scholarly journals The mineralocorticoid receptor leads to increased expression of EGFR and T-type calcium channels that support HL-1 cell hypertrophy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Katharina Stroedecke ◽  
Sandra Meinel ◽  
Fritz Markwardt ◽  
Udo Kloeckner ◽  
Nicole Straetz ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Mineralocorticoid Receptor ◽  
Molecular Mechanisms ◽  
Egf Receptor ◽  
Tumor Biology ◽  
Cell Diameter ◽  
Snp Analysis ◽  
Peak Current Density ◽  
Egfr Expression

AbstractThe EGF receptor (EGFR) has been extensively studied in tumor biology and recently a role in cardiovascular pathophysiology was suggested. The mineralocorticoid receptor (MR) is an important effector of the renin–angiotensin–aldosterone-system and elicits pathophysiological effects in the cardiovascular system; however, the underlying molecular mechanisms are unclear. Our aim was to investigate the importance of EGFR for MR-mediated cardiovascular pathophysiology because MR is known to induce EGFR expression. We identified a SNP within the EGFR promoter that modulates MR-induced EGFR expression. In RNA-sequencing and qPCR experiments in heart tissue of EGFR KO and WT mice, changes in EGFR abundance led to differential expression of cardiac ion channels, especially of the T-type calcium channel CACNA1H. Accordingly, CACNA1H expression was increased in WT mice after in vivo MR activation by aldosterone but not in respective EGFR KO mice. Aldosterone- and EGF-responsiveness of CACNA1H expression was confirmed in HL-1 cells by Western blot and by measuring peak current density of T-type calcium channels. Aldosterone-induced CACNA1H protein expression could be abrogated by the EGFR inhibitor AG1478. Furthermore, inhibition of T-type calcium channels with mibefradil or ML218 reduced diameter, volume and BNP levels in HL-1 cells. In conclusion the MR regulates EGFR and CACNA1H expression, which has an effect on HL-1 cell diameter, and the extent of this regulation seems to depend on the SNP-216 (G/T) genotype. This suggests that the EGFR may be an intermediate for MR-mediated cardiovascular changes and that SNP analysis can help identify subgroups of patients that will benefit most from MR antagonists.

scholarly journals Expression of renal distal tubule transporters TRPM6 and NCC in a rat model of cyclosporine nephrotoxicity and effect of EGF treatment

2011 ◽  
Vol 301 (3) ◽  
pp. F486-F493 ◽  
Author(s):  
Kristien J. Ledeganck ◽  
Gaëlle A. Boulet ◽  
Caroline A. Horvath ◽  
Marleen Vinckx ◽  
Johannes J. Bogers ◽  
...  
Keyword(s):  
Rat Model ◽  
Molecular Mechanisms ◽  
Egf Receptor ◽  
Distal Tubule ◽  
Fractional Excretion ◽  
Cyclosporine Nephrotoxicity ◽  
Final Adjustment ◽  
Underlying Mechanisms ◽  
Renal Expression

Renal magnesium (Mg2+) and sodium (Na+) loss are well-known side effects of cyclosporine (CsA) treatment in humans, but the underlying mechanisms still remain unclear. Recently, it was shown that epidermal growth factor (EGF) stimulates Mg2+ reabsorption in the distal convoluted tubule (DCT) via TRPM6 (Thébault S, Alexander RT, Tiel Groenestege WM, Hoenderop JG, Bindels RJ. J Am Soc Nephrol 20: 78–85, 2009). In the DCT, the final adjustment of renal sodium excretion is regulated by the thiazide-sensitive Na+-Cl− cotransporter (NCC), which is activated by the renin-angiotensin-aldosterone system (RAAS). The aim of this study was to gain more insight into the molecular mechanisms of CsA-induced hypomagnesemia and hyponatremia. Therefore, the renal expression of TRPM6, TRPM7, EGF, EGF receptor, claudin-16, claudin-19, and the NCC, and the effect of the RAAS on NCC expression, were analyzed in vivo in a rat model of CsA nephrotoxicity. Also, the effect of EGF administration on these parameters was studied. CsA significantly decreased the renal expression of TRPM6, TRPM7, NCC, and EGF, but not that of claudin-16 and claudin-19. Serum aldosterone was significantly lower in CsA-treated rats. In control rats treated with EGF, an increased renal expression of TRPM6 together with a decreased fractional excretion of Mg2+ (FE Mg2+) was demonstrated. EGF did not show this beneficial effect on TRPM6 and FE Mg2+ in CsA-treated rats. These data suggest that CsA treatment affects Mg2+ homeostasis via the downregulation of TRPM6 in the DCT. Furthermore, CsA downregulates the NCC in the DCT, associated with an inactivation of the RAAS, resulting in renal sodium loss.

scholarly journals Mouse Oocytes Enable LH-Induced Maturation of the Cumulus-Oocyte Complex via Promoting EGF Receptor-Dependent Signaling

2010 ◽  
Vol 24 (6) ◽  
pp. 1230-1239 ◽  
Author(s):  
You-Qiang Su ◽  
Koji Sugiura ◽  
Qinglei Li ◽  
Karen Wigglesworth ◽  
Martin M. Matzuk ◽  
...  
Keyword(s):  
Growth Factors ◽  
Granulosa Cells ◽  
Egf Receptor ◽  
Cumulus Cells ◽  
Wild Type ◽  
Paracrine Factors ◽  
Cumulus Oocyte Complex ◽  
Egfr Expression

Abstract LH triggers the maturation of the cumulus-oocyte complex (COC), which is followed by ovulation. These ovarian follicular responses to LH are mediated by epidermal growth factor (EGF)-like growth factors produced by granulosa cells and require the participation of oocyte-derived paracrine factors. However, it is not clear how oocytes coordinate with the EGF receptor (EGFR) signaling to achieve COC maturation. The aim of the present study was to test the hypothesis that oocytes promote the expression of EGFR by cumulus cells, thus enabling them to respond to the LH-induced EGF-like peptides. Egfr mRNA and protein expression were dramatically reduced in cumulus cells of mutant mice deficient in the production of the oocyte-derived paracrine factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15). Moreover, microsurgical removal of oocytes from wild-type COCs dramatically reduced expression of Egfr mRNA and protein, and these levels were restored by either coculture with oocytes or treatment with recombinant GDF9 or GDF9 plus recombinant BMP15. Blocking Sma- and Mad-related protein (SMAD)2/3 phosphorylation in vitro inhibited Egfr expression in wild-type COCs and in GDF9-treated wild-type cumulus cells, and conditional deletion of Smad2 and Smad3 genes in granulosa cells in vivo resulted in the reduction of Egfr mRNA in cumulus cells. These results indicate that oocytes promote expression of Egfr in cumulus cells, and a SMAD2/3-dependent pathway is involved in this process. At least two oocyte-derived growth factors, GDF9 and BMP15, are required for EGFR expression by cumulus cells.

scholarly journals P11.09 Pan-RTK inhibition of sLRIG1 mediates AXL downregulation in Glioblastoma

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii44-iii44
Author(s):  
V Neirinckx ◽  
A Hau ◽  
A Schuster ◽  
S Fritah ◽  
A Chevigné ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Growth Factor Receptor ◽  
Soluble Form ◽  
Growth Factor Signaling ◽  
Egfr Expression ◽  
Baculovirus Infection

Abstract INTRODUCTION Aberrant regulation of receptor tyrosine kinase (RTK) activity is characteristic of Glioblastoma (GBM). However, RTK-based targeted therapies have been largely unsuccessful in GBM patients, partially due to the complexity and redundance of RTK signaling. LRIG1 (Leucine-rich Repeats and ImmunoGlobulindomains protein 1) is known as an endogenous inhibitor of epidermal growth factor receptor (EGFR) during health and disease, however its mechanism of action is poorly understood. We previously showed that the soluble form of LRIG1 potently inhibits of GBM growth in vivo, irrespective of EGFR expression level and status, suggesting the involvement of other RTKs. Here, we aimed to shed light on the molecular mechanisms underlying its anti-cancer activity. MATERIAL AND METHODS We generated a recombinant human soluble LRIG1 protein by expressing LRIG1 ectodomain in insect cells via baculovirus infection and subsequent His-tag purification. rh-sLRIG1 was applied in the medium of classical GBM cell lines, patient-derived GBM stem-like cells and patient-derived 3D tumor organoids. Using different cell-based assays, cell proliferation, invasion, cell morphology, as well as protein expression and protein-protein interactions were investigated. RESULTS We find that recombinant sLRIG1 efficiently reduces proliferation, invasion and viability of GBM cells and patient-derived organoids, and modulates cytoskeleton proteins and cell shape. In line with previous data, the effect of recombinant sLRIG1 is independent of EGFR expression. Interestingly sLRIG1 regulates several RTKs by direct protein downregulation, including AXL, while EGFR expression is not affected. At the molecular level, we find that sLRIG1 interferes with AXL dimerization, while no protein interaction with EGFR is detected. CONCLUSION We identify AXL as a novel LRIG1 target and provide evidence that sLRIG1-mediated RTK downregulation requires direct protein-protein interaction. These data pave the way for a potential therapeutic application of recombinant sLRIG1 in the inhibition of growth factor signaling in GBM.

Enhanced expression of EGF receptor in a model of salt-sensitive hypertension

2005 ◽  
Vol 289 (2) ◽  
pp. F314-F321 ◽  
Author(s):  
Wei-Zhong Ying ◽  
Paul W. Sanders
Keyword(s):  
Egf Receptor ◽  
Primary Cultures ◽  
Growth Factor Receptor ◽  
Sprague Dawley ◽  
Renal Vasculature ◽  
Downstream Signaling ◽  
Egfr Expression ◽  
Enhanced Expression

Chronic kidney disease in the Dahl/Rapp salt-sensitive (S) rat is related to an arteriolopathic process that occurs following the onset of hypertension and involves vascular smooth muscle cell (VSMC) hyperplasia and luminal constriction. Because previous studies have shown that activation of the epidermal growth factor receptor (EGFR) produces a mitogenic stimulus in VSMC and the EGFR participates integrally in the vasoconstrictor responses of renal arterioles, the present study analyzed the expression of EGFR in these animals. Compared with Sprague-Dawley (SD) rats, renal cortical expression of EGFR was increased in both prehypertensive and hypertensive S rats. Immunohistochemistry using a polyclonal antibody to EGFR demonstrated that EGFR expression was prominent in the renal vasculature, particularly in the media of afferent and efferent arterioles and the aorta of S rats. When examined, primary cultures of VSMC from S rats showed increased expression of EGFR, compared with VSMC from SD and Dahl/Rapp salt-resistant rats. Following addition of EGF, autophosphorylation of the EGFR was enhanced in cells from S rats, as was the downstream signaling events that included activation of p42/44 MAPK and Akt pathways. Thus in vivo and in vitro studies demonstrated augmented expression and functional activity of the EGFR in S rats.

scholarly journals Allosteric activation of preformed EGF receptor dimers by a single ligand binding event

2021 ◽  
Author(s):  
Endang R. Purba ◽  
Ei-ichiro Saita ◽  
Reetesh R. Akhouri ◽  
Lars-Göran Öfverstedt ◽  
Gunnar Wilken ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Egf Receptor ◽  
Active Form ◽  
Growth Factor Receptor ◽  
Full Length ◽  
Receptor Dimer

Abstract Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after ligand binding. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilized by ligand binding. This conformational flexibility stabilization most likely accompanies rotation of the entire extracellular domain and the transmembrane a-helix, resulting in dissociation of the intracellular kinase dimer and, thus, rearranging it into an active form. Consistently, mutations of amino acid residues at the interface of the inactive, symmetric kinase dimer spontaneously activate the receptor in vivo. Optical single-molecule observation also demonstrated that binding of only one ligand activates the receptor dimer on the cell surface. Based on these results, we propose an allosteric model for the activation of EGFR dimers by ligand binding. Our results demonstrate how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

scholarly journals Generation of endogenous pH-sensitive EGF receptor and its application in high-throughput screening for proteins involved in clathrin-mediated endocytosis

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Mads Breum Larsen ◽  
Mireia Perez Verdaguer ◽  
Brigitte F Schmidt ◽  
Marcel P Bruchez ◽  
Simon C Watkins ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Gene Editing ◽  
Egf Receptor ◽  
Ph Sensitive ◽  
Fluorescence Excitation ◽  
High Throughput Analysis ◽  
Egfr Expression ◽  
High Throughput Assay

Previously we used gene-editing to label endogenous EGF receptor (EGFR) with GFP and demonstrate that picomolar concentrations of EGFR ligand drive signaling and endocytosis of EGFR in tumors in vivo (Pinilla-Macua et al., 2017). We now use gene-editing to insert a fluorogen activating protein (FAP) in the EGFR extracellular domain. Binding of the tandem dye pair MG-Bis-SA to FAP-EGFR provides a ratiometric pH-sensitive model with dual fluorescence excitation and a single far-red emission. The excitation ratio of fluorescence intensities was demonstrated to faithfully report the fraction of FAP-EGFR located in acidic endosomal/lysosomal compartments. Coupling native FAP-EGFR expression with the high method sensitivity has allowed development of a high-throughput assay to measure the rates of clathrin-mediated FAP-EGFR endocytosis stimulated with physiological EGF concentrations. The assay was utilized to screen a phosphatase siRNA library. These studies highlight the utility of endogenous pH-sensitive FAP-receptor chimeras in high-throughput analysis of endocytosis.

scholarly journals CSIG-04. PAN-RTK INHIBITION OF sLRIG1 MEDIATES AXL DOWNREGULATION IN GLIOBLASTOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi44-vi44
Author(s):  
Virginie Neirinckx ◽  
Ann-Christin Hau ◽  
Anne Schuster ◽  
Sabrina Fritah ◽  
Andy Chevigné ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Growth Factor Receptor ◽  
Soluble Form ◽  
Growth Factor Signaling ◽  
Egfr Expression ◽  
Baculovirus Infection

Abstract INTRODUCTION Aberrant regulation of receptor tyrosine kinase (RTK) activity is characteristic of Glioblastoma (GBM). However, RTK-based targeted therapies have been largely unsuccessful in GBM patients, partially due to the complexity and redundance of RTK signaling. LRIG1 (Leucine-rich Repeats and ImmunoGlobulin-like domains 1) is an important endogenous inhibitor of epidermal growth factor receptor (EGFR) during health and disease, however its mechanism of action is poorly understood. We previously showed that the soluble form of LRIG1 potently inhibits GBM growth in vivo, irrespective of EGFR expression level and status, suggesting the involvement of other RTKs. Here, we aimed to shed light on the molecular mechanisms underlying its anti-cancer activity. MATERIAL AND METHODS We generated a recombinant human soluble LRIG1 protein (rh-sLRIG1) by expressing LRIG1 ectodomain in insect cells via baculovirus infection and subsequent His-tag purification. rh-sLRIG1 was used to treat patient-derived GBM stem-like cells, classical GBM cell lines and patient-derived 3D tumor organoids. Using different cell-based assays, cell proliferation, invasion, cell morphology, as well as protein expression and protein-protein interactions were investigated. RESULTS We find that recombinant sLRIG1 efficiently reduced proliferation, invasion and viability of GBM cells and patient-derived organoids, and modulated cytoskeleton proteins and cell shape. In line with previous data, the effect of recombinant sLRIG1 was independent of EGFR expression. Interestingly sLRIG1 impacted multiple RTKs including AXL, by direct protein downregulation, while EGFR expression was not affected. At the molecular level, we find that sLRIG1 interfereed with AXL dimerization, while no protein interaction with EGFR was detected. CONCLUSION We identify AXL as a novel LRIG1 target and provide evidence that sLRIG1-mediated RTK downregulation requires direct protein-protein interaction. These data pave the way for a potential therapeutic application of recombinant sLRIG1 in the inhibition of growth factor signaling in GBM.

scholarly journals Metabolic reprogramming associated with aggressiveness occurs in the G-CIMP-high molecular subtypes of IDH1mut lower grade gliomas

2019 ◽  
Vol 22 (4) ◽  
pp. 480-492 ◽  
Author(s):  
Victor Ruiz-Rodado ◽  
Tathiane M Malta ◽  
Tomohiro Seki ◽  
Adrian Lita ◽  
Tyrone Dowdy ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Molecular Subtype ◽  
Tumor Biology ◽  
Metabolic Reprogramming ◽  
The Cancer Genome Atlas ◽  
Model Systems ◽  
Lower Grade ◽  
Molecular Features

Abstract Background Early detection of increased aggressiveness of brain tumors is a major challenge in the field of neuro-oncology because of the inability of traditional imaging to uncover it. Isocitrate dehydrogenase (IDH)-mutant gliomas represent an ideal model system to study the molecular mechanisms associated with tumorigenicity because they appear indolent and non-glycolytic initially, but eventually a subset progresses toward secondary glioblastoma with a Warburg-like phenotype. The mechanisms and molecular features associated with this transformation are poorly understood. Methods We employed model systems for IDH1 mutant (IDH1mut) gliomas with different growth and proliferation rates in vivo and in vitro. We described the metabolome, transcriptome, and epigenome of these models in order to understand the link between their metabolism and the tumor biology. To verify whether this metabolic reprogramming occurs in the clinic, we analyzed data from The Cancer Genome Atlas. Results We reveal that the aggressive glioma models have lost DNA methylation in the promoters of glycolytic enzymes, especially lactate dehydrogenase A (LDHA), and have increased mRNA and metabolite levels compared with the indolent model. We find that the acquisition of the high glycolytic phenotype occurs at the glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP)-high molecular subtype in patients and is associated with the worst outcome. Conclusion We propose very early monitoring of lactate levels as a biomarker of metabolic reprogramming and tumor aggressiveness.

scholarly journals Protein Inhibitor of Activated Signal Transducer and Activator of Transcription 1 Interacts with the N-Terminal Domain of Mineralocorticoid Receptor and Represses Its Transcriptional Activity: Implication of Small Ubiquitin-Related Modifier 1 Modification

2003 ◽  
Vol 17 (12) ◽  
pp. 2529-2542 ◽  
Author(s):  
Laurent Pascual-Le Tallec ◽  
Olivier Kirsh ◽  
Marie-Christine Lecomte ◽  
Say Viengchareun ◽  
Maria-Christina Zennaro ◽  
...  
Keyword(s):  
Mineralocorticoid Receptor ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Specific Interaction ◽  
Signal Transducer ◽  
Protein Inhibitor ◽  
E3 Ligases ◽  
Terminal Domain

Abstract Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood but seem to largely depend upon interactions with specific coregulators. To identify novel human MR (hMR) molecular partners, yeast two-hybrid screenings performed using the N-terminal domain as bait, allowed us to isolate protein inhibitor of activated signal transducer and activator of transcription (PIAS)1 and PIASxβ, described as SUMO (small ubiquitin-related modifier) E3-ligases. Specific interaction between PIAS1 and hMR was confirmed by glutathione-S-transferase pull-down experiments and N-terminal subdomains responsible for physical contacts were delineated. Transient transfections demonstrated that PIAS1 is a corepressor of aldosterone-activated MR transactivation but has no significant effect on human glucocorticoid receptor transactivation. The agonist or antagonist nature of the bound ligand also determines PIAS1 corepressive action. We provided evidence that PIAS1 conjugated SUMO-1 to hMR both in vitro and in vivo. Deciphering the unique sumoylation pattern of hMR, which possesses five consensus SUMO-1 binding sites, by combinatorial lysine substitutions, revealed a major impact of sumoylation on hMR properties. Using a murine mammary tumor virus promoter, PIAS1 action was independent of sumoylation whereas with glucocorticoid response element promoter, PIAS1 corepressive action depended on hMR sumoylation status. Taken together, our results identify a novel function for PIAS1 which interacts with the N-terminal domain of hMR and represses its ligand-dependent transcriptional activity, at least in part, through SUMO modifications.

scholarly journals TRIF/EGFR signalling mediates angiotensin-II-induced cardiac remodelling in mice

2020 ◽  
Vol 65 (2) ◽  
pp. 11-20
Author(s):  
Hong Zhu ◽  
Wei Cao ◽  
Peng Zhao ◽  
Jieyu Wang ◽  
Yuying Qian ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Egf Receptor ◽  
Renin Angiotensin System ◽  
Cardiac Remodelling ◽  
Ang Ii ◽  
Cardiac Inflammation ◽  
Egfr Ligands ◽  
Structural Disorders

The excessive activation of renin-angiotensin system (RAS) is one of key pathophysiological characteristics in the development of cardiac remodelling. Angiotensin (Ang) II, as a main active peptide in RAS, induces cardiac structural disorders and dysfunction. However, the molecular mechanisms are still not fully disclosed. Present study aimed to determine the role and potential mechanisms of cardiac TIR-domain-containing adapter-inducing interferon-β (TRIF) in Ang-II-mediated cardiac remodelling in mice. In vitro and in vivo studies showed Ang II and downstream aldosterone obviously increased the expression of TRIF, accompanied with cardiac structural abnormalities and functional injuries. Specific blockage of cardiac TRIF effectively decreased Ang-II/aldosterone-induced cardiac inflammation, fibrosis, hypertrophy and dysfunction in mice. Mechanistically, the TRIF triggered the activation of EGF receptor (EGFR) signalling by nuclear factor (NF)-κB transcriptional regulation and downstream EGFR ligands. Taken together, present study supported that cardiac TRIF was a potential therapeutic target for attenuating cardiac pathophysiological remodelling. The TRIF/EGFR axis partially explained the molecular mechanism of Ang-II/aldosterone-induced cardiac inflammation, fibrosis, hypertrophy and dysfunction in mice.

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