Er-Yuan Liao

Adiponectin Stimulates RANKL and Inhibits OPG Expression in Human Osteoblasts Through the MAPK Signaling Pathway

Our study indicates that recombinant adiponectin induced RANKL and inhibited OPG expression in human osteoblasts through the AdipoR1/p38 MAPK pathway, and these responses contributed to the adiponectin‐induced osteoclasts formation in the co‐culture of osteoblast and peripheral blood monocytes systems. These findings showed that adiponectin increased osteoclast formation indirectly through stimulating RANKL and inhibiting OPG production in osteoblasts. It also suggests the pharmacological nature of recombinant adiponectin that indirectly induces osteoclasts formation.

Insulin-Like Effects of Visfatin on Human Osteoblasts

Visfatin (also known as pre-B cell colony-enhancing factor or PBEF) is a novel adipocytokine that is highly expressed in visceral fat and upregulated in obesity and type 2 diabetes mellitus. Visfatin binds to and activates the insulin receptor (IR), thereby exerting insulin-mimetic effects in various cell lines. IR has been detected in osteoblasts, which is consistent with the role of insulin as an important osteotropic hormone. This study investigated the actions of visfatin on human primary osteoblasts. The expression and tyrosine phosphorylation of IR, IR substrate-1 (IRS-1), and IRS-2 were determined by immunoprecipitation and immunoblotting. Cell proliferation was determined by measuring [3H]thymidine incorporation and cell number. Glucose uptake was determined by measuring 2-[3H]deoxyglucose incorporation. Real-time quantitative reverse-transcription polymerase chain reaction (PCR) was used for determining alkaline phosphatase (ALP), osteocalcin, and type I collagen mRNA expression. Enzyme-linked immunosorbent assay and radioimmunoassay were used for measuring ALP activity, osteocalcin secretion, and type I collagen production. We found that visfatin induced tyrosine phosphorylation of IR, IRS-1, and IRS-2. Moreover, the effects of visfatin – glucose uptake, proliferation, and type I collagen enhancement of cultured human osteoblast-like cells – bore a close resemblance to those of insulin and were inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester, a specific inhibitor of IR tyrosine kinase activity. We also unexpectedly found that visfatin downregulated osteocalcin secretion from human osteoblast-like cells. These data indicate that the regulation of glucose uptake, proliferation, and type I collagen production by visfatin in human osteoblasts involves IR phosphorylation, the same signal-transduction pathway used by insulin.

PDGF-BB secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis

Osteogenesis during bone modeling and remodeling is coupled with angiogenesis. A recent study showed that a specific vessel subtype, strongly positive for CD31 and endomucin (CD31hiEmcnhi), couples angiogenesis and osteogenesis. Here, we found that platelet-derived growth factor-BB (PDGF-BB) secreted by preosteoclasts induces CD31hiEmcnhi vessel formation during bone modeling and remodeling. Mice with depletion of PDGF-BB in the tartrate-resistant acid phosphatase–positive cell lineage show significantly lower trabecular and cortical bone mass, serum and bone marrow PDGF-BB concentrations, and fewer CD31hiEmcnhi vessels compared to wild-type mice. In the ovariectomy (OVX)-induced osteoporotic mouse model, serum and bone marrow levels of PDGF-BB and numbers of CD31hiEmcnhi vessels are significantly lower compared to sham-operated controls. Treatment with exogenous PDGF-BB or inhibition of cathepsin K to increase the number of preosteoclasts, and thus the endogenous levels of PDGF-BB, increases CD31hiEmcnhi vessel number and stimulates bone formation in OVX mice. Thus, pharmacotherapies that increase PDGF-BB secretion from preosteoclasts offer a new therapeutic target for treating osteoporosis by promoting angiogenesis and thus bone formation.

A Comparison Study of the Reference Curves of Bone Mineral Density at Different Skeletal Sites in Native Chinese, Japanese, and American Caucasian Women

To understand the differences among reference curves for bone mineral density (BMD) for Chinese, Japanese, and American Caucasian women, we measured the BMD at the anteroposterior (AP) lumbar spine (L1–L4), lateral lumbar spine (L2–L4), hip (including the femoral neck, trochanter, intertrochanter, Ward’s triangle, and total hip), and ultradistal forearm by the dual-energy X-ray absorptiometry (DXA) in a total of 2728 healthy Chinese women, aged 5–96 years. Documented BMD data for Japanese women and device manufacturer’s BMD new reference databases (including the NHANES III dataset) for American Caucasian women were also used in this study. The cubic regression model was found to fit best in analyzing the age-associated variations of BMD at various sites in Chinese women, i.e., the equations had the largest coefficient of determination (R2). At the AP/Lat spine, trochanter, intertrochanter, and Ward’s triangle, BMD reference curves for Chinese women were lower than those for Japanese or Caucasian women, while at the femoral neck, total hip, and ultradistal forearm, the reference curves for Chinese women were higher than those for Japanese women, with overlaps and crossing of the curves for some age spans in comparing the Chinese and Caucasian women. There were significant differences in the peak BMD (PBMD) at various sites among the Chinese, Japanese, and Caucasian women (P = 0.000). The PBMDs for Chinese women at the lumbar spine and various sites of the hip were 5.7% ± 2.1% (mean ± SD, range, 2.7–7.9%) lower than those for Japanese women and 5.1% ± 2.7% (range, 0.5–7.2%) lower than those for Caucasian women; however, the PBMDs for Chinese women were 26.2% higher than those for Japanese women and 10% higher than those for Caucasian women at the ultradistal forearm. After the PBMD, average T-scores of Chinese women for losses at the AP lumbar spine with increasing age were nearly identical to those for Japanese women, but both were greater than those for Caucasian women. The average T-scores for BMD loss at various sites in Chinese women were higher than those for both Japanese and Caucasian women except at the femoral neck, where the T-scores of Chinese women were exceeded by those of both Japanese and Caucasian women. Estimated from the T-score curve of BMD loss, the age of osteoporosis occurrence at the femoral neck in Chinese women was about 10 years later than that in Japanese or Caucasian women; at the AP spine, Chinese women were similar to Japanese women; at the other sites, the age for occurrence of osteoporosis in Chinese women was about 5–15 years earlier than that in either Japanese or Caucasian women. There are differences in prevalence or odds ratio (OR) of osteoporosis at the same skeletal region for Chinese, Japanese, and Caucasian women aged ≥50 years or at different skeletal regions in women of the same race. The prevalences of osteoporosis at various regions of the hip in Chinese women are 10.1–19.8% and ORs are 22.0–32.3, of which prevalence at the femoral neck is the lowest (10.1%); the prevalences of osteoporosis in Japanese women are 11.6–16.8% and ORs are 21.1–26.3, of which prevalence at the femoral neck is the lowest (11.6%); and the prevalences of osteoporosis in Caucasian women are 13.0–20.0% and ORs are 19.4–48.9, of which prevalence at the femoral neck is the highest (20%). In conclusion, racial differences in BMD reference curves, prevalences, and risks of osteoporosis at various skeletal sites exist among native Chinese, Japanese, and American Caucasian women.

Application of micro-ct assessment of 3-d bone microstructure in preclinical and clinical studies

As the mechanical competence of trabecular bone is a function of its apparent density and 3-D distribution, assessment of 3-D trabecular structural characteristics may improve our ability to understand the pathophysiology of osteoporosis, to test the efficacy of pharmaceutical intervention, and to estimate bone biomechanical properties. We have studied ovariectomy-induced osteopenia in rats and its treatment with agents such as estrogen and sodium fluoride. We have demonstrated that 3-D micro-computed tomography (µCT) can directly quantify mouse trabecular and cortical bone structure with an isotropic resolution of 6 µm3. µCT is also useful for studying osteoporosis in mice and phenotypes of mice with gene manipulation, such as SHIP-knockout mice, which are severely osteoporotic due to increased numbers of hyperresorptive osteoclasts, PTHrP heterozygous-null mice, and mice with Zmpste24 deficiency. µCT can quantify osteogenesis in mouse Ilizarov leg-lengthening procedures, osteoconduction in a rat cranial defect model, and structural changes in arthritic rabbits, rats, and mice. In clinical studies, we evaluated longitudinal changes in the iliac crests. Paired bone biopsies from the same premenopausal and postmenopausal women showed the changes in 3-D trabecular structure, such as decreased trabecular thickness, shifting of trabecular model from platelike structure to rodlike structure, and decreased degree of anisotropy were remarkable. Treatment with PTH in postmenopausal women with osteoporosis significantly improved trabecular morphology with a shift toward a more platelike structure, increased trabecular connectivity density, and increased cortical thickness. Paired bone biopsy specimens from the iliac crest in postmenopausal women with osteoporosis before and an average of 2 years after beginning of estrogen replacement therapy demonstrated that posttreatment biopsies showed a significant change in the ratio of plates to rods and statistically insignificant changes in other 3-D trabecular parameters. Thus, µCT can characterize 3-D structure of various animal models, and the longitudinal changes in 3-D bone microarchitectural integrity that deteriorates in the transmenopausal period, is preserved with HRT, and is improved with PTH treatment in postmenopausal women.

miR-148a regulates osteoclastogenesis by targeting V-maf musculoaponeurotic fibrosarcoma oncogene homolog B.

MicroRNAs (miRNAs) play crucial roles in bone metabolism. In the present study, we found that miR‐148a is dramatically upregulated during osteoclastic differentiation of circulating CD14+ peripheral blood mononuclear cells (PBMCs) induced by macrophage colony stimulating factor (M‐CSF) and receptor activator of nuclear factor‐κB ligand (RANKL). Overexpression of miR‐148a in CD14+ PBMCs promoted osteoclastogenesis, whereas inhibition of miR‐148a attenuated osteoclastogenesis. V‐maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) is a transcription factor negatively regulating RANKL‐induced osteoclastogenesis. miR‐148a directly targeted MAFB mRNA by binding to the 3′ untranslated region (3′UTR) and repressed MAFB protein expression. In vivo, our study showed that silencing of miR‐148a using a specific antagomir‐inhibited bone resorption and increased bone mass in mice receiving ovariectomy (OVX) and in sham‐operated control mice. Furthermore, our results showed that miR‐148a levels significantly increased in CD14+ PBMCs from lupus patients and resulted in enhanced osteoclastogenesis, which contributed to the lower bone mineral density (BMD) in lupus patients compared with normal controls. Thus, our study provides a new insight into the roles of miRNAs in osteoclastogenesis, and contributes to a new therapeutic pathway for osteoporosis.

Apelin stimulates proliferation and suppresses apoptosis of mouse osteoblastic cell line MC3T3-E1 via JNK and PI3-K/Akt signaling pathways.

The aim of this study was to investigate the effects of apelin on proliferation and apoptosis of mouse osteoblastic MC3T3-E1 cells. APJ was expressed in MC3T3-E1 cells. Apelin did not affect Runx2 expression, alkaline phosphatase (ALP) activity, osteocalcin and type I collagen secretion, suggesting that it has no effect on osteoblastic differentiation of MC3T3-E1 cells. However, apelin stimulated MC3T3-E1 cell proliferation and inhibited cell apoptosis induced by serum deprivation. Our study also shows that apelin decreased cytochrome c release and caspase-3, capase-8 and caspase-9 activation in serum-deprived MC3T3-E1 cells. Apelin activated c-Jun N-terminal kinase (JNK) and Akt (phosphatidylinositol 3-kinase downstream effector), and the JNK inhibitor SP600125, the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 or the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO) inhibited its effects on proliferation and serum deprivation-induced apoptosis. Furthermore, apelin protected against apoptosis induced by the glucocorticoid dexamethasone or TNF-alpha. Apelin stimulates proliferation and suppresses serum deprivation-induced apoptosis of MC3T3-E1 cells and these actions are mediated via JNK and PI3-K/Akt signaling pathways.

Apelin suppresses apoptosis of human osteoblasts.

Objectives: Apelin is a recently discovered peptide that is the endogenous ligand for the orphan G-protein-coupled receptor APJ. Adipocytes can express and secrete apelin. Osteoblast can express apelin and APJ. The aim of this study was to investigate the action of apelin on apoptosis of human osteoblasts. Results: Apelin inhibited human osteoblasts apoptosis induced by serum deprivation. Suppression of APJ with small-interfering RNA (siRNA) abolished the anti-apoptotic activity of apelin. Our study also showed an increased Bcl-2 protein expression and decreased Bax protein expression under the treatment of apelin. Apelin decreased cytochrome c release and caspase-3 activation in human osteoblasts. Apelin activated phosphatidylinositol-3 kinase (PI-3 kinase) and Akt. The apelin-induced activation of Akt was blocked by suppression of APJ with siRNA. LY294002 (a PI-3 kinase inhibitor) or 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO; an Akt inhibitor) abolished apelin induced activation of Akt, and, LY294002 or HIMO abolished the anti-apoptotic activity of apelin. Furthermore, apelin protects against apoptosis induced by the glucocorticoid dexamethasone. Conclusions: Apelin suppresses serum deprivation-induced apoptosis of human osteoblasts and the anti-apoptotic action is mediated via the APJ/PI-3 kinase/Akt signaling pathway.

MiR‐503 Regulates Osteoclastogenesis via Targeting RANK

MicroRNAs (miRNAs) play important roles in osteoclastogenesis and bone resorption. However, no study has investigated the role of miRNA in postmenopausal osteoporosis. Here, we report that miR‐503 was markedly reduced in circulating progenitors of osteoclasts–CD14+ peripheral blood mononuclear cells (PBMCs) from postmenopausal osteoporosis patients compared with those from postmenopausal healthy women. Overexpression of miR‐503 in CD14+ PBMCs inhibited receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis. Conversely, silencing of miR‐503 in CD14+ PBMCs promoted osteoclastogenesis. RANK, which is activated by the binding of RANKL and inducing osteoclast differentiation, was confirmed to be a target of miR‐503. In vivo, silencing of miR‐503 using a specific antagomir in ovariectomy (OVX) mice increased RANK protein expression, promoted bone resorption, and decreased bone mass, whereas overexpression of miR‐503 with agomir inhibited bone resorption and prevented bone loss in OVX mice. Thus, our study revealed that miR‐503 plays an important role in the pathogenesis of postmenopausal osteoporosis and contributes to a new therapeutic way for osteoporosis.

Apelin and its receptor are expressed in human osteoblasts.

OBJECTIVES Apelin is a recently discovered peptide that is the endogenous ligand for the orphan G-protein-coupled receptor APJ. Adipocytes can express and secrete apelin. The aim of this study was to characterize apelin and APJ expression in human osteoblasts and to investigate the effects of apelin on osteoblasts. RESULTS Apelin and APJ were expressed in human osteoblasts. Apelin stimulated proliferation of human osteoblasts, but had no effect on alkaline phosphatase (ALP) activity, osteocalcin and type I collagen production in human osteoblasts. Suppression of APJ with small-interfering RNA (siRNA) abolished the apelin-induced cell proliferation. Apelin induced activation of Akt (Phosphatidylinositol-3 kinase downstream effector), but not MAPKs, such as c-jun N-terminal Kinase (JNK), p38 and ERK1/2 in human osteoblasts. This effect was blocked by suppression of APJ with siRNA. Furthermore, LY294002 (PI3 kinase inhibitor) blocked the activation of Akt by apelin and abolished the apelin-induced cell proliferation. CONCLUSIONS Human osteoblasts express apelin and APJ and apelin enhances human osteoblast proliferation, but has no effect on osteoblast differentiation, and APJ/PI3 kinase/Akt pathway is involved in the proliferation response. These findings suggest that apelin may function as a mitogenic agent for osteoblasts.