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The canonical Wnt/β-catenin signaling pathway in osteoblast-lineage cells inhibits osteoclastogenesis through the expression of osteoprotegerin (Opg), a decoy receptor of receptor activator of Nf-κb (Rank) ligands. Wnt5a, a typical non-canonical Wnt ligand, enhances the expression of Rank in osteoclast precursors, which, in turn, promotes the Rank ligand (Rankl)-induced formation of osteoclasts. In contrast, Wnt16 and Wnt4 have been shown to inhibit the Rankl-induced formation of osteoclasts through non-canonical Wnt signals. However, the relationships among these Wnt ligands in osteoclastogenesis remained to be elucidated. We herein showed that Wnt16, but not Wnt4, inhibited the Rankl-induced osteoclastogenesis in bone marrow-derived macrophage (BMM) cultures. Wnt3a and Wnt4 inhibited the 1α,25-dihydroxy vitamin D 3 (1,25D 3 )-induced osteoclastogenesis in co-cultures prepared from wild-type mice, but not in those from Opg –/– nice. Wnt16 inhibited the 1,25D 3 -induced formation of osteoclasts in both wild-type and Opg –/– co-cultures. Wnt16, Wnt4, and Wnt3a failed to inhibit the pit-forming activity of osteoclasts. Wnt16 failed to inhibit the Wnt5a-induced expression of Rank in osteoclast precursors. In contrast, Wnt5a abrogated the inhibitory effects of Wnt16 on Rankl-induced osteoclastogenesis. These results suggested that Wnt16 inhibited osteoclastogenesis, but not the function of osteoclasts and that Wnt16, an inhibitory Wnt ligand for osteoclastogenesis, regulates bone resorption in conjunction with Wnt5a. [ABSTRACT FROM AUTHOR]

The development of therapies that target cancer stem cells (CSCs) is an important challenge in cancer research. The antioxidant system is enhanced in CSCs, which may lead to resistance to existing therapies. Ascorbic acid (AA) has the potential to act as both an antioxidant and a pro-oxidant agent, but its effects on CSCs are a subject of current research. Here, we investigated the effect of AA focusing specifically on CSCs with the hepatocellular carcinoma cell line Li-7. The Li-7 cell line is heterogenous consisting of CD166- and CD166+ cells; CD166- cells include CSC-like cells (CD13+CD166- cells) and CD13−CD166- cells that can revert to CD13+CD166- cells. The addition of AA to the culture medium caused cell death in both cell populations in CD166- cells in a concentration dependent manner. In contrast, AA administration had a limited effect on CD166+ non-CSC cells. The level of reactive oxygen species after AA treatment was elevated only in CD166- cells. The effect of AA only occurred at low cell densities in 2D and 3D cultures. In a mouse tumor model injected with Li-7 cells, intraperitoneal administration of AA failed to prevent tumor formation but appeared to delay tumor growth. Our findings shed light on why AA administration has not become a mainstream treatment for cancer treatment; however, they also show the possibility that AA can be used in therapies to suppress CSCs. • Effects of ascorbic acid was tested in the hepatocellular carcinoma cell line Li-7. • Ascorbic acid increased the level of reactive oxygen species. • Ascorbic acid caused cell death predominantly in cancer stem cell-like cells. • Effects of AA occurred only at low cell densities in 2D and 3D cultures. • No tumor shrinkage effect of ascorbic acid was observed in a mouse xenograft model. [ABSTRACT FROM AUTHOR]

Abstract: Human umbilical cord blood (UCB) cells are an alternative source of hematopoietic stem cells for treatment of leukemia and other diseases. It is very difficult to assess the quality of UCB cells in the clinical situation. Here, we sought to assess the quality of UCB cells by transplantation to immunodeficient mice. Cryopreserved CD34+ UCB cells from twelve different human donors were transplanted into sublethally irradiated NOD/shi-scid Jic mice. In parallel, the gene expression profiles of the UCB cells were determined from oligonucleotide microarrays. UCB cells from three donors failed to establish an engraftment in the host mice, while the other nine succeeded to various extents. Gene expression profiling indicated that 71 genes, including HOXB4, C/EBP-β, and ETS2, were specifically overexpressed and 23 genes were suppressed more than 2-fold in the successful UCB cells compared to those that failed. Functional annotation revealed that cell growth and cell cycle regulators were more abundant in the successful UCB cells. Our results suggest that hematopoietic ability may vary among cryopreserved UCB cells and that this ability can be distinguished by profiling expression of certain sets of genes. [Copyright &y& Elsevier]

β-Thalassemia is an autosomal recessive genetic disease caused by defects in the production of adult hemoglobin (HbA, α 2 β 2), which leads to an imbalance between α- and non-α-globin chains. Reactivation of γ-globin expression is an effective strategy to treat β-thalassemia patients. Previously, it was demonstrated that hemoglobin subunit beta pseudogene 1 (HBBP1) is associated with elevated fetal hemoglobin (HbF, α 2 γ 2) in β-thalassemia patients. However, the mechanism underlying HBBP1 -mediated HbF production is unknown. In this study, using bioinformatics analysis, we found that HBBP1 is involved in γ-globin production, and then preliminarily confirmed this finding in K562 cells. When HBBP1 was overexpressed, γ-globin expression was increased at the transcript and protein levels in HUDEP-2 cells. Next, we found that ETS transcription factor ELK1 (ELK1) binds to the HBBP1 proximal promoter and significantly promotes its activity. Moreover, the synthesis of γ-globin was enhanced when ELK1 was overexpressed in HUDEP-2 cells. Surprisingly, ELK1 also directly bound to and activated the γ-globin proximal promoter. Furthermore, we found that HBBP1 and ELK1 can interact with each other in HUDEP-2 cells. Collectively, these findings suggest that HBBP1 can induce γ-globin by enhancing ELK1 expression, providing some clues for γ-globin reactivation in β-thalassemia. • HBBP1 is related to elevated γ-globin and promotes γ-globin expression by activating ELK1 in HUDEP-2 cells. • HBBP1 interacts with ELK1 and ELK1 directly binds to the proximal promoter of γ-globin in HUDEP-2 cells. • HBBP1-ablation significantly impairs erythroid differentiation in hematopoietic stem/progenitor cell. [ABSTRACT FROM AUTHOR]

Abstract Congenital scoliosis is a lateral curvature of the spine that is due to the presence of vertebral anomalies. Although genetic and environmental factors are involved in the pathogenesis of congenital scoliosis, the specific cause of only a small number of individuals has been identified to date. We identified a de novo missense mutation in the olfactomedin-like 1 (OLFML1) gene by whole-exome sequencing of a patient with congenital scoliosis. Then, we carried out further functional investigation in mice. An assessment of the tissue distribution of Olfml1 revealed it to be prominently expressed in developing skeletal tissues, specifically osteoblasts. Short hairpin RNA-mediated knockdown of Olfml1 in osteoblasts induced the translocation of Yes-associated protein (YAP) transcriptional coactivator from the cytoplasm to the nucleus, which accelerated the Hippo signaling pathway to promote osteoblast mineralization. In contrast, experimentally induced gain of function of Olfml1 retained YAP in the cytoplasm. There appears to exist a novel cell-autonomous mechanism by which osteoblasts avoid excess mineralization through Olfml1. Our results also indicate that mutation of OLFML1 leads to impaired osteoblast differentiation and abnormal development of bone tissue. Graphical abstract Image 1 Highlights • A de novo mutation was identified in OLFML1 in a patient with congenital scoliosis. • Olfml1 is strongly expressed in calvarial osteoblasts in mice. • Knockdown of Olfml1 accelerates osteoblast mineralization. • Olfml1 knockdown induces nuclear translocation of YAP and activates Hippo signaling. • Olfml1 expression vector transfection retains YAP in the cytoplasm. [ABSTRACT FROM AUTHOR]

Congenital sideroblastic anemia (CSA) is a hereditary disorder characterized by microcytic anemia and bone marrow sideroblasts. The most common form of CSA is attributed to mutations in the X-linked gene 5-aminolevulinic acid synthase 2 ( ALAS2 ). ALAS2 is a mitochondrial enzyme, which utilizes glycine and succinyl-CoA to form 5-aminolevulinic acid (ALA), a crucial precursor in heme synthesis. Therefore, ALA supplementation could be an effective therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. In a preclinical study, we examined the effects of ALA in human erythroid cells, including K562 cells and human induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells. ALA treatment resulted in significant dose-dependent accumulation of heme in the K562 cell line. Concomitantly, the treatment substantially induced erythroid differentiation as assessed using benzidine staining. Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes, such as the globin genes [hemoglobin alpha ( HBA ) and hemoglobin gamma ( HBG )] and the heme oxygenase 1 ( HMOX1 ) gene, in K562 cells. Next, to investigate the mechanism by which ALA is transported into erythroid cells, quantitative RT-PCR analysis was performed on previously identified ALA transporters, including solute carrier family 15 (oligopeptide transporter) , member (SLC15A) 1 , SLC15A2 , solute carrier family 36 (proton/amino acid symporter) , member (SLC36A1) , and solute carrier family 6 (neurotransmitter transporter) , member 13 (SLC6A13) . Our analysis revealed that SLC36A1 was abundantly expressed in erythroid cells. Thus, gamma-aminobutyric acid (GABA) was added to K562 cells to competitively inhibit SLC36A1-mediated transport. GABA treatment significantly impeded the ALA-mediated increase in the number of hemoglobinized cells as well as the induction of HBG , HBA , and HMOX1 . Finally, small-interfering RNA-mediated knockdown of ALAS2 in HiDEP cells considerably decreased the expression of HBA , HBG , and HMOX1 , and these expression levels were rescued with ALA treatment. In summary, ALA appears to be transported into erythroid cells mainly by SLC36A1 and is utilized to generate heme. ALA may represent a novel therapeutic option for CSA treatment, particularly for cases harboring ALAS2 mutations. [ABSTRACT FROM AUTHOR]