Design and Validation of a High-Throughput Reductive Catalytic Fractionation Method.

Reductive catalytic fractionation (RCF) is a promising method to extract and depolymerize lignin from biomass, and bench-scale studies have enabled considerable progress in the past decade. RCF experiments are typically conducted in pressurized batch reactors with volumes ranging between 50 and 1000 mL, limiting the throughput of these experiments to one to six reactions per day for an individual researcher. Here, we report a high-throughput RCF (HTP-RCF) method in which batch RCF reactions are conducted in 1 mL wells machined directly into Hastelloy reactor plates. The plate reactors can seal high pressures produced by organic solvents by vertically stacking multiple reactor plates, leading to a compact and modular system capable of performing 240 reactions per experiment. Using this setup, we screened solvent mixtures and catalyst loadings for hydrogen-free RCF using 50 mg poplar and 0.5 mL reaction solvent. The system of 1:1 isopropanol/methanol showed optimal monomer yields and selectivity to 4-propyl substituted monomers, and validation reactions using 75 mL batch reactors produced identical monomer yields. To accommodate the low material loadings, we then developed a workup procedure for parallel filtration, washing, and drying of samples and a 1H nuclear magnetic resonance spectroscopy method to measure the RCF oil yield without performing liquid-liquid extraction. As a demonstration of this experimental pipeline, 50 unique switchgrass samples were screened in RCF reactions in the HTP-RCF system, revealing a wide range of monomer yields (21-36%), S/G ratios (0.41-0.93), and oil yields (40-75%). These results were successfully validated by repeating RCF reactions in 75 mL batch reactors for a subset of samples. We anticipate that this approach can be used to rapidly screen substrates, catalysts, and reaction conditions in high-pressure batch reactions with higher throughput than standard batch reactors.

Catalytic carbon-carbon bond cleavage in lignin via manganese-zirconium-mediated autoxidation.

Efforts to produce aromatic monomers through catalytic lignin depolymerization have historically focused on aryl-ether bond cleavage. A large fraction of aromatic monomers in lignin, however, are linked by various carbon-carbon (C-C) bonds that are more challenging to cleave and limit the yields of aromatic monomers from lignin depolymerization. Here, we report a catalytic autoxidation method to cleave C-C bonds in lignin-derived dimers and oligomers from pine and poplar. The method uses manganese and zirconium salts as catalysts in acetic acid and produces aromatic carboxylic acids as primary products. The mixtures of the oxygenated monomers are efficiently converted to cis,cis-muconic acid in an engineered strain of Pseudomonas putida KT2440 that conducts aromatic O-demethylation reactions at the 4-position. This work demonstrates that autoxidation of lignin with Mn and Zr offers a catalytic strategy to increase the yield of valuable aromatic monomers from lignin.

Age-dependent genetic regulation of osteoarthritis: independent effects of immune system genes.

OBJECTIVES: Osteoarthritis (OA) is a joint disease with a heritable component. Genetic loci identified via genome-wide association studies (GWAS) account for an estimated 26.3% of the disease trait variance in humans. Currently, there is no method for predicting the onset or progression of OA. We describe the first use of the Collaborative Cross (CC), a powerful genetic resource, to investigate knee OA in mice, with follow-up targeted multi-omics analysis of homologous regions of the human genome. METHODS: We histologically screened 275 mice for knee OA and conducted quantitative trait locus (QTL) mapping in the complete cohort (> 8 months) and the younger onset sub-cohort (8-12 months). Multi-omic analysis of human genetic datasets was conducted to investigate significant loci. RESULTS: We observed a range of OA phenotypes. QTL mapping identified a genome-wide significant locus on mouse chromosome 19 containing Glis3, the human equivalent of which has been identified as associated with OA in recent GWAS. Mapping the younger onset sub-cohort identified a genome-wide significant locus on chromosome 17. Multi-omic analysis of the homologous region of the human genome (6p21.32) indicated the presence of pleiotropic effects on the expression of the HLA - DPB2 gene and knee OA development risk, potentially mediated through the effects on DNA methylation. CONCLUSIONS: The significant associations at the 6p21.32 locus in human datasets highlight the value of the CC model of spontaneous OA that we have developed and lend support for an immune role in the disease. Our results in mice also add to the accumulating evidence of a role for Glis3 in OA.

Autoxidation Catalysis for Carbon-Carbon Bond Cleavage in Lignin.

Selective lignin depolymerization is a key step in lignin valorization to value-added products, and there are multiple catalytic methods to cleave labile aryl-ether bonds in lignin. However, the overall aromatic monomer yield is inherently limited by refractory carbon-carbon linkages, which are abundant in lignin and remain intact during most selective lignin deconstruction processes. In this work, we demonstrate that a Co/Mn/Br-based catalytic autoxidation method promotes carbon-carbon bond cleavage in acetylated lignin oligomers produced from reductive catalytic fractionation. The oxidation products include acetyl vanillic acid and acetyl vanillin, which are ideal substrates for bioconversion. Using an engineered strain of Pseudomonas putida, we demonstrate the conversion of these aromatic monomers to cis,cis-muconic acid. Overall, this study demonstrates that autoxidation enables higher yields of bioavailable aromatic monomers, exceeding the limits set by ether-bond cleavage alone.

Osteoblast-derived EGFL6 couples angiogenesis to osteogenesis during bone repair.

Rationale: Angiogenesis and osteogenesis are highly coupled processes which are indispensable to bone repair. However, the underlying mechanism(s) remain elusive. To bridge the gap in understanding the coupling process is crucial to develop corresponding solutions to abnormal bone healing. Epidermal growth factor-like protein 6 (EGFL6) is an angiogenic factor specifically and distinctively up-regulated during osteoblast differentiation. In contrast with most currently known osteoblast-derived coupling factors, EGFL6 is highlighted with little or low expression in other cells and tissues. Methods: In this study, primary bone marrow mesenchymal stem cells (MSCs) and osteoblastic cell line (MC3T3-E1) were transduced with lentiviral silencing or overexpression constructs targeting EGFL6. Cells were induced by osteogenic medium, followed by the evaluation of mineralization as well as related gene and protein expression. Global and conditional knockout mice were established to examine the bone phenotype under physiological condition. Furthermore, bone defect models were created to investigate the outcome of bone repair in mice lacking EGFL6 expression. Results: We show that overexpression of EGFL6 markedly enhances osteogenic capacity in vitro by augmenting bone morphogenic protein (BMP)-Smad and MAPK signaling, whereas downregulation of EGFL6 diminishes osteoblastic mineralization. Interestingly, osteoblast differentiation was not affected by the exogenous addition of EGFL6 protein, thereby indicating that EGFL6 may regulate osteoblastic function in an intracrine manner. Mice with osteoblast-specific and global knockout of EGFL6 surprisingly exhibit a normal bone phenotype under physiological conditions. However, EGFL6-deficiency leads to compromised bone repair in a bone defect model which is characterized by decreased formation of type H vessels as well as osteoblast lineage cells. Conclusions: Together, these data demonstrate that EGFL6 serves as an essential regulator to couple osteogenesis to angiogenesis during bone repair.

Steroid-induced osteonecrosis of the femoral head reveals enhanced reactive oxygen species and hyperactive osteoclasts.

Steroid-induced osteonecrosis of the femoral head (ONFH) is a progressive bone disorder which typically results in femoral head collapse and hip joint dysfunction. It is well-accepted that abnormal osteoclast activity contributes to loss of bone structural integrity and subchondral fracture in ONFH. However, the pathophysiologic mechanisms underlying the recruitment and hyperactivation of osteoclasts in ONFH remain incompletely understood. We assessed the changes of reactive oxygen species (ROS) level and subsequent osteoclast alterations in steroid-induced osteonecrotic femoral heads from both patients and rat ONFH models. When compared with healthy neighboring bone, the necrotic region of human femoral head was characterized by robust up-regulated expression of osteoclast-related proteins [cathepsin K and tartrate-resistant acid phosphatase(TRAP)] but pronounced down-regulation of antioxidant enzymes (catalase, γ-glutamylcysteine synthetase [γ-GCSc], and superoxide dismutase 1 [SOD1]). In addition, the ratio of TNFSF11 (encoding RANKL)/TNFRSF11B (encoding OPG) was increased within the necrotic bone. Consistently, in rat ONFH models induced by methylprednisolone (MPSL) and imiquimod (IMI), significant bone loss in the femoral head was observed, attributable to increased numbers of TRAP positive osteoclasts. Furthermore, the decreased expression of antioxidant enzymes observed by immunoblotting was accompanied by increased ex-vivo ROS fluorescence signals of dihydroethidium (DHE) in rat ONFH models. Therefore, this study lends support to the rationale that antioxidant agents may be a promising therapeutic avenue to prevent or mitigate the progression of steroid-induced ONFH by inhibiting ROS level and hyperactive osteoclasts.

Arctiin abrogates osteoclastogenesis and bone resorption via suppressing RANKL-induced ROS and NFATc1 activation.

Osteoporosis, characterized by disrupted bone resorption and formation, is viewed as a global health challenge. Arctiin (ARC) is a main component of Arctium lappa L, which exerts chemopreventive effects against various tumor cells. However, the role of ARC in bone remodeling is still unclear. Here, we first demonstrated that ARC inhibits osteoclast formation and bone resorption function induced by the receptor activator of nuclear factor-κB ligand (RANKL) in a dose- and time-dependent manner without exerting cytotoxic effects. Mechanistic analysis revealed that ARC not only suppresses RANKL-induced mitogen-activated protein kinase (MAPK) and calcium signaling pathways, but also enhances the expression of cytoprotective enzymes that are involved in scavenging reactive oxygen species (ROS). Further, ARC inhibits the activation of the major transcription factor nuclear factor of activated T cells 1 (NFATc1) during RANKL-induced osteoclast formation. Preclinical studies showed that ARC protects bone loss in an ovariectomy (OVX) mouse model. Conclusively, our data confirmed that ARC could potentially inhibit osteoclastogenesis by abrogating RANKL-induced MAPK, calcium, and NFATc1 signaling pathway, as well as by promoting the expression of ROS scavenging enzymes in Nrf2/Keap1/ARE signaling pathway, thereby2 preventing OVX-induced bone loss. Thus, ARC may serve as a novel therapeutic agent for the treatment of osteoporosis.

Characterisation of genetic regulatory effects for osteoporosis risk variants in human osteoclasts.

BACKGROUND: Osteoporosis is a complex disease with a strong genetic contribution. A recently published genome-wide association study (GWAS) for estimated bone mineral density (eBMD) identified 1103 independent genome-wide significant association signals. Most of these variants are non-coding, suggesting that regulatory effects may drive many of the associations. To identify genes with a role in osteoporosis, we integrate the eBMD GWAS association results with those from our previous osteoclast expression quantitative trait locus (eQTL) dataset. RESULTS: We identify sixty-nine significant cis-eQTL effects for eBMD GWAS variants after correction for multiple testing. We detect co-localisation of eBMD GWAS and osteoclast eQTL association signals for 21 of the 69 loci, implicating a number of genes including CCR5, ZBTB38, CPE, GNA12, RIPK3, IQGAP1 and FLCN. Summary-data-based Mendelian Randomisation analysis of the eBMD GWAS and osteoclast eQTL datasets identifies significant associations for 53 genes, with TULP4 presenting as a strong candidate for pleiotropic effects on eBMD and gene expression in osteoclasts. By performing analysis using the GARFIELD software, we demonstrate significant enrichment of osteoporosis risk variants among high-confidence osteoclast eQTL across multiple GWAS P value thresholds. Mice lacking one of the genes of interest, the apoptosis/necroptosis gene RIPK3, show disturbed bone micro-architecture and increased osteoclast number, highlighting a new biological pathway relevant to osteoporosis. CONCLUSION: We utilise a unique osteoclast eQTL dataset to identify a number of potential effector genes for osteoporosis risk variants, which will help focus functional studies in this area.

Astilbin prevents bone loss in ovariectomized mice through the inhibition of RANKL-induced osteoclastogenesis.

Osteoporosis is the most common osteolytic disease characterized by excessive osteoclast formation and resultant bone loss, which afflicts millions of patients around the world. Astilbin, a traditional herb, is known to have anti-inflammatory, antioxidant and antihepatic properties, but its role in osteoporosis treatment has not yet been confirmed. In our study, astilbin was found to have an inhibitory effect on the RANKL-induced formation and function of OCs in a dose-dependent manner without cytotoxicity. These effects were attributed to its ability to suppress the activity of two transcription factors (NFATc1 and c-Fos) indispensable for osteoclast formation, followed by inhibition of the expression of bone resorption-related genes and proteins (Acp5/TRAcP, CTSK, V-ATPase-d2 and integrin ß3). Furthermore, we examined the underlying mechanisms and found that astilbin repressed osteoclastogenesis by blocking Ca2+ oscillations and the NF-κB and MAPK pathways. In addition, the therapeutic effect of MA on preventing bone loss in vivo was further confirmed in an ovariectomized mouse model. Therefore, considering its ability to inhibit RANKL-mediated osteoclastogenesis and the underlying mechanisms, astilbin might be a potential candidate for treating osteolytic bone diseases.

Loureirin B suppresses RANKL-induced osteoclastogenesis and ovariectomized osteoporosis via attenuating NFATc1 and ROS activities.

Rationale: Osteoporosis is a severe bone disorder that is a threat to our aging population. Excessive osteoclast formation and bone resorption lead to changes in trabecular bone volume and architecture, leaving the bones vulnerable to fracture. Therapeutic approaches of inhibiting osteoclastogenesis and bone resorption have been proven to be an efficient approach to prevent osteoporosis. In our study, we have demonstrated for the first time that Loureirin B (LrB) inhibits ovariectomized osteoporosis and explored its underlying mechanisms of action in vitro. Methods: We examined the effects of LrB on RANKL-induced osteoclast differentiation and bone resorption, and its impacts on RANKL-induced NFATc1 activation, calcium oscillations and reactive oxygen species (ROS) production in osteoclasts in vitro. We assessed the in vivo efficacy of LrB using an ovariectomy (OVX)-induced osteoporosis model, which was analyzed using micro-computed tomography (micro-CT) and bone histomorphometry. Results: We found that LrB represses osteoclastogenesis, bone resorption, F-actin belts formation, osteoclast specific gene expressions, ROS activity and calcium oscillations through preventing NFATc1 translocation and expression as well as affecting MAPK-NFAT signaling pathways in vitro. Our in vivo study indicated that LrB prevents OVX-induced osteoporosis and preserves bone volume by repressing osteoclast activity and function. Conclusions: Our findings confirm that LrB can attenuate osteoclast formation and OVX-induced osteoporosis. This novel and exciting discovery could pave the way for the development of LrB as a potential therapeutic treatment for osteoporosis.

Pseurotin A Inhibits Osteoclastogenesis and Prevents Ovariectomized-Induced Bone Loss by Suppressing Reactive Oxygen Species.

Rationale: Growing evidence indicates that intracellular reactive oxygen species (ROS) accumulation is a critical factor in the development of osteoporosis by triggering osteoclast formation and function. Pseurotin A (Pse) is a secondary metabolite isolated from Aspergillus fumigatus with antioxidant properties, recently shown to exhibit a wide range of potential therapeutic applications. However, its effects on osteoporosis remain unknown. This study aimed to explore whether Pse, by suppressing ROS level, is able to inhibit osteoclastogenesis and prevent the bone loss induced by estrogen-deficiency in ovariectomized (OVX) mice. Methods: The effects of Pse on receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis and bone resorptive function were examined by tartrate resistant acid phosphatase (TRAcP) staining and hydroxyapatite resorption assay. 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) was used to detect intracellular ROS production in vitro. Western blot assay was used to identify proteins associated with ROS generation and scavenging as well as ROS-mediated signaling cascades including mitogen-activated protein kinases (MAPKs), NF-κB pathways, and nuclear factor of activated T cells 1 (NFATc1) signaling. The expression of osteoclast-specific genes was assessed by qPCR. The in vivo potential of Pse was determined using an OVX mouse model administered with Pse or vehicle for 6 weeks. In vivo ROS production was assessed by intravenous injection of dihydroethidium (DHE) into OVX mice 24h prior to killing. After sacrifice, the bone samples were analyzed using micro-CT and histomorphometry to determine bone volume, osteoclast activity, and ROS level ex vivo. Results: Pse was demonstrated to inhibit osteoclastogenesis and bone resorptive function in vitro, as well as the downregulation of osteoclast-specific genes including Acp5 (encoding TRAcP), Ctsk (encoding cathepsin K), and Mmp9 (encoding matrix metalloproteinase 9). Mechanistically, Pse suppressed intracellular ROS level by inhibiting RANKL-induced ROS production and enhancing ROS scavenging enzymes, subsequently suppressing MAPK pathway (ERK, P38, and JNK) and NF-κB pathways, leading to the inhibition of NFATc1 signaling. Micro-CT and histological data indicated that OVX procedure resulted in a significant bone loss, with dramatically increased the number of osteoclasts on the bone surface as well as increased ROS level in the bone marrow microenvironment; whereas Pse supplementation was capable of effectively preventing these OVX-induced changes. Conclusion: Pse was demonstrated for the first time as a novel alternative therapy for osteoclast-related bone diseases such as osteoporosis through suppressing ROS level.

Cytochalasin Z11 inhibits RANKL-induced osteoclastogenesis via suppressing NFATc1 activation.

Excessive osteoclastogenesis and enhanced bone resorption are pathological hallmarks for bone diseases including osteolytic diseases, osteoporosis, and arthritis. Treatments targeting highly activated osteoclasts are regarded as promising therapies for osteoclast-related bone disorders. Cytochalasins are known as secondary metabolites of fungi and exhibit a variety of biological activities in cell biology and medicine. Cytochalasin Z11 (CytoZ11) was previously isolated from the Endothia gyrosa through solid substrate culture and showed therapeutic potential for leukaemia. However, the effects of CytoZ11 on osteoclasts currently remain unclear. Herein, CytoZ11 was found to be able to attenuate RANKL (receptor activator of nuclear factor-κB ligand)-induced osteoclastogenesis and bone resorptive activity dose-dependently. CytoZ11 could also inhibit mRNA expression of osteoclast-specific genes such as Ctr, Acp5, and Ctsk. Furthermore, CytoZ11 was demonstrated to suppress NFATc1 activation, which is due to the attenuation of two signaling pathways: c-Fos signaling and the NF-κB pathway. In summary, this study revealed that CytoZ11 may become a prospective drug for osteoclast-related disease by inhibiting osteoclast formation and function.

Madecassoside inhibits estrogen deficiency-induced osteoporosis by suppressing RANKL-induced osteoclastogenesis.

Osteoporosis is the most common osteolytic disease characterized by excessive osteoclast formation and resultant bone loss, which afflicts millions of patients around the world. Madecassoside (MA), isolated from Centella asiatica, was reported to have anti-inflammatory and antioxidant activities, but its role in osteoporosis treatment has not yet been confirmed. In our study, MA was found to have an inhibitory effect on the RANKL-induced formation and function of OCs in a dose-dependent manner without cytotoxicity. These effects were attributed to its ability to suppress the activity of two transcription factors (NFATc1 and c-Fos) indispensable for osteoclast formation, followed by inhibition of the expression of bone resorption-related genes and proteins (Acp5/TRAcP, CTSK, ATP6V0D2/V-ATPase-d2, and integrin ß3). Furthermore, we examined the underlying mechanisms and found that MA represses osteoclastogenesis by blocking Ca2+ oscillations and the NF-κB and MAPK pathways. In addition, the therapeutic effect of MA on preventing bone loss in vivo was further confirmed in an ovariectomized mouse model. Therefore, considering its ability to inhibit RANKL-mediated osteoclastogenesis and the underlying mechanisms, MA might be a potential candidate for treating osteolytic bone diseases.

Evodiamine inhibits RANKL-induced osteoclastogenesis and prevents ovariectomy-induced bone loss in mice.

Postmenopausal osteoporosis (PMO) is a progressive bone disease characterized by the over-production and activation of osteoclasts in elderly women. In our study, we investigated the anti-osteoclastogenic effect of evodiamine (EVO) in vivo and in vitro, as well as the underlying mechanism. By using an in vitro bone marrow macrophage (BMM)-derived osteoclast culture system, we found that EVO inhibited osteoclast formation, hydroxyapatite resorption and receptor activator of NF-κB ligand (RANKL)-induced osteoclast marker gene and protein expression. Mechanistically, we found that EVO inhibited the degradation and RANKL-induced transcriptional activity of IκBα. RANKL-induced Ca2+ oscillations were also abrogated by EVO. In vivo, an ovariectomized (OVX) mouse model was established to mimic PMO, and OVX mice received oral administration of either EVO (10 mg/kg) or saline every other day. We found that EVO can attenuate bone loss in OVX mice by inhibiting osteoclastogenesis. Taken together, our findings suggest that EVO suppresses RANKL-induced osteoclastogenesis through NF-κB and calcium signalling pathways and has potential value as a therapeutic agent for PMO.

Gene Expression Networks in the Murine Pulmonary Myocardium Provide Insight into the Pathobiology of Atrial Fibrillation.

The pulmonary myocardium is a muscular coat surrounding the pulmonary and caval veins. Although its definitive physiological function is unknown, it may have a pathological role as the source of ectopic beats initiating atrial fibrillation. How the pulmonary myocardium gains pacemaker function is not clearly defined, although recent evidence indicates that changed transcriptional gene expression networks are at fault. The gene expression profile of this distinct cell type in situ was examined to investigate underlying molecular events that might contribute to atrial fibrillation. Via systems genetics, a whole-lung transcriptome data set from the BXD recombinant inbred mouse resource was analyzed, uncovering a pulmonary cardiomyocyte gene network of 24 transcripts, coordinately regulated by chromosome 1 and 2 loci. Promoter enrichment analysis and interrogation of publicly available ChIP-seq data suggested that transcription of this gene network may be regulated by the concerted activity of NKX2-5, serum response factor, myocyte enhancer factor 2, and also, at a post-transcriptional level, by RNA binding protein motif 20. Gene ontology terms indicate that this gene network overlaps with molecular markers of the stressed heart. Therefore, we propose that perturbed regulation of this gene network might lead to altered calcium handling, myocyte growth, and contractile force contributing to the aberrant electrophysiological properties observed in atrial fibrillation. We reveal novel molecular interactions and pathways representing possible therapeutic targets for atrial fibrillation. In addition, we highlight the utility of recombinant inbred mouse resources in detecting and characterizing gene expression networks of relatively small populations of cells that have a pathological significance.

Neohesperidin suppresses osteoclast differentiation, bone resorption and ovariectomised-induced osteoporosis in mice.

Excessive bone resorption by osteoclasts plays an important role in osteoporosis. Bone loss occurs in ovariectomised (OVX) mice in a similar manner to that in humans, so this model is suitable for evaluating potential new therapies for osteoporosis. Neohesperidin (NE) is a flavonoid compound isolated from citrus fruits. Its role in bone metabolism is unknown. In this study we found that neohesperidin inhibits osteoclast differentiation, bone resorption and the expression of osteoclast marker genes, tartrate-resistant acid phosphatase and cathepsin K. In addition, neohesperidin inhibited receptor activator of NF-κB ligand (RANKL)-induced activation of NF-κB, and the degradation of inhibitor of kappa B-alpha (IκBα). Furthermore, neohesperidin inhibited RANKL induction of nuclear factor of activated T-cells (NFAT) and calcium oscillations. In vivo treatment of ovariectomised mice with neohesperidin protected against bone loss in mice. The results suggest neohesperidin has anti-osteoclastic effects in vitro and in vivo and possesses therapeutic potential as a natural anti-catabolic treatment in osteoporosis.

Morc3 mutant mice exhibit reduced cortical area and thickness, accompanied by altered haematopoietic stem cells niche and bone cell differentiation.

Morc3, a member of a highly conserved nuclear matrix protein super-family plays an important part in chromatin remodeling, DNA repair, epigenetic regulation and cellular senescence. However, its role in bone homeostasis is not known. In the present study, a phenotype-driven ENU mouse mutagenesis screen revealed that Morc3(mut +/-) mice exhibit reduced cortical area and thickness with increased cortical porosity. Morc3(mut +/-) mice displayed reduced osteoclast numbers and surface per bone surface as well as osteocyte numbers, concomitant with altered gene expressions such as Rankl/Opg and Sost in ex vivo long bones. In vitro experiments revealed a significant increase in the number of Sca-1(+)/c-kit(+) haematopoietic stem cells (HSCs), and a significant reduction in senescence associated ß-galactosidase activity in bone marrow macrophages (BMMs). In addition, we observed a decrease in osteoclastogenesis and bone resorption accompanied by upregulation of STAT1 expression in osteoclast lineage cells. Strikingly, Morc3 protein localization within the nuclear membrane was shifted to the cytoplasm in Morc3(mut +/-) osteoclasts. Further, Morc3(mut +/-) mice displayed increased osteoblast differentiation and altered gene expression. Collectively, our data show that Morc3 is a previously unreported regulator of cortical bone homeostasis and haematopoietic stem cells niche, accompanied by altered bone cell differentiation.

Loss of protein kinase C-δ protects against LPS-induced osteolysis owing to an intrinsic defect in osteoclastic bone resorption.

Bone remodeling is intrinsically regulated by cell signaling molecules. The Protein Kinase C (PKC) family of serine/threonine kinases is involved in multiple signaling pathways including cell proliferation, differentiation, apoptosis and osteoclast biology. However, the precise involvement of individual PKC isoforms in the regulation of osteoclast formation and bone homeostasis remains unclear. Here, we identify PKC-δ as the major PKC isoform expressed among all PKCs in osteoclasts; including classical PKCs (-α, -ß and -γ), novel PKCs (-δ, -ε, -η and -θ) and atypical PKCs (-ι/λ and -ζ). Interestingly, pharmacological inhibition and genetic ablation of PKC-δ impairs osteoclastic bone resorption in vitro. Moreover, disruption of PKC-δ activity protects against LPS-induced osteolysis in mice, with osteoclasts accumulating on the bone surface failing to resorb bone. Treatment with the PKC-δ inhibitor Rottlerin, blocks LPS-induced bone resorption in mice. Consistently, PKC-δ deficient mice exhibit increased trabeculae bone containing residual cartilage matrix, indicative of an osteoclast-rich osteopetrosis phenotype. Cultured ex vivo osteoclasts derived from PKC-δ null mice exhibit decreased CTX-1 levels and MARKS phosphorylation, with enhanced formation rates. This is accompanied by elevated gene expression levels of cathepsin K and PKC -α, -γ and -ε, as well as altered signaling of pERK and pcSrc416/527 upon RANKL-induction, possibly to compensate for the defects in bone resorption. Collectively, our data indicate that PKC-δ is an intrinsic regulator of osteoclast formation and bone resorption and thus is a potential therapeutic target for pathological osteolysis.