Epidermal Growth Factor Receptor Transactivation Research Articles

Exploring the Impact of Nitric Oxide and Epidermal Growth Factor Receptor on Vascular Remodeling Induced by Mechanical Stretch

Hypertension is a key risk factor in many cardiovascular diseases like heart failure, kidney failure and vascular hypertrophy. Mechanical stretch/hypertension, promotes vascular smooth muscle cells remodeling by inducing leptin expression and other hypertrophic proteins like ERK1/2, AKT, and cofilin. Also the Epidermal growth factor receptor (EGFR) transactivation has shown to mediate vascular remodeling by enhancing VSMC hypertrophy, migration, and proliferation. Nitric oxide (NO) has been shown to be a critical factor for many cellular physiological function, it has the ability to induce VSMC relaxation leading to reduce hypertension. Moreover NO has received extensive attention as an anti- hypertrophic agent. Several Studies have shown that nitric oxide does not only promote vasodilation but it also inhibits VSMC proliferation. The major aim of this study was to investigate the inhibitory effect of NO and the effect of blocking the EGFR in mechanical stretch induced vascular remodeling. Methods: Rat portal veins (RPVs) were isolated and pretreated with/without NO donor (SNAP) or EGFR kinase blocker (AG1478) and then stretched to study their effects on mechanical stretch induced leptin synthesis and VSMC remolding. Western blot analysis was done to detect protein expression for leptin, adiponectin, eNOS p-ERK1/2 and p-AKT. Immunocytochemistry was performed on rat aortic vascular smooth muscle (RASMC) to detect GATA-4 nuclear translocation. Moreover, we used laser confocal microscopy on frozen RPV sections to detect the level of ROS formation and leptin. Finally, we performed qPCR for adiponectin, adipoR1 and adipoR2 mRNA. Results: In this study we showed the effect of mechanical stretch at different time points (1hour and 24 hours). Mechanical stretch-induced leptin expression was mediated by ERK1/2, AKT activations and ROS formation. These effects were associated with eNOS and adiponectin downregulation. Mechanical stretch had no effect on the expression of adiponectin receptors (adipoR1 and adipoR2). RASMCs pretreated with 3.1 nmol/L for 3 hours had induced GATA-4 nuclear translocation. Pre-incubation the RPV with SNAP significantly decreased mechanical stretch induced leptin expression, ERK1/2 and AKT phosphorylation. Moreover, SNAP significantly inhibited the adiponectin downregulation by mechanical stretch. Leptin-induced GATA-4 nuclear translocation was inhibited by SNAP. The involvement of EGFR transactivation in mechanical stretch-induced leptin expression was investigated using AG1478. Pretreatment of RPVs with AG1478 had no effect on mechanical stretch-induced early leptin expression (1 hour) while it inhibited mechanical stretch-induced late leptin expression (24 hours), ROS formation, and adiponectin and eNOS downregulation. However, treatment with AG1478 showed no effect on leptin-induced GATA-4 nuclear translocation. Conclusion: This study showed that NO has a direct inhibitory effect on mechanical stretch-induced AKT and ERK1/2 pathways and inhibits ROS formation that leads to vascular remodeling. On the other hand, EGFR transactivation was able to mediate mechanical stretch-induced vascular hypertrophy via ROS formation and leptin production, thus making EGFR and NO a promising therapeutic target for hypertension induced hypertrophy. This work was funded by Qatar University grants award UREP27-050-3-020 from Qatar National Research Fund (a member of Qatar Foundation) to AZ. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Enhancement of southern flounder (Paralichthys lethostigma) sperm velocity and fertility by direct activation of sperm epidermal growth factor and adenylyl cyclase signaling pathways

Development of southern flounder commercial aquaculture is limited by poor sperm motility and fertility, but the underlying causes and mechanisms regulating sperm motility are unknown. In vitro treatment of southern flounder sperm with progestin hormones rapidly increases velocity (swimming speed), a major component of sperm motility, and also fertility through membrane progestin receptor alpha (mPRα) and increases in Acy (adenylyl cyclase)/cAMP signaling. The progestin-induced increase of Atlantic croaker sperm velocity, is also mediated through epidermal growth factor receptor (Egfr) transactivation resulting in extracellular-regulated kinase 1/2 (Erk1/2) signaling, but its role in regulating southern flounder sperm velocity is unknown. The hypotheses that Egfr transactivation and Erk1/2 signaling mediates progestin-induced increases in sperm velocity and calcium levels in southern flounder, and that direct stimulation of sperm Egfr/Erk1/2 and Acy/cAMP pathways increases sperm fertility in strip spawning trials were tested in the present study. Golf, Egfr, Erk1/2, and protein kinase A were detected on southern flounder sperm by immunocytochemistry. Involvement of Egfr/Erk1/2 signaling in increasing sperm velocity and calcium levels was investigated by preincubating sperm with two EGFR inhibitors, AG1478, and AG825, a matrix metalloproteinase inhibitor, Ilomastat, and two ERK1/2 inhibitors, PD98059, and U0126, for 30 min. Prior to 1 min. Treatment with progestins. Sperm swimming speed was observed under a microscope after activation with a hyperosmotic medium and recorded for 1 min. Whereas treatment with the inhibitors did not alter basal sperm velocity and calcium levels, all of them significantly attenuated velocity and calcium increases in response to progestins. Treatment in vitro for 1 min. With the Egfr agonist, recombinant human EGF (0.1 nM and 100 nM), mimicked the stimulatory effects of progestins on sperm velocity, the increase in calcium, and also fertility in a strip spawning trials with ovulated southern flounder eggs. The results suggest Egfr/Erk1/2 signaling is involved in sperm velocity and fertility responses to progestins in southern flounder. Direct in vitro treatment with the Acy activator, forskolin (10 μM) for 1 min. Mimicked the stimulatory action of progestins on sperm calcium levels, velocity, and fertility, suggesting that Acy/cAMP signaling also mediates increased sperm swimming speed and fertility. Importantly, both EGF and forskolin also directly stimulated southern flounder sperm velocity and fertility at the end of the reproductive season when sperm were no longer responsive to progestins. The results suggest that EGF and forskolin could potentially be used as pharmacological agents to enhance reproductive performance of male southern flounder broodstock.

EGF-receptor phosphorylation and downstream signaling are activated by genistein during subacute liver damage

The epidermal growth factor receptor (EGFR) plays an important role on hepatic protection in acute and chronic liver injury. The aim of this study was to investigate the role of genistein on EGFR expression, phosphorylation and signaling pathways in experimental subacute liver damage induced by carbon tetrachloride (CCl4). We used male Wistar rats that were randomly divided into four groups: (1) Control; (2) Genistein 5 mg/kg per oral; (3) Subacute liver damage induced by CCl4 4 mg/kg subcutaneously; and (4) Animals received CCl4 and genistein at the dosage indicated. The effect of genistein on EGFR expression, phosphorylation and signaling pathways were investigated by western blot and densitometric analyses. Histological changes were evaluated on slices stained with Hematoxylin-Eosin and Masson´s trichromic, as well as an immunohistochemical analysis for proliferating cell nuclear antigen (PCNA). Additionally, pro-inflammatory cytokines and liver enzymes were quantified. Our study showed that genistein increased EGFR expression, EGFR-specific tyrosine residues phosphorylation (pY1068-EGFR and pY84-EGFR), signal transducer and activator of transcription phosphorylation (pSTAT5), protein kinase B phosphorylation (pAKT) and PCNA in animals with CCl4-induced subacute liver damage. It was found a significant reduction of pro-inflammatory cytokines in serum from animals with subacute liver damage treated with genistein. Those effects were reflected in an improvement in the architecture and liver function. In conclusion, genistein can induce a transactivation of EGFR leading to downstream cell signaling pathways as early events associated with regeneration and hepatoprotection following subacute liver damage.

The Ca2+/CaM, Src kinase and/or PI3K-dependent EGFR transactivation via 5-HT2A and 5-HT1B receptor subtypes mediates 5-HT-induced vasoconstriction

G protein-coupled receptors (GPCR) and receptor tyrosine kinases (RTK) modulate vascular tone and contraction via rapid and long-term processes. Sustained activation of these receptor types can change vascular structure, and the ability of vasculature to adapt to high pressure. In this study, the interaction between serotonin (5-HT) receptors and epidermal growth factor receptors (EGFR) on vasoconstriction and the mechanisms of EGFR transactivation and its downstream mediators were investigated. We measured 5-HT-induced vasoconstriction in the aorta and the mesenteric artery; and the effects of EGFR, Src and PI3K, and their downstream mediators Erk1/2 and Akt phosphorylation on 5-HT-mediated vasoconstriction in the presence or absence of pharmacological inhibitors of Ca2+/CaM, EGFR, Src, and PI3K. Furthermore, we determined the contribution of 5-HT receptor subtypes to 5-HT-induced vasoconstriction and EGFR transactivation using selective 5-HT2A and 5-HT1B receptors ligands. Our results show that EGFR, Src, and PI3K are involved in 5-HT-induced vasoconstriction both in the aorta and the mesenteric artery, and that these kinases have a more prominent role in the mesenteric artery than the aorta. With regard to EGFR transactivation by 5-HT, Ca2+/CaM, Src and PI3K are upstream mediators, and transactivation is partly mediated by Erk1/2 and Akt activation. Furthermore, Ca2+/CaM, Src, and PI3K are the main regulators for Akt activation, however Src only has a prominent role for Erk1/2 activation. 5-HT2A and 5-HT1B receptors have different EGFR transactivation profiles through Src and/or PI3K, with 5-HT2A having a greater role than 5-HT1B receptors.

Opposing Effects of EGF Receptor Signaling on Proliferation and Differentiation Initiated by EGF or TSH/EGF Receptor Transactivation.

Regulation of thyroid cells by thyrotropin (TSH) and epidermal growth factor (EGF) has been known but different effects of these regulators on proliferation and differentiation have been reported. We studied these responses in primary cultures of human thyroid cells to determine whether TSH receptor (TSHR) signaling may involve EGF receptor (EGFR) transactivation. We confirm that EGF stimulates proliferation and de-differentiation whereas TSH causes differentiation in the absence of other growth factors. We show that TSH/TSHR transactivates EGFR and characterize it as follows: (1) TSH-induced upregulation of thyroid-specific genes is inhibited by 2 inhibitors of EGFR kinase activity, AG1478 and erlotinib; (2) the mechanism of transactivation is independent of an extracellular EGFR ligand by showing that 2 antibodies, cetuximab and panitumumab, that completely inhibited binding of EGFR ligands to EGFR had no effect on transactivation, and by demonstrating that no EGF was detected in media conditioned by thyrocytes incubated with TSH; (3) TSH/TSHR transactivation of EGFR is different than EGFR activation by EGF by showing that EGF led to rapid phosphorylation of EGFR whereas transactivation occurred in the absence of receptor phosphorylation; (4) EGF caused downregulation of EGFR whereas transactivation had no effect on EGFR level; (5) EGF and TSH stimulation converged on the protein kinase B (AKT) pathway, because TSH, like EGF, stimulated phosphorylation of AKT that was inhibited by EGFR inhibitors; and (6) TSH-induced upregulation of thyroid genes was inhibited by the AKT inhibitor MK2206. Thus, TSH/TSHR causes EGFR transactivation that is independent of extracellular EGFR ligand and in part mediates TSH regulation of thyroid hormone biosynthetic genes.

Augmented leptin-induced trefoil factor 3 expression and epidermal growth factor receptor transactivation differentially influences neoplasia progression in the stomach and colorectum of dietary fat-induced obese mice

Obesity is a risk factor for gastrointestinal malignancies and tumors. However, which factors either protect or predispose the gastrointestinal organs to high-fat diet (HFD)-induced neoplasia remains unclear. Here, we demonstrate that HFD impacts the stomach to a greater extent as compared to the colorectum, resulting in leptin receptor (LepR) signaling-mediated neoplasia in the tissues. HFD activated leptin signaling, which in turn, accelerates the pathogenesis in the gastric mucosa more than that in the colorectum along with ectopic TFF3 expression. Moreover, in the stomach, higher levels of phosphorylated epidermal growth factor receptor (EGFR) in addition to the activation of STAT3 and Akt were observed as compared to the colorectum. The mice with LepR deletion in the gastrointestinal epithelium exhibited a suppressed induction of leptin, TFF3, and phosphorylated EGFR in the stomach, whereas the levels in the colorectum were insignificant. In co-transfected COS-7 cells with LepR and EGFR plasmid DNA, leptin transactivated EGFR to accelerate TFF3 induction along with activation of STAT3, ERK1/2, Akt, and PI3K p85/p55. Furthermore, TFF3 could bind to EGFR but did not transactivate LepR. Leptin-induced TFF3 induction was markedly suppressed by inhibitors of PI3K (LY294002) and EGFR (Erlotinib). Together, these results suggest a novel role of LepR-mediated signaling in transactivating EGFR that leads to TFF3 expression via the PI3K-Akt pathway. Therefore, this study sheds light on the identification of potentially new therapeutic targets for the treatment of pre-cancerous symptoms in stomach and colorectum.

Tumor Necrosis Factor-α-Induced C-C Motif Chemokine Ligand 20 Expression through TNF Receptor 1-Dependent Activation of EGFR/p38 MAPK and JNK1/2/FoxO1 or the NF-κB Pathway in Human Cardiac Fibroblasts

Tumor necrosis factor (TNF)-α is involved in the pathogenesis of cardiac injury, inflammation, and apoptosis. It is a crucial pro-inflammatory cytokine in many heart disorders, including chronic heart failure and ischemic heart disease, contributing to cardiac remodeling and dysfunction. The implication of TNF-α in inflammatory responses in the heart has been indicated to be mediated through the induction of C-C Motif Chemokine Ligand 20 (CCL20). However, the detailed mechanisms of TNF-α-induced CCL20 upregulation in human cardiac fibroblasts (HCFs) are not completely defined. We demonstrated that in HCFs, TNF-α induced CCL20 mRNA expression and promoter activity leading to an increase in the secretion of CCL20. TNF-α-mediated responses were attenuated by pretreatment with TNFR1 antibody, the inhibitor of epidermal growth factor receptor (EGFR) (AG1478), p38 mitogen-activated protein kinase (MAPK) (p38 inhibitor VIII, p38i VIII), c-Jun amino N-terminal kinase (JNK)1/2 (SP600125), nuclear factor kappaB (NF-κB) (helenalin), or forkhead box O (FoxO)1 (AS1841856) and transfection with siRNA of TNFR1, EGFR, p38α, JNK2, p65, or FoxO1. Moreover, TNF-α markedly induced EGFR, p38 MAPK, JNK1/2, FoxO1, and NF-κB p65 phosphorylation which was inhibited by their respective inhibitors in these cells. In addition, TNF-α-enhanced binding of FoxO1 or p65 to the CCL20 promoter was inhibited by p38i VIII, SP600125, and AS1841856, or helenalin, respectively. Accordingly, in HCFs, our findings are the first to clarify that TNF-α-induced CCL20 secretion is mediated through a TNFR1-dependent EGFR/p38 MAPK and JNK1/2/FoxO1 or NF-κB cascade. We demonstrated that TNFR1-derived EGFR transactivation is involved in the TNF-α-induced responses in these cells. Understanding the regulation of CCL20 expression by TNF-α on HCFs may provide a potential therapeutic strategy in cardiac inflammatory disorders.

Endothelin-1 Stimulates PAI-1 Protein Expression via Dual Transactivation Pathway Dependent ROCK and Phosphorylation of Smad2L.

Objective:In addition to the carboxy region, Smad2 transcription factor can be phosphorylated in the linker region as well. Phosphorylation of Smad2 linker region (Smad2L) promotes the expression of plasminogen activator inhibitor type 1 (PAI-1) which leads to cardiovascular disorders such as atherosclerosis. The purpose of this study was to evaluate the role of dual transactivation of EGF and TGF-β receptors in phosphorylation of Smad2L and protein expression of PAI-1 induced by endothelin-1 (ET-1) in bovine aortic endothelial cells (BAECs). In addition, as an intermediary of G protein-coupled receptor (GPCR) signaling, the functions of ROCK and PLC were investigated in dual transactivation pathways.Materials and Methods:The experimental study is an in vitro study performed on BAECs. Proteins were investigated by western blotting using protein-specific antibodies against phospho-Smad2 linker region residues (Ser245/250/255), phospho-Smad2 carboxy residues (465/467), ERK1/(Thr202/Thr204), and PAI-1.Results:TGF (2 ng/ml), EGF (100 ng/ml) and ET-1 (100 nM) induced the phosphorylation of Smad2L. This response was blocked in the presence of AG1478 (EGFR antagonists), SB431542 (TGFR inhibitor), and Y27632 (Rho-associated protein kinase (ROCK antagonist). Moreover, ET-1-increased protein expression of PAI-1 was decreased in the presence of bosentan (ET receptor inhibitor), AG1478, SB431542, and Y27632.Conclusion:The results indicated that ET-1 increases the phosphorylation of Smad2L and protein expression of PAI-1 via induced the transactivation pathways of EGFR and TGFR. This study is the first attempt to scrutinize the significant role of ROCK in the protein expression of PAI-1.

Hepatocellular carcinoma chemoprevention by targeting the angiotensin-converting enzyme and EGFR transactivation.

Hepatocellular carcinoma (HCC) is a leading cause of death among cirrhotic patients, for which chemopreventive strategies are lacking. Recently, we developed a simple human cell-based system modeling a clinical prognostic liver signature (PLS) predicting liver disease progression and HCC risk. In a previous study, we applied our cell-based system for drug discovery and identified captopril, an approved angiotensin converting enzyme (ACE) inhibitor, as a candidate compound for HCC chemoprevention. Here, we explored ACE as a therapeutic target for HCC chemoprevention. Captopril reduced liver fibrosis and effectively prevented liver disease progression toward HCC development in a diethylnitrosamine (DEN) rat cirrhosis model and a diet-based rat model for nonalcoholic steatohepatitis–induced (NASH-induced) hepatocarcinogenesis. RNA-Seq analysis of cirrhotic rat liver tissues uncovered that captopril suppressed the expression of pathways mediating fibrogenesis, inflammation, and carcinogenesis, including epidermal growth factor receptor (EGFR) signaling. Mechanistic data in liver disease models uncovered a cross-activation of the EGFR pathway by angiotensin. Corroborating the clinical translatability of the approach, captopril significantly reversed the HCC high-risk status of the PLS in liver tissues of patients with advanced fibrosis. Captopril effectively prevents fibrotic liver disease progression toward HCC development in preclinical models and is a generic and safe candidate drug for HCC chemoprevention.

Dexmedetomidine-Induced Aortic Contraction Involves Transactivation of the Epidermal Growth Factor Receptor in Rats.

In this study, we examined whether aortic contraction, induced by the alpha-2 adrenoceptor agonist dexmedetomidine, is involved in the transactivation of the epidermal growth factor receptor (EGFR) in isolated endothelium-denuded rat aortas. Additionally, we aimed to elucidate the associated underlying cellular mechanisms. The effects of the alpha-2 adrenoceptor inhibitor rauwolscine, EGFR tyrosine kinase inhibitor AG1478, Src kinase inhibitors PP1 and PP2, and matrix metalloproteinase inhibitor GM6001 on EGFR tyrosine phosphorylation and c-Jun NH2-terminal kinase (JNK) phosphorylation induced by dexmedetomidine in rat aortic smooth muscles were examined. In addition, the effects of these inhibitors on dexmedetomidine-induced contraction in isolated endothelium-denuded rat aorta were examined. Dexmedetomidine-induced contraction was inhibited by the alpha-1 adrenoceptor inhibitor prazosin, rauwolscine, AG1478, PP1, PP2, and GM6001 alone or by a combined treatment with prazosin and AG1478. AG1478 (3 × 10−6 M) inhibited dexmedetomidine-induced contraction in isolated endothelium-denuded rat aortas pretreated with rauwolscine. Dexmedetomidine-induced EGFR tyrosine and JNK phosphorylation were inhibited by rauwolscine, PP1, PP2, GM6001, and AG1478. Furthermore, dexmedetomidine-induced JNK phosphorylation reduced upon EGFR siRNA treatment. Therefore, these results suggested that the transactivation of EGFR associated with dexmedetomidine-induced contraction, mediated by the alpha-2 adrenoceptor, Src kinase, and matrix metalloproteinase, caused JNK phosphorylation and increased calcium levels.

Neurokinin-1 receptor promotes non-small cell lung cancer progression through transactivation of EGFR

Despite the great advances in target therapy, lung cancer remains the top cause of cancer-related death worldwide. G protein-coupled receptor neurokinin-1 (NK1R) is shown to play multiple roles in various cancers; however, the pathological roles and clinical implication in lung cancer are unclarified. Here we identified NK1R as a significantly upregulated GPCR in the transcriptome and tissue array of human lung cancer samples, associated with advanced clinical stages and poor prognosis. Notably, NK1R is co-expressed with epidermal growth factor receptor (EGFR) in NSCLC patients’ tissues and co-localized in the tumor cells. NK1R can crosstalk with EGFR by interacting with EGFR, transactivating EGFR phosphorylation and regulating the intracellular signaling of ERK1/2 and Akt. Activation of NK1R promotes the proliferation, colony formation, EMT, MMP2/14 expression, and migration of lung cancer cells. The inhibition of NK1R by selective antagonist aprepitant repressed cell proliferation and migration in vitro. Knockdown of NK1R significantly slowed down the tumor growth in nude mice. The sensitivity of lung cancer cells to gefitinib/osimertinib is highly increased in the presence of the selective NK1R antagonist aprepitant. Our data suggest that NK1R plays an important role in lung cancer development through EGFR signaling and the crosstalk between NK1R and EGFR may provide a potential therapeutic target for lung cancer treatment.

Characterization of Type 1 Angiotensin II Receptor Activation Induced Dual-Specificity MAPK Phosphatase Gene Expression Changes in Rat Vascular Smooth Muscle Cells.

Activation of the type I angiotensin receptor (AT1-R) in vascular smooth muscle cells (VSMCs) plays a crucial role in the regulation of blood pressure; however, it is also responsible for the development of pathological conditions such as vascular remodeling, hypertension and atherosclerosis. Stimulation of the VSMC by angiotensin II (AngII) promotes a broad variety of biological effects, including gene expression changes. In this paper, we have taken an integrated approach in which an analysis of AngII-induced gene expression changes has been combined with the use of small-molecule inhibitors and lentiviral-based gene silencing, to characterize the mechanism of signal transduction in response to AngII stimulation in primary rat VSMCs. We carried out Affymetrix GeneChip experiments to analyze the effects of AngII stimulation on gene expression; several genes, including DUSP5, DUSP6, and DUSP10, were identified as upregulated genes in response to stimulation. Since various dual-specificity MAPK phosphatase (DUSP) enzymes are important in the regulation of mitogen-activated protein kinase (MAPK) signaling pathways, these genes have been selected for further analysis. We investigated the kinetics of gene-expression changes and the possible signal transduction processes that lead to altered expression changes after AngII stimulation. Our data shows that the upregulated genes can be stimulated through multiple and synergistic signal transduction pathways. We have also found in our gene-silencing experiments that epidermal growth factor receptor (EGFR) transactivation is not critical in the AngII-induced expression changes of the investigated genes. Our data can help us understand the details of AngII-induced long-term effects and the pathophysiology of AT1-R. Moreover, it can help to develop potential interventions for those symptoms that are induced by the over-functioning of this receptor, such as vascular remodeling, cardiac hypertrophy or atherosclerosis.

EGF Receptor Transactivation by Endothelin-1 Increased CHSY-1 Mediated by NADPH Oxidase and Phosphorylation of ERK1/2.

Objective Growth factors [transforming growth factor-β (TGF-β), epidermal growth factor (EGF), endothelin-1 (ET- 1)] stimulate proteoglycan synthesis resulting in retention and accumulation of low density lipoprotein (LDL) in vessel intima and leading to atherosclerosis development. This study investigated the role of ET-1 on the expression of CHSY1, proteoglycan synthesizing enzyme, through both EGF and TGF-β receptor transactivation in human vascular smooth muscle cells (VSMCs). Also, we explored the involvement of NADPH oxidase (NOX), an important intermediate of redox signaling, in ET-1 transactivated EGF receptor (EGFR) through endothelin receptors. Materials and Methods In this experimental study, phosphorylated ERK1/2 and CHSY1 protein levels in the human VSMCs were measured by Western blot analysis using anti phospho-ERK1/2 (Thr202/Tyr204) and anti CHSY1 antibodies. Results ET-1 (100 nM) and EGF (100 ng/ml) stimulated ERK1/2 phosphorylation and inhibited in the presence of bosentan (ET receptor inhibitor), AG1478 (EGFR inhibitor), and DPI (NOX antagonist). Also, ET-1 treatment increased CHSY1 enzyme level; this response was suppressed by bosentan, AG1478, DPI, and SB431542, TGF-β receptor antagonist. This study revealed that ET-1 increases expression of CHSY1 through transactivation of EGF and TGF-β receptors. ConclusionTransactivation through the EGF receptor mediated by phospho-ERK1/2 leads to expression of CHSY1 protein. EGF receptor transactivation by ET-1 is shown for the first time, to be dependent on NOX enzymes.

063 A new case series of Olmsted syndrome subjects confirms EGFR activation and shows remarkable efficacy of targeted systemic EGFR inhibition with acceptable side effects

Olmsted syndrome (OS) is a rare, painful, and severe form of palmoplantar keratoderma (PPK). It is most often caused by dominant mutations in the transient receptor potential vanilloid-3 (TRPV3) gene, resulting in the constitutive activation of this thermosensitive calcium channel. TRPV3 signaling leads to epidermal growth factor receptor (EGFR) activation and subsequent abnormal proliferation and differentiation of keratinocytes in mice and cell lines. We previously reported that blocking EGFR transactivation with oral Erlotinib treatment resulted in remarkable improvement in 4 young patients.

Lysophosphatidic Acid-Induced EGFR Transactivation Promotes Gastric Cancer Cell DNA Replication by Stabilizing Geminin in the S Phase.

Geminin, an inhibitor of the DNA replication licensing factor, chromatin licensing and DNA replication factor (Cdt) 1, is essential for the maintenance of genomic integrity. As a multifunctional protein, geminin is also involved in tumor progression, but the molecular details are largely unknown. Here, we found that lysophosphatidic acid (LPA)–induced upregulation of geminin was specific to gastric cancer cells. LPA acted via LPA receptor (LPAR) 3 and matrix metalloproteinases (MMPs) signaling to transactivate epidermal growth factor receptor (EGFR) (Y1173) and thereby stabilize geminin expression level during the S phase. LPA also induced the expression of deubiquitinating protein (DUB) 3, which prevented geminin degradation. These results reveal a novel mechanism underlying gastric cancer progression that involves the regulation of geminin stability by LPA-induced EGFR transactivation and provide potential targets for the signaling pathway and tumor cell–specific inhibitors.

β-Arrestin-Biased Allosteric Modulator Potentiates Carvedilol-Stimulated β Adrenergic Receptor Cardioprotection.

β 1 adrenergic receptors (β 1ARs) are central regulators of cardiac function and a drug target for cardiac disease. As a member of the G protein-coupled receptor family, β 1ARs activate cellular signaling by primarily coupling to Gs proteins to activate adenylyl cyclase, cAMP-dependent pathways, and the multifunctional adaptor-transducer protein β-arrestin. Carvedilol, a traditional β-blocker widely used in treating high blood pressure and heart failure by blocking β adrenergic receptor-mediated G protein activation, can selectively stimulate Gs-independent β-arrestin signaling of β adrenergic receptors, a process known as β-arrestin-biased agonism. Recently, a DNA-encoded small-molecule library screen against agonist-occupied β 2 adrenergic receptors (β 2ARs) identified Compound-6 (Cmpd-6) to be a positive allosteric modulator for agonists on β 2ARs. Intriguingly, it was further discovered that Cmpd-6 is positively cooperative with the β-arrestin-biased ligand carvedilol at β 2ARs. Here we describe the surprising finding that at β 1ARs unlike β 2ARs, Cmpd-6 is cooperative only with carvedilol and not agonists. Cmpd-6 increases the binding affinity of carvedilol for β 1ARs and potentiates carvedilol-stimulated, β-arrestin-dependent β 1AR signaling, such as epidermal growth factor receptor transactivation and extracellular signal-regulated kinase activation, whereas it does not have an effect on Gs-mediated cAMP generation. In vivo, Cmpd-6 enhances the antiapoptotic, cardioprotective effect of carvedilol in response to myocardial ischemia/reperfusion injury. This antiapoptotic role of carvedilol is dependent on β-arrestins since it is lost in mice with myocyte-specific deletion of β-arrestins. Our findings demonstrate that Cmpd-6 is a selective β-arrestin-biased allosteric modulator of β 1ARs and highlight its potential clinical utility in enhancing carvedilol-mediated cardioprotection against ischemic injury. SIGNIFICANCE STATEMENT: This study demonstrates the positive cooperativity of Cmpd-6 on β1ARs as a β-arrestin-biased positive allosteric modulator. Cmpd-6 selectively enhances the affinity and cellular signaling of carvedilol, a known β-arrestin-biased β-blocker for β1ARs, whereas it has minimal effect on other ligands tested. Importantly, Cmpd-6 enhances the β-arrestin-dependent in vivo cardioprotective effect of carvedilol during ischemia/reperfusion injury-induced apoptosis. The data support the potential therapeutic application of Cmpd-6 to enhance the clinical benefits of carvedilol in the treatment of cardiac disease.

GPCR-mediated EGFR transactivation ameliorates skin toxicities induced by afatinib.

Many G-protein-coupled receptor (GPCR) agonists have been studied for transactivating epidermal growth factor receptor (EGFR) signaling through extracellular or intracellular pathways. Accumulated evidence has confirmed that GPCR transactivation participates in various diseases. However, the clinical application of GPCR transactivation has not been explored, and more translational studies are needed to develop therapies to target GPCR-mediated EGFR transactivation. In cancer patients treated with EGFR inhibitors (EGFRi), especially afatinib, a unique acneiform rash is frequently developed. In this study, we first established the connection between GPCR transactivation and EGFRi-induced skin disease. We examined the ability of three different GPCR agonists to reverse signaling inhibition and ameliorate rash induced by EGFRi. The activation of different agonists follows unique time and kinase patterns. Rats treated with EGFRi show a similar skin phenotype, with rash occurring in the clinic; correspondingly, treatment with GPCR agonists reduced keratinocyte apoptosis, growth retardation and infiltration of inflammatory cytokines by transactivation. This phenomenon demonstrates that EGFR inhibition in keratinocytes regulates key factors associated with rash. Our findings indicate that maintaining EGFR signaling by GPCR agonists might provide a possible therapy for EGFR inhibitor-induced skin toxicities. Our study provides the first example of the translational application of GPCR transactivation in treating diseases.

Wnt signaling promotes tumor development in part through phosphofructokinase 1 platelet isoform upregulation.

The activation of Wnt signaling has been detected in various types of human cancer and has been shown to be associated with cancer development. In the present study, it was revealed that Wnt signaling induced the expression of phosphofructokinase 1 platelet isoform (PFKP), which has been reported to catalyze a rate‑limiting reaction in glycolysis and is important for the Warburg effect, proliferation, colony formation and cancer cell migration. Moreover, it was demonstrated that Wnt3A induced PFKP expression in a β‑catenin‑independent manner, resulting in increased PFK enzyme activity. Wnt3A‑induced epidermal growth factor receptor transactivation activated PI3K/AKT, which stabilized PFKP through PFKP S386 phosphorylation and subsequent PFKP upregulation. Wnt3A‑induced PFKP S386 phosphorylation increased PFKP expression and promoted the Warburg effect, cell proliferation, colony formation and the migratory ability of cancer cells. On the whole, the findings of the present study underscore the potential role of PFKP in Wnt signaling‑induced tumor development.

Interleukin-1β Induces Tissue Factor Expression in A549 Cells via EGFR-Dependent and -Independent Mechanisms

Tissue factor (TF) plays an important role in the progression and angiogenesis of tumor cells. The present study investigated the mechanism of interleukin-1β (IL-1β)-induced TF expression in A549 lung cancer cells. Based on mRNA and protein analyses, including appropriate inhibitor experiments, IL-1β was shown to induce TF expression in a time-dependent manner, mediated by IL-1 receptor-dependent phosphorylation of the mitogen-activated protein kinases (MAPK) p38, p42/44 and c-jun N-terminal kinase (JNK), as well as the Src kinase and the epidermal growth factor receptor (EGFR). Thereby, inhibition of EGFR transactivation by the Src inhibitor PP1 or direct EGFR inhibition by the EGFR tyrosine kinase inhibitor (TKI) erlotinib led to a reduction of IL-1β-induced TF expression and to a suppression of p42/44 MAPK and EGFR activation, while IL-1β-induced p38 MAPK and JNK activation remained unchanged. A knockdown of EGFR by siRNA was associated with decreased IL-1β-mediated p42/44 MAPK activation, which was no longer inhibitable by erlotinib. Concentration-dependent inhibition of IL-1β-induced TF expression was also observed in the presence of gefitinib and afatinib, two other EGFR TKIs. In summary, our results suggest that IL-1β leads to increased TF formation in lung cancer cells via both Src/EGFR/p42/44 MAPK-dependent and EGFR-independent signaling pathways, with the latter mediated via p38 MAPK and JNK.

Signal Crosstalk Promoted Proliferative Lesions in Mouse Mammary Glands As a Consequence of ET-1 Overexpression

Objective: As a distinct cellular signaling model, a receptor crosstalk between G protein-coupled receptors (GPCR) and epidermal growth factor receptor (EGFR) has been demonstrated in various in vitro studies. In addition, recent in vitro studies had focused on the signaling pathways of endothelin-1 (ET-1) in the pathophysiology of cancer. Accordingly, a growing interest in the analysis of the receptor crosstalk between ET-1 receptors and EGFR and functional consequences of EGFR activation of proliferative diseases evoked us to the analysis of this phenomenon in vivo. Materials and Methods: We performed a comparative study between ET-1 transgenic mice and control mice during the late pregnancy (n=7), early lactation (n=6) the mid of lactation (n=10) and involution day 14 (n=7) periods. Hematoxylin and eosin (HE)- stained parallel sections from mammary glands were microscopically examined. The key signal proteins (ETAR, ETBR, ERK1/2, pEGFR) in transactivation of EGFR were analyzed employing Western blot techniques. Genes (amphiregulin, TGFa, EGF, HB-EGF, ADAM 17) known to play an important role in these activities were analyzed using real-time PCR (RT-PCR) techniques. Results: ET-1 transgenic mice exhibited hyperproliferative lesions (lactational hyperplasia) during the middle of the lactation period. Our RT-PCR analyses showed a prominent up regulation of amphiregulin and ADAM 17 in ET-1 transgenic mice. Moreover, we found higher EGFR and ERKs activations in the transgenic mammary glands. Conclusion: This study highlights a causative effect of upregulated ET-1 gene expression on the induction of proliferative lesions via EGFR transactivation in mammary glands. Further, ET-1 overexpression induced an upregulation of amphiregulin and ADAM17 expressions in the transgenic mammary glands. These results suggests that the enhanced ET-1 gene expression and its receptors might have a crucial role in proliferative diseases maintaining EGFR activation.