論文

Osteoarthritis (OA) is characterized by progressive joint destruction, including synovial membrane alteration. EphB4 and its ligand ephrin-B2 were found in vitro to positively affect OA subchondral bone and cartilage. In vivo in an experimental mouse model overexpressing bone-specific Ephb4 (TgEphB4), a protective effect was found on both the subchondral bone and cartilage during OA. We investigated in the TgEphB4 mouse model the in vivo effect on synovial membrane during OA. Knee OA was surgically induced by destabilization of the medial meniscus (DMM). Synovial membrane was evaluated using histology, histomorphometry, IHC, and real-time PCR. Compared to DMM-wild-type (WT) mice, DMMTgEphB4 mice had a significant decrease in synovial membrane thickness, vascular endothelial growth factor, and the profibrotic markers fibrin, type 1 procollagen, type 3 collagen, connective tissue growth factor, smooth muscle actin-α, cartilage oligomeric matrix protein, and procollagen-lysine, and 2-oxoglutarate 5-dioxygenase 2. Moreover, factors known to modulate transforming growth factor-β signaling, transforming growth factor receptor 1/ALK1, phosphorylated Smad-1, and heat shock protein 90β were significantly decreased in DMM-TgEphB4 compared with DMM-WT mice. Ephb4 overexpression also exhibited a protective effect on synovial membrane thickness of aged (24-month-old) mice. Overexpression of bone-specific Ephb4 clearly demonstrated prevention of the development and/or progression of fibrosis in OA synovial membrane, reinforcing the hypothesis that protecting the subchondral bone prophylactically and during OA reduces the pathologic changes in other articular tissues.

Enhanced resistance to chemotherapy has been correlated with high levels of Delta-Np73 (DNp73), an anti-apoptotic protein of the p53 tumor-suppressor family which inhibits the pro-apoptotic members such as p53 and TAp73. Although genotoxic drugs have been shown to induce DNp73 degradation, lack of mechanistic understanding of this process precludes strategies to enhance the targeting of DNp73 and improve treatment outcomes. Antizyme (Az) is a mediator of ubiquitin-independent protein degradation regulated by the polyamine biosynthesis pathway. We show here that acetylpolyamine oxidase (PAOX), a catabolic enzyme of this pathway, upregulates DNp73 levels by suppressing its degradation via the Az pathway. Conversely, downregulation of PAOX activity by siRNA-mediated knockdown or chemical inhibition leads to DNp73 degradation in an Az-dependent manner. PAOX expression is suppressed by several genotoxic drugs, via selected members of the activator protein-1 (AP-1) transcription factors, namely c-Jun, JunB and FosB, which are required for stress-mediated DNp73 degradation. Finally, chemical- and siRNA-mediated inhibition of PAOX significantly reversed the resistant phenotype of DNp73-overexpressing cancer cells to genotoxic drugs. Together, these data define a critical mechanism for the regulation of DNp73 abundance, and reveal that inhibition of PAOX could widen the therapeutic index of cytotoxic drugs and overcome DNp73-mediated chemoresistance in tumors.

Medullary thymic epithelial cells (mTECs) expressing the autoimmune regulator AIRE and various tissue-specific antigens (TSAs) are critical for preventing the onset of autoimmunity and may attenuate tumor immunity. However, molecular mechanisms controlling mTEC development remain elusive. Here, we describe the roles of the transcription factor Spi-B in mTEC development. Spi-B is rapidly up-regulated by receptor activator of NF-κB ligand (RANKL) cytokine signaling, which triggers mTEC differentiation, and in turn up-regulates CD80, CD86, some TSAs, and the natural inhibitor of RANKL signaling, osteoprotegerin (OPG). Spi-B-mediated OPG expression limits mTEC development in neonates but not in embryos, suggesting developmental stage-specific negative feedback regulation. OPGmediated negative regulation attenuates cellularity of thymic regulatory T cells and tumor development in vivo. Hence, these data suggest that this negative RANKL-Spi-B-OPG feedback mechanism finely tunes mTEC development and function and may optimize the trade-off between prevention of autoimmunity and induction of antitumor immunity.

The human immune system can be reconstituted in experimental animals by transplanting human hematopoietic stem cells (hHSCs) into immunodeficient mice. To generate such humanized mice, further improvements are required, particularly to ensure that transplanted hHSCs are maintained in mice and proliferate long enough to follow prolonged immune responses to chronic diseases or monitor therapeutic effects. To prepare the relatively human bone marrow environment in mice, we generated nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor gamma chain null (NOG) mice expressing human Jagged1 (hJ1) in an osteoblast-specific manner (hJ1-NOG mice) to examine whether Notch signaling induced by hJ1 mediates hHSC proliferation and/or maintenance in mice. The established hJ1-NOG mice possess relatively larger bone marrow space and thinner cortical bone compared with nontransgenic littermates, but the number of c-kit+ Sca-1+ lineage- cells was not significantly different between hJ1-NOG and nontransgenic littermates. In the transplantation experiments of CD34+ cells obtained from human cord blood, CD34+CD38- cells (hHSCs) were more increased in hJ1-NOG recipient mice than in nontransgenic littermates in mouse bone marrow environment. In contrast, the transplanted mouse c-kit+ Sca-1+ lineage- cells did not show significant increase in the same hJ1-NOG mice. These results suggest that hJ1-NOG mice could contribute to the growth of transplanted human CD34+cells in a human-specific manner and be useful to study the invivo behavior and/or development of human stem cells, including cancer stem cells and immune cells.

Osteosarcoma is a malignant bone tumor in children and adolescents characterized by intrinsic therapeutic resistance. The IGF2 is expressed at elevated levels in osteosarcoma after treatment with chemotherapy, prompting an examination of its functional contributions to resistance. We found that continuous exposure to IGF2 or insulin in the absence of serum created a dormant growth state in osteosarcoma cells that conferred resistance to various chemotherapeutic drugs in vitro. Mechanistic investigations revealed that this dormant state correlated with downregulation of downstream signaling by the IGF1 receptor, heightened cell survival, enhanced autophagy, and the presence of extracellular glutamine. Notably, inhibiting autophagy or depleting glutamine was sufficient to increase chemotherapeutic sensitivity in osteosarcoma xenografts in mice. Clinically, we confirmed that IGF expression levels were elevated in human osteosarcoma specimens from patients who received chemotherapy. Together, our results suggest that activation of IGF or insulin signaling preserves the survival of osteosarcoma cells under chemotherapeutic stress, providing a drug-resistant population that may engender minimal residual disease. Attenuating this survival mechanism may help overcome therapeutic resistance in osteosarcoma.

The three-dimensional network of lacunae and canaliculi that regulates metabolism in bone contains osteocytes and their dendritic processes. We constructed a synchrotron radiation X-ray microscope for sequential tomography of mouse tibia first by using a Talbot interferometer to detect the degree of bone mineralization and then by using absorption contrast under a slightly defocused setting to enhance outline contrast thereby visualizing structures of the osteocyte lacuno-canalicular network. The resultant pair of tomograms was precisely aligned with each other, allowing evaluation of mineral density in the vicinity of each osteocyte lacuna and canaliculus over the entire thickness of the cortical bone. Thus, multiscan microscopic X-ray tomography is a powerful tool for analyzing bone mineralization in relation to the lacuno-canalicular network at the submicron resolution level.

Osteoclasts are bone-resorbing cells of monocytic origin. An imbalance between bone formation and resorption can lead to osteoporosis or osteopetrosis. Osteoclastogenesis is triggered by RANKL- and IP3-induced Ca2+ influx followed by activation of NFATc1, a master transcription factor for osteoclastogenic gene regulation. During differentiation, osteoclasts undergo cytoskeletal remodeling to migrate and attach to the bone surface. Simultaneously, they fuse with each other to form multinucleated cells. These processes require PI3-kinase-dependent cytoskeletal protein activation to initiate cytoskeletal remodeling, resulting in the formation of circumferential podosomes and fusion-competent protrusions. In multinucleated osteoclasts, circumferential podosomes mature into stabilized actin rings, which enables the formation of a ruffled border where intensive membrane trafficking is executed. Membrane lipids, especially phosphoinositides, are key signaling molecules that regulate osteoclast morphology and act as second messengers and docking sites for multiple important effectors. We examine the critical roles of phosphoinositides in the signaling cascades that regulate osteoclast functions.