Structural variants shape driver combinations and outcomes in pediatric high-grade glioma.

We analyzed the contributions of structural variants (SVs) to gliomagenesis across 179 pediatric high-grade gliomas (pHGGs). The most recurrent SVs targeted MYC isoforms and receptor tyrosine kinases (RTKs), including an SV amplifying a MYC enhancer in 12% of diffuse midline gliomas (DMG), indicating an underappreciated role for MYC in pHGG. SV signature analysis revealed that tumors with simple signatures were TP53 wild type (TP53WT) but showed alterations in TP53 pathway members PPM1D and MDM4. Complex signatures were associated with direct aberrations in TP53, CDKN2A and RB1 early in tumor evolution and with later-occurring extrachromosomal amplicons. All pHGGs exhibited at least one simple-SV signature, but complex-SV signatures were primarily restricted to subsets of H3.3K27M DMGs and hemispheric pHGGs. Importantly, DMGs with complex-SV signatures were associated with shorter overall survival independent of histone mutation and TP53 status. These data provide insight into the impact of SVs on gliomagenesis and the mechanisms that shape them.

A bioprinted human-glioblastoma-on-a-chip for the identification of patient-specific responses to chemoradiotherapy.

Patient-specific ex vivo models of human tumours that recapitulate the pathological characteristics and complex ecology of native tumours could help determine the most appropriate cancer treatment for individual patients. Here, we show that bioprinted reconstituted glioblastoma tumours consisting of patient-derived tumour cells, vascular endothelial cells and decellularized extracellular matrix from brain tissue in a compartmentalized cancer-stroma concentric-ring structure that sustains a radial oxygen gradient, recapitulate the structural, biochemical and biophysical properties of the native tumours. We also show that the glioblastoma-on-a-chip reproduces clinically observed patient-specific resistances to treatment with concurrent chemoradiation and temozolomide, and that the model can be used to determine drug combinations associated with superior tumour killing. The patient-specific tumour-on-a-chip model might be useful for the identification of effective treatments for glioblastoma patients resistant to the standard first-line treatment.

ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled.

Wnt signaling pathway plays critical roles in body axes patterning, cell fate specification, cell proliferation, cell migration, stem cell maintenance, cancer development and etc. Deregulation of this pathway can be causative of cancer, metabolic disease and neurodegenerative disease such as Parkinson`s disease. Among the core components of Wnt signaling pathway, we discovered that Dishevelled (Dsh) interacts with ULK1 and is phosphorylated by ULK1. Unexpectedly, the knockdown of ULK1 elicited a marked increase in Wnt/ß-catenin signaling. Multiple ULK1 phosphorylation sites existed on Dsh and many of them were located on the PDZ-DEP region. By using evolutionarily well conserved Drosophila Dsh, we found that S239, S247 and S254 in the PDZ-DEP region are involved in phosphorylation of Dsh by ULK1. Among these, S247 and S254 were conserved in human Dsh. When phospho-mimetic mutants (2D and 2E Dsh mutants) of these conserved residues were generated and expressed in the eyes of the fruit flies, the activity of Dsh was significantly decreased compared to wild type Dsh. Through additional alanine scanning, we further identified that S239, S247, S254, S266, S376, S554 and S555 on full length Dsh were phosphorylated by ULK1. In regards to the S266A mutation located in the PDZ domain among these phosphorylated residues, our results suggested that Dsh forms an SDS-resistant high molecular weight complex with ß-catenin and TCF in the nucleus in an S266 phosphorylation-dependent manner. Based on these results, we propose that ULK1 plays a pivotal role in the regulation of Wnt/ß-catenin signaling pathway by phosphorylating Dsh.

Induction of Lrp5 HBM-causing mutations in Cathepsin-K expressing cells alters bone metabolism.

High-bone-mass (HBM)-causing missense mutations in the low density lipoprotein receptor-related protein-5 (Lrp5) are associated with increased osteoanabolic action and protection from disuse- and ovariectomy-induced osteopenia. These mutations (e.g., A214V and G171V) confer resistance to endogenous secreted Lrp5/6 inhibitors, such as sclerostin (SOST) and Dickkopf homolog-1 (DKK1). Cells in the osteoblast lineage are responsive to canonical Wnt stimulation, but recent work has indicated that osteoclasts exhibit both indirect and direct responsiveness to canonical Wnt. Whether Lrp5-HBM receptors, expressed in osteoclasts, might alter osteoclast differentiation, activity, and consequent net bone balance in the skeleton, is not known. To address this, we bred mice harboring heterozygous Lrp5 HBM-causing conditional knock-in alleles to Ctsk-Cre transgenic mice and studied the phenotype using DXA, µCT, histomorphometry, serum assays, and primary cell culture. Mice with HBM alleles induced in Ctsk-expressing cells (TG) exhibited higher bone mass and architectural properties compared to non-transgenic (NTG) counterparts. In vivo and in vitro measurements of osteoclast activity, population density, and differentiation yielded significant reductions in osteoclast-related parameters in female but not male TG mice. Droplet digital PCR performed on osteocyte enriched cortical bone tubes from TG and NTG mice revealed that ~8-17% of the osteocyte population (depending on sex) underwent recombination of the conditional Lrp5 allele in the presence of Ctsk-Cre. Further, bone formation parameters in the midshaft femur cortex show a small but significant increase in anabolic action on the endocortical but not periosteal surface. These findings suggest that Wnt/Lrp5 signaling in osteoclasts affects osteoclastogenesis and activity in female mice, but also that some of the changes in bone mass in TG mice might be due to Cre expression in the osteocyte population.

Sclerostin neutralization unleashes the osteoanabolic effects of Dkk1 inhibition.

The WNT pathway has become an attractive target for skeletal therapies. High-bone-mass phenotypes in patients with loss-of-function mutations in the LRP5/6 inhibitor Sost (sclerosteosis), or in its downstream enhancer region (van Buchem disease), highlight the utility of targeting Sost/sclerostin to improve bone properties. Sclerostin-neutralizing antibody is highly osteoanabolic in animal models and in human clinical trials, but antibody-based inhibition of another potent LRP5/6 antagonist, Dkk1, is largely inefficacious for building bone in the unperturbed adult skeleton. Here, we show that conditional deletion of Dkk1 from bone also has negligible effects on bone mass. Dkk1 inhibition increases Sost expression, suggesting a potential compensatory mechanism that might explain why Dkk1 suppression lacks anabolic action. To test this concept, we deleted Sost from osteocytes in, or administered sclerostin neutralizing antibody to, mice with a Dkk1-deficient skeleton. A robust anabolic response to Dkk1 deletion was manifest only when Sost/sclerostin was impaired. Whole-body DXA scans, µCT measurements of the femur and spine, histomorphometric measures of femoral bone formation rates, and biomechanical properties of whole bones confirmed the anabolic potential of Dkk1 inhibition in the absence of sclerostin. Further, combined administration of sclerostin and Dkk1 antibody in WT mice produced a synergistic effect on bone gain that greatly exceeded individual or additive effects of the therapies, confirming the therapeutic potential of inhibiting multiple WNT antagonists for skeletal health. In conclusion, the osteoanabolic effects of Dkk1 inhibition can be realized if sclerostin upregulation is prevented. Anabolic therapies for patients with low bone mass might benefit from a strategy that accounts for the compensatory milieu of WNT inhibitors in bone tissue.

Inhibition of CaMKK2 Enhances Fracture Healing by Stimulating Indian Hedgehog Signaling and Accelerating Endochondral Ossification.

Approximately 10% of all bone fractures do not heal, resulting in patient morbidity and healthcare costs. However, no pharmacological treatments are currently available to promote efficient bone healing. Inhibition of Ca2+ /calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) reverses age-associated loss of trabecular and cortical bone volume and strength in mice. In the current study, we investigated the role of CaMKK2 in bone fracture healing and show that its pharmacological inhibition using STO-609 accelerates early cellular and molecular events associated with endochondral ossification, resulting in a more rapid and efficient healing of the fracture. Within 7 days postfracture, treatment with STO-609 resulted in enhanced Indian hedgehog signaling, paired-related homeobox (PRX1)-positive mesenchymal stem cell (MSC) recruitment, and chondrocyte differentiation and hypertrophy, along with elevated expression of osterix, vascular endothelial growth factor, and type 1 collagen at the fracture callus. Early deposition of primary bone by osteoblasts resulted in STO-609-treated mice possessing significantly higher callus bone volume by 14 days following fracture. Subsequent rapid maturation of the bone matrix bestowed fractured bones in STO-609-treated animals with significantly higher torsional strength and stiffness by 28 days postinjury, indicating accelerated healing of the fracture. Previous studies indicate that fixed and closed femoral fractures in the mice take 35 days to fully heal without treatment. Therefore, our data suggest that STO-609 potentiates a 20% acceleration of the bone healing process. Moreover, inhibiting CaMKK2 also imparted higher mechanical strength and stiffness at the contralateral cortical bone within 4 weeks of treatment. Taken together, the data presented here underscore the therapeutic potential of targeting CaMKK2 to promote efficacious and rapid healing of bone fractures and as a mechanism to strengthen normal bones. © 2018 American Society for Bone and Mineral Research.

Loss of mechanosensitive sclerostin may accelerate cranial bone growth and regeneration.

OBJECTIVE: Cranial defects can result from trauma, infection, congenital malformations, and iatrogenic causes and represent a surgical challenge. The current standard of care is cranioplasty, with either autologous or allogeneic material. In either case, the intrinsic vascularity of the surrounding tissues allows for bone healing. The objective of this study was to determine if mechanotransductive gene manipulation would yield non-weight-bearing bone regeneration in a critical size calvarial defect in mice. METHODS: A mouse model of Sost deletion in Sost knockout (KO) mice was created in which the osteocytes do not express sclerostin. A critical size calvarial defect (4 mm in diameter) was surgically created in the parietal bone in 8-week-old wild-type (n = 8) and Sost KO (n = 8) male mice. The defects were left undisturbed (no implant or scaffold) to simulate a traumatic calvariectomy model. Eight weeks later, the animals were examined at necropsy by planimetry, histological analysis of new bone growth, and micro-CT scanning of bone thickness. RESULTS: Defects created in wild-type mice did not fill with bone over the study period of 2 months. Genetic downregulation of sclerostin yielded animals that were able to regenerate 40% of the initial critical size defect area 8 weeks after surgery. A thin layer of bone covered a significant portion of the original defect in all Sost KO animals. A statistically significant increase in bone volume (p < 0.05) was measured in Sost KO mice using radiodensitometric analysis. Immunohistochemical analysis also confirmed that this bone regeneration occurred through the Wnt pathway and originated from the edge of the defect; BMP signaling did not appear to be affected by sclerostin. CONCLUSIONS: Mechanical loading is an important mechanism of bone formation in the cranial skeleton and is poorly understood. This is partially due to the fact that it is difficult to load bone in the craniomaxillofacial skeleton. This study suggests that modulation of the Wnt pathway, as is able to be done with monoclonal antibodies, is a potentially efficacious method for bone regeneration that requires further study.

Isocitrate protects DJ-1 null dopaminergic cells from oxidative stress through NADP+-dependent isocitrate dehydrogenase (IDH).

DJ-1 is one of the causative genes for early onset familiar Parkinson's disease (PD) and is also considered to influence the pathogenesis of sporadic PD. DJ-1 has various physiological functions which converge on controlling intracellular reactive oxygen species (ROS) levels. In RNA-sequencing analyses searching for novel anti-oxidant genes downstream of DJ-1, a gene encoding NADP+-dependent isocitrate dehydrogenase (IDH), which converts isocitrate into α-ketoglutarate, was detected. Loss of IDH induced hyper-sensitivity to oxidative stress accompanying age-dependent mitochondrial defects and dopaminergic (DA) neuron degeneration in Drosophila, indicating its critical roles in maintaining mitochondrial integrity and DA neuron survival. Further genetic analysis suggested that DJ-1 controls IDH gene expression through nuclear factor-E2-related factor2 (Nrf2). Using Drosophila and mammalian DA models, we found that IDH suppresses intracellular and mitochondrial ROS level and subsequent DA neuron loss downstream of DJ-1. Consistently, trimethyl isocitrate (TIC), a cell permeable isocitrate, protected mammalian DJ-1 null DA cells from oxidative stress in an IDH-dependent manner. These results suggest that isocitrate and its derivatives are novel treatments for PD associated with DJ-1 dysfunction.

Sost, independent of the non-coding enhancer ECR5, is required for bone mechanoadaptation.

Sclerostin (Sost) is a negative regulator of bone formation that acts upon the Wnt signaling pathway. Sost is mechanically regulated at both mRNA and protein level such that loading represses and unloading enhances Sost expression, in osteocytes and in circulation. The non-coding evolutionarily conserved enhancer ECR5 has been previously reported as a transcriptional regulatory element required for modulating Sost expression in osteocytes. Here we explored the mechanisms by which ECR5, or several other putative transcriptional enhancers regulate Sost expression, in response to mechanical stimulation. We found that in vivo ulna loading is equally osteoanabolic in wildtype and Sost-/- mice, although Sost is required for proper distribution of load-induced bone formation to regions of high strain. Using Luciferase reporters carrying the ECR5 non-coding enhancer and heterologous or homologous hSOST promoters, we found that ECR5 is mechanosensitive in vitro and that ECR5-driven Luciferase activity decreases in osteoblasts exposed to oscillatory fluid flow. Yet, ECR5-/- mice showed similar magnitude of load-induced bone formation and similar periosteal distribution of bone formation to high-strain regions compared to wildtype mice. Further, we found that in contrast to Sost-/- mice, which are resistant to disuse-induced bone loss, ECR5-/- mice lose bone upon unloading to a degree similar to wildtype control mice. ECR5 deletion did not abrogate positive effects of unloading on Sost, suggesting that additional transcriptional regulators and regulatory elements contribute to load-induced regulation of Sost.

Adult-Onset Deletion of ß-Catenin in (10kb)Dmp1-Expressing Cells Prevents Intermittent PTH-Induced Bone Gain.

ß-Catenin (ßcat) is a major downstream signaling node in canonical Wingless-related integration site (Wnt) signaling pathway, and its activity is crucial for canonical Wnt signal transduction. Wnt signaling has recently been implicated in the osteo-anabolic response to PTH, a potent calcium-regulating factor. We investigated whether ßcat is essential for the anabolic action of intermittent PTH by generating male mice with adult-onset deletion of ßcat in a subpopulation of bone cells (osteocytes and late-stage osteoblasts), treating them with an anabolic regimen of PTH, and measuring the skeletal responses. Male (10kb)Dmp1-CreERt2 transgenic mice that also harbored floxed loss-of-function ßcat alleles (ßcat(f/f)) were induced for Cre activity using tamoxifen, then injected daily with human PTH 1-34 (30 µg/kg) or vehicle for 5 weeks. Mice in which ßcat was deleted showed either total lack of bone mineral density (BMD) gain, or BMD loss, and did not respond to PTH treatment. However, bone mass measurements in the trabecular compartment of the femur and spine revealed PTH-induced bone gain whether ßcat was deleted or not. PTH-stimulated increases in periosteal and cancellous bone formation rates were not impaired by ßcat deletion, but resorption markers and cortical porosity were significantly increased in induced mice, particularly induced mice treated with PTH. These results suggest that ßcat is required for net-positive BMD effects of PTH therapy but that the anabolic effects per se of PTH treatment might not require osteocytic/osteoblastic ßcat.

A 3D-printed local drug delivery patch for pancreatic cancer growth suppression.

Since recurrence and metastasis of pancreatic cancer has a worse prognosis, chemotherapy has been typically performed to attack the remained malignant cells after resection. However, it is difficult to achieve the therapeutic concentration at the tumor site with systemic chemotherapy. Numerous local drug delivery systems have been studied to overcome the shortcomings of systemic delivery. However, because most systems involve dissolution of the drug within the carrier, the concentration of the drug is limited to the saturation solubility, and consequently cannot reach the sufficient drug dose. Therefore, we hypothesized that 3D printing of a biodegradable patch incorporated with a high drug concentration would provide a versatile shape to be administered at the exact tumor site as well as an appropriate therapeutic drug concentration with a controlled release. Here, we introduce the 3D-printed patches composed of a blend of poly(lactide-co-glycolide), polycaprolactone, and 5-fluorouracil for delivering the anti-cancer drug in a prolonged controlled manner and therapeutic dose. 3D printing technology can manipulate the geometry of the patch and the drug release kinetics. The patches were flexible, and released the drug over four weeks, and thereby suppressed growth of the subcutaneous pancreatic cancer xenografts in mice with minimized side effects. Our approach reveals that 3D printing of bioabsorbable implants containing anti-cancer drugs could be a powerful method for an effective local delivery of chemotherapeutic agents to treatment of cancers.

Postnatal ß-catenin deletion from Dmp1-expressing osteocytes/osteoblasts reduces structural adaptation to loading, but not periosteal load-induced bone formation.

Mechanical signal transduction in bone tissue begins with load-induced activation of several cellular pathways in the osteocyte population. A key pathway that participates in mechanotransduction is Wnt/Lrp5 signaling. A putative downstream mediator of activated Lrp5 is the nucleocytoplasmic shuttling protein ß-catenin (ßcat), which migrates to the nucleus where it functions as a transcriptional co-activator. We investigated whether osteocytic ßcat participates in Wnt/Lrp5-mediated mechanotransduction by conducting ulnar loading experiments in mice with or without chemically induced ßcat deletion in osteocytes. Mice harboring ßcat floxed loss-of-function alleles (ßcat(f/f)) were bred to the inducible osteocyte Cre transgenic (10)(kb)Dmp1-CreERt2. Adult male mice were induced to recombine the ßcat alleles using tamoxifen, and intermittent ulnar loading sessions were applied over the following week. Although adult-onset deletion of ßcat from Dmp1-expressing cells reduced skeletal mass, the bone tissue was responsive to mechanical stimulation as indicated by increased relative periosteal bone formation rates in recombined mice. However, load-induced improvements in cross sectional geometric properties were compromised in recombined mice. The collective results indicate that the osteoanabolic response to loading can occur on the periosteal surface when ß-cat levels are significantly reduced in Dmp1-expressing cells, suggesting that either (i) only low levels of ß-cat are required for mechanically induced bone formation on the periosteal surface, or (ii) other additional downstream mediators of Lrp5 might participate in transducing load-induced Wnt signaling.

Effects of electromagnetic field frequencies on chondrocytes in 3D cell-printed composite constructs.

In cartilage tissue engineering, electromagnetic field (EMF) therapy has been reported to have a modest effect on promoting cartilage regeneration. However, these studies were conducted using different frequencies of EMF to stimulate chondrocytes. Thus, it is necessary to investigate the effect of EMF frequency on cartilage formation. In addition to the stimulation, a scaffold is required to satisfy the characteristics of cartilage such as its hydrated and dense extracellular matrix, and a mechanical resilience to applied loads. Therefore, we 3D-printed a composite construct composed of a polymeric framework and a chondrocyte-laden hydrogel. Here, we observed frequency-dependent positive and negative effects on chondrogenesis using a 3D cell-printed cartilage tissue. We found that a frequency of 45 Hz promoted gene expression and secretion of extracellular matrix molecules of chondrocytes. In contrast, a frequency of 7.5 Hz suppressed chondrogenic differentiation in vitro. Additionally, the EMF-treated composite constructs prior to implantation showed consistent results with those of in vitro, suggesting that in vitro pre-treatment with different EMF frequencies provides different capabilities for the enhancement of cartilage formation in vivo. This correlation between EMF frequency and 3D-printed chondrocytes suggests the necessity for optimization of EMF parameters when this physical stimulus is applied to engineered cartilage. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1797-1804, 2016.

Development of a 3D cell printed construct considering angiogenesis for liver tissue engineering.

Several studies have focused on the regeneration of liver tissue in a two-dimensional (2D) planar environment, whereas actual liver tissue is three-dimensional (3D). Cell printing technology has been successfully utilized for building 3D structures; however, the poor mechanical properties of cell-laden hydrogels are a major concern. Here, we demonstrate the printing of a 3D cell-laden construct and its application to liver tissue engineering using 3D cell printing technology through a multi-head tissue/organ building system. Polycaprolactone (PCL) was used as a framework material because of its excellent mechanical properties. Collagen bioink containing three different types of cells-hepatocytes (HCs), human umbilical vein endothelial cells , and human lung fibroblasts--was infused into the canals of a PCL framework to induce the formation of capillary--like networks and liver cell growth. A co-cultured 3D microenvironment of the three types of cells was successfully established and maintained. The vascular formation and functional abilities of HCs (i.e., albumin secretion and urea synthesis) demonstrated that the heterotypic interaction among HCs and nonparenchymal cells increased the survivability and functionality of HCs within the collagen gel. Therefore, our results demonstrate the prospect of using cell printing technology for the creation of heterotypic cellular interaction within a structure for liver tissue engineering.

Short-term evaluation of electromagnetic field pretreatment of adipose-derived stem cells to improve bone healing.

An electromagnetic field is an effective stimulation tool because it promotes bone defect healing, albeit in an unknown way. Although electromagnetic fields are used for treatment after surgery, many patients prefer cell-based tissue regeneration procedures that do not require daily treatments. This study addressed the effects of an electromagnetic field on adipose-derived stem cells (ASCs) to investigate the feasibility of pretreatment to accelerate bone regeneration. After identifying a uniform electromagnetic field inside a solenoid coil, we observed that a 45 Hz electromagnetic field induced osteogenic marker expression via bone morphogenetic protein, transforming growth factor ß, and Wnt signalling pathways based on microarray analyses. This electromagnetic field increased osteogenic gene expression, alkaline phosphate activity and nodule formation in vitro within 2 weeks, indicating that this pretreatment may provide osteogenic potential to ASCs on three-dimensional (3D) ceramic scaffolds. This pretreatment effect of an electromagnetic field resulted in significantly better bone regeneration in a mouse calvarial defect model over 4 weeks compared to that in the untreated group. This short-term evaluation showed that the electromagnetic field pretreatment may be a future therapeutic option for bone defect treatment.

Involvement of Nrf2-GSH signaling in TGFß1-stimulated epithelial-to-mesenchymal transition changes in rat renal tubular cells.

Transforming growth factor-ß1 (TGFß1) induces epithelial-to-mesenchymal transition (EMT) in cultured renal tubular epithelial cells. This phenotypic transition has been known to be involved in the development of chronic kidney diseases by activating profibrotic gene expression. Since oxidative stress has been recognized as one of the contributors to this TGFß1-mediated pathology, we investigated the potential involvement of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which is a key transcription factor for the regulation of multiple antioxidant genes, in TGFß1-stimulated EMT gene changes using the rat proximal tubular epithelial cell line NRK52E. The treatment of NRK52E with TGFß1 led to changes in EMT gene expression, including increased α-Sma and decreased E-cadherin expression. In these cells, the TGFß1 treatment decreased the transcript level of the catalytic subunit of γ-glutamate cysteine ligase (Gclc), a glutathione (GSH) biosynthetic enzyme, and reduced the total GSH content with a concomitant decrease in Nrf2 transcription activity. Accordantly, pre-incubation with the GSH precursor N-acetylcysteine attenuated TGFß1-stimulated EMT gene changes. The involvement of Nrf2 in EMT gene changes has been demonstrated using NRK52E cells with nrf2 knockdown or pharmacological activation. When the expression of Nrf2 was stably silenced in NRK52E cells using interfering RNA administration, Gclc expression was significantly reduced and the increase in the levels of α-Sma and fibronectin-1 by TGFß1 was greater than those in the nonspecific RNA control group. Conversely, Nrf2 activation and subsequent Gclc increase by Nrf2-activating sulforaphane alleviated the TGFß1-stimulated α-Sma increase and E-cadherin decrease. Collectively, these results indicate that Nrf2-GSH signaling can modulate TGFß1-stimulated EMT gene changes and further suggest a beneficial role of Nrf2 inducers in renal pathogenesis.

Effect of redox modulating NRF2 activators on chronic kidney disease.

Chronic kidney disease (CKD) is featured by a progressive decline of kidney function and is mainly caused by chronic diseases such as diabetes mellitus and hypertension. CKD is a complex disease due to cardiovascular complications and high morbidity; however, there is no single treatment to improve kidney function in CKD patients. Since biological markers representing oxidative stress are significantly elevated in CKD patients, oxidative stress is receiving attention as a contributing factor to CKD pathology. Nuclear factor erythroid-2 related factor 2 (NRF2) is a predominant transcription factor that regulates the expression of a wide array of genes encoding antioxidant proteins, thiol molecules and their generating enzymes, detoxifying enzymes, and stress response proteins, all of which can counteract inflammatory and oxidative damages. There is considerable experimental evidence suggesting that NRF2 signaling plays a protective role in renal injuries that are caused by various pathologic conditions. In addition, impaired NRF2 activity and consequent target gene repression have been observed in CKD animals. Therefore, a pharmacological intervention activating NRF2 signaling can be beneficial in protecting against kidney dysfunction in CKD. This review article provides an overview of the role of NRF2 in experimental CKD models and describes current findings on the renoprotective effects of naturally occurring NRF2 activators, including sulforaphane, resveratrol, curcumin, and cinnamic aldehyde. These experimental results, coupled with recent clinical experiences with a synthetic triterpenoid, bardoxolone methyl, have brought a light of hope for ameliorating CKD progression by preventing oxidative stress and maintaining cellular redox homeostasis.

Electromagnetically controllable osteoclast activity.

The time-varying electromagnetic field (EMF) has been widely studied as one of the exogenous stimulation methods for improving bone healing. Our previous study showed that osteogenic differentiation of adipose-derived stem cells was accelerated by a 45-Hz EMF, whereas a 7.5-Hz EMF inhibited osteogenic marker expression. Accordingly, we hypothesized that each negative and positive condition for the osteogenic differentiation could inversely influence osteoclast formation and differentiation. Here, we demonstrated that osteoclast formation, differentiation, and activity can be regulated by altering the frequency of the electromagnetic stimulation, such as 7.5 (negative for osteogenic differentiation) and 45 Hz (positive for osteogenic differentiation). A 45 Hz EMF inhibited osteoclast formation whereas a 7.5-Hz EMF induced differentiation and activity. Osteoclastogenic markers, such as NFATc1, TRAP, CTSK, MMP9, and DC-STAMP were highly expressed under the 7.5-Hz EMF, while they were decreased at 45 Hz. We found that the 7.5-Hz EMF directly regulated osteoclast differentiation through ERK and p38 MAPK activation, whereas the EMF at 45 Hz suppressed RANKL-induced phosphorylation of IκB. Additionally, actin ring formation with tubules and bone resorptive activity were enhanced at 7.5 Hz through increased integrin ß3 expression. However, these were inhibited at 45 Hz. Although many questions remain unanswered, our study indicates that osteoclast formation and differentiation were controllable using physical tools, such as an EMF. It will now be of great interest to study the ill-defined correlation between electromagnetic conditions and osteoclast activities, which eventually could lead to determining the therapeutic characteristics of an EMF that will treat bone-related diseases.

Combined effect of three types of biophysical stimuli for bone regeneration.

Pretreatment using various types of biophysical stimuli could provide appropriate potential to cells during construction of the engineered tissue in vitro. We hypothesized that multiple combinations of these biophysical stimuli could enhance osteogenic differentiation in vitro and bone formation in vivo. Cyclic strain, an electromagnetic field, and ultrasound were selected and combined as effective stimuli for osteogenic differentiation using a developed bioreactor. Here we report the experimental evaluation of the osteogenic effects of various combinations of three different biophysical stimuli in vitro and in vivo using human adipose-derived stem cells (ASCs). Osteogenic differentiation of ASCs was accelerated by multiple-combination biophysical stimulation in vitro. However, both single stimulation and double-combination stimulation were sufficient to accelerate bone regeneration in vivo, while the osteogenic marker expression of those groups was not as high as that of triple-combination stimulation in vitro. We inferred from these data that ASCs appropriately differentiated into the osteogenic lineage by biophysical stimulation could be a better option for accelerating bone formation in vivo than relatively undifferentiated or completely differentiated ASCs. Although many questions remain about the mechanisms of combined effects of various biophysical stimuli, this approach could be a more powerful tool for bone tissue regeneration.

New Insights into Wnt-Lrp5/6-ß-Catenin Signaling in Mechanotransduction.

Mechanical loading is essential to maintain normal bone metabolism and the balance between bone formation and resorption. The cellular mechanisms that control mechanotransduction are not fully defined, but several key pathways have been identified. We discuss the roles of several components of the Wnt signaling cascade, namely Lrp5, Lrp6, and ß-catenin in mechanical loading-induced bone formation. Lrp5 is an important Wnt co-receptor for regulating bone mass and mechanotransduction, and appears to function principally by augmenting bone formation. Lrp6 also regulates bone mass but its action might involve resorption as well as formation. The role of Lrp6 in mechanotransduction is unclear. Studies addressing the role of ß-catenin in bone metabolism and mechanotransduction highlight the uncertainties in downstream modulators of Lrp5 and Lrp6. Taken together, these data indicate that mechanical loading might affect bone regulation triggering the canonical Wnt signaling (and perhaps other pathways) not only via Lrp5 but also via Lrp6. Further work is needed to clarify the role of the Wnt signaling pathway in Lrp5 and/or Lrp6-mediated mechanotransduction, which could eventually lead to powerful therapeutic agents that might mimic the anabolic effects of mechanical stimulation.