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KLF1 mutation E325K induces cell cycle arrest in erythroid cells differentiated from congenital dyserythropoietic anemia patient-specific induced pluripotent stem cells.
Kohara, H, Utsugisawa, T, Sakamoto, C, Hirose, L, Ogawa, Y, Ogura, H, Sugawara, A, Liao, J, Aoki, T, Iwasaki, T, et al
Experimental hematology. 2019;:25-37.e8
Abstract
Krüppel-like factor 1 (KLF1), a transcription factor controlling definitive erythropoiesis, is involved in sequential control of terminal cell division and enucleation via fine regulation of key cell cycle regulator gene expression in erythroid lineage cells. Type IV congenital dyserythropoietic anemia (CDA) is caused by a monoallelic mutation at the second zinc finger of KLF1 (c.973G>A, p.E325K). We recently diagnosed a female patient with type IV CDA with the identical missense mutation. To understand the mechanism underlying the dyserythropoiesis caused by the mutation, we generated induced pluripotent stem cells (iPSCs) from the CDA patient (CDA-iPSCs). The erythroid cells that differentiated from CDA-iPSCs (CDA-erythroid cells) displayed multinucleated morphology, absence of CD44, and dysregulation of the KLF1 target gene expression. In addition, uptake of bromodeoxyuridine by CDA-erythroid cells was significantly decreased at the CD235a+/CD71+ stage, and microarray analysis revealed that cell cycle regulator genes were dysregulated, with increased expression of negative regulators such as CDKN2C and CDKN2A. Furthermore, inducible expression of the KLF1 E325K, but not the wild-type KLF1, caused a cell cycle arrest at the G1 phase in CDA-erythroid cells. Microarray analysis of CDA-erythroid cells and real-time polymerase chain reaction analysis of the KLF1 E325K inducible expression system also revealed altered expression of several KLF1 target genes including erythrocyte membrane protein band 4.1 (EPB41), EPB42, glutathione disulfide reductase (GSR), glucose phosphate isomerase (GPI), and ATPase phospholipid transporting 8A1 (ATP8A1). Our data indicate that the E325K mutation in KLF1 is associated with disruption of transcriptional control of cell cycle regulators in association with erythroid membrane or enzyme abnormalities, leading to dyserythropoiesis.
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Molecular signatures and transcriptional regulatory networks of human immature decidual NK and mature peripheral NK cells.
Wang, F, Zhou, Y, Fu, B, Wu, Y, Zhang, R, Sun, R, Tian, Z, Wei, H
European journal of immunology. 2014;(9):2771-84
Abstract
Many differences exist between human immature and mature natural killer (NK) cells, but their respective molecular signatures and transcriptional regulators are relatively unknown. To gain new insights into the diversity and developmental regulation of human NK cells, we used data from high-resolution microarrays with independent verification to describe a comprehensive comparative analysis between immature decidual NK (idNK) cells with a CD56(bright) CD16(-) T-bet(-) phenotype and mature peripheral NK (mpNK) cells with a CD56(dim) CD16(+) T-bet(+) phenotype. This study shows that many novel growth factors, cytokines, and chemokines are expressed by NK cells, and they may regulate NK-cell development or function in an autocrine manner. Notably, we present that idNK and mpNK cells are enriched for homeobox and zinc-finger transcription factors (TFs), respectively. Additionally, many novel candidate transcriptional regulators are common to both idNK and mpNK cells. We further describe the transcriptional regulatory networks of NK cells and show that the endogenous growth factors, cytokines, and TFs enriched in idNK cells regulate each other and may contribute to idNK-cell immaturity. Together, these findings provide novel molecular signatures for immature and mature NK cells, and the novel candidate regulators identified here can be used to describe and further understand NK-cell differentiation and function.
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Dose-dependent effects of nicotine on proliferation and differentiation of human bone marrow stromal cells and the antagonistic action of vitamin C.
Shen, Y, Liu, HX, Ying, XZ, Yang, SZ, Nie, PF, Cheng, SW, Wang, W, Cheng, XJ, Peng, L, Xu, HZ
Journal of cellular biochemistry. 2013;(8):1720-8
Abstract
A range of biological and molecular effects caused by nicotine are considered to effect bone metabolism. Vitamin C functions as a biological antioxidant. This study was to evaluate the in vitro effects of nicotine on human bone marrow stromal cells and whether Vitamin C supplementation show the antagonism action to high concentration nicotine. We used CCK-8, alkaline phosphatase (ALP) activity assay, Von Kossa staining, real-time polymerase chain reaction and Western Blot to evaluate the proliferation and osteogenic differentiation. The results indicated that the proliferation of BMSCs increased at the concentration of 50, 100 ng/ml, got inhibited at 1,000 ng/ml. When Vitamin C was added, the OD for proliferation increased. For ALP staining, we found that BMSCs treated with 50 and 100 ng/ml nicotine showed a higher activity compared with the control, and decreased at the 1,000 ng/ml. Bone morphogenetic protein-2 (BMP-2) expression and the calcium depositions decreased at 100 and 1,000 ng/ml nicotine, while the addition of Vitamin C reversed the down regulation. By real-time PCR, we detected that the mRNA expression of collagen type I (COL-I) and ALP were also increased in 50 and 100 ng/ml nicotine groups (P < 0.05), while reduced at 1,000 ng/ml (P < 0.05). When it came to osteocalcin (OCN), the changes were similar. Taken all together, it is found that nicotine has a two-phase effect on human BMSCs, showing that low level of nicotine could promote the proliferation and osteogenic differentiation while the high level display the opposite effect. Vitamin C could antagonize the inhibitory effect of higher concentration of nicotine partly.
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Th17 helper cell and T-cell immunoglobulin and mucin domain 3 involvement in Guillain-Barré syndrome.
Liang, SL, Wang, WZ, Huang, S, Wang, XK, Zhang, S, Wu, Y
Immunopharmacology and immunotoxicology. 2012;(6):1039-46
Abstract
OBJECTIVE AND DESIGN We investigated the involvement of Th17 cells and T-cell immunoglobulin and mucin domain 3 (TIM-3) in Guillain-Barré syndrome (GBS) in comparison to healthy subjects. MATERIALS AND SUBJECTS Peripheral blood samples were obtained from 29 healthy subjects and 29 GBS patients. TREATMENT Peripheral blood mononuclear cells (PBMCs) and CD4(+) T cells were stimulated with anti-CD3 and anti-CD28 mAbs, in the absence or presence of anti-TIM-3 mAb. METHODS mRNA levels of TIM-3 and the transcription factor retinoic acid-related orphan receptor γt (RORγt) were determined by RT-PCR and were expressed relative to β-actin mRNA (housekeeping gene). Serum IFN-γ and IL-17 levels were determined by ELISA. RESULTS Compared to controls, relative TIM-3 mRNA levels were lower in both stimulated and unstimulated PBMCs from GBS patients. Unstimulated GBS CD4(+) T cells and GBS CD4+ T cells stimulated with anti-CD3 and CD28 mAbs had higher relative RORγt mRNA expression compared to controls. GBS CD4(+) T cells secreted significantly more IFN-γ and IL-17 in the presence of anti-TIM-3 mAb. GBS patients had (1) higher numbers of Th17, but not Th1 or Th2 cells in peripheral blood and (2) higher serum concentrations of IFN-γ and IL-17 compared to controls. CONCLUSION TIM-3 may inhibit Th17 cell activation, thereby modulating their cytokine secretion patterns. Th17 cell differentiation, IL-17 levels, and TIM-3 regulation may be involved in the pathogenesis of GBS.