Modulates Osteoclast Differentiation Research Articles

Astragalin mitigates inflammatory osteolysis by negatively modulating osteoclastogenesis via ROS and MAPK signaling pathway

Inflammatory bone destruction has gradually attracted attention worldwide and has been observed in several kinds of pathological bone diseases, such as osteoarthritis, osteomyelitis, rheumatic arthritis, and other infectious clinical trials in the skeletal system. In this regard, excessive osteoclasts and bone resorption activity participate in osteolytic processes. Thus, negatively modulating osteoclast differentiation and bone erosion has been considered an effective therapeutic strategy to limit the poor progression of inflammatory osteolysis. Astragalin (AST) is a bioactive component of traditional Chinese drugs, such as Rosa agrestis, which presents anti-inflammatory and antioxidant effects. However, it is unclear how AST may play an essential role in regulating the dynamic balance of the bone matrix by affecting osteoclastogenesis. This study found that AST could inhibit osteoclastic formation and bone resorption activity in a dose‐dependent manner without cytotoxicity. Administration of AST also inhibited the expression of cathepsin K, c-Fos, NFATc1, and TRAP at different stages of mRNA and protein levels during osteoclastogenesis. Reactive oxygen species (ROS) signalling could also be modulated by treatment with AST during RANKL-induced osteoclast differentiation through the Nrf2-HO1 signalling pathway. Additionally, AST could negatively regulate mitogen-activated protein kinase (MAPK) signalling in this process. In vivo, AST significantly reduced lipopolysaccharide (LPS)-induced bone loss in an osteolytic mouse model. AST might be a promising therapeutic candidate for treating osteolytic bone diseases in the future.

Vericiguat Modulates Osteoclast Differentiation and Bone Resorption via a Balance between VASP and NF-κB Pathways.

Bone homeostasis has been a dynamic equilibrium between osteoclasts (OCs) and osteoblasts (OBs). However, excessive activation of OCs could disturb the bone homeostasis. As a result, effective medical interventions for patients are greatly demanding. NO/guanylate cyclase (GC)/cGMP signaling cascade has been previously reported to regulate bone metabolism, and GC plays a significantly critical role. Vericiguat, as a novel oral soluble guanylate cyclase (sGC) stimulator, has been firstly reported in 2020 to treat patients with heart failure. Nevertheless, the biological effects of Vericiguat on the function of OCs have not yet been explored. In this present study, we found that Vericiguat with the concentration between 0 and 8 μM was noncytotoxic to bone marrow-derived monocyte-macrophage lineage (BMMs). Vericiguat could enhance the differentiation of OCs at concentration of 500 nM, whereas it inhibited OC differentiation at 8 μM. In addition, Vericiguat also showed dual effects on OC fusion and bone resorption in a dose-dependent manner. Furthermore, a molecular assay suggested that the dual regulatory effects of Vericiguat on OCs were mediated by the bidirectional activation of the IκB-α/NF-κB signaling pathway. Taken together, our present study demonstrated the dual effects of Vericiguat on the formation of functional OCs. The regulatory effects of Vericiguat on OCs were achieved by the bidirectional modulation of the IκB-α/NF-κB signaling pathway, and a potential balance between the IκB-α/NF-κB signaling pathway and sGC/cGMP/VASP may exist.

LncRNA SNHG15 as a ceRNA modulates Osteoclast Differentiation, Proliferation, and Metastasis by Sponging miR-381-3p/NEK2 Axis.

Background A growing number of studies have shown that long noncoding RNAs play an important role in osteoclast differentiation. However, there are few studies on the roles of lncRNA small nucleolar RNA host gene 15 (SNHG15) in osteoclast differentiation. Methods The expressions of SNHG15, miR-381-3p, and never in mitosis-related kinase 2 (NEK2) mRNA were detected by real-time quantitative polymerase chain reaction (RT-qPCR); Western blot detected NEK2 and osteoclast markers (Cathepsin K, CTSK), matrix metalloproteinase 9 (MMP9), nuclear factor of activated T cell 2 (NFAT2), and tartrate-resistant acid phosphatase (TRAP) protein levels; cell proliferation was detected by Cell Counting Kit-8 (CCK-8), and the formation of osteoclasts was observed by TRAP staining; the F-actin skeleton was stained with tetramethylrhodamine isothiocyanate (TRITC) phalloidin; cell migration rate was detected by Transwell; dual-luciferase reporter gene assay and RNA-binding protein immunoprecipitation (RIP) assay verified the targeting relationship between miR-381-3p, SNHG15, and NEK2. Results The expression of SNHG15 was increased in THP-1 cells stimulated by macrophage colony-stimulating factor (M-CSF)/receptor activator of nuclear factor-kappa B ligand (RANKL). Overexpression of SNHG15 significantly promoted the proliferation, migration, osteoclast differentiation, and expression of osteoclast markers CTSK, MMP9, NFAT2, and TRAP of THP-1 cells induced by M-CSF/RANKL. Knockdown of SNHG15 reversed this effect. Overexpression of SNHG15 downregulated the inhibitory effect of overexpression of miR-381-3p on the proliferation, migration, and differentiation of THP-1 cells induced by M-CSF/RANKL. Knockdown of miR-381-3p reversed the inhibitory effect of knockdown of NEK2 on the proliferation, migration, and differentiation of THP-1 cells induced by M-CSF/RANKL. Conclusion SNHG15 acted as a ceRNA promoted the proliferation, migration, and differentiation of THP-1 cells induced by M-CSF/RANKL through sponging miR-381-3p to promote the expression of NEK2.

Transcription Factor KLF7 Promotes Osteoclast Differentiation by Suppressing HO-1.

Background: Osteoporosis is a common orthopedic disease with high prevalence in patients older than 50 years. Osteoporosis is often detected only after the fracture and is hard to treat. Therefore, it is of great significance to explore the molecular mechanism of the occurrence of osteoporosis. Methods: The expression of Heme oxygenase-1 (HO-1) in people with different bone mineral density (BMD) was analyzed based on public databases. GenHacncer and JASPAR databases were adopted to search and verify the upstream transcription factor of HO-1. qRT-PCR, western blot and tartrate-resistant acid phosphatase assays were performed to explore the impact of HO-1 and Kruppel-like factor 7 (KLF7) on osteoclast differentiation. Chromatin immunoprecipitation (ChIP) assay confirmed the binding relationship between KLF7 and HO-1. Finally, Hemin, the agonist of HO-1, was applied in rescue assays, thereby verifying the mechanism of KLF7 modulating osteoclast differentiation by HO-1. Results: Bioinformatics analysis revealed that HO-1 was highly-expressed while KLF7 lowly-expressed in people with high BMD. Besides, a potential binding site of KLF7 was found on the promoter region of HO-1. ChIP assay further manifested the targeting relationship between HO-1 and KLF7. Western blot and TRAP staining unveiled that osteoclast differentiation was suppressed by HO-1, while facilitated by KLF7. Rescue experiments indicated that over-expressed HO-1 could reverse of the promoting effect of KLF7 on osteoclast differentiation. Conclusion: The study confirmed that osteoclast differentiation was promoted by KLF7 constraining HO-1, thereby facilitating osteoporosis. The cognation of the pathogenesis of osteoporosis was further enriched. New treatment could be developed on this basis.

Inhibition of NFAM1 suppresses phospho-SAPK/JNK signaling during osteoclast differentiation and bone resorption.

We have recently demonstrated NFAT activating protein with ITAM motif 1 (NFAM1) signaling increases osteoclast (OCL) formation/bone resorption associated with the Paget's disease of bone, however, the underlying molecular mechanisms of the NFAM1 regulation of OCL differentiation and bone resorption remains unclear. Here, we showed that RANK ligand stimulation enhances NFAM1 expression in preosteoclast cells. Conditioned media collected from RANKL stimulated RAW264.7 NFAM1 knockdown (KD) stable cells showed inhibition of interleukin-6 (2.5-fold), tumour necrosis factor-α (2.2-fold) and CXCL-5 (3-fold) levels compared to wild-type (WT) cells. Further, RANKL stimulation significantly increased p-STAT6 expression (5.5-fold) in WT cells, but no significant effect was observed in NFAM1-KD cells. However, no changes were detected in signal transducer and activator of transcription 3 levels in either of cell groups. Interestingly, NFAM1-KD suppressed the RANKL stimulated c-fos, p-c-Jun and c-Jun N-terminal kinase (JNK) activity in preosteoclasts. We further showed that the suppression of JNK activity is through inhibition of p-SAPK/JNK in these cells. In addition, NFATc1 expression, a critical transcription factor associated with osteoclastogenesis is significantly inhibited in NFAM1-KD preosteoclast cells. Interestingly, NFAM1 inhibition suppressed the OCL differentiation and bone resorption capacity in mouse bone marrow cell cultures. We also demonstrated inhibition of tartrate-resistant acid phosphatase expression in RANKL stimulated NFAM1-KD preosteoclast cells. Thus, our results suggest that NFAM1 control SAPK/JNK signaling to modulate osteoclast differentiation and bone resorption.

Pseudogene PTENP1 sponges miR-214 to regulate the expression of PTEN to modulate osteoclast differentiation and attenuate osteoporosis

Background aimsOsteoporosis (OP) is a common bone metabolic disease with a high incidence. Our study aimed to explore the pseudogene PTENP1/miR-214/PTEN axis to modulate the osteoclast differentiation in osteoporosis. MethodsPatients with osteoporosis were recruited in our study, and RANKL-induced osteoclast differentiation and ovariectomy-induced osteoporosis mouse model were established in vitro and in vivo, respectively. ResultsPseudogene PTENP1 and PTEN were significantly down-regulated and miR-214 was up-regulated in osteoporosis patients. In addition, overexpression of PTENP1 or silence of miR-214 inhibited the expression levels of osteoclast specific markers and osteoclast differentiation induced by RANKL. Overexpression of PTENP1 or silence of miR-214 also inhibited the levels of phosphorylation of PI3K and AKT, p65 nuclear translocation, IκBα degradation and the expression level of NFATc1. AlsoSilence of PTENP1 or overexpression of miR-214 induced the osteoclast differentiation under normal physiological condition. Pseudogene PTENP1 sponged miR-214 to regulate the expression of PTEN. ConclusionsIn an ovariectomy-induced osteoporosis mouse model, obvious pathological changes in bone tissues were found, and bone marrow mononuclear cells in this group were more likely to differentiate into osteoclasts. Therefore, pseudogene PTENP1 sponged miR-214 to regulate the expression of PTEN to inhibit osteoclast differentiation and attenuate osteoporosis by suppressing the PI3K/AKT/NF-κB signaling pathway.

METTL3 Modulates Osteoclast Differentiation and Function by Controlling RNA Stability and Nuclear Export.

Osteoclast differentiation and function are crucial for maintaining bone homeostasis and preserving skeletal integrity. N6-methyladenosine (m6A) is an abundant mRNA modification that has recently been shown to be important in regulating cell lineage differentiation. Nevertheless, the effect of m6A on osteoclast differentiation remains unknown. In the present study, we observed that the m6A level and methyltransferase METTL3 expression increased during osteoclast differentiation. Mettl3 knockdown resulted in an increased size but a decreased bone-resorbing ability of osteoclasts. The expression of osteoclast-specific genes (Nfatc1, c-Fos, Ctsk, Acp5 and Dcstamp) was inhibited by Mettl3 depletion, while the expression of the cellular fusion-specific gene Atp6v0d2 was upregulated. Mechanistically, Mettl3 knockdown elevated the mRNA stability of Atp6v0d2 and the same result was obtained when the m6A-binding protein YTHDF2 was silenced. Moreover, the phosphorylation levels of key molecules in the MAPK, NF-κB and PI3K-AKT signaling pathways were reduced upon Mettl3 deficiency. Depletion of Mettl3 maintained the retention of Traf6 mRNA in the nucleus and reduced the protein levels of TRAF6. Taken together, our data suggest that METTL3 regulates osteoclast differentiation and function through different mechanisms involving Atp6v0d2 mRNA degradation mediated by YTHDF2 and Traf6 mRNA nuclear export. These findings elucidate the molecular basis of RNA epigenetic regulation in osteoclast development.

High shear stress amplitude in combination with prolonged stimulus duration determine induction of osteoclast formation by hematopoietic progenitor cells

To date, it is unclear how fluid dynamics stimulate mechanosensory cells to induce an osteoprotective or osteodestructive response. We investigated how murine hematopoietic progenitor cells respond to 2 minutes of dynamic fluid flow stimulation with a precisely controlled sequence of fluid shear stresses. The response was quantified by measuring extracellular adenosine triphosphate (ATP), immunocytochemistry of Piezo1, and sarcoplasmic/endoplasmic Ca2+ reticulum ATPase 2 (SERCA2), and by the ability of soluble factors produced by mechanically stimulated cells to modulate osteoclast differentiation. We rejected our initial hypothesis that peak wall shear stress rate determines the response of hematopoietic progenitor cells to dynamic fluid shear stress, as it had only a minor correlation with the abovementioned parameters. Low stimulus amplitudes corresponded to activation of Piezo1, SERCA2, low concentrations of extracellular ATP, and inhibition of osteoclastogenesis and resorption area, while high amplitudes generally corresponded to osteodestructive responses. At a given amplitude (3 Pa) and waveform (square), the duration of individual stimuli (duty cycle) showed a strong correlation with the release of ATP and osteoclast number and resorption area. Collectively, our data suggest that hematopoietic progenitor cells respond in a viscoelastic manner to loading, since a combination of high shear stress amplitude and prolonged duty cycle is needed to trigger an osteodestructive response. PLAIN LANGUAGE SUMMARY: In case of painful joints or missing teeth, the current intervention is to replace them with an implant to keep a high-quality lifestyle. When exercising or chewing, the cells in the bone around the implant experience mechanical loading. This loading generally supports bone formation to strengthen the bone and prevent breaking, but can also stimulate bone loss when the mechanical loading becomes too high around orthopedic and dental implants. We still do not fully understand how cells in the bone can distinguish between mechanical loading that strengthens or weakens the bone. We cultured cells derived from the bone marrow in the laboratory to test whether the bone loss response depends on (i) how fast a mechanical load is applied (rate), (ii) how intense the mechanical load is (amplitude), or (iii) how long each individual loading stimulus is applied (duration). We mimicked mechanical loading as it occurs in the body, by applying very precisely controlled flow of fluid over the cells. We found that a mechanosensitive receptor Piezo1 was activated by a low amplitude stimulus, which usually strengthens the bone. The potential inhibitor of Piezo1, namely SERCA2, was only activated by a low amplitude stimulus. This happened regardless of the rate of application. At a constant high amplitude, a longer duration of the stimulus enhanced the bone-weakening response. Based on these results we deduce that a high loading amplitude tends to be bone weakening, and the longer this high amplitude persists, the worse it is for the bone.

CircRNA_28313/miR-195a/CSF1 axis modulates osteoclast differentiation to affect OVX-induced bone absorption in mice

ABSTRACTOsteoblastic bone formation and osteoclastic bone resorption dynamically maintain the bone homeostasis; in the present study, we attempt to investigate the mechanism of the excessive activation of osteoclasts inducing the deregulation of bone homeostasis from the perspective of non-coding RNA regulation. Differentially expressed patterns of circRNAs were examined in non-treated and RANKL + CSF1-treated bone marrow monocyte/macrophage (BMM) cells and differentially-expressed miRNAs during osteoclast differentiation were analyzed and identified. We found that circRNA_28313 was significantly induced by RANKL + CSF1 treatment. circRNA_28313 knockdown significantly inhibited RANKL + CSF1-induced differentiation of osteoclasts within BMM cells in vitro, while suppressed ovariectomized (OVX)-induced bone resorption in mice in vivo. Via bioinformatics analyses, it has been demonstrated that miR-195a might bind to circRNA_28313 and CSF1 and together form a circRNA-miRNA-mRNA network. circRNA_28313 relieves miR-195a-mediated suppression on CSF1 via acting as a ceRNA, therefore modulating the osteoclast differentiation in BMM cells. In conclusion, circRNA_28313, miR-195a, and CSF1 form a ceRNA network to function in RANKL + CSF1-induced osteoclast differentiation, thus affecting OVX-induced bone absorption in mice.

KBTBD11, a novel BTB-Kelch protein, is a negative regulator of osteoclastogenesis through controlling Cullin3-mediated ubiquitination of NFATc1

Kelch repeat and BTB domain-containing protein 11 (KBTBD11) is a member of the KBTBD subfamily of proteins that possess a BTB domain and Kelch repeats. Despite the presence of the Kbtbd11 gene in mammalian genomes, there are few reports about KBTBD11 at present. In this study, we identified the novel protein KBTBD11 as a negative regulator of osteoclast differentiation. We found that expression of KBTBD11 increased during osteoclastogenesis. Small-interfering-RNA-mediated knockdown of KBTBD11 enhanced osteoclast formation, and markedly increased the expression of several osteoclast marker genes compared with control cells. Conversely, KBTBD11 overexpression impaired osteoclast differentiation, and decreased the expression of osteoclast marker genes. Among six major signaling pathways regulating osteoclast differentiation, KBTBD11 predominantly influenced the nuclear factor of activated T cell cytoplasmic-1 (NFATc1) pathway. Mechanistically, KBTBD11 was found to interact with an E3 ubiquitin ligase, Cullin3. Further experiments involving immunoprecipitation and treatment with MG132, a proteasome inhibitor, showed that the KBTBD11–Cullin3 promotes ubiquitination and degradation of NFATc1 by the proteasome. Considering that NFATc1 is an essential factor for osteoclast differentiation, the KBTBD11 and Cullin3 probably regulate the levels of NFATc1 through the ubiquitin-proteasome degradation system. Thus, KBTBD11 negatively modulates osteoclast differentiation by controlling Cullin3-mediated ubiquitination of NFATc1.

Monomeric C-Reactive Protein Binds and Neutralizes Receptor Activator of NF-κB Ligand-Induced Osteoclast Differentiation.

C-reactive protein (CRP) is an established marker of rheumatoid arthritis (RA) but with ill-defined actions in the pathogenesis. Here, we show that CRP regulates the differentiation of osteoclasts, a central mediator of joint inflammation and bone erosion in RA, in a conformation- and receptor activator of NF-κB ligand (RANKL)-dependent manner. CRP in the native conformation is ineffective, whereas the monomeric conformation (mCRP) actively modulates osteoclast differentiation through NF-κB and phospholipase C signaling. Moreover, mCRP can bind RANKL, the major driver of osteoclast differentiation, and abrogate its activities. The binding and inhibition of RANKL are mediated by the cholesterol binding sequence (CBS) of mCRP. Corroborating the in vitro results, CRP knockout exacerbates LPS-induced bone resorption in mice. These results suggest that mCRP may be protective in joint inflammation by inhibiting pathological osteoclast differentiation and that the CBS peptide could be exploited as a potential RANKL inhibitor.

The emerging role of Hippo signaling pathway in regulating osteoclast formation

A delicate balance between osteoblastic bone formation and osteoclastic bone resorption is crucial for bone homeostasis. This process is regulated by the Hippo signaling pathway including key regulatory molecules RASSF2, NF2, MST1/2, SAV1, LATS1/2, MOB1, YAP, and TAZ. It is well established that the Hippo signaling pathway plays an important part in regulating osteoblast differentiation, but its role in osteoclast formation and activation remains poorly understood. In this review, we discuss the emerging role of Hippo-signaling pathway in osteoclast formation and bone homeostasis. It is revealed that specific molecules of the Hippo-signaling pathway take part in a stage specific regulation in pre-osteoclast proliferation, osteoclast differentiation and osteoclast apoptosis and survival. Upon activation, MST and LAST, transcriptional co-activators YAP and TAZ bind to the members of the TEA domain (TEAD) family transcription factors, and influence osteoclast differentiation via regulating the expression of downstream target genes such as connective tissue growth factor (CTGF/CCN2) and cysteine-rich protein 61 (CYR61/CCN1). In addition, through interacting or cross talking with RANKL-mediated signaling cascades including NF-κB, MAPKs, AP1, and NFATc1, Hippo-signaling molecules such as YAP/TAZ/TEAD complex, RASSF2, MST2, and Ajuba could also potentially modulate osteoclast differentiation and function. Elucidating the roles of the Hippo-signaling pathway in osteoclast development and specific molecules involved is important for understanding the mechanism of bone homeostasis and diseases.

Acute effects of dietary fatty acids on osteclastogenesis via RANKL/RANK/OPG system.

Postprandial state is directly linked with chronic diseases. We hypothesized that dietary fats may have acute effects on health status by modulating osteoclast differentiation and activation in a fatty acid-dependent manner. In healthy subjects, a fat-enriched meal increased plasma levels of the RANKL (receptor activator of nuclear factor κB ligand)/OPG (osteoprotegerin) ratio (SFAs > MUFAs = PUFAs) in the postprandial state. Postprandial TRL-SFAs enhanced tartrate-resistant acid phosphatase (TRAP) activity and the expression of osteoclast marker genes (TRAP, OSCAR, RANK, and CATHK) while downregulated the expression of OPG gene in human monocyte-derived osteoclasts. These effects were not observed with monounsaturated fatty acid (MUFA)-enriched postprandial triglyceride-rich lipoproteins (TRLs). Moreover, postprandial TRL-SFAs increased the release of osteoclastogenic cytokines (TNF-α, IL-1β, and IL-6) meanwhile TRL-MUFAs and TRL-PUFAs increased the release of anti-osteoclastogenic cytokines (IL-4 and IL-10) in the medium of human monocyte-derived osteoclasts. For the first time, we show that postprandial TRLs are metabolic entities with osteoclastogenic activity and that this property is related to the type of dietary fatty acid in the meal. The osteoclastogenic potency was as follows: SFAs >>> MUFAs = PUFAs. These exciting findings open opportunities for developing nutritional strategies with olive oil as the principal dietary source of MUFAs, notably oleic acid, to prevent development and progression of osteoclast-related diseases.

Pro-inflammatory Cytokines Modulating Osteoclast Differentiation and Function

In general, bone homeostasis is maintained through the balance between bone formation and resorption. Disruption in this balance results in bone-related diseases such as osteopetrosis, osteoporosis, and rheumatoid arthritis. Often, enhanced osteoclastogenesis is followed by accelerated bone resorption that is induced by pro-inflammatory cytokines in osteoporosis or rheumatoid arthritis, and leads to bone destruction. In this review study, factors involved in osteoclast differentiation and function are discussed, and how the prevention of such factors is effective in ameliorating bone loss in osteoporosis or rheumatoid arthritis. (J Rheum Dis 2016;23:148-153)

A short report: PAMM, a novel antioxidant protein, induced by oxidative stress

Reactive oxygen species (ROS) play a central role in estrogen deficiency-induced bone loss. We previously identified and characterized a novel member of the Peroxiredoxin (PRX) like 2 family that we called PAMM: Peroxiredoxin Activated in M-CSF stimulated Monocytes, a redox regulatory protein that modulates osteoclast differentiation in vitro. In this study, we report increased PAMM expression in H2O2-treated cells and in bones from ovariectomized (OVX) mice 4 weeks after surgery, models for oxidative stress in vitro and in vivo, respectively. We also detected increased PAMM abundance and phosphorylated Akt in OVX mice treated with estrogen. In addition, Wortmannin, a specific PI3Kinase inhibitor and Rapamycin, an inhibitor of the PI3Kinase/Akt pathway, blocked Akt phosphorylation and stimulation of PAMM expression by M-CSF. These results indicate that M-CSF-induced PAMM expression is mediated by Akt phosphorylation. Our data also suggest that estrogen-induced PAMM expression is mediated by phosphorylation of Akt. These findings point to PAMM as a potential candidate for Akt-mediated protection against oxidative stress.

Resolvin E1 and Chemokine-like Receptor 1 Mediate Bone Preservation

The polyunsaturated ω-3 fatty acid eicosapentaenoic acid-derived resolvin E1 (RvE1) enhances resolution of inflammation, prevents bone loss, and induces bone regeneration. Although the inflammation-resolving actions of RvE1 are characterized, the molecular mechanism of its bone-protective actions are of interest. To test the hypothesis that receptor-mediated events impact bone changes, we prepared transgenic mice overexpressing the RvE1 receptor chemokine-like receptor 1 (chemR23) on leukocytes. In zymosan-initiated peritonitis, neutrophil polymorphonuclear leukocyte infiltration in response to RvE1 was limited requiring log order lower doses in chemR23tg mice. Ligature-induced alveolar bone loss was diminished in chemR23tg mice. Local RvE1 treatment of uniform craniotomy in the parietal bone significantly accelerated regeneration of the bone defect. In in vitro bone cultures, RvE1 significantly enhanced expression of osteoprotegerin (OPG) without inducing change in receptor activator of NF-κB ligand levels, whereas the osteogenic markers alkaline phosphatase, bone sialoprotein, and Runt-related transcription factor 2 remained unchanged. These results indicate that RvE1 modulates osteoclast differentiation and bone remodeling by direct actions on bone, rescuing OPG production and restoring a favorable receptor activator of NF-κB ligand/OPG ratio, in addition to known anti-inflammatory and proresolving actions.

Insulin effect on RANKL and OPG expression in human osteoblast-like MG63 cells

To investigate the effect of insulin on receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) expression in human osteosarcoma MG63 cells using real-time polymerase chain reaction. Materials and methods: MG63 cells were exposed to different concentrations of human recombinant insulin (1, 10, and 100 nM) for 24 h, and then the expression of RANKL and OPG mRNA was identified. After that, MG63 cells were exposed to tensile stress combined with 0 or 10 nM insulin respectively for 0, 3, 6, and 12 h, and then the expression of RANKL and OPG mRNA was identified. Human monocytes were cultured with the collected culture media from MG63 cells plates that were exposed to different concentrations of human recombinant insulin (0 and 10 nM) for 7 days, and then were stained with an acid phosphatase kit. Results: After 24 h of treatment with 1, 10, and 100 nM doses of insulin, the expression of RANKL and OPG mRNA was suppressed (P < 0.05), and the ratio of OPG to RANKL also was suppressed (P < 0.05). Application of tensile stress to MG63 cells induced a decrease in RANKL mRNA expression and an increase in OPG mRNA expression at 6 and 12 h (P < 0.05); 10 nM insulin decreased the mechanical stress-induced RANKL and OPG mRNA expression in MG63 cells at 6 and 12 h (P < 0.05). The ratio of OPG to RANKL increased at 6 and 12 h in the 2 groups (P < 0.05), but insulin decreased the mechanical stress-induced OPG/RANKL ratio at 12 h (P < 0.05). For the group of monocytes cultured with DMEM collected from MG63 cells plates that were cultured with 0 nM human recombinant insulin, osteoclast formation was not observed. Osteoclast formation was observed in monocytes cultured with DMEM collected from MG63 cells plates that were cultured with 10 nM human recombinant insulin. Conclusion: These results suggest that insulin can modulate osteoclast differentiation via altering the OPG and RANKL expression in osteoblasts. The deficiency in insulin may decrease the number of osteoclasts and osteoclast-mediated bone resorption in patients with type 1 diabetes mellitus.

Early growth response 2 negatively modulates osteoclast differentiation through upregulation of Id helix–loop–helix proteins

Early growth response 2 (Egr2) is a zinc finger transcription factor that acts as an important modulator of various physiological processes. In this study, we show that Egr2 negatively regulates receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation. The overexpression of Egr2 in bone marrow-derived macrophages (BMMs) suppresses the formation of multinuclear osteoclasts and the expression of osteoclastogenic markers, including nuclear factor of activated T cells c1 (NFATc1). On the other hand, Egr2 overexpression does not impact the phagocytic activity of osteoclast precursors or the expression of macrophage-specific markers in the presence of the osteoclastogenic stimuli, RANKL and M-CSF. We further demonstrate that Egr2 induces the expression of the inhibitors of differentiation/DNA binding (Ids) helix–loop–helix (HLH) transcription factors, which are important repressors in RANKL-mediated osteoclastogenesis. Egr2 transactivates the Id2 promoter and increases its recruitment to the Id2 promoter region. In addition, Egr2-dependent induction of Id2 promoter activity, and its binding to the Id2 promoter is abrogated by the overexpression of the Egr2 repressor, NGFI-A binding protein 2 (Nab2). Accordingly, coexpression with Nab2 restores Egr2-mediated suppression of osteoclast differentiation. Furthermore, knockdown of Egr2 using shRNA enhances osteoclastogenesis and decreases Id2 gene expression. Ectopic expression of Id2 reverses the phenotype mediated by Egr2 silencing. Taken together, our results identify Egr2 as an important modulator of RANKL-induced osteoclast differentiation and provide the link between RANKL, Egr2 and Id proteins in osteoclast-lineage cells.

Growth/differentiation factor-15: prostate cancer suppressor or promoter?

Deregulation of expression and function of cytokines belonging to the transforming growth factor-β (TGF-β) family is often associated with various pathologies. For example, this cytokine family has been considered a promising target for cancer therapy. However, the detailed functions of several cytokines from the TGF-β family that could have a role in cancer progression and therapy remain unclear. One of these molecules is growth/differentiation factor-15 (GDF-15), a divergent member of the TGF-β family. This stress-induced cytokine has been proposed to possess immunomodulatory functions and its high expression is often associated with cancer progression, including prostate cancer (PCa). However, studies clearly demonstrating the mechanisms for signal transduction and functions in cell interaction, cancer progression and therapy are still lacking. New GDF-15 roles have recently been identified for modulating osteoclast differentiation and for therapy for PCa bone metastases. Moreover, GDF-15 is as an abundant cytokine in seminal plasma with immunosuppressive properties. We discuss studies that focus on the regulation of GDF-15 expression and its role in tissue homeostasis, repair and the immune response with an emphasis on the role in PCa development.

Role of DNA methylation in the regulation of the RANKL-OPG system in human bone

Osteoblasts are specialized cells that form new bone and also indirectly influence bone resorption by producing factors that modulate osteoclast differentiation. Although the methylation of CpG islands plays an important role in the regulation of gene expression, there is still scanty information about its role in human bone. The aim of this study was to investigate the influence of CpG methylation on the transcriptional levels of two osteoblast-derived critical factors in the regulation of osteoclastogenesis: the receptor activator of nuclear factor NF-κB ligand (RANKL) and its soluble decoy receptor osteoprotegerin (OPG). Quantitative methylation specific PCR (qMSP) and pyrosequencing analysis in various cell types showed that the methylation of regulatory regions of these genes, in the vicinity of the transcription start sites, repressed gene transcription, whereas an active transcription was associated with low levels of methylation. In addition, treatment with the DNA demethylating agent 5-azadeoxycitidine promoted a 170-fold induction of RANKL and a 20-fold induction of OPG mRNA expression in HEK-293 cells, which showed hypermethylation of the CpG islands and barely expressed RANKL and OPG transcripts at baseline. Transcriptional levels of both genes were also explored in bone tissue samples from patients with hip fractures and hip osteoarthritis. Although RANKL transcript abundance and the RANKL:OPG transcript ratio were significantly higher in patients with fractures than in those with osteoarthritis (RANKL: 0.76 ± 0.23 vs. 0.24 ± 0.08, p = 0.012; RANKL/OPG: 7.66 ± 2.49 vs. 0.92 ± 0.21, p = 0.002), there was no evidence for differential methylation across patient groups. In conclusion, the association between DNA methylation and the repression of RANKL and OPG expression strongly suggests that methylation-dependent mechanisms influence the transcription of these genes, which play a critical role in osteoclastogenesis. However, other mechanisms appear to be involved in the increased RANKL/OPG ratio of patients with osteoporotic fractures.