Mesenchymal Stem Cells Preconditioned with Hypoxia and Dexamethasone Promote Osteoblast Differentiation Under Stress Conditions.

Bone marrow-derived mesenchymal stem cells (MSCs), which are capable of differentiating into osteoblasts, are used in effective regenerative therapies. MSCs must be prompted to differentiate into osteoblasts for MSC transplantation to be effective. In this study, osteoblast differentiation markers involved in bone formation were evaluated to investigate the stress resistance of bone marrow-derived rat MSCs to dexamethasone and hypoxia and their ability to differentiate into osteoblasts. MSCs were allowed to differentiate into osteoblasts for 21 days in three different environments (dexamethasone treatment, hypoxic conditions [1% oxygen], or both). Osteoblast differentiation potential was evaluated according to alkaline phosphatase levels and a mineralisation assay. Immunofluorescence staining was used to determine the protein expression of the osteoblast differentiation markers type I collagen and osteopontin. MSCs differentiated into osteoblasts under hypoxic conditions but differentiated more slowly upon treatment with dexamethasone and dexamethasone plus hypoxia relative to the control. MSCs preconditioned with dexamethasone or hypoxia and then allowed to differentiate into osteoblasts under similar conditions differentiated comparably to control MSCs. MSCs that developed resistance to dexamethasone or hypoxia differentiated more quickly into osteoblasts than those that did not. The findings suggest that increasing the resistance of MSCs to stress by preconditioning them via dexamethasone or hypoxia exposure could result in more rapid differentiation into osteoblasts following transplantation.

A morphological study of adipose-derived stem cell sheets created with temperature-responsive culture dishes using scanning electron microscopy.

Adipose-derived stem cell (ADSC) sheets have potential to be effective in various therapies. In this study, we first demonstrated that a cell sheet composed of human ADSCs could be created using a new temperature-responsive culture dish from the DIC Corporation. The dish can cause detachment of adherent cells due to temperature changes, but a few morphological analyses have evaluated the presence or absence of damage on the detached surface of cell sheet. To characterize our ADSC sheet, we tried to observe the surface of ADSC sheets with scanning electron microscope (SEM) using the ionic liquid, which enables the rapid preparation of samples. No damage was found on the surface of the ADSC sheets on the side that had been in contact with the surface of the culture dishes. In addition, when the transcriptomes of the harvested cell sheets were compared with those of monolayer cultures, no up-regulation of cell death related genes were detected. These results propose that the detachment from temperature-responsive culture dish causes no serious damage on the prepared ADSC sheet. It is also suggested that the SEM with ionic liquids is a useful and rapid method for the analysis of ADSC sheets for therapy.

Resistance of bone marrow mesenchymal stem cells in a stressed environment - Comparison with osteocyte cells.

Introduction: Recently, the efficacy of mesenchymal stem cells (MSCs) mediated by their tissue repair and anti-inflammatory actions in the prevention and therapy of various disorders has been reported. In this research, our attention was focused specifically on the prevention and therapy of glucocorticoid-induced osteonecrosis. We investigated the stress resistance of MSC against glucocorticoid administration and hypoxic stress, which are factors known to induce osteocytic cell death. Materials and Methods: Mouse bone cells (MLO-Y4) and bone-marrow derived mouse MSCs were exposed to dexamethasone (Dex), hypoxia of 1% oxygen or both in vitro. Mitochondrial membrane potentials were estimated by mitochondria labeling with a cell-permeant probe (Mito tracker red); expression of these apoptosis-inducing molecules, oxidative stress marker (8-hydroxy-2'-deoxyguanosine), caspase-3, -9, and two apoptosis-inhibiting molecules, energy-producing ATP synthase (ATP5A) and X-linked inhibitor of apoptosis protein (XIAP), were analyzed by both immunofluorescence and western blot. Results: With exposure to either dexamethasone or hypoxia, MLO-Y4 showed reduced mitochondrial membrane potential, ATP5A and upregulation of 8-OHdG, cleaved caspases and XIAP. Those changes were significantly enhanced by treatment with dexamethasone plus hypoxia. In MSCs, however, mitochondrial membrane potentials were preserved, while no significant changes in the pro-apoptosis or anti-apoptosis molecules analyzed were found even with exposure to both dexamethasone and hypoxia. No such effects induced by treatment with dexamethasone, hypoxia, or both were demonstrated in MSCs at all. Discussion: In osteocyte cells subjected to the double stresses of glucocorticoid administration and a hypoxic environment osteocytic cell death was mediated via mitochondria. In contrast, MSC subjected to the same stressors showed preservation of mitochondrial function and reduced oxidative stress. Accordingly, even under conditions sufficiently stressful to cause the osteocytic cell death in vivo, it was thought that the function of MSC could be preserved, suggesting that in the case of osteonecrosis preventative and therapeutic strategies incorporating their intraosseous implantation may be promising.

Involvement of Autophagy in Rat Tail Static Compression-Induced Intervertebral Disc Degeneration and Notochordal Cell Disappearance.

The intervertebral disc is the largest avascular low-nutrient organ in the body. Thus, resident cells may utilize autophagy, a stress-response survival mechanism, by self-digesting and recycling damaged components. Our objective was to elucidate the involvement of autophagy in rat experimental disc degeneration. In vitro, the comparison between human and rat disc nucleus pulposus (NP) and annulus fibrosus (AF) cells found increased autophagic flux under serum deprivation rather in humans than in rats and in NP cells than in AF cells of rats (n = 6). In vivo, time-course Western blotting showed more distinct basal autophagy in rat tail disc NP tissues than in AF tissues; however, both decreased under sustained static compression (n = 24). Then, immunohistochemistry displayed abundant autophagy-related protein expression in large vacuolated disc NP notochordal cells of sham rats. Under temporary static compression (n = 18), multi-color immunofluorescence further identified rapidly decreased brachyury-positive notochordal cells with robust expression of autophagic microtubule-associated protein 1 light chain 3 (LC3) and transiently increased brachyury-negative non-notochordal cells with weaker LC3 expression. Notably, terminal deoxynucleotidyl transferase dUTP nick end labeling-positive apoptotic death was predominant in brachyury-negative non-notochordal cells. Based on the observed notochordal cell autophagy impairment and non-notochordal cell apoptosis induction under unphysiological mechanical loading, further investigation is warranted to clarify possible autophagy-induced protection against notochordal cell disappearance, the earliest sign of disc degeneration, through limiting apoptosis.

Mitochondrial Transcription Factor A added to Osteocytes in a Stressed Environment has a Cytoprotective Effect.

The main precipitant of glucocorticoid-associated femoral head osteonecrosis is widely accepted to be an ischemic-hypoxic event, with oxidative stress also as an underlying factor. Mitochondrial DNA is more vulnerable to oxidative injury than the nucleus, and mitochondrial transcription factor A (TFAM), which plays roles in its function, preservation, and regulation is being increasingly investigated. In the present study we focused on the impact of TFAM on the relation between the oxidative injury induced by the addition of glucocorticoid to a hypoxic environment and osteocytic cell necrosis. Using cultured osteocytes MLO-Y4 in a 1% hypoxic environment (hypoxia) to which 1µM dexamethasone (Dex) was added (Dex(+)/hypoxia(+)), an immunocytochemical study was conducted using 8-hydroxy-2'-deoxyguanosine (8-OHdG), an index of oxidative stress, and hypoxia inducible factor-1α (HIF-1α), a marker of hypoxia. Next, after adding TFAM siRNA, TFAM knockdown, cultured for 24h, and mitochondrial membrane potential were measured, they were stained with ATP5A which labels adenosine triphosphate (ATP) production. Dex was added to MLO-Y4 to which TFAM had been added, and cultured for 24h in hypoxia. The ratio of dead cells to viable cells was determined and compared. Enhanced expression of 8-OHdG, HIF-1α was found in osteocytes following the addition of glucocorticoid in a hypoxic environment. With TFAM knockdown, as compared to normoxia, mitochondrial function significantly decreased. On the other hand, by adding TFAM, the incidence of osteocytic cell necrosis was significantly decreased as compared with Dex(+)/hypoxia(+). TFAM was confirmed to be important in mitochondrial function and preservation, inhibition of oxidative injury and maintenance of ATP production. Moreover, prevention of mitochondrial injury can best be achieved by decreasing the development of osteocytic cell necrosis.

Taurine Inhibits Glucocorticoid-Induced Bone Mitochondrial Injury, Preventing Osteonecrosis in Rabbits and Cultured Osteocytes.

Mitochondrial injury has recently been implicated in the pathogenesis of glucocorticoid-induced osteonecrosis. Using cultured osteocytes and a rabbit model, we investigated the possibility that taurine (TAU), which is known to play a role in the preservation of mitochondrial function, might also prevent the development of osteonecrosis. To reduplicate the intraosseous environment seen in glucocorticoid-induced osteonecrosis, dexamethasone (Dex) was added to MLO-Y4 cultured in 1% hypoxia (H-D stress environment). An in vitro study was conducted in which changes in mitochondrial transcription factor A (TFAM), a marker of mitochondrial function, and ATP5A produced by mitochondria, induced by the presence/absence of taurine addition were measured. To confirm the effect of taurine in vivo, 15 Japanese White rabbits were administered methylprednisolone (MP) 20 mg/kg as a single injection into the gluteus muscle (MP+/TAU- group), while for 5 consecutive days from the day of MP administration, taurine 100 mg/kg was administered to 15 animals (MP+/TAU+ group). As a control 15 untreated rabbits were also studied. The rabbits in each of the groups were sacrificed on the 14th day after glucocorticoid administration, and the bilateral femora were harvested. Histopathologically, the incidence of osteonecrosis was quantified immunohistochemically by quantifying TFAM and ATP5A expression. In the rabbits exposed to an H-D stress environment and in MP+/TAU- group, TFAM and ATP5A expression markedly decreased. With addition of taurine in the in vitro and in vivo studies, the expression of TFAM and ATP5A was somewhat decreased as compared with Dex-/hypoxia- or MP-/TAU- group, while improvement was noted as compared with Dex+/hypoxia+ or MP+/TAU- group. In rabbits, the incidence of osteonecrosis was 80% in MP+/TAU- group, in contrast to 20% in the taurine administered group (MP+/TAU+), representing a significant decrease. Since taurine was documented to exert a protective effect on mitochondrial function by inhibiting the mitochondrial dysfunction associated with glucocorticoid administration, we speculated that it might also indirectly help to prevent the development of osteonecrosis in this context. Since taurine is already being used clinically, we considered that its clinical application would also likely be smooth.

Tensile Loads on Tethered Actin Filaments Induce Accumulation of Cell Adhesion-Associated Proteins in Vitro.

Focal adhesions (FAs) and adherens junctions (AJs), which serve as a mechanical interface of cell-matrix and cell-cell interactions, respectively, experience tensile force either originating from the deformation of the surrounding tissues or generated by the actomyosin machinery in the cell. These mechanical inputs cause enlargement of FAs and AJs, while the detailed mechanism for the force-dependent development of FAs and AJs remain unclear. Both FAs and AJs provide sites for tethering of actin filaments and actin polymerization. Here, we develop a cell-free system, in which actin filaments are tethered to glass surfaces, and show that application of tensile force to the tethered filaments in the cell extract induces accumulation of several FA and AJ proteins, associated with further accumulation of actin filaments via de novo actin polymerization. Decline in the tensile force results in a decrease in the amount of the accumulated proteins. These results suggest that the tensile force acting on the tethered actin filaments plays a crucial role in the accumulation of FA and AJ proteins.

DMPK is a New Candidate Mediator of Tumor Suppressor p53-Dependent Cell Death.

Tumor suppressor p53 plays an integral role in DNA-damage induced apoptosis, a biological process that protects against tumor progression. Cell shape dramatically changes when cells undergo apoptosis, which is associated with actomyosin contraction; however, it remains entirely elusive how p53 regulates actomyosin contraction in response to DNA-damaging agents. To identify a novel p53 regulating gene encoding the modulator of myosin, we conducted DNA microarray analysis. We found that, in response to DNA-damaging agent doxorubicin, expression of myotonic dystrophy protein kinase (DMPK), which is known to upregulate actomyosin contraction, was increased in a p53-dependent manner. The promoter region of DMPK gene contained potential p53-binding sequences and its promoter activity was increased by overexpression of the p53 family protein p73, but, unexpectedly, not of p53. Furthermore, we found that doxorubicin treatment induced p73 expression, which was significantly attenuated by downregulation of p53. These data suggest that p53 induces expression of DMPK through upregulating p73 expression. Overexpression of DMPK promotes contraction of the actomyosin cortex, which leads to formation of membrane blebs, loss of cell adhesion, and concomitant caspase activation. Taken together, our results suggest the existence of p53-p73-DMPK axis which mediates DNA-damage induced actomyosin contraction at the cortex and concomitant cell death.

MEKK1-dependent phosphorylation of calponin-3 tunes cell contractility.

MEKK1 (also known as MAP3K1), which plays a major role in MAPK signaling, has been implicated in mechanical processes in cells, such as migration. Here, we identify the actin-binding protein calponin-3 as a new MEKK1 substrate in the signaling that regulates actomyosin-based cellular contractility. MEKK1 colocalizes with calponin-3 at the actin cytoskeleton and phosphorylates it, leading to an increase in the cell-generated traction stress. MEKK1-mediated calponin-3 phosphorylation is attenuated by the inhibition of myosin II activity, the disruption of actin cytoskeletal integrity and adhesion to soft extracellular substrates, whereas it is enhanced upon cell stretching. Our results reveal the importance of the MEKK1-calponin-3 signaling pathway to cell contractility.

Planar compression of extracellular substrates induces S phase arrest via ATM-independent CHK2 activation.

Cell proliferation is regulated not only by soluble chemical factors but also by mechanical cues surrounding cells. Mechanical stretch of extracellular substrates is known to promote cell proliferation by driving exit from the G0 phase and entry into the S phase. Here, we report that planer compression of extracellular substrates induces cell cycle arrest in the S phase. The compression-induced S phase arrest is mediated by the checkpoint kinase 2 (CHK2)-p53 pathway. In contrast to the canonical S phase checkpoint pathway activated by DNA damage, CHK2 activation by the substrate compression is independent of ataxia telangiectasia mutated (ATM). We further find that disassembly of the actin cytoskeleton is required for the compression-induced S phase arrest. Notably, cancer cells do not exhibit S phase arrest upon the substrate compression. Our results suggest a novel mechanism for homeostatic control of cell growth under mechanical perturbations.