Hiroki Yokota

Size-Dependent Positioning of Human Chromosomes in Interphase Nuclei

By using a fluorescence in situ hybridization technique we revealed that for nine different q-arm telomere markers the positioning of chromosomes in human G(1) interphase nuclei was chromosome size-dependent. The q-arm telomeres of large chromosomes are more peripherally located than telomeres on small chromosomes. This highly organized arrangement of chromatin within the human nucleus was discovered by determining the x and y coordinates of the hybridization sites and calculating the root-mean-square radial distance to the nuclear centers in human fibroblasts. We demonstrate here that global organization within the G(1) interphase nucleus is affected by one of the most fundamental physical quantities-chromosome size or mass-and propose two biophysical models, a volume exclusion model and a mitotic preset model, to explain our finding.

Osteoblast Responses One Hour After Load-Induced Fluid Flow in a Three-Dimensional Porous Matrix

When bone is loaded, substrate strain is generated by the external force and this strain induces fluid flow that creates fluid shear stress on bone cells. Our current understanding of load-driven gene regulation of osteoblasts is based primarily on in vitro studies on planer two-dimensional tissue culture substrates. However, differences between a flat layer of cells and cells in 3-dimensional (3D) ECM are being recognized for signal transduction. Proliferation and differentiation of osteoblasts are affected by substrate geometry. Here we developed a novel 3D culture system that would mimic physiologically relevant substrate strain as well as strain-induced fluid flow in a 3D porous collagen matrix. The system allowed us to evaluate the responses of osteoblasts in a 3D stress-strain environment similar to a mechanical field to which bone is exposed. Using MC3T3-E1 osteoblasts grown in the 3D collagen matrix with and without hydroxyapatite deposition, we tested the role of strain and the strain-induced fluid flow in the expression of the load-responsive genes such as c-fos, egr1, cox2, osteopontin, and mmp1B involved in transcriptional regulation, osteogenesis, and rearrangement of ECM. Strain-induced fluid flow was visualized with a microspheres ~3 μm in diameter in real time, and three viscoelastic parameters were determined. The results obtained by semi-quantitative PCR, immunoblot assay, enzymatic activity assays for collagenase and gelatinase, and mechanical characterization of collagen matrices supported the dominant role of strain-induced fluid flow in expression of the selected genes one hour after the mechanical treatment.

Stress to Endoplasmic Reticulum of Mouse Osteoblasts Induces Apoptosis and Transcriptional Activation for Bone Remodeling

ATF4 is an essential regulator in osteogenesis as well as in stress responses to the endoplasmic reticulum (ER). We addressed a question: Does ER stress to osteoblasts upregulate ATF4 expression? If so, do they exhibit ATF4‐mediated bone remodeling or apoptosis? ER stress, induced by Thapsigargin and tunicamycin, elevated a phosphorylated form of eIF2α and ATF4, but the cellular fate depended on treatment duration. The treatment for 1 h, for instance, activated Runx2, and type I collagen, while the treatment for 24 h induced apoptosis. Our observations suggest that there is a threshold for ER stress and osteoblasts present a bi‐phasic pattern of their fate.

Reduction of cytokine-induced expression and activity of MMP-1 and MMP-13 by mechanical strain in MH7A rheumatoid synovial cells

Excessive mechanical load induces harmful outcomes for joint diseases, such as osteoarthritis and rheumatoid arthritis, but physical stimuli at appropriate intensity are essential for growth and maintenance of bone and articular cartilage. Using a fibroblast-like synoviocyte cell line derived from a patient with rheumatoid arthritis, we examined the effects of gentle cyclic strain, focusing on the expression and activity of matrix metalloproteinase-1 (MMP-1) and MMP-13. Synovial cells were cultured on a collagen-coated agar block and exposed to 2% cyclic strain at 6 rev./min for 1 h. Expression of MMP-1 and MMP-13 was assayed using semi-quantitative and real-time PCR, as well as immunoblotting. Their activity was measured using spectrofluorometry and zymography. The results showed that the cyclic strain reduced the mRNA and protein levels of MMP-1 and MMP-13, and that both collagenase and gelatinase activity was decreased under the strain. The reduction in MMP activity by the cyclic strain was not achieved by the transcriptional inhibitor, actinomycin D. In the presence of proinflammatory cytokines, such as IL-1 beta and TNF-alpha, the strain reduced the cytokine-induced expression and activities of MMPs. Interestingly, the strain elevated the mRNA level of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. These results support a potential role of mechanical strain in down-regulating the cytokine-mediated proteolytic processes in synoviocytes.

Osteogenic potentials with joint-loading modality

Osteogenic potentials with a novel joint-loading modality were examined, using mouse ulnae as a model system. Load-induced deformation of rigid bone is known to generate interstitial fluid flow and stimulate osteogenesis. However, in most of the previous studies, loads were applied to cortical bone. In the current study, we addressed the question of whether deformation of the epiphysis underneath the joint would enhance bone formation in the epiphysis and the diaphysis. In order to answer the question, we applied lateral loads to a mouse elbow and conducted a bone histomorphometric analysis, as well as measurements of strains and streaming potentials. Compared to the no-loading control, the histomorphometric results showed that 0.5-N loads, applied to the elbow at 2 Hz for 3 min/day for 3 consecutive days, increased the mineralizing surface (two- to threefold), the rate of mineral apposition (three- to fivefold), and the rate of bone formation (six- to eightfold) in the ulna. Strain measurements indicated that strains of around 30 µstrain, induced with the joint-loading modality, were under the minimum effective strain of around 1000 µstrain, which is considered necessary to achieve strain-driven bone formation. To evaluate the induction of fluid flow with the joint-loading modality, streaming potentials were measured in separate experiments, using mouse femurs ex vivo. We found that the streaming potentials correlated to the magnitude of the load applied to the epiphysis (r2 = 0.92), as well as the flow speed in the medullary cavity (r2 = 0.93). Taken together, the findings of the current study support the idea of joint-loading driven osteogenesis, through a mechanism that involves the induction of fluid flow in cortical bone.

A brief review of bone adaptation to unloading.

Weight-bearing bone is constantly adapting its structure and function to mechanical environments. Loading through routine exercises stimulates bone formation and prevents bone loss, but unloading through bed rest and cast immobilization as well as exposure to weightlessness during spaceflight reduces its mass and strength. In order to elucidate the mechanism underlying unloading-driven bone adaptation, ground-based in vitro and in vivo analyses have been conducted using rotating cell culturing and hindlimb suspension. Focusing on gene expression studies in osteoblasts and hindlimb suspension studies, this minireview introduces our recent understanding on bone homeostasis under weightlessness in space. Most of the existing data indicate that unloading has the opposite effects to loading through common signaling pathways. However, a question remains as to whether any pathway unique to unloading (and not to loading) may exist.

Mechanical Loading: Bone Remodeling and Cartilage Maintenance

Bone remodeling and cartilage maintenance are strongly influenced by biomechanical signals generated by mechanical loading. Although moderate loading is required to maintain bone mass and cartilage homeostasis, loading can cause deleterious effects such as bone fracture and cartilage degradation. Because a tight coupling exists between cartilage and bone, alterations in one tissue can affect the other. Bone marrow lesions are often associated with an increased risk of developing cartilage defects, and changes in the articular cartilage integrity are linked to remodeling responses in the underlying bone. Although mechanisms regulating the maintenance of these two tissues are different, compelling evidence indicates that the signal pathways crosstalk, particularly with the Wnt pathway. A better understanding of the complex tempero-spatial interplay between bone remodeling and cartilage degeneration will help develop a therapeutic loading strategy that prevents bone loss and cartilage degeneration.

Messenger-RNA expression of matrix metalloproteinases, tissue inhibitors of metalloproteinases, and transcription factors in rheumatic synovial cells under mechanical stimuli

In an effort to elucidate the role of mechanical stimuli in rheumatoid arthritis, we determined mRNA levels of matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, and three transcription factors (c-fos, ets-1, and ets-2) under two mechanical shearing conditions as well as simulated unloading. Human synovial cell cultures (MH7A and RA99-01), derived from rheumatoid arthritis patients, were grown for 1 h under mechanical stimuli and the transcript level was assayed by the reverse transcription-polymerase-chain reaction procedure. First, gentle shearing, estimated at approximately 1 dyn/cm(2), induced a consistent decrease in mRNA level of MMP-1, MMP-3, MMP-13, and ets-1 and an increase in the transcript level of TIMP-1, TIMP-2, c-fos, and ets-2. Second, intermediate shearing, estimated at approximately 6 dyn/cm(2), elevated the mRNA level of all MMPs, TIMPs, and the three transcription factors. Third, minimum mRNA level of c-fos, ets-1, and ets-2 was achieved under control conditions at rest, gentle shearing, and simulated unloading, respectively. These in vitro results support a stimulus-dependent transcriptional regulation of MMPs, TIMPs, and transcription factors in cell cultures, suggesting a potential role of shear stress in tissue degradation and prevention in rheumatic joints.

Involvement of p38 MAPK in Regulation of MMP13 mRNA in Chondrocytes in Response to Surviving Stress to Endoplasmic Reticulum

MMP13 is enriched in mature chondrocytes and considered a prime cause of ECM degradation in the osteoarthritic articular cartilage in temporomandibular joints. We asked whether surviving stress to the endoplasmic reticulum (ER) would upregulate transcription of MMP13, and if so, whether a cross-talk would exist between surviving ER stress and p38 MAPK pathways. Using C28/I2 chondrocyte cell line, ER stress was induced by thapsigargin and tunicamycin and upregulation of phosphorylated eIF2alpha and ATF4 protein was observed. Both thapsigargin and tunicamycin elevated the mRNA level of MMP13 and phosphorylation of p38 MAPK. Thapsigargin-induced MMP13 mRNA upregulation was significantly suppressed by SB203580, while its upregulation by tunicamycin was completely attenuated by SB203580. Those results support that homeostasis of chondrocytes is affected by the surviving ER stress through p38 MAPK pathways, suggesting a potential role of ER stress in joint diseases such as osteoarthritis.

Suppression of osteoclastogenesis through phosphorylation of eukaryotic translation initiation factor 2 alpha

In response to various stresses including viral infection, nutrient deprivation, and stress to the endoplasmic reticulum, eukaryotic translation initiation factor 2 alpha (eIF2α) is phosphorylated to cope with stress induced apoptosis. Although bone cells are sensitive to environmental stresses that alter the phosphorylation level of eIF2α, little is known about the role of eIF2α mediated signaling during the development of bone-resorbing osteoclasts. Using two chemical agents (salubrinal and guanabenz) that selectively inhibit de-phosphorylation of eIF2α, we evaluated the effects of phosphorylation of eIF2α on osteoclastogenesis of RAW264.7 pre-osteoclasts as well as development of MC3T3 E1 osteoblast-like cells. The result showed that salubrinal and guanabenz stimulated matrix deposition of osteoblasts through upregulation of activating transcription factor 4 (ATF4). The result also revealed that these agents reduced expression of the nuclear factor of activated T cells c1 (NFATc1) and inhibited differentiation of RAW264.7 cells to multi-nucleated osteoclasts. Partial silencing of eIF2α with RNA interference reduced suppression of salubrinal/guanabenz-driven downregulation of NFATc1. Collectively, we demonstrated that the elevated phosphorylation level of eIF2α not only stimulates osteoblastogenesis but also inhibit osteoclastogenesis through regulation of ATF4 and NFATc1. The results suggest that eIF2α-mediated signaling might provide a novel therapeutic target for preventing bone loss in osteoporosis.