Kazunori Hamamura

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.

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.

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.

RhoA-Mediated Signaling in Mechanotransduction of Osteoblasts

Osteoblasts play a pivotal role in load-driven bone formation by activating Wnt signaling through a signal from osteocytes as a mechanosensor. Osteoblasts are also sensitive to mechanical stimulation, but the role of RhoA, a small GTPase involved in the regulation of cytoskeleton adhesion complexes, in mechanotransduction of osteoblasts is not completely understood. Using MC3T3-E1 osteoblast-like cells under 1 hr flow treatment at 10 dyn/cm2, we examined a hypothesis that RhoA signaling mediates the cellular responses to flow-induced shear stress. To test the hypothesis, we conducted genome-wide pathway analysis and evaluated the role of RhoA in molecular signaling. Activity of RhoA was determined with a RhoA biosensor, which determined the activation state of RhoA based on a fluorescence resonance energy transfer between CFP and YFP fluorophores. A pathway analysis indicated that flow treatment activated phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling as well as a circadian regulatory pathway. Western blot analysis revealed that in response to flow treatment phosphorylation of Akt in PI3K signaling and phosphorylation of p38 and ERK1/2 in MAPK signaling were induced. FRET measurement showed that RhoA was activated by flow treatment, and an inhibitor to a Rho kinase significantly reduced flow-induced phosphorylation of p38, ERK1/2, and Akt as well as flow-driven elevation of the mRNA levels of osteopontin and cyclooxygenase-2. Collectively, the result demonstrates that in response to 1 hr flow treatment to MC3T3-E1 cells at 10 dyn/cm2, RhoA plays a critical role in activating PI3K and MAPK signaling as well as modulating the circadian regulatory pathway.

Effects of salubrinal on development of osteoclasts and osteoblasts from bone marrow-derived cells

BackgroundOsteoporosis is a skeletal disease leading to an increased risk of bone fracture. Using a mouse osteoporosis model induced by administration of a receptor activator of nuclear factor kappa-B ligand (RANKL), salubrinal was recently reported as a potential therapeutic agent. To evaluate the role of salubrinal in cellular fates as well as migratory and adhesive functions of osteoclast/osteoblast precursors, we examined the development of primary bone marrow-derived cells in the presence and absence of salubrinal. We addressed a question: are salubrinal’s actions more potent to the cells isolated from the osteoporotic mice than those isolated from the control mice?MethodsUsing the RANKL-injected and control mice, bone marrow-derived cells were harvested. Osteoclastogenesis was induced by macrophage-colony stimulating factor and RANKL, while osteoblastogenesis was driven by dexamethasone, ascorbic acid, and β-glycerophosphate.ResultsThe results revealed that salubrinal suppressed the numbers of colony forming-unit (CFU)-granulocyte/macrophages and CFU-macrophages, as well as formation of mature osteoclasts in a dosage-dependent manner. Salubrinal also suppressed migration and adhesion of pre-osteoclasts and increased the number of CFU-osteoblasts. Salubrinal was more effective in exerting its effects in the cells isolated from the RANKL-injected mice than the control. Consistent with cellular fates and functions, salubrinal reduced the expression of nuclear factor of activated T cells c1 (NFATc1) as well as tartrate-resistant acid phosphatase.ConclusionsThe results support the notion that salubrinal exhibits significant inhibition of osteoclastogenesis as well as stimulation of osteoblastogenesis in bone marrow-derived cells, and its efficacy is enhanced in the cells harvested from the osteoporotic bone samples.

Salubrinal reduces expression and activity of MMP13 in chondrocytes

OBJECTIVE Stress to the endoplasmic reticulum (ER) and inflammatory cytokines induce expression and activity of matrix metalloproteinase 13 (MMP13). Since a synthetic agent, salubrinal, is known to alleviate ER stress and attenuate nuclear factor kappa B (NFκB) signaling, we addressed a question whether upregulation of MMP13 by ER stress and cytokines is suppressed by administration of salubrinal. METHODS Using C28/I2 human chondrocytes, we applied ER stress with tunicamycin and inflammatory distress with tumor necrosis factor α (TNFα) and interleukin 1β (IL1β). RNA interference with siRNA specific to NFκB p65 (RelA) was employed to examine a potential involvement of NFκB signaling in salubrinals action in regulation of MMP13. We also employed primary human chondrocytes and evaluated MMP13 activity. RESULTS The result showed that tunicamycin activated p38 mitogen-activated protein kinase (MAPK), while inflammatory cytokines activated p38 MAPK and NFκB. In both cases, salubrinal significantly reduced expression and activity of MMP13. Silencing NFκB reduced inflammatory cytokine-driven upregulation of MMP13 activity. CONCLUSIONS The results demonstrate that salubrinal downregulates expression and activity MMP13 through p38 and NFκB signaling, suggesting its potential usage to treat degenerative diseases such as osteoarthritis.

In vitro and in silico analysis of an inhibitory mechanism of osteoclastogenesis by salubrinal and guanabenz.

Inactivating bone-resorbing osteoclasts is a prime therapeutic strategy for the prevention of bone loss in patients with osteopenia and osteoporosis. Synthetic agents such as salubrinal and guanabenz, which attenuate stress to the endoplasmic reticulum, are reported to inhibit development of osteoclasts. However, the mechanism of their inhibitory action on osteoclasts is largely unknown. Using genome-wide expression profiles, we predicted key transcription factors that downregulated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a master transcription factor for osteoclastogenesis. Principal component analysis (PCA) predicted a list of transcription factors that were potentially responsible for reversing receptor activator of nuclear factor kappa-B ligand (RANKL)-driven stimulation of osteoclastogenesis. A partial silencing of NFATc1 allowed a selection of transcription factors that were likely to be located upstream of NFATc1. We validated the predicted transcription factors by focusing on two AP-1 transcription factors (c-Fos and JunB) using RAW264.7 pre-osteoclasts as well as primary bone marrow cells. As predicted, their mRNA and protein levels were elevated by RANKL, and the elevation was suppressed by salubrinal and guanabenz. A partial silencing of c-Fos or JunB by RNA interference decreased NFATc1 as well as tartrate-resistant acid phosphatase (TRAP) mRNA. Collectively, a systems-biology approach allows the prediction of a RANKL-salubrinal/guanabenz-NFATc1 regulatory axis, and in vitro assays validate an involvement of AP-1 transcription factors in suppression of osteoclastogenesis.

Microarray analysis of thapsigargin - induced stress to the endoplasmic reticulum of mouse osteoblasts

Activating transcription factor 4 (ATF4) protein has a dual role in osteoblasts. It functions as a responder to stress to the endoplasmic reticulum (ER) as well as a transcription factor for bone formation. Little is known about molecular pathways that can potentially lead to stress-induced apoptosis or homeostasis of extracellular matrix (ECM) molecules. Based on microarray-derived mRNA expression data for mouse osteoblasts (MC3T3 E1 cells, clone 4), we analyzed the ER-stress responses in the presence of 10 nM Thapsigargin using two computational approaches: “Gene Set Enrichment Analysis (GSEA)” and “Ingenuity Pathways Analysis (IPA).” GSEA presented a strong linkage to an expression pattern observed in the responses to hypoxia, and IPA identified two molecular pathways: ATF4-unlinked connective tissue development and ATF4-linked organ morphology. Real-time polymerase chain reaction (PCR) and Western blot analyses validated eIF2α-driven translational regulation as well as ATF4-linked transcriptional activation of transcription factors and growth factors including FOS, FGF-9, and BMP-2. Consistent with the role of p38 MAPK in hypoxia, phosphorylation of p38 MAPK was activated in nonapoptotic osteoblasts under surviving ER stress. Furthermore, the level of phosphorylated PERK was elevated. These results support cross-talk between p38 MAPK and ER kinase, presenting a similarity to the responses to hypoxia as well as a pathway toward connective tissue development and organ morphology.

Chondroprotective effects of Salubrinal in a mouse model of osteoarthritis.

Objectives Salubrinal is a synthetic agent that elevates phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) and alleviates stress to the endoplasmic reticulum. Previously, we reported that in chondrocytes, Salubrinal attenuates expression and activity of matrix metalloproteinase 13 (MMP13) through downregulating nuclear factor kappa B (NFκB) signalling. We herein examine whether Salubrinal prevents the degradation of articular cartilage in a mouse model of osteoarthritis (OA). Methods OA was surgically induced in the left knee of female mice. Animal groups included age-matched sham control, OA placebo, and OA treated with Salubrinal or Guanabenz. Three weeks after the induction of OA, immunoblotting was performed for NFκB p65 and p-NFκB p65. At three and six weeks, the femora and tibiae were isolated and the sagittal sections were stained with Safranin O. Results Salubrinal suppressed the progression of OA by downregulating p-NFκB p65 and MMP13. Although Guanabenz elevates the phosphorylation level of eIF2α, it did not suppress the progression of OA. Conclusions Administration of Salubrinal has chondroprotective effects in arthritic joints. Salubrinal can be considered as a potential therapeutic agent for alleviating symptoms of OA. Cite this article: Bone Joint Res 2015;4:84–92