The transcription factor CREB is involved in sorafenib-inhibited renal cancer cell proliferation, migration and invasion

Our previous reports showed that the cyclic-AMP-response element-binding protein (CREB) served as a proto-oncogene in the process of tumorigenesis and mediated the growth and metastatic activity of renal cancer cells. Our study, therefore, explored the role of CREB in sorafenib-inhibited cell proliferation, migration and invasion. Renal cancer cells were cultured in medium containing sorafenib for 12, 24, 48 and 72 h. The MTT assay was used to study the cytotoxic effects of sorafenib. Cell invasion and migration were assayed in wound healing and transwell experiments, respectively. Protein expression levels were evaluated by western blotting. The results show that sorafenib treatment decreased cell viability in a dose- and time-dependent manner. Sorafenib inhibited cell migration and invasion and decreased the expression of MMP-2 and MMP-9. Moreover, addition of the recombinant plasmid pCI-neo/CREB (PN) reversed the sorafenib-induced inhibition of cell proliferation, migration and invasion. These results show that CREB is associated with the sorafenib-induced inhibition of proliferation, migration and invasion

Peptides identify multiple hotspots within the ligand binding domain of the TNF receptor 2

BACKGROUND: Hotspots are defined as the minimal functional domains involved in protein:protein interactions and sufficient to induce a biological response. RESULTS: Here we describe the use of complex and high diversity phage display libraries to isolate peptides (called Hotspot Ligands or HSPLs) which sub-divide the ligand binding domain of the tumor necrosis factor receptor 2 (TNFR2; p75) into multiple hotspots. We have shown that these libraries could generate HSPLs which not only subdivide hotspots on protein and non-protein targets but act as agonists or antagonists. Using this approach, we generated peptides which were specific for human TNFR2, could be competed by the natural ligands, TNFα and TNFβ and induced an unexpected biological response in a TNFR2-specific manner. CONCLUSIONS: To our knowledge, this is the first report describing the dissection of the TNFR2 into biologically active hotspots with the concomitant identification of a novel and unexpected biological activity

The Transcription Factor Creb is Involved in Sorafenib-Inhibited Renal Cancer Cell Proliferation, Migration and Invasion

Our previous reports showed that the cyclic-AMP-response element-binding protein (CREB) served as a proto-oncogene in the process of tumorigenesis and mediated the growth and metastatic activity of renal cancer cells. Our study, therefore, explored the role of CREB in sorafenib- -inhibited cell proliferation, migration and invasion. Renal cancer cells were cultured in medium containing sorafenib for 12, 24, 48 and 72 h. The MTT assay was used to study the cytotoxic effects of sorafenib. Cell invasion and migration were assayed in wound healing and transwell experiments, respectively. Protein expression levels were evaluated by western blotting. The results show that sorafenib treatment decreased cell viability in a dose- and time-dependent manner. Sorafenib inhibited cell migration and invasion and decreased the expression of MMP-2 and MMP-9. Moreover, addition of the recombinant plasmid pCI-neo/ CREB (PN) reversed the sorafenib-induced inhibition of cell proliferation, migration and invasion. These results show that CREB is associated with the sorafenib-induced inhibition of proliferation, migration and invasion

Disruption of Col9a2 expression leads to defects in osteochondral homeostasis and osteoarthritis-like phenotype in mice

Background/Objective: As one of the branched chains of Type IX collagen (Col9), Collagen IX alpha2 (Col9a2) has been reported to be associated with several orthopedic conditions. However, the relationship between Col9a2 and knee osteoarthritis (KOA) remains to be elucidated. Methods: To probe the relationship between Col9a2 and KOA, we performed a systematic analysis of Col9a2-deficient (Col9a2−/−) mice using whole-mount skeletal staining, Micro-CT (μCT), biomechanics, histomorphometry, immunohistochemistry (IHC), immunofluorescence (IF) and Elisa. Results: We found that the subchondral bone (SCB) in the knee joint of Col9a2−/− mice became sparse and deformed in the early stage, with altered bone morphometric parameters, reduced load-bearing capacity, dysfunctional bone homeostasis (decreased osteogenesis capacity and elevated bone resorption capacity), diminished cartilage proteoglycans and disrupted cartilage extracellular matrix (ECM) anabolism and catabolism compared with the Col9a2+/+ mice. In the late stage, the cartilage degeneration in Col9a2−/− mice were particularly pronounced compared to Col9a2+/+ mice, as evidenced by severe cartilage destruction and a marked reduction in cartilage thickness and area. Conclusion: Overall, Col9a2 is essential for maintaining osteochondral homeostasis in the knee joint of mice, and the absence of this gene is accompanied by distinct sclerosis of the SCB and a reduction in load-bearing capacity; in the late stage, in the lack of SCB stress inhibition, excessive load is consistently exerted on the cartilage, ultimately leading to osteoarthritic-like articular cartilage damage

Aberrant methylation and expression of TNXB promote chondrocyte apoptosis and extracullar matrix degradation in hemophilic arthropathy via AKT signaling

Recurrent joint bleeding in hemophilia patients frequently causes hemophilic arthropathy (HA). Drastic degradation of cartilage is a major characteristic of HA, but its pathological mechanisms has not yet been clarified. In HA cartilages, we found server matrix degradation and increased expression of DNA methyltransferase proteins. We thus performed genome-wide DNA methylation analysis on human HA (N=5) and osteoarthritis (OA) (N=5) articular cartilages, and identified 1228 differentially methylated regions (DMRs) associated with HA. Functional enrichment analyses revealed the association between DMR genes (DMGs) and extracellular matrix (ECM) organization. Among these DMGs, Tenascin XB (TNXB) expression was down-regulated in human and mouse HA cartilages. The loss of Tnxb in F8-/- mouse cartilage provided a disease-promoting role in HA by augmenting cartilage degeneration and subchondral bone loss. Tnxb knockdown also promoted chondrocyte apoptosis and inhibited phosphorylation of AKT. Importantly, AKT agonist showed chondroprotective effects following Tnxb knockdown. Together, our findings indicate that exposure of cartilage to blood leads to alterations in DNA methylation, which is functionally related to ECM homeostasis, and further demonstrate a critical role of TNXB in HA cartilage degeneration by activating AKT signaling. These mechanistic insights allow development of potentially new strategies for HA cartilage protection

Bushenhuoxue Formula Facilitates Articular Cartilage Repair and Attenuates Matrix Degradation by Activation of TGF-β Signaling Pathway

Objective. To investigate the effect and underlying mechanism of Bushenhuoxue (BSHX) formula on articular cartilage repair. Methods. Twenty-four full-thickness cartilage defect rats were divided into two groups: model group and BSHX group (treated with BSHX formula). Macroscopic observation and histopathological study were conducted after 4- and 8-week treatment. Additionally, we also evaluated chondrocyte proliferation, extracellular matrix (ECM) deposition, cartilage degradation, and chondrocyte hypertrophy-related genes expression in chondrogenic ATDC5 cells cultured in BSHX formula-mediated serum. Moreover, we assessed aforementioned genes expression and pSMAD2/3 protein level in Tgfβr2 siRNA transfected chondrogenic ATDC5 cells in order to address whether BSHX formula exerts cartilage repairing effect through TGF-β signaling. Results. Neocartilage regeneration promotion effect was observed in cartilage defect rats after BSHX formula treatment, with increases in Col2 and pSMAD2 and decreases in Mmp13 and Runx2. Moreover, cell proliferation, the elevated Col2a1, Aggrecan and pSMAD2/3, reduced Mmp13, Adamts5, Col10a1, and Runx2 expression were also observed in chondrogenic ATDC5 cells cultured in BSHX formula-mediated serum. Besides, the expression alteration of ECM deposition, cartilage degradation, chondrocyte hypertrophy-related genes, and pSMAD2/3 protein levels presented in Tgfβr2 downregulated chondrogenic ATDC5 cells couldn’t be adjusted by BSHX formula treatment. Conclusion. By activation of TGF-β signaling, BSHX formula can promote articular cartilage repair by accelerating chondrocyte proliferation and maintaining chondrocyte phenotype, upregulate ECM accumulation, and inhibit matrix degradation

The Fate and Distribution of Autologous Bone Marrow Mesenchymal Stem Cells with Intra-Arterial Infusion in Osteonecrosis of the Femoral Head in Dogs

This study aimed to investigate if autologous bone marrow mesenchymal stem cells (MSCs) could treat osteonecrosis of the femoral head (ONFH) and what the fate and distribution of the cells are in dogs. Twelve Beagle dogs were randomly divided into two groups: MSCs group and SHAM operated group. After three weeks, dogs in MSCs group and SHAM operated group were intra-arterially injected with autologous MSCs and 0.9% normal saline, respectively. Eight weeks after treatment, the necrotic volume of the femoral heads was significantly reduced in MSCs group. Moreover, the trabecular bone volume was increased and the empty lacunae rate was decreased in MSCs group. In addition, the BrdU-positive MSCs were unevenly distributed in femoral heads and various vital organs. But no obvious abnormalities were observed. Furthermore, most of BrdU-positive MSCs in necrotic region expressed osteocalcin in MSCs group and a few expressed peroxisome proliferator-activated receptor-γ (PPAR-γ). Taken together, these data indicated that intra-arterially infused MSCs could migrate into the necrotic field of femoral heads and differentiate into osteoblasts, thus improving the necrosis of femoral heads. It suggests that intra-arterial infusion of autologous MSCs might be a feasible and relatively safe method for the treatment of femoral head necrosis

Platelet-rich plasma promotes bone formation, restrains adipogenesis and accelerates vascularization to relieve steroids-induced osteonecrosis of the femoral head

Steroid-associated necrosis of the femoral head (SANFH) is one of the most common and refractory chronic diseases with increasing incidence. The typical pathological changes of SANFH include decreased osteogenic differentiation, enhanced intramedullary adipocytes deposition and impaired osseous circulation. In this study, we investigated the effects and potential mechanisms of Platelet-rich plasma (PRP) on SANFH. Sixty Sprague-Dawley rats were randomly divided into the control, PRP donor, model, and PRP groups. Compared to the model group, PRP treatment significantly increased the hemorheological indexes and serum levels of bone gla-protein (BGP) and vascular endothelial growth factor (VEGF), while decreased the levels of triglyceride (TG) and total cholesterol (TC). Meanwhile, Micro-CT and histopathological stain (Hematoxylin-eosin and Alcian blue-hematoxylin/orange G staining) were performed on the femoral head for morphological and histopathological evaluation, indicating that bone trabecular microstructure and bone mineral density (BMD) were significantly improved after PRP treatment. Immunohistochemical analysis revealed that PRP remarkably up-regulated the expression of osteogenic markers including β-catenin and alkaline phosphatase (ALP), angiogenic markers containing VEGF and platelet endothelial cell adhesion molecule-1 (CD31), while down-regulated adipogenic markers involving fatty acid-binding protein (FABP-4), and peroxisome proliferator-activated receptor gamma (PPAR-γ) in SANFH rat models. In summary, for the first time, PRP was demonstrated to prevent the development of SANFH through stimulating bone formation and vascularization as well as retarding adipogenesis