Redox Modulating NRF2: A Potential Mediator of Cancer Stem Cell Resistance

Tumors contain a distinct small subpopulation of cells that possess stem cell-like characteristics. These cells have been called cancer stem cells (CSCs) and are thought to be responsible for anticancer drug resistance and tumor relapse after therapy. Emerging evidence indicates that CSCs share many properties, such as self-renewal and quiescence, with normal stem cells. In particular, CSCs and normal stem cells retain low levels of reactive oxygen species (ROS), which can contribute to stem cell maintenance and resistance to stressful tumor environments. Current literatures demonstrate that the activation of ataxia telangiectasia mutated (ATM) and forkhead box O3 (FoxO3) is associated with the maintenance of low ROS levels in normal stem cells such as hematopoietic stem cells. However, the importance of ROS signaling in CSC biology remains poorly understood. Recent studies demonstrate that nuclear factor-erythroid 2-related factor 2 (NRF2), a master regulator of the cellular antioxidant defense system, is involved in the maintenance of quiescence, survival, and stress resistance of CSCs. Here, we review the recent findings on the roles of NRF2 in maintenance of the redox state and multidrug resistance in CSCs, focusing on how NRF2-mediated ROS modulation influences the growth and resistance of CSCs

Growth differentiation factor 11 locally controls anterior-posterior patterning of the axial skeleton.

Growth and differentiation factor 11 (GDF11) is a transforming growth factor β family member that has been identified as the central player of anterior-posterior (A-P) axial skeletal patterning. Mice homozygous for Gdf11 deletion exhibit severe anterior homeotic transformations of the vertebrae and craniofacial defects. During early embryogenesis, Gdf11 is expressed predominantly in the primitive streak and tail bud regions, where new mesodermal cells arise. On the basis of this expression pattern of Gdf11 and the phenotype of Gdf11 mutant mice, it has been suggested that GDF11 acts to specify positional identity along the A-P axis either by local changes in levels of signaling as development proceeds or by acting as a morphogen. To further investigate the mechanism of action of GDF11 in the vertebral specification, we used a Cdx2-Cre transgene to generate mosaic mice in which Gdf11 expression is removed in posterior regions including the tail bud, but not in anterior regions. The skeletal analysis revealed that these mosaic mice display patterning defects limited to posterior regions where Gdf11 expression is deficient, whereas displaying normal skeletal phenotype in anterior regions where Gdf11 is normally expressed. Specifically, the mosaic mice exhibited seven true ribs, a pattern observed in wild-type (wt) mice (vs. 10 true ribs in Gdf11-/- mice), in the anterior axis and nine lumbar vertebrae, a pattern observed in Gdf11 null mice (vs. six lumbar vertebrae in wt mice), in the posterior axis. Our findings suggest that GDF11, rather than globally acting as a morphogen secreted from the tail bud, locally regulates axial vertebral patterning

High Extracellular Calcium Increased Expression of Ank, PC-1 andOsteopontin in Mouse Calvarial Cells

In the process of bone remodeling, mineral phase of bone is dissolved by osteoclasts, resulting in elevation of calcium concentration in micro-environment. This study was performed to explore the effect of high extracellular calcium (Ca 2+ e) on mineralized nodule formation and on the expression of progressive ankylosis (Ank), plasma cell membrane glycoprotein-1 (PC-1) and osteopontin by primary cultured mouse calvarial cells. Osteoblastic differentiation and mineralized nodule formation was induced by culture of mouse calvarial cells in osteoblast differentiation medium containing ascorbic acid and β-glycerophosphate. Although Ank, PC-1 and osteopontin are well known inhibitors of mineralization, expression of these genes were induced at the later stage of osteoblast differentiation during when expression of osteocalcin, a late marker gene of osteoblast differentiation, was induced and mineralization was actively progressing. High Ca 2+ e (10 mM) treatment highly enhanced mRNA expression of Ank, PC-1 and osteopontin in the late stage of osteoblast differentiation but not in the early stage. Inhibition of p44/42 MAPK activation but not that of protein kinase C suppressed high Ca 2+ e-induced expression of Ank, PC-1 and osteopontin. When high Ca 2+ e (5 mM or 10 mM) was present in culture medium during when mineral deposition was actively progressing, matrix calcifiation was significantly increased by high Ca 2+ e. This stimulatory effect was abolished by pyrophosphate (5 mM) or levamisole (0.1-0.5 mM), an alkaline phosphatase inhibitor. In addition, probenecid (2mM), an inhibitor of Ank, suppressed matrix calcification in both control and high Ca 2+ e-treated group, suggesting the possible role of Ank in matrix calcification by osteoblasts. Taken together, these results showed that high Ca 2+ e stimulates expression of Ank, PC-1 and osteopontin as well as matrix calcification in late differentiation stage of osteoblasts and that p44/42 MAPK activation is involved in high Ca 2+ e- induced expression of Ank, PC-1 and osteopontin

The Expression of Matrix Metalloprotease 20 is Stimulated by Wild Type but not by 4 bp- or 2 bp- Deletion Mutant DLX3

Mutations in DLX3 are associated with both autosomal dominant hypoplastic hypomaturation amelogenesis imperfecta (ADHHAI) and tricho-dento-osseous (TDO) syndrome. ADHHAI is caused by a c.561_562delCT (2bpdel DLX3) mutation whereas TDO syndrome is associated with a c.571_574delGGGG (4bp-del DLX3) mutation. However, although the causal relationships between DLX3 and an enamel phenotype have been established, the pathophysiological role of DLX3 mutations in enamel development has not yet been clarified. In our current study, we prepared expression vectors for wild type and deletion mutant DLX3 products (4bp-del DLX3, 2bp-del DLX3) and examined the effects of their overexpression on the expression of the enamel matrix proteins and proteases. Wild type DLX3 enhanced the expression of matrix metalloprotease 20 (MMP20) mRNA and protein in murine ameloblast-like cells. However, neither a 4bp-del nor 2bpdel DLX3 increased MMP20 expression. Wild type DLX3, but not the above DLX3 mutants, also increased the activity of reporters containing 1.5 kb or 0.5 kb of the MMP20 promoter. An examination of protein stability showed that the half-life of wild type DLX3 protein was less than 12 h whilst that of both deletion mutants was longer than 24 h. Endogenous Dlx3 was also found to be continuously expressed during ameloblast differentiation. Since inactivating mutations in the gene encoding MMP20 are associated with amelogenesis imperfecta, the inability of 4bp-del or 2bp-del DLX3 to induce MMP20 expression suggests a possible involvement of such mutations in the enamel phenotype associated with TDO syndrome or ADHHAI

Tricho-dento-osseous Syndrome Mutant Dlx3 Shows Lower Transactivation Potential but Has Longer Half-life than Wild-type Dlx3

Dlx3 is a homeodomain protein and is known to play a role in development and differentiation of many tissues. Deletion of four base pairs in DLX3 (NT3198) is causally related to tricho-dento-osseous (TDO) syndrome (OMIM #190320), a genetic disorder manifested by taurodontism, hair abnormalities, and increased bone density in the cranium. The molecular mechanisms that explain the phenotypic characteristics of TDO syndrome have not been clearly determined. In this study, we examined phenotypic characteristics of wild type DLX3 (wtDlx3) and 4-BP DEL DLX3 (TDO mtDlx3) in C2C12 cells. To investigate how wtDlx3 and TDO mtDlx3 differentially regulate osteoblastic differentiation, reporter assays were performed by using luciferase reporters containing the promoters of alkaline phosphatase, bone sialoprotein or osteocalcin. Both wtDlx3 and TDO mtDlx3 enhanced significantly all the reporter activities but the effect of mtDlx3 was much weaker than that of wtDlx3. In spite of these differences in reporter activity, electrophoretic mobility shift assay showed that both wtDlx3 and TDO mtDlx3 formed similar amounts of DNA binding complexes with Dlx3 binding consensus sequence or with ALP promoter oligonucleotide bearing the Dlx3 binding core sequence. TDO mtDlx3 exhibits a longer half-life than wtDlx3 and it corresponds to PESTfind analysis result showing that potential PEST sequence was missed in carboxy terminal of TDO mtDlx3. In addition, co-immunoprecipitation demonstrated that TDO mtDlx3 binds to Msx2 more strongly than wtDlx3. Taken together, though TDO mtDlx3 acted as a weaker transcriptional activator than wtDlx3 in osteoblastic cells, there is possibility that during in vivo osteoblast differentiation TDO mtDlx3 may antagonize transcriptional repressor activity of Msx2 more effectively and for longer period than wtDlx3, resulting in enhancement of osteoblast differentiation

Surgical Correction of Hallermann-Streiff Syndrome: A Case Report of Esotropia, Entropion, and Blepharoptosis

We report a case of surgical treatment for Hallermann-Streiff syndrome in a patient with ocular manifestations of esotropia, entropion, and blepharoptosis. A 54-year-old man visited Yeouido St. Mary's Hospital complaining of ocular discomfort due to cilia touching the corneas of both eyes for several years. He had a bird-like face, pinched nose, hypotrichosis of the scalp, mandibular hypoplasia with forward displacement of the temporomandibular joints, a small mouth, and proportional short stature. His ophthalmic features included sparse eyelashes and eyebrows, microphthalmia, nystagmus, lower lid entropion in the right eye, and upper lid entropion with blepharoptosis in both eyes. There was esodeviation of the eyeball of more than 100 prism diopters at near and distance, and there were limitations in ocular movement on lateral gaze. The capsulopalpebral fascia was repaired to treat the right lower lid entropion, but an additional Quickert suture was required to prevent recurrence. Blepharoplasty and levator palpebrae repair were performed for blepharoptosis and dermatochalasis. Three months after lid surgery, the right medial rectus muscle was recessed 7.5 mm, the left medial rectus was recessed 7.25 mm, and the left lateral rectus muscle was resected 8.0 mm

Comparative Evaluation of Nanofibrous Scaffolding for Bone Regeneration in Critical-Size Calvarial Defects

In a previous study we found that nanofibrous poly(l-lactic acid) (PLLA) scaffolds mimicking collagen fibers in size were superior to solid-walled scaffolds in promoting osteoblast differentiation and bone formation in vitro. In this study we used an in vivo model to confirm the biological properties of nanofibrous PLLA scaffolds and to evaluate how effectively they support bone regeneration against solid-walled scaffolds. The scaffolds were implanted in critical-size defects made on rat calvarial bones. Compared with solid-walled scaffolds, nanofibrous scaffolds supported substantially more new bone tissue formation, which was confirmed by micro-computed tomography measurement and von Kossa staining. Goldner's trichrome staining showed abundant collagen deposition in nanofibrous scaffolds but not in the control solid-walled scaffolds. The cells in these scaffolds were immuno-stained strongly for Runx2 and bone sialoprotein (BSP). In contrast, solid-walled scaffolds implanted in the defects were stained weakly with trichrome, Runx2, and BSP. These in vivo results demonstrate that nanofibrous architecture enhances osteoblast differentiation and bone formation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78127/1/ten.tea.2008.0433.pd

Upregulation of smpd3 via BMP2 stimulation and Runx2.

Deletion of smpd3 induces osteogenesis and dentinogenesis imperfecta in mice. smpd3 is highly elevated in the parietal bones of developing mouse calvaria, but not in sutural mesenchymes. Here, we examine the mechanism of smpd3 regulation, which involves BMP2 stimulation of Runx2. smpd3 mRNA expression increased in response to BMP2 treatment and Runx2 transfection in C2C12 cells. The Runx2-responsive element (RRE) encoded within the -562 to -557 region is important for activation of the smpd3 promoter by Runx2. Electrophoretic mobility shift assays revealed that Runx2 binds strongly to the -355 to -350 RRE and less strongly to the -562 to -557 site. Thus, the smpd3 promoter is activated by BMP2 and is directly regulated by the Runx2 transcription factor. This novel description of smpd3 regulation will aid further studies of bone development and osteogenesis. [BMB reports 2009; 42(2): 86-90

Introduction of Transmembrane Inner Ear (tmie) Gene Can Recover the Hearing Impairment and Abnormal Behavior in the Circling Mouse

The spontaneous mutant circling mouse (cir/cir) shows a circling behavior and hearing loss. We produced transgenic mice overexpressing the causative gene, transmembrane inner ear (tmie), for the phenotypic rescue of the circling mouse. Through the continuous breeding with circling mice, the cir/cir homozygous mice carrying the transgene (cir/cir-tg) were produced. The rescued cir/cir -tg mice were able to swim in the water with proper orientation and did not show any circling behavior like wild type mice. Western blot and immunohistochemical analysis exhibited that the transgenic tmie was expressed in the inner ear. Inner and outer hair cells were recovered in the cochlea and spiral ganglion neurons were also recovered in the rescued mice. Auditory brainstem response (ABR) test demonstrated that the cir/cir -tg mice are able to respond to sound. This study demonstrates that tmie transgene can recover the hearing impairment and abnormal behavior in the circling mouse