EGFR Inhibitor AG1478 Research Articles

Neuropathological stage-dependent proteome mapping of the olfactory tract in Alzheimer's disease: From early olfactory-related omics signatures to computational repurposing of drug candidates.

Alzheimer's disease (AD) is the most common form of dementia, characterized by an early olfactory dysfunction, progressive memory loss, and behavioral deterioration. Albeit substantial progress has been made in characterizing AD-associated molecular and cellular events, there is an unmet clinical need for new therapies. In this study, olfactory tract proteotyping performed in controls and AD subjects (n = 17/group) showed a Braak stage-dependent proteostatic impairment accompanied by the progressive modulation of amyloid precursor protein and tau functional interactomes. To implement a computational repurposing of drug candidates with the capacity to reverse early AD-related olfactory omics signatures (OMSs), we generated a consensual OMSs database compiling differential omics datasets obtained by mass-spectrometry or RNA-sequencing derived from initial AD across the olfactory axis. Using the Connectivity Map-based drug repurposing approach, PKC, EGFR, Aurora kinase, Glycogen synthase kinase, and CDK inhibitors were the top pharmacologic classes capable to restore multiple OMSs, whereas compounds with targeted activity to inhibit PI3K, Insulin-like growth factor 1 (IGF-1), microtubules, and Polo-like kinase (PLK) represented a family of drugs with detrimental potential to induce olfactory AD-associated gene expression changes. To validate the potential therapeutic effects of the proposed drugs, in vitro assays were performed. These validation experiments revealed that pretreatment of human neuron-like SH-SY5Y cells with the EGFR inhibitor AG-1478 showed a neuroprotective effect against hydrogen peroxide-induced damage while the pretreatment with the Aurora kinase inhibitor Reversine reduced amyloid-beta (Aβ)-induced neurotoxicity. Taken together, our data pointed out that OMSs may be useful as substrates for drug repurposing to propose novel neuroprotective treatments against AD.

Optical spectra of EGFR inhibitor AG-1478 for benchmarking DFT functionals

Optical spectroscopy (UV–vis and fluorescence spectroscopy) is sensitive to the chemical environment and conformation of fluorophores and therefore, serves as an ideal probe for the conformation and solvent responses. Tyrosine kinase inhibitors (TKI) such as AG-1478 of epidermal growth factor receptor when containing a quinazolinamine scaffold are fluorophores. It is, however, very important to benchmark density functional theory (DFT) method against optical spectral measurements, when time-dependent DFT is applied. In this study, the performance of up to 22 DFT functionals is benchmarked with respect to the measured optical spectra of AG-1478 in dimethyl sulfoxide (DMSO) solvent. It is discovered when combined with the 6–311++G(d, p) basis set, there are top seven functionals; B3PW91, B3LYP, B3P86, PBE1PBE, APFD, HSEH1PBE, and N12SX DFT-VXC functionals are identified as the top performers. Becke’s three-parameter exchange functional (B3) tends to generate accurate optical spectra to form the best three functionals, B3LYP, B3PW91 and B3P86. Specifically, B3PW91 was recommended for studying the optical properties of 4-quinazolinamine TKIs, B3LYP was found to be excellent for absorption spectrum, while B3P86 was identified as the best for emission spectrum. Any further corrections to B3LYP, such as CAM-B3LYP, LC-B3LYP, and B3LYP-D3 result in larger errors in the optical spectra of AG-1478 in DMSO solvent. These best three (B3Vc) functionals are reliable tools for optical properties of the TKIs and therefore the design of new agents with larger Stokes shift for medical image applications. To obtain reliable optical spectra for this class of 4-quinazolinamine based TKIs, it is important to include the electron correlation energy.

Cryptosporidium parvum maintains intracellular survival by activating the host cellular EGFR-PI3K/Akt signaling pathway

Autophagy is a critical cellular mechanism in helping infected cells remove intracellular pathogens and is countered by pathogens maintaining intracellular survival by regulating autophagy through the manipulation of the host cellular signal transduction pathway. Cryptosporidium parvum is a zoonotic intracellular but extracytoplasmic protozoon that causes diarrhea in infants and young children worldwide. However, it is still unclear how Cryptosporidium adapts to intracellular survival. In the present study, we demonstrated that C. parvum could activate the EGFR-PI3K/Akt signaling pathway to promote intracellular survival in HCT-8 cells. The western blot results showed that C. parvum induced EGFR and Akt phosphorylation in HCT-8 cells. The EGFR inhibitor AG1478 decreased EGFR and Akt phosphorylation, and the PI3K inhibitor LY294002 impaired Akt phosphorylation induced by C. parvum in HCT-8 cells. Inhibition of EGFR or Akt decreased the number of intracellular parasites. Second, low-dose infection of C. parvum triggered complete autophagy and enhanced autophagic flux in HCT-8 cells. The expressions of mTOR and p62 were decreased, and the expressions of LC3 and Beclin1 were increased in C. parvum-infected HCT-8 cells. Transfection with siBeclin1 or siATG7 reduced LC3 accumulation, while lysosome inhibitor E64d+pepA increased LC3 accumulation induced by C. parvum in HCT-8 cells. Intracellular parasite proliferation was decreased when treated with autophagy inducer rapamycin, whereas autophagy inhibitor 3-MA, E64d+pep A, siBeclin1 or siATG7 increased intracellular parasites. Third, C. parvum inhibited autophagy killing to promote its own intracellular survival by activating EGFR-Akt signaling pathway. The EGFR inhibitor AG1478 enhanced autophagic flux, and Akt inhibitor IV increased LC3 accumulation and inhibited C. parvum proliferation in HCT-8 cells. Akt inhibitor IV-inhibited C. parvum proliferation was attenuated by E64d+pepA. In summary, C. parvum could maintain intracellular survival by inhibiting autophagy via EGFR-PI3K/Akt pathway. These results revealed a new mechanism for the interaction of C. parvum with host cells.

Blockade of the P2Y2 Receptor Attenuates Alcoholic Liver Inflammation by Targeting the EGFR-ERK1/2 Signaling Pathway.

PurposeIt is well known that inflammation plays a key role in complex pathological progressions of alcohol-associated liver disease (ALD). To date, effective therapy for ALD is lacking. P2Y2 receptor (P2Y2R), a G protein-coupled P2Y purinergic receptor, represents a novel pharmacological target in many inflammations.MethodsThe alcohol-associated liver injury and inflammation mouse model was established. The effect of P2Y2R on alcohol-induced liver injury and inflammation was evaluated using quantitative real-time PCR, Western blot and immunohistochemical assay. An alcohol-stimulated (100 mmol/L, for 24 h) AML-12 cell model was established. Different agonists, antagonists and P2Y2R siRNA were used to explore the possible mechanisms of P2Y2R.ResultsIn vivo, results showed that the hepatoprotective effect of P2Y2R blockade by significantly suppressed liver structural abnormalities and lipid infiltration, and decreased levels of ALT/AST and TNF-α/IL-1β in the high dosage group of suramin (20 mg/kg) compared to control diet (CD)-fed mice. At the same time, we found that alcohol feeding promoted the phosphorylation of EGFR and ERK1/2, both of which were effectively inhibited by suramin (20 mg/kg). In vitro, suramin or P2Y2R silencing effectively inhibited the phosphorylation of EGFR and ERK1/2, similar to the down-regulated effects of their corresponding inhibitors (EGFR inhibitor AG1478 and ERK1/2 inhibitor U0126) accompanied by reduced levels of TNF-α and IL-1β compared to alcohol-induced AML-12 cell. In addition, we found that silencing P2Y2R attenuated the apoptosis of hepatocyte.ConclusionOur findings suggest that P2Y2R regulates alcoholic liver inflammation by targeting the EGFR-ERK1/2 signaling pathway and plays an important role in hepatocyte apoptosis, which may provide new ideas for the development of methods to treat ALD.

PI3K inhibitor 3-MA promotes the antiproliferative activity of esomeprazole in gastric cancer cells by downregulating EGFR via the PI3K/FOXO3a pathway

Gastric cancer is a common gastrointestinal malignancy worldwide, with a high mortality rate and poor prognosis. Esomeprazole (ESO) has been shown to have anticancer activity by affecting cell growth and autophagy and its mechanism in gastric cancer cells is evident. The PI3K/AKT/FOXO3a pathway is central in cancers. 3-Methyladenine (3-MA), a dual inhibitor of PI3K and autophagy, plays a synergistic role in combination with antitumor agents. In this study, we assessed the role of ESO on the PI3K/AKT/FOXO3a pathway and the beneficial effects of ESO combined with 3-MA in gastric cancer cells. Cell viability, proliferation, invasion, migration, apoptosis, autophagy, and protein expression were detected by CCK-8, EdU, Transwell, flow cytometry, immunofluorescence assay, and western blot. ESO decreased cell viability in a concentration- and time-dependent manner and increased autophagy with upregulation of LC3II and P62. Additionally, ESO inhibited the proliferation, migration, and invasion and induced the apoptosis of gastric cancer cells in a concentration-dependent manner. ESO inhibited PI3K/AKT/FOXO3a signaling and EGFR and SKP2 expression concentration-dependent. 3-MA enhanced the antiproliferative activity of ESO and synergistically inhibited PI3K/FOXO3a signaling and the expression of EGFR but not SKP2. Furthermore, pretreatment with the EGFR inhibitor AG1478 enhanced the antiproliferative activity of ESO in gastric cancer cells. In conclusion, our results suggested that the PI3K inhibitor 3-MA promotes the antiproliferative activity of ESO in gastric cancer cells by synergistically downregulating EGFR via the PI3K/FOXO3a pathway.

Extracellular AGR2 activates neighboring fibroblasts through endocytosis and direct binding to β-catenin that requires AGR2 dimerization and adhesion domains

Anterior gradient 2 (AGR2) is often overexpressed in several types of cancer. AGR2 is cytoplasmic or secreted as an extracellular signal. Intracellular AGR2 properties and role in cancer have been well studied, but its extracellular function is largely unclear. It has been shown that extracellular AGR2 activates endothelial cells and fibroblasts in culture, but the mechanism of AGR2 signaling is not well elucidated. Here, we report that tumor secreted or externally added AGR2 translocates into cytoplasm by endocytosis, binds to β-catenin and further co-translocates to the nucleus in NIH3T3 fibroblasts. Externally added AGR2 also increased β-catenin expression, stability, and accumulation in the nucleus in both fibroblasts and cancer cells. External AGR2 rescued the expression of β-catenin, which was suppressed by EGFR inhibitor AG1478 indicating an alternative pathway to regulate β-catenin independent of EGFR signal. These effects were abolished when a monoclonal antibody against AGR2 was added to the experiments, confirming the effects are caused by AGR2 only. Putting together, our results show that extracellular AGR2 signaling pathway involves endocytosis mediated cellular translocation, direct binding and regulating β-catenin nuclear accumulation. It is also a target against tumor initiated AGR2 signaling to form and maintain tumor microenvironment.

Amphiregulin-EGFR Autocrine Signaling Mediates Neutrophil Elastase-Triggered Prostate Cancer Progression

Abstract Neutrophil elastase (NE) is a serine protease stored in neutrophil azurophilic granules. Growing evidence indicates that NE is intimately involved in the activities of proinflammatory cytokines / chemokines, growth factors, and cell surface receptors. These molecular regulations can modulate innate immune responses as well as directly promote cancer cell outgrowth. To date, however, little is known regarding the molecular mechanisms underlying the stimulatory properties of NE in cancer cells. Here we examine NE effects on prostate cells, demonstrating that NE triggers proliferative signals and cell migration in six prostate cell lines representing the spectrum of prostate cell malignancy, including normal prostatic epithelium, benign prostatic hypertrophy, and metastatic prostate cancer. Using ERK activation as a read-out, we show that NE promotes ERK phosphorylation in a dose dependent manner, and time course study further reveal a sustained ERK activation upon NE treatment. Western blot evaluation demonstrates strong EGFR expression in cell lines derived from normal and benign prostatic gland, and preferential expression in hormone resistant versus hormone responsive cells. In agreement with EGFR-dependent mitogenic signaling, activation of ERK is abrogated by siRNA-mediated EGFR knockdown, as well as by pretreatment of cells with irreversible EGFR inhibitor AG1478. Importantly, NE evokes cancer cell migration at a lower range of NE concentrations relative to nonneoplastic cells. In prostate cells, from a total of seven EGFR ligands, amphiregulin (AREG) is predominantly expressed, and the addition of NE results in the release of AREG. Moreover, AREG gene silencing by siRNA or inhibition of AREG biological activity by neutralizing antibody, prevents NE-induced ERK phosphorylation and cell migration. Together, our study reveals a distinct and essential role of AREG-EGFR signaling axis in NE-triggered prostatic cellular response.

SAT-138 Neutrophil Elastase Promotes Proliferative Signals in Prostate Cells Through EGFR and DDR1

Studies examining many different cancers have demonstrated that inflammation plays a critical role in tumor progression, in part through the release of proteases from stromal cells that function to either remodel the tumor microenvironment or to directly stimulate cancer cells to grow. One specific protease, neutrophil elastase (NE), has been shown to be a critical regulator of cancer growth in several mouse models. Accordingly, our laboratory demonstrated that NE, most likely from granulocytic myeloid-derived suppressor cells, potentially promotes prostate cancer progression in several different in-vivo and in-vitro models. To date, however, little is known regarding the mechanisms utilized by NE to promote tumor growth. It has been suggested that NE might cleave epidermal growth factor (EGF) or transforming growth factor-α from the cell surface to induce activation of EGFR/ERK signal transduction in an autocrine fashion. Alternatively, NE has been shown to enter into early endosomes to degrade insulin receptor substrate-I, ultimately resulting in phosphoinositol 3-kinase hyperactivity and subsequent tumor cell proliferation. Here we demonstrate that NE triggered proliferative signals in six prostate cell lines representing the spectrum of prostate cell differentiation, including normal prostatic epithelium, benign prostatic hypertrophy, and metastatic prostate cancer. Focusing on ERK signaling, we found that the stimulatory effect of NE on ERK phosphorylation was dose dependent and was abrogated by small interfering RNA induced EGFR knockdown, as well as by pretreatment of cells with irreversible EGFR inhibitor AG1478. Unlike EGF, however, NE-initiated EGFR phosphorylation was minimal. Thus, while EGFR appears to be critical for NE-induced ERK activation, perhaps it is not extensively activated directly by NE. Notably, discoidin domain receptor-1 (DDR1) was strongly expressed in normal prostate epithelium cells, but gradually decreased and had little expression in benign and metastatic prostate cancer cells sequentially. Nevertheless, similar to EGFR knockdown, silencing of DDR1 in all cell types inhibited NE mediated pERK upregulation, suggesting that DDR1 may also be important for NE-induced action. Together, our data suggest that NE, in concert with low level signals from the EGFR and DDR1, play an important role in promoting prostate cell proliferation both in normal and cancerous prostate epithelial cells.

Urolithin A, a Product of the Microbiome, Attenuates the Palmitate‐TLR4 Inflammatory Pathway in Renal Tubule Cells

Obesity is a major risk factor for cardiovascular disease and chronic renal injury. Tubulointerstial inflammation may reflect increased exposure of saturated fatty acids (sFA) to the renal epithelium and activation of the toll‐like receptor 4 (TLR4). Obesity‐induced inflammation may also facilitate gut microbiome dysbiosis and potentially alter the processing of exogenous polyphenolic compounds to metabolites with enhanced protective effects. Indeed, Urolithin A, the primary product of the microbiome metabolism of ellagitanins, promotes gut barrier function and attenuates colonic inflammation. However, the role of Urolithin A to attenuate tubular injury and inflammation has not been assessed. We propose that Urolithin A has anti‐inflammatory actions in rat proximal tubule cells (PTCs) chronically exposed to the sFA palmitate.PTCs (NRK‐52e cells) were exposed to palmitate [200 μM] for a 24 hours in the presence or absence of Urolithin A [50 μM], the TLR4 inhibitor TAK242 [1 μM], the EGFR inhibitor AG1478 [1 μM] and cSRC kinase inhibitor Dasatinib [10 μM] as both EGFR and cSRC may be integral to TLR4 activation. Cytokine expression in the PTCs was quantified by separate ELISAs.Palmitate exposure stimulated the robust release of PAI‐1 6‐fold [4.3 ± 0.3 vs. 24.2 ± 0.4 ng/ml; P<0.001, n=3], CCL2 150‐fold [0.04 ± 0.01 vs. 6.2 ± 0.4 ng/ml; P<0.001, n=3] and CCL5 10‐fold [23.4 ± 0.1 vs. 255 ± 11 pg/ml; P<0.001, n=3]. The TLR4 inhibitor abolished the increase in CCL2 and CCL5, and markedly attenuated PAI‐1 release [70%, P<0.01]. The EGFR inhibitor partially inhibited all three cytokines [40–50%, P<0.01] while the cSRC inhibitor abolished PAI‐1 and partially inhibited CCL5, but failed to inhibit CCL2 release. Urolithin A essentially abolished the increase in PAI‐1 and CCL5, and markedly inhibited CCL2 release to palmitate [70%, P<0.01].We conclude that Urolithin A may be renoprotective in obesity‐induced injury by targeting activation of TLR4 within the renal epithelium to attenuate the cytokine release. Moreover, a deficit in the microbiome‐dependent generation of Urolithin A may potentially promote renal inflammation.Support or Funding InformationChronic Disease Research Fund, AHA Transformative Grant 18TPA34170522; NIH R01HL146818, the Farley Hudson Foundation, the Hypertension & Vascular Research Center

EGFR inhibitor AG1478 blocks the formation of 3D structures mainly through ERK signaling pathway in Matrigel-induced 3D reconstruction of eccrine sweat gland-like structures.

EGFR signaling plays important roles in the development of eccrine sweat glands. We previously demonstrate that Matrigel induces eccrine sweat gland cells to reconstruct the three-dimensional (3D) structures of eccrine sweat glands, but the mechanisms are still unknown. In the study, eccrine sweat gland cells were cultured within a 3D Matrigel, and EGFR inhibitor AG1478, or MEK1/2 inhibitor U0126, were added to the medium respectively. The morphology of the 3D-reconstructed eccrine sweat gland-like structures was observed, the localization of phospho-EGFR was detected, and protein levels of EGFR, phospho-EGFR, phospho-JAK, phospho-AKT and phospho-ERK were examined. The results showed that cells treatment with AG1478 from Day 0 of 3D cultures blocked formation of spheroid-like structures. AG1478 administration caused reduced phospho-EGFR, concomitant with downregulation of phospho-ERK1/2, but not phospho-JAK or phospho-AKT. Phospho-EGFR and phospho-ERK were reduced, and only a small number of 3D-structures were formed following treatment with U0126. We conclude that EGFR plays important roles in Matrigel-induced 3D structures of eccrine sweat gland-like structures, and ERK1/2 signaling is responsible, at least in part, for the effect of EGFR.

Stimulation of neuroendocrine differentiation in prostate cancer cells by GHRH and its blockade by GHRH antagonists.

Prostate cancer is the second leading cause of cancer-related deaths among men in developed countries. Neuroendocrine prostate cancer, in particular, is associated with an aggressive phenotype and a poor prognosis. Neuroendocrine cells produce and secrete peptide hormones and growth factors in a paracrine/autocrine manner which promote the progression of the disease. Recent studies have demonstrated that extracellular vesicles or exosomes are released by prostate cancer cells, supporting the spread of prostate cancer. Hence, the aim of this study was to investigate the effect of growth hormone-releasing hormone (GHRH) on neuroendocrine differentiation (NED) in the androgen-dependent prostate cancer cell line LNCaP and the molecular mechanisms underlying these effects. GHRH induced an increase in the percentage of neurite-bearing cells and in the protein levels of Neuron-Specific Enolase. Both effects were blocked by the GHRH receptor antagonist MIA-690. In addition, pretreatment of these cells with the calcium chelator BAPTA, the EGFR inhibitor AG-1478 or the HER2 inhibitor AG-825 reduced the effectof GHRH, suggesting that the GHRH-induced stimulation of NED involves calcium channel activation and EGFR/HER2 transactivation. Finally, PC3-derived exosomes led to an increase in NED, cell proliferation and cell adhesion. Altogether, these findings suggest that GHRH antagonists should be considered for in the management of neuroendocrine prostate cancer.

SUN-479 Role of β-arrestin2 in ERK 1/2 Activation Mediated by Corticotropin-Releasing Factor Receptor Type 1

The Corticotropin-Releasing Factor (CRF) is a neuropeptide released by the hypothalamus in response to stress. CRF activates the CRF1 and CRF2 receptors, which are members of the G protein-coupled receptor (GPCR) superfamily. Although it has been shown that the CRF1 receptor activates ERK 1/2 cascade in different cell models [1] however, to date is not well understood the roles of upstream pathways associated with this effects. β-arrestin2 is an adapter protein that participates in the regulation of GPCRs and has important functions in cell signaling by mediating activation of multiple transductional pathways, including Src and MAPK [2]. In the present work, we investigated the role of β-arrestin2 in the activation of MAPK by induction of 100 nM CRF. We used mouse embryonic fibroblast cells that lack β-arrestin2 (MEF KO-β-arrestin2), transfected with the CRF1 receptor that lack β-arrestin2 (MEF KO-β-arrestin2) as model. Our data revealed that β-arrestin2 is involved in the activation of ERK 1/2 since we detected lower (P<0.05) phosphorylation of ERK 1/2 in the KO-β-arrestin2 cells after 5 min CRF of stimulationCRF. Using MEF cells from double-knockout mice, lacking β-arrestin1 and β-arrestin2 (MEF 2KO-β-arrestin1/2), we found that β-arrestin1 is not involved in ERK 1/2 activation induced by CRF because there was no change on phosphorylation of ERK 1/2 between MEF KO-β-arrestin2 and 2KO-β-arrestin1/2 cells. The pretreatment with the EGFR inhibitor AG1478 promoted a decreased (P<0.05) ERK 1/2 phosphorylation in control cells. Interestingly, no EGFR activation was detected in the KO-β-arrestin2 cells. These results indicate that β-arrestin2 is necessary for the CRF1 receptor-mediated EGFR transactivation mechanism that induces ERK 1/2 phosphorylation. We also found that the transactivation of EGFR by CRF stimulation is independent of metalloproteinase activity through the pretreatment with the metalloproteinase inhibitor GM6001. To determine the role of Src in the CRF-mediated ERK activation, we pretreated the cells with the Src-specific inhibitor PP2. We observed that PP2 promoted a significant decrease (P<0.05) in CRF-mediated ERK activation. We also found a reduction (P<0.05) in Src phosphorylation at Y416 in MEF KO-β-arrestin2 cells stimulated with 5 min of CRF, suggesting that β-arrestin2 is involved in Src activation. Together, our data demonstrate that β-arrestin2 is an important player during the CRF1-dependent activation of the ERK 1/2 pathway. Reference: [1]Hauger et al., Ann. N. Y. Acad. Sci. (2009) 1179: 120-143.[2]Luttrell et al., Proc. Natl. Acad. Sci. (2001) 98: 2449-2454Sources of Research Support: UC MEXUS-CONACYT grant for collaborative projects to JAO-R and RLH; CONACYT grant to JAO-R; Merit Review grant; MIRECC of VISN22, and CESMH from the Department of VA; NIH grant to RLH. GKP-M was supported by CONACYT fellowship grant.

Roles for HB-EGF in Mesenchymal Stromal Cell Proliferation and Differentiation During Skeletal Growth.

ABSTRACTHB‐EGF, a member of the EGF superfamily, plays important roles in development and tissue regeneration. However, its functions in skeletal stem cells and skeleton development and growth remain poorly understood. Here, we used the Cre/LoxP system to ablate or express HB‐EGF in Dermo1+ mesenchymal stromal cells and their progenies, including chondrocytes and osteoblast lineage cells, and bone marrow stromal cells (BMSCs). Dermo1‐Cre; HB‐EGFf/f mice only showed a modest increase in bone mass, whereas Dermo1‐HB‐EGF mice developed progressive chondrodysplasia, chondroma, osteoarthritis‐like joint defects, and loss of bone mass and density, which were alleviated by treatment with EGFR inhibitor AG1478. The cartilage defects were recapitulated in chondrocyte‐specific HB‐EGF overexpression (Col2‐HB‐EGF) mice with a lesser severity. Dermo1‐HB‐EGF mice showed an increase in proliferation but defects in differentiation of chondrocytes and osteoblasts. HB‐EGF promoted BMSC proliferation via the Akt1 and Erk pathways but inhibited BMSC differentiation via restraining Smad1/5/8 activation. However, Dermo1‐HB‐EGF mice showed normal osteoclastogenesis and bone resorption. These results reveal an important function of autocrine or paracrine HB‐EGF in mesenchymal stromal cell proliferation and differentiation and suggest that EGF signaling needs to be tightly controlled to maintain bone and articular cartilage integrity. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Abstract 100: Mechanistic Insight into Tannic Acid Mediated Angiotensin Type 1 Receptor and PCSK 9 Downregulation

Deregulated angiotensin type 1 receptor (AT1R) and low-density lipoprotein receptor (LDLR) are implicated in cardiovascular disorders. AT1R is over expressed in hypertension and LDLR is involved in lowering plasma LDL cholesterol. In this study we investigate the role of tannic acid (TA), a hydrolyzable polyphenol, on AT1R and LDLR in rat aortic smooth muscle cells (RASMC) and rat liver epithelial cells (RLEC) respectively. TA (10 μg/ml) treatment exhibited down-regulation of AT1R specific [3H] AngII binding compared to untreated control (3628.32 ± 289.42 vs. 7244.66 ± 40.26 DPM/mg protein, P &lt; 0.001). TA mediated AT1R downregulation was dose (0-40 μg/ml) and time dependent (0-24 hours) without significant change in receptor affinity (K d 1.302 ± 0.152 nM for TA treated vs. K d 1.391 ± 0.048 nM for untreated). Consistently, TA inhibited mRNA expression (TA 0.023± 0.0012 vs. control 0.051± 0.0045 mRNA fold). Furthermore, TA activated of ERK p42/44, which was inhibited by MEK inhibitor PD98059 (20μM) and an EGFR inhibitor AG1478 (5μM) suggesting a role for EGFR induced ERK p42/44 in AT1R downregulation. A similar effect was observed with EGF (0.5 μg/ml) alone (EGF; 3788.04 ± 206.51 vs. Control; 6549.22 ± 115.48 DPM/mg protein, P &lt; 0.001). LY294002 (10μM), an inhibitor of phosphoinositide 3-kinase (PI3K), inhibited both TA and EGF induced pERK and reversed AT1R downregulation suggesting that TA mediated AT1R downregulation is through EGFR-PI3K-ERK pathway. Consistent with the receptor downregulation significant inhibition of AngII induced intracellular calcium (TA 203.4 ± 7.79nM vs control 408.5 ± 5.33 nM) was observed. Under similar conditions proprotein convertase subtilisin/kexin type 9 (PCSK9), a crucial enzyme responsible for LDLR turn over, was decreased 2.8 fold at the protein level and mRNA levels (reduced 46.78± 2.3%) in RLEC. Alternatively, we observed 3.6 fold increased cell surface expression of LDLR and induced LDL-c uptake in human hepatocyte cells (2677 ± 26.39 untreated vs. 3894 ± 40.1 for TA treated). Our study provides a mean to control hypertension and hypercholesteremia associated cardiovascular disorders.

The epidermal growth factor receptor inhibitor AG1478 inhibits eosinophilic inflammation in upper airways

Mucus hypersecretion and eosinophil infiltration are important characteristics of eosinophilic inflammation in upper airways, such as allergic rhinitis and chronic rhinosinusitis with nasal polyp. EGFR transactivation induces mucus and inflammatory cytokine secretion from airway epithelial cells. However, the roles of EGFR in eosinophilic inflammation in upper airways are still unknown. The purpose of the study is to elucidate the effects of the EGFR inhibitor AG1478 on eosinophilic airway inflammation. AG1478 significantly inhibited thrombin-induced GM-CSF secretion from nasal epithelial cells and thrombin-induced secretion of eotaxin-1 and RANTES from nasal fibroblasts. Intranasal instillation of AG1478 inhibited OVA-induced goblet cell metaplasia, mucus production and eosinophil/neutrophil infiltration in rat nasal epithelium, as did intraperitoneal injection of AG1478. These results indicate that EGFR transactivation plays an important role in eosinophilic airway inflammation. Intranasal instillation of an EGFR inhibitor may be a new therapeutic approach for the treatment of intractable eosinophilic inflammation in upper airways.

Protein Disulfide Isomerase increases Nox1 activation through EGFR signaling in hypertension

We have previously shown that expression of protein disulfide isomerase (PDI) and Nox1 in resistance arteries correlates with the development of hypertension. Epidermal growth factor (EGF)-like ligands are increased in vascular disease and their levels correlate with higher levels of vascular Nox1. EGFR can be activated through metalloproteinase (MMP) mediated cleavage and release of EGF-ligands. Here, we sought to investigate the molecular mechanisms through which PDI regulates Nox1 expression in hypertension. Pro-and mature heparin bound (HB)-EGF ligands were increased in the aorta of 8-week-old spontaneously hypertensive rats (SHR) as compared to control (Wistar) rats. In contrast, the mature form of the ligand was lower in aortae, and increased in the plasma, of 12-week-old SHR. We next assessed if shedding of EGF ligands was responsible for the increase in Nox1 expression. Overexpression of PDI increased the expression of Nox1 in rat VSMCs and this effect was inhibited by the EGFR inhibitor AG1478, the MMP inhibitor TAP and the flavoenzyme inhibitor diphenylene iodonium (DPI). In contrast, overexpression of a PDI mutated in its 4 redox active cysteines (PDI mut) failed to increase Nox1 expression. We confirmed these observations following expression of a Nox1-promoter luciferase plasmid in mouse VSMCs. HB-EGF stimulated cells transfected with WT PDI showed an increase in Nox1 promoter activity as compared to cells transfected with PDI mut. Finally, cells deficient in Nox1 did not respond to HB-EGF when transfected with PDI WT, suggesting that Nox1 activation and PDI redox cysteines are essential for inducing the activation of EGFR. Altogether, our findings indicate that PDI increases the redox-mediated expression of Nox1 via EGFR signaling in hypertension.

432 The epidermal growth factor receptor is critical for the Staphylococcus aureus-mediated induction of human beta-defensin-3 in keratinocytes

Antimicrobial peptides (AMP) contribute to cutaneous defense due to their potent antimicrobial activity. A feature of many AMP expressed by keratinocytes is their inducibility by cytokines and microbial factors. There is increasing evidence that several skin infections may be associated with a dysregulation of AMP. Patients receiving anti-EGFR (epidermal growth factor receptor) therapy often suffer from cutaneous infection caused by Staphylococcus (S.) aureus. This is in line with recent studies indicating a pivotal role of the EGFR to mediate the expression of various AMP in keratinocytes. Human beta-defensin-3 (hBD-3) is an important AMP expressed by keratinocytes to control the growth of S. aureus. Since S. aureus is able to induce the expression of hBD-3 in keratinocytes we analyzed if the EGFR is involved in this induction. To this end we stimulated human primary keratinocytes with S. aureus in the presence of the anti-EGFR antibody cetuximab. Inactivation of the EGFR by cetuximab already resulted in a decreased constitutive hBD-3 gene expression as measured by real-time PCR. Simulation of the keratinocytes with living S. aureus or their culture supernatants resulted in an increased hBD-3 gene expression which was abrogated in the presence of cetuximab. Accordingly, the EGFR inhibitor AG1478 also inhibited the S. aureus-mediated hBD-3 induction. Induction of hBD-3 in keratinocytes treated with the EGFR ligand TGF-alpha confirmed the EGFR-dependent induction of hBD-3. Treatment of a 3D skin equivalent with living S. aureus also resulted in an increased expression of hBD-3. This S. aureus-mediated hBD-3 induction was decreased in skin equivalents treated with cetuximab. Together, these data further highlight the importance of the EGFR-pathway in cutaneous defense against S. aureus. Our data suggest that a failure of S. aureus-mediated hBD-3 induction in patients receiving anti-EGFR therapy may contribute to the increased S. aureus infection rate seen in these patients.

High glucose induces HGF-independent activation of Met receptor in human renal tubular epithelium

Context: The role of hepatocyte growth factor (HGF) in diabetic kidney damage remains controversial.Objective: To test the hypothesis that high glucose levels activate pathways related to HGF and its receptor Met and that this could participate in glucose-induced renal cell damage.Materials and methods: HK2 cells, a human proximal tubule epithelial cell line, were stimulated with high glucose for 48 hours. Levels of pMet/Met, pEGFR/EGFR, pSTAT3/STAT3, pAkt/Akt and pERK1/2/ERK1/2 were studied by immunoblotting. Absence of HGF was verified by qRT-PCR and ELISA.Results: High glucose level activated Met and its downstream pathways STAT3, Akt and ERK independently of HGF. High glucose induced an integrin ligand fibronectin. HGF-independent Met phosphorylation was prevented by inhibition of integrin α5β1, Met inhibitor crizotinib, Src inhibitors PP2 and SU5565, but not by EGFR inhibitor AG1478. High glucose increased the expression of TGFβ-1, CTGF and the tubular damage marker KIM-1 and increased apoptosis of HK2 cells, effects inhibited by crizotinib.Conclusion: High glucose activated Met receptor in HK2 cells independently of HGF, via induction of integrin α5β1 and downstream signaling. This mode of Met activation was associated with tubular cell damage and apoptosis and it may represent a novel pathogenic mechanism and a treatment target in diabetic nephropathy.

Migration of oral squamous cell carcinoma cells are induced by HGF/c-Met signalling via lamellipodia and filopodia formation.

The activation of receptor tyrosine kinases (RTKs) results in cellular effects including cell proliferation, survival, migration and invasion; RTKs also play an important role in tumourigenesis. It has been reported that EGFR signalling controls the migration of oral squamous cell carcinoma (OSCC) SAS and HSC3 cells but not of HSC4 cells, although the proliferation of HSC4 cells is regulated by EGF/EGFR. In the present study, we investigated the roles of EGFR and the c-Met signalling pathway in cell migration via filopodia and lamellipodia formation, which may be prerequisites for migration. To explore the role of c-Met in cell migration, we inhibited c-Met RTK activity using the c-Met inhibitor SU11274 and activated c-Met using hepatocyte growth factor (HGF) in three OSCC cell lines HSC4, SAS and Ca9-22 and investigated migration potency using a wound healing assay. We showed that inhibition of c-Met significantly suppressed, and activation of c-Met significantly promoted, the migration of OSCC cells. Additionally, the migration of SAS and Ca9-22 cells was inhibited by the EGFR inhibitors AG1478 and cetuximab and promoted by EGF treatment. Moreover, migration potency was correlated with lamellipodia formation. Furthermore, western blot analyses demonstrated that SU11274 decreased and HGF increased lamellipodin protein levels as well as phosphorylated c-Met levels. Collectively, we demonstrated that c-Met signalling induced lamellipodia formation by upregulating lamellipodin, thereby promoting the migration of OSCC cells.

Icariside II activates EGFR-Akt-Nrf2 signaling and protects osteoblasts from dexamethasone.

The potential effect of icariside II on dexamethasone-induced osteoblast cell damages was evaluated here. In MC3T3-E1 osteoblastic cells and the primary murine osteoblasts, co-treatment with icariside II dramatically attenuated dexamethasone- induced cell death and apoptosis. Icariside II activated Akt signaling, which is required for its actions in osteoblasts. Akt inhibitors (LY294002, perifosine and MK-2206) almost abolished icariside II-induced osteoblast cytoprotection against dexamethasone. Further studies showed that icariside II activated Nrf2 signaling, downstream of Akt, to inhibit dexamethasone-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary osteoblasts. On the other hand, Nrf2 shRNA knockdown inhibited icariside II-induced anti-dexamethasone cytoprotection in MC3T3-E1 cells. Finally, we showed that icariside II induced heparin-binding EGF (HB-EGF) production and EGFR trans-activation in MC3T3-E1 cells. EGFR inhibition, via anti-HB-EGF antibody, EGFR inhibitor AG1478 or EGFR shRNA knockdown, almost blocked icariside II-induced Akt-Nrf2 activation in MC3T3-E1 cells. Collectively, we conclude that icariside II activates EGFR-Akt-Nrf2 signaling and protects osteoblasts from dexamethasone. Icariside II might have translational value for the treatment of dexamethasone-associated osteoporosis/osteonecrosis.