scholarly journals HYAL1 Is Downregulated in Idiopathic Pulmonary Fibrosis and Inhibits HFL-1 Fibroblast Proliferation When Upregulated

2020 ◽  
Vol 2020 ◽  
pp. 1-15 ◽  
Author(s):  
Dong Leng ◽  
Xiaoxi Huang ◽  
Jiawen Yi ◽  
Hongying Zhao ◽  
Yuhui Zhang
Keyword(s):  
Data Mining ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Western Blot ◽  
Microarray Data ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Lung Fibroblast ◽  
Fibroblast Proliferation ◽  
Qrt Pcr

Background. Idiopathic pulmonary fibrosis (IPF), the most common interstitial lung disease, arises from transforming growth factor beta 1- (TGFβ1-) induced aberrant fibroproliferation in response to epithelial injury. The TGFβ1 antagonists—hyaluronidases (HYALs)—have been used to clinically treat pulmonary fibrosis. This study focused on characterizing the effect of HYAL1, the main enzyme in hyaluronan degradation, on human lung fibroblast proliferation and apoptosis, and elucidating its potential underlying mechanism of action. Methods. We first performed microarray data mining of previously published gene expression datasets to identify key gene signatures in IPF lung tissues. HYAL1 expression levels in IPF and normal lung tissues were then characterized using immunohistochemistry followed by real-time quantitative reverse transcription-PCR (qRT-PCR) and western blot analysis on isolated fibroblasts from fresh lung tissues of IPF and healthy donors. A human fetal lung fibroblast HFL-1 cell line, which was used in place of primary lung fibroblasts, was used to assess the proliferative or apoptotic effects associated with lentiviral-induced HYAL1 overexpression using CCK-8 cell proliferation assay and Annexin V-APC staining. The identification of potentially associated molecular pathways was performed using microarray analysis followed by qRT-PCR and western blot analysis. Results. Lung tissue microarray data mining and immunohistochemistry revealed significantly downregulation of HYAL1 in IPF lung tissue. However, HYAL1 expression level in IPF fibroblasts was significantly upregulated at the mRNA level, but not altered at the protein level. HYAL1 overexpression in HFL-1 fibroblasts reduced fibroproliferation modestly but did not promote apoptosis. In addition, HYAL1 overexpression led to concomitant transcription factor downregulation, bone morphogenetic protein receptor 2 (BMPR2) signaling activation, but had no effect on TGFβ receptor 2 (TGFβR2) signaling. Conclusions. We showed that HYAL1 overexpression could prevent HFL-1 fibroproliferation. Furthermore, our findings suggest that transcriptional regulators and BMP receptor signaling may be involved in HYAL1 modulation in IPF therapy.

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 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.

PIP4KIIα Gene Silencing by Lentivirus-Mediated Delivery of shRNA in Human K562 Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3200-3200
Author(s):  
Tânia Regina Zaccariotto ◽  
Daniela Maria Ribeiro ◽  
Joao Machado-Neto ◽  
Magnun N N Santos ◽  
Carolina Lanaro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Globin Gene ◽  
K562 Cells ◽  
Globin Genes ◽  
Qrt Pcr ◽  
Globin Gene Expression

Abstract Abstract 3200 Background: Phosphatidylinositol-phosphate-kinase type II alpha (PIP4KIIα) belongs to a family of lipid kinases responsible for the production of a variety of lipid second messengers, such as PI4,5P2 (phosphatidylinositol 4,5-biphosphate), and appears to be implicated in the regulation of gene expression, pre-mRNA processing and mRNA export. In a previous study, two transcripts, PIP4KIIα and β-globin, were found to be overexpressed in reticulocytes from two siblings with Hb H disease, suggesting a possible relationship between this enzyme and the production of globins, particularly β-globin. Recently, we established a gene expression pattern for PIP4KIIα in healthy individuals during in vitro erythropoiesis and observed a gradual increase in the expression of this gene during erythroid differentiation similar to that observed for globin genes, reinforcing the hypothesis of a relationship between PIP4KIIα and globin expression. Aim: To investigate the effects of PIP4KIIα gene silencing on the expression of α- and γ-globin genes in human K562 cells. Methods: Two different human K562 cells cultures were transduced with a lentiviral vector encoding PIP4KIIα-specific shRNA or non-relevant control shRNA. After transduction the positive cells were selected by adding puromycin to the culture and collected 2, 6, 8 and 10 days later to analyze gene and protein expression. PIP4KIIα and α- and γ-globin gene expression was assessed by qRT-PCR and quantified using the equation RQ=2−ΔΔCt. Western blot analysis was performed to determine PIP4KIIα protein expression. β-actin and GAPDH were used as endogenous controls in the qRT-PCR, and β-actin in the Western blot. Results: Analysis of the results showed that there was a statistically significant reduction in PIP4KIIα mRNA levels in knockdown cells (79%) (0.208 ± 0.048; p<0.0001) compared with the control culture. Western blot analysis corroborated these findings. PIP4KIIα silencing resulted in an 18% (0.927 ± 0.244; p=0.09) and 44% (0.625 ± 0.124; p=0.03) reduction in the expression of α- and γ-globin genes, respectively, compared with the control. Conclusion: Although the reduction in α-globin gene expression did not achieve statistical significance, our results revealed alterations in α- and γ-globin gene expression in PIP4KIIα knockdown cells, suggesting a parallelism between the expression of PIP4KIIα and globin genes and reinforcing the hypothesis that the former may be involved in regulation of the latter. This work was supported by FAPESP, CNPq and INCTS. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The role of activating transcription factor 6 in hydroxycamptothecin-induced fibroblast autophagy and apoptosis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jin Tao ◽  
Hui Chen ◽  
Xiaolei Li ◽  
Jingcheng Wang
Keyword(s):  
Transcription Factor ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Counting ◽  
Immunofluorescent Staining ◽  
Fibroblast Proliferation ◽  
Activating Transcription Factor ◽  
Related Proteins

Abstract Background The over-proliferation of fibroblasts is considered to be the main cause of scar adhesion after joint surgery. Hydroxycamptothecin (HCPT), though as a potent antineoplastic drug, shows preventive effects on scar adhesion. This study aimed to investigate the role of activating transcription factor 6 (ATF-6) in the HCPT-induced inhibition of fibroblast viability. Methods The cell counting kit-8 (CCK-8) assay, western blot analysis, lentivirus-mediated gene silencing, transmission electron microscopy (TEM) analysis, immunofluorescent staining for autophagy-related protein light chain 3 (LC3) were used to explore the effect of HCPT on triggering fibroblast apoptosis and inhibiting fibroblast proliferation, and the involvement of possible signaling pathways. Results It was found that HCPT exacerbated fibroblast apoptosis and repressed its proliferation. Subsequently, endoplasmic reticulum stress (ERS)-related proteins were determined by western blot prior to ATF6 p50 was screened out and reexamined after it was silenced. As a result, ATF6-mediated ERS played a role in HCPT-induced fibroblast apoptosis. Autophagy-related proteins and autophagosomes were detected after the HCPT administration using western blot and TEM analyses, respectively. Autophagy was activated after the HCPT treatment. With the co-treatment of autophagy inhibitor 3-methyladenine (3-MA), both the western blot analysis and the CCK-8 assay showed inhibited autophagy, which indicated that the effect of HCPT on fibroblast proliferation was partially reversed. Besides, the LC3 immunofluorescence staining revealed suppressed autophagy after silencing ATF6 p50. Conclusion Our results demonstrate that HCPT acts as a facilitator of fibroblast apoptosis and inhibitor of fibroblast proliferation for curbing the postoperative scar adhesion, in which the ATF6-mediated ERS pathway and autophagy are involved.

Functional Analysis of Cyclin D1 in Mantle Cell Lymphoma (MCL) by Specific Lentiviral shRNA Mediated Knockdown.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1584-1584
Author(s):  
Margit Klier ◽  
Natasa Anastasov ◽  
Daniela Angermeier ◽  
Mark Raffeld ◽  
Falko Fend ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Infection Rates ◽  
Facs Analysis ◽  
Cyclin D2 ◽  
Qrt Pcr

Abstract Introduction: Cyclin D1 overexpression is the hallmark of MCL. However, the importance of cyclin D1 for the maintenance of MCL still remains to be defined. Therefore, the aim of this study is to elucidate the role of cyclin D1 overexpression using the siRNA technology in well-characterized MCL cell lines, as a model system. Material and Methods: A highly efficient cyclin D1-shRNA (96% knockdown) was identified using a lacZ-cyclin D1 fusion gene reporter system in HEK-293T cells. This shRNA was cloned into a lentiviral transfer vector carrying GFP as a reporter gene, which enables the detection of infected cells by FACS analysis. Seven MCL cell lines were analyzed (Granta 519, Jeko-1, Rec-1, Z-138, UPN-1, Hbl-2 and JVM-2), using appropriate controls. Western Blot analysis and qRT-PCR were performed to quantitate the knockdown effect. The effect of cyclin D1 knockdown on proliferation, cell cycle, and viability was analyzed by MTT assay and FACS analysis. Results: The infection rates varied among the different MCL cell lines. Rec-1 and Hbl-2 showed low infection rates (50%) even at high MOI’s (multiplicity of infection), whereas UPN-1 and JVM-2 had moderate infection rates (80%). Jeko-1, Granta 519 and Z-138 showed high infection rates (almost 100% of the cells). Despite the good tranfection rate, the downregulation of cyclin D1, as measured by Western Blot and qRT-PCR, was about 80% in Granta 519, and 65% in Jeko-1 and Z-138. No IFN response, as secondary effect was identified. Interestingly, no apoptosis was observed, and there was only a moderate retardation of growth (60% of control cells) with 10% shift from the S phase to G1 phase of the cell cycle when compared to the controls, suggesting that other cell cycle proteins might compensate, at least partially, for the loss of cyclin D1. Accordingly, cyclin D2 showed upregulation in Western blot analysis and qRT-PCR, whereas the phosphorylation status of retinoblastoma protein on Ser780 was reduced and the expression of the CDK inhibitor p27Kip1 increased. No changes were observed in the expression of cyclin D3, Cyclin E, CDK4 and CDK2. Conclusions: In this study, a system that enables the specific downregulation of cyclin D1 in MCL cell lines was established. Surprisingly, the downregulation of cyclin D1 in MCL cell lines resulted in only a moderate inhibition on cell growth with no apoptosis. The reasons for this might be 1) that the upregulation of cyclin D2 compensates for cyclin D1 downregulation, and/or 2) that the chromosomal translocation leading to cyclin D1 overexpression is an initiating event in MCL lymphomagenesis followed by secondary genetic events at later stages of the disease, which make cyclin D1 dispensable. This finding has important implications for MCL therapy, as strategies targeting only cyclin D1 might be hampered by the redundancy of the system, resulting in a low probability of treatment response.

Transcriptional Profiling and Cytokine Signaling In The Pathogenesis Of Diamond-Blackfan Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2474-2474
Author(s):  
Yoan Konto-Ghiorghi ◽  
Elena Bibikova ◽  
Bertil Glader ◽  
Anupama Narla ◽  
Kathleen Sakamoto
Keyword(s):  
Western Blot ◽  
Cord Blood ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Diamond Blackfan Anemia ◽  
Qrt Pcr ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract Diamond-Blackfan Anemia (DBA) is a pediatric bone marrow failure syndrome, characterized by anemia and congenital abnormalities. Ribosomal protein S19 (RPS19) is mutated in approximately 25% of DBA patients, resulting in haploinsufficiency. Eighty percent of patients will initially respond to corticosteroids; however, this drug is associated with significant toxicity, including immunosuppression and growth delay. Identification of novel pathways could lead to new approaches to treat DBA that minimize the toxicities observed with current therapies. To recapitulate the effects of RPS19 deficiency, we transduced CD34+ cells purified from fetal liver or cord blood with two different shRNA lentiviral constructs against RPS19. GFP was used as a selection marker for cells transduced with RPS19 or control scrambled shRNA. RPS19 knockdown was confirmed by Western blot analysis and by quantitative real-time PCR (qRT-PCR) (73%, p< 0.01 for shRNA1, and 87%, p< 0.01 for shRNA2, respectively), compared with scrambled shRNA control. To further characterize the transcriptional landscape of RPS19-deficient cells, we performed RNA-sequencing analysis with mRNA from fetal liver CD34+ cells transduced with lentivirus RPS19 shRNA. Our results showed that genes involved in cytokine/chemokine signaling, including GDF15, CCL1, and CD70, are overexpressed in RPS19-deficient cells. We hypothesize that genes regulating the expression of these cytokines could contribute to red cell progenitor defects observed in DBA patients. qRT-PCR confirmed the three cytokines GDF15, CCL1, and CD70 to be overexpressed in both fetal liver CD34+ RPS19-deficient cells (more than 10-fold for the three genes, p< 0.01, using each of the two RPS19 shRNAs) and cord blood CD34+ RPS19-deficient cells (more than 10-fold for GDF15 and CCL1, and 3-fold for CD70, p< 0.01, using each of the two RPS19 shRNAs). To test whether GDF15 is a modulator of erythropoiesis, we transduced fetal liver CD34+ cells with lentiviral shRNA against GDF15 (50% knock-down, p<0.01) and assessed their erythroid differentiation potential in methylcellulose. Methylcellulose colony assays indicated that the numbers of erythroid colonies (BFU-E and CFU-E) are decreased by 60% in fetal liver CD34+ cells transduced with shRNA-GDF15 compared to fetal liver CD34+ cells transduced with a control scrambled shRNA, thus suggesting a role for GDF15 in erythroid differentiation. To understand the mechanisms that lead to GDF15, CCL1, and CD70 overexpression in RPS19-deficient CD34+ cells, we compared in silico the promoter sequences of these three cytokine genes. Similar putative transcription factor binding sites were identified in all three promoters, for regulators that have already been shown to play major roles in hematopoiesis: GATA1, GATA2, IKAROS1, IKAROS4, and E2F1. We then performed Western Blot analysis to measure the expression of GATA1, IKAROS1, and E2F1 at the protein level in RPS19-deficient CD34+ cells. These results showed that GATA1 expression was decreased by 50% in RPS19-deficient fetal liver CD34+ cells and by 25% in RPS19-deficient cord blood CD34+ cells. Likewise, IKAROS1 expression in Western Blot analysis was decreased by 20% in RPS19-deficient fetal liver CD34+ cells and by 15% in RPS19-deficient cord blood CD34+ cells. We are currently studying the mechanisms by which these pathways contribute to DBA pathogenesis and to identify potentially novel targets for DBA therapy. Disclosures: No relevant conflicts of interest to declare.

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.

Alendronate promotes the gene expression of extracellular matrix mediated by SP-1/SOX-9

2021 ◽  
pp. 096032712098887
Author(s):  
L Wang ◽  
B Mi ◽  
Y Zhang ◽  
H Yan ◽  
H Zhu
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Specific Inhibitor ◽  
Expression Level ◽  
Dependent Manner ◽  
Qrt Pcr ◽  
Tnf Α

Background and purpose: Osteoarthritis (OA) is a disease with significant degenerative changes of articular cartilage, which is reported to be closely related to the integrity of chondrocytes extracellular matrix (ECM). Alendronate belongs to the family of bisphosphonates with promising cartilage repair function. In the present study, the effects of Alendronate on the gene expression of chondrocytes ECM and the potential mechanism will be investigated to explore the potential therapeutic property of Alendronate on OA. Methods: Human SW1353 chondrocytes were stimulated with 1 and 2 μM Alendronate for 12 h. The gene expression of Col2α1, COL9α2, and Acan in the treated chondrocytes was determined by qRT-PCR. QRT-PCR and western blot analysis were used to evaluate the expression level of SOX-9 in the treated chondrocytes. The expression level of SP-1 was checked by qRT-PCR and immunostaining. SiRNA against SP-1 was transfected into chondrocytes to knockdown the expression of SP-1. The levels of p-ERK1/2 and total ERK1/2 were examined using western blot analysis. TNF-α was used to induce an OA-like in vitro model in the chondrocytes for therapeutic evaluations. Results: Treatment with Alendronate increased the levels of ECM related genes ( Col2α1, COL9α2, and Acan) in a dose-dependent manner through increasing the expression of SOX-9, a central regulator of ECM genes. Additionally, our findings demonstrate that the effects of Alendronate in the expression of SOX-9 are mediated by SP-1 as silencing of SP-1 abolished these effects. Notably, Alendronate increased the phosphorylation of ERK1/2 and inhibition of ERK1/2 using its specific inhibitor U0126 blocked the expression of SP-1. Finally, we found that treatment with Alendronate could rescue TNF-α-induced reduction of Col2α1, COL9α2, Acan and SOX-9. Conclusion: Our data indicated that Alendronate might promote the gene expression of extracellular matrix through SOX-9 mediated by the ERK1/2/SP1 signaling pathway.

scholarly journals Identification of the Multifaceted Chemopreventive Activity of Curcumin Against the Carcinogenic Potential of the Food Additive, KBrO3

2018 ◽  
Vol 24 (5) ◽  
pp. 595-614 ◽  
Author(s):  
Ismael Obaidi ◽  
Michael Higgins ◽  
Bojlul Bahar ◽  
Jessica L. Davis ◽  
Tara McMorrow
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Food Additive ◽  
Dna Adduct ◽  
Potassium Bromate ◽  
Protective Agent ◽  
Pcr Array ◽  
Qrt Pcr ◽  
Carcinogenic Potential

Background: Potassium bromate (KBrO3), a food additive, has been used in many bakery products as an oxidizing agent. It has been shown to induce renal cancer in many in-vitro and in-vivo experimental models. Objectives: This study evaluated the carcinogenic potential of potassium bromate (KBrO3) and the chemopreventive mechanisms of the anti-oxidant and anti-inflammatory phytochemical, curcumin against KBrO3-induced carcinogenicity. Method: Lactate dehydrogenase (LDH) cytotoxicity assay and morphological characteristics were used to assess curcumin's cytoprotective potential against KBrO3 toxicity. To assess the chemopreventive potential of curcumin against KBrO3-induced oxidative insult, intracellular H2O2 and the nuclear concentration of the DNA adduct 8- OHdG were measured. PCR array, qRT-PCR, and western blot analysis were used to identify dysregulated genes by KBrO3 exposure. Furthermore, immunofluorescence was used to evaluate the ciliary loss and the disturbance of cellular tight junction induced by KBrO3. Results: Oxidative stress assays showed that KBrO3 increased the levels of intracellular H2O2 and the DNA adduct 8-OHdG. Combination of curcumin with KBrO3 efficiently reduced the level of H2O2 and 8-OHdG while upregulating the expression of catalase. PCR array, qRT-PCR, and western blot analysis revealed that KBrO3 dysregulated multiple genes involved in inflammation, proliferation, and apoptosis, namely CTGF, IL-1, and TRAF3. Moreover, qRT-PCR and immunofluorescence studies showed that KBrO3 negatively affected the tight junctional protein (ZO-1) and induced a degeneration of primary ciliary proteins. The negative impact of KBrO3 on cilia was markedly repressed by curcumin. Conclusion: Curcumin could potentially be used as a protective agent against carcinogenicity of KBrO3.

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