Pyrroloquinoline Quinine Inhibits RANKL-Mediated Expression of NFATc1 in Part via Suppression of c-Fos in Mouse Bone Marrow Cells and Inhibits Wear Particle-Induced Osteolysis in Mice

The effects of pyrroloquinoline quinine (PQQ) on RANKL-induced osteoclast differentiation and on wear particle-induced osteolysis were examined in this study. PQQ inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) in a dose-dependent manner without any evidence of cytotoxicity. The mRNA expression of c-Fos, NFATc1, and TRAP in RANKL-treated BMMs was inhibited by PQQ treatment. Moreover, RANKL-induced c-Fos and NFATc1 protein expression was suppressed by PQQ. PQQ additionally inhibited the bone resorptive activity of differentiated osteoclasts. Further a UHMWPE-induced murine calvaria erosion model study was performed to assess the effects of PQQ on wear particle-induced osteolysis in vivo. Mice treated with PQQ demonstrated marked attenuation of bone erosion based on Micro-CT and histologic analysis of calvaria. These results collectively suggested that PQQ demonstrated inhibitory effects on osteoclast differentiation in vitro and may suppress wear particle-induced osteolysis in vivo, indicating that PQQ may therefore serve as a useful drug in the prevention of bone loss.     

ER Stress Mediates TiAl6V4 Particle-Induced Peri-Implant Osteolysis by Promoting RANKL Expression in Fibroblasts

Wear particle-induced osteolysis is a major cause of aseptic loosening, which is one of the most common reasons for total hip arthroplasty (THA) failure. Previous studies have shown that the synovial fibroblasts present in the periprosthetic membrane are important targets of wear debris during osteolysis. However, the interaction mechanisms between the wear debris and fibroblasts remain largely unknown. In the present study, we investigated the effect of ER (endoplasmic reticulum) stress induced by TiAl₆V₄ particles (TiPs) in human synovial fibroblasts and calvarial resorption animal models. The expression of ER stress markers, including IRE1-α, GRP78/Bip and CHOP, were determined by western blot in fibroblasts that had been treated with TiPs for various times and concentration. To address whether ER stress was involved in the expression of RANKL, the effects of ER stress blockers (including 4-PBA and TUDCA) on the expression of RANKL in TiPs-treated fibroblasts were examined by real-time PCR, western blot and ELISA. Osteoclastogenesis was assessed by tartrate resistant acid phosphatase (TRAP) staining. Our study demonstrated that ER stress markers were markedly upregulated in TiPs-treated fibroblasts. Blocking ER stress significantly reduced the TiPs-induced expression of RANKL both in vitro and in vivo. Moreover, the inhibition of ER stress ameliorated wear particle-induced osteolysis in animal models. Taken together, these results suggested that the expression of RANKL induced by TiPs was mediated by ER stress in fibroblasts. Therefore, down regulating the ER stress of fibroblasts represents a potential therapeutic approach for wear particle-induced periprosthetic osteolysis.     

Prevention of wear particle-induced osteolysis by a novel V-ATPase inhibitor saliphenylhalamide through inhibition of osteoclast bone resorption

Wear particle-induced peri-implant loosening (Aseptic prosthetic loosening) is one of the most common causes of total joint arthroplasty. It is well established that extensive bone destruction (osteolysis) by osteoclasts is responsible for wear particle-induced peri-implant loosening. Thus, inhibition of osteoclastic bone resorption should prevent wear particle induced osteolysis and may serve as a potential therapeutic avenue for prosthetic loosening. Here, we demonstrate for the first time that saliphenylhalamide, a new V-ATPase inhibitor attenuates wear particle-induced osteolysis in a mouse calvarial model. In vitro biochemical and morphological assays revealed that the inhibition of osteolysis is partially attributed to a disruption in osteoclast acidification and polarization, both a prerequisite for osteoclast bone resorption. Interestingly, the V-ATPase inhibitor also impaired osteoclast differentiation via the inhibition of RANKL-induced NF-κB and ERK signaling pathways. In conclusion, we showed that saliphenylhalamide affected multiple physiological processes including osteoclast differentiation, acidification and polarization, leading to inhibition of osteoclast bone resorption in vitro and wear particle-induced osteolysis in vivo. The results of the study provide proof that the new generation V-ATPase inhibitors, such as saliphenylhalamide, are potential anti-resorptive agents for treatment of peri-implant osteolysis.     

MiR‐106b inhibition suppresses inflammatory bone destruction of wear debris‐induced periprosthetic osteolysis in rats

Aseptic loosening caused by periprosthetic osteolysis (PPO) is the main reason for the primary artificial joint replacement. Inhibition of inflammatory osteolysis has become the main target of drug therapy for prosthesis loosening. MiR‐106b is a newly discovered miRNA that plays an important role in tumour biology, inflammation and the regulation of bone mass. In this study, we analysed the in vivo effect of miR‐106b on wear debris‐induced PPO. A rat implant loosening model was established. The rats were then administrated a lentivirus‐mediated miR‐106b inhibitor, miR‐106b mimics or an equivalent volume of PBS by tail vein injection. The expression levels of miR‐106b were analysed by real‐time PCR. Morphological changes in the distal femurs were assessed via micro‐CT and histopathological analysis, and cytokine expression levels were examined via immunohistochemical staining and ELISA. The results showed that treatment with the miR‐106b inhibitor markedly suppressed the expression of miR‐106b in distal femur and alleviated titanium particle‐induced osteolysis and bone loss. Moreover, the miR‐106b inhibitor decreased TRAP‐positive cell numbers and suppressed osteoclast formation, in addition to promoting the activity of osteoblasts and increasing bone formation. MiR‐106b inhibition also significantly regulated macrophage polarization and decreased the inflammatory response as compared to the control group. Furthermore, miR‐106b inhibition blocked the activation of the PTEN/PI3K/AKT and NF‐κB signalling pathways. Our findings indicated that miR‐106b inhibition suppresses wear particles‐induced osteolysis and bone destruction and thus may serve as a potential therapy for PPO and aseptic loosening.     

Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-β Peptide and APP-CTFβ Levels in Neuronal Cells

Autophagy is a vital pathway for the removal of β-amyloid peptide (Aβ) and the aggregated proteins that cause Alzheimer’s disease (AD). We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ) by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1–42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly. Pretreatment with cilostazol (10–30 μM) or resveratrol (20 μM) prevented these Aβ1–42 evoked suppressions. LC3-II (a marker of mammalian autophagy) levels were significantly increased by cilostazol, and this increase was reduced by 3-methyladenine. To evoke endogenous Aβ overproduction, N2aSwe cells (N2a cells stably expressing human APP containing the Swedish mutation) were cultured in medium with or without tetracycline (Tet⁺ for 48 h and then placed in Tet^- condition). Aβ and APP-CTFβ expressions were increased after 12~24 h in Tet^- condition, and these increased expressions were significantly reduced by pretreating cilostazol. Cilostazol-induced reductions in the expressions of Aβ and APP-CTFβ were blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion). After knockdown of the SIRT1 gene (to ~40% in SIRT1 protein), cilostazol failed to elevate the expressions of beclin-1, Atg5, and LC3-II, indicating that cilostazol increases these expressions by up-regulating SIRT1. Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy. In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.     

PKCβ Positively Regulates RANKL-Induced Osteoclastogenesis by Inactivating GSK-3β

Protein kinase C (PKC) family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. However, the role of PKC in receptor activator of NF-κB ligand (RANKL) signaling has remained elusive. We now demonstrate that PKCβ acts as a positive regulator which inactivates glycogen synthase kinase-3β (GSK-3β) and promotes NFATc1 induction during RANKL-induced osteoclastogenesis. Among PKCs, PKCβ expression is increased by RANKL. Pharmacological inhibition of PKCβ decreased the formation of osteoclasts which was caused by the inhibition of NFATc1 induction. Importantly, the phosphorylation of GSK-3β was decreased by PKCβ inhibition. Likewise, down-regulation of PKCβ by RNA interference suppressed osteoclast differentiation, NFATc1 induction, and GSK-3β phosphorylation. The administration of PKC inhibitor to the RANKL-injected mouse calvaria efficiently protected RANKL-induced bone destruction. Thus, the PKCβ pathway, leading to GSK-3β inactivation and NFATc1 induction, has a key role in the differentiation of osteoclasts. Our results also provide a further rationale for PKCβ’s therapeutic targeting to treat inflammation-related bone diseases.     

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, −β 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.     

SIRT3 Overexpression Attenuates Palmitate-Induced Pancreatic β-Cell Dysfunction

Abnormally high levels of circulating free fatty acids can lead to pancreatic islet β-cell dysfunction and apoptosis, contributing to β-cell failure in Type 2 diabetes. The NAD⁺-dependent protein deacetylase Sirtuin-3 (SIRT3) has been implicated in Type 2 diabetes. In this study, we tested whether SIRT3 overexpression affects palmitate-induced β-cell dysfunction in cells of line NIT1, which are derived from mouse pancreatic β-cells. Two different lengths of SIRT3 were overexpressed: full length SIRT3 (SIRT3LF), which was preferentially targeted to mitochondria and partially to the nucleus, and its N-terminal truncated form (SIRT3SF), which was located in the nucleus and cytoplasm. Overexpression of SIRT3LF and SIRT3SF using an adenoviral system alleviated palmitate-induced lipotoxicity such as reduction of cell viability and mitogen-activated protein kinase (MAPK) activation. Chronic exposure to low concentrations of palmitate suppressed glucose-stimulated insulin secretion, but the suppression was effectively reversed by overexpression of SIRT3LF or SIRT3SF. The mRNA levels of the endoplasmic reticulum (ER) stress responsive genes ATF4, GRP94 and FKBP11 were increased by palmitate treatment, but the increases were completely inhibited by SIRT3LF overexpression and less effectively inhibited by SIRT3SF overexpression. This result suggests that overexpression of SIRT3 inhibits induction of ER stress by palmitate. Collectively, we conclude that overexpression of SIRT3 alleviates palmitate-induced β-cell dysfunction.     

Inverse Regulation of the Cytosolic Ca2+ Buffer Parvalbumin and Mitochondrial Volume in Muscle Cells via SIRT1/PGC-1α Axis

Skeletal muscles show a high plasticity to cope with various physiological demands. Different muscle types can be distinguished by the force, endurance, contraction/relaxation kinetics (fast-twitch vs. slow-twitch muscles), oxidative/glycolytic capacity, and also with respect to Ca²⁺-signaling components. Changes in Ca²⁺ signaling and associated Ca²⁺-dependent processes are thought to underlie the high adaptive capacity of muscle fibers. Here we investigated the consequences and the involved mechanisms caused by the ectopic expression of the Ca²⁺-binding protein parvalbumin (PV) in C2C12 myotubes in vitro, and conversely, the effects caused by its absence in in fast-twitch muscles of parvalbumin null-mutant (PV−/−) mice in vivo. The absence of PV in fast-twitch muscle tibialis anterior (TA) resulted in an increase in the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and of its positive regulator, the deacetylase sirtuin 1 (SIRT1). TA muscles from PV−/− mice also have an increased mitochondrial volume. Mild ionophore treatment of control (PV-devoid) C2C12 myotubes causing a moderate elevation in [Ca²⁺]_c resulted in an increase in mitochondrial volume, together with elevated PGC-1α and SIRT1 expression levels, whilst it increased PV expression levels in myotubes stably transfected with PV. In PV-expressing myotubes the mitochondrial volume, PGC-1α and SIRT1 were significantly lower than in control C2C12 myotubes already at basal conditions and application of ionophore had no effect on either one. SIRT1 activation causes a down-regulation of PV in transfected myotubes, whilst SIRT1 inhibition has the opposite effect. We conclude that PV expression and mitochondrial volume in muscle cells are inversely regulated via a SIRT1/PGC-1α signaling axis.     

SIRT1 Is a Regulator in High Glucose-Induced Inflammatory Response in RAW264.7 Cells

Sepsis is defined as a systemic inflammatory response syndrome that disorders the functions of host immune system, including the imbalance between pro- and anti-inflammatory responses mediated by immune macrophages. Sepsis could also induce acute hyperglycemia. Studies have shown that the silent mating type information regulation 2 homolog 1 (SIRT1), an NAD⁺-dependent deacetylase, mediates NF-κb deacetylation and inhibits its function. Therefore, SIRT1 is likely to play an important role in high glucose-mediated inflammatory signalings. Here we demonstrate that high glucose significantly downregulates both the mRNA and protein levels of SIRT1 and upregulates the mRNA level and the release of two pro-inflammatory cytokines, IL-1β and TNF-α, in RAW264.7 macrophages. Interestingly, the reduced level of SIRT1 by high glucose is remarkably upregulated by SIRT1 activator SRT1720, while the level and the release of IL-1β and TNF-α significantly decrease with the use of SRT1720. However, when the function of SIRT1 is inhibited by EX527 or its expression is suppressed by RNAi, the upregulated level and release of IL-1β and TNF-α by high glucose are further increased. Taken together, these findings collectively suggest that SIRT1 is an important regulator in many high glucose-related inflammatory diseases such as sepsis.     

Talin1 and Rap1 Are Critical for Osteoclast Function

To determine talin1''s role in osteoclasts, we mated TLN1^fl/fl mice with those expressing cathepsin K-Cre (CtsK-TLN1) to delete the gene in mature osteoclasts or with lysozyme M-Cre (LysM-TLN1) mice to delete TLN1 in all osteoclast lineage cells. Absence of TLN1 impairs macrophage colony-stimulating factor (M-CSF)-stimulated inside-out integrin activation and cytoskeleton organization in mature osteoclasts. Talin1-deficient precursors normally express osteoclast differentiation markers when exposed to M-CSF and receptor activator of nuclear factor κB (RANK) ligand but attach to substrate and migrate poorly, arresting their development into mature resorptive cells. In keeping with inhibited resorption, CtsK-TLN1 mice exhibit an ∼5-fold increase in bone mass. Osteoclast-specific deletion of Rap1 (CtsK-Rap1), which promotes talin/β integrin recognition, yields similar osteopetrotic mice. The fact that the osteopetrosis of CtsK-TLN1 and CtsK-Rap1 mice is substantially more severe than that of those lacking αvβ3 is likely due to added failed activation of β1 integrins. In keeping with osteoclast dysfunction, mice in whom talin is deleted late in the course of osteoclastogenesis are substantially protected from ovariectomy-induced osteoporosis and the periarticular osteolysis attending inflammatory arthritis. Thus, talin1 and Rap1 are critical for resorptive function, and their selective inhibition in mature osteoclasts retards pathological bone loss.     

Osteocytic cells exposed to titanium particles increase sclerostin expression and inhibit osteoblastic cell differentiation mostly via direct cell‐to‐cell contact

The mechanism underlying induction of periprosthetic osteolysis by wear particles remains unclear. In this study, cultured MLO‐Y4 osteocytic cells were exposed to different concentrations of titanium (Ti) particles. The results showed that Ti particles increased expression of the osteocytic marker SOST/sclerostin in a dose‐dependent manner, accelerated apoptosis of MLO‐Y4 cells, increased the expression of IL‐6, TNF‐α and connexin 43. SOST silence alleviated the increase of MLO‐Y4 cells apoptosis, decreased the expression of IL‐6, TNF‐α and connexin 43 caused by Ti particles. The different co‐culture systems of MLO‐Y4 cells with MC3T3‐E1 osteoblastic cells were further used to observe the effects of osteocytic cells'' changes induced by Ti particles on osteoblastic cells. MLO‐Y4 cells treated with Ti particles inhibited dramatically differentiation of MC3T3‐E1 cells mostly through direct cell‐to‐cell contact. SOST silence attenuated the inhibition effects of Ti‐induced MLO‐Y4 on MC3T3‐E1 osteoblastic differentiation, which ALP level and mineralization of MC3T3‐E1 cells increased and the expression of ALP, OCN and Runx2 increased compared to the Ti‐treated group. Taken together, Ti particles had negative effects on MLO‐Y4 cells and the impact of Ti particles on osteocytic cells was extensive, which may further inhibit osteoblastic differentiation mostly through intercellular contact directly. SOST/sclerostin plays an important role in the process of mutual cell interaction. These findings may help to understand the effect of osteocytes in wear particle‐induced osteolysis.     

Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation

The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene) has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol’s anti-aging effects both in vitro and in vivo attributed to activation of a (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. In mammals seven members (SIRT1-7) of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ) and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated β-galactosidase (SA-β-gal) activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21^CIP1 and p16^INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-β-gal activity, γ-H2AX phosphorylation and p53, p21^CIP1 and p16^INK4A levels. Interestingly DNA damaging agent doxorubicin also induced senescence in BJ fibroblasts associated with decreased SIRT1/2 levels. In conclusion our data reveal that resveratrol induced premature senescence is associated with SIRT1 and SIRT2 down regulation in human dermal fibroblasts. Here we suggest that the concomitant decline in SIRT1/2 expression in response to resveratrol treatment may be a cause for induction of senescence, which is most likely mediated by a regulatory mechanism activated by DNA damage response.     

NAMPT regulates mitochondria biogenesis via NAD metabolism and calcium binding proteins during skeletal muscle contraction

[Purpose]

The purpose of this study was to investigate the effect that muscle contraction induced NAD metabolism via NAMPT has on mitochondrial biogenesis.

[Methods]

Primary skeletal muscle cells were isolated from the gastrocnemius in C57BL/6 mice. The muscle cells were stimulated by electrical current at 1Hz for 3 minutes in conditions of normal or NAD metabolism related inhibitor treatment. NAD/NADH level, Sirt1 and mitochondria biogenesis related signal factor’s changes were examined in normal or NAD metabolism related inhibitor treated cells.

[Results]

Electrical stimulation (ES) induced muscle contractions significantly increased NAD/NADH levels, NAMPT inhibitor FK-866 inhibited ES-induced NAD formation, which caused SIRT1 expression and PGC-1α deacetylation to decrease. Moreover, NAMPT inhibition decreased mitochondrial biogenesis related mRNA, COX-1 and Tfam levels. Along with AMPK inhibitor, compound C decreases SIRT1 expression, PGC-1α deacetylation and muscle contraction induced mitochondrial biogenesis related mRNA increment. These results indicated that the AMPK-NAMPT signal is a key player for muscle contraction induced SIRT1 expression and PGC-1α deacetylation, which influences mitochondrial biogenesis. Inhibition of the AMPK upregulator, Camkkβ, STO-609 decreased AMPK phosphorylation and SIRT1 expression but did not decrease PGC-1α deacetylation. However, CAMKII inhibition via AIP decreased PGC-1α deacetylation.

[Conclusion]

In conclusion, the results indicate that NAMPT plays an important role in NAD metabolism and mitochondrial biogenesis. However, mitochondrial biogenesis is also controlled by different calcium binding protein signals including Camkkβ and CAMKII. [Keyword] Muscle contraction, NAD metabolism, SIRT1, PGC-1 α, mitochondria biogenesis.