Showing papers on "Tartrate-resistant acid phosphatase published in 2019"

Pseurotin A Inhibits Osteoclastogenesis and Prevents Ovariectomized-Induced Bone Loss by Suppressing Reactive Oxygen Species.

Abstract: Rationale: Growing evidence indicates that intracellular reactive oxygen species (ROS) accumulation is a critical factor in the development of osteoporosis by triggering osteoclast formation and function. Pseurotin A (Pse) is a secondary metabolite isolated from Aspergillus fumigatus with antioxidant properties, recently shown to exhibit a wide range of potential therapeutic applications. However, its effects on osteoporosis remain unknown. This study aimed to explore whether Pse, by suppressing ROS level, is able to inhibit osteoclastogenesis and prevent the bone loss induced by estrogen-deficiency in ovariectomized (OVX) mice. Methods: The effects of Pse on receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis and bone resorptive function were examined by tartrate resistant acid phosphatase (TRAcP) staining and hydroxyapatite resorption assay. 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) was used to detect intracellular ROS production in vitro. Western blot assay was used to identify proteins associated with ROS generation and scavenging as well as ROS-mediated signaling cascades including mitogen-activated protein kinases (MAPKs), NF-κB pathways, and nuclear factor of activated T cells 1 (NFATc1) signaling. The expression of osteoclast-specific genes was assessed by qPCR. The in vivo potential of Pse was determined using an OVX mouse model administered with Pse or vehicle for 6 weeks. In vivo ROS production was assessed by intravenous injection of dihydroethidium (DHE) into OVX mice 24h prior to killing. After sacrifice, the bone samples were analyzed using micro-CT and histomorphometry to determine bone volume, osteoclast activity, and ROS level ex vivo. Results: Pse was demonstrated to inhibit osteoclastogenesis and bone resorptive function in vitro, as well as the downregulation of osteoclast-specific genes including Acp5 (encoding TRAcP), Ctsk (encoding cathepsin K), and Mmp9 (encoding matrix metalloproteinase 9). Mechanistically, Pse suppressed intracellular ROS level by inhibiting RANKL-induced ROS production and enhancing ROS scavenging enzymes, subsequently suppressing MAPK pathway (ERK, P38, and JNK) and NF-κB pathways, leading to the inhibition of NFATc1 signaling. Micro-CT and histological data indicated that OVX procedure resulted in a significant bone loss, with dramatically increased the number of osteoclasts on the bone surface as well as increased ROS level in the bone marrow microenvironment; whereas Pse supplementation was capable of effectively preventing these OVX-induced changes. Conclusion: Pse was demonstrated for the first time as a novel alternative therapy for osteoclast-related bone diseases such as osteoporosis through suppressing ROS level.

Bone formation activity of an osteogenic dodecapeptide from blue mussels (Mytilus edulis).

Abstract: A novel osteogenic dodecapeptide peptide (PIE), IEELEEELEAER, was purified from the protein hydrolysate of blue mussels (Mytilus edulis). PIE was identified using a capillary electrophoresis electrospray ionization-quadrupole-time of flight mass spectrometer. PIE showed a good reduction in the bone loss in ovariectomized mice, and it also increased the bone mineral density of the ovariectomized mice. PIE has a high affinity with integrins (PDB: , ). There are 8 and 12 amino acid residues from PIE that interact with integrins and , respectively. PIE accelerates the transformation of G0/G1 phase cells into G2 M phase cells, which promotes the growth of osteoblasts. PIE (100 μg mL-1) can enhance alkaline phosphatase (ALP) activity by 26.48% compared with the control, and it also inhibits the growth of osteoclasts and tartrate resistant acid phosphatase (TRAP) activity. Therefore, PIE may contribute to preventing osteoporosis both in vitro and in vivo.

Preventive Effects of Evodiamine on Dexamethasone-Induced Osteoporosis in Zebrafish

Abstract: The aim of this study was to investigate the effect of evodiamine (EV) on dexamethasone-induced osteoporosis in zebrafish. Zebrafish larvae were exposed to different concentrations of dexamethasone to obtain the osteoporosis in zebrafish. Calcium, phosphorus, and alizarin red staining determination were performed to evaluate the effects of EV on bone mineralization. Alkaline phosphatase (ALP), hydroxyproline (HP), and tartrate resistant acid phosphatase (TRAP) were also measured by commercial kits. The expression of MMP3-OPN-MAPK pathway in zebrafish was measured by Western blot. RT-PCR was used to determine mRNA levels of MMP3, OPN, and MAPK. EV could significantly increase the content of calcium and phosphorus. The results of alizarin red staining showed that EV could significantly increase the calcium sink of horse fish, increasing the area of bone formation. EV could increase the content of hydroxyproline in zebrafish. EV also increased ALP and TRAP in zebrafish. Western blot and RT-PCR results showed that EV restored the MMP3-OPN-MAPK pathway in zebrafish. In conclusion, we found that EV can alleviate dexamethasone-induced osteoporosis in zebrafish. The mechanism is related to activating MMP3-OPN-MAPK pathway and then activating bone remodeling.

JAK2‐STAT3 signaling pathway is involved in rat periapical lesions induced by Enterococcus faecalis

Abstract: Objectives This study aimed to investigate the role of JAK2-STAT3 (Janus kinase 2/signal transducer and activator of transcription 3) in periapical disease caused by Enterococcus faecalis, as well as the correlation between lipoteichoic acid (LTA) in E. faecalis and the activity of the JAK2-STAT3 signaling pathway and osteoclast formation. Materials and methods A rat model of periapical periodontitis induced by E. faecalis was established. Periapical bone resorption was confirmed by HE staining. The expression of JAK2, p-JAK2, STAT3, and p-STAT3 was assessed with immunohistochemical staining. Osteoclasts were observed through enzyme histochemical staining. LTA acted on mouse osteoclast precursor cells (RAW264.7 cells); a JAK2 inhibitor (AG490) was used to inhibit the JAK2-STAT3 pathway in RAW264.7 cells. The expression of proteins in the JAK2-STAT3 pathway and TRAP (tartrate resistant acid phosphatase) in RAW264.7 cells was also detected. Results Rat periapical periodontitis was successfully established and bone resorption peaked at day 21. The expression of critical components in the JAK2-STAT3 pathway increased with the progression of inflammation. LTA promoted the differentiation of RAW264.7 cells into osteoclasts. NFATc1 was highly expressed and was inhibited by AG490. Conclusions JAK2-STAT3 signaling pathway plays an important role in the process of periapical bone resorption and osteoclastogenesis.

Vav1 inhibits RANKL-induced osteoclast differentiation and bone resorption.

Abstract: Vav1 is a Rho/Rac guanine nucleotide exchange factor primarily expressed in hematopoietic cells. In this study, we investigated the potential role of Vav1 in osteoclast (OC) differentiation by comparing the ability of bone marrow mononuclear cells (BMMCs) obtained from Vav1-deficient (Vav1-/-) and wild-type (WT) mice to differentiate into mature OCs upon stimulation with macrophage colony stimulating factor and receptor activator of nuclear kappa B ligand in vitro. Our results suggested that Vav1 deficiency promoted the differentiation of BMMCs into OCs, as indicated by the increased expression of tartrate-resistant acid phosphatase, cathepsin K, and calcitonin receptor. Therefore, Vav1 may play a negative role in OC differentiation. This hypothesis was supported by the observation of more OCs in the femurs of Vav1-/- mice than in WT mice. Furthermore, the bone status of Vav1-/- mice was analyzed in situ and the femurs of Vav1-/- mice appeared abnormal, with poor bone density and fewer number of trabeculae. In addition, Vav1-deficient OCs showed stronger adhesion to vitronectin, an αvβ3 integrin ligand important in bone resorption. Thus, Vav1 may inhibit OC differentiation and protect against bone resorption. [BMB Reports 2019; 52(11): 659-664].

Cigarette Smoke Extract Activates Tartrate-Resistant Acid Phosphatase-Positive Macrophage.

Abstract: Background It has been reported that smoking is one of the strongest positive risk factors for abdominal aortic aneurysms (AAAs). Although many studies have been directed to decipher the effect of smoking on AAA, its effect on macrophage activation has not yet been explored. Objectives We have reported the importance of osteoclastogenesis (OCG) in aneurysm formation. Therefore, we examined the effect of cigarette smoking on OCG and arterial aneurysmal formation by using cigarette smoke extract (CSE) in this study. Methods Macrophage cell lines were stimulated with CSE, and their activation and differentiation were examined in vitro. Since macrophages activated through the OCG pathway are identified by tartrate-resistant acid phosphatase (TRAP) expression, these cells are referred to as TRAP-positive macrophages (TPMs) in this study. We also applied CSE-contained PBS in the calcium chloride-induced mouse carotid aneurysm model in vivo. Results Macrophages stimulated with CSE expressed significantly higher levels of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), TRAP, cathepsin K, matrix metalloproteinase-9 and membrane-type metalloproteinase (MT1-MMP). CSE-treated mouse aneurysms showed increased aneurysm size with increased TPM infiltration and protease expression compared to non-CSE-treated mouse aneurysms. Conclusions These results suggest that CSE intensifies OCG in macrophages and promotes arterial aneurysmal progression.

Vitamin D Inhibition of TRPV5 Expression During Osteoclast Differentiation.

Abstract: Background Vitamin D is an important steroid that can regulate bone metabolism including osteoclast (OC) differentiation. Transient receptor potential cation channel subfamily V member 5 (TRPV5), is a calcium channel protein involved in OC differentiation. However, the impact of vitamin D on TRPV5 expression during OC differentiation is not clear. Objectives To determine if 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates the expression of TRPV5 during OC differentiation. Methods Bone marrow mononuclear macrophage (BMMs) were induced to differentiate into OC with or without treatment with 10 nM 1,25(OH)2D3. The expression levels of vitamin D receptor (VDR) and TRPV5 were examined. The expression of several OC markers, including tartrate resistant acid phosphatase (TRAP), carbonic anhydrase II (Ca II), cathepsin K (CTSK), and vacuolar-type H+-ATPase (V-ATPase) were also detected. Results We found that the VDR was expressed in murine bone marrow-derived macrophages at the early stage of OC differentiation. TRPV5 expression was increased during OC differentiation, which was down-regulated by 1,25(OH)2D3 after a prolonged exposure. The 1,25(OH)2D3 and TRPV5 inhibitors inhibited OC differentiation. Conclusions 1,25(OH)2D3 can inhibit TRPV5 expression as well as TRPV5 inhibitors during OC differentiation. This suggests that 1,25(OH)2D3 may suppress OC differentiation by inhibiting TRPV5 expression.

A simple and robust reporter gene assay for measuring the bioactivity of anti-RANKL therapeutic antibodies

Abstract: RANKL (receptor activator of nuclear factor κB ligand) plays a key role in the differentiation, activation and survival of osteoclasts. Denosumab, which targets RANKL, is approved for osteoporosis or bone loss that has a high risk for fracture and bone metastases from solid tumors. Bioactivity determination is essential for the safety and efficacy of therapeutic antibodies. At present, the mechanism of action (MOA) based bioassay for anti-RANKL monoclonal antibodies (mAbs) is the measurement of tartrate resistant acid phosphatase (TRAP) activity, which takes about five days and has complex operation and relatively high variation. In this study, we developed a reporter gene assay (RGA) based on a RAW264.7 cell line stably expressing luciferase reporter under the control of nuclear factor-κB (NF-κB) response elements. After optimizing the key parameters, the validation results based on ICH-Q2 not only show superior specificity, precision, linearity, accuracy and passage stability, but also a short duration and simple operation. These results demonstrate the RGA based on the RANKL–RANK–NF-κB pathway can be an excellent alternative for measuring the bioactivity of anti-RANKL mAbs.