Ligand activation of overexpressed epidermal growth factor receptors transforms NIH 3T3 mouse fibroblasts.

The cell surface receptor for the mitogenic peptide epidermal growth factor (EGF) is involved in control of normal cell growth and may play a role in the genesis of human neoplasia such as squamous carcinoma and glioblastoma. Soft-agar growth and focus-formation experiments with NIH 3T3 mouse fibroblasts transfected with an expression plasmid demonstrated the ligand-dependent transforming potential of the human EGF receptor without structural alterations. Activation of overexpressed normal receptor alone appears to be sufficient for transformation of NIH 3T3 cells in vitro.     

In vivo activation of rat aortic platelet-derived growth factor and epidermal growth factor receptors by angiotensin II and hypertension

It is unclear whether the previous in vitro evidence of a link between angiotensin II (Ang II) and growth factor receptors can apply to the in vivo situation. In this study, we examined vascular platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptor activation in stroke-prone spontaneously hypertensive rats (SHRSP) and the role of Ang II. Tyrosyl phosphorylation of the growth factor receptors was determined by Western blot analysis coupled with immunoprecipitation. Tyrosyl phosphorylation of the aortic PDGF beta-receptor, but not the EGF receptor, was chronically increased in SHRSP with hypertension, compared with normotensive rats, being accompanied by increased extracellular signal-regulated kinase (ERK) activity. Treatment of SHRSP with ACE inhibitors (perindopril or enalapril) significantly reduced aortic PDGF beta-receptor tyrosyl phosphorylation and ERK activity, whereas treatment with hydralazine failed to reduce these activities. Therefore, these aortic changes in SHRSP were mediated by Ang II in response to vascular ACE. Ang II was infused into rats to examine the effects on aortic growth factor receptors. Chronic Ang II infusion, via the angiotensin type 1 receptor, significantly increased activation of the aortic PDGF beta-receptor but not the EGF receptor. Thus, the aortic PDGF beta-receptor, activated by ACE-mediated Ang II, seems to be responsible for vascular remodeling in hypertensive rats.     

Expression of human epidermal growth factor precursor cDNA in transfected mouse NIH 3T3 cells.

Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 microM ZnCl2. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is approximately 150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with 125I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself.     

Molecular and mechanical synergy: cross-talk between integrins and growth factor receptors

The full repertoire of molecules and mechanisms which lead to cardiac hypertrophy are poorly understood. Many studies over the last several decades have shown how various growth factors are involved in the hypertrophic response. It has also been intuitively obvious that mechanical mechanisms which impose hemodynamic loads on the working myocardium must also be involved in this process. Integrins are cell adhesion receptors that are potent bi-directional signaling molecules. They are cellular mechanoreceptors in many cells and are clearly one of the molecules which orchestrate mechano-biochemical coupling in the heart. In recent years they too have been shown to be involved in the hypertrophic response pathway. This review will detail background information on integrins in general, discuss integrins in the myocardium and illustrate how integrin and growth factor signaling pathways might combinatorially function in the heart.     

Functional cross-talk between receptors for peptide and steroid hormones

Communication between a cell surface peptide hormone receptor and an intracellular steroid hormone receptor can take various routes, as dictated by the physiology of a particular cell type. There is increasing evidence for a novel route which requires that a peptide hormone receptor pathway converge on a steroid hormone receptor, leading to its activation. One consequence of such a process can be signal amplification for the peptide hormone receptor agonist. This is exemplified by the self-potentiating action of GnRH, which is a critical component in events leading to a surge in LH secretion and ovulation. One signaling pathway stimulated by the GnRH receptor may entail a phosphorylation cascade resulting in progesterone-independent modulation of progesterone receptor activity.     

The multi-faceted cross-talk between the insulin and angiotensin II signaling systems

Insulin and angiotensin II are hormones that play pivotal roles in the control of two vital and closely related systems, the metabolic and the circulatory systems, respectively. A failure in the proper action of each of these hormones results, to a variable degree, in the development of two highly prevalent and commonly overlapping diseases-diabetes mellitus and hypertension. In recent years, a series of studies has revealed a tight connection between the signal transduction pathways that mediate insulin and angiotensin II actions in target tissues. This molecular cross-talk occurs at multiple levels and plays an important role in phenomena that range from the action of anti-hypertensive drugs to cardiac hypertrophy and energy acquisition by the heart. At the extracellular level, the angiotensin-converting enzyme controls angiotensin II synthesis but also interferes with insulin signaling through the proper regulation of angiotensin II and through the accumulation of bradykinin. At an early intracellular level, angiotensin II, acting through JAK-2/IRS-1/PI3-kinase, JNK and ERK, may induce the serine phosphorylation and inhibition of key elements of the insulin-signaling pathway. Finally, by inducing the expression of the regulatory protein SOCS-3, angiotensin II may impose a late control on the insulin signal. This review will focus on the main advances obtained in this field and will discuss the implications of this molecular cross-talk in the common clinical association between diabetes mellitus and hypertension.     

Nuclear localization of epidermal growth factor and epidermal growth factor receptors in human thyroid tissues.

Epidermal growth factor (EGF) has widespread growth effects, and in some tissues proliferation is associated with the nuclear localization of EGF and epidermal growth factor receptor (EGFR). In the thyroid, EGF promotes growth but differs from thyrotropin (TSH) in inhibiting rather than stimulating functional parameters. We have therefore studied the occurrence and cellular distribution of EGF and EGFR in normal thyroid, in Graves' disease, where growth is mediated through the thyrotropin receptor (TSHR), and in a variety of human thyroid tumors. In the normal gland the staining was variable, but largely cytoplasmic, for both EGF and EGFR. In Graves' disease there was strong cytoplasmic staining for both EGF and EGFR, with frequent positive nuclei. Nuclear positivity for EGF and particularly for EGFR was also a feature of both follicular adenomas and follicular carcinomas. Interestingly, nuclear staining was almost absent in papillary carcinomas. These findings document for the first time the presence of nuclear EGF and EGFR in thyroid. Their predominant occurrence in tissues with increased growth (Graves' disease, follicular adenoma, and carcinoma) may indicate that nuclear EGF and EGFR play a role in growth regulation in these conditions. The absence of nuclear EGF and EGFR in papillary carcinomas would suggest that the role played by EGF in growth control differs between papillary carcinoma and follicular adenomas/carcinomas of the thyroid.     

Colchicine inhibits epidermal growth factor degradation in 3T3 cells.

Colchicine (2 microM) did not affect the initial rate of association of 125I-labeled epidermal growth factor (125I-EGF) to Swiss 3T3 cells but continued incubation (up to 24 hr) led to an increase in cell-associated radioactivity. The effect is also produced by Colcemid, vinblastine, and podophyllotoxin but not by lumicolchicine. Disruption of microtubules with colchicine does not alter the rate of "down regulation" of EGF receptors, suggesting the binding and internalization of the factor proceed unchanged. However, colchicine markedly decreases the rate of appearance of acid-soluble radioactivity from cells either incubated continuously with 125I-EGF for 24 hr or exposed to the radioactive peptide for only 1 or 3 hr. The results indicate that colchicine decreases the rate of degradation of internalized 125I-EGF. Because antitubulin agents enhance the mitogenic effect of EGF our results suggest that peptide degradation can be dissociated from the long-term biological effect.     

Caveolin-1 negatively regulates a metalloprotease-dependent epidermal growth factor receptor transactivation by angiotensin II

A metalloprotease, ADAM17, mediates the generation of mature ligands for the epidermal growth factor receptor (EGFR). This is the key signaling step by which angiotensin II (AngII) induces EGFR transactivation leading to hypertrophy and migration of vascular smooth muscle cells (VSMCs). However, the regulatory mechanism of ADAM17 activity remains largely unclear. Here we hypothesized that caveolin-1 (Cav1), the major structural protein of a caveolae, a membrane microdomain, is involved in the regulation of ADAM17. In cultured VSMCs, infection of adenovirus encoding Cav1 markedly inhibited AngII-induced EGFR ligand shedding, EGFR transactivation, ERK activation, hypertrophy and migration, but not intracellular Ca²⁺ elevation. Methyl-β-cyclodextrin and filipin, reagents that disrupt raft structure, both stimulated an EGFR ligand shedding and EGFR transactivation in VSMCs. In addition, non-detergent sucrose gradient membrane fractionations revealed that ADAM17 cofractionated with Cav1 in lipid rafts. These results suggest that lipid rafts and perhaps caveolae provide a negative regulatory environment for EGFR transactivation linked to vascular remodeling induced by AngII. These novel findings may provide important information to target cardiovascular diseases under the enhanced renin angiotensin system.     

Increased epidermal growth factor receptors in gastric carcinomas

The epidermal growth factor and the homologous alpha-tumor growth factor are mitogenic polypeptides that act by binding to the epidermal growth factor receptor. The present study investigated whether increased production of epidermal growth factor/alpha-tumor growth factor or increased density of epidermal growth factor receptors may occur in gastric carcinomas as compared with normal mucosa from the same individuals. Epidermal growth factor receptors were measurable by (125I)EGF-binding assays in 13 of 15 normal mucosas and in 15 of 15 carcinomas. The epidermal growth factor-binding capacity was significantly higher in carcinomas than in mucosa. A comparison of pairs of mucosa and carcinomas showed an increase of epidermal growth factor receptors in 9 of 15 carcinomas, no change in 3, and a decrease in 2 carcinomas. One mucinous adenocarcinoma contained extreme numbers of epidermal growth factor receptors (2445 fmol/mg protein) corresponding to a 320-fold increase over normal mucosa. Epidermal growth factor-like activity was increased in 2 of 22 carcinomas compared with mucosa. We conclude that relative overexpression of epidermal growth factor receptors occurs in a fraction of gastric carcinomas. Whether increased expression of epidermal growth factor receptors is associated with particular patterns of tumor progression needs to be investigated.     

Effector function of type II collagen-stimulated T cells from rheumatoid arthritis patients: cross-talk between T cells and synovial fibroblasts

OBJECTIVE: To investigate the effector function exerted by type II collagen (CII)-stimulated T cells on rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), and to determine their contribution to RA pathogenesis. METHODS: We used enzyme-linked immunosorbent assays to measure the levels of interleukin-15 (IL-15), tumor necrosis factor alpha (TNFalpha), and IL-18 production by FLS that were cocultured with antigen-activated T cells. Likewise, we analyzed the levels of interferon-gamma (IFN gamma) and IL-17 production by RA T cells coincubated with FLS. To investigate the cross-talk between CII-stimulated T cells and RA FLS, we examined the effect of using a transwell membrane to separate T cells and FLS in a culture chamber, as well as the effect of adding an antibody to block CD40 ligation. RESULTS: The levels of IL-15, TNF alpha, IFN gamma, and IL-17 were all significantly increased in the serum of RA patients compared with normal control serum. Among the patients, the group with a stronger T cell proliferation response to CII showed higher levels of these inflammatory mediators. When coincubated with RA FLS, these T cells induced the production of IL-15, TNF alpha, and IL-18 by FLS with an intensity that increased in proportion to the duration of CII stimulation. T cells, in turn, responded to FLS stimulation by secreting higher amounts of IL-17 and IFN gamma in coculture. Interestingly, T cells that were activated by CII for longer periods of time showed stronger induction of these cytokines. The cross-talk between T cells and FLS appeared to require direct cell-cell contact as well as CD40 ligation, at least in part. CONCLUSION: Through repeated stimulation by CII, RA synovial T cells became trained effector cells that induced the production of proinflammatory mediators by FLS, while in the process the T cells becoming more sensitized to the activation signal from FLS.     

VEGF induces S1P1 receptors in endothelial cells: Implications for cross-talk between sphingolipid and growth factor receptors

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that binds to S1P1 (EDG-1) receptors and activates the endothelial isoform of NO synthase (eNOS). S1P and the polypeptide growth factor vascular endothelial growth factor (VEGF) act independently to modulate angiogenesis and activate eNOS. In these studies, we explored the cross-talk between S1P and VEGF signaling pathways. When cultured bovine aortic endothelial cells were treated with VEGF (10 ng/ml), the expression of S1P1 protein and mRNA increased by approximately 4-fold. S1P1 up-regulation by VEGF was seen within 30 min of VEGF addition and reached a maximum after 1.5 h. By contrast, expression of neither bradykinin B2 receptors nor the scaffolding protein caveolin-1 was altered by VEGF treatment. The EC50 for VEGF-promoted induction of S1P1 expression was approximately 2 ng/ml, within its physiological concentration range. S1P1 induction by VEGF was attenuated by the tyrosine kinase inhibitor genistein and by the PKC inhibitor calphostin C. Preincubation of bovine aortic endothelial cells with VEGF (10 ng/ml for 90 min) markedly enhanced subsequent S1P-dependent eNOS activation. VEGF pretreatment of cultured endothelial cells also markedly potentiated S1P-promoted eNOS phosphorylation at Ser-1179, as well as S1P-mediated activation of kinase Akt. In isolated rat arteries, VEGF pretreatment markedly potentiated S1P-mediated vasorelaxation and eNOS Ser-1179 phosphorylation. Taken together, these data indicate that VEGF specifically induces expression of S1P1 receptors, associated with enhanced intracellular signaling responses to S1P and the potentiation of S1P-mediated vasorelaxation. We suggest that VEGF acts to sensitize the vascular endothelium to the effects of lipid mediators by promoting the induction of S1P1 receptors, representing a potentially important point of cross-talk between receptor-regulated eNOS signaling pathways in the vasculature.     

Compartmentalization of signaling-competent epidermal growth factor receptors in endosomes

In this study, the preparation of detergent-resistant membranes (DRMs) and the immunoisolation of intracellular vesicles enriched in raft markers were used to investigate the effect of physiological doses of epidermal growth factor (EGF) in vivo on the compartmentalization and activation of EGF receptor (EGFR) in rat liver endosomes. Both of these techniques show that after EGF administration, a distinctive population of intracellular EGFR, which was characterized by a high level of tyrosine phosphorylation, accumulated in endosomes. EGFR recruited to early endosomes were more tyrosine phosphorylated than those from late endosomes. However, the level of tyrosine phosphorylation of EGFR in DRMs isolated from early and late endosomes was comparable, suggesting that EGFR in endosomal DRMs are more resistant to tyrosine dephosphorylation. In accordance with the higher level of Tyr phosphorylation, EGF induced an augmented recruitment of Grb2 and Shc to endosomal DRMs compared with whole endosomes. Furthermore, a proteomic analysis identified a selective increase of many alpha-subunits of heterotrimeric G proteins in endosomal DRMs in response to EGF. These observations suggest that a distinctive pool of endocytic EGFR, potentially competent for signaling, is actively trafficking through intracellular compartments with the characteristic of lipid rafts.     

表皮生长因子对类风湿关节炎滑膜细胞的作用

目的 观察表皮生长因子（ＥＧＦ）对类风湿关节炎（ＲＡ）滑膜细胞的作用和细胞中有丝分裂素激活的蛋白激酶（ＭＡＰＫ）的激活情况。方法 ３～５代体外培养的ＲＡ滑膜细胞,＾３Ｈ－ＴｄＲ掺入检测ＥＧＦ对细胞ＤＮＡ合成的影响;ＥＧＦ刺激后裂解细胞,收获蛋白,Ｗｅｓｔｅｒｎ ｂｌｏｔ检测ＭＡＰＫ的活化。结果 ＥＧＦ刺激组和对照组每分钟计数值差异有显著性（Ｐ〈０．００１）。ＥＧＦ能引起细胞内明显ＭＡＰＫ活化。结论     

Suppressor of cytokine signaling-3 Provides a novel interface in the cross-talk between angiotensin II and insulin signaling systems

Angiotensin II inhibits insulin-induced activation of phosphatidylinositol 3-kinase through a mechanism, at least in part, dependent on serine phosphorylation of the insulin receptor and insulin receptor substrates (IRS)-1/2. Recent evidence shows that suppressor of cytokine signaling-3 (SOCS-3) is induced by insulin and angiotensin II and participates in the negative control of further stimulation of each of these signaling systems independently. In the present study, we evaluated the interaction of angiotensin II-induced SOCS-3 with the insulin signaling pathway in the heart of living rats. A single iv dose of angiotensin II promotes a significant increase of SOCS-3 in heart, an effect that lasts up to 180 min. Once induced, SOCS-3 interacts with the insulin receptor, JAK-2, IRS-1, and IRS-2. The inhibition of SOCS-3 expression by a phosphorthioate-modified antisense oligonucleotide partially restores angiotensin II-induced inhibition of insulin-induced insulin receptor, IRS-1 and IRS-2 tyrosine phosphorylation, and IRS-1 and IRS-2 association with p85-phosphatidylinositol 3-kinase and [Ser473] phosphorylation of Akt. Moreover, the inhibition of SOCS-3 expression partially reverses angiotensin II-induced inhibition of insulin-stimulated glucose transporter-4 translocation to the cell membrane. These results are reproduced in isolated cardiomyocytes. Thus, SOCS-3 participates, as a late event, in the negative cross-talk between angiotensin II and insulin, producing an inhibitory effect on insulin-induced glucose transporter-4 translocation.     

An angiotensin II type 1 receptor mutant lacking epidermal growth factor receptor transactivation does not induce angiotensin II-mediated cardiac hypertrophy

We have shown previously that tyrosine 319 in a conserved YIPP motif in the C terminus of angiotensin II (Ang II) type 1 receptors (AT(1)Rs) is essential for transactivation of epidermal growth factor receptor (EGFR) in vitro. We hypothesized that the signaling mechanism mediated through the specific amino acid sequence in the G protein-coupled receptor plays an important role in mediating cardiac hypertrophy in vivo. Transgenic mice with cardiac-specific overexpression of wild-type AT(1)R (Tg-WT) and an AT(1)R with a mutation in the YIPP motif (Tg-Y319F) were studied. Tg-Y319F mice developed no significant cardiac hypertrophy, in contrast to the significant development of hypertrophy in Tg-WT mice. Expression of fetal-type genes, such as atrial natriuretic factor, was also significantly lower in Tg-Y319F than in Tg-WT mice. Infusion of Ang II caused an enhancement of hypertrophy in Tg-WT mice but failed to induce hypertrophy in Tg-Y319F mice. Left ventricular myocardium in Tg-Y319F mice developed significantly less apoptosis and fibrosis than that in Tg-WT mice. EGFR phosphorylation was significantly inhibited in Tg-Y319F mice, confirming that EGFR was not activated in Tg-Y319F mouse hearts. In contrast, activation/phosphorylation of protein kinase C, STAT3, extracellular signal-regulated kinase, and Akt and translocation of Galphaq/11 to the cytosolic fraction were maintained in Tg-Y319F hearts. Furthermore, a genetic cross between Tg-WT and transgenic mice with cardiac-specific overexpression of dominant negative EGFR mimicked the phenotype of Tg-Y319F mice. In conclusion, overexpression of AT(1)-Y319F in cardiac myocytes diminished EGFR transactivation and inhibited a pathological form of cardiac hypertrophy. The YIPP motif in the AT(1)R plays an important role in mediating cardiac hypertrophy in vivo.