β2-Adrenergic receptor-induced transactivation of epidermal growth factor receptor and platelet-derived growth factor receptor via Src kinase promotes rat cardiomyocyte survival

Chronic stimulation of the β-AR (adrenergic receptor) promotes apoptosis of cardiomyocytes, which is implicated in cardiac dysfunction. β1-AR and β2-AR are the main subtypes of β-AR that exert distinct effects on the survival of cardiomyocytes. To clarify the physiological roles of β1-AR and β2-AR in cardiomyocytes, the effects of β1-AR or β2-AR knockdown on the survival of H9c2 cardiomyocytes was investigated. Knockdown of β2-AR, but not β1-AR, suppressed the phosphorylation of EGFR (epidermal growth factor receptor) and PDGFR (platelet-derived growth factor receptor) induced by ISO (isoprenaline). The EGFR inhibitor, AG1478, attenuated ERK (extracellular-signal-regulated kinase) activation and partially decreased cell survival. Pretreatment with AG1296, a PDGFR inhibitor, abolished ISO-induced Akt (also known as protein kinase B) phosphorylation and led to a decrease in cell viability. In addition, the Src tyrosine kinase inhibitor, PP2, blocked ISO-mediated both Akt and ERK activation and heavily suppressed viability. Accordingly, in primary neonatal rat cardiomyocytes, the β2-AR inhibitor, but not the β1-AR inhibitor, abrogated the transactivation of EGFR and PDGFR, which was respectively related to Akt and ERK activation. The results show that β2-AR transactivates PDGFR and EGFR, thereby promoting survival of cardiomyocytes.     

Enhancement of epidermal growth factor receptor expression on glioma cells by recombinant tumor necrosis factor α

Summary Recombinant tumor necrosis factor (rTNF; optimal dose 1000 U/ml) significantly increased the density of epidermal growth factor receptor (EGF-R) in three of four glioma cell lines in culture as determined by binding analysis of anti-EGF-R monoclonal antibody (mAb) 425. Since enhancement of EGF-R expression by rTNF- was inhibited when cells were treated with the protein synthesis inhibitor cycloheximide, the effects of rTNF may be protein-synthesis-dependent. The dose of rTNF that was optimal for up-regulation of EGF-R on glioma cells did not inhibit the growth of these cells.¹²⁵I-labeled mAb 425 lysed glioma cells in culture following its internalization into the cells. After glioma cells had been treated with rTNF, the growth-inhibitory effects of the mAb were significantly enhanced, probably a reflection of the increase in EGF-R density on the tumor cell surfaces. The rTNF effects were specific to the EGF-R and did not affect unrelated glioma-associated antigens. In our previous clinical trials,¹²⁵I-labeled mAb 425 showed immunotherapeutic effects in glioma patients. The present study provides the basis for considerations of combined immunotherapy of glioma patients with¹²⁵I-labeled mAb 425 and rTNF.     

Engineered high-affinity affibody molecules targeting platelet-derived growth factor receptor β in vivo

Platelet-derived growth factor receptor (PDGFR) β is a marker of stromal pericytes and fibroblasts and represents an interesting target for both diagnosis and therapy of solid tumors. A receptor-specific imaging agent would be a useful tool for further understanding the prognostic role of this receptor in vivo. Affibody molecules constitute a class of very small binding proteins that are highly suited for in vivo imaging applications and that can be selected to specifically recognize a desired target protein. Here we describe the isolation of PDGFRβ-specific Affibody molecules with subnanomolar affinity. First-generation Affibody molecules were generated from a large naive library using phage display selection. Subsequently, sequences from binders having a desired selectivity profile and competing with the natural ligand for binding were used in the design of an affinity maturation library, which was created using a single partially randomized oligonucleotide. From this second-generation library, Affibody molecules with a 10-fold improvement in affinity (K_d = 0.4-0.5 nM) for human PDGFRβ and a 4-fold improvement in affinity (K_d = 6-7 nM) for murine PDGFRβ were isolated and characterized. Complete reversible folding after heating to 90 °C, as demonstrated by circular dichroism analysis, supports tolerance to labeling conditions for molecular imaging. The binders were highly specific, as verified by dot blot showing staining reactivity only with human and murine PDGFRβ, but not with human PDGFRα, or a panel of control proteins including 16 abundant human serum proteins. The final binder recognized the native conformation of PDGFRβ expressed in murine NIH-3T3 fibroblasts and human AU565 cells, and inhibited ligand-induced receptor phosphorylation in PDGFRβ-transfected porcine aortic endothelial cells. The PDGFRβ-specific Affibody molecule also accumulated around tumoral blood vessels in a model of spontaneous insulinoma, confirming a potential for in vivo targeting.     

Molecular cloning and developmental expression of rat fibroblast growth factor receptor 3

Fibroblast growth factors (FGFs) are involved in the control of a variety of biological functions including regulation and differentiation of various cell types. Furthermore, they play important roles in the processes of regeneration, angiogenesis, and chemotaxis. The family of FGF receptors (FGFRs) comprises four members, FGFR-1 to -4, which exist in several differentially expressed splice variants. Except for FGFR-3, primary structures and expression of the three other FGFRs have been described in the rat system. Although expression studies with heterologous probes of FGFR-3 from mice have been performed in the rat system, these analyses were limited and the complete set of receptors has not yet been revealed. To understand the developmental functions of FGFR-3, it is important to elucidate the expression pattern in embryos of different stages. In this study, we have isolated a cDNA of FGFR-3 from rat brain. Expression analyses by RT-PCR of adult rat revealed expression in several tissues, however, expression levels were highest in lung and brain. During embryonic development, FGFR-3 displays a diffuse expression in most tissues at embryonic day 14 (E14), as observed by in situ hybridization experiments. In E18 the expression pattern is more restricted, showing strong signals in spinal cord, dorsal root ganglia, cortex, chondrocytes, and endothelial cells. The temporal and spatial pattern of FGFR-3 expression suggests specific functions in several tissues during development.     

Hyaluronan facilitates transforming growth factor-β1-dependent proliferation via CD44 and epidermal growth factor receptor interaction

Fibroblast proliferation is an early feature of progressive tissue fibrosis and is largely regulated by the cytokine transforming growth factor-β1 (TGF-β1). In the oral mucosa, fibroblasts have a unique phenotype and demonstrate healing with no fibrosis/scarring. Our previous studies show that whereas dermal fibroblasts proliferate in response to TGF-β1, oral fibroblasts have an antiproliferative response to this cytokine. Hyaluronan (HA) was directly linked to this TGF-β1-dependent response. The aim of this study was to understand the underlying mechanism through which HA regulates TGF-β-dependent responses. Using patient-matched oral and dermal fibroblasts, we show that TGF-β1-dependent proliferation is mediated through the HA receptor CD44, whereas the TGF-β1-mediated antiproliferative response is CD44-independent. Furthermore, overexpression of HAS2 (HA synthase-2) in oral cells modifies their response, and they subsequently demonstrate a proliferative, CD44-dependent response to TGF-β1. We also show that epidermal growth factor (EGF) and its receptor (EGFR) are essential for TGF-β1/HA/CD44-dependent proliferation. Increased HA levels promote EGFR and CD44 coupling, potentiating signal transduction through the MAPK/ERK pathway. Thus, in a HA-rich environment, late ERK1/2 activation results from EGFR/CD44 coupling and leads to a proliferative response to TGF-β1. In comparison, in a non-HA-rich environment, only early ERK1/2 activation occurs, and this is associated with an antiproliferative response to TGF-β1. In summary, HA facilitates TGF-β1-dependent fibroblast proliferation through promoting interaction between CD44 and EGFR, which then promotes specific MAPK/ERK activation, inducing cellular proliferation.     

c-Kit-negative fibroblast-like cells express platelet-derived growth factor receptor α in the murine gastrointestinal musculature

Platelet-derived growth factor receptors (PDGFRs) belong to the same kinase group as c-Kit receptor tyrosine kinase that is specifically expressed in the interstitial cells of Cajal (ICC) in the gastrointestinal tract. In this study, we examined PDGFRα immunoreactivity in the murine gastrointestinal tract. PDGFRα-immunopositive (PDGFRα-ip) cells were observed in the musculature in all parts of the gastrointestinal tract. Although PDGFRα-ip cells were distinct from ICC and neurons, these cells were closely associated with intramuscular ICC and enteric nerve fibers. In the myenteric layer, PDGFRα-ip cells formed a cellular network with their ramified processes and encompassed myenteric ganglia. Numerous PDGFRα-ip cells were observed in the subserosal plane and showed a multipolar shape. The distribution pattern of the PDGFRα-ip cells in the ICC-deficient W ^v /W ^v mutant mice was the same as that in normal mice. PDGFRα-ip cells that showed intense immunoreactivity of SK3 potassium channel were considered to correspond to fibroblast-like cells or non-Cajal interstitial cells. Our observations suggest that PDGFRα-ip cells are basic cellular elements throughout the gastrointestinal musculature and are involved in the gastrointestinal functions.     

Heterobicyclic inhibitors of transforming growth factor beta receptor I (TGFβRI)

The TGFβ-TGFβR signaling pathway has been reported to play a protective role in the later stages of tumorigenesis via increasing immunosuppressive Treg cells and facilitating the epithelial to mesenchymal transition (EMT). Therefore, inhibition of TGFβR has the potential to enhance antitumor immunity. Herein we disclose the identification and optimization of novel heterobicyclic inhibitors of TGFβRI that demonstrate potent inhibition of SMAD phosphorylation. Application of structure-based drug design to the novel pyrrolotriazine chemotype resulted in improved binding affinity (Ki apparent?=?0.14?nM), long residence time (T_1/2?>?120?min) and significantly improved potency in the PSMAD cellular assay (IC₅₀?=?24?nM). Several analogs inhibited phosphorylation of SMAD both in vitro and in vivo. Additionally, inhibition of TGFβ-stimulated phospho-SMAD was observed in primary human T cells.     

Platelet-derived growth factor receptor α-positive cells in the tunica muscularis of human colon

An obstacle to understanding motor pathologies of the gastrointestinal (GI) tract is that the physiology of some of the cellular components of the gut wall is not understood. Morphologists identified fibroblast-like cells in the tunica muscularis many years ago, but little is known about these interstitial cells because of inadequate techniques to identify these cells. Recent findings have shown that fibroblast-like cells express platelet-derived growth factor receptor α (PDGFRα) in mice and that antibodies for these receptors can be used to label the cells. We used immunohistochemical techniques to study the phenotype and intercellular relationships of fibroblast-like cells in the human colon. Fibroblast-like cells are labelled specifically with antibodies to PDGFRα and widely distributed through the tunica muscularis of human colon. These cells form discrete networks in the region of the myenteric plexus and within the circular and longitudinal muscle layers. Platelet-derived growth factor receptor α(+) cells are distinct from c-Kit(+) interstitial cells of Cajal and closely associated with varicose processes of neurons expressing substance P (excitatory motor neurons) or neuronal nitric oxide synthase (nNOS) (inhibitory motor neurons). Platelet-derived growth factor receptor α(+) cells express small conductance Ca(2+)-activated K(+) channels (SK3), which are likely to mediate purinergic neural regulation of colonic muscles. Our data suggest that PDGFRα(+) cells may have an important role in transducing inputs from enteric motor neurons. This study identifies reagents and techniques that will allow investigation of this class of interstitial cells and help develop an understanding of the role of PDGFRα(+) cells in the human GI tract in health and disease.     

Epidermal growth factor, transforming growth factor-α, and epidermal growth factor receptor expression and localization in the canine endometrium during the estrous cycle and in bitches with pyometra

Gene expression and immunohistochemical localization of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and epidermal growth factor receptor (EGF-R) were compared between the endometrium of bitches (Canis familiaris) with pyometra accompanied by cystic endometrial hyperplasia (CEH) and that of healthy bitches at similar stages of the estrous cycle. In normal bitches, endometrial TGF-α mRNA levels were highest at proestrus and gradually decreased as the cycle progressed to anestrus. Epidermal growth factor receptor mRNA levels were not significantly affected by the stage of the estrous cycle. Epidermal growth factor mRNA levels were higher at Day 35 of diestrus than at other stages of the estrous cycle (P < 0.05). In bitches with pyometra, endometrial TGF-α and EGF-R mRNA levels did not differ significantly from those at diestrus in normal bitches, but EGF mRNA levels were lower than those at Day 35 of diestrus in normal bitches (P < 0.05). In normal bitches, positive immunohistochemical staining for TGF-α, EGF, and EGF-R was mainly present in the glandular and luminal epithelial cells of the endometrium. In contrast, in bitches with pyometra, immunoreactivity for EGF was clearly present in endometrial stromal cells. Inflammatory cells that had infiltrated the endometrial stroma stained strongly for TGF-α and EGF-R. Luminal and glandular epithelial cells also stained positive for EGF-R. In conclusion, expression of TGF-α by inflammatory cells and a low level of expression and differential localization of EGF may be involved in aberrant growth of endometrial glands and development of CEH.     

Identification of mannose receptor as receptor for hepatocyte growth factor β-chain: novel ligand-receptor pathway for enhancing macrophage phagocytosis

Hepatocyte growth factor (HGF), a heterodimer composed of the α-chain and β-chain, exerts multifunctional actions for tissue repair and homeostasis via its receptor, MET. HGF is cleaved by proteases secreted from inflammatory cells, and NK4 and β-chain remnant (HGF-β) are generated. Here, we provide evidence that HGF-β binds to a new receptor other than MET for promoting a host cell clearance system. By an affinity cross-linking, radiolabeled HGF-β was bound to liver non-parenchymal cells, particularly to Kupffer cells and sinusoidal endothelial cells, but not to parenchymal hepatocytes. The cross-linked complex was immunoprecipitated by anti-HGF antibody, but not anti-MET antibody, implying that HGF-β binds to non-parenchymal cells at a site distinct from MET. Mass spectrometric detection of the ligand receptor complex revealed that the binding site of HGF-β was the mannose receptor (MR). Actually, an ectopic expression of MR in COS-7 cells, which express no endogenous MR or MET, enabled HGF-β to bind these cells at a K(D) of 89 nM, demonstrating that MR is the new receptor for HGF-β. Interaction of HGF-β and MR was diminished by EGTA, and by an enzymatic digestion of HGF-β sugar chains, suggesting that MR may recognize the glycosylation site(s) of HGF-β in a Ca(2+)-dependent fashion. Notably, HGF-β, but not other MR ligands, enhanced the ingestion of latex beads, or of apoptotic neutrophils, by Kupffer cells, possibly via an F-actin-dependent pathway. Thus, the HGF-β·MR complex may provide a new pathway for the enhancement of cell clearance systems, which is associated with resolution of inflammation.     

Gefitinib induces epidermal growth factor receptor dimers which alters the interaction characteristics with ¹²⁵I-EGF

The tyrosine kinase inhibitor gefitinib inhibits growth in some tumor types by targeting the epidermal growth factor receptor (EGFR). Previous studies show that the affinity of the EGF-EGFR interaction varies between hosting cell line, and that gefitinib increases the affinity for some cell lines. In this paper, we investigate possible mechanisms behind these observations. Real-time interaction analysis in LigandTracer® Grey revealed that the HER2 dimerization preventing antibody pertuzumab clearly modified the binding of ¹²⁵I-EGF to EGFR on HER2 overexpressing SKOV3 cells in the presence of gefitinib. Pertuzumab did not affect the binding on A431 cells, which express low levels of HER2. Cross-linking measurements showed that gefitinib increased the amount of EGFR dimers 3.0–3.8 times in A431 cells in the absence of EGF. In EGF stimulated SKOV3 cells the amount of EGFR dimers increased 1.8–2.2 times by gefitinib, but this effect was cancelled by pertuzumab. Gefitinib treatment did not alter the number of EGFR or HER2 expressed in tumor cell lines A431, U343, SKOV3 and SKBR3. Real-time binding traces were further analyzed in a novel tool, Interaction Map, which deciphered the different components of the measured interaction and supports EGF binding to multiple binding sites. EGFR and HER2 expression affect the levels of EGFR monomers, homodimers and heterodimers and EGF binds to the various monomeric/dimeric forms of EGFR with unique binding properties. Taken together, we conclude that dimerization explains the varying affinity of EGF – EGFR in different cells, and we propose that gefitinib induces EGFR dimmers, which alters the interaction characteristics with ¹²⁵I-EGF.     

Developmental expression of transforming growth factor-α and epidermal growth factor receptor proteins in the human pancreas and digestive tract

This study was designed to localize transforming growth factor alpha (TGF-) and epidermal growth factor receptor (EGFR) expression in the developing human gastrointestinal tract and pancreas. Immunohistochemical techniques using specific antibodies against human TGF- and EGFR were performed on digestive tissues of fetuses from 9 to 10 to 24 weeks of gestation, children and adults. In fetuses, TGF- and EGFR proteins were expressed in all epithelial tissues studied with a good correlation and from an age as early as 9 to 10 weeks of gestation, except for TGF- in the esophagus. The strongest TGF- immunostaining was noted in the stomach and the proximal colon. Unexpectedly, immunoreactive gut endocrine cells were observed with the two antibodies used. Relatively numerous in fetuses, they decreased in number with age and were rare in adults particularly along the colon. Enteroglucagon-secreting cells were shown to express TGF- while some gastrin, somatostatin and pancreatic glucagon cells were immunostained with EGFR antibodies. The presence of TGF- and of its recetor in digestive tract epithelium and pancreatic tissues early in fetal life suggests a functional role for TGF- during the developmental process of the digestive system. We demonstrate that TGF- is also produced by endocrine cells and might have an additional mode of action other than paracrine, at least during fetal life.     

Sperm epidermal growth factor receptor (EGFR) mediates α7 acetylcholine receptor (AChR) activation to promote fertilization

To attain fertilization the spermatozoon binds to the egg zona pellucida (ZP) via sperm receptor(s) and undergoes an acrosome reaction (AR). Several sperm receptors have been described in the literature; however, the identity of this receptor is not yet certain. In this study, we suggest that the α7 nicotinic acetylcholine receptor (α7nAChR) might be a sperm receptor activated by ZP to induce epidermal growth factor receptor (EGFR)-mediated AR. We found that isolated ZP or α7 agonists induced the AR in sperm from WT but not α7-null spermatozoa, and the induced AR was inhibited by α7 or EGFR antagonists. Moreover, α7-null sperm showed very little binding to the egg, and microfluidic affinity in vitro assay clearly showed that α7nAChR, as well as EGFR, interacted with ZP3. Induction of EGFR activation and the AR by an α7 agonist was inhibited by a Src family kinase (SFK) inhibitor. In conclusion we suggest that activation of α7 by ZP leads to SFK-dependent EGFR activation, Ca(2+) influx, and the acrosome reaction.     

Selective deletion of hepatocyte platelet-derived growth factor receptor α and development of liver fibrosis in mice

Background

Platelet-derived growth factor receptor α (PDGFRα) expression is increased in activated hepatic stellate cells (HSCs) in cirrhotic liver, while normal hepatocytes express PDGFRα at a negligible level. However, cancerous hepatocytes may show upregulation of PDGFRα, and hepatocellular carcinoma is preceded by chronic liver injury. The role of PDGFRα in non-cancerous hepatocytes and liver fibrosis is unclear. We hypothesized that upon liver injury, PDGFRα in insulted hepatocytes contributes to liver fibrosis by facilitating intercellular crosstalk between hepatocytes and HSCs.

Methods

Hepatocytes were isolated from normal and thioacetamide (TAA)-induced cirrhotic livers for assessment of PDGFRα expression. Conditional knock-out (KO) C57BL/6 mice, in which PDGFRα was selectively deleted in hepatocytes, were generated. Liver fibrosis was induced by injecting TAA for 8?weeks. Hep3B cells were transfected with a small interfering RNA (siRNA) (PDGFRα or control) and co-cultured with LX2 cells.

Results

PDGFRα expression was increased in hepatocytes from fibrotic livers compared to normal livers. Conditional PDGFRα KO mice had attenuated TAA-induced liver fibrosis with decreased HSC activation and proliferation. Immunoblot analyses revealed decreased expression of phospho-p44/42 MAPK in TAA-treated KO mice; these mice also showed almost complete suppression of the upregulation of mouse double minute 2. Although KO mice exhibited increased expression of transforming growth factor (TGF)-β and Smad2/3, this was compensated for by increased expression of inhibitory Smad7. LX2 cells co-cultured with PDGFRα siRNA-infected Hep3B cells showed decreased PDGFRα, α smooth muscle actin, collagen α1(I), TGFβ, and Smad2/3 expression. LX2/PDGFRα-deleted hepatocyte co-culture medium showed decreased PDGF-BB and PDGF-CC levels.

Conclusions

Deletion of PDGFRα in hepatocytes attenuated the upregulation of PDGFRα in HSCs after TAA treatment, resulting in decreased liver fibrosis and HSC activation. This suggests that in the event of chronic liver injury, PDGFRα in hepatocytes plays an important role in liver fibrosis by affecting PDGFRα expression in HSCs.

     

Epidermal growth factor–stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis

Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits—membrane structures containing clathrin and other proteins—eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation.