Either Epidermal Growth Factor Research Articles

Abstract 156: Dynamic live-cell visualization and quantification of Akt activity using a genetically-encoded, fluorescent kinase translocation reporter

Abstract Dysregulation of signal transduction pathways is associated with cancer initiation, progression, and recurrence. The PI3K/Akt signaling pathway has been extensively investigated as a therapeutic target due to its mechanistic association with several hallmarks of cancer. Studying dynamic changes in kinase activity can be difficult, and assays to measure these changes in live cells in a physiologically relevant environment are lacking. Standard approaches to monitoring Akt kinase activity are limited to end point assays which lack the ability to monitor the effects of treatments over time. Here we demonstrate the utility of the Incucyte® Kinase Akt Lentivirus Reagent, encoding a kinase translocation reporter based on a green fluorescent protein-tagged Akt substrate whose subcellular localization is phosphorylation-dependent, and a red fluorescent nuclear protein to denote the nuclear/cytoplasmic boundary. To demonstrate inhibition of Akt, A549 cells stably expressing the reporter were treated with compounds targeting the PI3K/Akt kinase pathway, including allosteric Akt inhibitors MK2206 and API-1, competitive Akt inhibitors AZD5363 and Ipatasertib, and upstream PI3K kinase inhibitors LY294002 and PI-103. Quantification of treatment responses using the Incucyte® Live Cell Analysis System showed concentration-dependent inhibition of Akt activity for all compounds with varying kinetic profiles over 24 hours. To study activation of Akt, HeLa cells were first cultured in the absence of serum to reduce Akt activity. After 4 hours of incubation in serum-free conditions, the cells were treated with either epidermal growth factor (EGF) or recombinant insulin-like growth factor (R3-IGF-1) to activate Akt. An increase in Akt activity was observed for both treatments. While R3-IGF-1-induced activation was sustained over the 12-hour time course, activation by EGF diminished over time. Cell lines with mutations in the tumor suppressor PTEN showed no response to serum starvation or activation with EGF or R3-IGF-1. In addition to monitoring Akt activity over time, the integrated red nuclear restricted protein of the reporter enables concurrent measurements of proliferation. The selective Akt inhibitor MK2206 decreased Akt activity in a similar concentration-dependent manner in both T-47D and MDA-MB-231 cell lines. In contrast, measurements of red object count from the same cells reveal differential effects of MK2206 on proliferation between the two cell lines, with concentration-dependent inhibition of T-47D cell growth but little effect on MDA-MB-231 cells. Overall, these data highlight the utility of the Incucyte® Kinase Akt Lentivirus Reagent to provide valuable kinetic measurements of Akt activity using live cells within a physiologically relevant environment. Citation Format: John N. Rauch, Susan K. Foltin, Libuse Oupicka, Matthew Dilsaver, Grigory S. Filonov, Gillian Lovell, Jasmine Trigg, Cicely L. Schramm. Dynamic live-cell visualization and quantification of Akt activity using a genetically-encoded, fluorescent kinase translocation reporter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 156.

Mechanical disruption of E-cadherin complexes with epidermal growth factor receptor actuates growth factor–dependent signaling

Increased intercellular tension is associated with enhanced cell proliferation and tissue growth. Here, we present evidence for a force-transduction mechanism that links mechanical perturbations of epithelial (E)-cadherin (CDH1) receptors to the force-dependent activation of epidermal growth factor receptor (EGFR, ERBB1)-a key regulator of cell proliferation. Here, coimmunoprecipitation studies first show that E-cadherin and EGFR form complexes at the plasma membrane that are disrupted by either epidermal growth factor (EGF) or increased tension on homophilic E-cadherin bonds. Although force on E-cadherin bonds disrupts the complex in the absence of EGF, soluble EGF is required to mechanically activate EGFR at cadherin adhesions. Fully quantified spectral imaging fluorescence resonance energy transfer further revealed that E-cadherin and EGFR directly associate to form a heterotrimeric complex of two cadherins and one EGFR protein. Together, these results support a model in which the tugging forces on homophilic E-cadherin bonds trigger force-activated signaling by releasing EGFR monomers to dimerize, bind EGF ligand, and signal. These findings reveal the initial steps in E-cadherin-mediated force transduction that directly link intercellular force fluctuations to the activation of growth regulatory signaling cascades.

Metabolic syndrome in rats is associated with erectile dysfunction by impairing PI3K/Akt/eNOS activity

Metabolic syndrome (MetS) is a risk factor for erectile dysfunction (ED), but the underlying mechanisms are unclear. The aims of this study were to determine the underlying mechanisms of metabolic syndrome-related ED (MED). Sprague Dawley (SD) rats were fed a high-fat diet for 6 months, and metabolic parameters were then assessed. An apomorphine test was conducted to confirm MED. Only rats with MED were administered an intracavernosal injection of either epidermal growth factor (EGF) or vehicle for 4 weeks. Erectile responses were evaluated by determining the mean arterial blood pressure (MAP) and intracavernosal pressure (ICP). Levels of protein expression were examined by western blotting and immunohistochemistry. Body weight, fasting blood glucose, plasma insulin and plasma total cholesterol were increased in the MetS rats compared with those in control rats (each p < 0.05). The maximum ICP/MAP, total ICP/MAP and concentration of cyclic guanosine mono-phosphate (cGMP) were significantly decreased in MED rats (each p < 0.05). The expression levels of p110α, p-Akt1 (Tyr308)/Akt1 and p-eNOS (Ser1177)/eNOS were reduced in MED rats (each p < 0.05). Activation of the PI3K/Akt/eNOS signaling cascade (intracavernosal injection of EGF) reversed these changes (each p < 0.05). The present study demonstrates that downregulation of the PI3K/Akt/eNOS signaling pathway is involved in MED.

Effect of growth factors on oocyte maturation and allocations of inner cell mass and trophectoderm cells of cloned bovine embryos.

This study was conducted to determine the additive effects of exogenous growth factors during in vitro oocyte maturation (IVM) and the sequential culture of nuclear transfer (NT) embryos. Oocyte maturation and culture of reconstructed embryos derived from bovine granulosa cells were performed in culture medium supplemented with either epidermal growth factor (EGF) alone or a combination of EGF with insulin-like growth factor-I (IGF-I). The maturation rates of oocytes matured in the presence of EGF or the EGF + IGF-I combination were significantly higher than those of oocytes matured in the presence of only fetal calf serum (FCS) (P 0.05). IGF-I alone or in combination with EGF in sequential embryo culture medium significantly increased the ratio of inner cell mass (ICM) to total blastocyst cells (P < 0.05). Our results showed that the addition of growth factors to IVM and sequential culture media of cloned bovine embryos increased the ICM without changing the total cell number. These unknown and uncontrolled effects of growth factors can alter the allocation of ICM and trophectoderm cells (TE) in NT embryos. A decrease in TE cell numbers could be a reason for developmental abnormalities in embryos in the cloning system.

Abstract B11: A microfluidic platform to evaluate soluble signaling in the metastasis microenvironment

Abstract Cancer mortality varies widely across different cancer types, but over 90% of cancer deaths, regardless of tumor origin, are caused by metastasis. Recent work to elucidate the mechanisms of metastasis has focused on the early stages of metastasis, specifically migration within a primary tumor and intravasation into vessels of the circulatory system. What is still relatively unknown is how these circulating cancer cells extravasate from the vessels to form micrometastases in distant organs. Chemokine gradients have long been associated with cell migration in embryogenesis and have also been implicated in promoting migration of cancer cells in vitro. This study was designed to investigate how competing gradients of chemotactic cytokines produced by cells in the metastatic niche communicate with cancer cells trapped in the vasculature to promote extravasation and colonization of new tumors. A microfluidic channel having five inlets and outlets was fabricated from polydimethylsiloxane (PDMS) bound to glass, and metastatic breast cancer cells (MDA-MB-231-4175 cell line, ATCC) were seeded into the device. Following cell attachment, a syringe pump supplied cells with a constant gradient of either epidermal growth factor (EGF) or stromal cell-derived factor 1 (SDF-1)-two known breast cancer chemoattractants-in serum-free media. Cells in the device were imaged regularly for 8 hours, and the timelapse data was used to quantify velocity, directionality, and chemotactic index of the cells. Following single-chemokine gradient studies, EGF and SDF-1 gradients of equal strength but opposite direction were introduced to the chip simultaneously. Cells in the device were again imaged for 8 hours, and velocity, directionality, and chemotactic index of cells in the competing gradients were quantified. To study chemokine gradients involved specifically in hematogenous metastasis, an extravasation microenvironment was built inside a microfluidic channel similar to the device previously described. Evenly spaced PDMS posts extending the height of the channel separated the channel into 5 lanes. A collagen gel was polymerized in two lanes to simulate niche extracellular matrix and to provide structural support for endothelial cells grown to confluence in the shape of a vessel. The shape of the EGF diffusion gradient formed across the endothelial monolayer was characterized using fluorescein isothiocyanate (FITC)–dextran as a diffusion marker. These experiments test the ability of both EGF and SDF-1 to promote directional migration of MDA-MB-231-4175 both individually and when applied in opposition, allowing us to test the hypotheses that chemotaxis of metastatic breast cancer cells can be controlled by chemokine gradients and that cells can sense and respond to multiple gradients simultaneously. In regard to hematogenous metastasis, characterization of an EGF gradient across an endothelial monolayer in both the absence and presence of cancer cells will assess the function of endothelial cell tight junctions in establishing chemokine gradients in the metastasis microenvironment. Combined, these studies will evaluate the hypotheses that: (1) Metastatic cancer cells trapped in the vasculature must initiate communication by modifying tight junctions between endothelial cells and (2) Once chemokines are free to diffuse into the vessel, extravasation and colonization are influenced by communication via a variety of competing chemokines that the cancer cells can sense and respond to simultaneously. Which chemokines ultimately “win” the competition and succeed in attracting cancer cells to colonize a target organ is the driving question of this research and will be the focus of future work. Citation Format: Laura Blaha, Chentian Zhang, Byungwoo Ryu, Rhoda Alani, Mario Cabodi, Joyce Wong. A microfluidic platform to evaluate soluble signaling in the metastasis microenvironment. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B11. doi:10.1158/1538-7445.CHTME14-B11

Abstract C145: Compounds that inhibit the activation of Grb2 by EGFR, HER2, and HER3 complexes.

Abstract Purpose: To demonstrate that ORIL003 (Glc4-Rha2-Glc-diosgenin; full structure will be disclosed) inhibits the activation of Grb2 by EGFR, HER2 and HER3 complexes using the Receptor-Heteromer Investigation Technology (Receptor-HIT) on the bioluminescence resonance energy transfer (BRET) platform. Introduction: The unique property of inhibiting a range of receptor tyrosine kinases (RTKs) in the cell plasma membrane has been demonstrated in a family of steroid saponins. As the compounds have a pan-inhibitory effect, they are effective in inhibiting a wide range of cancer cell types, including lung, breast, prostate, colorectal, melanoma, ovarian and leukemia. Methods: Receptor-HIT, previously published in the form of G protein-coupled receptor heteromer identification technology (GPCR-HIT; See et al, 2011 Assay Drug Dev Technol 9:21–30), was used to determine close proximity and activation of ErbB growth factor receptors and Grb2. EGFR, HER2 and HER3 were tagged with Rluc8 (a variant of Renilla luciferase) as donor; Grb2 was tagged with GFP2 (a variant of green fluorescent protein) as acceptor. Human embryonic kidney 293 cells were stimulated with either epidermal growth factor (EGF) or heregulin (HRG) at a concentration of 20–50 nM. ORIL003 (as an example of the steroid saponin family) was applied to the cells at a concentration of 10 μM, which was known from in vitro studies to completely inhibit cancer cell growth. Results: Stimulation by EGFR or HRG resulted in recruitment of Grb2. This recruitment was inhibited by the use of ORIL003 in a dose-dependent manner. Furthermore, constitutive interactions between the ErbB growth factor receptors and Grb2 also appear to be inhibited by ORIL003. Conclusions: ORIL003 is effective in inhibiting the activation of Grb2 by EGFR, HER2 and HER3 complexes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C145.

Nitric oxide is the primary mediator of cytotoxicity induced by GSH depletion in neuronal cells

Glutathione (GSH) levels progressively decline during aging and in neurodegenerative disorders. However, the contribution of such event in mediating neuronal cell death is still uncertain. In this report, we show that, in neuroblastoma cells as well as in primary mouse cortical neurons, GSH decrease, induced by buthionine sulfoximine (BSO), causes protein nitration, S-nitrosylation and DNA strand breaks. Such alterations are also associated with inhibition of cytochrome c oxidase activity and microtubule network disassembly, which are considered hallmarks of nitric oxide (NO) toxicity. In neuroblastoma cells, BSO treatment also induces cell proliferation arrest through the ERK1/2-p53 pathway that finally results in caspase-independent apoptosis, as evident from the translocation of apoptosis-inducing factor from mitochondria towards nuclei. A deeper analysis of the signaling processes indicates that the NO-cGMP pathway is involved in cell proliferation arrest and death. In fact, these events are completely reversed by L-NAME, a specific NO synthase inhibitor, indicating that NO, rather than the depletion of GSH per se, is the primary mediator of cell damage. In addition, the guanylate cyclase (GC) inhibitor LY83583 is able to completely block activation of ERK1/2 and counteract BSO toxicity. In cortical neurons, NMDA (N-methyl-D-aspartic acid) treatment results in GSH decrease and BSO-mediated NO cytotoxicity is enhanced by either epidermal growth factor (EGF) or NMDA. These findings support the idea that GSH might represent the most important buffer of NO toxicity in neuronal cells, and indicate that the disruption of cellular redox buffering controlled by GSH makes neuronal cells susceptible to endogenous physiological flux of NO.

A cell-based high-throughput screen for epidermal growth factor receptor pathway inhibitors

Epidermal growth factor receptor (EGFR) is a valid drug target for development of target-based therapeutics against non-small-cell lung cancer. In this study, we established a high-throughput cell-based assay to screen for compounds that may inhibit EGFR activation and/or EGFR-mediated downstream signaling pathway. This drug screening platform is based on the characterization of an EGFR-transfected 32D cell line (32D-EGFR). The expression of EGFR in 32D cells allowed cell proliferation in the presence of either epidermal growth factor (EGF) or interleukin 3 (IL-3) and provided a system for both screening and counterscreening of EGFR pathway-inhibitory compounds. After the completion of primary and secondary screenings in which 32D-EGFR cells were grown under the stimulation of either EGF or IL-3, 9 of 20,000 compounds were found to selectively inhibit the EGF-dependent proliferation, but not the IL-3-dependent proliferation, of 32D-EGFR cells. Subsequent analysis showed that 3 compounds of the 9 initial hits directly inhibited the kinase activity of recombinant EGFR in vitro and the phosphorylation of EGFR in H1299 cells transfected with EGFR. Thus, this 32D-EGFR assay system provides a promising approach for identifying novel EGFR and EGFR signaling pathway inhibitors with potential antitumor activity.

Gene expression profiling defined pathways correlated with fibroblast cell proliferation induced by Opisthorchis viverrini excretory/secretory product

To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory (ES) product. NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA (cDNA) array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semi-quantitative SYBR-based real-time RT-PCR. Among a total of 15,000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serine-threonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O. viverrini ES product. The expression level of signal transduction genes; pkC, pdgfr alpha, jak 1, eps 8, tgf beta 1i4, strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC, eps 8 and tgfbeta 1i4 which are the downstream signaling molecules of either epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta) showed statistical significance (P < 0.05). O. viverrini ES product stimulates the significant changes of gene expression in several functional categories and these mainly include transcripts related to cell proliferation. The TGF-beta and EGF signal transduction pathways are indicated as the possible pathways of O. viverrini-driven cell proliferation.

A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors.

Invasion of tumor cells into the surrounding connective tissue and blood vessels is a key step in the metastatic spread of breast tumors. Although the presence of macrophages in primary tumors is associated with increased metastatic potential, the mechanistic basis for this observation is unknown. Using a chemotaxis-based in vivo invasion assay and multiphoton-based intravital imaging, we show that the interaction between macrophages and tumor cells facilitates the migration of carcinoma cells in the primary tumor. Gradients of either epidermal growth factor (EGF) or colony-stimulating factor 1 (CSF-1) stimulate collection into microneedles of tumor cells and macrophages even though tumor cells express only EGF receptor and macrophages express only CSF-1 receptor. Intravital imaging shows that macrophages and tumor cells migrate toward microneedles containing either EGF or CSF-1. Inhibition of either CSF-1- or EGF-stimulated signaling reduces the migration of both cell types. This work provides the first direct evidence for a synergistic interaction between macrophages and tumor cells during cell migration in vivo and indicates a mechanism for how macrophages may contribute to metastasis.

IGF-I stimulates proliferation of spontaneously immortalized human keratinocytes (HACAT) by autocrine/paracrine mechanisms.

HaCaT keratinocytes are derived from adult human skin and although spontaneously immortalized, remain highly related to their normal counterparts. We observed that HaCaT cells can proliferate in serum-free medium (SFM), in contrast to normal human keratinocytes whose growth in vitro requires a feeder layer and/or the supplementation with hormones and growth factors. Since autocrine production of growth factors has been proposed as the pathway that cells may exploit to escape growth regulation, we have investigated whether this is occurring in HaCaT cultured in SFM. Either epidermal growth factor (EGF) or insulin-like growth factor-1 (IGF-I) was effective and dose-dependently stimulated HaCaT replication. The ability of these keratinocytes to express EGF and IGF-I and their receptors was investigated by northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR). We report that HaCaT cells synthesize mRNAs for IGF-I, IGF-II, IGF-IR and EGF-R but not EGF mRNA. Immunoneutralization of IGF-I with specific monoclonal antibodies blocked spontaneous HaCaT proliferation in SFM, as did incubation with antibodies against IGF-IR. These data demonstrate that an autocrine/paracrine loop based on IGF-I may allow HaCaT keratinocytes to proliferate autonomously in culture in contrast to keratinocytes in primary culture. A similar mechanism may be involved in the development of hyperproliferative diseases of human skin and its functional disruption may represent the target for therapeutic approaches.

Proliferative generation of mammalian auditory hair cells in culture

Hair cell (HC) and supporting cell (SC) productions are completed during early embryonic development of the mammalian cochlea. This study shows that acutely dissociated cells from the newborn rat organ of Corti, developed into so-called otospheres consisting of 98% nestin (+) cells when plated on a non-adherent substratum in the presence of either epidermal growth factor (EGF) or fibroblast growth factor (FGF2). Within cultured otospheres, nestin (+) cells were shown to express EGF receptor (EGFR) and FGFR2 and rapidly give rise to newly formed myosin VIIA (+) HCs and p27 KIP1 (+) SCs. Myosin VIIA (+) HCs had incorporated bromodeoxyuridine (BrdU) demonstrating that they were generated by a mitotic process. Ultrastructural studies confirmed that HCs had differentiated within the otosphere, as defined by the presence of both cuticular plates and stereocilia. This work raises the hypothesis that nestin (+) cells might be a source of newly generated HCs and SCs in the injured postnatal organ of Corti.

Activation of Elk-1, an Ets transcription factor, by glucose and EGF treatment of insulinoma cells.

Elk-1, a member of the ternary complex factor family of Ets domain proteins that bind serum response elements, is activated by phosphorylation in a cell-specific manner in response to growth factors and other agents. The purpose of the current study was to determine whether Elk-1 activation contributes to glucose-/depolarization-induced Ca(2+)-dependent induction of immediate early response genes in pancreatic islet beta-cells. The results of experiments in insulinoma (MIN6) cells demonstrated that Elk-1-binding sites (Ets elements) in the Egr-1 gene promoter contribute to transcriptional activation of the gene. Treatment with either epidermal growth factor (EGF), a known inducer of beta-cell hyperplasia, glucose, or KCl-induced depolarization resulted in Ser(383) phosphorylation and transcriptional activation of Elk-1 (4 +/- 0.3-, P = 0.003, 2.3 +/- 0.19-, P = 0.002, and 2.2 +/- 0.1- fold, P = 0.001 respectively). The depolarization response was inhibited by the Ca(2+) channel blocker verapamil and by the MEK inhibitor PD98059 (53 +/- 6 and 55 +/- 0.5%, respectively). EGF-induced activation of Elk-1 was also inhibited by PD98059 (60 +/- 5%). A dominant negative Ras produced partial inhibition (42%) of the depolarization-induced Elk-1 transcriptional activation. Transfection with a constitutively active Ca(2+)/calmodulin kinase IV plasmid also resulted in Elk-1 transcriptional activation. Experiments with p38, phosphatidylinositol 3-kinase, and protein kinase A inhibitors indicated that these pathways are not involved. We conclude that Elk-1 activation contributes to glucose-/depolarization-induced Ca(2+)-dependent induction of immediate early growth response genes in pancreatic islet beta-cells. Furthermore, the results demonstrated a convergence of nutrient- and growth factor-mediated signaling pathways on Elk-1 activation through induction of Ras/mitogen-activated protein kinase ERK-1 and -2. The role of these pathways in the glucose-induced proliferation of islet beta-cells can now be assessed.

EGF Infusion Stimulates the Proliferation and Migration of Embryonic Progenitor Cells Transplanted in the Adult Rat Striatum

Immature progenitor cells (generated by in vitro propagation) may provide a useful alternative to primary cells (from dissected embryonic tissue) for transplantation if their migratory and proliferative and differentiation properties can be controlled and directed in vivo. In this study E15 murine EGF-responsive progenitor cells were transplanted to the striatum of adult rats. Simultaneously, these animals received continuous infusion of either epidermal growth factor (EGF) or vehicle, to the lateral ventricle, for 8 days. In animals that received EGF, the transplanted progenitors migrated toward the lateral ventricle and proliferated, as evidenced by bromodeoxyuridine incorporation. Progenitor cells transplanted to rats that received vehicle infusions showed neither of these responses. In all animals, transplanted progenitors expressed an immature astrocyte or oligodendrocyte phenotype, the majority of cells being astrocytes. We conclude that EGF stimulates the migration and proliferation of murine progenitor cells in vivo, either directly or indirectly, but does not influence their phenotypic differentiation.

Involvement of p38 mitogen-activated protein kinase activation in bromocriptine-induced apoptosis in rat pituitary GH3 cells.

Bromocriptine, a dopamine D(2) receptor agonist, is a therapeutic agent for patients with prolactinoma and hyperprolactinemia. In this study we demonstrated that bromocriptine induced activation of p38 mitogen-activated protein (MAP) kinase, with concomitant induction of apoptosis in rat pituitary adenoma cell line GH3 cells. Treatment of GH3 cells for 48 h with bromocriptine increased the p38 MAP kinase activity up to 3- to 5-fold and simultaneously increased the number of apoptotic cells. Inclusion in the medium of SB212090 or SB203580, specific p38 MAP kinase inhibitors, completely abolished the bromocriptine-induced activation of p38 MAP kinase and significantly reduced the number of apoptotic cells. The bromocriptine-induced p38 MAP kinase activation was not prevented by S(-)-eticropride hydrochloride, a specific D(2) receptor antagonist. Treatment with either epidermal growth factor (EGF) or thyrotropin-releasing hormone (TRH), which stimulates p44/42 MAP kinase, rescued cells from the bromocriptine-induced apoptosis, with concomitant inhibition of the bromocriptine-induced p38 MAP kinase activation. These results suggest that bromocriptine induces apoptosis in association with p38 MAP kinase activation, and that the p44/42 MAP kinase signaling through EGF and TRH receptors has an opposing effect on p38 MAP kinase activation as well as on apoptosis induced with bromocriptine in GH3 cells.

Novel cardiovascular actions of the activins.

Proliferation and directed migration of vascular cells are key components in vascular diseases such as atherosclerosis and restenosis following percutaneous transluminal coronary angioplasty. However, the precise cellular and molecular mechanisms involved in the control of vascular cell proliferation or migration at the tissue level remain largely undefined. Molecules contributing to these processes are elaborated by distinct cell types and act in both autocrine and paracrine modes. They include two broad classes, polypeptide growth factors and vasoactive G-protein-coupled receptor (GPCR) agonists. Examples of the former, such as platelet-derived growth factor, bind to and activate cell surface receptor tyrosine kinases, initiating intracellular biochemical signaling pathways associated with cell proliferation or migration. In contrast, recent evidence suggests that vasoactive GPCR agonists (e.g. angiotensin II, endothelin-1, alpha-thrombin) elicit cell growth indirectly by inducing the production of autocrine or paracrine factors in vascular cells. Recent studies have identified activin A as a novel component of conditioned medium obtained from GPCR agonist-stimulated vascular smooth muscle cells (SMCs). Although activin A alone only weakly stimulated rat aortic SMC DNA synthesis, it demonstrated a potent co-mitogenic effect in combination with either epidermal growth factor (EGF) or heparin binding EGF-like growth factor in these cells, increasing DNA synthesis by up to 5- and 4-fold respectively. Furthermore, in a rat carotid-injury model, activin A mRNA was upregulated within 6 h after injury, followed by increases in immunoreactive protein detected in the expanding neointima 7 to 14 days later. Taken together, these results indicate that activin A is a common vascular SMC-derived growth factor induced by vasoactive agonists that may, either alone or in combination with other factors, contribute to fibroproliferative vascular diseases.

Regulation of c-fos transcription by chemopreventive isoflavonoids and lignans in MDA-MB-468 breast cancer cells.

Isoflavonoids and lignans are diet constituents with chemopreventive properties. We compared the ability of the isoflavonoids genistein and equol, the lignans enterodiol, enterolactone and nordihydroguaiaretic acid (NDGA) and the lignan metabolite methyl p-hydroxyphenyllactate to interfere with mitogenic and tumour promotional signal transduction pathways. Their effects on c-fos mRNA levels after induction by either epidermal growth factor (EGF) or the tumour promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was measured in human breast cancer-derived MDA-MB-468 cells. Of the six agents, only genistein decreased EGF-induced, c-fos transcription (by 63% compared to control at 100 mumol/l). In contrast, both genistein and equol at 100 mumol/l decreased TPA-induced c-fos levels, by 75 and 67%, respectively. NDGA and methyl p-hydroxyphenyllactate did not inhibit TPA mediated c-fos transcription and enterolactone and enterodiol had only a weak inhibitory effect. NDGA at 0.1-10 mumol/l increased c-fos mRNA levels. None of the agents inhibited protein kinase C and only genistein inhibited EGF receptor-linked protein tyrosine kinase obtained from MDA-MB-468 cells, with an IC50 of 60 mumol/l. NDGA and genistein arrested cell colony formation potently, genistein was 15-fold more growth-inhibitory than equol. The results suggest that both genistein and equol interfere similarly with TPA-induced signal transduction pathways. Inhibition by genistein of EGF-induced c-fos mRNA transcription is probably related to its interruption of EGF receptor-linked protein tyrosine kinase, whereas genistein-induced growth arrest is not. If ability to antagonise phorbol ester effects is important for chemopreventive efficacy, equol and genistein might be equi-efficacious chemopreventors, whereas enterolactone, enterodiol and NDGA should be much less potent. If phorbol ester antagonism together with antimitogenic activity determine optimal chemopreventive activity of this type of agent, genistein would be more potent than equol.

Estrogen receptor transactivation in MCF-7 breast cancer cells by melatonin and growth factors

The pineal hormone, melatonin, inhibits proliferation of estrogen receptor (ER)-positive MCF-7 human breast cancer cells, modulates both ER mRNA and protein expression, and appears to be serum dependent, indicating interaction between melatonin and serum components. To examine the effects of melatonin on ER activity, ER transactivation assays were performed by transiently transfecting MCF-7 cells with an ERE-luciferase reporter construct. MCF-7 cells pre-treated with melatonin for as little as 5 min followed by either epidermal growth factor (EGF) or insulin resulted in the estrogen-independent transactivation of the ER. None of the compounds when used alone transactivated the ER. The ability of melatonin and EGF to transactivate the ER was abolished by the addition of the antiestrogen, ICI 164 384, suggesting that melatonin and EGF co-operate to transactivate the ER. The modulation of ER transactivation was associated with changes in mitogen activated protein kinase activity and ER phosphorylation. This ER transactivation was blocked by pertussis toxin, a G αi -protein-coupled receptor inhibitor, suggesting cross talk between the G-protein-coupled melatonin receptor pathway and the EGF/insulin tyrosine kinase receptor pathways in modulating ER transactivation. Exactly how the ability of melatonin in combination with EGF to transactivate the ER relates to melatonin’s observed growth suppressive effects is not clear. It is possible that, although melatonin and EGF transactivate the ER, this transactivation does not result in the full transcription of estrogen-responsive genes, but rather, makes the ER refractory to activation by estradiol, thus, blocking the mitogenic actions of estradiol.

Pp90rsk1 regulates estrogen receptor-mediated transcription through phosphorylation of Ser-167.

The estrogen receptor alpha (ER), a member of the steroid receptor superfamily, contains an N-terminal hormone-independent transcriptional activation function (AF-1) and a C-terminal hormone-dependent transcriptional activation function (AF-2). Here, we used in-gel kinase assays to determine that pp90rsk1 activated by either epidermal growth factor (EGF) or phorbol myristate acetate specifically phosphorylates Ser-167 within AF-1. In vitro kinase assays demonstrated that pp90rsk1 phosphorylates the N terminus of the wild-type ER but not of a mutant ER in which Ser-167 was replaced by Ala. In vivo, EGF stimulated phosphorylation of Ser-167 as well as Ser-118. Ectopic expression of active pp90rsk1 increased the level of phosphorylation of Ser-167 compared to that of either a mutant pp90rsk1, which is catalytically inactive in the N-terminal kinase domain, or to that of vector control. The ER formed a stable complex with the mutant pp90rsk1 in vivo. Transfection of the mutant pp90rsk1 depressed ER-dependent transcription of both a wild-type ER and a mutant ER that had a defective AF-2 domain (ER TAF-1). Furthermore, replacing either Ser-118 or Ser-167 with Ala in ER TAF-1 showed similar decreases in transcription levels. A double mutant in which both Ser-118 and Ser-167 were replaced with Ala demonstrated a further decrease in transcription compared to either of the single mutations. Taken together, our results strongly suggest that pp90rsk1 phosphorylates Ser-167 of the human ER in vivo and that Ser-167 aids in regulating the transcriptional activity of AF-1 in the ER.

Stimulation of p85/RING3 kinase in multiple organs after systemic administration of mitogens into mice.

Using an autophosphorylation membrane assay, we examined activation of kinases in different organs after intraperitoneal injections of mitogens and cytokines into mice. In the multiple organs examined administration of either epidermal growth factor (EGF), phorbol 12-myristate 13-acetate (PMA) or interleukin-1beta (IL-1beta) activated a number of kinases. Most notably among those was a kinase of approximately 85 kDa (p85) that was activated by EGF, PMA and IL-1beta in the lung, kidney, brain, liver and heart. The size and properties of this enzyme are indistinguishable from the RING3 kinase that has a very high activity in leukocytes of patients with leukemia. In animals treated with PMA, antibodies against RING3 kinase immunoprecipitated PMA-responsive p85 activity from the lung and brain suggesting that p85 and RING3 kinases are the same enzymes. Activation of p85/RING3 kinase by growth factors in multiple organs might reflect involvement of this enzyme in the pathogenesis of leukemias and other proliferative diseases.