Noggin as a regulator of bone remodelling

Bone Morphogenetic Protein 2 (BMP2) is used in orthopaedic surgery to promote bone healing. The endogenous synthesis of BMP-2 antagonist family members, however, may limit the efficacy of exogenous BMP2. Noggin is one of these inhibitors that blocks the effects of BMP on the differentiation and activation of osteoblast (OB) in vitro and in vivo and inhibits OB-mediated osteoclast (OC) development. Furthermore, Noggin was found to modulate osteoclastogenesis through a direct effect on OC lineage cells. The present study aimed at elucidating the underlying mechanisms of these effects. Direct (conventional culture dishes) and indirect (transwell culture dishes) co-cultures of murine OB/OPC (Osteoclast Progenitor Cells) and cultures of OPC alone were supplemented with combinations of Noggin, BMP2, L51P (engineered, inactive variant of BMP2) and DMH1 (BMP receptor 1 inhibitor). In cultures of OPC, Noggin but not DMH1 caused an increase in the number of OC by a factor of 3 (p< 0.01). This effect could not be reversed by BMP2 and L51P, respectively. In contrast, in co-cultures of OB/OPC, exposure to Noggin attenuated OC development. In direct co-cultures, this inhibitory effect of Noggin was blocked by BMP2 and L51P. In both direct and indirect co-culture systems, exposure to Noggin induced the release of GM-CSF, a potent inhibitor of osteoclastogenesis, by a factor of 6 and 4, respectively (p< 0.01). Treatment of the cultures with αGM-CSF Ab, however, restored OC development in the indirect co-culture system only. The data suggests a previously unknown function of Noggin directly acting pro-differentiation on OC lineage cells independently of BMP signalling. In co-cultures, besides GM-CSF, cell-cell contact between OB and OPC is required for mediation of the maximal inhibitory effects of Noggin on OC development. The nature of potential interaction partners for Noggin, however, remains to be elucidated

Inositol Phosphatase SHIP1 – a Regulator of Osteoclast Lineage Cell Development and Activity

Introduction: Src-homology (SH) 2 domain-containing inositol-5-phosphatase 1 (SHIP1) is a negative regulator of the PI3K/Akt pathway that is expressed in hematopoietic cells. Osteoclast (OC) development depends on two essential pathways activated by receptor activator of NF-κB ligand (RANKL) and colony-stimulating factor-1 (CSF-1). Both pathways involve PI3K in their signalling and may therefore be regulated by SHIP1. SHIP1-deficient mice ((SHIPstyx/styx) are characterized by low bone density that has been suggested to be caused by an increased number of hyperactive OC. Purpose: This study aimed to investigate cellular mechanisms leading to low bone mass in SHIP1-deficient mice. Methods: MicroCT analysis of vertebrae and femora was performed to evaluate bone structure in vivo. To study OC development in vitro, progenitor cells (OPC) from SHIP1-deficient SHIPstyx/styx and control mice were cultured with RANKL and CSF-1. Osteoclastogenesis was assessed using an XTT cell viability assay and by determining TRAP activity. Furthermore, the capacity of OC to dissolve amorphous calcium phosphate (CaP) was determined. Results: In vivo, BV/TV of vertebrae and femora of SHIPstyx/styx mice was decreased compared to wt animals (40% and 35%, respectively, p<0.01). Trabecular number in vertebrae from SHIPstyx/styx mice was increased by 26%, while thickness was decreased by 30% (p<0.01). In femora from SHIPstyx/styx, trabecular thickness was reduced by 25% (p<0.05), whereas trabecular number remained unchanged. In vitro, SHIPstyx/styx OPC showed a 1.5-fold increased proliferation compared to controls (p<0.001), yet the number of OPC-derived OC was reduced by 40%. The capacity of SHIPstyx/styx OC to dissolve CaP was decreased by 60% compared to controls (p<0.001). Conclusions: Our data indicates a central role for SHIP1 in OC development and activity in vitro. The low bone mass phenotype in SHIPstyx/styx mice, however, may be caused by reduced bone formation or by the wasting disease and systemic inflammatory condition characteristic of SHIP1-deficient mice

The inositol phosphatase SHIP1 regulates skeletal development

Background/Introduction: Src-homology (SH) 2 domain-containing inositol-5-phosphatase 1 (SHIP1) is a lipid phosphatase expressed mainly in hematopoietic cells. SHIP1 regulates cell proliferation, differentiation, and survival via the PI3K/Akt signaling pathway. SHIP1-deficient (Styx) mice are osteoporotic, which is associated with an increased number of osteoclasts (OC). Purpose: This study aimed to investigate the underlying mechanisms through which SHIP1 controls osteoporosis. Methods: Osteoclast progenitor cells (OPC) were generated by incubating bone marrow cells with CSF-1. To develop OC, OPC from Styx, Styx het (heterozygous) and wt (wild type) mice were cultured with RANKL and CSF-1. Osteoclastogenesis was evaluated using an XTT cell viability assay, TRAP activity (OC marker) and qRT-PCR. Micro-computed tomography (Micro-CT) of vertebrae and femora were performed to evaluate the bone structure. Results: Deficiency in SHIP1 affected several aspects of bone. Compared to Styx het and wt controls, OPC-derived Styx OC presented several developmental defects, including a lower TRAP/XTT ratio and a 52% decrease in Calcr transcripts (encoding for the Calcitonin Receptor) (p<0.001). In vivo, there was a strong reduction of BV/TV in vertebrae and femora of Styx versus wt animals (39.6% and 35%, respectively, p<0.01). In particular, trabeculae in Styx vertebrae were increased by 8% (p<0.05) in numbers while decreased by 37% in thickness (p<0.001). In contrast, in Styx femora both the number and thickness of the trabeculae were decreased by 16% and 14%, respectively. These different phenotypes in Styx femora versus vertebrae indicate different paths to osteoporosis in bones with primary or secondary spongiosa. Conclusion(s): Taken together, our data indicate a central role for SHIP1-dependent PI3K/Akt signalling in bone remodeling. Further investigation will address the role of osteoblasts in the development of osteoporosis in SHIP1-deficient Styx mice

SHIP1 deficiency causes inflammation-dependent retardation in skeletal growth.

Inflammation and skeletal homeostasis are closely intertwined. Inflammatory diseases are associated with local and systemic bone loss, and post-menopausal osteoporosis is linked to low-level chronic inflammation. Phosphoinositide-3-kinase signalling is a pivotal pathway modulating immune responses and controlling skeletal health. Mice deficient in Src homology 2-containing inositol phosphatase 1 (SHIP1), a negative regulator of the phosphoinositide-3-kinase pathway, develop systemic inflammation associated with low body weight, reduced bone mass, and changes in bone microarchitecture. To elucidate the specific role of the immune system in skeletal development, a genetic approach was used to characterise the contribution of SHIP1-controlled systemic inflammation to SHIP1-dependent osteoclastogenesis. Lymphocyte deletion entirely rescued the skeletal phenotype in Rag2 -/- /Il2rg -/- /SHIP1 -/- mice. Rag2 -/- /Il2rg -/- /SHIP1 -/- osteoclasts, however, displayed an intermediate transcriptomic signature between control and Rag2 +/+ /Il2rg +/+ /SHIP1 -/- osteoclasts while exhibiting aberrant in vitro development and functions similar to Rag2 +/+ /Il2rg +/+ /SHIP1 -/- osteoclasts. These data establish a cell-intrinsic role for SHIP1 in osteoclasts, with inflammation as the key driver of the skeletal phenotype in SHIP1-deficient mice. Our findings demonstrate the central role of the immune system in steering physiological skeletal development

Refeeding-Syndrom: Was wissen Humanmedizinstudierende im letzten Studiensemester?

Hintergrund Das Refeeding-Syndrom (RFS) ist eine Stoffwechselreaktion verursacht durch eine inadäquate Wiederernährung nach einer Hungerphase. Das RFS ist gekennzeichnet durch eine Serumelektrolyt- und Flüssigkeitsverschiebung, welche schwerwiegende Folgen wie Herzrhythmusstörungen verursachen können und gelegentlich zum Tod führen. Zur Prävention sind eine frühzeitige Risikoerkennung sowie ein angepasstes Ernährungsmanagement notwendig. Verschiedene Studien weisen auf ein unzureichendes Wissen der Ärzteschaft hin. Für die Schweiz existieren zum heutigen Zeitpunkt keine Daten. Mit dieser Studie wurde der Wissensstand der Humanmedizinstudierenden der Deutschschweiz im letzten Semester zum RFS untersucht. Material und Methodik Das Wissen wurde mittels Onlinefragebogen erfasst. Dieser enthielt 15 Multiple-Choice-Fragen. Die maximal mögliche Gesamtpunktzahl betrug 20 Punkte. Die Ergebnisse wurden gemäß Schweizer Notenberechnung beurteilt (beste Note 6, schlechteste Note 1). 60 % der Punkte mussten erreicht werden, um ein genügendes Ergebnis (Note 4) zu erreichen. Ergebnisse 79 von 540 Studierenden beantworteten den Fragebogen, was einer Rücklaufquote von 14,6 % entspricht. Die Studierenden erreichten durchschnittlich 9,9 Punkte (50 % der Gesamtpunktzahl). Das Resultat von 53 % der Studierenden war ungenügend. Neun von 79 Studierenden (11,4 %) war das RFS unbekannt. Schlussfolgerung Das Wissen der Schweizer Humanmedizinstudierenden zum RFS ist nicht zufriedenstellend und sollte unbedingt optimiert werden. In stationären Einrichtungen ist ein flächendeckendes Ernährungsscreening mit einem validierten Instrument empfohlen. Risikopatienten für eine Mangelernährung sollten an eine Ernährungsberatung (ERB) überwiesen werden. Die ERB erkennt im Ernährungsassessment das Risiko für ein RFS und kann in Zusammenarbeit mit der behandelnden Ärzteschaft den Ernährungsaufbau planen. Eine gute multiprofessionelle Zusammenarbeit ist unerlässlich, um Komplikationen zu verhindern.Background Refeeding syndrome (RFS) is a metabolic reaction caused by inadequate refeeding after starvation. It is associated with electrolyte and fluid shifts. Severe consequences such as cardiac arrhythmia and death are possible. Prevention entails risk detection and adequate nutritional management. Several studies point to inadequate knowledge of physicians. No data is available for Switzerland. This trial aimed to investigate the knowledge on RFS of medical students in their last semester in German speaking Switzerland. Material and Methods The knowledge of medical students was tested using an online questionnaire including 15 multiple-choice questions. The maximum total score was 20 points. Results were evaluated using standard Swiss educational grading system (best grade 6, worst grade 1). A minimum of 60 % total score was needed for a satisfactory result (grade 4). Results Ninety-seven of 540 students answered the questionnaire, which corresponds to a response rate of 14.6 %. The students achieved an average score of 9.9 points (50 % of the total score). 53 % of the students showed insufficient knowledge. The RFS was unknown to nine of 79 students (11.4 %). Conclusion The knowledge of Swiss medical students on RFS is unsatisfactory and should be optimized. In clinical practice, a nutritional screening with a validated tool is recommended in all inpatients. Patients at risk for malnutrition should be referred to a dietitian for nutritional assessment. The dietitian recognizes the risk of RFS and may subsequently plan nutritional management in collaboration with the treating physicists. A well-functioning multiprofessional collaboration is essential to prevent complications

Extracellular Iron is a Modulator of the Differentiation of Osteoclast Lineage Cells.

Osteoclasts originate from the hematopoietic stem cell and share a differentiation pathway with the cells of the monocyte/macrophage lineages. Development and activation of osteoclasts, and as a consequence regulation of bone resorption, depend on two growth factors: macrophage colony-stimulating factor and receptor activator of NF-κB ligand. Furthermore, cell development and activity are modulated by a microenvironment composed of cytokines and growth factors and of the extracellular matrix. Membrane transporters are a means for cells to interact with their environment. Within this study, the expression of proteins regulating cellular iron homeostasis in osteoclast-like cells grown from bone marrow-derived progenitors was compared to the expression of this set of proteins by monocyte/macrophage lineage cells. In differentiating osteoclasts, levels of transcripts encoding transferrin receptor 1 and divalent metal transporter 1 (Slc11A2) were increased, while levels of transcripts encoding ferroportin (Slc40A1) and natural resistance-associated macrophage protein 1 (Slc11A1) were decreased. Supplementation of the culture media with exogenous iron led to an increase in the proliferation of osteoclast progenitor cells and to the expression of a macrophage-like phenotype, while the development of osteoclasts was reduced. Upon transfer of mature OC onto a CaP substrate, iron depletion of the medium with the Fe(3+)-chelator Deferoxamine Mesylate decreased CaP dissolution by ~30 %, which could be restored by addition of exogenous iron. During the 24 h of the assay, no effects were observed on total TRAP activity. The data demonstrate transcriptional regulation of the components of cellular iron transporters during OC development and suggests that iron homeostasis may contribute to fine-tuning of the RANKL-induced OC development

Glutamate Receptor Agonists and Glutamate Transporter Antagonists Regulate Differentiation of Osteoblast Lineage Cells

Development and function of osteoblast lineage cells are regulated by a complex microenvironment consisting of the bone extracellular matrix, cells, systemic hormones and cytokines, autocrine and paracrine factors, and mechanical load. Apart from receptors that transduce extracellular signals into the cell, molecular transporters play a crucial role in the cellular response to the microenvironment. Transporter molecules are responsible for cellular uptake of nutritional components, elimination of metabolites, ion transport, and cell-cell communication. In this report, the expression of molecular transporters in osteoblast lineage cells was investigated to assess their roles in cell development and activity. Low-density arrays, covering membrane and vesicular transport molecules, were used to assess gene expression in osteoblasts representing early and late differentiation states. Receptors and transporters for the amino acid glutamate were found to be differentially expressed during osteoblast development. Glutamate is a neurotransmitter in the central nervous system, and the mechanisms of its release, signal transduction, and cellular reabsorption in the synaptic cleft are well understood. Less clear, however, is the control of equivalent processes in peripheral tissues. In primary osteoblasts, inhibition of glutamate transporters with nonselective inhibitors leads to an increase in the concentration of extracellular glutamate. This change was accompanied by a decrease in osteoblast proliferation, stimulation of alkaline phosphatase, and the expression of transcripts encoding osteocalcin. Enzymatic removal of extracellular glutamate abolished these pro-differentiation effects, as did the inhibition of PKC- and Erk1/2-signaling pathways. These findings demonstrate that glutamate signaling promotes differentiation and activation of osteoblast lineage cells. Consequently, the glutamate system may represent a putative therapeutic target to induce an anabolic response in the skeletal system. Known antagonists of glutamate transporters will serve as lead compounds in developing new and specific bioactive molecules

TNFα inhibits the development of osteoclasts through osteoblast-derived GM-CSF

Inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) are potent stimulators of osteoclast formation and bone resorption and are frequently associated with pathologic bone metabolism. The cytokine exerts specific effects on its target cells and constitutes a part of the cellular microenvironment. Previously, TNFα was demonstrated to inhibit the development of osteoclasts in vitro via an osteoblast-mediated pathway. In the present study, the molecular mechanisms of the inhibition of osteoclastogenesis were investigated in co-cultures of osteoblasts and bone marrow cells (BMC) and in cultures of macrophage-colony stimulating factor (M-CSF) dependent, non-adherent osteoclast progenitor cells (OPC) grown with M-CSF and receptor activator of NF-κB ligand (RANKL). Granulocyte-macrophage colony stimulating factor (GM-CSF), a known inhibitor of osteoclastogenesis was found to be induced in osteoblasts treated with TNFα and the secreted protein accumulated in the supernatant. Dexamethasone (Dex), an anti-inflammatory steroid, caused a decrease in GM-CSF expression, leading to partial recovery of osteoclast formation. Flow cytometry analysis revealed that in cultures of OPC, supplemented with 10% conditioned medium (CM) from osteoblasts treated with TNFα/1,25(OH)(2)D(3), expression of RANK and CD11c was suppressed. The decrease in RANK expression may be explained by the finding, that GM-CSF and the CM from wt osteoblasts were found to suppress the expression of c-Fos, Fra-1, and Nfatc-1. The failure of OPC to develop into CD11c(+) dendritic cells suggests that cell development is not deviated to an alternative differentiation pathway, but rather, that the monocytes are maintained in an undifferentiated, F4/80(+), state. The data further implies possible interactions among inflammatory cytokines. GM-CSF induced by TNFα acts on early hematopoietic precursors, inhibiting osteoclastogenesis while acting as the growth factor for M-CSF independent inflammatory macrophages. These in turn may condition a microenvironment enhancing osteoclast differentiation and bone resorption upon migration of the OPC from circulation to the bone/bone marrow compartment

Effect of monoterpenes on the formation and activation of osteoclasts in vitro

Monoterpenes, present in aromatic plants, are known to inhibit bone resorption in vivo. In this in vitro study, they inhibited the activation of osteoclasts only at high concentrations but inhibited the formation at much lower concentrations. Therefore, monoterpenes may act in vivo directly on osteoclastogenesis. INTRODUCTION: Monoterpenes are the major components of essential oils, which are formed in many plants. Typically, they are found in herbs and certain fruits. When fed to rats, they inhibit bone resorption by an unknown mechanism. In this study, their effect on the activity and formation of osteoclasts in vitro was studied. MATERIALS AND METHODS: The effect of monoterpenes on the development of osteoclasts was studied in co-cultures of bone marrow cells and osteoblasts and in cultures of spleen cells grown with colony stimulating factor (CSF)-1 and RANKL. In cultures of primary osteoblasts, alkaline phosphatase activity and levels of mRNA encoding RANKL and osteoprotegerin (OPG) mRNA (RT-PCR), and in osteoblast and spleen cell cultures, lactate dehydrogenase activity, a measure of toxicity, were determined. The activity of isolated rat osteoclasts was determined by counting the osteoclasts with actin rings using histofluorometry. RESULTS: The monoterpenes inhibited the formation of osteoclasts more strongly in co-cultures (> or = 1 microM) than in cultures of spleen cells (> or = 10 microM). They had a minor effect on osteoblasts. Toxic effects were not observed. The inhibition of the formation of osteoclasts was not reversed by the addition of farnesol and geranylgeraniol, excluding an effect of the monoterpenes through the mevalonate pathway. A high concentration of 1 mM was required to inhibit the activation of osteoclasts. This effect, shown for menthol and borneol, was reversible. CONCLUSIONS: The results suggest that the monoterpenes inhibit bone resorption in vivo through a direct effect on the formation of osteoclasts acting mainly on the hemopoietic cells