BMP2 induces osteogenic differentiation through ACKR3 in mesenchymal stem cells.

In recent years, bone loss related diseases have attracted more and more attention, such as osteoporosis and osteonecrosis of the femoral head exhibited symptoms of osteopenia or insufficient bone mass in a certain stage. Mesenchymal stem cells (MSCs), which can be induced to differentiate into osteoblasts under certain conditions can provide a new solution bone disease. Herein, we deciphered the possible mechanism by which BMP2 drives the transduction of MSCs to the osteoblast lineage through ACKR3/p38/MAPK signaling. The levels of ACKR3 in femoral tissues of samples from humans with different ages and sexes were measured firstly and found that ACKR3 protein levels increase with age. In vitro cellular assays showed that ACKR3 inhibits BMP2-induced osteo-differentiation and promotes adipo-differentiation of MSCs, whereas siACKR3 exhibited the opposite effects. In vitro embryo femur culture experiment showed that inhibition of ACKR3 enhanced BMP2-induced trabecular bone formation in C57BL6/J mouse. In terms of molecular mechanisms, we found that p38/MAPK signaling might play the key role. ACKR3 agonist TC14012 suppressed the phosphorylation of p38 and STAT3 in BMP2 induced MSCs differentiation. Our findings suggested that ACKR3 might be a novel therapeutic target for the treatment of bone-associated diseases and bone-tissue engineering.

Evolution of the CBL and CIPK gene families in Medicago: genome-wide characterization, pervasive duplication, and expression pattern under salt and drought stress.

BACKGROUND: Calcineurin B-like proteins (CBLs) are ubiquitous Ca2+ sensors that mediate plant responses to various stress and developmental processes by interacting with CBL-interacting protein kinases (CIPKs). CBLs and CIPKs play essential roles in acclimatization of crop plants. However, evolution of these two gene families in the genus Medicago is poorly understood. RESULTS: A total of 68 CBL and 135 CIPK genes have been identified in five genomes from Medicago. Among these genomes, the gene number of CBLs and CIPKs shows no significant difference at the haploid genome level. Phylogenetic and comprehensive characteristic analyses reveal that CBLs and CIPKs are classified into four clades respectively, which is validated by distribution of conserved motifs. The synteny analysis indicates that the whole genome duplication events (WGDs) have contributed to the expansion of both families. Expression analysis demonstrates that two MsCBLs and three MsCIPKs are specifically expressed in roots, mature leaves, developing flowers and nitrogen fixing nodules of Medicago sativa spp. sativa, the widely grown tetraploid species. In particular, the expression of these five genes was highly up-regulated in roots when exposed to salt and drought stress, indicating crucial roles in stress responses. CONCLUSIONS: Our study leads to a comprehensive understanding of evolution of CBL and CIPK gene families in Medicago, but also provides a rich resource to further address the functions of CBL-CIPK complexes in cultivated species and their closely related wild relatives.

Effects of RNA interference-mediated knockdown of livin and survivin using monomethoxypolyethylene glycol-chitosan nanoparticles in MG-63 osteosarcoma cells.

MG-63 human osteosarcoma cells were transfected with short hairpin RNA (shRNA) against livin and survivin using monomethoxypolyethylene glycol­chitosan (mPEG­CS) nanoparticles (NPs) as carriers, with the aim of evaluating the effect on cell proliferation and apoptosis. mPEG­CS NPs sized ~100 nm were prepared by ionic crosslinking. mPEG­CS­livin shRNA, mPEG­CS­survivin shRNA and mPEG­CS­(livin shRNA + survivin shRNA) NPs were constructed by electrostatic adsorption at NP suspension/gene solution ratios of 3:1 to transfect MG­63 cells. The expression levels of livin and survivin mRNA and protein were measured by reverse transcription­polymerase chain reaction and western blotting, respectively. The inhibitory effects of downregulated livin and survivin expression on cell proliferation were measured using an MTT assay. The apoptosis­inducing effects of livin and surivin knockdown were investigated using a Hoechst staining kit. All shRNA groups resulted in reduced expression of livin and survivin mRNA and protein in MG­63 cells. The MTT assay and Hoechst staining indicated that simultaneous knockdown of livin and survivin genes inhibited the proliferation of MG­63 cells and promoted their apoptosis, to a greater extent than knocking down either gene individually. The simultaneous interference mediated by mPEG­CS NPs significantly reduced livin and survivin expression in MG­63 cells, suppressed proliferation and facilitated apoptosis, to a greater extent than knockdown of either livin or survivin alone were. Thus the results indicate a synergistic effect of livin and survivin.