Epidermal Growth Factor Receptor Transactivation Research Articles

Molecular mechanisms involved in the protective effect of pituitary adenylate cyclase-activating polypeptide in an in vitro model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons. Based on transcriptional profiles of motor cortex samples, in a previous work, we were able to classify two subgroups of sporadic ALS (SALS) patients, named SALS1 and SALS2. A further meta-analysis study has revealed sixteen drug targets commonly deregulated in SALS2 and superoxide dismutase 1 (SOD1) G93A mice. The identified candidate drug targets included pituitary adenylate cyclase-activating polypeptide (PACAP), epidermal growth factor receptor (EGFR) and matrix metallopeptidase-2 (MMP-2). By using a motor neuron-like hybrid cell line (NSC-34) expressing human SOD1 G93A as an in vitro model of ALS, here we investigated the functional correlation among these three genes. Our results have shown that PACAP increases cell viability following serum deprivation. This effect is induced through EGFR transactivation mediated by protein kinase A stimulation. Furthermore, EGFR phosphorylation activates mitogen-activated protein kinases/extracellular signal-regulated kinases 1 and 2 survival signaling pathway and increases MMP-2 expression, significantly reduced by serum starvation. These results suggest that a deeper characterization of mechanisms involved in PACAP/EGFR/MMP-2 axis activation in G93A SOD1 mutated neurons may allow identifying new targets for ALS therapy.

Positive crosstalk between EGFR and the TF-PAR2 pathway mediates resistance to cisplatin and poor survival in cervical cancer.

Cisplatin-based chemoradiation is the standard treatment for cervical cancer, but chemosensitizing strategies are needed to improve patient survival. EGFR (Epidermal Growth Factor Receptor) is an oncogene overexpressed in cervical cancer that is involved in chemoresistance. Recent studies showed that EGFR upregulates multiple elements of the coagulation cascade, including tissue factor (TF) and the protease-activated receptors (PAR) 1 and 2. Moreover, many G protein-coupled receptors, including PARs, have been implicated in EGFR transactivation. However, the role of coagulation proteins in the progression of cervical cancer has been poorly investigated. Herein we employed cervical cancer cell lines and The Cancer Genome Atlas (TCGA) database to evaluate the role of EGFR, TF and PAR2 in chemoresistance. The SLIGKL-NH2 peptide (PAR2-AP) and coagulation factor VIIa (FVIIa) were used as PAR2 agonists, while cetuximab was used to inhibit EGFR. The more aggressive cell line CASKI showed higher expression levels of EGFR, TF and PAR2 than that of C33A. PAR2 transactivated EGFR, which further upregulated cyclooxygenase-2 (COX2) expression. PAR2-AP decreased cisplatin-induced apoptosis through an EGFR- and COX2-dependent mechanism. Furthermore, treatment of CASKI cells with EGF upregulated TF expression, while treatment with cetuximab decreased the TF protein levels. The RNA-seq data from 309 TCGA samples showed a strong positive correlation between EGFR and TF expression (P = 0.0003). In addition, the increased expression of EGFR, PAR2 or COX2 in cervical cancer patients was significantly correlated with poor overall survival. Taken together, our results suggest that EGFR and COX2 are effectors of the TF/FVIIa/PAR2 signaling pathway, promoting chemoresistance.

Matrix metalloproteinase-2-induced epidermal growth factor receptor transactivation impairs redox balance in vascular smooth muscle cells and facilitates vascular contraction

Increased reactive oxygen species (ROS) formation may enhance matrix metalloproteinase (MMP)-2 activity and promote cardiovascular dysfunction. We show for the first time that MMP-2 is upstream of increased ROS formation and activates signaling mechanisms impairing redox balance. Incubation of vascular smooth muscle cells (VSMC) with recombinant MMP-2 increased ROS formation assessed with dihydroethidium (DHE) by flow cytometry. This effect was blocked by the antioxidant apocynin or by polyethylene glycol-catalase (PEG-catalase), and by MMP inhibitors (doxycycline or GM6001). Next, we showed in HEK293 cells that MMP-2 transactivates heparin-binding epidermal growth factor (HB-EGF) leading to EGF receptor (EGFR) activation and increased ROS concentrations. This effect was prevented by the EGFR kinase inhibitor Ag1478, and by phospholipase C (PLC) or protein kinase C (PKC) inhibitors (A778 or chelerythrine, respectively), confirming the involvement of EGFR pathway in MMP-2-induce responses. Next, we showed that intraluminal exposure of aortas to MMP-2 increased vascular MMP-2 levels detected by immunofluorescence and gelatinolytic activity (by in situ zimography) in association with increased ROS formation. This effect was inhibited by MMP inhibitors (phenanthroline or doxycycline) and by apocynin or PEG-catalase. MMP-2 also increased aortic contractility to phenylephrine and this effect was prevented by MMP inhibitor GM6001 and by apocynin or PEG-catalase, showing again that increased ROS formation mediates functional effects of MMP-2. These results show that MMP-2 activates the EGFR and triggers downstream signaling pathways increasing ROS formation and promoting vasoconstriction. These findings may have various implications for cardiovascular diseases.

Abstract 242: Large pathway and gene set analysis of GWAS data identifies novel associations for pancreatic cancer

Abstract Objective Genome-wide association studies (GWAS) identify associations of individual SNPs with cancer risk but usually only explain a fraction of the inherited variability. Pathway analysis of genetic variants has been shown for many disorders to be a powerful tool in discovering novel networks of susceptibility genes. Design We have conducted a large agnostic pathway-based meta-analysis of GWAS data using the summary-based adaptive rank truncated product (sARTP) method to identify novel gene sets and pathways associated with pancreatic ductal adenocarcinoma (PDA) in 9,040 cases and 12,495 controls. We performed expression quantitative trait loci (eQTL) analysis and functional annotation of the top SNPs in genes identified in PDA-associated gene sets and pathways. Results We identified 14 gene sets and pathways associated with PDA at FDR < 0.05. Five of the strongest signals (P-value ≤ 1.3x10-5 Bonferroni corrected) were in genes from KEGG maturity onset diabetes of the young, Reactome regulation of beta cell development, Biocarta role of epidermal growth factor (EGF) receptor transactivation by G-protein-coupled receptors in cardiac hypertrophy pathway, Nikolsky breast cancer chr17q11-q21 amplicon gene set, and Pujana ATM Pearson correlation coefficient (PCC) network gene set. We identified and validated rs876493 and three correlated SNPs (PGAP3) and rs3124737 (CASP7) as eQTLs in two normal derived pancreas tissue datasets. Conclusion Our agnostic gene set and pathway analysis integrated with functional annotation and eQTL analysis provides novel insight into genes and pathways that may be biologically relevant for risk of PDA. Citation Format: Naomi Walsh, Han Zhang, Paula L. Hyland, Qi Yang, Evelina Mocci, Mingfeng Zhang, Erica J. Childs, Zhaoming Wang, Stephen Chanock, Patricia Hartge, Robert Hoover, Peter Kraft, Donghui Li, Eric J. Jacobs, Gloria M. Petersen, Brian M. Wolpin, Harvey A. Risch, Laufey T. Amundadottir, Kai Yu, Alison P. Klein, Rachael Z. Stolzenberg-Solomon. Large pathway and gene set analysis of GWAS data identifies novel associations for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 242.

ADAM17 participates in the protective effect of paeoniflorin on mouse brain microvascular endothelial cells.

Paeoniflorin (PF), the most abundant active ingredient of traditional Chinese herbal medicine Paeoniae Radix, has been recognized as a potential neuroprotectant due to its remarkable efficacy on mitigating cerebral infarction and preventing the neurodegenerative diseases. However, the precise mechanisms of PF remain incompletely understood. In this study, we first provided evidence for the protective effect of PF on hydrogen peroxide-induced injury on mouse brain microvascular endothelial bEnd.3 cells, and for transactivation of the epidermal growth factor receptor (EGFR) signal induced by PF, suggesting that EGFR transactivation might be involved in the beneficial role of PF. Next, by detecting the phosphorylation of a disintegrin and metalloprotease 17 (ADAM17) at Thr 735 and performing loss-of-function experiments with the ADAM17 inhibitor and ADAM 17-siRNA, we showed that PF-induced transactivation of EGFR and downstream ERKs and AKT signaling pathways were dependent on ADAM17. Furthermore, PF-induced phosphorylation of ADAM17 and the EGFR transactivation were inhibited by the inhibitors of adenosine A1 receptor (A1R) or Src kinase that were applied to cells prior to PF treatment, implying the involvement of A1R, and Src in the activation of ADAM17. Finally, PF reduced the cell surface level of TNF-receptor 1 (TNFR1) and increased the content of soluble TNFR1 (sTNFR1) in the culture media, indicating that PF might enhance the shedding of sTNFR1. Taken together, we conclude that A1R and Src-dependent activation of ADAM17 participates in PF-induced EGFR transactivation and TNFR1 shedding on mouse brain microvascular endothelial cells, which may contributes to the neuroprotective effects of PF.

Supplementation of p40, a Lactobacillus rhamnosus GG-derived protein, in early life promotes epidermal growth factor receptor-dependent intestinal development and long-term health outcomes.

The beneficial effects of the gut microbiota on growth in early life are well known. However, knowledge about the mechanisms underlying regulating intestinal development by the microbiota is limited. p40, a Lactobacillus rhamnosus GG-derived protein, transactivates epidermal growth factor receptor (EGFR) in intestinal epithelial cells for protecting the intestinal epithelium against injury and inflammation. Here, we developed p40-containing pectin/zein hydrogels for targeted delivery of p40 to the small intestine and the colon. Treatment with p40-containing hydrogels from postnatal day 2 to 21 significantly enhanced bodyweight gain prior to weaning and functional maturation of the intestine, including intestinal epithelial cell proliferation, differentiation, and tight junction formation, and IgA production in early life in wild-type mice. These p40-induced effects were abolished in mice with specific deletion of EGFR in intestinal epithelial cells, suggesting that transactivation of EGFR in intestinal epithelial cells may mediate p40-regulated intestinal development. Furthermore, neonatal p40 treatment reduced the susceptibility to intestinal injury and colitis and promoted protective immune responses, including IgA production and differentiation of regulatory T cells, in adult mice. These findings reveal novel roles of neonatal supplementation of probiotic-derived factors in promoting EGFR-mediated maturation of intestinal functions and innate immunity, which likely promote long-term beneficial outcomes.

Roles of PDZ‐dependent Interactions and N‐glycosylation in G Protein‐coupled Estrogen Receptor 1 (GPER1)/GPR30‐mediated Stimulation of ERK1/2 Activity

G protein‐coupled receptor 30 (GPR30) is a G protein‐coupled receptor (GPCR) that is attracting considerable attention in breast cancer and cardiometabolic regulation. Following reports that GPR30 is required for some rapid estrogen responses, e.g. increased cAMP production and ERK1/2 activity, in estrogen receptor (ER)‐negative cells, GPR30 was renamed G protein‐coupled estrogen receptor 1 (GPER1). However, many questions remain about the identity of the cognate receptor ligand, receptor‐effector coupling, and receptor membrane trafficking. To address the mechanism by which human GPR30 activates ERK1/2, we used HEK293 cells with and without ectopic expression of GPR30. Specifically, we investigated the role of the type I PSD‐95/Discs‐large/ZO‐1 homology (PDZ) motif at the receptor C terminus (‐SSAV) and three consensus sites for N glycosylation (N‐X‐S/T) in the receptor N‐terminal domain (N25, N32, N44). We found previously that the C‐terminal PDZ motif enables the receptor to interact with SAP97 and protein kinase A (PKA)‐anchoring protein (AKAP) 5, and this interaction is necessary for retaining the receptor in the plasma membrane and mediating a constitutive decrease in cAMP production that is not inhibited by pertussis toxin, thus independent of Gi/o. Here, we found that the receptor also constitutively increases ERK1/2 activity. Interestingly, this increase was inhibited by PTX as well as by wortmannin, but not by AG1478, indicating it is mediated by Gi/o and phosphoinositide 3‐kinase (PI3K) but not epidermal growth factor receptor (EGFR) transactivation. Deleting the receptor PDZ motif or knocking down AKAP5 also inhibited the increase, showing that the PDZ interaction is also necessary for this response. Interestingly, the proposed GPR30 agonist G‐1 increased ERK1/2 activity in a GPR30‐dependent manner, but this increase was only observed at very low levels of receptor expression below that required for the constitutive increase. Furthermore, deleting the PDZ motif, which completely inhibited the constitutive increase in ERK1/2 activity, did not inhibit the G‐1‐stimulated increase. Mutating the potential N‐glycosylation residues N25 or N32 to I in the GPR30 N‐terminal domain did not prevent receptor plasma membrane expression or ERK1/2 activation. On the other hand, mutating N44 to I completely prevented both plasma membrane expression and ERK1/2 activation, and caused receptor degradation. Thus, the PDZ‐dependent receptor interaction with SAP97 and AKAP5, and therefore plasma membrane retention, is necessary for constitutive GPR30‐mediated stimulation of ERK1/2 activation, whereas G‐1‐stimulated ERK1/2 activation may remain following constitutive internalization. On the other hand, N‐glycosylation of N44 appears to be necessary for maturation of the receptor to the plasma membrane.Support or Funding InformationSwedish Research Council and Swedish Cancer FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Loss of Caveolin‐3 (Cav‐3) Promotes G‐protein‐regulated Matrix Metalloprotease 14 (MMP14) Activation in the Aged Heart

Age‐associated cardiovascular disease significantly correlates with morbidity and mortality worldwide; therefore, investigation of cellular signaling pathways in the heart that change with age may help identify novel therapeutic strategies for mitigating the progression of cardiovascular pathologies. Our lab has uncovered a membrane‐delimited receptor transactivation pathway in the heart, wherein G‐protein coupled receptors (GPCRs) signal via heterotrimeric G‐proteins to activate membrane‐tethered matrix metalloprotease 14 (MMP14/MT1‐MMP) and consequently induce “shedding” of membrane‐bound receptor ligands (J Biol Chem 290: 9941‐7). Specifically, G‐protein‐regulated MMP14 releases heparin‐binding EGF‐like growth factor (HB‐EGF), which then triggers activation of epidermal growth factor receptor (EGFR) signaling. We hypothesized that the components of this mechanism are compartmentalized within caveolar lipid raft domains (i.e. cholesterol‐rich membranes containing caveolin‐1 (Cav‐1) and caveolin‐3 (Cav‐3) isoforms), wherein co‐localization with caveolin proteins serves to repress MMP14 enzymatic activity. Furthermore, we hypothesized that age‐dependent loss of Cav‐3 in the heart increases MMP14 activity and thereby alters the signaling pathway to contribute to deleterious cardiac aging phenotypes. We thus sought to compare changes in G‐protein‐regulated MMP14 activity and subsequent activation of HB‐EGF/EGFR signaling in membrane fractions isolated from cardiac myocytes (CMs) and cardiac fibroblasts (CFs) of young (2–3 month) and aged (18–21 month) mice, with or without transgenic CM‐specific overexpression of Cav‐3 (i.e., Cav‐3 OE), to determine if preserved Cav‐3 expression prevents or lessens harmful age‐associated membrane signaling. We observed approximately a two‐fold increase of MMP14 activity in membranes from CMs of aged wild‐type (WT) mice compared to young controls (p< 0.05). Immunoblot analysis of buoyant fractions isolated by differential centrifugation of cardiac tissue in a discontinuous sucrose density gradient confirmed that MMP14, G‐proteins, HB‐EGF, and EGFR co‐localize with caveolin protein isoforms in lipid rafts. Work in progress will test if co‐localization of these proteins is reduced in aged wild‐type (WT) animals but not in Cav‐3 OE animals, and if MMP14 and Cav‐3 co‐immunoprecipitate (co‐IP) within lipid raft domains and thus form a functional signaling module that is disrupted in aged WT subjects but remains intact in Cav‐3 OE mice. Taken together, the results suggest that GPCR transactivation of EGFR and regulation by signaling components in caveolar lipid rafts may be a feature of membrane signaling that is altered in the aged heart.Support or Funding InformationSupported by NIH grants R21AG053568 and R21AG52914.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Understanding Angiotensin II Type 1 Receptor Signaling in Vascular Pathophysiology.

Angiotensin II is the most important endocrine ligand in the renin–angiotensin system (RAS), contributing to the development of several cardiovascular diseases including hypertension.1 Angiotensin II mediates its signal transduction and functions via the angiotensin II receptors.2 Historically, the presence of 2 subtypes of angiotensin II receptors were pharmacologically recognized based on the sensitivity to the first orally active nonpeptide angiotensin II receptor antagonist, losartan. The losartan-sensitive receptor was termed AT1 (angiotensin II receptor type 1) receptor. It was assumed to be a heterotrimeric GPCR (G protein–coupled receptor) because it generates inositol triphosphate and diacylglycerol, leading to intracellular Ca2+ elevation and protein kinase C activation, respectively. Most known physiological and pathophysiological functions of angiotensin II, including stimulation of vasoconstriction and salt and water reabsorption, are mediated through the AT1 receptor. The losartan-insensitive receptor was termed AT2 receptor, whereas its G protein coupling remains unclear.1,3,4 In 1991, 2 research groups in the United States independently isolated cDNA (termed AGTR1) encoding the mammalian AT1 receptor.5,6 Subsequently, rat AT2 receptor cDNA (AGTR2) was cloned in 1993.7,8 This pioneer work revealed complete amino acid sequences of the angiotensin II receptor subtypes belonging to the 7-transmembrane GPCR superfamily. In the early 1990s, several studies reported that AT1 receptor elicits tyrosine phosphorylation of multiple proteins and activation of MAPK (mitogen-activated protein kinase; eg, p42/p44 MAPK)/ERK (extracellular signal–regulated kinase; eg, ERK1/2) in various cell types including vascular smooth muscle cells (VSMC). The early 1990s also saw the establishment of the concept that angiotensin II via the AT1 receptor has a direct action on cardiac myocytes, fibroblasts, and VSMCs causing hypertrophic and fibrotic cardiovascular remodeling.9,10 The cardiovascular remodeling caused by angiotensin II seemed to be …

Thrombin promotes PAI-1 expression and migration in keratinocytes via ERK dependent Smad linker region phosphorylation

Keratinocyte proliferation and migration is essential during re-epithelialisation for the restoration of the epithelial barrier during skin wound healing. Numerous growth factors are involved in the stimulation of keratinocyte proliferation and migration. The signalling pathways that drive these processes during wound healing are not well defined. This study investigated thrombin-mediated signalling in keratinocytes. The thrombin receptor, protease-activated receptor 1 (PAR-1) is a seven transmembrane G-protein coupled receptor that is known to transactivate the epidermal growth factor receptor (EGFR). Immortalized human keratinocytes (HaCaT cells) were treated with thrombin and selective inhibitors to EGFR and MAP kinases. Whole cell lysates were separated on SDS-PAGE and analysed by Western blot using antibodies against transcription factor Smad2. Quantitative real-time polymerase chain reaction was used to measure the mRNA expression of PAI-1 while scratch wound assays were used to measure keratinocyte migration. Western blot data showed that thrombin mediates PAR-1 transactivation of EGFR and the downstream phosphorylation of the transcription factor Smad2 linker (Smad2L) region. ERK1/2 inhibition by UO126 caused a decrease in Smad2L phosphorylation while the p38 inhibitor SB202190 and JNK inhibitor SP600125 did not. Smad2L Ser250 was specifically phosphorylated by this thrombin mediated pathway while Ser245 and Ser255 were not. Thrombin increased PAI-1 mRNA expression and keratinocyte migration and this was reduced when either EGFR or ERK1/2 were blocked. Taken together these results show that thrombin mediated mRNA expression of PAI-1 in keratinocytes and migration occurs via EGFR transactivation and involves signalling intermediates ERK1/2 and Smad2 and may be a key pathway in skin wound healing.

Osteoglycin attenuates cardiac fibrosis by suppressing cardiac myofibroblast proliferation and migration through antagonizing lysophosphatidic acid 3/matrix metalloproteinase 2/epidermal growth factor receptor signalling.

Cardiac myofibroblasts (CMFs) play a crucial role in the progression of pathological fibrotic cardiac remodelling. The expression of osteoglycin (OGN) is increased in diseased hearts; however, the role of OGN in pathological cardiac remodelling is not understood. Here, we sought to determine the effect of OGN on cardiac interstitial fibrosis and investigate the molecular mechanisms of OGN in CMF activation and matrix production. We found that OGN expression was significantly upregulated in mouse hearts in response to chronic 14-day angiotensin II (Ang II) infusion. Mice lacking OGN (OGN-/-) exhibited enhanced cardiac interstitial fibrosis and significantly more severe cardiac dysfunction following Ang II infusion compared to wild-type mice. OGN deficiency did not alter blood pressure, nor had effect on transforming growth factor-beta signalling activation, but presented with increased proliferative activity in hearts. In vitro studies with isolated CMFs revealed that OGN deficiency significantly increased proliferation and migration and enhanced the transactivation of epidermal growth factor receptor (EGFR) signalling by Ang II. On the other hand, OGN overexpression in CMFs decreased their proliferation and migration via reducing EGFR activation. Overexpression of OGN also suppressed the shedding of membrane anchored EGFR ligand. Moreover, OGN was found to interact with a lysophosphatidic acid (LPA) receptor isoform 3 and thus to attenuate EGFR transactivation through blocking cell surface translocation of membrane type 1 matrix metalloproteinase (MT1-MMP) and subsequent pro-MMP-2 activation in a Ras homolog gene family, member A (RhoA)/Rho-associated, coiled-coil containing protein kinase (ROCK)-dependent manner. These findings suggest that OGN negatively regulates cardiac fibrotic remodelling by attenuating CMF proliferation and migration through LPA3-mediated and Rho/ROCK-dependent inhibition of MT1-MMP translocation, MMP2 activation and EGFR transactivation.

TGF-β transactivates EGFR and facilitates breast cancer migration and invasion through canonical Smad3 and ERK/Sp1 signaling pathways.

Transforming growth factor‐beta (TGF‐β) functions as a potent proliferation inhibitor and apoptosis inducer in the early stages of breast cancer, yet promotes cancer aggressiveness in the advanced stages. The dual effect of TGF‐β on cancer development is known as TGF‐β paradox, and the remarkable functional conversion of TGF‐β is a pivotal and controversial phenomenon that has been widely investigated for decades. This phenomenon may be attributed to the cross talk between TGF‐β signaling and other pathways, including EGF receptor (EGFR) signaling during cancer progression. However, the underlying mechanism by which TGF‐β shifts its role from a tumor suppressor to a cancer promoter remains elusive. In this study, TGF‐β is positively correlated with EGFR expression in breast cancer tissues, and a functional linkage is observed between TGF‐β signaling and EGFR transactivation in breast cancer cell lines. TGF‐β promotes the migration and invasion abilities of breast cancer cells, along with the increase in EGFR expression. EGFR is also essential for TGF‐β‐induced enhancement of these abilities of breast cancer cells. Canonical Smad3 signaling and ERK/Sp1 signaling pathways mediate TGF‐β‐induced EGFR upregulation. Hence, our study provided insights into a novel mechanism by which TGF‐β supports breast cancer progression.

2-Methoxyestradiol causes matrix metalloproteinase 9-mediated transactivation of epidermal growth factor receptor and angiotensin type 1 receptor downregulation in rat aortic smooth muscle cells.

Studies have demonstrated the therapeutic potential of estrogen metabolite 2-methoxyestradiol (2ME2) in several cardiovascular disorders, including hypertension. However, the exact mechanism(s) remains unknown. In this study, primary rat aortic smooth muscle cells (RASMCs) were exposed to 2ME2, and angiotensin type 1 receptor (AT1R) expression, function, and associated signaling pathways were evaluated. In RASMCs, 2ME2 downregulated AT1R expression in a concentration- and time-dependent manner, which was correlated with reduced mRNA expression. The 2ME2 effect was through G protein-coupled receptor 30 (GPR30) that inhibits second messenger cAMP. Moreover, 2ME2 exposure phosphorylated ERK1/2 that was sensitive to MEK inhibitor PD98059. Selective epidermal growth factor receptor (EGFR) inhibitor AG1478 blocked 2ME2-induced EGFR transactivation and attenuated subsequent phosphorylation of ERK1/2 preventing AT1R downregulation. The transactivation was dependent on 2ME2-induced release of matrix metalloproteinase 9 (MMP9) and epidermal growth factor demonstrated by ELISA. Furthermore, transfection with small interfering (si) RNA targeting MMP9 impeded ERK1/2 activation and AT1R downregulation in response to 2ME2 and G1 stimulation. Interestingly, under similar conditions, stimulation of GPR30 with the selective agonist G1 elicited similar signaling pathways and downregulated the AT1R expression that was reversed by GPR30 antagonist G15. Furthermore, 2ME2 and G1 inhibited angiotensin II (ANG II) induced Ca2+ release, a response consistent with AT1R downregulation. Collectively, our study demonstrates for the first time that 2ME2 binding to GPR30 induces MMP9 specific transactivation of EGFR that mediates ERK1/2-dependent downregulation of AT1R in RASMCs. The study provides critical insights into the newly discovered role and signaling pathways of 2ME2 in the regulation of AT1R in vascular cells and its potential to be developed as a therapeutic agent that ameliorates hypertension.

The activation of G protein-coupled estrogen receptor induces relaxation via cAMP as well as potentiates contraction via EGFR transactivation in porcine coronary arteries.

Estrogen exerts protective effects against cardiovascular diseases in premenopausal women, but is associated with an increased risk of both coronary heart disease and stroke in older postmenopausal women. Studies have shown that activation of the G-protein-coupled estrogen receptor 1 (GPER) can cause either relaxation or contraction of arteries. It is highly likely that these dual actions of GPER may contribute to the seemingly paradoxical effects of estrogen in regulating coronary artery function. The objective of this study was to test the hypothesis that activation of GPER enhances agonist-stimulated porcine coronary artery contraction via epidermal growth factor receptor (EGFR) transactivation and its downstream extracellular signal-regulated kinases (ERK1/2) pathway. Isometric tension studies and western blot were performed to determine the effect of GPER activation on coronary artery contraction. Our findings demonstrated that G-1 caused concentration-dependent relaxation of ET-1-induced contraction, while pretreatment of arterial rings with G-1 significantly enhanced ET-1-induced contraction. GPER antagonist, G-36, significantly inhibited both the G-1-induced relaxation effect and G-1-enhanced ET-1 contraction. Gallein, a Gβγ inhibitor, significantly increased G-1-induced relaxation, yet inhibited G-1-enhanced ET-1-mediated contraction. Similarly, inhibition of EGFR with AG1478 or inhibition of Src with phosphatase 2 further increased G-1-induced relaxation responses in coronary arteries, but decreased G-1-enhanced ET-1-induced contraction. Western blot experiments in porcine coronary artery smooth muscle cells (PCASMC) showed that G-1 increased tyrosine phosphorylation of EGFR, which was inhibited by AG-1478. Furthermore, enzyme-linked immunosorbent assays showed that the level of heparin-binding EGF (HB-EGF) released by ET-1 treatment increased two-fold; whereas pre-incubation with G-1 further increased ET-1-induced HB-EGF release to four-fold over control conditions. Lastly, the role of ERK1/2 was determined by applying the MEK inhibitor, PD98059, in isometric tension studies and detecting phospho-ERK1/2 in immunoblotting. PD98059 potentiated G-1-induced relaxation response, but blocked G-1-enhanced ET-1-induced contraction. By western blot, G-1 treatment decreased phospho-ERK1/2, however, in the presence of the adenylyl cyclase inhibitor, SQ22536, G-1 significantly increased ERK1/2 phosphorylation in PCASMC. These data demonstrate that activation of GPER induces relaxation via cAMP as well as contraction via a mechanism involving transactivation of EGFR and the phosphorylation of ERK1/2 in porcine coronary arteries.

Helicobacter pylori-Induced HB-EGF Upregulates Gastrin Expression via the EGF Receptor, C-Raf, Mek1, and Erk2 in the MAPK Pathway.

Helicobacter pylori is associated with hypergastrinemia, which has been linked to the development of gastric diseases. Although the molecular mechanism is not fully understood, H. pylori is known to modulate the Erk pathway for induction of gastrin expression. Herein we found that an epidermal growth factor (EGF) receptor kinase inhibitor significantly blocked H. pylori-induced gastrin promoter activity, suggesting involvement of EGF receptor ligands. Indeed, H. pylori induced mRNA expression of EGF family members such as amphiregulin, EGF, heparin-binding EGF-like growth factor (HB-EGF), and transforming growth factor-α. Of these, specific siRNA targeting of HB-EGF significantly blocked H. pylori-induced gastrin expression. Moreover, H. pylori induced HB-EGF ectodomain shedding, which we found to be a critical process for H. pylori-induced gastrin expression. Thus, we demonstrate a novel role for human mature HB-EGF in stimulating gastrin promoter activity during H. pylori infection. Further investigation using specific siRNAs targeting each isoform of Raf, Mek, and Erk elucidated that the mechanism underlying H. pylori-induced gastrin expression can be delineated as the sequential activation of HB-EGF, the EGF receptor, C-Raf, Mek1, and the Erk2 molecules in the MAPK pathway. Surprisingly, whereas Erk2 acts as a potent activator of gastrin expression, siRNA knockdown of Erk1 induced gastrin promoter activity, suggesting that Erk1 typically acts as a repressor of gastrin expression. Elucidation of the mechanism of gastrin modulation by HB-EGF-mediated EGF receptor transactivation should facilitate the development of therapeutic strategies against H. pylori-related hypergastrinemia and consequently gastric disease development, including gastric cancers.

Prostaglandin E2 Impairs P2Y2/P2Y4 Receptor Signaling in Cerebellar Astrocytes via EP3 Receptors.

Prostaglandin E2 (PGE2) is an important bioactive lipid that accumulates after tissue damage or inflammation due to the rapid expression of cyclooxygenase 2. PGE2 activates specific G-protein coupled EP receptors and it mediates pro- or anti-inflammatory actions depending on the cell-context. Nucleotides can also be released in these situations and they even contribute to PGE2 production. We previously described the selective impairment of P2Y nucleotide signaling by PGE2 in macrophages and fibroblasts, an effect independent of prostaglandin receptors but that involved protein kinase C (PKC) and protein kinase D (PKD) activation. Considering that macrophages and fibroblasts influence inflammatory responses and tissue remodeling, a similar mechanism involving P2Y signaling could occur in astrocytes in response to neuroinflammation and brain repair. We analyzed here the modulation of cellular responses involving P2Y2/P2Y4 receptors by PGE2 in rat cerebellar astrocytes. We demonstrate that PGE2 inhibits intracellular calcium responses elicited by UTP in individual cells and that inhibiting this P2Y signaling impairs the astrocyte migration elicited by this nucleotide. Activation of EP3 receptors by PGE2 not only impairs the calcium responses but also, the extracellular regulated kinases (ERK) and Akt phosphorylation induced by UTP. However, PGE2 requires epidermal growth factor receptor (EGFR) transactivation in order to dampen P2Y signaling. In addition, these effects of PGE2 also occur in a pro-inflammatory context, as evident in astrocytes stimulated with bacterial lipopolysaccharide (LPS). While we continue to investigate the intracellular mechanisms responsible for the inhibition of UTP responses, the involvement of novel PKC and PKD in cerebellar astrocytes cannot be excluded, kinases that could promote the internalization of P2Y receptors in fibroblasts.

EGFR transactivation is involved in TNF-α-induced expression of thymic stromal lymphopoietin in human keratinocyte cell line

BackgroundThymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine involved in the pathology of inflammatory skin diseases, such as atopic dermatitis and psoriasis. Tumor necrosis factor (TNF)-α, a key cytokine in inflammatory skin diseases, is a known TSLP inducer. TNF-α activates NF-κB and induces transactivation of epidermal growth factor receptor (EGFR) in epithelial cells. However, the detailed mechanism of TSLP induction by TNF-α has remained unclear. ObjectiveWe investigated the involvement of TNF-α-induced EGFR transactivation in TSLP expression. MethodsHaCaT cells were stimulated with TNF-α or EGF in the presence or absence of an EGFR kinase inhibitor or other signaling inhibitors. The expression of TSLP mRNA was analyzed by RT-PCR and the phosphorylation level of signal proteins was analyzed by western blot. TSLP promoter and NF-κB transcription activities were analyzed by luciferase assay. ResultsTNF-α-induced TSLP expression was inhibited by the EGFR kinase inhibitor AG1478. While TSLP expression was induced by EGF, it was inhibited by the MEK inhibitor, U0126. Inhibitors of p38 and ADAM proteases suppressed the TNF-α-induced TSLP expression and EGFR phosphorylation, but not the EGF-induced expression. ConclusionTNF-α-induced EGFR transactivation results in TSLP induction through ERK activation. The activation of p38 and ADAM proteases mediates TNF-α-induced EGFR phosphorylation. These findings suggested that the TNF-α-induced EGFR transactivation pathway could be a target for the treatment of inflammatory skin diseases.

Investigation on the regulatory effect of PGE2 on ESCC cells through the trans-activation of EGFR by EP2 and the relevant mechanism.

To determine whether the combination of prostaglandin E2 (PGE2) and EP2, the subtype receptor of PGE2, could trans-activate the epidermal growth factor receptor (EGFR). In this experiment, we selected epithelial cells from normal esophageal mucosa as the negative control group, and the ESCC EC109 and TE-1 cell strain as the observation group. Real-time PCR and Western-blotting were used to detect the expression of EP2, EGFR and phosphorylated EGFR (p-EGFR). The pre-treatment of ESCC cell strains was carried out using Butaprost (special agonist of PGE2 and EP2) and RNAi of EP2, and we observed the expression of EP2, EGFR, and p-EGFR. WST-8 (CCK-8) was applied for the detection of the cell proliferation rate. The transwell invasion experiment was conducted for the detection of the invasion capability of cells. The expression of MMP-9 (matrix metalloproteinase-9), VEGF (vascular endothelial growth factor), pro-inflammatory factors (IL-6 and TNF-α) in the cell supernatant were detected using ELISA. The high mRNA and protein expression of EP2, EGFR, and p-EGFR were found in the EC109 and TE-1 cell strains in the observation group, which were higher than those in the control group (p < 0.05). After the intervention of PGE2, EP2 expression was decreased and the p-EGFR expression was increased (p < 0.05). There was no variation found in the expression of EGFR (p > 0.05). After cells were intervened using Butaprost, the expressions of EP2 and p-EGFR were increased (p < 0.05), and there were no changes identified in the expression of EGFR (p > 0.05). After the intervention of RNAi, the expression of EP2 and p-EGFR was decreased (p < .05), and no changes were identified in the expression of EGFR (p > 0.05). After the intervention of PGE2 and Butaprost, great increases were seen in the cell proliferation rate, invasion capability, and the expression of MMP-9, VEGF, IL-6, and TNF-α in EC109 and TE-1 cell strains (p < 0.05), however, the intervention of RNAi could reduce above indexes (p < 0.05). Through cell experiments, we verified that the combination of prostaglandin E2 (PGE2) and EP2, the subtype receptor of PGE2, could trans-activate the epidermal growth factor receptor (EGFR) to regulate the proliferation and invasion capability of esophageal squamous cell carcinoma (ESCC) cells, and secrete and express multiple cytokines, thus discovering the pathological mechanism of inflammation to carcinoma transition in the occurrence of ESCC, and providing the experimental evidence for the search of new target in the treatment of ESCC. ESCC cells can highly express the receptor subtype EP2 of PGE2 that can transactivate the EGFR, through which PGE2 is involved in the transition mechanism from inflammation to cancer.

Intracrine prostaglandin E2 pro-tumoral actions in prostate epithelial cells originate from non-canonical pathways.

Prostaglandin E2 (PGE2 ) increases cell proliferation and stimulates migratory and angiogenic abilities in prostate cancer cells. However, the effects of PGE2 on non-transformed prostate epithelial cells are unknown, despite the fact that PGE2 overproduction has been found in benign hyperplastic prostates. In the present work we studied the effects of PGE2 in immortalized, non-malignant prostate epithelial RWPE-1 cells and found that PGE2 increased cell proliferation, cell migration, and production of vascular endothelial growth factor-A, and activated in vitro angiogenesis. These actions involved a non-canonic intracrine mechanism in which the actual effector was intracellular PGE2 (iPGE2 ) instead of extracellular PGE2 : inhibition of the prostaglandin uptake transporter (PGT) or antagonism of EP receptors prevented the effects of PGE2 , which indicated that PGE2 activity depended on its carrier-mediated translocation from the outside to the inside of cells and that EP receptors located intracellularly (iEP) mediated the effects of PGE2 . iPGE2 acted through transactivation of epidermal growth factor-receptor (EGFR) by iEP, leading to increased expression and activity of hypoxia-inducible factor-1α (HIF-1α). Interestingly, iPGE2 also mediates the effects of PGE2 on prostate cancer PC3 cells through the axis iPGE2 -iEP receptors-EGFR-HIF-1α. Thus, this axis might be responsible for the growth-stimulating effects of PGE2 on prostate epithelial cells, thereby contributing to prostate proliferative diseases associated with chronic inflammation. Since this PGT-dependent non-canonic intracrine mechanism of PGE2 action operates in both benign and malignant prostate epithelial cells, PGT inhibitors should be tested as a novel therapeutic modality to treat prostate proliferative disease.

Biphasic Regulation of Caveolin-1 Gene Expression by Fluoxetine in Astrocytes: Opposite Effects of PI3K/AKT and MAPK/ERK Signaling Pathways on c-fos.

Previously, we reported that fluoxetine acts on 5-HT2B receptor and induces epidermal growth factor receptor (EGFR) transactivation in astrocytes. Recently, we have found that chronic treatment with fluoxetine regulates Caveolin-1 (Cav-1)/PTEN/PI3K/AKT/glycogen synthase kinase 3β (GSK-3β) signaling pathway and glycogen content in primary cultures of astrocytes with bi-phasic concentration dependence. At low concentrations fluoxetine down-regulates Cav-1 gene expression, decreases membrane content of PTEN, increases PI3K activity and increases phosphorylation of GSK-3β and increases its activity; at high concentrations fluoxetine acts on PTEN/PI3K/AKT/GSK-3β in an inverse fashion. Here, we present the data indicating that acute treatment with fluoxetine at lower concentrations down-regulates c-Fos gene expression via PI3K/AKT signaling pathway; in contrast at higher concentrations fluoxetine up-regulates c-Fos gene expression via MAPK/extracellular-regulated kinase (ERK) signaling pathway. However, acute treatment with fluoxetine has no effect on Cav-1 protein content. Similarly, chronic effects of fluoxetine on Cav-1 gene expression are suppressed by inhibitor of PI3K at lower concentrations, but by inhibitor of MAPK at higher concentrations, indicating that the mechanism underlying bi-phasic regulation of Cav-1 gene expression by fluoxetine is opposing effects of PI3K/AKT and MAPK/ERK signal pathways on c-Fos gene expression. The effects of fluoxetine on Cav-1 gene expression at both lower and higher concentrations are abolished by AG1478, an inhibitor of EGFR, indicating the involvement of 5-HT2B receptor induced EGFR transactivation as we reported previously. However, PP1, an inhibitor of Src only abolished the effect by lower concentrations, suggesting the relevance of Src with PI3K/AKT signal pathway during activation of EGFR.