findings reveal a previously undefined role of magnesium in promoting CGRP-mediated osteogenic differentiation, which suggests the therapeutic potential of this ion in orthopedics.

Differentiation of mesenchymal stem cells into a particular lineage is tightly regulated, and malfunction of this regulation could lead to pathological consequences. Patients with osteoporosis have increased adipocyte accumulation, but the mechanisms involved remain to be defined. In this study, we aimed to investigate if microRNAs regulate mesenchymal progenitor cells and bone marrow stromal cell (BMSC) differentiation through modulation of Runx2, a key transcription factor for osteogenesis. We found that miR-204 and its homolog miR-211 were expressed in mesenchymal progenitor cell lines and BMSCs and their expression was induced during adipocyte differentiation, whereas Runx2 protein expression was suppressed. Retroviral overexpression of miR-204 or transfection of miR-204 oligo decreased Runx2 protein levels and miR-204 inhibition significantly elevated Runx2 protein levels, suggesting that miR-204 acts as an endogenous attenuator of Runx2 in mesenchymal progenitor cells and BMSCs. Mutations of putative miR-204 binding sites upregulated the Runx2 3 0 -UTR reporter activity, suggesting that miR-204/211 bind to Runx2 3 0 -UTR. Perturbation of miR-204 resulted in altered differentiation fate of mesenchymal progenitor cells and BMSCs: osteoblast differentiation was inhibited and adipocyte differentiation was promoted when miR-204 was overexpressed in these cells, whereasosteogenesis was upregulated and adipocyte formation was impaired when miR-204 was inhibited. Together, our data demonstrated that miR-204/211 act as important endogenous negative regulators of Runx2, which inhibit osteogenesis and promote adipogenesis of mesenchymal progenitor cells and BMSCs.

Self-incompatibility S-locus–encoded F-box (SLF) proteins have been identified in Antirrhinum and several Prunus species. Although they appear to play an important role in self-incompatible reaction, functional evidence is lacking. Here, we provide several lines of evidence directly implicating a role of AhSLF-S2 in self-incompatibility in Antirrhinum. First, a nonallelic physical interaction between AhSLF-S2 and S-RNases was demonstrated by both coimmunoprecipitation and yeast two-hybrid assays. Second, AhSLF-S2 interacts with ASK1- and CULLIN1-like proteins in Antirrhinum, and together, they likely form an Skp1/Cullin or CDC53/F-box (SCF) complex. Third, compatible pollination was specifically blocked after the treatment of the proteasomal inhibitors MG115 and MG132, but they had little effect on incompatible pollination both in vitro and in vivo, indicating that the ubiquitin/26S proteasome activity is involved in compatible pollination. Fourth, the ubiquitination level of style proteins was increased substantially after compatible pollination compared with incompatible pollination, and coimmunoprecipitation revealed that S-RNases were ubiquitinated after incubating pollen proteins with compatible but not with incompatible style proteins, suggesting that non-self S-RNases are possibly degraded by the ubiquitin/26S proteasome pathway. Fifth, the S-RNase level appeared to be reduced after 36 h of compatible pollination. Taken together, these results show that AhSLF-S2 interacts with S-RNases likely through a proposed SCFAhSLF-S2 complex that targets S-RNase destruction during compatible rather than incompatible pollination, thus providing a biochemical basis for the inhibition of pollen tube growth as observed in self-incompatible response in Antirrhinum

Recently, we have provided evidence that the polymorphic self-incompatibility (S) locus-encoded F-box (SLF) protein AhSLF-S 2 plays a role in mediating a selective S-RNase destruction during the self-incompatible response in Antirrhinum hispanicum. To investigate its role further, we first transformed a transformation-competent artificial chromosome clone (TAC26) containing both AhSLF-S 2 and AhS 2 -RNase into a self-incompatible (SI) line of Petunia hybrida. Molecular analyses showed that both genes are correctly expressed in pollen and pistil in four independent transgenic lines of petunia. Pollination tests indicated that all four lines became self-compatible because of the specific loss of the pollen function of SI. This alteration was transmitted stably into the T1 progeny. We then transformed AhSLF-S 2 cDNA under the control of a tomato (Lycopersicon esculentum) pollen-specific promoter LAT52 into the self-incompatible petunia line. Molecular studies revealed that AhSLF-S 2 is specifically expressed in pollen of five independent transgenic plants. Pollination tests showed that they also had lost the pollen function of SI. Importantly, expression of endogenous SLF or SLF-like genes was not altered in these transgenic plants. These results phenocopy a well-known phenomenon called competitive interaction whereby the presence of two different pollen S alleles within pollen leads to the breakdown of the pollen function of SI in several solanaceaous species. Furthermore, we demonstrated that AhSLF-S 2 physically interacts with PhS 3 -RNase from the P. hybrida line used for transformation. Together with the recent demonstration of PiSLF as the pollen determinant in P. inflata, these results provide direct evidence that the polymorphic SLF including AhSLF-S 2 controls the pollen function of S-RNase-based self-incompatibility.

Patients with chronic inflammatory disorders, such as rheumatoid arthritis, often have osteoporosis due to a combination of Tumor necrosis factor-induced increased bone resorption and reduced bone formation. To test if TNF inhibits bone formation by affecting the commitment and differentiation of mesenchymal stem cells (MSCs) into osteoblasts, we examined the osteogenic potential of MSCs from TNF transgenic (TNF-Tg) mice, a model of chronic inflammatory arthritis. MSC-enriched cells were isolated from bone marrow stromal cells using negative selection with anti-CD45 antibody coated magnetic beads. The expression profile of MSC surface markers the osteogenic, chondrogenic, and adipogenic properties of CD45− cells were confirmed by FACS and cell differentiation assays. MSC-enriched CD45− cells from TNF-Tg mice formed significantly decreased numbers of fibroblast and ALP+ colonies and had a decreased expression of osteoblast marker genes. As TNF may upregulate ubiquitin ligases, which negatively regulate osteoblast differentiation, we examined the expression levels of several ubiquitin ligases and found that Wwp1 expression was significantly increased in MSC-enriched CD45− cells of TNF-Tg mice. Wwp1 knockdown rescued impaired osteoblast differentiation of TNF-Tg CD45− cells. Wwp1 promotes ubiquitination and degradation of JunB, an AP-1 transcription factor that positively regulates osteoblast differentiation. Injection of TNF into wild-type mice resulted in decreased osteoblast differentiation of MSCs and increased JunB ubiquitination, which was completely blocked in Wwp1−/− mice. Thus, Wwp1 targets JunB for ubiquitination and degradation in MSCs after chronic exposure to TNF, and inhibition of Wwp1 in MSCs could be a new mechanism to limit inflammation-mediated osteoporosis by promoting their differentiation into osteoblasts.

Chronic inflammatory disorders, such as rheumatoid arthritis, are often accompanied by systemic bone loss, which is thought to occur through inflammatory cytokine-mediated stimulation of osteoclast resorption and inhibition of osteoblast function. However, the mechanisms involved in osteoblast inhibition remain poorly understood. Here we test the hypothesis that increased Smad ubiquitin regulatory factor 1 (Smurf1)-mediated degradation of the bone morphogenetic protein pathway signaling proteins mediates reduced bone formation in inflammatory disorders. Osteoblasts derived from bone marrow or long bone samples of adult tumor necrosis factor (TNF) transgenic (TNF-Tg) mice were used in this study. TNF decreased the steady-state levels of Smad1 and Runx2 protein similarly to those in long bones of TNF-Tg mice. In the presence of the proteasome inhibitor MG132, TNF increased accumulation of ubiquitinated Smad1 protein. TNF administration over calvarial bones caused decreases in Smad1 and Runx2 protein levels and mRNA expression of osteoblast marker genes in wild-type, but not in Smurf1 ؊/؊ mice. Vertebral bone volume and strength of TNF-Tg/Smurf1 ؊/؊ mice were examined by a combination of micro-CT, bone histomorphometry, and biomechanical testing and compared with those from TNF-Tg littermates. TNF-Tg mice had significantly decreased bone volume and biomechanical properties, which were partially rescued in TNF-Tg/Smurf1 ؊/؊ mice. We conclude that in chronic inflammatory disorders where TNF is increased, TNF induces the expression of ubiquitin ligase Smurf1 and promotes ubiquitination and proteasomal degradation of Smad1 and Runx2, leading to systemic bone loss. Inhibition of ubiquitin-mediated Smad1 and Runx2 degradation in osteoblasts could help to treat inflammation-induced osteoporosis.Osteoporosis and fragility fractures are common and preventable complications of rheumatoid arthritis (RA).2 For example, one study has reported that 53.3% of RA patients had osteoporosis and 19.3% had vertebral fractures, rates that are much higher than those of the general population (1). These complications are thought to occur through inflammatory stimulation of osteoclast bone resorption and inhibition of osteoblast function (2) mediated by cytokines, such as TNF (3-5). TNF and other cytokines are overproduced by various cells in the inflamed joints of RA patients, which lead to severe local erosion of cartilage and bone, as well as periarticular osteopenia and systemic osteoporosis.TNF is a strong inhibitor of osteoblast functions in vitro, but the molecular mechanisms that mediate the inhibitory effects of TNF on osteoblasts have not been fully investigated. TNF inhibits the recruitment of osteoblast progenitors, reduces expression of genes produced by mature osteoblasts, and promotes osteoblast apoptosis through the NF-B pathway (6 -10). It decreases the expression and DNA binding activity of runt-related transcription factor 2 (Runx2), which is partially through suppression of Runx2 gene transcription and destabilization o...

ObjectivesIn this study, we aim to determine the effect of metformin on osteoarthritis (OA) development and progression.MethodsDestabilisation of the medial meniscus (DMM) surgery was performed in 10-week-old wild type and AMP-activated protein kinase (AMPK)α1 knockout (KO) mice. Metformin (4 mg/day in drinking water) was given, commencing either 2 weeks before or 2 weeks after DMM surgery. Mice were sacrificed 6 and 12 weeks after DMM surgery. OA phenotype was analysed by micro-computerised tomography (μCT), histology and pain-related behaviour tests. AMPKα1 (catalytic alpha subunit of AMPK) expression was examined by immunohistochemistry and immunofluorescence analyses. The OA phenotype was also determined by μCT and MRI in non-human primates.ResultsMetformin upregulated phosphorylated and total AMPK expression in articular cartilage tissue. Mild and more severe cartilage degeneration was observed at 6 and 12 weeks after DMM surgery, evidenced by markedly increased Osteoarthritis Research Society International scores, as well as reduced cartilage areas. The administration of metformin, commencing either before or after DMM surgery, caused significant reduction in cartilage degradation. Prominent synovial hyperplasia and osteophyte formation were observed at both 6 and 12 weeks after DMM surgery; these were significantly inhibited by treatment with metformin either before or after DMM surgery. The protective effects of metformin on OA development were not observed in AMPKα1 KO mice, suggesting that the chondroprotective effect of metformin is mediated by AMPK signalling. In addition, we demonstrated that treatment with metformin could also protect from OA progression in a partial medial meniscectomy animal model in non-human primates.ConclusionsThe present study suggests that metformin, administered shortly after joint injury, can limit OA development and progression in injury-induced OA animal models.

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