Benefits of lumican on human bone health: clinical evidence using bone marrow aspirates.

BACKGROUND/AIMS: Lumican, a small leucine-rich proteoglycan, has shown osteoprotective effects by synchronously stimulating bone formation and suppressing bone resorption. To clarify the role of lumican in human bone metabolism, the association between lumican concentrations and osteoporosis-related phenotypes was evaluated using bone marrow (BM) samples directly reflecting local microenvironments. METHODS: BM aspirates were obtained from 77 patients during hip surgery for either fragility hip fractures (HF) (n = 29) or osteoarthritis (n = 48) and centrifuged. Concentrations of lumican and biochemical bone markers in BM supernatants were measured using enzyme linked immunosorbent assays. RESULTS: After considering confounders, lumican concentrations in BM supernatants were 16.9% lower in patients with HF than in controls, with each increase in the standard deviation of lumican concentration being associated with a 61% lower likelihood of HF. The odds ratios for HF decreased linearly with increasing lumican tertiles in BM, with the odds of having fragility HF markedly lower in participants in the highest than in the lowest lumican tertile. Higher lumican level correlated significantly with higher femur neck bone mineral density and higher bone-specific alkaline phosphatase levels, but not with tartrate-resistant acid phosphatase 5b concentrations, in BM supernatants. CONCLUSION: These data clinically validate previous in vitro and animal experiments showing the beneficial roles of lumican for bone homeostasis and suggest that lumican may contribute to a reduction in fracture risk in humans mainly through its stimulation of bone formation.

Aldosterone Inhibits In Vitro Myogenesis by Increasing Intracellular Oxidative Stress via Mineralocorticoid Receptor.

BACKGROUND: Despite clinical evidence indicating poor muscle health in subjects with primary aldosteronism (PA), it is still unclear whether the role of aldosterone in muscle metabolism is direct or mediated indirectly via factors, such as electrolyte imbalance or impaired glucose uptake. As one approach to clarify this issue, we investigated the effect of aldosterone on in vitro myogenesis and the potential mechanism explaining it. METHODS: Myogenesis was induced in mouse C2C12 myoblasts with 2% horse serum. Immunofluorescence, quantitative reversetranscription polymerase chain reaction, Western blot, viability, and migration analyses were performed for experimental research. RESULTS: Recombinant aldosterone treatment suppressed muscle differentiation from mouse C2C12 myoblasts in a dose-dependent manner, and consistently reduced the expression of myogenic differentiation markers. Furthermore, aldosterone significantly increased intracellular reactive oxygen species (ROS) levels in myotubes, and treatment with N-acetyl cysteine, a potent biological thiol antioxidant, reversed the decrease of myotube area, myotube area per myotube, nucleus number per myotube, and fusion index due to aldosterone through decreasing oxidative stress. A binding enzyme-linked immunosorbent assay confirmed that mineralocorticoid receptor (MR) interacted with aldosterone in C2C12 myoblasts, while eplerenone, an MR inhibitor, blocked aldosterone-stimulated intracellular ROS generation during myogenesis and markedly attenuated the suppression of in vitro myogenesis by aldosterone. CONCLUSION: These findings support the hypothesis that hypersecretion of aldosterone, like PA, directly contributes to muscular deterioration and suggest that antioxidants and/or MR antagonists could be effective therapeutic options to reduce the risk of sarcopenia in these patients.

Lumican Inhibits Osteoclastogenesis and Bone Resorption by Suppressing Akt Activity.

Lumican, a ubiquitously expressed small leucine-rich proteoglycan, has been utilized in diverse biological functions. Recent experiments demonstrated that lumican stimulates preosteoblast viability and differentiation, leading to bone formation. To further understand the role of lumican in bone metabolism, we investigated its effects on osteoclast biology. Lumican inhibited both osteoclast differentiation and in vitro bone resorption in a dose-dependent manner. Consistent with this, lumican markedly decreased the expression of osteoclastogenesis markers. Moreover, the migration and fusion of preosteoclasts and the resorptive activity per osteoclast were significantly reduced in the presence of lumican, indicating that this protein affects most stages of osteoclastogenesis. Among RANKL-dependent pathways, lumican inhibited Akt but not MAP kinases such as JNK, p38, and ERK. Importantly, co-treatment with an Akt activator almost completely reversed the effect of lumican on osteoclast differentiation. Taken together, our findings revealed that lumican inhibits osteoclastogenesis by suppressing Akt activity. Thus, lumican plays an osteoprotective role by simultaneously increasing bone formation and decreasing bone resorption, suggesting that it represents a dual-action therapeutic target for osteoporosis.

Elevated ceramides 18:0 and 24:1 with aging are associated with hip fracture risk through increased bone resorption.

We assessed whether circulating ceramides, which play a role in a number of degenerative changes with aging, significantly differed according to fragility hip fracture (HF) status. We also performed a human study using bone marrow (BM) aspirates, directly reflecting the bone microenvironment, in addition to in vitro experiments. Peripheral blood and BM samples were simultaneously collected from 74 patients 65 years or older at hip surgery for either HF (n = 28) or for other causes (n = 46). Ceramides were measured by liquid chromatography-tandem mass spectrometry. Age was correlated positively with circulating C16:0, C18:0, and C24:1 ceramide levels. Patients with fragility HF had 21.3%, 49.5%, 34.3%, and 22.5% higher plasma C16:0, C18:0, C18:1, and C24:1 ceramide levels, respectively, than those without HF. Higher C16:0, C18:0, C18:1, and C24:1 ceramide levels were positively related to bone resorption markers in both blood and BM samples. Furthermore, in vitro studies showed that C18:0 and C24:1 ceramides directly increased osteoclastogenesis, bone resorption, and expression levels of osteoclast differentiation markers. These results suggested that the association of increased ceramides, especially C18:0 and C24:1, with adverse bone phenotypes in elderly people could be explained mainly by the increase in osteoclastogenesis and bone resorption.

SLIT2 inhibits osteoclastogenesis and bone resorption by suppression of Cdc42 activity.

Axon guidance molecules, originally found to mediate the positioning of axons during nerve development, have been receiving great attention due to their critical roles in regulating bone metabolism. Recently, SLITs, another group of neuronal guidance proteins, were found to be significantly expressed in bone cells. Furthermore, we had provided experimental evidence that SLIT3 is an osteoclast-secreted coupling factor playing an osteoprotective role. Therefore, we hypothesized that SLIT2, a member of the SLIT family, may also affect bone homeostasis. SLIT2 suppressed osteoclast differentiation in a dose-dependent manner and in vitro bone resorption by more than 80%. Consistently, the expression of osteoclast differentiation markers, such as tartrate-resistant acid phosphatase (Trap) and calcitonin receptor (Ctr), was decreased by SLIT2. The migration and fusion of preosteoclasts were markedly reduced in the presence of SLIT2, suggesting that SLIT2 mainly functions in the early stage of osteoclastogenesis. SLIT2 suppressed Cdc42 activity among small GTPases, whereas Cdc42 overexpression almost completely reversed the SLIT2-mediated suppression of osteoclast differentiation. Among ROBO1-4, the SLIT receptors, ROBO1 and ROBO3 were known to be predominantly expressed in osteoclast lineages. A binding ELISA experiment in mouse bone marrow-derived macrophages showed that ROBO1, rather than ROBO3, was directly associated with SLIT2, and gene silencing with Robo1 siRNA blocked the SLIT2-mediated suppression of osteoclastogenesis. Taken together, our results indicated that SLIT2 inhibits osteoclastogenesis and the resultant bone resorption by decreasing Cdc42 activity, suggesting that this was a potential therapeutic target in metabolic bone diseases related to high bone turnover states.

Sirt6 cooperates with Blimp1 to positively regulate osteoclast differentiation.

Global deletion of the gene encoding a nuclear histone deacetylase sirtuin 6 (Sirt6) in mice leads to osteopenia with a low bone turnover due to impaired bone formation. But whether Sirt6 regulates osteoclast differentiation is less clear. Here we show that Sirt6 functions as a transcriptional regulator to directly repress anti-osteoclastogenic gene expression. Targeted ablation of Sirt6 in hematopoietic cells including osteoclast precursors resulted in increased bone volume caused by a decreased number of osteoclasts. Overexpression of Sirt6 led to an increase in osteoclast formation, and Sirt6-deficient osteoclast precursor cells did not undergo osteoclast differentiation efficiently. Moreover, we showed that Sirt6, induced by RANKL-dependent NFATc1 expression, forms a complex with B lymphocyte-induced maturation protein-1 (Blimp1) to negatively regulate expression of anti-osteoclastogenic gene such as Mafb. These findings identify Sirt6 as a novel regulator of osteoclastogenesis by acting as a transcriptional repressor.

Sirtuin 3 (SIRT3) maintains bone homeostasis by regulating AMPK-PGC-1ß axis in mice.

The mitochondrial sirtuin 3 (SIRT3) is involved in suppressing the onset of multiple pathologies, including cardiovascular disease, fatty liver, age-related hearing loss, and breast cancer. But a physiological role of SIRT3 in bone metabolism is not known. Here we show that SIRT3 is a key regulatory molecule to maintain bone homeostasis. Mice deficient in SIRT3 exhibited severe osteopenia owing to increased numbers of osteoclasts. Osteoclast precursors from Sirt3-/- mice underwent increased osteoclastogenesis in response to receptor activator of nuclear factor-κB ligand (RANKL), an essential cytokine for osteoclast differentiation. SIRT3 expression from RANKL induction depended on the transcription coactivator PGC-1ß (peroxisome proliferator-activated receptor-γ co-activator-1ß) and the nuclear receptor ERRα (estrogen receptor-related receptor α), and that SIRT3 inhibited the differentiation by interfering with the RANKL-induced expression of PGC-1ß. Thus an auto-regulatory feedback mechanism operates to induce its own inhibitor SIRT3 by PGC-1ß. Moreover, Sirt3-/- osteoclast precursors reduced AMP-activated protein kinase (AMPK) phosphorylation through down-regulating the expression of AMPK. Our results suggest that a mitochondrial SIRT3 is an intrinsic inhibitor for RANKL-mediated osteoclastogenesis.

Improving Flavonoid Bioaccessibility using an Edible Oil-Based Lipid Nanoparticle for Oral Delivery.

To enhance the oral bioaccessibility of flavonoids, including quercetin, naringenin, and hesperetin, we prepared an edible oil-based lipid nanoparticle (LNP) system. Flavonoid-loaded LNPs were similar to the blank LNP in physicochemical characteristics (z average <154.8 nm, polydispersity index <0.17, and ζ potential < -40.8 mV), and their entrapment efficiency was >81% at 0.3 wt % flavonoid concentration of the lipid phase. In the simulated digestion assay (mouth, stomach, and small intestine), LNPs were hydrolyzed under small intestine conditions and protected successfully incorporated flavonoids (≥94%). Moreover, the relative bioaccessibility of flavonoids was >71%, which was otherwise <15%, although flavonoids were released rapidly from LNPs into the medium. In conclusion, since the flavonoids incorporated in LNPs were preserved well during oral digestion and had improved bioaccessibility, the designed LNP system may serve as an encapsulation strategy to enhance the bioavailability of nonbioaccessible nutraceuticals in foods.

2-(trimethylammonium) ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate suppresses osteoclast maturation and bone resorption by targeting macrophage-colony stimulating factor signaling.

2-(Trimethylammonium) ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate [(R)-TEMOSPho], a derivative of an organic chemical identified from a natural product library, promotes highly efficient megakaryopoiesis. Here, we show that (R)-TEMOSPho blocks osteoclast maturation from progenitor cells of hematopoietic origin, as well as blocking the resorptive function of mature osteoclasts. The inhibitory effect of (R)-TEMOSPho on osteoclasts was due to a disruption of the actin cytoskeleton, resulting from impaired downstream signaling of c-Fms, a receptor for macrophage-colony stimulating factor linked to c-Cbl, phosphoinositol-3-kinase (PI3K), Vav3, and Rac1. In addition, (R)-TEMOSPho blocked inflammation-induced bone destruction by reducing the numbers of osteoclasts produced in mice. Thus, (R)-TEMOSPho may represent a promising new class of antiresorptive drugs for the treatment of bone loss associated with increased osteoclast maturation and activity.

SIRT2 directs the replication stress response through CDK9 deacetylation.

Sirtuin 2 (SIRT2) is a sirtuin family deacetylase that directs acetylome signaling, protects genome integrity, and is a murine tumor suppressor. We show that SIRT2 directs replication stress responses by regulating the activity of cyclin-dependent kinase 9 (CDK9), a protein required for recovery from replication arrest. SIRT2 deficiency results in replication stress sensitivity, impairment in recovery from replication arrest, spontaneous accumulation of replication protein A to foci and chromatin, and a G2/M checkpoint deficit. SIRT2 interacts with and deacetylates CDK9 at lysine 48 in response to replication stress in a manner that is partially dependent on ataxia telangiectasia and Rad3 related (ATR) but not cyclin T or K, thereby stimulating CDK9 kinase activity and promoting recovery from replication arrest. Moreover, wild-type, but not acetylated CDK9, alleviates the replication stress response impairment of SIRT2 deficiency. Collectively, our results define a function for SIRT2 in regulating checkpoint pathways that respond to replication stress through deacetylation of CDK9, providing insight into how SIRT2 maintains genome integrity and a unique mechanism by which SIRT2 may function, at least in part, as a tumor suppressor protein.

Development of a Korean version of the Cancer Therapy Satisfaction Questionnaire (CTSQ): cross-cultural adaptation, reliability, and validity.

PURPOSE: (1) To translate original English Cancer Therapy Satisfaction Questionnaire (CTSQ) into Korean and perform validation, (2) to compare CTSQ domains of expectations of therapy (ET), feelings about side effects (FSE), and satisfaction with therapy (SWT) by cancer therapy type. METHODS: Cross-cultural adaptation was performed according to guidelines: translation, back translation, focus-group, and field test. We performed validation with internal consistency by Cronbach's alpha and construct validity by exploratory factor analysis (EFA) with varimax rotation method. We compared each CTSQ domain between traditional Korean Medicine (TKM) and integrative cancer therapy (ICT) of combining western and TKM by two-sample t test. RESULTS: Cross-cultural adaptation produced no major modifications in the items and domains. A total of 102 outpatients were participated. Mean age was 51.9 ± 12.4. Most were stage 4 (74.4 %) cancer. Mean scores of ET, FSE, and SWT were 81.2 ± 15.7, 79.5 ± 22.9, and 75.7 ± 14.8, respectively. Cronbach's alpha of ET, FSE, and SWT were 0.86, 0.78, and 0.74, respectively. EFA loaded items on the three domains, which is very close to that of the original CTSQ. ET and SWT was similar, but FSE was significantly higher in TKM than ICT (87.5 ± 19.3 vs. 74.9 ± 23.5; p = 0.0054). CONCLUSIONS: Cross-cultural adaptation was successful, and the adapted Korean CTSQ demonstrated good internal consistency and construct validity. Similar expectation and satisfaction was shown between the two types of therapy, but patient's reported feelings about side effects was significantly lower in patients receiving TKM than receiving ICT. Korean version of CTSQ can be used to evaluate Korean cancer patient's experiences receiving various cancer therapy types.