Endogenous Epidermal Growth Factor Research Articles

EGF-Enhanced GnRH-II Regulation in Decidual Stromal Cell Motility through Twist and N-Cadherin Signaling.

Crucial roles in embryo implantation and placentation in humans include the invasion of the maternal decidua by extravillous trophoblasts and the motile behavior of decidual endometrial stromal cells. The effects of the epidermal growth factor (EGF) and GnRH-II in the endometrium take part in early pregnancy. In the present study, we demonstrated the coaction of EGF- and GnRH-II-promoted motility of human decidual endometrial stromal cells, indicating the possible roles of EGF and GnRH-II in embryo implantation and early pregnancy. After obtaining informed consent, we obtained human decidual endometrial stromal cells from decidual tissues from normal pregnancies at 6 to 12 weeks of gestation in healthy women undergoing suction dilation and curettage. Cell motility was evaluated with invasion and migration assays. The mechanisms of EGF and GnRH-II were performed using real-time PCR and immunoblot analysis. The results showed that human decidual tissue and stromal cells expressed the EGF and GnRH-I receptors. GnRH-II-mediated cell motility was enhanced by EGF and was suppressed by the knockdown of the endogenous GnRH-I receptor and EGF receptor with siRNA, revealing that GnRH-II promoted the cell motility of human decidual endometrial stromal cells through the GnRH-I receptor and the activation of Twist and N-cadherin signaling. This new concept regarding the coaction of EGF- and GnRH-promoted cell motility suggests that EGF and GnRH-II potentially affect embryo implantation and the decidual programming of human pregnancy.

Precision Targeting of Endogenous Epidermal Growth Factor Receptor (EGFR) by Structurally Aligned Dual-Modifier Labeling.

Covalent modification of endogenous proteins by chemical probes is used for proteome-wide profiling of cellular protein function and drug discovery. However, probe selectivity in the complex cellular environment is a challenge, and new probes with better target selectivity are continuously needed. On the basis of the success of monocovalent activity-based and reactivity-based probes, an approach of structurally aligned dual-modifier labeling (SADL) was investigated here on its potential in improving target precision. Two reactive groups, based on the acrylamide and NHS ester chemistry, were linked with structural alignment to be under the same anilinoquinazoline ligand-directive for targeting the epidermal growth factor receptor (EGFR) protein kinase as the model system for proteome-wide profiling. The SADL approach was compared with its monocovalent precursors in a label-free MaxLFQ workflow using MDA-MB-468 triple negative breast cancer cells. The dual-modifier probe consistently showed labeling of EGFR with improved precision over both monocovalent precursors under various controls. The workflow also labeled endogenous USP34 and PKMYT1 with high selectivity. Precision labeling with two covalent modifiers under a common ligand directive may broaden protein identification opportunities in the native environment to complement genetic and antibody-based approaches for elucidating biological or disease mechanisms, as well as accelerating drug target discovery.

EGF Downregulates Presynaptic Maturation and Suppresses Synapse Formation In Vitro and In Vivo.

Neuronal differentiation, maturation, and synapse formation are regulated by various growth factors. Here we show that epidermal growth factor (EGF) negatively regulates presynaptic maturation and synapse formation. In cortical neurons, EGF maintained axon elongation and reduced the sizes of growth cones in culture. Furthermore, EGF decreased the levels of presynaptic molecules and number of presynaptic puncta, suggesting that EGF inhibits neuronal maturation. The reduction of synaptic sites is confirmed by the decreased frequencies of miniature EPSCs. In vivo analysis revealed that while peripherally administrated EGF decreased the levels of presynaptic molecules and numbers of synaptophysin-positive puncta in the prefrontal cortices of neonatal rats, EGF receptor inhibitors upregulated these indexes, suggesting that endogenous EGF receptor ligands suppress presynaptic maturation. Electron microscopy further revealed that EGF decreased the numbers, but not the sizes, of synaptic structures in vivo. These findings suggest that endogenous EGF and/or other EGF receptor ligands negatively modulates presynaptic maturation and synapse formation.

VEGF Upregulates EGFR Expression to Stimulate Chemotactic Behaviors in the rMC-1 Model of Müller Glia.

Progressive vision loss in adults has become increasingly prevalent worldwide due to retinopathies associated with aging, genetics, and epigenetic factors that damage the retinal microvasculature. Insufficient supply of oxygen and/or nutrients upregulates factors such as vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), which can induce abnormal angiogenesis and damage the structural arrangement of the retinal blood barrier (BRB). Müller glia (MG) regulate the diffusion of essential compounds across the BRB and respond to retinal insults via reactive gliosis, which includes cell hypertrophy, migration, and/or proliferation near areas of elevated VEGF concentration. Increasing concentrations of exogenous VEGF, upregulated by retinal pigmented epithelium cells, and endogenous epidermal growth factor receptor (EGF-R) stimulation in MG, implicated in MG proliferative and migratory behavior, often lead to progressive and permanent vision loss. Our project examined the chemotactic responses of the rMC-1 cell line, a mammalian MG model, toward VEGF and EGF signaling fields in transwell assays, and within respective concentration gradient fields produced in the glia line (gLL) microfluidic system previously described by our group. rMC-1 receptor expression in defined ligand fields was also evaluated using quantitative polymerase chain reaction (qPCR) and immunocytochemical staining. Results illustrate dramatic increases in rMC-1 chemotactic responses towards EGF gradient fields after pre-treatment with VEGF. In addition, qPCR illustrated significant upregulation of EGF-R upon VEGF pre-treatment, which was higher than that induced by its cognate ligand, EGF. These results suggest interplay of molecular pathways between VEGF and EGF-R that have remained understudied in MG but are significant to the development of effective anti-VEGF treatments needed for a variety of retinopathies.

Interaction of epidermal growth factor with COX-2 products and peroxisome proliferator-activated receptor-γ system in experimental rat Barrett's esophagus.

Mixed acidic-alkaline refluxate is a major pathogenic factor in chronic esophagitis progressing to Barrett's esophagus (BE). We hypothesized that epidermal growth factor (EGF) can interact with COX-2 and peroxisome proliferator-activated receptor-γ (PPARγ) in rats surgically prepared with esophagogastroduodenal anastomosis (EGDA) with healthy or removed salivary glands to deplete salivary EGF. EGDA rats were treated with 1) vehicle, 2) EGF or PPARγ agonist pioglitazone with or without EGFR kinase inhibitor tyrphostin A46, EGF or PPARγ antagonist GW9662 respectively, 3) ranitidine or pantoprazole, and 4) the selective COX-2 inhibitor celecoxib combined with pioglitazone. At 3 mo, the esophageal damage and the esophageal blood flow (EBF) were determined, the mucosal expression of EGF, EGFR, COX-2, TNFα, and PPARγ mRNA and phospho-EGFR/EGFR protein was analyzed. All EGDA rats developed chronic esophagitis, esophageal ulcerations, and intestinal metaplasia followed by a fall in the EBF, an increase in the plasma of IL-1β, TNFα, and mucosal PGE2 content, the overexpression of COX-2-, and EGF-EGFR mRNAs, and proteins, and these effects were aggravated by EGF and attenuated by pioglitazone. The rise in EGF and COX-2 mRNA was inhibited by pioglitazone but reversed by pioglitazone cotreated with GW9662. We conclude that 1) EGF can interact with PG/COX-2 and the PPARγ system in the mechanism of chronic esophagitis; 2) the deleterious effect of EGF involves an impairment of EBF and the overexpression of COX-2 and EGFR, and 3) agonists of PPARγ and inhibitors of EGFR may be useful in the treatment of chronic esophagitis progressing to BE.NEW & NOTEWORTHY Rats with EGDA exhibited chronic esophagitis accompanied by a fall in EBF and an increase in mucosal expression of mRNAs for EGF, COX-2, and TNFα, and these effects were exacerbated by exogenous EGF and reduced by removal of a major source of endogenous EGF with salivectomy or concurrent treatment with tyrphostin A46 or pioglitazone combined with EGF. Beneficial effects of salivectomy in an experimental model of BE were counteracted by PPARγ antagonist, whereas selective COX-2 inhibitor celecoxib synergistically with pioglitazone reduced severity of esophageal damage and protected esophageal mucosa from reflux. We propose the cross talk among EGF/EGFR, PG/COX-2, and proinflammatory cytokines with PPARγ pathway in the mechanism of pathogenesis of chronic esophagitis progressing to BE and EAC.

MICAL-L2 potentiates Cdc42-dependent EGFR stability and promotes gastric cancer cell migration.

Enhanced migration potential is a common characteristic of cancer cells induced by mechanisms that are incompletely defined. The present study was designed to investigate relationship of a new discovered cytoskeleton regulator MICAL‐L2 and the endogenous epidermal growth factor receptor (EGFR) signalling pathways in gastric cancer cell migration. Increased expression of MICAL‐L2 in gastric cancer cells up‐regulated EGFR protein level, accompanied by the increase of cell migration, whereas silencing MICAL‐L2 down‐regulated EGFR and inhibited cell migration. Expression of MICAL‐L2 was also shown positively correlated with the activation of HSP27/cytoskeleton and HSP27/β‐catenin signalling pathways that provide key mechanisms controlling cell migration. The up‐regulating effect of MICAL‐L2 on EGFR is mediated through a transcription‐independent mechanism that involves inhibiting EGFR protein degradation in lysosome. Further analysis indicated that Cdc42 activation contributed in maintaining the effect of MICAL‐L2 on EGFR stability. Furthermore analysis of clinic specimens revealed increased expression of MICAL‐L2 in carcinoma tissues and a positive correlation between MICAL‐L2 and EGFR expression levels. The above results indicate that MICAL‐L2 potentiates gastric cell migration via inhibiting EGFR degradation in lysosome via a Cdc42‐dependent manner that leads to the activation of EGFR/HSP27 signalling pathways.

Therapeutic effect of the epidermal growth factor on diabetic foot ulcer and the underlying mechanisms.

The present study aimed to explore the therapeutic effect and underlying mechanism of epidermal growth factor (EGF) on the wound healing of diabetic foot ulcers (DFU). A total of 48 rabbits with DFU were randomly divided into 2 groups, comprising the treatment and control groups. Full-thickness skin (10×10 mm) was excised from the thigh of each rabbit. The wounds in the treatment group were treated with 100 mg/l EGF once a day for 1 month. The control group received no treatment. At 20 days following treatment, new granulation tissues that formed beyond the edge of the wound were collected for subsequent analysis. Tissues from rabbits in the treatment group produced a greater number of fibroblasts, which exhibited a fibroblastic morphology when compared with those in the control group. In the treatment group, a larger number of these fibroblasts were observed as clusters, and there were numerous blood vessels when compared with the control group. The fibroblasts in the control group exhibited an irregular morphology, contained fewer organelles and the surrounding collagenous fibers were sparse. These fibroblasts also demonstrated a disordered arrangement and it was revealed that the wound healed at a slower rate compared with the treatment group. Endogenous EGF mRNA detection revealed that there was a significant difference (P<0.05) in the relative gray value of EGF mRNA between the treatment (103.27±4.27) and control (63.88±4.36) groups. In conclusion, EGF may accelerate the healing of DFU, and exogenous EGF treatment may upregulate the expression of EGF mRNA in newly generated tissues.

Imaging of protein-specific glycosylation by glycan metabolic tagging and in situ proximity ligation

Glycosylation is an important posttranslational modification, which regulates a number of critical biological processes including cell-cell recognition, signal transduction and disease progression. Probing the glycosylation status on a specific protein of interest enables an in-depth understanding of the role of glycosylation on protein structure and function. However, methods for monitoring protein-specific glycosylation are largely lacking. Here we describe a highly sensitive fluorescence imaging strategy to visualize the protein-specific glycosylation by combining glycan metabolic tagging and in situ proximity ligation (termed GPLA). We demonstrate the visualization of sialylation, fucosylation and GalNAcylation on several important membrane proteins. Notably, the high spatial resolution of this method allows subcellular localization of the glycosylated fraction of the proteins. We further show that our strategy can be applied to image the dimerization of endogenous epidermal growth factor receptor. Thus, our study provides a unique tool to monitor the protein-specific glycosylation in a dynamic cellular context.

Interdependency of EGF and GLP-2 Signaling in Attenuating Mucosal Atrophy in a Mouse Model of Parenteral Nutrition.

Background & AimsTotal parenteral nutrition (TPN), a crucial treatment for patients who cannot receive enteral nutrition, is associated with mucosal atrophy, barrier dysfunction, and infectious complications. Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) improve intestinal epithelial cell (IEC) responses and attenuate mucosal atrophy in several TPN models. However, it remains unclear whether these 2 factors use distinct or overlapping signaling pathways to improve IEC responses. We investigated the interaction of GLP-2 and EGF signaling in a mouse TPN model and in patients deprived of enteral nutrition.MethodsAdult C57BL/6J, IEC-Egfrknock out (KO) and IEC-pik3r1KO mice receiving TPN or enteral nutrition were treated with EGF or GLP-2 alone or in combination with reciprocal receptor inhibitors, GLP-2(3-33) or gefitinib. Jejunum was collected and mucosal atrophy and IEC responses were assessed by histologic, gene, and protein expression analyses. In patients undergoing planned looped ileostomies, fed and unfed ileum was analyzed.ResultsEnteral nutrient deprivation reduced endogenous EGF and GLP-2 signaling in mice and human beings. In the mouse TPN model, exogenous EGF or GLP-2 attenuated mucosal atrophy and restored IEC proliferation. The beneficial effects of EGF and GLP-2 were decreased upon Gefitinib treatment and in TPN-treated IEC-EgfrKO mice, showing epidermal growth factor–receptor dependency for these IEC responses. By contrast, in TPN-treated IEC-pi3kr1KO mice, the beneficial actions of EGF were lost, although GLP-2 still attenuated mucosal atrophy.ConclusionsUpon enteral nutrient deprivation, exogenous GLP-2 and EGF show strong interdependency for improving IEC responses. Understanding the differential requirements for phosphatidylinositol 3-kinase/phosphoAKT (Ser473) signaling may help improve future therapies to prevent mucosal atrophy.

The role of endogenous epidermal growth factor receptor ligands in mediating corneal epithelial homeostasis.

To provide a comprehensive study of the biological role and therapeutic potential of six endogenous epidermal growth factor receptor (EGFR) ligands in corneal epithelial homeostasis. Kinetic analysis and dose response curves were performed by using in vitro and in vivo wound-healing assays. Biochemical assays were used to determine receptor expression and activity. Human tears were collected and quantitatively analyzed by multianalyte profiling for endogenous EGFR ligands. Epidermal growth factor receptor ligands improved wound closure and activated EGFR, but betacellulin (BTC) was the most efficacious promoter of wound healing in vitro. In contrast, only epidermal growth factor (EGF) promoted wound healing in vivo. Human tears from 25 healthy individuals showed EGFR ligands at these average concentrations: EGF at 2053 ± 312.4 pg/mL, BTC at 207 ± 39.4 pg/mL, heparin-binding EGF at 44 ± 5.8 pg/mL, amphiregulin at 509 ± 28.8 pg/mL, transforming growth factor-α at 84 ± 19 pg/mL, and epiregulin at 52 ± 15 pg/mL. Under unwounded conditions, only EGF was present at concentrations near the ligand's Kd for the receptor, indicating it is the primary mediator of corneal epithelial homeostasis. Other ligands were present but at concentrations 11- to 7500-fold less their Kd, preventing significant ligand binding. Further, the high levels of EGF and its predicted binding preclude receptor occupancy by exogenous ligand and can explain the discrepancy between the in vitro and in vivo data. Therefore, therapeutic use of EGFR ligands may be unpredictable and impractical.

Fast Spatiotemporal Correlation Spectroscopy to Determine Protein Lateral Diffusion Laws in Live Cell Membranes

Fast Spatiotemporal Correlation Spectroscopy to Determine Protein Lateral Diffusion Laws in Live Cell Membranes

Single Molecule Diagnostic Method to Reveal Cancer-Related EGFR Signaling

Receptor tyrosine kinases (RTK) regulate cell fate such as differentiation, proliferation, survival and migration via recruiting SH2 containing downstream proteins. Dysregulated interaction between RTK and SH2 containing downstream protein incessantly activates MAPK or PI3K-Akt pathway which will cause a cancer in various organs. Real-time single-molecule co-immunoprecipitation (co-IP) is able to reveal quantitatively these protein-protein interactions using endogenous proteins at single molecule resolution. Here we develop a single molecule diagnostic method measuring endogenous epidermal growth factor receptor (EGFR) signaling extracted from cancer cell lines or lung tissues of cancer patients with eGFP labeled Grb2 as a probe, an adaptor protein containing SH2 involved in various EGFR signaling pathways using real-time single-molecule co-IP. This method distinguishes cell lines or tissues expressing highly activated EGFR. We also demonstrate that even cell lines have the same EGFR mutation, αA746-E750, protein interaction network can be altered, which is not revealed by genome sequencing based diagnosis. Our approach allows us to investigate signaling network of proteins at single molecule resolution. It also suggests a concept of a molecular diagnostic method at protein-protein interaction level.

Mitogen‐inducible gene 6 potentiates glucolipotoxicity‐induced pancreatic beta cell death

Destruction of the pancreatic insulin‐secreting beta cells, triggered by endoplasmic reticulum (ER) stress and glucolipotoxicity (GLT), is a common feature of type 2 diabetes. We previously identified that the endogenous epidermal growth factor inhibitor mitogen‐inducible gene 6 (Mig6) is induced by ER stress to promote beta cell apoptosis. In this study, we examined: 1) whether GLT also stimulates Mig6 expression, and 2) the cellular signaling cascades that interplay with Mig6 to facilitate GLT‐induced beta cell death. Using INS‐1‐derived 832/13 beta cells, we discovered that glucose, but not palmitate, stimulates Mig6 expression in a dose‐ and time‐dependent manner. Utilizing adenoviral‐mediated delivery of Mig6 to 832/13 cells, we found that Mig6 exacerbated GLT‐induced pro‐apoptotic JNK activation and caspase 3 activation. Furthermore, Mig6 might promote beta cell death by blocking cellular pro‐survival signaling pathways, as we have demonstrated that Mig6 overexpression inhibits the Akt and Erk signaling pathways in 832/13 cells. In conclusion, high glucose induces Mig6 expression in the pancreatic beta cells, which might facilitate GLT‐induced cell death. Supported by NIH DK078732(To PTF)

Endogenous cGMP-dependent protein kinase reverses EGF-induced MAPK/ERK signal transduction through phosphorylation of VASP at Ser239

In our previous study, we demonstrated that type II cGMP-dependent protein kinase (PKG II) was expressed at lower levels in different human cancer cell lines and that exogenous PKG II inhibited epidermal growth factor (EGF)-induced MAPK/ERK signaling. In order to investigate its functions further in this signaling pathway, it is necessary to elucidate whether endogenous PKG has the same effect or not. This study aimed to investigate the possible inhibitory effect of endogenous PKG activity on EGF-induced MAPK/ERK signal transduction in human lung cancer cells and its mechanism. Human small cell lung carcinoma cells (SCLCs) were treated with the PKG-selective cGMP analog 8-pCPT-cGMP to activate endogenous PKG, EGF and cGMP followed by EGF, respectively. The results showed that increased endogenous PKG activity inhibited the EGF-induced phosphorylation of the epidermal growth factor receptor (EGFR) and the binding between Sos1 and Grb2. In addition, EGF-triggered Ras activation was reversed by increased endogenous PKG activity. While the EGF-induced phosphorylation of MEK and ERK were inhibited by increased endogenous PKG activity, there was a significant increase of phosphorylated vasodilator-stimulated phosphoprotein (p-VASP) at Ser239. Furthermore, we investigated whether endogenous PKG exerted its effects on EGF-induced MAPK/ERK signaling through phosphorylation of VASP at Ser239. Downregulation of the levels of p-VASP Ser239 by point mutation blocked the effects of endogenous PKG on EGF-induced MAPK/ERK signal transduction. The data shown here suggest that endogenous PKG reverses the EGF-induced MAPK/ERK signaling pathway by phosphorylating VASP at Ser239.

Domain-Based Biosensor Assay to Screen for Epidermal Growth Factor Receptor Modulators in Live Cells

Traditional drug discovery efforts have resulted in the approval of a handful of receptor tyrosine kinase (RTK) inhibitors; however, their discovery relied solely on screening recombinant kinases, often with poor cellular activity outcome. The ability to screen RTKs in their natural environment is sought as an alternative approach. We have adapted a novel strategy utilizing a green fluorescent protein-labeled SRC homology 2 domain-based biosensor as a surrogate reporter of endogenous epidermal growth factor receptor (EGFR) activity in A549 cells. Upon activation of the receptor, EGFR function in live cells is measured by the number of green granules that form. Here we describe assay miniaturization and demonstrate specificity for EGFR through its chemical inhibition and RNAi-dependent knockdown resulting in complete abrogation of granule formation. Gefitinib and PD 153035 were identified as hits in a pilot screen. This approach allows for the identification of novel EGFR modulators in high-throughput formats for screening chemical and RNAi libraries.

Aromatic Aldehyde and Hydrazine Activated Peptide Coated Quantum Dots for Easy Bioconjugation and Live Cell Imaging

We present a robust scheme for preparation of semiconductor quantum dots (QDs) and cognate partners in a conjugation ready format. Our approach is based on bis-aryl hydrazone bond formation mediated by aromatic aldehyde and hydrazinonicotinate acetone hydrazone (HyNic) activated peptide coated quantum dots. We demonstrate controlled preparation of antibody--QD bioconjugates for specific targeting of endogenous epidermal growth factor receptors in breast cancer cells and for single QD tracking of transmembrane proteins via an extracellular epitope. The same approach was also used for optical mapping of RNA polymerases bound to combed genomic DNA in vitro.

Pressure activates epidermal growth factor receptor leading to the induction of iNOS via NFκB and STAT3 in human proximal tubule cells

Ureteral obstruction leads to increased pressure and inducible nitric oxide synthase (iNOS) expression. This study examined the involvement of epidermal growth factor (EGF) receptor (EGFR), nuclear factor-kappaB (NFkappaB), and signal transducers and activators of transcription 3 (STAT3) in iNOS induction in human proximal tubule (HKC-8) cells in response to pressure or EGF. HKC-8 cells were subjected to 60 mmHg pressure or treated with EGF for 0-36 h. iNOS was more rapidly induced in response to EGF than pressure. The addition of EGFR, NFkappaB, and STAT3 inhibitors significantly suppressed pressure- or EGF-stimulated iNOS mRNA and protein expression. Analysis of the activated states of EGFR, NFkappaB p65, and STAT3 after exposure to both stimuli demonstrated phosphorylation within 2.5 min. Anti-EGF antibody inhibited iNOS induction in pressurized HKC-8 cells, providing evidence that endogenous EGF mediates the response to pressure. In ureteral obstruction, when pressure is elevated, phosphorylated EGFR was detected in the apical surface of the renal tubules, validating the in vitro findings. These data indicate that EGFR, NFkappaB, and STAT3 are required for human iNOS gene induction in response to pressure or EGF, indicating a similar mechanism of activation.

Essential Role of c-Cbl in Amphiregulin-Induced Recycling and Signaling of the Endogenous Epidermal Growth Factor Receptor

The intracellular processing of the epidermal growth factor receptor (EGFR) induced by epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) has been studied meticulously, with the former resulting in EGFR degradation and the latter in EGFR recycling to the plasma membrane. However, little is known about how other EGF family growth factors affect the trafficking of the EGFR. Additionally, although both EGF and TGF-alpha have been shown to effectively induce initial c-Cbl (ubiquitin ligase)-mediated ubiquitination of the EGFR, limited information is available regarding the role of c-Cblin the trafficking and signaling of recycling EGFR. Thus, in this study, we investigated the roles of c-Cblin endogenous EGFR trafficking and signaling after stimulation with amphiregulin (AR). We demonstrated that a physiological concentration of AR induced recycling of the endogenous EGFR to the plasma membrane, which correlated closely with transient association of the EGFR with c-Cbl and transient EGFR ubiquitination. Most importantly, we used c-Cbl small interfering RNA (siRNA) duplexes and ac-Cbl dominant negative mutant to show that c-Cbl is critical for the efficient transition of the EGFR from early endosomes to a recycling pathway and that c-Cbl regulates the duration of extracellular signal regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) phosphorylation. These data support novel functions of c-Cbl in mediating recycling of EGF receptors to the plasma membrane, as well as in mediating the duration of activation (transient vs sustained) of ERK1/2 MAPK phosphorylation.

Egr‐1 and serum response factor are involved in growth factors‐ and serum‐mediated induction of E2‐EPF UCP expression that regulates the VHL‐HIF pathway

E2-EPF ubiquitin carrier protein (UCP) has been shown to be highly expressed in common human cancers and target von Hippel-Lindau (VHL) for proteosomal degradation in cells, thereby stabilizing hypoxia-inducible factor (HIF)-1alpha. Here, we investigated cellular factors that regulate the expression of UCP gene. Promoter deletion assay identified binding sites for early growth response-1 (Egr-1) and serum response factor (SRF) in the UCP promoter. Hepatocyte or epidermal growth factor (EGF), or phorbol 12-myristate 13-acetate induced UCP expression following early induction of Egr-1 expression in HeLa cells. Serum increased mRNA and protein levels of SRF and UCP in the cell. By electrophoretic mobility shift and chromatin immunoprecipitation assays, sequence-specific DNA-binding of Egr-1 and SRF to the UCP promoter was detected in nuclear extracts from HeLa cells treated with EGF and serum, respectively. Overexpression of Egr-1 or SRF increased UCP expression. RNA interference-mediated depletion of endogenous Egr-1 or SRF impaired EGF- or serum-mediated induction of UCP expression, which was required for cancer cell proliferation. Systemic delivery of EGF into mice also increased UCP expression following early induction of Egr-1 expression in mouse liver. The induced UCP expression by the growth factors or serum increased HIF-1alpha protein level under non-hypoxic conditions, suggesting that the Egr-1/SRF-UCP-VHL pathway is in part responsible for the increased HIF-1alpha protein level in vitro and in vivo. Thus, growth factors and serum induce expression of Egr-1 and SRF, respectively, which in turn induces UCP expression that positively regulates cancer cell growth.

Heparin-binding epidermal growth factor and Src family kinases in proliferation of renal epithelial cells

Our recent studies have shown that proliferation of renal proximal tubular cells (RPTC) in the absence of growth factors requires activation of the epidermal growth factor (EGF) receptor. We sought to identify the endogenous EGF receptor ligand and investigate the mechanism(s) by which RPTC proliferate in different models. RPTC expressed both pro- and cleaved forms of heparin-binding epidermal growth factor (HB-EGF) and several metalloproteinases (MMP-2, -3, -9, and ADAM10, ADAM17) that have been reported to cleave HB-EGF. Treatment of RPTC with CRM 197, an inhibitor of HB-EGF binding to the EGF receptor, or downregulation of HB-EGF with small interfering RNA inhibited RPTC proliferation following plating. Furthermore, GM6001 (pan-MMP inhibitor), tumor-necrosis factor protease inhibitor-1 (TAPI-1; MMP and ADAM17 inhibitor), and GW280264X (ADAM10 and -17 inhibitor), but not GI254023X (ADAM10 inhibitor), attenuated the proliferation after plating. Although EGF receptor activation is required for RPTC proliferation after oxidant injury, CRM197, GM6001, and TAPI-1 did not block this response. In contrast, inhibition of Src with PP1 blocked EGF receptor activation and RPTC proliferation after oxidant injury. In addition, PP1 treatment attenuated HB-EGF-enhanced RPTC proliferation. We suggest that RPTC proliferation after plating is mediated by HB-EGF produced through an autocrine/paracrine mechanism and RPTC proliferation following oxidant injury is mediated by Src without involvement of HB-EGF.