Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular stromal potential.

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

CA-074Me compound inhibits osteoclastogenesis via suppression of the NFATc1 and c-FOS signaling pathways.

The osteoclast is an integral cell of bone resorption. Since osteolytic disorders hinge on the function and dysfunction of the osteoclast, understanding osteoclast biology is fundamental to designing new therapies that curb osteolytic disorders. The identification and study of lysosomal proteases, such as cathepsins, have shed light on mechanisms of bone resorption. For example, Cathepsin K has already been identified as a collagen degradation protease produced by mature osteoclasts with high activity in the acidic osteoclast resorption pits. Delving into the mechanisms of cathepsins and other osteoclast related compounds provides new targets to explore in osteoclast biology. Through our anti-osteoclastogenic compound screening experiments we encountered a modified version of the Cathepsin B inhibitor CA-074: the cell membrane-permeable CA-074Me (L-3-trans-(Propylcarbamoyl) oxirane-2-carbonyl]-L-isoleucyl-L-proline Methyl Ester). Here we confirm that CA-074Me inhibits osteoclastogenesis in vivo and in vitro in a dose-dependent manner. However, Cathepsin B knockout mice exhibited unaltered osteoclastogenesis, suggesting a more complicated mechanism of action than Cathepsin B inhibition. We found that CA-074Me exerts its osteoclastogenic effect within 24 h of osteoclastogenesis stimulation by suppression of c-FOS and NFATc1 pathways.

Effects of biomechanical forces on signaling in the cortical collecting duct (CCD).

An increase in tubular fluid flow rate (TFF) stimulates Na reabsorption and K secretion in the cortical collecting duct (CCD) and subjects cells therein to biomechanical forces including fluid shear stress (FSS) and circumferential stretch (CS). Intracellular MAPK and extracellular autocrine/paracrine PGE2 signaling regulate cation transport in the CCD and, at least in other systems, are affected by biomechanical forces. We hypothesized that FSS and CS differentially affect MAPK signaling and PGE2 release to modulate cation transport in the CCD. To validate that CS is a physiological force in vivo, we applied the intravital microscopic approach to rodent kidneys in vivo to show that saline or furosemide injection led to a 46.5 ± 2.0 or 170 ± 32% increase, respectively, in distal tubular diameter. Next, murine CCD (mpkCCD) cells were grown on glass or silicone coated with collagen type IV and subjected to 0 or 0.4 dyne/cm(2) of FSS or 10% CS, respectively, forces chosen based on prior biomechanical modeling of ex vivo microperfused CCDs. Cells exposed to FSS expressed an approximately twofold greater abundance of phospho(p)-ERK and p-p38 vs. static cells, while CS did not alter p-p38 and p-ERK expression compared with unstretched controls. FSS induced whereas CS reduced PGE2 release by ∼40%. In conclusion, FSS and CS differentially affect ERK and p38 activation and PGE2 release in a cell culture model of the CD. We speculate that TFF differentially regulates biomechanical signaling and, in turn, cation transport in the CCD.

Proteomic identification of 14-3-3ϵ as a linker protein between pERK1/2 inhibition and BIM upregulation in human osteosarcoma cells.

Despite advancements in multimodality chemotherapy, conventional cytotoxic treatments still remain ineffective for a subset of patients with aggressive metastatic or multifocal osteosarcoma. It has been shown that pERK1/2 inhibition enhances chemosensitivity to doxorubicin and promotes osteosarcoma cell death in vivo and in vitro. One of the pro-apoptotic mechanisms is upregulation of Bim by pERK1/2 inhibitors. To this end, we examined proteomic changes of 143B human osteosarcoma cells with and without treatment of PD98059, pERK1/2 inhibitor. Specifically, we identified 14-3-3ϵ protein as a potential mediator of Bim expression in response to inhibition of pERK1/2. We hypothesized that 14-3-3ϵ mediates upregulation of Bim expression after pERK1/2 inhibition. We examined the expression of Bim after silencing 14-3-3ϵ using siRNA. The 14-3-3ϵ gene silencing resulted in downregulation of Bim expression after PD98059 treatment. These data indicate that 14-3-3ϵ is required for Bim expression and that it has an anti-cancer effect under pERK1/2 inhibition in 143B cells. By playing an essential role upstream of Bim, 14-3-3ϵ may potentially be a coadjuvant factor synergizing the effect of pERK1/2 inhibitors in addition to conventional cytotoxic agents for more effective osteosarcoma treatments.

Aggravation of inflammatory response by costimulation with titanium particles and mechanical perturbations in osteoblast- and macrophage-like cells.

The interface between bone tissue and metal implants undergoes various types of mechanical loading, such as strain, compression, fluid pressure, and shear stress, from daily activities. Such mechanical perturbations create suboptimal environments at the host bone-implant junction, causing an accumulation of wear particles and debilitating osseous integration, potentially leading to implant failure. While many studies have focused on the effect of particles on macrophages or osteoprogenitor cells, differential and combined effects of mechanical perturbations and particles on such cell types have not been extensively studied. In this study, macrophages and osteoprogenitor cells were subjected to physiological and superphysiological mechanical stimuli in the presence and absence of Ti particles with the aim of simulating various microenvironments of the host bone-implant junction. Macrophages and osteoprogenitor cells were capable of engulfing Ti particles through actin remodeling and also exhibited changes in mRNA levels of proinflammatory cytokines under certain conditions. In osteoprogenitor cells, superphysiological strain increased proinflammatory gene expression; in macrophages, such mechanical perturbations did not affect gene expression. We confirmed that this phenomenon in osteoprogenitor cells occurred via activation of the ERK1/2 signaling pathway as a result of damage to the cytoplasmic membrane. Furthermore, AZD6244, a clinically relevant inhibitor of the ERK1/2 pathway, mitigated particle-induced inflammatory gene expression in osteoprogenitor cells and macrophages. This study provides evidence of more inflammatory responses under mechanical strains in osteoprogenitor cells than macrophages. Phagocytosis of particles and mechanical perturbation costimulate the ERK1/2 pathway, leading to expression of proinflammatory genes.

Actin and ERK1/2-CEBPß signaling mediates phagocytosis-induced innate immune response of osteoprogenitor cells.

Wear particles at the host bone-implant interface are a major challenge for successful bone implant arthoplasties. Current understanding of aseptic loosening consists of macrophage-mediated inflammatory responses and increasing osteoclastogenesis, which lead to an imbalance between bone formation and resorption. Despite its significant role in bone regeneration and implant osteointegration, the osteoprogenitor response to wear particles has been examined recent years. More specifically, the intracellular mechanism of osteoprogenitor mediated inflammation has not been fully elucidated. In this study, we examined the role of osteoprogenitors and the cellular mechanism by which metal wear particles elicit an inflammatory cascade. Through both in vivo and in vitro experiments, we have demonstrated that osteoprogenitor cells are capable of initiating inflammatory responses by phagocytosing wear particles, which lead to subsequent accumulation of macrophages and osteoclastogenesis, and the ERK_CEBP/ß intracellular signaling is a key inflammatory pathway that links phagocytosis of wear particles to inflammatory gene expression in osteoprogenitors. AZD6244 treatment, a potent inhibitor of the ERK pathway, attenuated particle mediated inflammatory osteolysis both in vivo and in vitro. This study advances our understanding of the mechanisms of osteoprogenitor-mediated inflammation, and provides further evidence that the ERK_CEBP/ß pathway may be a suitable therapeutic target in the treatment of inflammatory osteolysis.