scholarly journals LEFTY2 Controls Migration of Human Endometrial Cancer Cells via Focal Adhesion Kinase Activity (FAK) and miRNA-200a

2016 ◽  
Vol 39 (3) ◽  
pp. 815-826 ◽  
Author(s):  
Nour Alowayed ◽  
Madhuri S. Salker ◽  
Ni Zeng ◽  
Yogesh Singh ◽  
Florian Lang
Keyword(s):  
Cell Proliferation ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Annexin V ◽  
Cellular Adhesion ◽  
Negative Regulator ◽  
Migratory Activity ◽  
Ishikawa Cells ◽  
And Migration ◽  
E Cadherin

Background: LEFTY2, a suppressor of cell proliferation, tumor growth, regulator of stemness and embryonic differentiation, is a negative regulator of cancer cell reprogramming. Malignant transformation may lead to migration requiring loss of adhesion and gain of migratory activity. Signaling involved in the orchestration of migration, proliferation and spreading of cells include focal adhesion kinase (FAK) and adhesion molecule E-cadherin. Aims: The present study explored whether LEFTY2 influences the proliferation marker MKi67, FAK activity, E-cadherin abundance and migration of Ishikawa human endometrial carcinoma cells. Moreover, the study explored the involvement of microRNA-200a (miR-200a), which is known to regulate cellular adhesion by targeting E-Cadherin. Methods: FAK activity was estimated from FAK phosphorylation quantified by Western blotting, migration utilizing a wound healing assay, miR-200a and MKi67 expression levels utilizing qRT-PCR, cell proliferation and apoptosis using BrdU and Annexin V staining, respectively, and E-Cadherin (E-Cad) abundance, using confocal microscopy. Results: LEFTY2 (25 ng/ml, 48 hours) treatment was followed by decrease of MKi67 expression, FAK activity and migration. LEFTY2 upregulated miRNA-200a and E-Cad protein level in Ishikawa cells. The effect of LEFTY2 on migration was mimicked by FAK inhibitor PF 573228 (50 µM). Addition of LEFTY2 in the presence of PF-573228 did not result in a further significant decline of migration. Conclusion: In conclusion, LEFTY2 down-regulates MKi67 expression and FAK activity, up-regulates miR-200a and E-cadherin, and is thus a powerful negative regulator of endometrial cell proliferation and migration.

scholarly journals Glycogen synthase kinase 3β promotes osteosarcoma invasion and migration via regulating PTEN and phosphorylation of focal adhesion kinase

2021 ◽  
Author(s):  
Wei Mai ◽  
Lingyu Kong ◽  
Hongwei Yu ◽  
Junjie Bao ◽  
Chunyu Song ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Western Blotting ◽  
Focal Adhesion ◽  
Glycogen Synthase ◽  
Migration And Invasion ◽  
Glycogen Synthase Kinase 3Β ◽  
Sirna Transfection ◽  
Invasion And Migration ◽  
Human Osteosarcoma ◽  
And Migration

Aim: Typical features of human osteosarcoma are highly invasive and migratory capacities. Our study aimed to investigate the roles of glycogen synthase kinase 3β (GSK3β) in human osteosarcoma metastasis. Methods: GSK3β expressions in clinical osteosarcoma tissues with or without metastasis were examined by immunohistochemical staining. The expressions of GSK3β, p-GSK3βSer9, and p-GSK3βTyr216 in human osteoblast cells (hFOB1.19) and human osteosarcoma cells (MG63, SaOS-2 and U2-OS) were detected by western blotting. The GSK3β activity was measured by non-radio isotopic in vitro kinase assay. Migration and invasion abilities of MG-63 cells treated with small-molecular GSK3β inhibitors were respectively examined by monolayer-based wound-healing assay and transwell assay. The mRNA expressions of GSK3β, matrix metalloproteinase-2 (MMP-2), MMP-9, phosphatase with tensin homology (PTEN), and focal adhesion kinase (FAK) were detected after siRNA transfection for 72 h. Meanwhile, protein expressions of GSK3β, FAK, p-FAKY397, PTEN, MMP-2, and MMP-9 were measured by western blotting. Results: Clinical osteosarcoma tissues with metastasis showed higher GSK3β expressions. MG63 and U2-OS cells which were easy to occur metastasis showed significantly higher expressions and activities of GSK3β than SaOS-2 cells. Inhibition of GSK3β with small-molecular GSK3β inhibitors in MG63 cells significantly attenuated cell migration and invasion. These effects were associated with reduced expressions of MMP-2 and MMP-9. Moreover, increased PTEN and decreased p-FAKY397 expressions were observed following GSK3b knock-down by siRNA transfection. Conclusion: GSK3β might promote osteosarcoma invasion and migration via pathways associated with PTEN and phosphorylation of FAK.

scholarly journals The effects of c-Src kinase on EMT signaling pathway in human lens epithelial cells associated with lens diseases

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xingyu Li ◽  
Fang Wang ◽  
Meixia Ren ◽  
Minjuan Du ◽  
Jian Zhou
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cells ◽  
Western Blot ◽  
Src Kinase ◽  
Human Lens ◽  
Lens Epithelial Cells ◽  
Protein Levels ◽  
Human Lens Epithelial Cells ◽  
And Migration ◽  
E Cadherin

Abstract Background The signaling pathway of epithelial to mesenchymal transition (EMT) is regulated by c-Src kinase in many cells. The purpose of this study was to investigate the effects of c-Src kinase on EMT of human lens epithelial cells in vivo stimulated by different factors. Methods Human lens epithelial cells, HLE-B3, were exposed to either an inflammatory factor, specifically IL-1α, IL-6, TNF-α or IL-1β, at 10 ng/mL or high glucose (35.5 mM) for 30 mins. Activity of c-Src kinase was evaluated by the expression of p-Src418 with western blot assay. To investigate the effects of activation of c-Src on EMT, HLE-B3 cells were transfected with pCDNA3.1-SrcY530F to upregulate activity of c-Src kinase, and pSlience4.1-ShSrc to knock it down. The expressions of c-Src kinase and molecular markers of EMT such as E-cadherin, ZO-1, α-SMA, and Vimentin were examined at 48 h by RT-PCR and western blot. At 48 h and 72 h of transfection, cell proliferation was detected by MTT, and cell mobility and migration were determined by scratch and transwell assays. Results Activity of c-Src kinase, which causes the expression of p-Src418, was upregulated by different inflammatory factors and high glucose in HLE-B3 cells. When HLE-B3 cells were transfected with pCDNA3.1-SrcY530F, the expression of c-Src kinase was upregulated on both mRNA and protein levels, and activity of c-Src kinase, expression of p-Src418 increased. The expressions of both E-cadherin and ZO-1 were suppressed, while the expressions of vimentin and α-SMA were elevated on both mRNA and protein levels at the same time. Cell proliferation, mobility and migration increased along with activation of c-Src kinase. Conversely, when HLE-B3 cells were transfected with pSlience4.1-ShSrc, both c-Src kinase and p-Src418 expressions were knocked down. The expressions of E-cadherin and ZO-1 increased, but the expressions of Vimentin and α-SMA decreased; meanwhile, cell proliferation, mobility and migration reduced. Conclusions The c-Src kinase in lens epithelial cells is easily activated by external stimuli, resulting in the induction of cell proliferation, mobility, migration and EMT.

Circ_0085296 suppresses trophoblast cell proliferation, invasion, and migration via modulating miR-144/E-cadherin axis

Placenta ◽  
2020 ◽  
Vol 97 ◽  
pp. 18-25
Author(s):  
Hailing Zhu ◽  
Xia Niu ◽  
Qinghua Li ◽  
Yuehua Zhao ◽  
Xue Chen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Trophoblast Cell ◽  
Invasion And Migration ◽  
And Migration ◽  
E Cadherin

scholarly journals Cardiac fibroblasts require focal adhesion kinase for normal proliferation and migration

2009 ◽  
Vol 296 (3) ◽  
pp. H627-H638 ◽  
Author(s):  
Ana Maria Manso ◽  
Seok-Min Kang ◽  
Sergey V. Plotnikov ◽  
Ingo Thievessen ◽  
Jaewon Oh ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Adult Mouse ◽  
Cardiac Fibroblasts ◽  
Focal Adhesions ◽  
Protein Levels ◽  
Tyrosine Kinase 2 ◽  
A Cell ◽  
And Migration ◽  
Proliferation And Migration

Migration and proliferation of cardiac fibroblasts (CFs) play an important role in the myocardial remodeling process. While many factors have been identified that regulate CF growth and migration, less is known about the signaling mechanisms involved in these processes. Here, we utilized Cre-LoxP technology to obtain focal adhesion kinase (FAK)-deficient adult mouse CFs and studied how FAK functioned in modulating cell adhesion, proliferation, and migration of these cells. Treatment of FAKflox/flox CFs with Ad/Cre virus caused over 70% reduction of FAK protein levels within a cell population. FAK-deficient CFs showed no changes in focal adhesions, cell morphology, or protein expression levels of vinculin, talin, or paxillin; proline-rich tyrosine kinase 2 (Pyk2) expression and activity were increased. Knockdown of FAK protein in CFs increased PDGF-BB-induced proliferation, while it reduced PDGF-BB-induced migration. Adhesion to fibronectin was not altered. To distinguish between the function of FAK and Pyk2, FAK function was inhibited via adenoviral-mediated overexpression of the natural FAK inhibitor FAK-related nonkinase (FRNK). Ad/FRNK had no effect on Pyk2 expression, inhibited the PDGF-BB-induced migration, but did not change the PDGF-BB-induced proliferation. FAK deficiency had only modest effects on increasing PDGF-BB activation of p38 and JNK MAPKs, with no alteration in the ERK response vs. control cells. These results demonstrate that FAK is required for the PDGF-BB-induced migratory response of adult mouse CFs and suggest that FAK could play an essential role in the wound-healing response that occurs in numerous cardiac pathologies.

scholarly journals Roles of focal adhesion kinase (FAK) in megakaryopoiesis and platelet function: studies using a megakaryocyte lineage–specific FAK knockout

Blood ◽  
2008 ◽  
Vol 111 (2) ◽  
pp. 596-604 ◽  
Author(s):  
Ian S. Hitchcock ◽  
Norma E. Fox ◽  
Nicolas Prévost ◽  
Katherine Sear ◽  
Sanford J. Shattil ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Platelet Function ◽  
Therapeutic Benefit ◽  
Negative Regulator ◽  
Null Mouse ◽  
Wild Type ◽  
Platelet Recovery ◽  
Lyn Kinase

Focal adhesion kinase (FAK) plays a key role in mediating signaling downstream of integrins and growth factor receptors. In this study, we determined the roles of FAK in vivo by generating a megakaryocyte lineage–specific FAK-null mouse (Pf4-Cre/FAK-floxed). Megakaryocyte and platelet FAK expression was ablated in Pf4-Cre/FAK-floxed mice without affecting expression of the FAK homologue PYK2, although PYK2 phosphorylation was increased in FAK−/− megakaryocytes in response to fibrinogen. Megakaryopoiesis is greatly enhanced in Pf4-Cre/FAK-floxed mice, with significant increases in megakaryocytic progenitors (CFU-MK), mature megakaryocytes, megakaryocyte ploidy, and moderate increases in resting platelet number and platelet recovery following a thrombocytopenic stress. Thrombopoietin (Tpo)–mediated activation of Lyn kinase, a negative regulator of megakaryopoiesis, is severely attenuated in FAK-null megakaryocytes compared with wild-type controls. In contrast, Tpo-mediated activation of positive megakaryopoiesis regulators such as ERK1/2 and AKT is increased in FAK-null megakaryocytes, providing a plausible explanation for the observed increases in megakaryopoiesis in these mice. In Pf4-Cre/FAK-floxed mice, rebleeding times are significantly increased, and FAK-null platelets exhibit diminished spreading on immobilized fibrinogen. These studies establish clear roles for FAK in megakaryocyte growth and platelet function, setting the stage for manipulation of this component of the Tpo signaling apparatus for therapeutic benefit.

scholarly journals Decrease of Store-Operated Ca2+ Entry and Increase of Na+/Ca2+ Exchange by Pharmacological JAK2 Inhibition

2016 ◽  
Vol 38 (2) ◽  
pp. 683-695 ◽  
Author(s):  
Jing Yan ◽  
Zohreh Hosseinzadeh ◽  
Bingbing Zhang ◽  
Mirjam Froeschl ◽  
Klaus Schulze-Osthoff ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Abundance ◽  
Transcript Levels ◽  
Migratory Activity ◽  
Jak2 Inhibitor ◽  
Whole Cell Patch Clamp ◽  
And Migration ◽  
The Impact ◽  
Powerful Mechanism ◽  
Jak2 Inhibition

Background/Aims: Cell proliferation and migration are regulated by cytosolic Ca2+ activity ([Ca2+]i). Mechanisms modifying [Ca2+]i include store-operated Ca2+-entry (SOCE) accomplished by the pore-forming ion channel unit Orai1 and its regulator STIM1, as well as Ca2+ extrusion by Na+/Ca2+ exchanger NCX1. Kinases participating in the orchestration of cell proliferation include the Janus activated kinase JAK2. The present study explored the impact of pharmacological JAK2 inhibition on SOCE and Na+/Ca2+ exchange. Methods: MCF-7 breast carcinoma cells were studied in the absence and presence of the JAK2 inhibitors TG101348 (250 nM - 1 µM) or of AG490 (20 - 40 µM). Transcript levels were quantified with RT-PCR, protein abundance with immunoblotting, [Ca2+]i with Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and Na+/Ca2+ exchanger activity from increase of [Ca2+]i as well as Ca2+ current in whole cell patch clamp following extracellular Na+ removal. Migratory activity was determined by a wound healing assay. Results: JAK2 inhibitor TG101348 (1 µM) decreased Orai1 and STIM1 protein abundance, increased NCX1 transcript levels, decreased Ca2+ release from intracellular stores, decreased SOCE, increased Ca2+ entry as well as Ca2+-current following extracellular Na+-removal, and decreased migration. Similar effects on Ca2+ release, SOCE, and Ca2+-entry following extracellular Na+-removal were observed following treatment with AG490. Conclusion: The present observations disclose a novel powerful mechanism regulating intracellular Ca2+ release, cellular Ca2+ entry, cellular Ca2+ extrusion and cell migration.

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