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Hyperhomocystinemia is a modifiable risk factor for osteoporosis and fracture. Physiologic concentrations of Hcy directly activate osteoclast formation and activity through stimulation of p38 MAPK and integrin β3. The effects of Hcy were mediated by generation of intracellular ROS.
Introduction: Hyperhomocysteinemia is a modifiable risk factor for osteoporosis and its related bone fractures. It has been reported that bone resorption and turnover rate were increased in hyperhomocystinemia. Using mouse bone marrow cells, we examined the direct effects of homocysteine (Hcy) on osteoclast formation and activity.
Materials and Methods: Osteoclast formation was determined by TRACP staining and TRACP activity. Intracellular reactive oxygen species (ROS) generation was measured using a fluorescent probe, dichlorodihydrofluorescein diacetate. Intracellular signaling cascades of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and NF-κB were measured by Western blotting. Integrin β3 mRNA levels were measured by RT-PCR. Actin ring formation and bone resorption assays were also performed.
Results: Physiologic concentrations of Hcy upregulated TRACP+ multinucleated cells and TRACP activity, stimulated actin ring formation, and increased the number of nuclei per cell and the level of expression of integrin β3 mRNA. In addition, Hcy increased bone resorption and stimulated p38 MAPK activity and intracellular reactive oxygen species (ROS) generation. All of these Hcy-induced changes were blocked by pretreatment with the antioxidant, N-acetyl cysteine.
Conclusions: Hcy directly activates osteoclast formation and activity through increased generation of intracellular ROS. These findings suggest that, in individuals with mild to moderate hyperhomocystinemia, increased bone resorption by osteoclasts may contribute to osteoporosis and that an antioxidant may attenuate bone loss in these individuals.
Homocysteine (Hcy) is a sulfur-containing amino acid that functions as a key intermediate in methionine metabolism.(1) The link between Hcy and osteoporosis was provided by hereditary homocystinuria, a rare genetic disorder characterized by severe hyperhomocystinemia and early onset of atherosclerosis and osteoporosis.(2) Interestingly, mild to moderate hyperhomocystinemia is quite prevalent in elderly individuals(3,4) and has been shown to be associated with lower bone mass(5,6) and higher fracture risk.(7,8) In addition, a polymorphism of the gene encoding methylenetetrahydrofolate reductase, which catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate, the substrate for Hcy methylation, was found to be associated with osteoporosis and increased fracture risk.(9–12) Furthermore, treatment with folate and vitamin B12, which lower Hcy, was found to reduce the risk of hip fracture in patients with stroke.(13) Collectively, these findings strongly suggest that hyperhomocystinemia is associated with increased risk of osteoporosis in the general population.
In patients with congenital homocystinuria, a condition characterized by extremely high plasma Hcy concentrations, Hcy is thought to interfere with collagen cross-linking, resulting in poor quality of bone and increased susceptibility to fracture.(14,15) It is not clear, however, whether these results are directly applicable to normal variations of Hcy and whether hyperhomocystinemia induces osteoporosis by a similar mechanism in the general population. In patients with mild to moderate hyperhomocystinemia, blood Hcy levels were found to positively correlate with urinary deoxypyridinoline, a marker for bone resorption, suggesting that increased bone resorption may be involved in Hcy-induced osteoporosis.(16,17)
Hcy has been shown to be a potent pro-oxidant in vivo(18,19) and in vitro.(20) In addition, osteoclast lineages are very sensitive to oxidative stress, with osteoclast formation stimulated by increased generation of intracellular reactive oxygen species (ROS).(21–23) We therefore hypothesized that Hcy may stimulate osteoclast formation and osteoclast activity by increasing the generation of intracellular ROS. To test this hypothesis, we examined the direct effects of Hcy on osteoclast formation and osteoclast activity and its mechanism of action. We also tested whether an antioxidant, N-acetyl cysteine (NAC), could block the effects induced by Hcy.
MATERIALS AND METHODS
Reagents and materials
Hcy, NAC, the leukocyte acid phosphatase kit, and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St Louis, MO, USA). Macrophage-colony stimulating factor (M-CSF), soluble RANKL, and the mouse JE/MCP-1 (monocyte chemoattractant protein-1) immunoassay kit were obtained from R&D Systems (Minneapolis, MN, USA). Antibodies directed against p38, phospho-p38, extracellular signal-regulated kinase (ERK), c-jun N-terminal protein kinase (JNK), phospho-JNK, inhibitor of nuclear κBα (IκBα), and phospho-IκBα were purchased from Cell Signaling Technology (Beverly, MA, USA), and an antibody directed against phopho-ERK was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent probe was purchased from Molecular Probes (Leiden, The Netherlands). The Osteologic Multitest Slide was purchased from BD Biosciences (Bedford, MA, USA).
Culture of bone marrow cells and differentiation into osteoclasts
Primary mouse bone marrow cells (BMCs) were obtained by flushing femora and tibias of 5- to 6-week-old ICR mice. After culture for 24 h at 37°C in α-MEM (Sigma-Aldrich) containing 10% FBS (Gibco, Grand Island, NY, USA), 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO2, the nonadherent cells were collected and cultured in 48-well plates at a density of 1 × 105/well. The cells were differentiated into osteoclasts by culturing in the presence of M-CSF (30 ng/ml) and soluble RANKL (50 ng/ml) for 5 days, changing the culture medium every 3 days.
Primary mouse osteoblasts were obtained by sequential collagenase digestion from the calvariae of newborn mice in α-MEM containing 10% FBS and cultured for 1 day in 48-well plates at a density of 1.2 × 104/well. BMCs (1 × 105/well) were added to each well, and BMCs were co-cultured with osteoblasts for 5 days in the presence of 10−8 M 1α,25-dihydroxyvitamin D3 (Calbiochem, La Jolla, CA, USA) and 10−6 M prostaglandin E2 (Calbiochem), changing the culture medium every 3 days. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Asan Institute for Life Sciences.
TRACP staining and activity assay
After 5 days in culture, the adherent cells were fixed and stained for TRACP, a marker enzyme of osteoclasts, using the leukocyte acid phosphatase kit following the manufacturer's instructions. TRACP+ multinucleated cells containing three or more nuclei were considered to be osteoclasts and were counted under a light microscope (Olympus Optical, Tokyo, Japan).
TRACP activity was measured as described.(24) Briefly, adherent cells were gently washed twice with PBS and lysed with 100 μl of 0.2% Triton X-100, and TRACP activity in the cell lysate was determined by incubating an aliquot of lysate in 50 μl of substrate solution (50 mM citrate buffer [pH 4.5], 5.5 mM p-nitrophenol phosphate, 10 mM sodium tartrate) for 30 minutes at 37°C. The reaction was stopped by adding 100 μl of 0.1N NaOH, and the absorbance at 405 nm was measured using a microplate reader (SPECTRAmax 340 PC; Molecular Devices, Palo Alto, CA, USA).
Osteoclast apoptosis assay
After culture of BMCs in the presence of M-CSF (30 ng/ml) and soluble RANKL (50 ng/ml) for 5 days, the cells were incubated without the soluble RANKL for 14 h. After rinsing once with PBS, cells were fixed with 10% formaldehyde for 5 minutes at 20°C and incubated with 1 μg/ml DAPI in PBS for 20 minutes. Cells displaying condensed chromatin or fragmented nuclei were considered apoptotic.
BMCs (1 × 106/well) were seeded in 6-well plates and cultured in α-MEM containing 10% FBS and M-CSF (10 ng/ml) for 3 days. After incubation for 3 h in α-MEM containing 0.2% FBS and M-CSF (10 ng/ml), the cells were incubated in the presence or absence of Hcy (100 μM) and/or soluble RANKL (30 ng/ml) for the indicated times. The cells were washed with PBS and incubated in lysis buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM NaF, and protease inhibitor mixture) at 4°C for 20 minutes. The protein concentration in each lysate was determined using BCA reagent (Pierce Chemical Co., Rockford, IL, USA). Cell lysates (30∼40 μg of total protein per lane) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell BioSciences, Dassel, Germany). After blocking with 5% skim milk, the membranes were incubated with primary antibodies, followed by incubation with horseradish peroxidase–conjugated secondary antibodies and visualization using enhanced chemiluminescence detection reagent (Amersham BioSciences, Seoul, Korea). Band intensity was quantified densitometrically using Quantity One Software (VersaDoc Model 3000 Imaging System; BioRad).
Actin ring formation assay
BMCs were cultured on glass coverslips for 5 days with M-CSF (30 ng/ml) and soluble RANKL (50 ng/ml), fixed with 2% paraformaldehyde for 10 minutes, washed twice with 20 mM glycine-PBS, and stained with 0.1 μg/ml rhodamine-conjugated phalloidin (Molecular Probes) for 10 minutes. Fluorescent images were collected using a confocal microscope (Leica Microsystems, Wetzlar, Germany).
RT-PCR for integrin β3 mRNA
Total RNA was purified from BMCs using TRIzol reagent (Invitrogen, Rockville, MD, USA) according to the manufacturer's instruction, and cDNA was synthesized from 5-μg aliquots of total RNA using the Superscript III First-Strand Synthesis System (Invitrogen). All PCR amplifications were performed using 5 U Taq polymerase (Bioneer, Daejeon, Korea), 15 mM MgCl2, and 2.5 mM dNTP mixture (Bioneer) in a final volume of 30 μl. The specific primers for integrin β3 were 5′-ACTGCAACTGTACTACACGCAC-3′ (sense) and 5′-AGTAGCTTCCAGATGAGCAGAG-3′ (antisense), and specific primers for GAPDH were 5′-ACTTTGTCAAGCTCATTTCC-3′ (sense) and 5′-TGCAGCGAACTTTATTGATG-3′ (antisense), which was amplified as a control for RNA loading and variations in cDNA synthesis efficiency. The amplification protocol consisted of 30 cycles of denaturation at 94°C for 1 minutes, annealing at 60°C for 1 minute and extension at 72°C for 1 minute. The PCR products (10 μl) were separated on 1% agarose gels, which were stained with ethidium bromide and visualized under UV light.
Measurement of MCP-1 concentrations
BMCs (1 × 105/well) were seeded in 48-well plates and cultured in α-MEM containing 10% FBS in the presence of M-CSF (10 ng/ml) for 3 days, and the cells were incubated with or without Hcy (100 μM) for 2, 6, 24, and 48 h in the presence of M-CSF (10 ng/ml) and soluble RANKL (30 ng/ml). The conditioned media were collected and MCP-1 concentrations were assayed using a commercially available immunoassay kit (Mouse JE/MCP-1 Immunoassay), according to the manufacturer's instructions. The standard curve was generated using recombinant mouse MCP-1 at concentrations from 0 to 1000 pg/ml, and sensitivity was <2 pg/ml.
Measurement of intracellular ROS levels
Intracellular ROS levels were measured using the fluorescent probe, H2DCFDA, which, after crossing the plasma membrane, is deacetylated to H2DCF and oxidized to the fluorescent product, DCF. BMCs (3 × 104/well in 96-well plates) were cultured for 3 days in α-MEM containing 10% FBS and M-CSF (10 ng/ml), washed with Hanks' balanced salt solution buffer (HBSS, Sigma-Aldrich), and incubated for 20 minutes at 37°C in HBSS containing 20 μM H2DCFDA. The medium was changed to fresh HBSS containing M-CSF (10 ng/ml), with or without Hcy (100 μM) and/or soluble RANKL (30 ng/ml), and the cells were incubated for 10 minutes. Fluorescence intensity was measured using a fluorometer (SPECTRAmax GEMINI-XS; Molecular Devices), with excitation at 480 nm and emission at 530 nm.
BMCs (1 × 104/well) were plated on Osteologic Multitest Slides in the presence of M-CSF (30 ng/ml) and soluble RANKL (50 ng/ml), following the manufacturer's instructions. After 10 days in culture, changing the medium every 3 days, the slides were washed with 5% sodium hypochlorite solution to remove the cells. The area of resorption pits was analyzed using Quantity One Software (VersaDoc Model 3000 Imaging System; BioRad).
All data are expressed as mean ± SD. Between-group differences were assessed using the Mann-Whitney U-test, and differences among three or more groups were assessed by ANOVA with posthoc analysis by Duncan's multiple range test. A p value <0.05 was considered statistically significant. The SPSS 11.5 package (SPSS, Chicago, IL, USA) was used for statistical procedures.
Hcy directly upregulates osteoclast formation induced by RANKL and suppresses osteoclast apoptosis
Treatment of BMCs with 10∼100 μM Hcy, but not 5 μM Hcy, dose-dependently increased RANKL-induced differentiation into osteoclasts, based on TRACP staining (Fig. 1A) and activity (Fig. 1B). Incubation of 10 and 100 μM Hcy with co-cultures of BMCs and osteoblasts also showed dose-dependent stimulation of osteoclast formation (Fig. 1C). In both culture systems, the effects of Hcy were observed on days 3–5 but not on days 1–3 (Figs. 1D and 1E). In addition, we determined whether Hcy might stimulate osteoclast apoptosis, because it was reported that Hcy partially protected clodronate-induced apoptosis of osteoclasts.(25) We observed that 10 and 100 μM Hcy significantly suppressed osteoclast apoptosis in a dose-dependent manner (Fig. 1F). These data suggest that Hcy stimulates differentiation of BMCs into osteoclasts and also suppresses apoptosis of osteoclasts. In this study, we focused here on Hcy-stimulated differentiation into osteoclasts.
Hcy activates p38 mitogen-activated protein kinase signaling
Mitogen-activated protein kinases (MAPKs) and NF-κB pathways are intracellular signaling cascades involved in osteoclast formation.(26) As expected, soluble RANKL increased the phosphorylation of p38, ERK, JNK, and IκBα compared with the RANKL-untreated controls (Fig. 2A). In both the presence and absence of soluble RANKL, Hcy activated p38 MAPK (Fig. 2B), but had no effect on the phosphorylation of ERK, JNK, and IκBα (Fig. 2A).
Hcy stimulates osteoclastic activity
Because actin ring formation is a marker for osteoclastic activity,(27,28) we tested the effect of Hcy on actin ring formation in the presence of M-CSF and RANKL. We found that Hcy increased actin ring formation in a dose-dependent manner (Fig. 3A). When we tested the effect of Hcy on the numbers of osteoclast nuclei (Fig. 3B), we found that Hcy had no effect on the numbers of osteoclasts with three to five nuclei but significantly increased the number of larger osteoclasts with more than six nuclei in a dose-dependent manner, suggesting that Hcy mainly affects the fusion of pre-osteoclasts to induce formation of more mature osteoclasts. The expression of integrin β3 mRNA was upregulated by 10 and 100 μM Hcy (1.7 ± 0.1- and 3.0 ± 0.2-fold, respectively) or RANKL (1.5 ± 0.1-fold; Fig. 3C). In addition, 10 and 100 μM Hcy enhanced the upregulation shown by soluble RANKL alone (2.6 ± 0.2- and 3.5 ± 0.3-fold, respectively; Fig. 3C).
Hcy increases intracellular ROS generation in BMCs
To determine the effect of Hcy on intracellular ROS generation, we measured DCF content in the presence or absence of RANKL and/or Hcy (Fig. 4). Hcy significantly increased intracellular ROS levels by 154.4 ± 16.2%, and Hcy enhanced the generation of ROS shown by soluble RANKL alone by 159.3 ± 9.0%. Treatment with NAC alone, a potent biological thiol antioxidant, significantly reduced intracellular ROS levels, and pretreatment with NAC blocked Hcy-induced stimulation of intracellular ROS generation in both the presence and absence of RANKL.
Hcy has been reported to increase MCP-1 expression in monocyte lineage cells,(29) and the MCP-1 is a potent stimulator of osteoclasts.(30) However, 100 μM Hcy did not stimulated MCP-1 excretion in the culture of M-CSF-stimulated osteoclast precursors compared with Hcy-untreated control (data not shown).
NAC blocks Hcy-induced upregulation of osteoclast formation and stimulation of p38 MAPK and integrin β3
When we tested the effects of NAC on RANKL-induced osteoclast formation (Fig. 5A), we found that NAC alone markedly reduced RANKL-induced osteoclast formation to 28.9 ± 5.2% of the control level, and that Hcy did not stimulate osteoclast formation in the presence of NAC. In addition, pretreatment with NAC almost completely blocked Hcy-induced activation of p38 MAPK (Fig. 5B) and integrin β3 (Fig. 5C) and Hcy-induced actin ring formation (data not shown). In addition, the pretreatment of p38 MAPK inhibitor blocked Hcy-stimulated osteoclast formation (Fig. 6A) and stimulated Hcy-induced ROS generation (Fig. 6B). These suggest that intracellular ROS generation may mediate Hcy-induced osteoclast formation, at least in part, through p38 MAPK activation. However, integrin β3 activation by Hcy was not affected by the p38 MAPK inhibitor pretreatment (Fig. 6C), suggesting that Hcy-stimulated integrin β3 mRNA may be mediated by intracellular ROS generation, independently of p38 MAPK activation.
Hcy stimulates resorption potency
Hcy-induced stimulation of osteoclast formation and osteoclast activity should increase the potency of bone resorption. When BMCs were incubated with M-CSF and soluble RANKL on Osteologic Multitest Slides, Hcy markedly increased bone resorption in a dose-dependent manner (Fig. 7A), with 10 and 100 μM Hcy increasing the resorbed area to 3.3 ± 0.4- and 8.3 ± 0.6-fold of the control level, respectively (Figs. 7A and 7B). NAC pretreatment blocked Hcy-stimulated bone resorption.
We have shown here that Hcy upregulates both osteoclast formation and osteoclast activity, resulting in ∼8-fold elevation of bone resorption compared with control. This was mediated by an increase in intracellular ROS through the stimulation of p38 MAPK activity and integrin β3. To our knowledge, this is the first report showing that Hcy-induced increases in ROS generation mediate the upregulation of osteoclast formation and activity. Interestingly, all of these effects were noted at Hcy concentrations as low as 10 μM, which is within the range of its plasma concentrations in the general population.(7) We also found that NAC, which blocked Hcy-induced ROS generation, also blocked the Hcy-induced upregulation of osteoclast formation, actin ring formation, and bone resorption, as well as p38 MAPK activity and integrin β3 mRNA levels. These results suggest that increased bone resorption by Hcy may be related to increased osteoporosis(5,6) and higher fracture risk(7,8) in individuals with mild to moderate hyperhomocystinemia. These findings also suggest that treatment with NAC may decrease bone resorption and increase bone mass in these subjects.
It is generally accepted that two critical steps occur in osteoclast formation: commitment of progenitor cells to osteoclast precursor cells, and fusion of the TRACP+ mononuclear cells to form multinucleated osteoclasts.(26) We found that incubation of BMCs with Hcy for days 3–5 of the culture period stimulated osteoclast formation, whereas Hcy had no effect during the earlier period, suggesting that Hcy mainly acts on the later stages of differentiation. The activation of RANK, the receptor for RANKL, is a key step in stimulating the differentiation of mature osteoclasts during later stages.(31) The binding of RANKL to its receptor leads to the activation of distinct signaling cascades, such as NF-κB and MAPKs, including p38 MAPK, JNK, and ERK, through several adaptor proteins and cofactors.(32) Although increased generation of ROS stimulates osteoclast formation and activity, the precise signaling cascades activated by ROS are still not known.(21–23) Estrogen deprivation has been reported to activate NFκB through ROS generation,(23,33) and RANKL-induced ROS has been found to recruit TNF receptor-associated factor 6.(34) Antioxidants, including NAC and glutathione, have been found to block the RANKL-induced activation of Akt, NF-κB, and ERK.(35) We have shown here that Hcy stimulates osteoclast formation, at least in part, through activation of p38 MAPK, which is one of the enzymes activated by ROS,(36,37) but not NF-κB, JNK, or ERK.
To resorb bone, it is essential that active osteoclasts form actin rings,(27) which correspond exactly to clear zones in bone resorbing osteoclasts, making this unique cytoskeletal organization a functional marker of activated osteoclasts.(27,28) Multiple nuclei also are characteristic of more mature and active osteoclasts.(26) In addition, integrin β3 expression, which is stimulated during osteoclast differentiation,(38) is critical for cell fusion(39,40) and actin ring formation,(41) making it another marker for osteoclastic activity. We showed that Hcy stimulated the formation of actin rings and number of osteoclasts with more than six nuclei, as well as stimulating integrin β3 expression, which was blocked by NAC. Taken together, these findings indicate that Hcy also stimulated osteoclastic activity through ROS generation. However, the Hcy-stimulated integrin β3 was not blocked by p38 MAPK inhibitor, indicating that activation of osteoclasts by Hcy may be mediated by another signaling instead of p38 MAPK activation.
RANKL itself has been reported to increase intracellular ROS during the process of osteoclast formation(34,35); however, our result showed that RANKL alone did not have a significant effect on ROS generation. This discrepancy may be caused by dissimilarities in RANKL concentration and/or assay methods. We also found that Hcy stimulated ROS generation, p38 MAPK activation, and integrin β3 expression in the absence of soluble RANKL, suggesting that Hcy may stimulate ROS generation, at least in part, by a RANKL-independent pathway. In the absence of RANKL, however, Hcy did not induce the formation of TRACP+ multinucleated cells, actin rings, and bone resorption (data not shown), suggesting that Hcy alone is not sufficient to enhance osteoclast formation and activity.
While this paper was in preparation, Herrmann et al.(42) reported that Hcy stimulated TRACP, cathepsin K, and bone resorption in a human peripheral blood mononuclear cell culture system, although they did not study its detailed mechanisms. These findings provide further support for our results, showing that Hcy increased bone resorption in both human and murine cells.
In conclusion, we have shown here that Hcy directly activates osteoclast formation and activity through increased intracellular ROS generation in vitro. These findings suggest that increased bone resorptive action by osteoclasts may contribute to osteoporosis in individuals with mild to moderate hyperhomocystinemia and that an antioxidant may have therapeutic implications in attenuating bone loss in these individuals.
This work was supported by the Korea Health 21 R& D Project from the Korean Ministry of Health & Welfare (01-PJ3-PG6-01GN11-0002 and A050491) and by the 21C Frontier Proteomics Project from the Korean Ministry of Science & Technology (FPR05C2-281).