scholarly journals Immune Cell Induced Migration of Osteoprogenitor Cells Is Mediated by TGF-β Dependent Upregulation of NOX4 and Activation of Focal Adhesion Kinase

2018 ◽  
Vol 19 (8) ◽  
pp. 2239 ◽  
Author(s):  
Sabrina Ehnert ◽  
Caren Linnemann ◽  
Romina Aspera-Werz ◽  
Daria Bykova ◽  
Sara Biermann ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Immune Cell ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Conditioned Media ◽  
Dependent Manner ◽  
Osteoprogenitor Cells ◽  
Nadph Oxidase 4

The cytokines secreted by immune cells have a large impact on the tissue, surrounding a fracture, e.g., by attraction of osteoprogenitor cells. However, the underlying mechanisms are not yet fully understood. Thus, this study aims at investigating molecular mechanisms of the immune cell-mediated migration of immature primary human osteoblasts (phOBs), with transforming growth factor beta (TGF-β), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) and focal adhesion kinase (FAK) as possible regulators. Monocyte- and macrophage (THP-1 cells ± phorbol 12-myristate 13-acetate (PMA) treatment)-conditioned media, other than the granulocyte-conditioned medium (HL-60 cells + dimethyl sulfoxide (DMSO) treatment), induce migration of phOBs. Monocyte- and macrophage (THP-1 cells)-conditioned media activate Smad3-dependent TGF-β signaling in the phOBs. Stimulation with TGF-β promotes migration of phOBs. Furthermore, TGF-β treatment strongly induces NOX4 expression on both mRNA and protein levels. The associated reactive oxygen species (ROS) accumulation results in phosphorylation (Y397) of FAK. Blocking TGF-β signaling, NOX4 activity and FAK signaling effectively inhibits the migration of phOBs towards TGF-β. In summary, our data suggest that monocytic- and macrophage-like cells induce migration of phOBs in a TGF-β-dependent manner, with TGF-β-dependent induction of NOX4, associated production of ROS and resulting activation of FAK as key mediators.

scholarly journals NOX4 regulates TGFβ-induced proliferation and self-renewal in glioblastoma stem cells

2019 ◽  
Author(s):  
P García-Gómez ◽  
M Dadras ◽  
C Bellomo ◽  
A Mezheyeuski ◽  
K Tzavlaki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Dependent Manner ◽  
Ros Production ◽  
Self Renewal ◽  
Glioblastoma Stem Cells ◽  
Nadph Oxidase 4

ABSTRACTGlioblastoma (GBM) is the most aggressive and common glioma subtype with a median survival of 15 months after diagnosis. Current treatments have limited therapeutic efficacy, thus more effective approaches are needed. The glioblastoma tumoral mass is characterized by a small cellular subpopulation, the Glioblastoma stem cells (GSCs), which has been held accountant for initiation, invasion, proliferation, relapse and resistance to chemo- and radiotherapy. Targeted therapies against GSCs are crucial, and so is the understanding of the molecular mechanisms that govern the GSCs. Transforming growth factor β (TGFβ), platelet growth factor (PDGF) signalling and Reactive Oxygen Species (ROS) production govern and regulate cancer-stem cell biology. In this work, we focus on the role of the NADPH oxidase 4 (NOX4) downstream of TGFβ signalling in the GSCs. NOX4 utilises NADPH to generate ROS; TGFβ induces NOX4 expression, thus increasing ROS production. Interestingly, NOX4 itself regulates GSC self-renewal and modulates Since TGFβ regulates PDGFB in GSC, we analysed how PDGFB modulates NOX4 expression and increases ROS production. Both TGFβ and PDGF signalling regulate GSC proliferation in a NOX4/ROS-dependent manner. The transcription factor NRF2, involved in the transcriptional regulation of antioxidant and metabolic responses, is regulated by both TGFβ and NOX4. This results in an antioxidant response, which positively contributes to GSC self-renewal and proliferation. In conclusion, this work functionally establishes NOX4 as a key mediator of GSC biology.

scholarly journals LPHN2 inhibits vascular permeability by differential control of endothelial cell adhesion

2021 ◽  
Vol 220 (11) ◽  
Author(s):  
Chiara Camillo ◽  
Nicola Facchinello ◽  
Giulia Villari ◽  
Giulia Mana ◽  
Noemi Gioelli ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Focal Adhesion ◽  
Nuclear Localization ◽  
Molecular Mechanisms ◽  
Transcriptional Regulators ◽  
Dependent Manner ◽  
Knock Out ◽  
Dynamic Modulation ◽  
Differential Control ◽  
G Protein Coupled

Dynamic modulation of endothelial cell-to-cell and cell–to–extracellular matrix (ECM) adhesion is essential for blood vessel patterning and functioning. Yet the molecular mechanisms involved in this process have not been completely deciphered. We identify the adhesion G protein–coupled receptor (ADGR) Latrophilin 2 (LPHN2) as a novel determinant of endothelial cell (EC) adhesion and barrier function. In cultured ECs, endogenous LPHN2 localizes at ECM contacts, signals through cAMP/Rap1, and inhibits focal adhesion (FA) formation and nuclear localization of YAP/TAZ transcriptional regulators, while promoting tight junction (TJ) assembly. ECs also express an endogenous LPHN2 ligand, fibronectin leucine-rich transmembrane 2 (FLRT2), that prevents ECM-elicited EC behaviors in an LPHN2-dependent manner. Vascular ECs of lphn2a knock-out zebrafish embryos become abnormally stretched, display a hyperactive YAP/TAZ pathway, and lack proper intercellular TJs. Consistently, blood vessels are hyperpermeable, and intravascularly injected cancer cells extravasate more easily in lphn2a null animals. Thus, LPHN2 ligands, such as FLRT2, may be therapeutically exploited to interfere with cancer metastatic dissemination.

scholarly journals MON-647 Mechanisms of Insulin Resistance in Skeletal Muscle in Women with PCOS

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Alba Moreno-Asso ◽  
Luke C McIlvenna ◽  
Rhiannon K Patten ◽  
Andrew J McAinch ◽  
Raymond J Rodgers ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Glucose Uptake ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Reproductive Age ◽  
Whole Body ◽  
Dependent Manner ◽  
Cultured Myotubes ◽  
Tgfβ Superfamily

Abstract Polycystic ovary syndrome (PCOS) is the most common female endocrine disorder affecting metabolic, reproductive and mental health of 8-13% of reproductive-age women. Insulin resistance (IR) appears to underpin the pathophysiology of PCOS and is present in approximately 85% of women with PCOS. This underlying IR has been identified as unique from, but synergistic with, obesity-induced IR (1). Skeletal muscle accounts for up to 85% of whole body insulin-stimulated glucose uptake, however, in PCOS this is reduced about 27% when assessed by hyperinsulinemic euglycemic clamp (2). Interestingly, this reduced insulin-stimulated glucose uptake observed in skeletal muscle tissue is not retained in cultured myotubes (3), suggesting that environmental factors may play a role in this PCOS-specific IR. Yet, the molecular mechanisms regulating IR remain unclear (4). Previous work suggested that Transforming Growth Factor Beta (TGFβ) superfamily ligands may be involved in the metabolic morbidity associated with PCOS (5). In this study, we investigated the effects of TGFβ1 (1, 5ng/ml), and the Anti-Müllerian hormone (AMH; 5, 10, 30ng/ml), a novel TGFβ superfamily ligand elevated in women with PCOS, as causal factors of IR in cultured myotubes from women with PCOS (n=10) and healthy controls (n=10). AMH negatively affected glucose uptake and insulin signalling increasing p-IRS1 (ser312) in a dose-dependent manner in myotubes from both women with and without PCOS. AMH did not appear to activate the canonical TGFβ/BMP signalling pathway. Conversely, TGFβ1 had an opposite effect in both PCOS and control myotubes cultures, decreasing phosphorylation of IRS1 (ser312) and enhancing glucose uptake via Smad2/3 signalling. In conclusion, these results suggest that AMH may play a role in skeletal muscle IR observed in PCOS, however, further research is required to elucidate its mechanisms of action and broader impact in this syndrome. References: (1) Stepto et al. Hum Reprod 2013 Mar;28(3):777-784. (2) Cassar et al. Hum Reprod 2016 Nov;31(11):2619-2631. (3) Corbould et al., Am J Physiol-Endoc 2005 May;88(5):E1047-54. (4) Stepto et al. J Clin Endocrinol Metab, 2019 Nov 1;104(11):5372-5381. (5) Raja-Khan et al. Reprod Sci 2014 Jan;21(1):20-31.

scholarly journals Suppression of the TGF-β pathway by a macrolide antibiotic decreases fibrotic responses by ocular fibroblasts in vitro

2020 ◽  
Vol 7 (9) ◽  
pp. 200441
Author(s):  
Thomas Stahnke ◽  
Beata Gajda-Deryło ◽  
Anselm G. Jünemann ◽  
Oliver Stachs ◽  
Katharina A. Sterenczak ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Macrolide Antibiotic ◽  
Drug Repositioning ◽  
Dependent Manner ◽  
Glaucoma Filtration Surgery ◽  
Concentration Dependent Manner ◽  
Device Implantation

To elucidate and to inhibit post-surgical fibrotic processes after trabeculectomy in glaucoma therapy, we measured gene expression in a fibrotic cell culture model, based on transforming growth factor TGF-β induction in primary human tenon fibroblasts (hTFs), and used Connectivity Map (CMap) data for drug repositioning. We found that specific molecular mechanisms behind fibrosis are the upregulation of actins, the downregulation of CD34, and the upregulation of inflammatory cytokines such as IL6, IL11 and BMP6 . The macrolide antibiotic Josamycin (JM) reverses these molecular mechanisms according to data from the CMap, and we thus tested JM as an inhibitor of fibrosis. JM was first tested for its toxic effects on hTFs, where it showed no influence on cell viability, but inhibited hTF proliferation in a concentration-dependent manner. We then demonstrated that JM suppresses the synthesis of extracellular matrix (ECM) components. In hTFs stimulated with TGF-β1, JM specifically inhibited α-smooth muslce actin expression, suggesting that it inhibits the transformation of fibroblasts into fibrotic myofibroblasts. In addition, a decrease of components of the ECM such as fibronectin, which is involved in in vivo scarring, was observed. We conclude that JM may be a promising candidate for the treatment of fibrosis after glaucoma filtration surgery or drainage device implantation in vivo .

scholarly journals TIMP-1 Induces α-Smooth Muscle Actin in Fibroblasts to Promote Urethral Scar Formation

2015 ◽  
Vol 35 (6) ◽  
pp. 2233-2243 ◽  
Author(s):  
Yinglong Sa ◽  
Chao Li ◽  
Hongbin Li ◽  
Hailin Guo
Keyword(s):  
Smooth Muscle ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Human Fibroblasts ◽  
Mapk Signaling ◽  
Scar Formation ◽  
Dependent Manner ◽  
Muscle Actin ◽  
Mesenchymal Transition

Background/Aims: Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been reported to upregulate in urethral scar. However, the underlying molecular mechanisms remain undefined. Methods: Here, we studied levels of TIMP-1 and α-smooth muscle actin (α-SMA) in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Then we either overexpressed TIMP-1, or inhibited TIMP-1 by lentiviruses carrying a transgene or a short hairpin small interfering RNA for TIMP-1 in human fibroblasts. We examined the effects of modulation of TIMP-1 on α-SMA, and on epithelial-mesenchymal transition (EMT)-related genes. We also studied the underlying mechanisms. Results: We detected significantly higher levels of TIMP-1 and α-smooth muscle actin (α-SMA) in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Moreover, the levels of TIMP-1 and α-SMA strongly correlated. Moreover, we found that TIMP-1 significantly increased levels of α-SMA, transforming growth factor β 1 (TGFβ1), Collagen I and some other key factors related to an enhanced EMT, suggesting that TIMP-1 may induce transformation of fibroblasts into myofibroblasts to promote tissue EMT to enhance the formation of urethral scar. Moreover, increases in TIMP-1 also induced an increase in fibroblast cell growth and cell invasion, in an ERK/MAPK-signaling-dependent manner. Conclusion: Our study thus highlights a pivotal role of TIMP-1 in urethral scar formation.

Focal Adhesion Kinase Inactivation Reduces the Development of Acute Leukemia and Partially Rescues Hematopoietic Stem Cell Defects in Pten-Knockout Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 864-864
Author(s):  
Dewen You ◽  
Andrew Volk ◽  
Clare Sun ◽  
Junping Xin ◽  
Geunhyoung Ha ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Phosphatase Activity ◽  
Focal Adhesion ◽  
Protein Phosphatase ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Polycytidylic Acid

Abstract Abstract 864 Phosphatase and tensin homolog on chromosome 10 (Pten) is a tumor suppressor which possesses both lipid and protein phosphatase activities. Mutations and epigenetic inactivations of the Pten gene are commonly detected in a large number of tissue malignancies, including leukemias and lymphomas. Studies using Hematopoietic Pten-knockout in adult mice (Pten−/−) have demonstrated that Pten plays a critical role in maintaining the homeostasis of bone marrow (BM) hematopoiesis. Pten inactivation promotes the proliferation and peripheral mobilization of BM hematopoietic stem cells (HSCs). Pten−/− mice develop myeloproliferative disorders (MPD) within days, followed by acute leukemic transformation. Most previous studies attributed such phenotypic changes observed in Pten−/− mice to excessive activation of the PI3K/AKT/mTOR signal, a consequence of the loss of Pten's lipid phosphatase activity. However, the role of Pten's protein phosphatase activity in the regulation of HSCs and leukemogenesis is not well studied. Focal adhesion kinase (Fak) is a critical substrate for the protein phosphatase activity of Pten. Dysregulation of Fak has been observed in many cancers, including acute myeloid leukemias (AML) and acute lymphocytic leukemias (ALL). Therefore, we postulated that Fak might play a pivotal role in the development and progression of leukemia following Pten deletion. To test this hypothesis, we generated Mx1-Cre+Ptenfl/flFakfl/fl mice (an interferon-inducible Pten and Fak compound-knockout, Pten−/−Fak−/−) in which both the Pten and Fak genes in the hematopoietic system are deleted upon injection of polyinosinic-polycytidylic acid (pI-pC). Our results showed that the genetic inactivation of Fak can partially rescue HSC defects associated with Pten deficiency. We found that peripheral mobilization of HSCs in Pten−/−Fak−/− mice is significantly reduced compared to Pten−/− mice. As a consequence, more long-term HSCs (LT-HSCs) are preserved in the BM of Pten−/−Fak−/− mice compared to Pten−/− mice. Transplantation studies suggested that the hematopoietic reconstitutive capacity of Pten−/−Fak−/− HSCs is significantly improved compared to Pten−/− HSCs. Although Fak deletion fails to prevent the development of MPD in Pten−/− mice, Fak deletion does significantly reduce the frequency of AML/ALL, also significantly delays the onset of AML/ALL in comparison to Pten−/− mice. This study suggests that Fak might be a potential target for preventing the MPD-to-AML/ALL transformation and therefore blocking the Fak activity may hold a promise for a novel anti-leukemia therapy. The molecular mechanisms underlying the phenotype restoration of Pten−/− mice by Fak deletion in the hematopoietic system are actively being studied in our laboratory. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Platelet-derived serotonin links vascular disease and tissue fibrosis

2011 ◽  
Vol 208 (5) ◽  
pp. 961-972 ◽  
Author(s):  
Clara Dees ◽  
Alfiya Akhmetshina ◽  
Pawel Zerr ◽  
Nicole Reich ◽  
Katrin Palumbo ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Tissue Remodeling ◽  
Transforming Growth Factor Β ◽  
Vascular Damage ◽  
Skin Fibrosis ◽  
Dependent Manner ◽  
Dermal Fibrosis ◽  
The Central Nervous System

Vascular damage and platelet activation are associated with tissue remodeling in diseases such as systemic sclerosis, but the molecular mechanisms underlying this association have not been identified. In this study, we show that serotonin (5-hydroxytryptamine [5-HT]) stored in platelets strongly induces extracellular matrix synthesis in interstitial fibroblasts via activation of 5-HT2B receptors (5-HT2B) in a transforming growth factor β (TGF-β)–dependent manner. Dermal fibrosis was reduced in 5-HT2B−/− mice using both inducible and genetic models of fibrosis. Pharmacologic inactivation of 5-HT2B also effectively prevented the onset of experimental fibrosis and ameliorated established fibrosis. Moreover, inhibition of platelet activation prevented fibrosis in different models of skin fibrosis. Consistently, mice deficient for TPH1, the rate-limiting enzyme for 5-HT production outside the central nervous system, showed reduced experimental skin fibrosis. These findings suggest that 5-HT/5-HT2B signaling links vascular damage and platelet activation to tissue remodeling and identify 5-HT2B as a novel therapeutic target to treat fibrotic diseases.

scholarly journals Steel Factor Enhances Integrin-Mediated Tyrosine Phosphorylation of Focal Adhesion Kinase (pp125FAK) and Paxillin

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1574-1584 ◽  
Author(s):  
Hiroyuki Takahira ◽  
Akihiko Gotoh ◽  
Alec Ritchie ◽  
Hal E. Broxmeyer
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Direct Action ◽  
Human Growth ◽  
Dependent Manner ◽  
Integrin Receptors ◽  
Steel Factor ◽  
Extracellular Matrix Components ◽  
Dependent Cell

Integrin-mediated interaction of hematopoietic progenitor cells with bone marrow stromal extracellular matrix components is important in hematopoiesis. Focal adhesion kinase (pp125FAK) plays a central role in signal transduction through integrin receptors. We studied matrix-integrin interaction and subsequent signaling in human growth factor-dependent cell line, TF-1. Adherence of unstimulated TF-1 cells to fibronectin-coated wells was blocked by antiintegrin β1 and combination of anti-α4 with anti-α5 antibodies, indicating α4β1 and α5β1 integrin mediated adherence. Steel factor (SLF) increased TF-1 adhesion to fibronectin dose-dependently and 10−7 mol/L wortmannin suppressed SLF-induced adhesion. Immunoprecipitation and immunoblotting with antiphosphotyrosine antibody showed that adherence of TF-1 cells to fibronectin without cytokine caused tyrosine phosphorylation of several proteins identified as pp125FAK and paxillin. SLF induced spreading of adherent TF-1 cells and enhanced tyrosine phosphorylation of pp125FAK and paxillin in a dose-dependent manner. Treatment with SLF without plating on fibronectin did not induce tyrosine phosphorylation of pp125FAK. Wortmannin, at 10−7 mol/L, completely abolished SLF-induced enhancement of pp125FAK tyrosine phosphorylation, while c-kit autophosphorylation was not affected. This suggests that increase of pp125FAK tyrosine phosphorylation was mediated through a wortmannin sensitive pathway, rather than by direct action on c-kit tyrosine kinase. Treatment of adherent TF-1 cells with RGDS peptide plus anti-α4 antibody also inhibited SLF-induced enhancement of pp125FAK tyrosine phosphorylation without detachment of TF-1 cells. These data suggest that SLF enhances integrin-fibronectin-dependent tyrosine phosphorylation of pp125FAK through activation of integrin (“inside-out” signaling) and following integrin occupancy. This establishes a novel linkage between c-kit/SLF pathway and integrin fibronectin signaling.

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