Knockdown Of Epidermal Growth Factor Receptor Research Articles

Lead acetate induces EGFR activation upstream of SFK and PKCα linkage to the Ras/Raf-1/ERK signaling

Lead acetate (Pb), a probable human carcinogen, can activate protein kinase C (PKC) upstream of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Yet, it remains unclear whether Pb activation of PKC → ERK1/2 involves receptor/non-receptor tyrosine kinases and the Ras signaling transducer. Here we demonstrate a novel mechanism elicited by Pb for transmitting ERK1/2 signaling in CL3 human non-small-cell lung adenocarcinoma cells. Pb induction of higher steady-state levels of Ras-GTP was essential for increasing phospho-Raf-1 S338 and phospho-ERK1/2. Pre-treatment of the cells with a conventional PKC inhibitor Gö6976 or depleting PKCα using specific small interfering RNA blocked Pb induction of Ras-GTP. Pb also activated cellular tyrosine kinases. Specific pharmacological inhibitors, PD153035 for epidermal growth factor receptor (EGFR) and SU6656 for Src family tyrosine kinases (SFK), but not AG1296 for platelet-derived growth factor receptor, could suppress the Pb-induced tyrosine kinases, PKCα, Ras-GTP, phospho-Raf-1 S338 and phospho-ERK1/2. Furthermore, phosphorylation of tyrosines on the EGFR multiple autophosphorylation sites and the conserved SFK autophosphorylation site occurred during exposure of cells to Pb for 1–5 min and 5–30 min, respectively. Intriguingly, Pb activation of EGFR required the intrinsic kinase activity but not dimerization of the receptor. Inhibition of SFK or PKCα activities did not affect EGFR phosphorylation, while knockdown of EGFR blocked SFK phosphorylation and PKCα activation following Pb. Together, these results indicate that immediate activation of EGFR in response to Pb is obligatory for activation of SFK and PKCα and subsequent the Ras–Raf-1–MKK1/2–ERK1/2 signaling cascade.

Helicobacter pyloriactivate epidermal growth factor receptor- and phosphatidylinositol 3-OH kinase-dependent Akt and glycogen synthase kinase 3β phosphorylation

The signalling pathways leading to the development of Helicobacter pylori-induced gastric cancer remain poorly understood. We tested the hypothesis that H. pylori infections involve the activation of Akt signalling in human gastric epithelial cancer cells. Immunoblot, immunofluorescence and kinase assays show that H. pylori infection of gastric epithelial cells induced phosphorylation of Akt at Ser 473 and Thr 308. Mutations in the H. pylori virulence factor OipA dramatically reduced phosphorylation of Ser 473, while the cag pathogenicity island mutants predominantly inhibited phosphorylation of Thr 308. As the downstream of Akt activation, H. pylori infection inactivated the inactivation of glycogen synthase kinase 3beta at Ser 9 by its phosphorylation. As the upstream of Akt activation, H. pylori infection activated epidermal growth factor receptor (EGFR) at Tyr 992, phosphatidylinositol 3-OH kinase (PI3K) p85 subunit and PI3K-dependent kinase 1 at Ser 241. Pharmacologic inhibitors of PI3K or mitogen-activated protein kinase kinase (MEK), Akt knock-down and EGFR knock-down showed that H. pylori infection induced the activation of EGFR-->PI3K-->PI3K-dependent kinase 1-->Akt-->extracellular signal-regulated kinase signalling pathways, the inactivation of glycogen synthase kinase 3beta and interleukin-8 production. The combined functions of cag pathogenicity island and OipA were necessary and sufficient for full activation of signalling at each level. We propose activation of these pathways as a novel mechanism for H. pylori-mediated carcinogenesis.

Two Pathways Are Better Than One

Glutamate is an excitatory neurotransmitter that activates ionotropic and metabotropic glutamate receptors (mGluRs) on neurons and glia to mediate such functions as synaptic plasticity, proliferation, and survival. mGluR5 activates the transcription factor nuclear factor κB (NF-κB), but the mechanisms involved are unclear. Sitcheran et al . performed gel shift assays to demonstrate that glutamate induced the binding of the p65 and p50 NF-κB subunits to DNA in the human glioma cell line, H4. The authors also examined the signaling of the epidermal growth factor receptor (EGFR), which is found on astrocytes and is also known to activate NF-κB. Reporter assays showed that glutamate-induced activation of NF-κB was comparable to that induced by EGF. Western blotting analyses demonstrated that glutamate induced the phosphorylation and activation of inhibitor of κB kinase α and β (IKKα and IKKβ) and of p65. Knockdown of both IKKα and IKKβ in H4 cells by small inhibitory RNA (siRNA) suppressed glutamate-induced phosphorylation of p65. In canonical NF-κB signaling, IKKβ phosphorylates IκBα, which leads to its degradation and the release of active NF-κB subunits. In this study, however, the authors found no evidence of glutamate-induced (or EGF-induced) phosphorylation or degradation of IκBα, although coimmunoprecipitation assays showed that glutamate substantially reduced the interaction between IκBα and p65 compared with that in control cells. Knockdown of EGFR blocked mGluR5-stimulated phosphorylation of p65. The authors found that mGluR5 stimulation led to the phosphorylation of specific tyrosine residues in EGFR and to its physical association with mGluR5. A cell-permeable Ca 2+ chelator blocked mGluR5-mediated NF-κB activation, and an inhibitor of EGFR activity reduced mGluR5-stimulated Ca 2+ signaling. Together, these data suggest that EGFR signaling is critical for the noncanonical activation of NF-κB by glutamate and that this crosstalk may have implications for the treatment of glioblastoma, in which EGFR abundance is increased. R. Sitcheran, W. C. Comb, P. C. Cogswell, A. S. Baldwin, Essential role for epidermal growth factor receptor in glutamate receptor signaling to NF-κB. Mol. Cell. Biol. 28 , 5061-5070 (2008). [Abstract] [Full Text]

A cross-talk between the androgen receptor and the epidermal growth factor receptor leads to p38MAPK-dependent activation of mTOR and cyclinD1 expression in prostate and lung cancer cells

In androgen sensitive LNCaP prostate cancer cells, the proliferation induced by the epidermal growth factor (EGF) involves a cross-talk between the EGF receptor (EGFR) and the androgen receptor (AR). In lung cancer the role of the EGF–EGFR transduction pathway has been documented, whereas androgen activity has received less attention. Here we demonstrate that in LNCaP and A549 non-small cell lung cancer (NSCLC), AR and EGFR are required for either 5α-dihydrotestosterone (DHT) or EGF-stimulated cell growth. Only EGF activated ERK signaling and up-regulated early gene expression, while DHT triggered the expression of classical AR-responsive genes with the exception of the EGF-induced PSA transcript in A549 cells. DHT and EGF up-regulated cyclinD1 (CD1) at both mRNA and protein levels in A549 cells, while in LNCaP cells each mitogen increased only CD1 protein expression. In both cell contexts, CD1 up-regulation was prevented by selective inhibitors as well as by knock-down of either AR or EGFR and also inhibiting p38MAPK and the mammalian target of rapamycin (mTOR) pathways. Interestingly, p38MAPK and mTOR repression prevented the activation of the mTOR target ribosomal p70S6 kinase induced by DHT and EGF, indicating that p38MAPK acts as an upstream mTOR regulator. In addition, the proliferative effects promoted by both DHT and EGF in LNCaP and A549 cancer cells were no longer observed blocking either p38MAPK or mTOR activity. Hence, our data suggest that p38MAPK-dependent activation of the mTOR/CD1 pathway may represent a mechanism through which AR and EGFR cross-talk contributes to prostate and lung cancer progression.

Epidermal growth factor receptor regulates osteoclast differentiation and survival through cross‐talking with RANK signaling

The epidermal growth factor receptor (EGFR) functions in various cellular physiological processes such as proliferation, differentiation, and motility. Although recent studies have reported that EGFR signaling is involved in osteoclast recruitment and formation, the molecular mechanism of EGFR signaling for the regulation of osteoclastogenesis remains unclear. We investigated the role of the EGFR in osteoclast differentiation and survival and show that the expression of the EGFR was highly up-regulated by receptor activator of nuclear factor-kappaB ligand (RANKL) during osteoclast differentiation. EGFR-specific tyrosine kinase inhibitors and EGFR knockdown blocked RANKL-dependent osteoclast formation, suggesting that EGFR signaling plays an important role in osteoclastogenesis. EGFR inhibition impaired the RANKL-mediated activation of osteoclastogenic signaling pathways, including c-Jun N-terminal kinase (JNK), NF-kappaB, and Akt/protein kinase B (PKB). In addition, EGFR inhibition in differentiated osteoclasts caused apoptosis through caspase activation. Inhibition of the phosphoinositide-3 kinase (PI3K)-Akt/PKB pathway and subsequent activation of BAD and caspases-9 and -3 may be responsible for the EGFR inhibition-induced apoptosis. The EGFR physically associated with receptor activator of nuclear factor-kappaB (RANK) and Grb2-associated binder 2 (Gab2). Moreover, RANKL transactivated EGFR. These data indicate that EGFR regulates RANKL-activated signaling pathways by cross-talking with RANK, suggesting that the EGFR may play a crucial role as a RANK downstream signal and/or a novel type of RANK co-receptor in osteoclast differentiation and survival.

Mitochondrially localized EGFR is subjected to autophagic regulation and implicated in cell survival

Although generally acknowledged as a plasma membrane protein, the epidermal growth factor (EGF) receptor has been found in the nucleus and subcellular organelles. Recently, the mitochondrial localization of the EGF receptor (EGFR) was reported; nevertheless, the molecular mechanism underlying EGFR localization in mitochondria is largely unknown. Using immunofluorescence and immunoelectron microscopy, we observed that EGFR did localize within mitochondria. Moreover, EGFR mitochondrial translocation can be increased by rapamycin treatment in A431 cells and greatly reduced by the presence of 3-methyladenine (3-MA), an inhibitor of autophagy. The reduction of mitochondrial EGFR via autophagy inhibition is further confirmed by small interference RNA (siRNA), through which the essential protein Beclin 1 was depleted. Knocking down Beclin 1 markedly decreased the mitochondrial translocation of EGFR that was induced by rapamycin. We also noticed that the content of mitochondrial EGFR transfer is decreased when the cells are exposed to the apoptotic inducer etoposide. Additionally, either EGF treatment or EGFR knockdown by siRNA results in a greater decline of cell viability in cells possessing more mitochondrial EGFRs. Taken together, we conclude that EGFR mitochondrial localization is regulated by either autophagy or programmed cell death and is correlated with cell survival.

The EGF receptor interacts with the type 1 IGF receptor and regulates its stability

Both the epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGF1R) require homo- and hetero-dimerisation with their own family members to acquire full function. We recently showed that IGF1R gene silencing led to EGFR hyper-phosphorylation in human breast cancer cells, and hypothesised that this crosstalk might be associated with direct IGF1R:EGFR interaction. Indeed we could detect reciprocal co-precipitation between the IGF1R and EGFR when overexpressed in SKUT-1 cells, and between endogenous IGF1R and EGFR in MDA-MB-468 breast carcinoma cells, two squamous cancer cell lines, and clinical samples of breast cancer. Interaction was abolished by knockdown of either receptor, and we noted that EGFR knockdown also suppressed IGF1R protein levels. Further investigation revealed that EGFR depletion induced enhancement of IGF1R ubiquitylation and degradation. These results indicate novel evidence of crosstalk between two key cancer treatment targets, capable of modifying the stability of IGF1R protein.

Adenylate cyclase 5 and KCa1.1 channel are required for EGFR up‐regulation of PCNA in native contractile rat basilar artery smooth muscle

In synthetic phenotype vascular smooth muscle cells (VSMC), activation of epidermal growth factor (EGF) receptor (EGFR) induces a sustained increase in intermediate conductance K(Ca) (int-K(Ca); K(Ca)3.1) channels that is essential for proliferation. However, a comparable mechanism has not been identified in native contractile phenotype VSMC, which express large conductance K(Ca) (maxi-K(Ca); K(Ca)1.1) channels, not int-K(Ca) channels. Using patch clamp of freshly isolated contractile VSMC from rat basilar artery, we found that EGF (100 ng ml(-1)) caused hyperpolarization (7.9 +/- 3.9 mV) due to activation of iberiotoxin-sensitive, maxi-K(Ca) channels. The EGFR ligands EGF (100 ng ml(-1)), transforming growth factor alpha (0.4 ng ml(-1)) and heparin-binding EGF (100 ng ml(-1)) all caused a 20% increase in maxi-K(Ca) channel current that was blocked by AG-1478 or by knock-down of EGFR expression using cisterna magna infusion of antisense oligodeoxynucleotide (AS-ODN). In controls, EGFR knock-down, and EGFR gain-of-expression (angiotensin II hypertension), the increase in maxi-K(Ca) current correlated with the abundance of EGFR protein expressed. The EGFR-mediated increase in maxi-K(Ca) channel activity was blocked by inhibiting cAMP-dependent protein kinase (cAK) using KT-5720 or Rp-cAMP, or by inhibiting adenylate cyclase type 5 (AC-5) using 2',5'-dideoxyadenosine or knock-down of AC-5 expression by intracisternal AS-ODN. Direct infusion of EGF into cisterna magna caused up-regulation of proliferating cell nuclear antigen (PCNA) in VSMC that was prevented by coinfusion of iberiotoxin or of AG-1478. Our data, which are consistent with the hypothesis that hyperpolarization is critical for a proliferative response, are the first to implicate AC-5 and maxi-K(Ca) channels in gene activation related to EGFR signalling in native contractile VSMC.

Herpes Simplex Virus 1 Amplicon Vector-Mediated siRNA Targeting Epidermal Growth Factor Receptor Inhibits Growth of Human Glioma Cells in Vivo

In primary glioblastomas and other tumor types, the epidermal growth factor receptor (EGFR) is frequently observed with alterations, such as amplification, structural rearrangements, or overexpression of the gene, suggesting an important role in glial tumorigenesis and progression. In this study, we investigated whether posttranscriptional gene silencing by vector-mediated RNAi to inhibit EGFR expression can reduce the growth of cultured human gli36 glioma cells. To “knock down” EGFR expression, we have created HSV-1-based amplicons that contain the RNA polymerase III-dependent H1 promoter to express double-stranded hairpin RNA directed against EGFR at two different locations (pHSVsiEGFR I and pHSVsiEGFR II). We demonstrate that both pHSVsiEGFR I and pHSVsiEGFR II mediated knock-down of transiently transfected full-length EGFR or endogenous EGFR in a dose-dependent manner. The knock-down of EGFR resulted in the growth inhibition of human glioblastoma (gli36-luc) cells both in culture and in athymic mice in vivo. Cell cycle analysis and annexin V staining revealed that siRNA-mediated suppression of EGFR induced apoptosis. Overall HSV-1 amplicons can mediate efficient and specific posttranscriptional gene silencing.

Involvement of NADPH Oxidase Isoforms and Src Family Kinases in CD95-dependent Hepatocyte Apoptosis

CD95 ligand (CD95L) triggers a rapid formation of reactive oxygen species (ROS) as an upstream event of CD95 activation and apoptosis induction in rat hepatocytes. This ROS response was sensitive to inhibition by diphenyleneiodonium, apocynin, and neopterin, suggestive of an involvement of NADPH oxidases. In line with this, hepatocytes expressed mRNAs not only of the phagocyte gp91phox (Nox 2), but also of the homologs Nox 1 and 4 and Duox 1 and 2, as well as the regulatory subunit p47phox. gp91phox (Nox 2) and p47phox were also identified at the protein level in rat hepatocytes. CD95L induced within 1 min ceramide formation and serine phosphorylation of p47phox, which was sensitive to inhibitors of sphingomyelinase and protein kinase Czeta (PKCzeta). These inhibitors and p47phox protein knockdown inhibited the early CD95L-induced ROS response, suggesting that ceramide and PKCzeta are upstream events of the CD95L-induced Nox/Duox activation. CD95L also induced rapid activation of the Src family kinase Yes, being followed by activation of c-Src, Fyn, and c-Jun-N-terminal kinases (JNK). Only Yes and JNK activation were sensitive to N-acetylcysteine, inhibitors of NADPH oxidase, PKCzeta, or sphingomyelinase, indicating that the CD95L-induced ROS response is upstream of Yes and JNK but not of Fyn and c-Src activation. Activated Yes rapidly associated with the epidermal growth factor receptor (EGFR), which became phosphorylated at Tyr845 and Tyr1173 but not at Tyr1045. Activated EGFR then triggered an AG1478-sensitive CD95-tyrosine phosphorylation, which was a signal for membrane targeting of the EGFR/CD95 complex, subsequent recruitment of Fas-associated death domain and caspase 8, and apoptosis induction. All of these events were significantly blunted by inhibitors of sphingomyelinase, PKCzeta, NADPH oxidases, Yes, or EGFR-tyrosine kinase activity and after protein knockdown of either p47phox, Yes, or EGFR. The data suggest that CD95L-induced apoptosis involves a sphingomyelinase- and PKCzeta-dependent activation of NADPH oxidase isoforms, which is required for Yes/EGFR/CD95 interactions as upstream events of CD95 activation.

NADPH Oxidase-Dependent Reactive Oxygen Species are Important to the Early Stage of CD95 Engagement in Hepatocytes *

Abstract CD95 ligand (CD95L) triggers a rapid formation of reactive oxygen species (ROS) as an upstream event of CD95 activation and apoptosis induction in rat hepatocytes. This ROS response was sensitive to inhibition by diphenyleneiodonium, apocynin, and neopterin, suggestive of an involvement of NADPH oxidases. In line with this, hepatocytes expressed mRNAs not only of the phagocyte gp91 phox (Nox 2), but also of the homologs Nox 1 and 4 and Duox 1 and 2, as well as the regulatory subunit p47 phox . gp91 phox (Nox 2) and p47 phox were also identified at the protein level in rat hepatocytes. CD95L induced within 1 min ceramide formation and serine phosphorylation of p47 phox , which was sensitive to inhibitors of sphingomyelinase and protein kinase Cζ (PKCζ). These inhibitors and p47 phox protein knockdown inhibited the early CD95L-induced ROS response, suggesting that ceramide and PKCζ are upstream events of the CD95Linduced Nox/Duox activation. CD95L also induced rapid activation of the Src family kinase Yes, being followed by activation of c-Src, Fyn, and c-Jun-N-terminal kinases (JNK). Only Yes and JNK activation were sensitive to N -acetylcysteine, inhibitors of NADPH oxidase, PKCζ, or sphingomyelinase, indicating that the CD95L-induced ROS response is upstream of Yes and JNK but not of Fyn and c-Src activation. Activated Yes rapidly associated with the epidermal growth factor receptor (EGFR), which became phosphorylated at Tyr845 and Tyr1173 but not at Tyr1045. Activated EGFR then triggered an AG1478-sensitive CD95-tyrosine phosphorylation, which was a signal for membrane targeting of the EGFR/CD95 complex, subsequent recruitment of Fas-associated death domain and caspase 8, and apoptosis induction. All of these events were significantly blunted by inhibitors of sphingomyelinase, PKCζ, NADPH oxidases, Yes, or EGFR-tyrosine kinase activity and after protein knockdown of either p47 phox , Yes, or EGFR. The data suggest that CD95L-induced apoptosis involves a sphingomyelinase- and PKCζ-dependent activation of NADPH oxidase isoforms, which is required for Yes/EGFR/CD95 interactions as upstream events of CD95 activation. (Hepatology 2005;42:956–958.)

Involvement of the Src family kinase yes in bile salt-induced apoptosis

Hydrophobic bile acids induce CD95 (Fas, APO-1)-dependent hepatocyte apoptosis, which involves epidermal growth factor receptor (EGFR)-catalyzed CD95 tyrosine phosphorylation. The mechanisms underlying bile salt-induced EGFR activation remain unclear. Bile acid-induced EGFR activation was studied in 24-hour cultured rat hepatocytes and perfused rat liver. The proapoptotic bile salts taurolithocholate-3-sulfate (TLCS), glycochenodesoxycholate (GCDC) and taurochenodeoxycholate (TCDC), but not taurocholate (TC), activate within 1 minute the Src kinase family member Yes, followed by an association of Yes with EGFR and subsequent EGFR activation. EGFR phosphorylation by TLCS involves tyrosines 845 and 1173 but not 1045. Yes/EGFR association and EGFR activation were sensitive to inhibition by SU6656 but not by PP-2. cAMP had no effect on TLCS and GCDC-induced Yes activation but induced Ser/Thr phosphorylation of Yes and prevented Yes/EGFR association and subsequent EGFR activation. Both SU6656 and cAMP had no effect on bile salt-induced c-Jun N-terminal kinase activation, but blocked bile salt-induced CD95 tyrosine phosphorylation, membrane trafficking of CD95, formation of the death-inducing signaling complex, and apoptosis. In 4-day cultured hepatocytes, knockdown of either Yes or EGFR strongly attenuated bile salt-induced CD95 activation and apoptosis. The data identify the Src kinase Yes as an upstream target of proapoptotic bile acids, which triggers EGFR activation, subsequent CD95 tyrosine phosphorylation, and apoptosis. The antiapoptotic effect of cAMP involves a protein kinase A-dependent inhibition of Yes/EGFR association, thereby preventing EGFR activation, which is required for CD95 activation.

The Src family kinase Yes triggers hyperosmotic activation of the epidermal growth factor receptor and CD95.

Hyperosmotic exposure of rat hepatocytes triggers epidermal growth factor receptor (EGFR) activation, which results in an activation of the CD95 system and sensitizes the cells toward apoptosis (Reinehr, R., Schliess, F., and Haüssinger, D. (2003) FASEB J. 17, 731-733). The mechanisms underlying the hyperosmotic EGFR activation were studied. Hyperosmotic exposure (405 mosm) resulted in a rapid activation of the Src kinase family members Yes, Fyn, and Lck. Hyperosmotic Yes, but not Fyn activation, was antioxidant-sensitive and was followed by a rapid Yes/EGFR association. PP-2 abolished the hyperosmotic activation of Fyn and Lck but not activation of Yes and EGFR and their association. However, these latter processes were prevented in the presence of SU6656. SU6656 and antioxidants, but not PP-2 and AG1478, also inhibited the hyperosmotic JNK activation. Cyclic AMP had no effect on hyperosmotic Yes and JNK activation but prevented EGFR/Yes association and EGFR activation in an H89-sensitive way. When the hyperosmolarity-induced Yes-EGFR protein complex started to disappear after 30 min, an association between EGFR and CD95 became apparent, which was followed by CD95 tyrosine phosphorylation and activation. SU6656 but not PP-2 also inhibited EGFR/CD95 association, CD95 tyrosine phosphorylation, CD95 membrane trafficking, and death-inducing signaling complex (DISC) formation. EGFR knockdown had no effect on hyperosmotic Yes activation but prevented CD95 tyrosine phosphorylation, membrane targeting, and DISC formation. Hyperosmotic EGFR and CD95 activation was also largely blunted following Yes knockdown. The data suggest that hyperosmotic signaling triggers an oxidative stress-dependent Yes activation, which is followed by JNK and EGFR activation and subsequent activation of the CD95 system. However, the functional relevance of hyperosmolarity-induced Fyn and Lck activation remains to be elucidated.