The mitogenic action of insulin-like growth factor I in normal human mammary epithelial cells requires the epidermal growth factor receptor tyrosine kinase

The signals used by insulin-like growth factor I (IGF-I) to stimulate proliferation in human mammary epithelial cells have been investigated. IGF-I caused the activation of both ERKs and Akt. Activation of ERKs was slower and more transient than that of Akt. ZD1839, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, prevented activation of ERKs but not Akt by IGF-I. Inhibition of the EGFR with function-blocking monoclonal antibodies also specifically blocked IGF-I-induced ERK activation. These effects occurred in primary mammary epithelial cells and in two cell lines derived from normal mammary epithelium but not in mammary fibroblasts or IGF-I-responsive breast carcinoma cell lines. Although IGF-I stimulated the proliferation of both normal and carcinoma cell lines, ZD1839 blocked this only in the normal line. ZD1839 had no effect on IGF-I receptor (IGF-IR) autophosphorylation in intact cells. IGF-I-induced ERK activation was insensitive to a broad spectrum matrix-metalloproteinase inhibitor and to CRM-197, an inhibitor of the EGFR ligand heparin-bound epidermal growth factor. EGFR was detectable within IGF-IR immunoprecipitates from normal mammary epithelial cells. Treatment of cells with IGF-I led to an increase in the amount of tyrosine-phosphorylated EGFR within these complexes. ZD1839 had no effect on complex formation but completely abolished their associated EGFR tyrosine phosphorylation. These findings indicate that IGF-I utilizes a novel EGFR-dependent signaling pathway involving the formation of a complex between the IGF-IR and the EGFR to activate the ERK pathway and to stimulate proliferation in normal human mammary epithelial cells. This form of regulation may be lost during malignant progression.     

Autocrine epidermal growth factor signaling stimulates directionally persistent mammary epithelial cell migration.

Cell responses to soluble regulatory factors may be strongly influenced by the mode of presentation of the factor, as in matrix-bound versus diffusible modes. The possibly diverse effect of presenting a growth factor in autocrine as opposed to exogenous (or paracrine) mode is an especially important issue in cell biology. We demonstrate here that migration behavior of human mammary epithelial cells in response to stimulation by epidermal growth factor (EGF) is qualitatively different for EGF presented in exogenous (paracrine), autocrine, and intracrine modes. When EGF is added as an exogenous factor to the medium of cells that express EGF receptor (EGFR) but not EGF, cell migration speed increases while directional persistence decreases. When these EGFR-expressing cells are made to also express via retroviral transfection EGF in protease-cleaveable transmembrane form on the plasma membrane, migration speed similarly increases, but directional persistence increases as well. Addition of exogenous EGF to these cells abrogates their enhanced directional persistence, reducing their directionality to a level similar to wild-type cells. If the EGFR-expressing cells are instead transduced with a gene encoding EGF in a soluble form, migration speed and directional persistence were unaffected. Thus, autocrine presentation of EGF at the plasma membrane in a protease-cleavable form provides these cells with an enhanced ability to migrate persistently in a given direction, consistent with their increased capability for organizing into gland-like structures. In contrast, an exogenous/paracrine mode of EGF presentation generates a "scattering" response by the cells. These findings emphasize the functional importance of spatial restriction of EGFR signaling, and suggest critical implications for growth factor-based therapeutic treatments.     

Autophagy regulation in pancreatic acinar cells is independent of epidermal growth factor receptor signaling

Autophagy is an intracellular degradation system in eukaryotic cells that occurs at a basal level. It can also be induced in response to environmental signals including nutrients, hormones, microbial pathogens, and growth factors, although the mechanism is not known in detail. We previously demonstrated that excessive autophagy is induced within pancreatic acinar cells deficient in Spink3, which is a trypsin inhibitor. SPINK1, the human homolog of murine Spink3, has structural similarity to epidermal growth factor (EGF), and can bind and stimulate the EGF receptor (EGFR). To analyze the role of the EGFR in pancreatic development, in the regulation of autophagy in pancreatic acinar cells, and in cerulein-induced pancreatitis, we generated and examined acinar cell-specific Egfr-deficient (Egfr^−/−) mice. Egfr^−/− mice showed no abnormalities in pancreatic development, induction of autophagy, or cerulein-induced pancreatitis, suggesting that Egfr is dispensable for autophagy regulation in pancreatic acinar cells.     

蛋白酪氨酸磷酸酶-PEST在生理调节及相关疾病中的作用

蛋白质分子中酪氨酸残基可逆性的磷酸化是细胞内信号分子传导的基本方式。两类作用相反的酶参与磷酸化的调节:蛋白酪氨酸激酶(protein tyrosinekinase,PTK)和蛋白酪氨酸磷酸酶(protein tyrosine phosphatase,PTP)。含脯氨酸-谷氨酸-丝氨酸-苏氨酸(P-E-S-T)结构域的蛋白酪氨酸磷酸酶(PTP-PEST)属于非受体型酪氨酸磷酸酶类,其本身能与多种蛋白质相互作用,并在细胞迁移、免疫细胞活化和胚胎发育等生理过程中发挥重要作用。本文对PTP-PEST的结构特点、生理功效、介导的信号传导途径和近年来PTP-PEST在疾病中的作用作一综述。     

The major vault protein is a novel substrate for the tyrosine phosphatase SHP-2 and scaffold protein in epidermal growth factor signaling

The catalytic activity of the Src homology 2 (SH2) domain-containing tyrosine phosphatase, SHP-2, is required for virtually all of its signaling effects. Elucidating the molecular mechanisms of SHP-2 signaling, therefore, rests upon the identification of its target substrates. In this report, we have used SHP-2 substrate-trapping mutants to identify the major vault protein (MVP) as a putative SHP-2 substrate. MVP is the predominant component of vaults that are cytoplasmic ribonucleoprotein complexes of unknown function. We show that MVP is dephosphorylated by SHP-2 in vitro and it forms an enzyme-substrate complex with SHP-2 in vivo. In response to epidermal growth factor (EGF), SHP-2 associates via its SH2 domains with tyrosyl-phosphorylated MVP. MVP also interacts with the activated form of the extracellular-regulated kinases (Erks) in response to EGF and a constitutive complex between tyrosyl-phosphorylated MVP, SHP-2, and the Erks was detected in MCF-7 breast cancer cells. Using MVP-deficient fibroblasts, we demonstrate that MVP cooperates with Ras for optimal EGF-induced Elk-1 activation and is required for cell survival. We propose that MVP functions as a novel scaffold protein for both SHP-2 and Erk. The regulation of MVP tyrosyl phosphorylation by SHP-2 may play an important role in cell survival signaling.     

Induced autocrine signaling through the epidermal growth factor receptor contributes to the response of mammary epithelial cells to tumor necrosis factor alpha

In contrast to the well known cytotoxic effects of tumor necrosis factor (TNF) alpha in many mammary cancer cells, we have found that TNF stimulates the proliferation and motility of human mammary epithelial cells (HMECs). Since the response of HMECs to TNF is similar to effects mediated by epidermal growth factor receptor (EGFR) activation, we explored the potential role of cross-talk through the EGFR signaling pathways in mediating cellular responses to TNF. Using a microarray enzyme-linked immunoassay, we found that exposure to TNF stimulated the dose-dependent shedding of the EGFR ligand transforming growth factor alpha (TGFalpha). Both proliferation and motility of HMECs induced by TNF was prevented either by inhibiting membrane protein shedding with a metalloprotease inhibitor, by blocking epidermal growth factor receptor (EGFR) kinase activity, or by limiting ligand-receptor interactions with an antagonistic anti-EGFR antibody. EGFR activity was also necessary for TNF-induced release of matrix metalloprotease-9, thought to be an essential regulator of mammary cell migration. The cellular response to TNF was associated with a biphasic temporal pattern of extracellular signal-regulated kinase (ERK) phosphorylation, which was EGFR-dependent and modulated by inhibition of metalloprotease-mediated shedding. Significantly, the late phase of ERK phosphorylation, detectable within 4 h after exposure, was blocked by the metalloprotease inhibitor batimastat, indicating that autocrine signaling through ligand shedding was responsible for this secondary wave of ERK activity. Our results indicate a novel and important role for metalloprotease activation and EGFR transmodulation in mediating the cellular response to TNF.     

Heparin binding epidermal growth factor-like growth factor is a transforming growth factor beta-regulated gene in intestinal epithelial cells.

Heparin binding epidermal growth factor-like growth factor (HB-EGF) is an EGF-related peptide with prominent effects on cell growth and migration. We explored potentially unique characteristics of HB-EGF in the intestinal epithelial cell line RIE-1. HB-EGF stimulated [(3)H]thymidine incorporation to a level equivalent to transforming growth factor alpha (TGFalpha). HB-EGF also rapidly activated MAPK and induced cyclin D1 in mid-G1 with kinetics similar to TGFalpha. Unlike TGFalpha, HB-EGF mRNA was induced within 1 h by a variety of stimuli, including TGFbeta1. Maximal induction by TGFbeta (7-fold) occurred within 2 h of treatment. Actinomycin D decay curves showed that TGFbeta1 had no effect on HB-EGF mRNA half-life (T(1/2) 20 min). Induction of HB-EGF by TGFbeta1 was not affected by pretreatment with the MEK inhibitor PD-98059 while inhibition of protein kinase C either partially (calphostin C) or completely (staurosporin) blocked induction. Our results suggest that major differences exist in the regulation of the closely related EGF family members TGFalpha and HB-EGF. TGFbeta and HB-EGF, structurally unrelated peptides with potent effects on wound healing, may function coordinately to mediate responses to wounding or cell injury in the intestinal epithelium.     

Prolactin inhibits epidermal growth factor (EGF)-stimulated signaling events in mouse mammary epithelial cells by altering EGF receptor function.

We have previously shown that lactogenic hormones stimulate epidermal growth factor (EGF) mRNA accumulation in mouse mammary glands in vivo and in mouse mammary epithelial cells (NMuMG line). However, our in vitro studies indicate that the lactogenic hormone prolactin (PRL) completely inhibits EGF-stimulated DNA synthesis. PRL does not alter cholera toxin or insulin-like growth factor-1-stimulated cell growth, thus the inhibition appears to be specific for EGF. Our current studies are designed to evaluate the effects of PRL on EGF-stimulated signaling events in the NMuMG cell line. Cells treated with PRL for 30 min demonstrated a loss of high affinity EGF-binding ability. After long-term PRL treatment (18 h) there was a decrease in EGF receptor (R) number, as determined by [125I]EGF binding. PRL treatment (8 h) also decreased EGF-R mRNA levels. An EGF-stimulated increase in EGF-R mRNA observed 2-4 h after treatment was decreased when PRL was added to the cultures. Furthermore, levels of EGF-stimulated tyrosine phosphorylation of the EGF-R (170 kDa) and phospholipase C gamma (145 kDa) are dramatically decreased in cells treated with PRL. Also of great interest was a decrease in EGF-stimulated c-myc mRNA in PRL-treated cells. We conclude that PRL is acting to down-regulate the EGF-R, thus limiting EGF-stimulated cell signaling in mammary tissue.     

Suppression of protein tyrosine kinase activity of the epidermal growth factor receptor by epidermal growth factor

Epidermal growth factor (EGF) receptor protein kinase activity, estimated by the use of peptide substrates, was reduced by as much as 70% after the treatment of intact A431 human carcinoma cells with EGF. The apparent decrease in protein kinase activity was observed after immunoprecipitation of the receptor or after purification of the receptor by lectin chromatography. By the use of [35S]methionine, it was determined that the total amount of receptor obtained was the same whether or not cells were treated with EGF. EGF stimulated the purified receptor protein kinase activity in vitro; however, the EGF-stimulated activity of receptor from EGF-treated cells continued to be reduced by as much at 70% compared to the EGF-stimulated activity from untreated cells. The reduction in receptor protein kinase activity induced by EGF may represent a feedback mechanism by which responsiveness to the growth factor is regulated.     

The epidermal growth factor receptor tyrosine kinase in liver epithelial cells. The effect of ligand-dependent changes in cellular location

Epidermal growth factor (EGF) activates the intrinsic tyrosine-specific protein kinase of its receptor (EGF-R). We studied the effect of EGF-dependent EGF-R internalization on receptor autophosphorylation and on the appearance of tyrosine phosphoproteins in rat liver epithelial cells. Peak receptor autophosphorylation activity (3- to 6-fold over basal) was found in homogenates of EGF-treated cells at times when the majority of receptors (greater than 90%) had been internalized but not yet degraded (15 to 30 min). Stimulated activity persisted for at least 2 h if EGF-R degradation was blocked by methylamine or 18 degrees C incubation. Detection of stimulated autophosphorylation in homogenates of cells treated with EGF in culture required detergent in the assay. Detergent was not necessary to detect stimulated autophosphorylation when EGF was added directly to homogenates of untreated cells. Immunoblots using antibodies against phosphotyrosine (p-Tyr) demonstrated that EGF treatment of intact cells increased the p-Tyr content of at least seven proteins (EGF-R, 115, 100, 75, 66, 57, and 52 kDa) within 5 s. Incubation of intact cells with EGF at 0 degrees C to prevent endocytosis still resulted in tyrosine phosphorylation of these seven proteins. In contrast, several substrates (120, 78, and 38 kDa) showed delayed increases (45-90 s) in tyrosine phosphorylation at 37 degrees C; their phosphorylation was even slower at 18 degrees C and did not occur at 0 degrees C. In cells incubated with EGF at 18 degrees C or in the presence of methylamine, EGF-R p-Tyr in the intact cell was lost by 2 h even though receptor was not degraded and still exhibited enhanced autophosphorylation in the homogenate assay. These findings suggest that tyrosine phosphorylation in response to EGF occurs predominantly during the initial stages of endocytosis and is mediated for the most part by ligand-receptor complexes at the cell surface. A subset of phosphorylations may require intracellular movement.     

Multiple mechanisms are responsible for transactivation of the epidermal growth factor receptor in mammary epithelial cells

The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps.     

Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells.

We have previously reported that antibodies to phosphotyrosine recognize the phosphorylated forms of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors (Zippel et al., Biochim. Biophys. Acta 881:54-61, 1986, and Sturani et al., Biochem. Biophys. Res. Commun. 137:343-350, 1986). In this report, the time course of receptor phosphorylation is investigated. In normal human fibroblasts, ligand-induced phosphorylation of PDGF and EGF receptors is followed by rapid dephosphorylation. However, in A431 cells the tyrosine-phosphorylated form of EGF receptor persists for many hours after EGF stimulation, allowing a detailed analysis of the conditions affecting receptor phosphorylation and dephosphorylation. In A431 cells, the number of receptor molecules phosphorylated on tyrosine was quantitated and found to be about 10% of total EGF receptors. The phosphorylated receptor molecules are localized on the cell surface, and they are rapidly dephosphorylated upon removal of EGF from binding sites by a short acid wash of intact cells and upon a mild treatment with trypsin. ATP depletion also results in rapid dephosphorylation, indicating that continuous phosphorylation-dephosphorylation reactions occur in the ligand-receptor complex at steady state. Phorbol 12-myristate 13-acetate added shortly before EGF reduces the rate and the final extent of receptor phosphorylation. Moreover, it also reduces the amount of phosphorylated receptors if it is added after EGF. Down-regulation of protein kinase C by chronic treatment with phorbol dibutyrate increases the receptor phosphorylation induced by EGF, suggesting a homologous feedback regulation of EGF receptor functions.     

The RhoA-Rok-myosin II pathway is involved in extracellular matrix-mediated regulation of prolactin signaling in mammary epithelial cells

In mammary epithelial cells (MECs), prolactin-induced signaling and gene expression requires integrin-mediated cell adhesion to basement membrane (BM). In the absence of proper cell-BM interactions, for example, culturing cells on collagen-coated plastic dishes, signal propagation is substantially impaired. Here we demonstrate that the RhoA-Rok-myosin II pathway accounts for the ineffectiveness of prolactin signaling in MECs cultured on collagen I. Under these culture conditions, the RhoA pathway is activated, leading to downregulation of prolactin receptor expression and reduced prolactin signaling. Enforced activation of RhoA in MECs cultured on BM suppresses prolactin receptor levels, and prevents prolactin-induced Stat5 tyrosine phosphorylation and β-casein expression. Overexpression of dominant negative RhoA in MECs cultured on collagen I, or inhibiting Rok activity, increases prolactin receptor expression, and enhances prolactin signaling. In addition, inhibition of myosin II ATPase activity by blebbistatin also exerts a beneficial effect on prolactin receptor expression and prolactin signaling, suggesting that tension exerted by the collagen substratum, in collaboration with the RhoA-Rok-myosin II pathway, contributes to the failure of prolactin signaling. Furthermore, MECs cultured on laminin-coated plastic have similar morphology and response to prolactin as those cultured on collagen I. They display high levels of RhoA activity and are inefficient in prolactin signaling, stressing the importance of matrix stiffness in signal transduction. Our results reveal that RhoA has a central role in determining the fate decisions of MECs in response to cell-matrix interactions.     

PRL modulates cell cycle regulators in mammary tumor epithelial cells.

PRL is essential for normal lobulo-alveolar growth of the mammary gland and may contribute to mammary cancer development or progression. However, analysis of the mechanism of action of PRL in these processes is complicated by the production of PRL within mammary epithelia. To examine PRL actions in a mammary cell-specific context, we selected MCF-7 cells that lacked endogenous PRL synthesis, using PRL stimulation of interferon-gamma-activated sequence-related PRL response elements. Derived clones exhibited a greater proliferative response to PRL than control cells. To understand the mechanism, we examined, by Western analysis, levels of proteins essential for cell cycle progression as well as phosphorylation of retinoblastoma protein. The expression of cyclin D1, a critical regulator of the G1/S transition, was significantly increased by PRL and was associated with hyperphosphorylation of retinoblastoma protein at Ser(780). Cyclin B1 was also increased by PRL. In contrast, PRL decreased the Cip/Kip family inhibitor, p21, but not p16 or p27. These studies demonstrate that PRL can stimulate the cell cycle in mammary epithelia and identify specific targets in this process. This model system will enable further molecular dissection of the pathways involved in PRL-induced proliferation, increasing our understanding of this hormone and its interactions with other factors in normal and pathogenic processes.     

Tyrosine phosphorylation of mitogen-activated protein kinase in cells with tyrosine kinase-negative epidermal growth factor receptors.

Epidermal growth factor (EGF) treatment of cells expressing the human EGF receptor (EGFr) results in rapid tyrosine phosphorylation of several cellular proteins including mitogen-activated protein (MAP) kinase. EGF treatment of cells expressing a tyrosine kinase-inactive EGFr failed to induce the tyrosine phosphorylation of endogenous substrates in response to EGF; however, the tyrosine phosphorylation and activation of MAP kinase did occur. This observation indicates that MAP kinase is activated in response to a signal other than the tyrosine kinase activity of the EGFr. Because EGF does not stimulate cells expressing the inactive EGFr to proliferate, phosphorylation of MAP kinase may not be sufficient for the EGF-dependent mitogenesis.     

Secretion of an epidermal growth factor-like growth factor by epidermal growth factor-independent rat mammary carcinoma cells.

Rat mammary carcinoma (RMC) cells derived from serially transplantable mammary tumors are independent of epidermal growth factor (EGF) for long-term growth in serum-free medium. This phenotype is in contrast to that of normal mammary epithelial cells or cells derived from nontransplantable tumors that express an absolute requirement for EGF for growth in culture. The results of the experiments reported here indicate that EGF-independent RMC cells secrete a growth factor with potent EGF-like mitogenic activity. Conditioned media obtained from these cells can substitute for EGF for the growth of the EGF-dependent cell line MCF-10. This growth factor is neither EGF nor transforming growth factor alpha and does not compete with 125I-EGF for binding to EGF receptors. Phosphotyrosine Western blot analysis of lysates obtained from EGF-independent RMC cells revealed the presence of a 190 kilodalton (kDa) protein that was distinct from the EGF receptor. Similarly, growth of MCF-10 cells to confluence in serum-free medium supplemented with conditioned medium growth factor in place of EGF resulted in the disappearance of the EGF receptor band and appearance of the 190 kDa band in phosphotyrosine Western blots. The 190 kDa tyrosine-phosphorylated protein detected in cells stimulated by the conditioned medium factor is unlikely to be the c-erbB-2 protein, as indicated by negative results in immunoprecipitation experiments and in vitro kinase assays. In summary, EGF-independent RMC cells secrete a factor with potent EGF-like mitogenic activity. This suggests that an autocrine loop involving this growth factor mediates EGF independence in these cells.     

Inhibition of protein tyrosine phosphatase activity by diamide is reversed by epidermal growth factor in fibroblasts.

Diamide (azodicarboxylic acid bis(dimethylamide] inhibits protein tyrosine phosphatase activity in fibroblasts without altering protein tyrosine kinase activity associated with the epidermal growth factor receptor. The loss of protein tyrosine phosphatase activity caused by diamide is reversed by 2-mercaptoethanol or epidermal growth factor.     

Linoleate metabolites enhance the in vitro proliferative response of mouse mammary epithelial cells to epidermal growth factor

Linoleic acid, arachidonic acid, prostaglandin E1, and prostaglandin E2 stimulated the proliferation of mammary epithelial cells in serum-free primary cultures only in the presence of epidermal growth factor. Linoleate-stimulated growth was manifest later in culture when proliferation, initiated by epidermal growth factor only, reached a plateau while linoleate-supplemented epidermal growth factor cultures continued to proliferate. The cultures in the plateau phase of growth could be restimulated to grow by adding either linoleic acid or prostaglandin E2 to the media. While the linoleate response could be abolished by the cyclooxygenase inhibitor, indomethacin, prostaglandin E2-stimulated growth remained unaffected. Linoleic acid was metabolized to arachidonic acid and prostaglandin E2, both in the growing and resting cultures. Proliferating cells metabolized linoleate and prostaglandin E2 extensively so that neither the fatty acid nor prostaglandin E2 accumulated in large quantities in the proliferating cultures. The concentrations of prostaglandin E2 in growing cultures supplemented with linoleic acid were much higher than in cultures without it. These results suggest that the metabolism of linoleic acid leading to prostaglandin production, not its contribution to membrane polyunsaturation, is necessary for sustained growth of mammary epithelial cells in the presence of epidermal growth factor.