Receptor Activator of NF-κB (RANK) Cytoplasmic IVVY535–538 Motif Plays an Essential Role in Tumor Necrosis Factor-α (TNF)-mediated Osteoclastogenesis

Tumor necrosis factor-α (TNF) enhances osteoclast formation and activity leading to bone loss in various pathological conditions, but its precise role in osteoclastogenesis remains controversial. Although several groups showed that TNF can promote osteoclastogenesis independently of the receptor activator of NF-κB (RANK) ligand (RANKL), others demonstrated that TNF-mediated osteoclastogenesis needs permissive levels of RANKL. Here, we independently reveal that although TNF cannot stimulate osteoclastogenesis on bone slices, it can induce the formation of functional osteoclasts on bone slices in the presence of permissive levels of RANKL or from bone marrow macrophages (BMMs) pretreated by RANKL. TNF can still promote the formation of functional osteoclasts 2 days after transient RANKL pretreatment. These data have confirmed that TNF-mediated osteoclastogenesis requires priming of BMMs by RANKL. Moreover, we investigated the molecular mechanism underlying the dependence of TNF-mediated osteoclastogenesis on RANKL. RANK, the receptor for RANKL, contains an IVVY^535–538 motif that has been shown to play a vital role in osteoclastogenesis by committing BMMs to the osteoclast lineage. We show that TNF-induced osteoclastogenesis depends on RANKL to commit BMMs to the osteoclast lineage and RANKL regulates the lineage commitment through the IVVY motif. Mechanistically, the IVVY motif controls the lineage commitment by reprogramming osteoclast genes into an inducible state in which they can be activated by TNF. Our findings not only provide important mechanistic insights into the action of RANKL in TNF-mediated osteoclastogenesis but also establish that the IVVY motif may serve as an attractive therapeutic target for bone loss in various bone disorders.     

Oleanolic acid exerts inhibitory effects on the late stage of osteoclastogenesis and prevents bone loss in osteoprotegerin knockout mice

Postmenopausal women undergo rapid bone loss, which caused by the accelerated osteoclastic bone resorption. Receptor activator of nuclear factor kappa-B ligand (RANKL) plays critical and essential roles on varied stages of osteoclastogenesis. Oleanolic acid (OA), a naturally derived small compound, has been found suppress osteoclastogenesis in early stage of bone marrow macrophages (BMMs). However, whether OA also regulates the late stage of osteoclastogenesis remains unclear. Here, the regulatory effect of OA on the late stage of osteoclastogenesis was investigated in vitro using RANKL-pretreated BMMs and in vivo using osteoprotegerin (OPG) knockout mice. Our in vitro studies demonstrate that OA inhibits the late stage of osteoclastogenesis from RANKL-pretreated BMMs. For in vivo animal investigation, OA attenuates the bone loss phenotypes in OPG-knockout mice by decreasing the densities of osteoclast, which are in consistent with the finding with in vitro osteoclastogenesis. Mechanistic investigations found that OA largely inhibit the activity of c-Fos and Nuclear factor of activated T-cells c1 (NFATc1) with RANKL-pretreated BMMs and OPG-knockout mice. Furthermore, OA suppresses the activities of osteoclast genes, such as Tartrate resistant acid phosphatase (TRAP), CathepsinK (Ctsk), and Matrix metalloproteinase 9 (MMP9). Taken together these findings, they have not only defined an inhibitory effect of OA in the late stage of osteoclastogenesis but have also gained new molecular mechanisms underlying the process of osteoclast formation.     

CPTP: A sphingolipid transfer protein that regulates autophagy and inflammasome activation†

The macroautophagy/autophagy and inflammasome pathways are linked through their roles in innate immunity and chronic inflammatory disease. Ceramide-1-phosphate (C1P) is a bioactive sphingolipid that regulates pro-inflammatory eicosanoid production. Whether C1P also regulates autophagy and inflammasome assembly/activation is not known. Here we show that CPTP (a protein that traffics C1P from its site of phosphorylation in the trans-Golgi to target membranes) regulates both autophagy and inflammasome activation. In human epithelial cells, knockdown of CPTP (but not GLTP [glycolipid transfer protein]) or expression of C1P binding-site point mutants, stimulated an 8- to 10-fold increase in autophagosomes and altered endogenous LC3-II and SQSTM1/p62 protein expression levels. CPTP depletion-induced autophagy elevated early markers of autophagosome formation (Golgi-derived ATG9A-vesicles, WIPI1), required key phagophore assembly and elongation factors (ATG5, ATG7, ULK1), and suppressed MTOR phosphorylation and that of its downstream target, RPS6KB1/p70S6K. Wild-type CPTP overexpression exerted a protective effect against starvation-induced autophagy. In THP-1 macrophage-like surveillance cells, CPTP knockdown induced not only autophagy but also elevated CASP1/caspase-1 levels, and strongly increased IL1B/interleukin-1β and IL18 release via a NLRP3 (but not NLRC4) inflammasome-based mechanism, while only moderately increasing inflammatory (pyroptotic) cell death. Inflammasome assembly and activation stimulated by CPTP depletion were autophagy dependent. Elevation of intracellular C1P by exogenous C1P treatment (instead of CPTP inhibition) also induced autophagy and IL1B release. Our findings identify human CPTP as an endogenous regulator of early-stage autophagosome assembly and inflammasome-driven, pro-inflammatory cytokine generation and release.     

Autophagic receptors Nbr1 and p62 coregulate skeletal remodelling

Skeletal remodelling is an ongoing process requiring the coordinated action of different cell types to maintain homeostatic control of bone synthesis and degradation. Mutations in p62/SQSTM1 are associated with sporadic and 5q35-linked Paget’s Disease of Bone (PDB), characterized by focal increased bone turnover. These mutations cluster in the ubiquitin associated (UBA) domain and are thought to lead to enhancement of NFκB pathway activation involved in osteoclastogenesis and hyper-responsiveness to receptor activator of nuclear factor-κB ligand (RANKL). The structurally similar selective autophagic receptor, Nbr1, binds to LC3 and p62, and is sequestered into autophagosomes, whereas it accumulates in autophagic-deficient tissues. We have shown that truncation of Nbr1 in a murine model, where it can still interact with p62 but not LC3, leads to increased osteoblast differentiation and activity in vivo. This results in an age-dependent increase in bone mass and bone mineral density. This is a molecular consequence of loss of autophagy receptor function via deletion of its C-terminal UBA domain, and/or modulation of the p38 MAPK cellular signalling pathway.     

DDIT3/CHOP promotes autophagy in chondrocytes via SIRT1-AKT pathway

Endoplasmic reticulum (ER) stress can initiate autophagy via unfolded protein response (UPR). As a key downstream gene of UPR, DDIT3/CHOP is expressed in chondrocytes. However, the regulation mechanism of DDIT3/CHOP on autophagy in chondrocytes remains unclear. In this study, the expression levels of autophagic markers Beclin1 and LC3B were found to decrease while p62 increase in the tibial growth plate and costal primary chondrocytes from DDIT3/CHOP KO mice. In vitro, overexpressing DDIT3/CHOP induced autophagy in ATDC5 chondrocytes, displaying an elevated immunofluorescence signal of LC3B and elevated numbers of autophagosomes and autolysosomes. Analysis of the gain- and loss-of-function indicated that the protein level of Beclin1 and the ratio of LC3BII/I increased in DDIT3/CHOP overexpression cells, whereas decreased in DDIT3/CHOP knockdown cells. The decreased level of p62 and additional accumulation of LC3BII caused by chloroquine (CQ) further indicated that DDIT3/CHOP enhanced autophagic flux. Mechanistically, we found that DDIT3/CHOP binds directly to the promoter of SIRT1 to promote its expression by CHIP, qRT-PCR, and Western blot analysis. In addition, SIRT1 enhanced autophagic activity in ATDC5 cells, and inhibition or activation of SIRT1 partially reversed the effect of overexpressing or downregulating DDIT3/CHOP on autophagy. Furthermore, AKT signaling was found to be responsible for DDIT3/CHOP-regulated autophagy in ATDC5 cells. SIRT1 knockdown reversed the effect of DDIT3/CHOP overexpression on AKT signaling. In conclusion, our data clarifies that DDIT3/CHOP promotes autophagy in ATDC5 chondrocytes through the SIRT1-AKT pathway. These results were also confirmed in the primary chondrocytes.     

Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy.     

PFKP facilitates ATG4B phosphorylation during amino acid deprivation-induced autophagy

ATG4B facilitates autophagy by promoting autophagosome maturation through the reversible lipidation and delipidation of LC3. Recent reports have shown that phosphorylation of ATG4B regulates its activity and LC3 processing, leading to modulate autophagy activity. However, the mechanism about how ATG4B phosphorylation is involved in amino acid deprivation-induced autophagy is unclear. Here, we combined the tandem affinity purification with mass spectrometry (MS) and identified the ATG4B-interacting proteins including its well-known partner gamma-aminobutyric acid receptor-associated protein (GABARAP, a homolog of LC3) and phosphofructokinase 1 platelet isoform (PFKP). Further immunoprecipitation assays showed that amino acid deprivation strengthened the interaction between ATG4B and PFKP. By genetic depletion of PFKP using CRISPR/Cas9, we uncovered that PFKP loss reduced the degradation of LC3-II and p62 due to a partial block in autophagic flux. Furthermore, MS analysis of Flag-tagged ATG4B immunoprecipitates identified phosphorylation of ATG4B serine 34 residue (S34) and PFKP serine 386 residue (S386) under amino acid deprivation condition. In vitro kinase assay validated that PFKP functioning as a protein kinase phosphorylated ATG4B at S34. This phosphorylation could enhance ATG4B activity and p62 degradation. In addition, PFKP S386 phosphorylation was important to ATG4B S34 phosphorylation and autophagy in HEK293T cells. In brief, our findings describe that PFKP, a rate-limiting enzyme in the glycolytic pathway, functions as a protein kinase for ATG4B to regulate ATG4B activity and autophagy under amino acid deprivation condition.     

Suppressors of cytokine signaling-1 and -3 regulate osteoclastogenesis in the presence of inflammatory cytokines

Bone metabolism and the immune system have a correlative relationship, and both are controlled by various common cytokines, such as IFNs and ILs, produced in the bone microenvironments. The suppressor of cytokine signaling-1 (SOCS1) and SOCS3 are negative regulators of such cytokines. Although SOCSs are shown to be induced during osteoclast differentiation, their physiological roles in osteoclast differentiation and function have not been clarified. Thus, we examined the roles of SOCS1 and SOCS3 in osteoclastogenesis using SOCS1- and SOCS3-deficient mice. IFN-gamma-mediated inhibition of osteoclast differentiation from bone marrow-derived monocytes (BMMs) was strongly enhanced in SOCS1-deficient BMMs, but was diminished in SOCS1-overexpressing BMMs. Moreover, LPS-induced osteoclastogenesis and bone destruction in vivo were suppressed in SOCS1(+/-) mice compared with those in wild-type mice, suggesting that SOCS1 antagonizes the inhibitory effect of IFN-gamma on osteoclastogenesis. SOCS3 did not alter the inhibitory effect of IFNs in osteoclastogenesis in both gain and loss of functional assays; however, the suppressive effect of IL-6 on osteoclast differentiation was greater in SOCS3-deficient BMMs than in wild-type BMMs in vitro. In addition, IL-6 significantly prevented LPS-induced bone destruction in SOCS3-deficient mice, although it failed in wild-type mice in vivo. In SOCS3-deficient BMMs, expression levels of TNF-receptor-associated factor-6 and IkappaB were drastically reduced and receptor activator of the NF-kappaB ligand-induced IkappaB phosphorylation was severely impaired in the presence of IL-6. These data suggest that both SOCS1 and SOCS3 regulate osteoclastogenesis by blocking the inhibitory effect of inflammatory cytokines on receptor activator of the NF-kappaB ligand-mediated osteoclast differentiation signals. Selective suppression of SOCS1 and SOCS3 in osteoclast precursors may be a possible therapeutic strategy for inflammatory bone destruction.     

D,L-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) increases endoplasmic reticulum stress, autophagy and apoptosis accompanying ceramide accumulation via ceramide synthase 5 protein expression in A549 cells

In A549 cells, the addition of D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) led to marked autophagy with massive microtubule-associated protein 1 light chain 3B (LC3B)-II protein expression as an indication of autophagy and a steep decrease of p62 protein as a co-indication of autophagy. The addition of DL-PDMP caused massive autophagy with an increase of CAAT/enhancer binding protein homologous protein (CHOP) expression as the marker of endoplasmic reticulum (ER) stress, lactate dehydrogenase (LDH) release without caspase 3 activation and many autophagic vacuoles/devoid of a cell membrane on morphology. On the other hand, the addition of DL-PDMP caused an increase in cellular or subcellular ceramides (Cers), especially palmitoyl-Cer, based on de novo synthesis of Cer, and led to caspase-independent apoptosis. Marked increases of Cer levels in the nuclear envelope were observed 17 h after the addition. The elevations of Cer synthase activity and longevity-assurance homologue (LASS)5 protein expression were observed in subcellular fractions from 30 min until 2 h after the addition. However, the elevations of Cer synthase activity were independent of reactive oxygen species generation or cytochrome P450 4F2 activity. Since an increase in LASS5 protein expression in subcellular fraction occur in preference to the variation of LC3B-II protein expression via CHOP expression after the addition and Cer accumulation induced by the addition contributes to ER stress, it is thought that an elevation of Cer synthase activity via LASS5 protein expression associate to autophagy via CHOP expression (ER stress) with the addition.     

miR-134-5p inhibits osteoclastogenesis through a novel miR-134-5p/Itgb1/MAPK pathway

Osteoporosis affects approximately 200 million people and severely affects quality of life, but the exact pathological mechanisms behind this disease remain unclear. Various miRNAs have been shown to play a predominant role in the regulation of osteoclast formation. In this study, we explored the role of miR-134-5p in osteoclastogenesis both in vivo and in vitro. We constructed an ovariectomized (OVX) mouse model and performed microarray analysis using bone tissue from OVX mice and their control counterparts. Quantitative RT-PCR data from bone tissue and bone marrow macrophages (BMMs) confirmed the decreased expression of miR-134-5p in OVX mice observed in microarray analysis. In addition, a decrease in miR-134-5p was also observed during induced osteoclastogenesis of BMMs collected from C57BL/6N mice. Through transfection with miR-134-5p agomirs and antagomirs, we found that miR-134-5p knockdown significantly accelerated osteoclast formation and cell proliferation and inhibited apoptosis. Furthermore, a luciferase reporter assay showed that miR-134-5p directly targets the integrin surface receptor gene Itgb1. Cotransfection with Itgb1 siRNA reversed the effect of the miR-134-5p antagomir in promoting osteoclastogenesis. Moreover, the abundance levels of MAPK pathway proteins phosphorylated-p38 (p-p38) and phosphorylated-ERK (p-ERK) were significantly increased after transfection with the miR-134-5p antagomir but decreased after transfection with the miR-134-5p agomir or Itgb1 siRNA, which indicated a potential relationship between the miR-134-5p/Itgb1 axis and the MAPK pathway. Collectively, these results revealed that miR-134-5p inhibits osteoclast differentiation of BMMs both in vivo and in vitro and that the miR-134-5p/Itgb1/MAPK pathway might be a potential target for osteoporosis therapy.     

Nicotinate-Curcumin Impedes Foam Cell Formation from THP-1 Cells through Restoring Autophagy Flux

Our previous studies have indicated that a novel curcumin derivate nicotinate-curcumin (NC) has beneficial effects on the prevention of atherosclerosis, but the precise mechanisms are not fully understood. Given that autophagy regulates lipid metabolism, the present study was designed to investigate whether NC decreases foam cell formation through restoring autophagy flux in oxidized low-density lipoprotein (ox-LDL)-treated THP-1 cells. Our results showed that ox-LDL (100 μg/ml) was accumulated in THP-1 cells and impaired autophagy flux. Ox-LDL-induced impairment of autophagy was enhanced by treatment with the autophagy inhibitor chloroquine (CQ) and rescued by the autophagy inducer rapamycin. The aggregation of ox-LDL was increased by CQ, but decreased by rapamycin. In addition, colocalization of lipid droplets with LC3-II was remarkably reduced in ox-LDL group. In contrast, NC (10 μM) rescued the impaired autophagy flux by significantly increasing level of LC3-II, the number of autophagolysosomes, and the degradation of p62 in ox-LDL-treated THP-1 cells. Inhibition of the PI3K-Akt-mTOR signaling was required for NC-rescued autophagy flux. Notably, our results showed that NC remarkably promoted the colocalization of lipid droplets with autophagolysosomes, increased efflux of cholesterol, and reduced ox-LDL accumulation in THP-1 cells. However, treatment with 3-methyladenine (3-MA) or CQ reduced the protective effects of NC on lipid accumulation. Collectively, the findings suggest that NC decreases lipid accumulation in THP-1 cells through restoring autophagy flux, and further implicate that NC may be a potential therapeutic reagent to reverse atherosclerosis.     

Autophagy regulates hypoxia-induced osteoclastogenesis through the HIF-1α/BNIP3 signaling pathway

Previous studies have implicated that hypoxic stress could enhance osteoclast differentiation; however, the underlying mechanism remains poorly understood. Autophagy is a dynamic lysosomal degradation process that has emerged as an important regulator under hypoxic environment. In the present study, we demonstrate for the first time that autophagy regulates hypoxia-induced osteoclastogenesis in vitro. We found that exposure of RAW264.7 cells to hypoxia (0.2% oxygen) resulted in enhanced osteoclast differentiation, accompanied by the observation of several specific features of autophagy, including appearance of membranous vacuoles, formation of acidic vesicular organelles, cleavage and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, increase in autophagic flux, as well as up-regulation of autophagy-related gene (Atg) expression. Moreover, suppression of autophagy with DN-Atg5(K130R) or 3-methyladenine (3-MA) significantly attenuated the osteoclast differentiation under hypoxic conditions, indicating the functional significance of autophagy in hypoxia-induced osteoclastogenesis. The data also showed that the activation of autophagy under hypoxic conditions was caused by up-regulated expression of hypoxia-inducible factor-1α (HIF-1α)-dependent Bcl-2 adenovirus E1a 19 kDa interacting protein 3 (BNIP3). Importantly, knockdown of HIF-1α or BNIP3 obviously abrogated hypoxia-induced autophagy activation and osteoclastogenesis enhancement. Collectively, our results highlight the fact that autophagy is a pivotal regulator for hypoxia-induced osteoclast differentiation, which may provide new insight into the pathological processes of osteoclastogenesis under hypoxic stress and help develop new therapeutic strategies for abnormal osteoclastogenesis.     

The role of autophagy in the pathogenesis of exposure keratitis

Incomplete tear film spreading and eyelid closure can cause defective renewal of the ocular surface and air exposure‐induced epithelial keratopathy (EK). In this study, we characterized the role of autophagy in mediating the ocular surface changes leading to EK. Human corneal epithelial cells (HCECs) and C57BL/6 mice were employed as EK models, respectively. Transmission electron microscopy (TEM) evaluated changes in HCECs after air exposure. Each of these models was treated with either an autophagy inhibitor [chloroquine (CQ) or 3‐methyladenine (3‐MA)] or activator [Rapamycin (Rapa)]. Immunohistochemistry assessed autophagy‐related proteins, LC3 and p62 expression levels. Western blotting confirmed the expression levels of the autophagy‐related proteins [Beclin1 and mammalian target of rapamycin (mTOR)], the endoplasmic reticulum (ER) stress‐related proteins (PERK, eIF2α and CHOP) and the PI3K/Akt/mTOR signalling pathway‐related proteins. Real‐time quantitative PCR (qRT‐PCR) determined IL‐1β, IL‐6 and MMP9 gene expression levels. The TUNEL assay detected apoptotic cells. TEM identified autophagic vacuoles in both EK models. Increased LC3 puncta formation and decreased p62 immunofluorescent staining and Western blotting confirmed autophagy induction. CQ treatment increased TUNEL positive staining in HCECs, while Rapa had an opposite effect. Similarly, CQ injection enhanced air exposure‐induced apoptosis and inflammation in the mouse corneal epithelium, which was inhibited by Rapa treatment. Furthermore, the phosphorylation status of PERK and eIF2α and CHOP expression increased in both EK models indicating that ER stress‐induced autophagy promoted cell survival. Taken together, air exposure‐induced autophagy is indispensable for the maintenance of corneal epithelial physiology and cell survival.     

Autophagy dysregulation caused by ApoM deficiency plays an important role in liver lipid metabolic disorder

Autophagy is thought to be a key mechanism in maintaining the balance of liver lipid metabolism. However, the relationship between apolipoprotein M (ApoM) and autophagy has not been reported, and the role of ApoM in triglyceride metabolism is still unclear. In this study, we investigated the correlation between ApoM and autophagy and liver triglyceride metabolism in ApoM-knockout animal and cellular models. First, we observed that spontaneous hepatic steatosis developed in the liver of adult ApoM^?/? mice, which was presented as the accumulation of large quantities of lipid droplets in hepatocytes under electron microscopy; Oil Red O staining showed significant accumulation of triglycerides. At the molecular level, the expression of lipid synthesis-associated proteins (primarily triglyceride synthesis) as well as acetyl-CoA carboxylase alpha (ACACA), fatty acid synthase (FASN) and sterol regulatory element-binding protein 1 (SREBP1) was upregulated. Moreover, lipid metabolic disorder and accumulation were accompanied by dysfunction in autophagy, which displayed predominantly as inhibition of the degradation pathway; for example, P62 protein accumulated and key proteins involved in the initiation of autophagy including ATG7, ATG5-12, Beclin1 and the LC3BII/LC3BI ratio were upregulated as a feedback response. When the autophagy dysfunction was ameliorated by the activation of autophagy pathways induced by starvation, the lipid metabolic disorder was corrected to a certain extent. This suggests that the autophagy dysfunction caused by the deficiency of ApoM is an important factor in hepatic steatosis (triglyceride accumulation). ApoM plays a key role in normal autophagy activity in the liver and thereby further regulates the metabolism of liver lipids, particularly triglycerides.     

Compressive mechanical force augments osteoclastogenesis by bone marrow macrophages through activation of c‐Fms‐mediated signaling

Little is known about the effects of mechanical forces on osteoclastogenesis by bone marrow macrophages (BMMs) in the absence of mechanosensitive cells, including osteoblasts and fibroblasts. In this study, we examined the effects of mechanical force on osteoclastogenesis by applying centrifugal force to BMMs using a horizontal microplate rotor. Our findings, as measured by an in vitro model system, show that tumor necrosis factor (TNF)‐α is capable of inducing osteoclast differentiation from BMMs and bone resorption in the presence of macrophage‐colony stimulating factor (M‐CSF) and is further facilitated by receptor activator of nuclear factor‐kappaB (NF‐κB) ligand (RANKL). Application of force to BMMs accelerated TNF‐α‐induced osteoclastogenesis; this was inhibited either by anti‐TNF‐α or anti‐TNF‐α receptor but not by OPG. TNF‐α also increased c‐Fms expression at both mRNA and protein levels in BMMs. An anti‐c‐Fms antibody completely inhibited osteoclast differentiation and bone resorption induced by TNF‐α but partially blocked osteoclastogenesis stimulated in combination with RANKL. These results suggest that TNF‐α (in the presence of M‐CSF) is capable of inducing osteoclastogenesis from BMMs, and that osteoclastogenesis is significantly stimulated by force application through the activation of c‐Fms‐mediated signaling. Overall, the present study reveals the facilitating effect of mechanical force on osteoclastic differentiation from BMMs without the addition of mechanosensitive cells. J. Cell. Biochem. 111: 1260–1269, 2010. © 2010 Wiley‐Liss, Inc.