scholarly journals TGF-β1 is Involved in Vitamin D-Induced Chondrogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells by Regulating the ERK/JNK Pathway

2017 ◽  
Vol 42 (6) ◽  
pp. 2230-2241 ◽  
Author(s):  
Xiaorui Jiang ◽  
Botao Huang ◽  
Huiying Yang ◽  
Guishi Li ◽  
Chunlei Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Vitamin D ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Chondrogenic Differentiation ◽  
Jnk Pathway ◽  
Qrt Pcr ◽  
Tgf Β1

Background/Aims: Osteoarthritis (OA) is characterized by degradation of cartilage, sole cell type of which is chondrocytes. Bone marrow-derived mesenchymal stem cells (BMSCs) possess multipotency and can be directionally differentiated into chondrocytes under stimulation. This study was aimed to explore the possible roles of vitamin D and transforming growth factor-β1 (TGF-β1) in the chondrogenic differentiation of BMSCs. Methods: BMSCs were isolated from femurs and tibias of rats and characterized by flow cytometry. After stimulation with vitamin D, BMSC proliferation and migration were measured by Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. Chondrogenic differentiation was estimated through expression levels of specific markers by qRT-PCR and Western blot analysis. After stable transfection, the effects of aberrantly expressed TGF-β1 on vitamin D-induced alterations, including BMSC viability, migration and chondrogenic differentiation, were all evaluated utilizing CCK-8 assay, Transwell assay, qRT-PCR and Western blot analysis. Finally, the phosphorylation levels of key kinases in the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways were determined by Western blot analysis. Results: Vitamin D remarkably promoted BMSC viability, migration and chondrogenic differentiation. These alterations of BMSCs induced by vitamin D were reinforced by TGF-β1 overexpression while were reversed by TGF-β1 silencing. Additionally, the phosphorylation levels of ERK, JNK and c-Jun were enhanced by TGF-β1 overexpression but were reduced by TGF-β1 knockdown. Conclusion: Vitamin D promoted BMSC proliferation, migration and chondrogenic differentiation. TGF-β1 might be implicated in the vitamin D-induced alterations of BMSCs through regulating ERK/JNK pathway.

scholarly journals The regulatory mechanism of p38/MAPK in the chondrogenic differentiation from bone marrow mesenchymal stem cells

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Ning Ma ◽  
Xiao Teng ◽  
Qi Zheng ◽  
Peng Chen
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Western Blot ◽  
P38 Mapk ◽  
Chondrogenic Differentiation ◽  
Marrow Mesenchymal Stem Cells ◽  
Collagen Ii ◽  
Tgf Β1 ◽  
P38 Pathway

Abstract Background Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation and joint inflammation, in which growth factors are significantly involved. The extracellular signal-regulated p38 MAPK pathways play important roles in the regulation of osteogenic and chondrogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). However, the exact mechanism remains unclear. Methods In this study, the chondrogenic differentiation of human BMSCs was initiated in micromass culture in the presence of TGF-β1 for 14 days. Quantitative RT-PCR and Western blot were performed to detect the transfection effect of shRNA-p38 interfering plasmid in BMSCs. The protein expressions of p/t-p38, SOX9, collagen II, Aggrecan, p/t-Smad1, and p/t-Smad4, as well as the kinase activities of p38/ERK/JNK pathway, were investigated using Western blot analysis. Additionally, the level of chondroitin sulfate and glycosaminoglycans (GAG) expression were measured by Alcian blue staining and GAG assay kit via qualitative and quantitative methods, respectively. Results The results demonstrated that p38 pathway was activated in the chondrogenic differentiation of BMSCs induced by TGF-β1. Cartilage-specific genes and chondrogenic regulators, such as SOX9, collagen II, Aggrecan, and GAG, were upregulated by TGF-β1, which could be reversed by predisposed with shRNA-p38 interfering plasmid and p38-MAPK inhibitors (SB203580). Moreover, the activation of p38/ERK/JNK pathways in the presence of TGF-β1 was suppressed by shRNA-p38 and SB203580 treatment. Conclusion Collectively, the activation of p38/ERK/JNK/Smad pathways plays a facilitated role in the chondrogenic differentiation induced by TGF-β1. After suppressing the p38 pathway, the chondrogenesis can be inhibited, which can be used to guide the treatment of osteoarthritis.

Abstract 663: Identification of 2 Novel Candidate Genes of Atherosclerosis Susceptibility on Mouse Chromosome 3: Pla2g12a and Elovl6

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Alexandros Nicolaou ◽  
Kristina Sass ◽  
Bernd H Northoff ◽  
Daniel Teupser ◽  
Lesca M Holdt
Keyword(s):  
Western Blot ◽  
Candidate Genes ◽  
Mouse Chromosome ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ldl Receptor ◽  
Atherosclerotic Lesion ◽  
Lesion Size ◽  
Specific Expression ◽  
Qrt Pcr

Quantitative trait locus (QTL) mapping in an F2 intercross (n=452) of atherosclerosis-susceptible C57BL/6 (B6) and atherosclerosis-resistant FVB mice on the LDL-receptor deficient background revealed a novel atherosclerosis susceptibility locus on mouse chromosome (Chr) 3. In previous work the susceptible genetic region on Chr3 was narrowed to 80 - 160 MB and validated by congenic FVB.Chr3 B6/B6 mice. We hypothesized that underlying genetic variation in this region leads to differential expression of causal genes, thereby affecting atherosclerosis susceptibility. We performed transcriptome-wide expression analyses in livers of congenic FVB.Chr3 B6/B6 and FVB mice (n=4/4) using Illumina Ref-8 arrays followed by validation in livers of congenic FVB.Chr3 B6/B6 and FVB mice (n=8/9) as well as in livers of B6 and FVB mice (n=5/5) by quantitative real-time PCR (qRT-PCR). C is -regulation was investigated in F2 livers (n=47) by correlating the expression to the genotype. Tissue-specific expression of genes was examined by qRT-PCR in parental B6 and FVB mice. Western blot analysis and immunohistochemical staining (IHC) were performed. Mechanisms of atherogenesis were investigated by RNAi. Pla2g12a and Elovl6 were identified as candidate genes co-segregating with the atherosclerosis QTL at marker rs13464244. Pla2g12a mRNA expression was inversely correlated (r 2 =0.2, p=0.002) with atherosclerotic lesion size in F2 mice while Elovl6 expression was positively correlated (r 2 =0.18, p=0.002). qRT-PCR revealed a strong expression of Pla2g12a in muscle and fat tissues whereas Elovl6 was highly expressed in liver and fat tissues. Western blot analysis revealed significantly decreased protein expression of Pla2g12a in livers of B6 compared to FVB and an increased expression of Elovl6 in B6 mice. IHC staining of Pla2g12a and Elovl6 in aortic roots indicated high expression in macrophages and predominantly in endothelial cells. siRNA knockdown of Elovl6 was associated with reduced adhesion and increased apoptosis. In conclusion, we identified Elovl6 and Pla2g12a as promising candidate genes of atherosclerosis susceptibility on mouse Chr3. Further work is necessary to better understand the influence of these two genes on atherosclerosis development.

scholarly journals Synergistic stimulation of osteoblast differentiation of rat mesenchymal stem cells by leptin and 25(OH)D3 is mediated by inhibition of chaperone-mediated autophagy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qiting He ◽  
Ruixi Qin ◽  
Julie Glowacki ◽  
Shuanhu Zhou ◽  
Jie Shi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Vitamin D ◽  
Mesenchymal Stem Cells ◽  
Osteoblast Differentiation ◽  
Signal Pathway ◽  
Calcium Deposition ◽  
Qrt Pcr ◽  
Alp Activity ◽  
Chaperone Mediated Autophagy

Abstract Background Vitamin D is important for the mineralization of bones by stimulating osteoblast differentiation of bone marrow mesenchymal stem cells (BMMSCs). BMMSCs are a target of vitamin D action, and the metabolism of 25(OH)D3 to biologically active 1α,25(OH)2D3 in BMMSCs promotes osteoblastogenesis in an autocrine/paracrine manner. Our previous study with human BMMSCs showed that megalin is required for the 25(OH)D3-DBP complex to enter cells and for 25(OH)D3 to stimulate osteoblast differentiation in BMMSCs. Furthermore, we reported that leptin up-regulates megalin in those cells. Leptin is a known inhibitor of PI3K/AKT-dependent chaperone-mediated autophagy (CMA). In this study, we tested the hypothesis that leptin acts synergistically with 25(OH)D3 to promote osteoblastogenesis in rat BMMSCs by a mechanism that entails inhibition of PI3K/AKT-dependent CMA. Methods BMMSCs were isolated from rat bone marrow (4-week-old male SD rats); qRT-PCR and western immunoblots or immunofluorescence were used to evaluate the expression of megalin, ALP, COL1A1, RUNX2, OSX, OSP, and CMA in rBMMSCs. The osteoblast differentiation was evaluated by ALP activity, ALP staining, and calcium deposition. The viability of rBMMSCs was assessed with the CCK-8 kit. Biosynthesis of 1α,25(OH)2D3 was measured by a Rat 1α,25(OH)2D3 ELISA Kit. Results The combination of leptin and 25(OH)D3 treatment significantly enhanced osteoblast differentiation as shown by ALP activity, ALP staining, and calcium deposition, the expression of osteogenic genes ALP, COL1A1, RUNX2, OSX, and OSP by qRT-PCR and western immunoblots in rBMMSCs. Leptin enhanced the expression of megalin and synthesis of 1α,25(OH)2D3 in rBMMSCs. Our data showed that leptin inhibited CMA activity of rBMMSCs by activating PI3K/AKT signal pathway; the ability of leptin to enhance 25(OH)D3 promoted osteoblast differentiation of rBMMSCs was weakened by the PI3K/AKT signal pathway inhibitor. Conclusions Our data reveal the mechanism by which leptin and 25(OH)D3 promote osteoblast differentiation in rBMMSCs. Leptin promoted the expression of megalin by inhibiting CMA, increased the utilization of 25(OH)D3 by rBMMSCs, and enhanced the ability of 25(OH)D3 to induce osteoblast differentiation of rBMMSCs. PI3K/AKT is at least partially involved in the regulation of CMA. These data indicate the importance of megalin in BMMSCs for vitamin D’s role in skeletal health.

scholarly journals Integrin β3 Mediates the Endothelial-to-Mesenchymal Transition via the Notch Pathway

2018 ◽  
Vol 49 (3) ◽  
pp. 985-997 ◽  
Author(s):  
Weisen Wang ◽  
Zhi Wang ◽  
Dingyuan Tian ◽  
Xi Zeng ◽  
Yangdong Liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Notch Signaling ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Notch Signaling Pathway ◽  
Rt Pcr ◽  
Mesenchymal Transition ◽  
Integrin Β3 ◽  
Endothelial To Mesenchymal Transition ◽  
Tgf Β1

Background/Aims: Neointimal hyperplasia is responsible for stenosis, which requires corrective vascular surgery, and is also a major morphological feature of many cardiovascular diseases. This hyperplasia involves the endothelial-to-mesenchymal transition (EndMT). We investigated whether integrin β3 can modulate the EndMT, as well as its underlying mechanism. Methods: Integrin β3 was overexpressed or knocked down in human umbilical vein endothelial cells (HUVECs). The expression of endothelial markers and mesenchymal markers was determined by real-time reverse transcription PCR (RT-PCR), immunofluorescence staining, and western blot analysis. Notch signaling pathway components were detected by real-time RT-PCR and western blot analysis. Cell mobility was evaluated by wound-healing, Transwell, and spreading assays. Fibroblast-specific protein 1 (FSP-1) promoter activity was determined by luciferase assay. Results: Transforming growth factor (TGF)-β1 treatment or integrin β3 overexpression significantly promoted the EndMT by downregulating VE-cadherin and CD31 and upregulating smooth muscle actin α and FSP-1 in HUVECs, and by enhancing cell migration. Knockdown of integrin β3 reversed these effects. Notch signaling was activated after TGF-β1 treatment of HUVECs. Knockdown of integrin β3 suppressed TGF-β1-induced Notch activation and expression of the Notch downstream target FSP-1. Conclusion: Integrin β3 may promote the EndMT in HUVECs through activation of the Notch signaling pathway.

scholarly journals Fucoxanthin Inhibits Myofibroblast Differentiation and Extracellular Matrix Production in Nasal Polyp-Derived Fibroblasts via Modulation of Smad-Dependent and Smad-Independent Signaling Pathways

Marine Drugs ◽  
2018 ◽  
Vol 16 (9) ◽  
pp. 323 ◽  
Author(s):  
Hyun Jung ◽  
Dae-Sung Lee ◽  
Seong Park ◽  
Jung Choi ◽  
Won-Kyo Jung ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathways ◽  
Nasal Polyp ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Type I ◽  
Molecular Signaling ◽  
Tgf Β1

Nasal polyps (NPs) are a multifactorial disorder associated with a chronic inflammatory state of the nasal mucosa. Fucoxanthin (Fx) is a characteristic orange carotenoid obtained from brown algae and has diverse immunological properties. The present study investigated whether Fx inhibits fibrosis-related effects in nasal polyp-derived fibroblasts (NPDFs) and elucidated the molecular signaling pathways involved. The production of collagen type I (Col-1) was investigated in NP tissue via immunohistochemistry and western blot analysis. NPDFs were treated with transforming growth factor (TGF)-β1 (1 ng/mL) in the presence or absence of Fx (5–30 µM). The levels of α-smooth muscle actin (α-SMA), Col-1, and phosphorylated (p)-Smad 2/3, signal protein-1 (SP-1), MAPKs (mitogen-activated protein kinases), and Akt were measured by western blot analysis. The expression of Col-1 was detected in NP tissues. TGF-β1 stimulated the production of α-SMA and Col-1, and stimulated the contraction of collagen gel. However, pretreatment with Fx attenuated these effects. Furthermore, these inhibitory effects were mediated through modulation of both Smad 2/3 and Akt/SP-1 signaling pathways in TGF-β1-induced NPDFs. The results from the present study suggest that Fx may be a novel anti-fibrotic agent for the treatment of NP formation.

Requirement of CREB in Normal and Malignant Hematopoiesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1168-1168
Author(s):  
Jerry C. Cheng ◽  
Deepa Shankar ◽  
Stanley F. Nelson ◽  
Kathleen M. Sakamoto
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Western Blot ◽  
Blot Analysis ◽  
Hematopoietic Cells ◽  
Wild Type ◽  
Qrt Pcr ◽  
T315i Mutation

Abstract CREB is a nuclear transcription factor that plays an important role in regulating cellular proliferation, memory, and glucose homeostasis. We previously demonstrated that CREB is overexpressed in bone marrow cells from a subset of patients with acute leukemia at diagnosis. Furthermore, CREB overexpression is associated with an increased risk of relapse and decreased event-free survival in adult AML patients. Transgenic mice that overexpress CREB in myeloid cells developed myeloproliferative/myelodysplastic syndrome after one year. To further understand the role of CREB in leukemogenesis and in normal hematopoiesis, we employed RNA interference methods to inhibit CREB expression. To achieve sustained, CREB-specific gene knockdown in leukemia and normal hematopoietic cells, a lentiviral-based small hairpin (shRNA) approach was taken. Three CREB specific shRNAs were generated and tested for efficiency of gene knockdown in 293T cells. Knockdown efficiency approached 90 percent by Western blot analysis compared to vector alone and luciferase controls. Human myeloid leukemia cell lines, K562, TF1, and MV411, were then infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed using quantitative real time (qRT)-PCR, Western blot analysis, and growth and viability assays. Lentiviral CREB-shRNA achieved between 50 to 90 percent knockdown of CREB compared to control shRNAs at the protein and mRNA levels. To control for non-specific effects, we performed qRT-PCR analysis of the interferon response gene, OAS1, which was not upregulated in cells transduced with CREB shRNA constructs. Within 72 hours, cells transduced with CREB shRNA had decreased proliferation and survival. Similar results were obtained with murine leukemia cells (NFS60 and BA/F3 bcr-abl).To study the role of CREB in normal hematopoiesis, both primary murine and human hematopoietic cells were transduced with our shRNA constructs, and methylcellulose-based colony assays were performed. Primary hematopoietic cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in colony number compared to control virus-infected cells (p<0.05). Bone marrow colonies consisted of myeloid progenitor cells that were mostly Mac-1+ by FACs analysis. Interestingly, there were fewer differentiated cells in the CREB shRNA transduced cells compared to vector control or wild type cells, suggesting that CREB is critical for both myeloid cell proliferation and differentiation. To study the in vivo effects of CREB knockdown on leukemia progression, we studied mice injected with BA/F3 cells that express both bcr/abl with the T315I mutation and a luciferase reporter gene. BA/F3 cells expressing the T315I mutation have a 2-fold increase in CREB overexpression compared to wild-type cells. Disease progression was monitored using bioluminescence imaging with luciferin. CREB knockdown was 90 percent after transduction and prior to injection into SCID mice. We observed improved survival of mice injected with CREB shRNA transduced BA/F3 bcr-abl (T315I) compared to vector control cells. To understand the mechanism of growth suppression resulting from CREB downregulation, we performed microarray analysis with RNA from CREB shRNA transduced K562 and TF1 cells. Several genes were downregulated using a Human Affymetrix chip. Most notable was Beclin1, a tumor suppressor gene often deleted in prostate and breast cancer that has been implicated in autophagy. Our results demonstrate that CREB is required for normal and leukemic cell proliferation both in vitro and in vivo.

Thrombin-Releasable Protein Z-Dependent Protease Inhibitor Identified in Platelets and Is Likely Due to Uptake from Plasma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1751-1751
Author(s):  
Xin Han ◽  
Martin H. Nguyen ◽  
Michael Fernandez ◽  
George J. Broze ◽  
Carlos E. Bueso-Ramos
Keyword(s):  
Bone Marrow ◽  
Western Blot ◽  
Protease Inhibitor ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Factor Xa ◽  
Human Populations ◽  
Rt Pcr ◽  
Protein Z ◽  
Platelet Factor

Abstract Protein Z (PZ) is a vitamin K-dependent protein, whereas PZ-dependent protease inhibitor (ZPI) is a member of the serine protease inhibitor superfamily. ZPI rapidly inhibits factor Xa in the presence of PZ, calcium, and phospholipids and inhibits factor XIa in a PZ-independent fashion (Blood2000; 96:3049–3055). PZ circulates as a complex with ZPI in plasma and deletion of either the PZ or the ZPI gene is associated with the prothrombotic phenotype in mice. In addition, W303X or R67X nonsense mutations in the ZPI gene are reportedly associated with deep venous thrombosis in certain human populations. Western blot analysis of platelets stimulated with thrombin (0–200 mU/mL) showed they contained and released ZPI (approximately 200 ng/109 platelets) with the same molecular weight as plasma ZPI (72 kDa). The majority of the ZPI was released within 1 min. by 25 mU/mL thrombin. PZ was not detected in platelets by western blot analysis. Immunohistochemical staining using a monoclonal anti-ZPI antibody demonstrated a cytoplasmic fine granular staining pattern in maturing megakaryocytes in bone marrow aspirates and in circulating platelets, suggesting that ZPI may be stored in alpha granules. ZPI mRNA, however, was not detected by reverse transcriptase polymerase chain reaction (RT-PCR) in platelets or bone marrow aspirates, but was detected in human liver cDNA. RT-PCR for platelet factor 4 and glyceraldehyde 3-phosphate dehydrogenase mRNA showed amplified products with expected sizes. In conclusion, thrombin-releasable ZPI, but not PZ, is present in platelets and is most likely derived from the uptake of ZPI from plasma. ZPI released from activated platelets may play a role in the regulation of local coagulation at a site of injury.

Identification of a Novel Epigenetic Regulator That Acts As a Tumor Suppressor in the Development of MLL AML Leukemic Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3923-3923
Author(s):  
Hangyu Yi ◽  
Halina Leung ◽  
Aliaksei Holik ◽  
Florida Voli ◽  
Marie-Liesse Asselin-Labat ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tumor Suppressor ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Histone Demethylase ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Promising Therapeutic Approach

Abstract Acute myeloid leukemia (AML) is still a deadly form of leukemia due to frequent relapse caused by the persistence of drug-resistant leukemic stem cells (LSCs). We have previously demonstrated a crucial role for β-catenin signaling in regulating LSCs and identified GPR84 as an important β-catenin regulator in the maintenance of mixed-lineage leukemia (MLL) LSCs (Wang et al., Science 2010; Dietrich et al., Blood 2014). Hence, targeting LSCs by pharmacological inhibition of GPR84/β-catenin signaling represents a promising therapeutic approach. In collaboration with a pharmaceutical company that has developed a novel GPR84 antagonist (GP), we investigated the effect of GP in MLL pre-leukemic stem cell (pre-LSC) function. GP (20 μM) significantly inhibited the colony forming ability of MLL pre-LSCs (P < 0.0001) but had little effect on normal hematopoietic stem cells. Quantitative RT-PCR and western blot analysis confirmed GP-induced downregulation of GPR84 target genes, including Hoxa5, Hoxa7 and Meis1a, indicating GP-induced inhibition of GPR84 signaling. To further examine the mechanism of GPR84 inhibition on MLL pre-LSCs, we evaluated several epigenetic regulators (i.e. JMJD1c and EZH2) known to promote leukemogenesis (Zhu et al., ‎J Clin Invest 2016; Tanaka et al., Blood 2012). Western blot analysis showed that inhibition of GPR84 signaling did not alter the expression of JMJD1c or EZH2. However, we observed a significant increase in the expression of a novel and not-yet-characterized histone demethylase (HD) in AML. To investigate the role of HD in AML leukemogenesis, we overexpressed HD in MLL pre-LSCs and subsequent serial replating assay showed a marked reduction in colony forming ability (P < 0.005), indicating impaired self-renewal in vitro. Consistent with our in vitro observations, in vivo transplantation in syngeneic mice revealed a significant delay in leukemia onset and increase in mouse survival (P < 0.001). We next performed western blot analysis to examine the demethylase activity of HD, and our data revealed that HD overexpression caused a substantial reduction in global histone 3 lysine 36 dimethylation (H3K36me2), an epigenetic mark normally associated with transcriptional activation and elongation. In order to identify genes regulated by HD through demethylation of H3K36me2, we performed H3K36me2 ChIP-seq on HD overexpressing MLL pre-LSCs. Our analysis identified several genes including anti-apoptotic protein Mcl-1 and angiogenic receptor Nrp1, which are known to be involved in AML leukemogenesis, with decreased H3K36me2 mark on both the transcriptional start site and gene body. Subsequent western blot analysis confirmed the decreased expression of both Mcl-1 and Nrp1 in HD overexpressing pre-LSCs. Given the prominent roles of anti-apoptosis and angiogenesis in the development of hematologic malignancies such as leukemia, we are currently evaluating these mechanisms caused by HD overexpression in an important subtype of AML. Taken together, our study identifies a novel histone demethylase that acts downstream of GPR84 signaling to function as a potent tumor suppressor in the development of MLL LSCs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibition of NHE1 Induced Apoptosis in Cytarabine Resistant Leukemia Cell Lines and Primary Leukemia Cells from AML Patients, Which Showed Increased Intracellular pH and NHE1 Activity

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3614-3614 ◽  
Author(s):  
Shin Young Hyun ◽  
Young Kyung Kim ◽  
Ji Eun Jang ◽  
Yundeok Kim ◽  
Yu Ri Kim ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Qrt Pcr ◽  
Nhe1 Activity ◽  
Induced Apoptosis ◽  
Primary Leukemia

Abstract Background: Na/H exchanger 1 (NHE1), an important participant in the precise regulation system of intracellular pH (pHi), is known to be involved in pathological processes such as cell transformation, maintenance and active progression of the neoplastic process. Some studies have showed that leukemic cells showed higher pHi than normal cells, and NHE1 inhibitor could induce acidification and apoptosis of the leukemic cells. In this study, we tried to elucidate the role of NHE1 in leukemic cells according to cytarabine (AraC) resistance. Materials and Methods: Two human AML cell lines, AraC sensitive (AS)-OCI-AML2 cells and AraC resistant (AR)-OCI-AML2 cells, primary leukemic cells from AML patients, and normal bone marrow mononuclear cells (BMMNC) from healthy donor were analyzed. The pH-sensitive fluorescent dye, 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) was used to measure pHi and NHE1 activity. The fluorescent ratio of the 490/440 nm was calibrated intracellularly. The expression of NHE1 was measured by qRT-PCR and western blot analysis. To inhibit the NHE1, the amiloride analogue, 5-(N,N-hexamethylene) amiloride (HMA) (10 µM, 20 µM, 30 µM) was used. Results: To confirmed AraC sensitivity, cell lines were treated with 10 µM AraC for 24 hours, and apoptosis fraction in AS-OCI-AML2 cells and AR-OCI-AML2 cells were 53.1±7.2 % and 4.0±0.8 %, respectively. The pHi of AR-OCI-AML2 cells was significantly higher than AS-OCI-AML2 cells (7.839±0.033 vs. 7.589±0.129, P=0.045) and BMMNC (7.839±0.033 vs. 7.578±0.035, P=0.083), and these differences were associated with higher NHE1 activity. Compared AS-OCI-AML2 cells, AR-OCI-AML2 cells showed significantly higher NHE1 expression by western blot analysis (Figure 1), and NHE1 mRNA levels (0.039±0.014 vs. 1.565±0.070, P<.001) by qRT-PCR. Treatment with HMA (20 µM) could induce apoptosis both on AS-OCI-AML2 cells (26.9±2.8%) and AR-OCI-AML2 cells (37.4±18.8%). Interestingly, induction of apoptosis by HMA was dose-dependent both in AS-OCI-AML2 cells and AR-OCI-AML2 cells, and higher concentration of HMA (30 µM) could induce apoptosis on most of AR-OCI-AML2 cells (68.7±20.2%). Co-treatment experiment with 10 µM AraC and 20 µM HMA in AS-OCI-AML2 cells showed additive effect on inducing apoptosis (AraC vs. HMA vs. HMA+AraC = 53.1±12.4 vs. 53.1±12.4 vs. 67.20±4.3%, Figure 2), but in AR-OCI-AML2 cells, co-treatment did not show additional or synergistic effect on inducing apoptosis (AraC vs. HMA vs. HMA+AraC = 4.0±0.1 vs. 27.1±2.2 vs. 28.1±2.0%, Figure 2). As in the cell lines, primary leukemia cells from patients with AraC resistance showing higher pHi and NHE activity than those from patients without. HMA could induce apoptosis on primary cell lines regardless AraC sensitivity. Conclusions: In this study, we first showed that NHE1 inhibition could induce apoptosis in leukemia cells regardless AraC sensitivity. Apoptotic activity was related with higher pHi and NHE activity in AraC resistant cell lines and primary leukemic cells. NHE inhibition induced apoptosis may be independent with AraC induced apoptosis. The heterogeneity in pHi and NHE activity within leukemic cells may be related to alteration in drug delivery machinery or dormant status of leukemia cells. Further experimental and clinical studies are needed to elucidate the therapeutic application of NHE1 inhibitor to AraC resistant AML. Figure 1. Western blot analysis showed higher level of expression of Na/H exchanger I in AR-AML-OCI2 cells than AS-AML-OCI2 cells. Figure 1. Western blot analysis showed higher level of expression of Na/H exchanger I in AR-AML-OCI2 cells than AS-AML-OCI2 cells. Figure 2. Percentage of apoptotic cells after treatment with 20 µM HMA and/or 10 µM AraC. Figure 2. Percentage of apoptotic cells after treatment with 20 µM HMA and/or 10 µM AraC. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effect of Sox9 on TGF-β1-mediated atrial fibrosis

2021 ◽  
Author(s):  
Hechuan Wang ◽  
Yiqi Chen ◽  
Shuting Zhao ◽  
Xiaowen Wang ◽  
Kai Lu ◽  
...  
Keyword(s):  
Cell Migration ◽  
Western Blot ◽  
Blot Analysis ◽  
Protein Level ◽  
Western Blot Analysis ◽  
Transforming Growth Factor ◽  
Quantitative Polymerase Chain Reaction ◽  
Right Atrial ◽  
Atrial Fibrosis ◽  
Tgf Β1

Abstract Atrial fibrosis is a crucial mechanism responsible for atrial fibrillation (AF). Sex-determining region Y-box containing gene 9 (Sox9) plays a pivotal role in fibrosis of many organs such as the skin, kidney, and liver. However, there are few studies about the occurrence and maintenance of Sox9 in atrial fibrosis. In this study, we investigated the role of Sox9 in the fibrotic phenotype of human atrial tissues and rat atrial fibroblasts in vitro. In the human right atrial tissue, Masson’s trichrome staining, immunofluorescence, real-time quantitative polymerase chain reaction, and western blot analysis were carried out to explore the relationship between Sox9 and atrial fibrosis at the morphological, functional, and molecular levels. In cultured atrial fibroblasts, Sox9 was overexpressed by adenovirus or depleted by siRNA, and then, recombinant human transforming growth factor (TGF)-β1 was added. Immunofluorescence analysis, western blot analysis, Transwell assay, and scratch assay were used to analyze the cells. In patient atrial tissues, Sox9 was increased with worsened atrial fibrosis, and this increase was related to AF severity. In rat atrial fibroblasts, Sox9 was promoted by TGF-β1, and the α-smooth muscle actin (α-SMA) protein level and the ability of cell migration were increased after Sox9 overexpression by adenovirus, while the α-SMA protein level and the cell migration ability were decreased after Sox9 depletion by siRNA. In conclusion, Sox9 is involved in the regulation of fibrosis in the atria and may be located downstream of TGF-β1. Our findings may provide a new perspective to treat atrial fibrosis during AF.

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