Niandong Qian

MicroRNA-17-92a upregulation by estrogen leads to Bim targeting and inhibition of osteoblast apoptosis.

Summary Anti-apoptotic effects of estrogen on osteoblasts are very important in the etiology of estrogen protection of the adult skeleton against bone loss. The mechanisms of this process are still not fully understood. Recent studies implicated an important role of microRNAs in estrogen-mediated responses in various cellular processes, including cell apoptosis and proliferation. Therefore, we hypothesized that these regulatory molecules might be involved with estrogen in protecting osteoblasts from apoptosis. Western blotting, quantitative real-time PCR, flow cytometry and luciferase assays were employed to investigate the role of microRNAs in this process. The microRNA cluster miR-17-92a, a post-transcriptional regulator, was significantly reduced during dexamethasone, etoposide and tumor necrosis factor alpha (TNF-&agr;)-induced osteoblasts apoptosis. The repression of miR-17-92a was significantly attenuated by estrogen. To delineate the role of miR-17-92a in apoptosis, we silenced and overexpressed miR-17-92a in osteoblasts. We found that miR-17-92a depletion significantly enhanced dexamethasone-induced apoptosis and overexpressing miR-17-92a remarkably increased the anti-apoptotic effects of estrogen on osteoblasts. Mechanistic studies showed that miR-17-92a inhibited Bim expression through a microRNA-17-92a-binding site within the 3′-untranslated region of Bim. The post-transcriptional repression of Bim was further confirmed by a luciferase reporter assay. These results showed that miR-17-92a, plays a significant role in the process of estrogen protection of osteoblasts against apoptosis, by regulating Bim expression.

Bone Selective Protective Effect of a Novel Bone-seeking Estrogen on Trabecular Bone in Ovariectomized Rats

The drawbacks of estrogen restrict the clinical use of hormone replacement therapy, and it would be most helpful to explore new estrogenic substances that could prevent bone loss and be free from any adverse effects. We synthesized a new compound named bone-seeking estrogen (SE2) by combining 17β-estradiol (E2) with iminodiacetic acid through the Mannich reaction. E2 and SE2 were labeled with isotope 3H, and the tissue distribution tests of E2-3H and SE2-3H were analyzed by the radioactivity. The specific nuclear binding of E2 and SE2 in osteoblasts was measured. SE2 exhibited significantly greater affinity for bone but lower affinity for ovary and uterus than did E2, and SE2 maintained a high affinity for the estrogen receptor alpha similar to that of E2. SE2 administration did not induce uterine hypertrophy. Body weight increase was significantly suppressed by treatment with E2 but not by SE2 after ovariectomy (OVX). SE2 decreased bone turnover as E2 after OVX detected by serum biochemical markers. Bone histology and micro-CT analysis revealed that SE2 administration, similar to E2, could improve bone mass and trabecular architecture after OVX. Biomechanical analyses showed that SE2 treatment effectively increased mechanical properties after OVX. The results suggested that SE2 was effective in preventing OVX-induced bone loss and exhibited few side effects on body weight and uterine hypertrophy, which was beneficial in reducing the adverse effects caused by E2. SE2 may be a better choice than E2 for the prevention of postmenopausal osteoporosis.

Glucocorticoids impair bone formation of bone marrow stromal stem cells by reciprocally regulating microRNA-34a-5p

SummaryThe inhibitory effects of glucocorticoids (GCs) on bone marrow stromal stem cell (BMSC) proliferation and osteoblastic differentiation are an important pathway through which GCs decrease bone formation. We found that microRNA-34a-5p was a critical player in dexamethasone (Dex)-inhibited BMSC proliferation and osteogenic differentiation. MicroRNA-34a-5p might be used as a therapeutic target for GC-impaired bone formation.IntroductionThe inhibitory effects of glucocorticoids (GCs) on bone marrow stromal stem cell (BMSC) proliferation and osteoblastic differentiation are an important pathway through which GCs decrease bone formation. The mechanisms of this process are still not completely understood. Recent studies implicated an important role of microRNAs in GC-mediated responses in various cellular processes, including cell proliferation and differentiation. Therefore, we hypothesized that these regulatory molecules might be implicated in the process of GC-decreased BMSC proliferation and osteoblastic differentiation.MethodsWestern blot, quantitative real-time PCR, and cell proliferation and osteoblastic differentiation assays were employed to investigate the role of microRNAs in GC-inhibited BMSC proliferation and osteoblastic differentiation.ResultsWe found that microRNA-34a-5p was reciprocally regulated by Dex during the process of BMSC proliferation and osteoblastic differentiation. Furthermore, we confirmed that microRNA-34a-5p was a critical player in Dex-inhibited BMSC proliferation and osteogenic differentiation. Mechanistic studies showed that Dex inhibited BMSC proliferation by microRNA-34a-5p targeting cell cycle factors, including CDK4, CDK6, and Cyclin D1. Furthermore, downregulation of microRNA-34a-5p by Dex leads to Notch signaling activation, resulting in inhibition of BMSC osteogenic differentiation.ConclusionsThese results showed that microRNA-34a-5p, a crucial regulator for BMSC proliferation and osteogenic differentiation, might be used as a therapeutic target for GC-impaired bone formation.

Desferrioxamine reduces ultrahigh-molecular-weight polyethylene-induced osteolysis by restraining inflammatory osteoclastogenesis via heme oxygenase-1

As wear particles-induced osteolysis still remains the leading cause of early implant loosening in endoprosthetic surgery, and promotion of osteoclastogenesis by wear particles has been confirmed to be responsible for osteolysis. Therapeutic agents targeting osteoclasts formation are considered for the treatment of wear particles-induced osteolysis. In the present study, we demonstrated for the first time that desferrioxamine (DFO), a powerful iron chelator, could significantly alleviate osteolysis in an ultrahigh-molecular-weight polyethylene (UHMWPE) particles-induced mice calvaria osteolysis model. Furthermore, DFO attenuated calvaria osteolysis by restraining enhanced inflammatory osteoclastogenesis induced by UHMWPE particles. Consistent with the in vivo results, we found DFO was also able to inhibit osteoclastogenesis in a dose-dependent manner in vitro, as evidenced by reduction of osteoclasts formation and suppression of osteoclast specific genes expression. In addition, DFO dampened osteoclasts differentiation and formation at early stage but not at late stage. Mechanistically, the reduction of osteoclastogenesis by DFO was due to increased heme oxygenase-1 (HO-1) expression, as decreased osteoclasts formation induced by DFO was significantly restored after HO-1 was silenced by siRNA, while HO-1 agonist COPP treatment enhanced DFO-induced osteoclastogenesis inhibition. In addition, blocking of p38 mitogen-activated protein kinase (p38MAPK) signaling pathway promoted DFO-induced HO-1 expression, implicating that p38 signaling pathway was involved in DFO-mediated HO-1 expression. Taken together, our results suggested that DFO inhibited UHMWPE particles-induced osteolysis by restraining inflammatory osteoclastogenesis through upregulation of HO-1 via p38MAPK pathway. Thus, DFO might be used as an innovative and safe therapeutic alternative for treating wear particles-induced aseptic loosening.

Activation of HIFa Pathway in Mature Osteoblasts Disrupts the Integrity of the Osteocyte/Canalicular Network

The hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, are the central mediators of the homeostatic response that enables cells to survive and differentiate in low-oxygen conditions. Previous studies indicated that disruption of the von Hippel-Lindau gene (Vhl) coincides with the activation of HIFα signaling. Here we show that inactivation of Vhl in mature osteoblasts/osteocytes induces their apoptosis and disrupts the cell/canalicular network. VHL-deficient (ΔVHL) mice exhibited a significantly increased cortical bone area resulting from enhanced proliferation and osteogenic differentiation of the bone marrow stromal cells (BMSCs) by inducing the expression of β-catenin in the BMSC. Our data suggest that the VHL/HIFα pathway in mature osteoblasts/osteocytes plays a critical role in the bone cell/canalicular network and that the changes of osteocyte morphology/function and cell/canalicular network may unleash the bone formation, The underlying mechanism of which was the accumulation of β-catenin in the osteoblasts/osteoprogenitors of the bone marrow.

Increased 15-lipoxygenase-1 expression in chondrocytes contributes to the pathogenesis of osteoarthritis

15-Lipoxygenase-1 (15-LO-1) is involved in many pathological processes. The purpose of this study was to determine the potential role of 15-LO-1 in osteoarthritis (OA). The levels of 15-LO-1 expression were measured by western blotting and quantitative real-time PCR in articular cartilage from the OA rat models and OA patients. To further investigate the effects of 15-LO-1 on chondrocyte functions, such as extracellular matrix (ECM) secretion, the release of matrix-degrading enzymes, the production of reactive oxygen species (ROS), cell proliferation and apoptosis, we decreased or increased 15-LO-1 expression in chondrocytes by means of transfecting with siRNA targeting 15-LO-1 and plasmid encoding 15-LO-1, respectively. The results showed that 15-LO-1 expression was obviously increased in articular cartilage from OA rats and OA patients. It was also found that many factor-related OA, such as mechanical loading, ROS, SNP and inflammatory factor, significantly promoted 15-LO-1 expression and activity in chondrocytes. Silencing 15-LO-1 was able to markedly alleviate mechanical loading-induced cartilage ECM secretion, cartilage-degrading enzyme secretion and ROS production. Overexpression of 15-LO-1 could inhibit chondrocyte proliferation and induce chondrocyte apoptosis. In addition, reduction of 15-LO-1 in vivo significantly alleviated OA. Taken together, these results indicate that 15-LO-1 has an important role in the disease progression of OA. Thus 15-LO-1 may be a good target for developing drugs in the treatment of OA.

Estrogen inhibits osteoclasts formation and bone resorption via microRNA-27a targeting PPARγ and APC: GUO et al.

Inhibition of osteoclasts formation and bone resorption by estrogen is very important in the etiology of postmenopausal osteoporosis. The mechanisms of this process are still not fully understood. Recent studies implicated an important role of microRNAs in estrogen‐mediated responses in various cellular processes, including cell differentiation and proliferation. Thus, we hypothesized that these regulatory molecules might be implicated in the process of estrogen‐decreased osteoclasts formation and bone resorption. Western blot, quantitative real‐time polymerase chain reaction, tartrate‐resistant acid phosphatase staining, pit formation assay and luciferase assay were used to investigate the role of microRNAs in estrogen‐inhibited osteoclast differentiation and bone resorption. We found that estrogen could directly suppress receptor activator of nuclear factor B ligand/macrophage colony‐stimulating factor‐induced differentiation of bone marrow‐derived macrophages into osteoclasts in the absence of stromal cell. MicroRNA‐27a was significantly increased during the process of estrogen‐decreased osteoclast differentiation. Overexpressing of microRNA‐27a remarkably enhanced the inhibitory effect of estrogen on osteoclast differentiation and bone resorption, whereas which were alleviated by microRNA‐27a depletion. Mechanistic studies showed that microRNA‐27a inhibited peroxisome proliferator‐activated receptor gamma (PPARγ) and adenomatous polyposis coli (APC) expression in osteoclasts through a microRNA‐27a binding site within the 3′‐untranslational region of PPARγ and APC. PPARγ and APC respectively contributed to microRNA‐27a‐decreased osteoclast differentiation and bone resorption. Taken together, these results showed that microRNA‐27a may play a significant role in the process of estrogen‐inhibited osteoclast differentiation and function.

The prevention of latanoprost on osteoclastgenesis in vitro and lipopolysaccharide-induced murine calvaria osteolysis in vivo

Identification of agents that inhibit osteoclast formation and function is important for the treatment of osteolytic diseases which feature excessive osteoclast formation and bone resorption. Latanoprost (LTP), an analog of prostaglandin F2α, is a medication which works to lower pressure inside the eyes. Prostaglandin F2α was reported to regulate bone metabolism, however, the effect of LTP in osteoclastogenesis is still unknown. Here, we found that LTP suppressed RANKL‐induced osteoclastogenesis in a dose‐dependent manner as illustrated by TRAP activity and TRAP staining. In addition, the osteoclast function was also reduced by LTP treatment, as indicated in less osteoclastic resorption pit areas. Furthermore, LTP inhibited the mRNA expressions of osteoclast marker genes such as TRAP and cathepsin K. In order to illustrate its molecular mechanism, we examined the changing of mRNA and protein levels of NFATc1 and c‐fos by LTP treatment, as well as the phosphorylation of ERK, AKT, JNK, and p38. The results suggested that LTP inhibited RANKL‐induced osteoclastgenesis and function by inhibiting ERK, AKT, JNK, and p38 cascade, following by the c‐fos/NFATc1 pathway. In agreement with in vitro results, using an in vivo lipopolysaccharide‐induced murine calvaria osteolysis mouse model, we found that administration of LTP was able to reverse the lipopolysaccharide‐induced bone loss. Together, these data demonstrated that LTP attenuated the bone loss in lipopolysaccharide‐induced murine calvaria osteolysis mice through inhibiting osteoclast formation and function. Our study thus provided the evidences that LTP was a potential treatment option against osteolytic bone diseases.

A novel rhein derivative modulates bone formation and resorption and ameliorates oestrogen-dependent bone loss

Osteoporosis, an osteolytic disease that affects millions of people worldwide, features a bone remodeling imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Identifying dual target‐directed agents that inhibit excessive bone resorption and increase bone formation is considered an efficient strategy for developing new osteoporosis treatments. Rhein, a natural anthraquinone, can be isolated from various Asian herbal medicines. Rhein and its derivatives have been reported to have various beneficial pharmacological effects, especially their bone‐targeting ability and anti‐osteoclastogenesis activity. Moreover, hydrogen sulfide (H2S) was reported to prevent ovariectomy‐ (OVX‐) induced bone loss by enhancing bone formation, and sulfur replacement therapy has been considered a novel and plausible therapeutic option. Based on this information, we synthesized a rhein‐derived thioamide (RT) and investigated its effects on bone resorption and bone formation in vitro and in vivo. It has been found that the RT‐inhibited receptor activator of the nuclear factor‐κB (NF‐κB) ligand‐ (RANKL‐) induced osteoclastogenesis and bone resorption in a dose‐dependent manner. The expression of osteoclast marker genes was also suppressed by RT treatment. Furthermore, exploration of signal transduction pathways indicated that RT markedly blocked RANKL‐induced osteoclastogenesis by attenuating MAPK pathways. However, RT treatment in an osteoblastic cell line, MC3TE‐E1, indicated that RT led to an increase in the deposition of minerals and the expression of osteoblast marker genes, as demonstrated by Alizarin Red staining and alkaline phosphatase activity. Importantly, an OVX mouse model showed that RT could attenuate the bone loss in estrogen deficiency‐induced osteoporosis in vivo with a smart H2S‐releasing property and that there was a considerable improvement in the biomechanical properties of bone. Accordingly, our current work highlights the dual regulation of bone remodeling by the rhein‐derived molecule RT. This may be a highly promising approach for a new type of anti‐osteoporosis agent.