Anmin Chen

miR‑142‑3p promotes osteoblast differentiation by modulating Wnt signaling

Canonical Wnt signaling is critical for the control of osteoblast differentiation in human mesenchymal stem cells. MicroRNAs (miRs) are essential regulators of cell differentiation by post‑transcriptional regulation of target gene expression. The aim of the present study was to investigate the molecular mechanism by which miR‑142‑3p promotes osteoblastic differentiation using the human fetal osteoblastic 1.19 (hFOB1.19), real-time PCR and western blot analysis. Results showed an increased expression of miR‑142‑3p during osteoblast differentiation in the mesenchymal precursor cell line, hFOB1.19. In addition, the ectopic over-expression of miR‑142‑3p promoted hFOB1.19 differentiation, whereas the inhibition of miR‑142‑3p repressed differentiation. The expression of miR‑142‑3p was positively correlated with β‑catenin, an important protein in Wnt signaling. The adenomatous polyposis coli (APC) gene was a direct target of miR‑142‑3p, whereby miR‑142‑3p promoted Wnt signaling through inhibition of APC, leading to accumulation and nuclear translocation of β‑catenin. Therefore, miR‑142‑3p may be an essential mediator of osteoblast differentiation and a new therapeutic strategy for osteogenesis disorders.

A short-hairpin RNA targeting osteopontin downregulates MMP-2 and MMP-9 expressions in prostate cancer PC-3 cells.

Osteopontin (OPN), a secreted phosphoglycoprotein, is frequently associated with cell proliferation and tumor metastatic spread in a variety of cancers. It has been reported that OPN induce matrix metalloproteinase (MMP)-2 and MMP-9 activations through nuclear factor kappaB (NF-kappaB)-mediated signaling pathways. In this study, we investigated the roles of OPN in human prostate cancer cells and provided clues about the possible functions of IkappaB kinase (IKK) in NF-kappaB-mediated OPN-induced activations of MMP-2 and MMP-9. Short-hairpin RNA (shRNA) expression vectors were used to inhibit OPN expression in PC-3 cells, human prostate cancer cell line, and IKK inhibitor VII were applied to inhibit the activities of IKK-1 and IKK-2. The results showed that OPN shRNA-mediated RNA interference can downregulate OPN, MMP-2 and MMP-9 expressions, thereby resulting in suppression of the proliferation, migration and invasion of PC-3 cells in vitro and tumor growth in vivo. Moreover, the inhibition of IKK-2 can suppress MMP-2 and MMP-9 expressions, in contrast, the inhibition of IKK-1 has no effects on the OPN, MMP-2 and MMP-9 expression levels. Thus, this study demonstrated that OPN knockdown could downregulate MMP-2 and MMP-9 expressions result in inhibiting the malignant physiological behaviors of PC-3 cell and that IKK-2 may play a crucial role in OPN-induced MMP-2 and MMP-9 expressions via NF-kappaB-mediated signaling pathways.

The Fascinating Effects of Baicalein on Cancer: A Review

Cancer is one of the leading causes of death worldwide and a major global health problem. In recent decades, the rates of both mortality and morbidity of cancer have rapidly increased for a variety of reasons. Despite treatment options, there are serious side effects associated with chemotherapy drugs and multiple forms of drug resistance that significantly reduce their effects. There is an accumulating amount of evidence on the pharmacological activities of baicalein (e.g., anti-inflammatory, antioxidant, antiviral, and antitumor effects). Furthermore, there has been great progress in elucidating the target mechanisms and signaling pathways of baicalein’s anti-cancer potential. The anti-tumor functions of baicalein are mainly due to its capacities to inhibit complexes of cyclins to regulate the cell cycle, to scavenge oxidative radicals, to attenuate mitogen activated protein kinase (MAPK), protein kinase B (Akt) or mammalian target of rapamycin (mTOR) activities, to induce apoptosis by activating caspase-9/-3 and to inhibit tumorinvasion and metastasis by reducing the expression of matrix metalloproteinase-2/-9 (MMP-2/-9). In this review, we focused on the relevant biological mechanisms of baicalein involved in inhibiting various cancers, such as bladder cancer, breast cancer, and ovarian cancer. Moreover, we also summarized the specific mechanisms by which baicalein inhibited the growth of various tumors in vivo. Taken together, baicalein may be developed as a potential, novel anticancer drug to treat tumors.

Epoxyeicosanoids suppress osteoclastogenesis and prevent ovariectomy-induced bone loss

Epoxyeicosatrienoic acids (EETs) are products of arachidonic acid metabolism catalyzed by cytochrome P450 epoxygenases. These small molecules are autocrine and paracrine lipid mediators with important roles in inflammation, cardiovascular function, and angiogenesis. Recent evidence has highlighted EETs as potent promoters of organ regeneration and malignant metastasis. We speculated that EETs might impact osteoclastogenesis and bone loss. Using both in vitro and in vivo studies, we observed that EETs significantly attenuated bone loss and inhibited osteoclast formation and activity, which were associated with a decreased receptor activator of NF‐κB ligand (RANKL): osteoprotegerin ratio and serum levels of TNF‐α and IL‐1β. At the molecular level, EETs abrogated RANKL‐induced activation of NF‐κB, activator protein‐1 (AP‐1), and MAPKs, including ERK and JNK, but not p38, during osteoclast formation. EETs also prevented the production of reactive oxygen species (ROS) following RANKL stimulation. As a result, EETs suppressed osteoclast‐specific gene expression, including tartrate resistant acid phosphatase (TRAP), cathepsin K (CK), matrix metalloproteinase (MMP)‐9, and receptor activator of NF‐κB (RANK). In conclusion, our findings demonstrate that EETs inhibit osteoclastogenesis through modulation of multiple pathways both upstream and downstream of RANKL signaling. The administration or stabilized endogenous levels of EETs could represent a novel therapeutic strategy for osteoclast‐related disorders, such as rheumatoid arthritis and postmenopausal osteoporosis.—Guan, H., Zhao. L., Cao, H., Chen, A., Xiao, J., Epoxyeicosanoids suppress osteoclastogenesis and prevent ovariectomy‐induced bone loss. FASEB J. 29, 1092–1101 (2015). www.fasebj.org

Inhibitory effects of high glucose/insulin environment on osteoclast formation and resorption in vitro

Patients with type 2 diabetes mellitus (T2DM) exhibit hyperglycemia and hyperinsulinemia and increased risk of fracture at early stage, but they were found to have normal or even enhanced bone mineral density (BMD). This study was aimed to examine the molecular mechanisms governing changes in bone structure and integrity under both hyperglycemic and hyperinsulinemic conditions. Monocytes were isolated from the bone marrow of the C57BL/6 mice, induced to differentiate into osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) and exposed to high glucose (33.6 mmol/L), high insulin (1 μmol/L), or a combination of high glucose/high insulin (33.6 mmol/L glucose and 1 μmol/L insulin). Cells cultured in α-MEM alone served as control. After four days of incubation, the cells were harvested and stained for tartrate resistant acid phosphatase (TRAP). Osteoclast-related genes including RANK, cathepsin K and TRAP were determined by using real-time PCR. The resorptive activity of osteoclasts was measured by using a pit formation assay. Osteoclasts that were derived from monocytes were of multinucleated nature and positive for TRAP, a characteristic marker of osteoclasts. Cell counting showed that the number of osteoclasts was much less in high glucose and high glucose/high insulin groups than in normal glucose and high insulin groups. The expression levels of RANK and cathepsin K were significantly decreased in high glucose, high insulin and high glucose/high insulin groups as compared with normal glucose group, and the TRAP activity was substantially inhibited in high glucose environment. The pit formation assay revealed that the resorptive activity of osteoclasts was obviously decreased in high glucose group and high glucose/high insulin group as compared with normal group. It was concluded that osteoclastogenesis is suppressed under hyperglycemic and hyperinsulinemic conditions, suggesting a disruption of the bone metabolism in diabetic patients.SummaryPatients with type 2 diabetes mellitus (T2DM) exhibit hyperglycemia and hyperinsulinemia and increased risk of fracture at early stage, but they were found to have normal or even enhanced bone mineral density (BMD). This study was aimed to examine the molecular mechanisms governing changes in bone structure and integrity under both hyperglycemic and hyperinsulinemic conditions. Monocytes were isolated from the bone marrow of the C57BL/6 mice, induced to differentiate into osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) and exposed to high glucose (33.6 mmol/L), high insulin (1 μmol/L), or a combination of high glucose/high insulin (33.6 mmol/L glucose and 1 μmol/L insulin). Cells cultured in α-MEM alone served as control. After four days of incubation, the cells were harvested and stained for tartrate resistant acid phosphatase (TRAP). Osteoclast-related genes including RANK, cathepsin K and TRAP were determined by using real-time PCR. The resorptive activity of osteoclasts was measured by using a pit formation assay. Osteoclasts that were derived from monocytes were of multinucleated nature and positive for TRAP, a characteristic marker of osteoclasts. Cell counting showed that the number of osteoclasts was much less in high glucose and high glucose/high insulin groups than in normal glucose and high insulin groups. The expression levels of RANK and cathepsin K were significantly decreased in high glucose, high insulin and high glucose/high insulin groups as compared with normal glucose group, and the TRAP activity was substantially inhibited in high glucose environment. The pit formation assay revealed that the resorptive activity of osteoclasts was obviously decreased in high glucose group and high glucose/high insulin group as compared with normal group. It was concluded that osteoclastogenesis is suppressed under hyperglycemic and hyperinsulinemic conditions, suggesting a disruption of the bone metabolism in diabetic patients.

Proliferation and chondrogenic differentiation of CD105-positive enriched rat synovium-derived mesenchymal stem cells in three-dimensional porous scaffolds

Stem cell-based tissue engineering has provided an alternative strategy to treat cartilage lesions, and synovium-derived mesenchymal stem cells (SMSCs) are considered as a promising cell source for cartilage repair. In this study, the SMSCs were isolated from rat synovium, and CD105-positive (CD105(+)) cells were enriched using magnetic activated cell sorting. Sorted cells were subsequently seeded onto the chitosan-alginate composite three-dimensional (3D) porous scaffolds and cultured in chondrogenic culture medium in the presence of TGF-β₃ and BMP-2 for 2 weeks in vitro. After 2 weeks in culture, scanning electron microscopy results showed that cells attached and proliferated well on scaffolds, and secreted extracellular matrix were also observed. From day 7 to day 14, the total DNA and glucosaminoglycan content of the cells cultured in scaffolds were found to have increased significantly, and cell cycle analyses revealed that the percentage of cells in the S and G2/M phases increased and the percentage of cells in the G0/G1 phase decreased. Compared with non-sorted cells, the sorted cells cultured in scaffolds underwent more chondrogenic differentiation, as evidenced by higher expression of type II collagen and Sox9 at the protein and mRNA levels. The results suggest that CD105(+) enriched SMSCs may be a potential cell source for cartilage tissue engineering, and the chitosan-alginate composite 3D porous scaffold could provide a favorable microenvironment for supporting proliferation and chondrogenic differentiation of cells.

Osteogenic effect of low intensity pulsed ultrasound on rat adipose-derived stem cells in vitro

The osteogenic in vitro effect of low intensity pulsed ultrasound (LIPUS) on SD rat adipose-derived stem cells (ADSCs) was investigated. Rat ADSCs underwent LIPUS (intensity=100 mW/cm2) or sham exposure for 8 min per treatment once everyday in vitro, and then the alkaline phosphatase (ALP) activity and mineralized nodule formation were assessed to evaluate the osteogenic effect of LIPUS on ADSCs. To further explore the underlying mechanism, the osteogenic-related gene mRNA expression was determined by using reverse transcriptase-polymerase chain reaction (RT-PCR) at 1st, 3rd, 5th, 7th day after exposure repectively. Westen blot was used to evaluate the protein expression levels of two osteogenic differentiation associated genes at 7th and 14th day repectively. It was found that ALP activity was increased after LIPUS exposure and LIPUS resulted in mineralized nodule formation of ADSCs in vitro. LIPUS-treated ADSCs displayed higher mRNA expression levels of runt-related transcription factor 2 (Runx2), osteocalcin (OCN), ALP and bone sialoprotein (BSP) genes than controls, and the protein levels of Runx2 and BSP were also increased. The results suggested that LIPUS may induce the osteogenic differentiation of ADSCs in vitro.SummaryThe osteogenic in vitro effect of low intensity pulsed ultrasound (LIPUS) on SD rat adipose-derived stem cells (ADSCs) was investigated. Rat ADSCs underwent LIPUS (intensity=100 mW/cm2) or sham exposure for 8 min per treatment once everyday in vitro, and then the alkaline phosphatase (ALP) activity and mineralized nodule formation were assessed to evaluate the osteogenic effect of LIPUS on ADSCs. To further explore the underlying mechanism, the osteogenic-related gene mRNA expression was determined by using reverse transcriptase-polymerase chain reaction (RT-PCR) at 1st, 3rd, 5th, 7th day after exposure repectively. Westen blot was used to evaluate the protein expression levels of two osteogenic differentiation associated genes at 7th and 14th day repectively. It was found that ALP activity was increased after LIPUS exposure and LIPUS resulted in mineralized nodule formation of ADSCs in vitro. LIPUS-treated ADSCs displayed higher mRNA expression levels of runt-related transcription factor 2 (Runx2), osteocalcin (OCN), ALP and bone sialoprotein (BSP) genes than controls, and the protein levels of Runx2 and BSP were also increased. The results suggested that LIPUS may induce the osteogenic differentiation of ADSCs in vitro.

Inhibition of SDF-1α/CXCR4 Signalling in Subchondral Bone Attenuates Post-Traumatic Osteoarthritis

Previous studies showed that SDF-1α is a catabolic factor that can infiltrate cartilage, decrease proteoglycan content, and increase MMP-13 activity. Inhibiting the SDF-1α/CXCR4 signalling pathway can attenuate the pathogenesis of osteoarthritis (OA). Recent studies have also shown that SDF-1α enhances chondrocyte proliferation and maturation. These results appear to be contradictory. In the current study, we used a destabilisation OA animal model to investigate the effects of SDF-1α/CXCR4 signalling in the tibial subchondral bone and the OA pathological process. Post-traumatic osteoarthritis (PTOA) mice models were prepared by transecting the anterior cruciate ligament (ACLT), or a sham surgery was performed, in a total of 30 mice. Mice were treated with phosphate buffer saline (PBS) or AMD3100 (an inhibitor of CXCR4) and sacrificed at 30 days post ACLT or sham surgery. Tibial subchondral bone status was quantified by micro-computed tomography (μCT). Knee-joint histology was analysed to examine the articular cartilage and joint degeneration. The levels of SDF-1α and collagen type I c-telopeptidefragments (CTX-I) were quantified by ELISA. Bone marrow mononuclear cells (BMMCs) were used to clarify the effects of SDF-1α on osteoclast formation and activity in vivo. μCT analysis revealed significant loss of trabecular bone from tibial subchondral bone post-ACLT, which was effectively prevented by AMD3100. AMD3100 could partially prevent bone loss and articular cartilage degeneration. Serum biomarkers revealed an increase in SDF-1α and bone resorption, which were also reduced by AMD3100. SDF-1α can promote osteoclast formation and the expression oftartrate resistant acid phosphatase (TRAP), cathepsin K (CK), and matrix metalloproteinase (MMP)-9 in osteoclasts by activating the MAPK pathway, including ERK and p38, but not JNK. In conclusion, inhibition of SDF-1α/CXCR4signalling was able to prevent trabecular bone loss and attenuated cartilage degeneration in PTOA mice.

Dose-dependent inhibitory effects of zoledronic acid on osteoblast viability and function in vitro

Zoledronic acid (ZA), which is one of the most potent and efficacious bisphosphonates, has been commonly used in clinical practice for the treatment of various bone disorders. The extensive use of ZA has been associated with increasing occurrence of jaw complications, now known as bisphosphonate-associated osteonecrosis of the jaw (BRONJ). However, the mechanism underlying BRONJ remains to be fully elucidated. The aim of the present study was to investigate the effects of different concentrations of ZA on the MC3T3-E1 murine preosteoblast cell line cells and examine the possible pathogenesis of BRONJ. In the present study, the effect of ZA on the viability, apoptosis, differentiation and maturation of MC3T3-E1 cells, as well as its relevant molecular mechanism, were examined The results of a Cell Counting Kit 8 assay, a flow cytometric Annexin-V/propidium iodide assay and western blot analysis demonstrated that ZA exhibited a significant inhibition of cell viability and induction of apoptosis at concentrations >10 µM. Subsequently, the effect of ZA on cell differentiation at concentrations <1 µM were investigated. In this condition, ZA inhibited bone nodule formation and decreased the activity of alkaline phosphatase. The results of reverse transcription-quantitative polymerase chain reaction and western blot analyses indicated that ZA downregulated the expression levels of the marker genes and proteins associated with osteogenic differentiation. Further investigation revealed that the suppression of differentiation by ZA was associated with decreased expression of bone morphogenetic protein-2 (BMP-2) and downregulation of the phosphorylation levels in the downstream extracellular signal-regulated kinase 1/2 and p38 pathways. These adverse effects of ZA were observed to be concentration-dependent. The results from the present study suggested that ZA at higher concentrations induces cytotoxicity towards osteoblasts, and ZA at lower concentrations suppresses osteoblast differentiation by downregulation of BMP-2. These results assist in further understanding the mechanisms of BRONJ.

Adipocytes regulate the bone marrow microenvironment in a mouse model of obesity

Obesity is markedly associated with abnormal bone density indicating the importance of adipocytes in bone metabolism. However, the specific function of adipocytes remains unclear, with marked discrepancies in observations of previous studies. In the present study, the effect of adipocytes on osteoblasts/osteoclasts was analyzed. A mouse model of obesity was established and an in vitro co-culture system was utilized containing adipocyte and MC3T3/RAW 264.7 cells in a Transwell plate. Compared with control mice, obese mice exhibited low body weight and bone mineral density of the tibia and fat cells were observed to accumulate in bone marrow. MC3T3/RAW 264.7 cells were co-cultured with adipocytes and the mRNA and protein expression of alkaline phosphatase and osteocalcin was found to be decreased in MC3T3-E1 cells and mRNA and protein expression of tartrate-resistant acid phosphatase and cathepsin K was significantly increased in RAW 264.7 cells. In addition, the effect of adipocytes on the osteoprotegerin (OPG)/receptor activator of nuclear factor κB ligand (RANKL)/RANK system indicated that the RANKL/OPG ratio secreted by osteoblasts increased and RANK expression by osteoclasts increased, leading to increased osteoclastogenesis. These results indicate that bone metabolism is impaired in obese mice leading to decreased osteoblastogenesis and marked increases in osteoclastogenesis and low bone mass.