Alendronate sodium induces G1 phase arrest and apoptosis in human umbilical vein endothelial cells by inhibiting ROS-mediated ERK1/2 signaling.

Bisphosphonates are potent bone resorption inhibitors, among which alendronate sodium (ALN) is commonly prescribed for most osteoporosis patients, but long-term application of ALN can cause bisphosphonate-related osteonecrosis of jaw (BRONJ), the pathogenesis of which remains unclear. Previous studies have suggested that bisphosphonates cause jaw ischemia by affecting the biological behavior of vascular endothelial cells, leading to BRONJ. However, the impacts of ALN on vascular endothelial cells and its mechanism remain unclear. The purpose of this work is to assess the influence of ALN on human umbilical vein endothelial cells (HUVECs) and clarify the molecular pathways involved. We found that high concentration of ALN induced G1 phase arrest in HUVECs, demonstrated by downregulation of Cyclin D1 and Cyclin D3. Moreover, high concentration of ALN treatment showed pro-apoptotic effect on HUVECs, demonstrated by increased levels of the cleaved caspase-3, the cleaved PARP and Bax, along with decreased levels of anti-apoptotic protein Bcl-2. Further experiments showed that ERK1/2 phosphorylation was decreased. Additionally, ALN provoked the build-up of reactive oxygen species (ROS) in HUVECs, leading to ERK1/2 pathway suppression. N-acetyl-L-cysteine (NAC), a ROS scavenger, efficiently promoted the ERK1/2 phosphorylation and mitigated the G1 phase arrest and apoptosis triggered by ALN in HUVECs. PD0325901, an inhibitor of ERK1/2 that diminishes the ERK1/2 phosphorylation enhanced the ALN-induced G1 phase arrest and apoptosis in HUVECs. These findings show that ALN induces G1 phase arrest and apoptosis through ROS-mediated ERK1/2 pathway inhibition in HUVECs, providing novel insights into the pathogenic process, prevention and treatment of BRONJ in individuals receiving extended use of ALN.

Infusion of GMSCs relieves autoimmune arthritis by suppressing the externalization of neutrophil extracellular traps via PGE2-PKA-ERK axis.

INTRODUCTION: Rheumatoid arthritis (RA) is a systemic autoimmune disease with limited treatment success, characterized by chronic inflammation and progressive cartilage and bone destruction. Accumulating evidence has shown that neutrophil extracellular traps (NETs) released by activated neutrophils are important for initiating and perpetuating synovial inflammation and thereby could be a promising therapeutic target for RA. K/B × N serum transfer-induced arthritis (STIA) is a rapidly developed joint inflammatory model that somehow mimics the inflammatory response in patients with RA. Human gingival-derived mesenchymal stem cells (GMSCs) have been previously shown to possess immunosuppressive effects in arthritis and humanized animal models. However, it is unknown whether GMSCs can manage neutrophils in autoimmune arthritis. OBJECTIVES: To evaluate whether infusion of GMSCs can alleviate RA by regulating neutrophils and NETs formation. If this is so, we will explore the underlying mechanism(s) in an animal model of inflammatory arthritis. METHODS: The effects of GMSCs on RA were assessed by comparing the symptoms of the K/B × N serum transfer-induced arthritis (STIA) model administered either with GMSCs or with control cells. Phenotypes examined included clinical scores, rear ankle thickness, paw swelling, inflammation, synovial cell proliferation, and immune cell frequency. The regulation of GMSCs on NETs was examined through immunofluorescence and immunoblotting in GMSCs-infused STIA mice and in an in vitro co-culture system of neutrophils with GMSCs. The molecular mechanism(s) by which GMSCs regulate NETs was explored both in vitro and in vivo by silencing experiments. RESULTS: We found in this study that adoptive transfer of GMSCs into STIA mice significantly ameliorated experimental arthritis and reduced neutrophil infiltration and NET formation. In vitro studies also showed that GMSCs inhibited the generation of NETs in neutrophils. Subsequent investigations revealed that GMSCs secreted prostaglandin E2 (PGE2) to activate protein kinase A (PKA), which ultimately inhibited the downstream extracellular signal-regulated kinase (ERK) pathway that is essential for NET formation. CONCLUSION: Our results demonstrate that infusion of GMSCs can ameliorate inflammatory arthritis mainly by suppressing NET formation via the PGE2-PKA-ERK signaling pathway. These findings further support the notion that the manipulation of GMSCs is a promising stem cell-based therapy for patients with RA and other autoimmune and inflammatory diseases.

Characterization of different osteoclast phenotypes in the progression of bone invasion by oral squamous cell carcinoma.

The present study aimed to characterize different phenotypes of osteoclasts in the progression of bone invasion by oral squamous cell carcinoma (OSCC). A local bone invasion model of OSCC was established by injecting SCC25 human OSCC cells into the center of calvariae in nude mice, and all mice were found to have a typical bone resorption area. Staining for tartrate-resistant acid phosphatase (TRAP) revealed various types of giant osteoclasts in the tumour-bone interface. Bone marrow cells (BMCs) were isolated from the nude mice for primary osteoclast culture, but only a few giant osteoclasts were generated. Additionally, special blood centrifuge tubes were utilized to obtain large numbers of peripheral blood mononuclear cells (PBMCs). Using magnetic activated cell sorting (MACS) and the cytokines colony-stimulating factor (CSF) and receptor activator of nuclear factor-κb ligand (RANKL), we differentiated human osteoclasts from CD14+ monocytes of PBMCs. Bone resorption was further confirmed by a bone resorption assay. Finally, Transwell inserts were used for indirect cell co-culture of SCC25 cells and CD14+ monocytes. Expression of specific osteoclast markers was detected by real-time PCR and western blotting. After co-culture for 3 and 6 days, conditioned medium (CM) of SCC25 cells stimulated the expression of osteoclast markers, and additional osteoclasts were detected through staining of TRAP and F-actin. In the present study distinct osteoclast phenotypes were observed in the established bone invasion animal model, and were confirmed using various primary osteoclast cultures. CM of OSCC cells may promote the expression of osteoclast markers and induce the differentiation of monocytes to mature osteoclasts, which can resorb adjacent bone tissue.

Utilization of E-cadherin by monocytes from tumour cells plays key roles in the progression of bone invasion by oral squamous cell carcinoma.

E-cadherin (E-cad) is recently reported to be expressed in early stages of osteoclastogenesis, and blocking E-cad with neutralizing antibodies decreases osteoclast differentiation. Since our previous research demonstrates the loss of E-cad protein in the bone invasion by oral squamous cell carcinoma (OSCC), we hypothesize that E-cad may be utilized by monocytes to fuse and differentiate into osteoclasts. Two research models are used in the present study to explore our hypothesis. On one hand, we use OSCC cells of SCC25 to establish an animal model of bone invasion by OSCC, and investigate whether E-cad protein disappears in vivo; on the other hand, we use the indirect co-culture model of SCC25 and RAW 264.7 cells, with the treatment of transforming growth factor-ß1 (TGF-ß1), and observe whether the decreased E-cad protein is 'hijacked' in vitro. Results showed the animal model of OSCC with bone invasion was successfully established. Immunohistochemistry (IHC) found similar changes of E-cad protein, which was weakly stained by tumour cells. By using 5 ng/ml of TGF-ß1, we confirmed the artificial epithelial-mesenchymal transition (EMT) of SCC25 cells, with changes of EMT marker expression and cell morphology. Real-time PCR showed E-cad mRNA decreased in SCC25 while increased in RAW 264.7 of the indirect cell co-culture model, and immunofluoresence (IF) observed the evident switch of E-cad staining from SCC25 to RAW 264.7. With the supplement of receptor activator of NF-κB ligand (RANKL), tartrate-resistant acid phosphatase (TRAP) and F-actin staining confirmed the increased number of osteoclasts. Taken together, our study found the switch of E-cad protein in the progression of bone invasion by OSCC. The loss of E-cad in tumour cells may be utilized by monocytes to differentiate into osteoclasts, thus further explaining the underlying mechanisms of bone invasion by OSCC, which may supply clues for future molecular biotherapies.