EGF-induced EGF-receptor and MAP kinase phosphorylation in goat cumulus cells during in vitro maturation

EGF has been shown to influence meiotic maturation and development competence of oocyte in various mammalian species. We previously reported, in goat, that the EGF receptor (EGF-R) was present both on cumulus cells and oocytes. Here, EGF-induced signaling was investigated during the in vitro maturation process in goat cumulus-oocyte complexes (COCs). Cumulus cells and oocytes were subjected to Western immunoblotting analysis using anti-MAP kinase, anti-phosphotyrosine, anti-phospho MAP kinase, and anti-phospho EGF-R antibodies. We demonstrated that treatment with EGF during the in vitro maturation process induced rapid tyrosine phosphorylation of EGF-R in a time and concentration dependent manner in cumulus cells. A similar pattern of activation by phosphorylation was observed for MAP kinase upon EGF stimulation. AG 1478, an inhibitor of the EGF kinase, suppressed EGF-stimulated phosphorylation of EGF-R and also affected the MAP kinase activation. Treatment with the MEK inhibitor PD 98059 abolished EGF-induced MAP kinase activation. We did not observe oocyte EGF-R phosphorylation in our experiments during the in vitro maturation process. Our data indicate, in goat cumulus cells, that activation of EGF-R by EGF triggers signaling through the MAP kinase pathway during in vitro maturation. This supports the hypothesis that the major site of action for EGF, that regulates oocyte maturation, is the cumulus cell.     

Oxygen concentration regulates NO-dependent relaxation of aortic smooth muscles

Nitric oxide (NO) functions as an endothelium-derived relaxation factor and regulates vascular resistance. Recent studies in this laboratory (Arch. Biochem. Biophys. 323, 27–32, 1995) revealed that the lifetime of NO significantly increased at physiologically low levels of oxygen concentrations and, hence, this gaseous radical strongly inhibited mitochondrial electron transport for a fairly long duration at low oxygen concentrations. The present work describes the effect of oxygen concentration on NO-induced relaxation and guanylate cyclase (GC) activity of endothelium-denuded aorta of the rat. Both NO and 2,2′-hydroxynitrosohydrazono)bis-ethanamine (NOC18), an NO donor, induced the relaxa-tion of endothelium-denuded helical segments of rat aorta which were contracted by norepinephrine. NO-dependent relaxation of arterial specimens was enhanced by lowering oxygen concentration in the medium with concomitant increase in their cGMP levels. Anoxia induced the relaxation of the aorta by some NO-enhanceable and methylene blue-insensitive mechanism. These results suggested that local concentrations of oxygen might play important roles in the regulation of NO-dependent GC activity and vascular tonus of resistance arteries.     

Oxygen concentration regulates NO-dependent relaxation of aortic smooth muscles

Nitric oxide (NO) functions as an endothelium-derived relaxation factor and regulates vascular resistance. Recent studies in this laboratory (Arch. Biochem. Biophys. 323, 27-32, 1995) revealed that the lifetime of NO significantly increased at physiologically low levels of oxygen concentrations and, hence, this gaseous radical strongly inhibited mitochondrial electron transport for a fairly long duration at low oxygen concentrations. The present work describes the effect of oxygen concentration on NO-induced relaxation and guanylate cyclase (GC) activity of endothelium-denuded aorta of the rat. Both NO and 2,2'-hydroxynitrosohydrazono)bis-ethanamine (NOC18), an NO donor, induced the relaxa-tion of endothelium-denuded helical segments of rat aorta which were contracted by norepinephrine. NO-dependent relaxation of arterial specimens was enhanced by lowering oxygen concentration in the medium with concomitant increase in their cGMP levels. Anoxia induced the relaxation of the aorta by some NO-enhanceable and methylene blue-insensitive mechanism. These results suggested that local concentrations of oxygen might play important roles in the regulation of NO-dependent GC activity and vascular tonus of resistance arteries.     

EGF-receptor regulates salivary gland branching morphogenesis by supporting proliferation and maturation of epithelial cells and survival of mesenchymal cells

Epidermal growth factor receptor (EGF-R) regulates epithelial morphogenesis during development and is important for the proper branching of the lung, mammary gland, and pancreas. We analyzed the salivary gland phenotype of EGF-R-deficient mice and showed impaired growth, branching, and maturation of the epithelium. Furthermore, treatment of wild-type E13 salivary glands with gefitinib, a small molecular inhibitor of EGF-R, led to apoptosis of the mesenchyme. Interestingly, MMP2 and plasminogen activators were upregulated upon inhibition of EGF-R signaling. To summarize, we show that EGF-R is a physiological regulator of salivary gland development and its main function is to support the proliferation and maturation of the epithelium and the survival of the mesenchyme.     

Oxygen regulates mitogen-stimulated proliferation of fetal human airway smooth muscle cells

Increased airway smooth muscle (ASM) content is characteristic of infants with chronic lung disease of prematurity/bronchopulmonary dysplasia. Oxygen therapy, reactive oxygen species (ROS), and immature antioxidant defenses are major risk factors in chronic lung disease of prematurity/bronchopulmonary dysplasia, but their interrelationship is unclear. The direct effects of raised Po2 and modulation of ROS were examined on proliferation of cultured fetal human ASM cells. A bell-shaped relationship was found between Po2 and DNA synthesis induced by fetal bovine serum, platelet-derived growth factor, and basic fibroblastic growth factor, with peak responses occurring at 10-kPa Po2. Changes in DNA synthesis by Po2 did not occur in the absence of mitogen. ROS generation, estimated by dichlorodihydrofluorescein oxidation, was increased by mitogens but was unaffected by nonmitogens (bradykinin, histamine). There was an inverse relationship between ROS generation and Po2, and mitogen-induced ROS generation was substantially potentiated as the Po2 fell. H2O2 mimicked the effect of Po2 on fetal bovine serum-stimulated proliferation, whereas treatment with antioxidants (GSH, N-acetylcysteine) reduced it. These data demonstrate that increases in Po2 above levels found in utero modulate proliferation of fetal ASM cells but only in the presence of growth factors. They also strongly suggest that, under these conditions, proliferation is mediated in part by generation of ROS.     

RAFTK/Pyk2 regulates EGF-induced PC12 cell spreading and movement

The protein tyrosine kinase RAFTK, also termed Pyk2, is a member of the focal adhesion kinase (FAK) subfamily. In this report, we show the role of RAFTK in neuroendocrine PC12 cells upon epidermal growth factor (EGF) stimulation. Following EGF treatment, we observed that RAFTK was tyrosine-phosphorylated in a time- and dose-dependent manner, while FAK was constitutively phosphorylated and primarily regulated by cell adhesion. Moreover, we found that RAFTK associated with the phosphorylated EGF receptor (EGFR) upon EGF stimulation. RAFTK phosphorylation was mediated primarily through PLCgamma-IP3-Ca(2+) signaling and partially through PI3-Kinase. Furthermore, overexpression of PRNK, a specific dominant-negative construct of RAFTK, was sufficient to block EGF-induced cell spreading and movement. Paxillin, a key modulator of the actin cytoskeleton and an RAFTK substrate, was also phosphorylated following EGF treatment. EGF induced a dynamic reorganization of RAFTK and paxillin at neuronal adhesion sites, with the specific localization of paxillin at the inner juxtaposition of RAFTK. Additionally, we observed that RAFTK associated with the scaffold protein c-Cbl and mediated its phosphorylation. Our data demonstrate that while FAK mediated cell adhesion, RAFTK was localized at the cytoplasm where it mediated inside-out signaling through intracellular Ca(2+), thus leading to cell spreading and movement upon EGF stimulation.     

Oxygen concentration regulates the proliferative response of human fibroblasts to serum and growth factors

This report demonstrates that oxygen concentration within the physiologic range of 2.5 to 20% controls the pattern of proliferation of human diploid fibroblasts by modulating their response to serum and purified growth factors. Reducing oxygen concentration from 20 to 2.5% increased the division rate and final density of fibroblasts cultured in serum-containing medium. DNA synthesis in response to serum, as well as to EGF and PDGF, was enhanced significantly. Exposing quiescent cells to reduced oxygen enhanced serum-induced DNA synthesis in a time-dependent manner. The stimulatory effect persisted when the oxygen concentration was raised to ambient levels before the addition of serum. These results suggest that oxygen concentration within the physiologic range may control proliferation indirectly by altering the activity of a stable intermediate that regulates the cellular response to growth factors.     

Arf6 regulates EGF-induced internalization of E-cadherin in breast cancer cells

E-cadherin internalization facilitates dissolution of adherens junctions and promotes tumor cell epithelial-mesenchymal transition (EMT) and migration. Our previous results have shown that Arf6 exerts pro-migratory action in breast cancer cells after EGF stimulation. Despite the fact that EGF signaling stimulates EMT of breast cancer cells, the effect of Arf6 on internalization of E-cadherin of breast cancer cells under EGF treatment remains to be determined. Here, we showed that EGF dose-dependently stimulated E-cadherin internalization by MCF-7 cells with the maximal effect at 50 ng/ml. Meanwhile, EGF treatment markedly increased Arf6 activation. Arf6 was involved in complexes of E-cadherin, and more E-cadherin was pulled down with Arf6 when the activity of the latter was increased. Immunoblotting and immunofluorescence assays showed that transfection breast cancer cells with Arf6-T27N or Arf6 siRNA suppressed EGF-induced E-cadherin internalization. Taken together, our study demonstrated that Arf6 activation plays a potential role in EGF-induced E-cadherin internalization, providing new mechanism underlying the effect of Arf6 on promoting breast cancer cell metastasis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12935-015-0159-3) contains supplementary material, which is available to authorized users.     

Docosahexaenoic acid regulates serum amyloid A protein to promote lipolysis through down regulation of perilipin

Docosahexaenoic acid (DHA) increases lipolysis and decreases lipogenesis through several pathways. DHA also enhances the expression of serum amyloid A protein (SAA), a possible lipid metabolism related gene. The question of whether DHA regulates the expression of SAA to affect lipid metabolism and increase lipolysis needs to be demonstrated in human adipocytes. We designed experiments to determine the role of SAA in regulating lipid metabolism in HepG2 cells using microarray technology. In human hepatocytes, recombinant human SAA1 (hSAA1) inhibited the expression of genes related to lipogenesis and promoted the expression of those involved in lipolysis. When human breast adipocytes were treated with hSAA1 or DHA in vitro, the expression of peroxisome proliferator-activated receptor γ and other lipogenic genes was decreased, whereas the expression of several lipolytic genes was increased. Glycerol release was increased by both SAA and DHA treatments, suggesting that they increased lipolytic activity in human adipocytes. The expression of perilipin, a lipid droplet-protective protein, was decreased, and hormone-sensitive lipase was increased by both of hSAA1 and DHA treatment. We speculate that the mechanism of lipolysis by DHA or SAA is at least partially the result of increased expression of hormone-sensitive lipase and decreased expression of perilipin. Whereas DHA treatment increased expression of hSAA1 in human adipocytes, the DHA-mediated reduction in expression of lipogenesis genes and enhancement of lipolysis may be through the activity of hSAA1. These results may be useful in developing new approaches to reduce body fat deposition.     

EBP50 inhibits EGF-induced breast cancer cell proliferation by blocking EGFR phosphorylation

Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) suppresses breast cancer cell proliferation, potentially through its regulatory effect on epidermal growth factor receptor (EGFR) signaling, although the mechanism by which this occurs remains unknown. Thus in our studies, we aimed to determine the effect of EBP50 expression on EGF-induced cell proliferation and activation of EGFR signaling in the breast cancer cell lines, MDA-MB-231 and MCF-7. In MDA-MB-231 cells, which express low levels of EBP50, EBP50 overexpression inhibited EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. In MCF-7 cells, which express high levels of EBP50, EBP50 knockdown promoted EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. Knockdown of EBP50 in EBP50-overexpressed MDA-MB-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation and restoration of EBP50 expression in EBP50-knockdown MCF-7 cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation, further confirming that the activation of EGF-induced downstream molecules could be specifically inhibited by EBP50 expression. Since EGFR signaling was triggered by EGF ligands via EGFR phosphorylation, we further detected the phosphorylation status of EGFR in the presence or absence of EBP50 expression. Overexpression of EBP50 in MDA-MB-231 cells inhibited EGF-stimulated EGFR phosphorylation, whereas knockdown of EBP50 in MCF-7 cells enhanced EGF-stimulated EGFR phosphorylation. Meanwhile, total expression levels of EGFR were unaffected during EGF stimulation. Taken together, our data shows that EBP50 can suppress EGF-induced proliferation of breast cancer cells by inhibiting EGFR phosphorylation and blocking EGFR downstream signaling in breast cancer cells. These results provide further insight into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer.     

NADPH oxidase controls EGF-induced proliferation via an ERK1/2-independent mechanism

We have used HyPer, a ratiometric GFP-based biosensor, to follow H₂O₂ dynamics in live cells. We have found that activation of the EGF receptor in epithelial cells leads to sustained generation of intracellular H₂O₂, which is blocked by apocynin, an inhibitor of the plasma membrane NADPH oxidase assembly. Apocynin also blocked HeLa cell proliferation induced by EGF, indicating that NADPH oxidase is critically involved. However, apocynin failed to alter the kinetics of EGF-stimulated ERK1/2 activation. We conclude that NADPH oxidase and intracellular H₂O₂ are important downstream targets of EGF receptor that mediate the proliferation response by mechanisms distinct from activation of the classical ERK1/2 MAP-kinase pathway.     

Genistein and erbstatin inhibition of normal mammary epithelial cell proliferation is associated with EGF-receptor down-regulation

Epidermal growth factor (EGF) is a potent mitogen for normal mouse mammary epithelial cells grown in primary culture. EGF activation of the EGF-receptor (EGF-R) induces intrinsic tyrosine kinase activity which results in EGF-R autophosphorylation and tyrosine phosphorylation of other intracellular substrates involved in EGF-R signal transduction. Genistein and erbstatin are anticancer agents which have been shown to be potent tyrosine kinase inhibitors. However, the effects of these compounds in modulating EGF-dependent normal mammary epithelial cell proliferation is presently unknown. Therefore, studies were conducted to determine the effects of genistein and erbstatin on EGF-dependent proliferation, and EGF-R levels and autophosphorylation in normal mouse mammary epithelial cells grown in primary culture and maintained in serum-free media. Chronic treatment with 6.25–100 μM genistein or 1–16 μM erbstatin significantly decreased EGF-dependent mammary epithelial cell proliferation in a dose-responsive manner. However, the highest doses of genistein (100 μM ) and erbstatin (16 μM ) were found to be cytotoxic. Additional studies showed that acute treatment with 6.25–400 μM genistein did not affect EGF-R levels or EGF-induced EGF-R autophosphorylation, while acute treatment with 1–64 μM erbstatin caused a slight reduction in EGF-R levels, but had no effect on EGF-dependent EGF-R autophosphorylation in these cells. In contrast, chronic treatment with similar doses of genistein or erbstatin resulted in a large dose-responsive decrease in EGF-R levels, and a corresponding decrease in total cellular EGF-R autophosphorylation intensity. These results demonstrate that the inhibitory effects of chronic genistein and erbstatin treatment on EGF-dependent mammary epithelial cell proliferation is not due to a direct inhibition of EGF-R tyrosine kinase activity, but results primarily from a down-regulation in EGF-R levels and subsequent decrease in mammary epithelial cell mitogenic-responsiveness to EGF stimulation.     

BCAR3 regulates EGF-induced DNA synthesis in normal human breast MCF-12A cells

BCAR3 (breast cancer anti-estrogen resistance 3) is a signal transducer containing an SH2 domain, a proline/serine-rich domain and a GDP-exchange factor homologous domain, whose role in signaling pathways is currently unclear. Furthermore, BCAR3 is implicated in anti-estrogen resistance of breast cancer cells. In the present study, we investigated the functional role of BCAR3 in a mitogenic signaling pathway of EGF in non-tumorigenic human breast epithelial MCF-12A cells. Microinjection of an anti-BCAR3 antibody, siRNAs targeting BCAR3 and an SH2 domain of BCAR3 inhibited EGF-induced DNA synthesis. Direct association of BCAR3 with activated EGF receptor and Cas was observed. Lastly, microinjection of a BCAR3 expression plasmid induced DNA synthesis. These findings suggest that the BCAR3 protein, through its SH2 domain, is involved in the signaling pathways of EGF leading to cell cycle progression, and that BCAR3 itself is part of a mitogenic signaling pathway.     

NADPH oxidase controls EGF-induced proliferation via the ERK1/2-independent mechanism

Using HyPer, a ratiometric GFP-based biosensor, the dynamics of H2O2 in living cells has been studied. It was found that activation of the receptor of the epidermal growth factor (EGF) in epithelial cells leads to sustained generation of intracellular H2O2, which is blocked by apocynin, an inhibitor of the assembly of plasma membrane NADPH oxidase. Apocynin also blocked HeLa cell proliferation induced by EGF, indicating that NADPH oxidase should be involved in the process. However, apocynin failed to alter the kinetics of the EGF-stimulated ERK1/2 activation. It was concluded that NADPH oxidase and intracellular H2O2 are the important downstream targets of the EGF receptor, which mediate the proliferation response by mechanisms distinct from the activation of the classical ERK1/2 MAP-kinase pathway.     

MicroRNA参与调控睾丸支持细胞的增殖与粘附功能

精子发生过程需要生精细胞及睾丸体细胞的共同参与,这两种细胞也决定着睾丸的发育及雄性生育力。支持细胞是生精小管中唯一的体细胞,在正常精子发生过程中发挥重要的作用。支持细胞增殖与粘附功能的异常将导致精子发生异常,进而引发雄性不育。近年来研究发现,microRNA (miRNA)可调控支持细胞的增殖与粘附功能,其表达水平在激素、内分泌干扰素和营养状况等多种因素作用下发生特异性变化。本文总结了与睾丸支持细胞增殖与粘附功能相关的miRNA及其作用机制,以期发现并鉴定更多与支持细胞相关的miRNA,进而为探索与支持细胞相关不育症的病因提供理论依据。     

Integrin-linked kinase regulates chondrocyte shape and proliferation

The interaction of chondrocytes with the extracellular-matrix environment is mediated mainly by integrins. Ligated integrins are recruited to focal adhesions (FAs) together with scaffolding proteins and kinases, such as integrin-linked kinase (Ilk). Ilk binds the cytoplasmic domain of β1-, β2- and β3-integrins and recruits adaptors and kinases, and is thought to stimulate downstream signalling events through phosphorylation of protein kinase B/Akt (Pkb/Akt) and glycogen synthase kinase 3-β (GSK3-β). Here, we show that mice with a chondrocyte-specific disruption of the gene encoding Ilk develop chondrodysplasia, and die at birth due to respiratory distress. The chondrodysplasia was characterized by abnormal chondrocyte shape and decreased chondrocyte proliferation. In addition, Ilk-deficient chondrocytes showed adhesion defects, failed to spread and formed fewer FAs and actin stress fibres. Surprisingly, phosphorylation of Pkb/Akt and GSK3-β is unaffected in Ilk-deficient chondrocytes. These findings suggest that Ilk regulates actin reorganization in chondrocytes and modulates chondrocyte growth independently of phosphorylation of Pkb/Akt and GSK3-β.     

Insulin receptor down regulation in human erythrocytes

Insulin receptor processing in human erythrocytes was investigated. Insulin binding to the cell surface was found to decrease by 70% in cells which had first been incubated with insulin for 3 h, then washed for 3 h. After an additional 16-h incubation without insulin, the level of cell surface insulin binding was restored to control values, even in the presence of cyclohexamide. Our results suggest that erythrocyte insulin receptors are internalized in response to insulin and that receptors are subsequently recycled to the surface of the cell.