Proteomics methods for probing molecular mechanisms in signal transduction.

mRNA splicing and various posttranslational modifications to proteins result in a larger number of proteins than genes. Assessing the dynamic nature of this proteome is the challenge of modern proteomics. Recent advances in high throughput methods greatly facilitate the analysis of proteins involved in signal transduction, their production, posttranslational modifications and interactions. Highly reproducible two dimensional polyacrylamide gel electrophoresis (2D-PAGE) methods, coupled with matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS) allow rapid separation and identification of proteins. These methods, alone or in conjunction with other techniques such as immunoprecipitation, allow identification of various critical posttranslational modifications, such as phosphorylation. High throughput identification of important protein-protein interactions is accomplished by yeast two hybrid approaches. In vitro and in vivo pulldown assays, coupled with MALDI-TOF-MS, provide an important alternative to two hybrid approaches. Emerging advances in production of protein-based arrays promise to further increase throughput of proteomics-based approaches to signal transduction.

Production and regulation of leptin in bovine mammary epithelial cells.

Previously, leptin has been found in human and rodent mammary tissue. The present research was conducted to determine (1) if leptin is produced by bovine mammary epithelial cells and (2) if leptin production in bovine mammary epithelial cells is hormonally regulated. Western blot analysis indicated the presence of leptin in bovine milk, while reverse transcription-polymerase chain reaction (RT-PCR) indicated the presence of leptin mRNA in mammary tissue and cultured bovine mammary epithelial cells (MAC-T cell line). A real time RT-PCR method was developed that allowed quantitative assessment of bovine leptin mRNA over approximately 3 orders of magnitude. Time course studies indicated a rapid increase in leptin mRNA in response to insulin or insulin-like growth factor-I (IGF-I). When normalized against bovine GAPDH as an internal control, 0.5 or 1h treatment with 10 ng/mL insulin gave 39+/-4 and 64+/-2-fold increase in leptin mRNA compared with 0 h control. Leptin mRNA was increased 257+/-9 and 75+/-23-fold by 0.5 or 1h treatment with 10 ng/mL IGF-I. Dose response studies indicated significant increases in leptin mRNA in response to as little as 1 ng/mL insulin or 0.1 ng/mL IGF-I. Maximum increase in leptin mRNA was observed in response to 10 ng/mL insulin and 10 ng/mL IGF-I. These results indicate that production of leptin by bovine mammary epithelial cells can be regulated by factors known to alter mammary function and nutrient partitioning. This suggests that leptin may be an autocrine/paracrine signal in the bovine mammary gland.

Regional distribution of cortical microglia parallels that of neurofibrillary tangles in Alzheimer's disease.

It has been postulated that microglia contribute to the development of neurofibrillary tangles (NFT) in Alzheimer's disease (AD). We compared the distribution of microglia with that of NFT in both AD and non-AD cases. In AD cases, we found that the extent of area covered by Ricinus communic agglutinin-1 labeled microglia generally paralleled NFT frequency and distribution. Microglia occupied the greatest area in tangle-rich periallocortex/allocortex, a lesser area in association cortex, and the smallest area in tangle-poor primary cortex. Interestingly, this pattern was also present in non-AD cases where there were few to no NFT. These findings suggest that regional variations in microglial distribution may constitute, at least in part, a template for the development of NFT.

Epidermal growth factor as an autocrine modulator of stress response in mammary epithelial cells.

Mouse mammary epithelial cells (NMuMG line) were heat shocked at 42 degreesC for up to 2 h, with continuous culture at 37 degreesC used as controls. EGF mRNA expression, determined by Northern blot hybridization, was increased 4- to 5-fold in heat-shocked cells compared with controls. Heat shock also induced accumulation of EGF immunoreactive protein in both media and cell lysates of NMuMG cells. Oligonucleotides antisense to the EGF mRNA blocked the accumulation of EGF protein, while sense controls were without effect. Antisense oligonucleotide concentrations that inhibited EGF production by NMuMG cells during heat shock dramatically reduced the ability of cells to survive heat shock, while sense oligonucleotide did not affect cell survival. The antisense oligonucleotide inhibition of cell survival was reversed by adding EGF during the heat shock period. Thus the production of EGF or EGF-like peptides during periods of cellular stress may be an important survival mechanism in mammary epithelium.

Hormonal regulation of epidermal growth factor receptor content and signaling in bovine mammary tissue.

Mammary tissue from midpregnant heifers was cultured with epidermal growth factor (EGF) or transforming growth factor alpha for 1-3 days. After 1 day, 10 nM EGF or transforming growth factor alpha doubled DNA synthesis, whereas lower concentrations (0.1 or 1 nM) increased DNA synthesis 2- to 3-fold after 2-3 days in culture. In other studies, bovine mammary tissue was transplanted to ovariectomized athymic mice and treated for 10 days with saline, estradiol (1 microg/day), progesterone (1 mg/day), or estradiol + progesterone. Subsequent explant culture of the bovine tissue indicated that estradiol + progesterone augmented the ability of EGF to stimulate DNA synthesis. The increased response to EGF was associated with increased EGF binding and with increased EGF-induced tyrosine kinase that paralleled the increased EGF binding. In other studies, athymic mice bearing xenografted bovine mammary tissue were primed for 10 days with estradiol and progesterone, followed by 2-day treatment with saline (control), hydrocortisone (200 microg/day), PRL (1 mg/day), or hydrocortisone + PRL. Hydrocortisone and PRL alone decreased, and PRL + hydrocortisone eliminated, EGF-induced DNA synthesis. EGF receptor content was unaffected by hydrocortisone but was reduced by PRL or hydrocortisone + PRL. Furthermore, the ability of EGF to induce tyrosine kinase activity was decreased by PRL and by hydrocortisone + PRL. The decreased kinase activity was greater than the decrease in receptor binding, suggesting a specific modulation of EGF receptor kinase activity in response to lactogenic hormones.

C-Src activation by ErbB2 leads to attachment-independent growth of human breast epithelial cells.

Nontumorigenic human mammary epithelial cells (184.A1 line) were stably transfected with ErbB2 or with Ha-Ras. Transformation with ErbB2, but not ras, resulted in a 5-6 fold increase in c-src activity without affecting c-src content of cells. Similar activation of c-src by ErbB2 was also observed in other non-tumorigenic mammary epithelial cells, including the human line MCF10A and the mouse line NMuMG. Activation of c-src appeared to be dependent on active ErbB2 tyrosine kinase, as the ErbB2 inhibitor tyrphostin AG 825 blocked the induction of c-src kinase activity, as well as the ability of transformed cells to grow on soft agar, but not plastic. The src-selective inhibitor PP1 effectively reduced c-src activity, as well as growth of ErbB2-transformed cells on soft agar, but not on plastic. These results indicate that activation of c-src is a consequence of ErbB2 kinase activity in human breast cancer cells overexpressing ErbB2, and that increased activity of c-src may be responsible for attachment-independent growth of the cells.

Regulation of epidermal growth factor expression in mammary epithelial cells by a Yin-Yang-1-like element.

The hormone combination of insulin, dexamethasone and prolactin induced accumulation of preproepidermal growth factor (EGF) mRNA in HC11 mouse mammary epithelial cells 16-24 h after the hormones were added to the cultures. Individual hormones or combinations of two of the hormones had no effect on EGF mRNA concentrations. The same hormone combination was capable of inducing expression of a reporter gene construct containing -888 to +25 bp of the EGF gene fused with luciferase. Deletions of the promoter between -888 and -271 bp had no detectable effect on basal or hormone-induced reporter gene expression. However, further deletion from -270 to -74 bp increased baseline to approximately equal hormone-induced reporter gene expression. This deletion also abolished the hormone-induced increase in reporter gene expression. Sequence analysis suggested that this region contained a binding site for Yin-Yang-1 (YY1), which was confirmed by gelshift analysis. Mutation of the YY1 binding site increased baseline reporter gene expression to the same level as induced by insulin, dexamethasone and prolactin in the wild-type promoter. These results indicate that expression of the EGF gene in mammary epithelium is repressed by the YY1 site, and that removal of repression may play a part in regulating EGF gene expression in lactating mammary tissue.

Adenosine stimulation of DNA synthesis in mammary epithelial cells.

Adenosine increased the DNA synthesis rate and the percentage of S-phase cells 2-3-fold in mouse mammary epithelial cells (NMuMG), with an optimum concentration of 10-100 microM. This effect was not mimicked by adenosine metabolites adenine, hypoxanthine, or inosine. N-ethylcarboxamidoadenosine (NECA, a relatively nonselective adenosine receptor agonist) and 2-p-(2-carboxyethyl) phenethylamino-5'-N ethylcarbox-amidoadenosine (CGS-21680, an A2 selective agonist) also increased DNA synthesis by mammary epithelial cells. However, N6-cyclohexyladenosine (CHA, an agonist for A1 type receptors) decreased DNA synthesis. The A1 selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) had no effect on basal or adenosine-induced DNA synthesis, whereas the A2 selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) decreased adenosine-induced DNA synthesis. Similar effects were observed in another nontumorigenic mouse mammary epithelial line, HC11, as well as the nontumorigenic human lines MCF-10A and 184.A1. Binding studies indicated that NMuMG cells contained approximately 3200 A1 receptors and about 5300 A2 receptors per cell. Both CGS-21680 and CHA increased GTPase activity in isolated cell membranes, whereas only CGS-21680 increased activity of adenylyl cyclase. Adenosine and CGS-21680 increased expression of a cyclic AMP responsive reporter gene. In addition, the protein kinase A inhibitor H-89 blocked the ability of adenosine and CGS-21680 to induce DNA synthesis, but did not affect EGF-induced DNA synthesis. These results indicate that adenosine appears to be a possible growth promoting agent in mammary tissue, and this effect may be mediated by extracellular receptors of the A2 type.

Microglial expression of MHC class II increases in normal aging of nonhuman primates.

The search for factors that influence age-related behavioral and cognitive slowing is ongoing. Because microglia are involved in many neurological disorders, they may also contribute to changes in normal aging. To assess increases in microglial activity, we used an antibody against MHC class II to label microglia in three groups of brains from female Macaca nemestrina: juvenile (2-5 years), young adult (5-11 years), and mature (11-19 years). Image analysis was completed on four white matter and three gray matter regions in a single coronal plane. Microglial expression of MHC class II increased with age, and was highest in the white matter regions of the mature, or middle-aged, monkeys. The higher expression of this antigen may indicate that the cells will more easily respond to stimulation. Their location in the white matter suggest that they may influence myelin loss and the eventual cognitive decline in aged human and nonhuman primates.

Prolactin decreases epidermal growth factor receptor kinase activity via a phosphorylation-dependent mechanism.

Previously, we have shown that prolactin inhibits epidermal growth factor (EGF)-induced mitogenesis in mouse mammary epithelial cells without altering the response to other growth promoting agents. This effect has been associated with reduced EGF-induced EGF receptor (EGFR) tyrosine phosphorylation, Grb-2 association, and Ras activation. Our current hypothesis is that prolactin induces an alteration in EGFR kinase activity via a phosphorylation-dependent mechanism. To test this hypothesis, we treated normal murine mammary gland cells with or without 100 ng/ml prolactin. EGFR isolated by wheat germ agglutinin affinity chromatography from nontreated cells exhibited substantial ligand-induced phosphorylation, and EGFR isolated from prolactin-treated cells displayed minimal EGF-induced EGFR phosphorylation, as well as decreased kinase activity toward exogenous substrates. The observed decrease in ligand-induced EGFR phosphorylation could not be attributed to either differential amounts of EGFR, decreased EGF binding affinity, or the presence of a phosphotyrosine phosphatase or ATPase. EGFR isolated from prolactin-treated cells exhibited increased phosphorylation on threonine. Removal of this phosphorylation with alkaline phosphatase restored EGFR kinase activity to levels observed in nontreated cells. Therefore, these results suggest that prolactin antagonizes EGF signaling by increasing EGFR threonine phosphorylation and decreasing EGF-induced EGFR tyrosine phosphorylation.

Gradient of microglial activation in the brain of SIV infected macaques.

Brains of macaques inoculated with macrophage-tropic, neurovirulent virus 7F, with lymphocyte-tropic SIV mac239, or with dual-tropic SIVmac239/1yE, were examined for microglial activation, astrocyte activation, apoptosis and neuron loss. The brain one animal inoculated with neurovirulent virus 7f showed massive microglial activation as assessed by expression of the major histo-compatibility complex class II (MHC-II). In this animal very numerous, large microglial nodules expressing MHC-II were concentrated in the basal pons and internal capsule. These microglial nodules contained cells undergoing apoptosis detected by in situ end labeling of fragmented DNA. In this animal, neuron loss was apparent near the microglial nodules. In the animals inoculated with SIVmac239 or SIVmac239/17E, pathologic changes such as perivascular cuffing and formation of microglial nodules were absent. However, increased expression of MHC-11 by microglial cells was also concentrated in white matter of the basal pons, midbrain and internal capsule. These results indicate the microglial activation in SIV-infected macaques follows a ventral to dorsal gradient regardless of viral tropism. These results also show that the type and severity of neuropathological changes in SIV-infected macaques is highly dependent on the tropism of the inoculated virus.

Prolactin inhibits EGF-induced DNA synthesis in mammary epithelium via early signaling mechanisms: possible involvement of protein kinase C.

Prolactin and prolactin agonists inhibited EGF-induced DNA synthesis in mammary epithelium, whereas other pituitary hormones had no effect on EGF-induced DNA synthesis. The inhibitory effect of prolactin was seen for EGF and TGF-alpha, but not for IGF-I or cholera toxin. Autoradiography indicated that prolactin decreased the ability of EGF to induce cells to progress to S phase of the cell cycle, and time course studies indicated that the effects of prolactin were not due to an altered timing of DNA synthesis induction. Prolactin addition within 30 min of adding EGF was necessary to inhibit EGF-induced DNA synthesis. Conditioned media from prolactin-treated cells from which prolactin had been neutralized with the extracellular domain of the prolactin receptor had no effect on EGF-induced DNA synthesis, suggesting that the effect was due to prolactin, not an autocrine factor induced by prolactin. Prolactin induced a rapid association of protein kinase C with the membrane fraction of NMuMG cells, as well as increased threonine phosphorylation of the EGF receptor. Protein kinase C inhibitors eliminated most of the inhibitory effect of prolactin on EGF-induced DNA synthesis. The protein kinase C inhibitor Calphostin C restored high-affinity EGF binding in prolactin-treated cells and reversed the inhibitory effect of prolactin on EGF-induced EGF receptor tyrosine phosphorylation.

Mastitis increases growth factor messenger ribonucleic acid in bovine mammary glands.

Multiparous Holstein cows were infected in two quarters by intramammary infusion with Streptococcus agalactiae and slaughtered approximately 36 h later. Mammary tissue was removed from the infected quarters, uninfected contralateral quarters, and from pair-slaughtered uninfected controls; the tissue was frozen in liquid nitrogen. The RNA was extracted, and Northern blot analysis was performed for a variety of growth factors, stress-induced genes, milk protein genes, and control genes. Infection increased levels of mRNA coding for heat shock proteins 89 alpha, 89 beta, 70, 60, and 27. Simultaneously, concentrations of alpha-lactalbumin and casein mRNA decreased; alpha-lactalbumin mRNA showed a greater decline. The mRNA for several growth factors, including acidic fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor, transforming growth factor-alpha, IGF-I, and IGF-II, were also increased as was the apoptosis marker, testosterone-repressed prostate mucin-2. Concentrations of mRNA for controls, beta-actin, and glyceraldehyde-3-phosphate dehydrogenase were unaffected. These results indicate that mastitis induces changes in the levels of mRNA encoding for a variety of peptide growth factors. Such changes in growth factors could be important in a variety of processes that occur during infection, such as protection against injury or tissue repair and recovery processes.

Effects of epidermal growth factor (EGF) and prolactin on EGF receptor cytoskeletal association in mammary epithelial cells.

Prolactin treatment of NMuMG mammary epithelial cells inhibits the ability of epidermal growth factor (EGF) to transduce a variety of signals, possibly by interfering with receptor tyrosine phosphorylation. However, the mechanism by which prolactin inhibits EGF receptor signaling is unclear. The objective of this study was to evaluate the effects of prolactin on the dynamics of EGF receptor degradation and resynthesis, and on the association of the receptor with the cytoskeleton. EGF decreased the EGF receptor content of NMuMG cells, and this decrease was unaffected by prolactin treatment. Subsequent to the decrease in EGF receptors, cells reaccumulated EGF receptors, and this re-accumulation was also unaffected by prolactin. In other studies, EGF induced a rapid association of EGF receptor with Triton X-100-insoluble (cytoskeletal) elements. The cytoskeletally associated receptors were more heavily tyrosine phosphorylated than soluble receptors in the absence of prolactin. In the presence of prolactin, similar amounts of EGF receptor associated with the cytoskeleton, but both cytoskeletal and soluble receptors exhibited decreased tyrosine phosphorylation. These studies indicate that the effects of prolactin on EGF receptor signaling are not likely to be due to altered receptor dynamics or cytoskeletal association but are more likely due to an alteration in receptor kinase activity.

Involvement of c-src in beta-casein expression by mammary epithelial cells.

HC11 mouse mammary epithelial cells were stably transfected with c-src or a dominant negative mutant of c-src driven by a MMLV promoter. Prolactin increased c-src activity in control and c-src transfected cells, while dominant negative c-src reduced the prolactin-induced increase in c-src activity. Dominant negative c-src also reduced the ability of insulin, hydrocortisone, and prolactin to induce beta-casein accumulation to levels one-half to one-third that of control cells. This effect was uncorrelated with any change in cell proliferation or laminin accumulation and was observed both in cultures grown to confluency in the presence of EGF and in cultures grown on laminin.

Prolactin inhibits epidermal growth factor-induced Ras-MAPK signaling in mammary epithelial cells.

Previously, our laboratory has shown that prolactin (PRL) inhibits epidermal growth factor (EGF)-induced DNA synthesis. One pathway for the initiation of DNA synthesis is EGF-receptor (EGF-R) signaling through Ras and mitogen-activated protein kinase (MAPK). To determine the effects of PRL on EGF-induced MAPK activation and phosphorylation, MAPK or phosphotyrosine (Tyr(P)) was immunoprecipitated from normal murine mammary epithelial (NMuMG) cells treated with PRL (100 ng/ml) and/or EGF (10 ng/ml) for 10-min periods. EGF-induced phosphorylation and activation were then examined by Western analysis and a myelin basic protein (MBP)-specific kinase assay. The p42 isoform of MAPK showed a distinct decrease in activity and phosphorylation when cells were treated with PRL. Concluding that PRL affects EGF signaling upstream of MAPK, we examined the effect of PRL on EGF-induced Ras activity. NMuMG cells were incubated with [32P]orthophosphoric acid, treated as described above, immunoprecipitated with an antibody specific to Ras, and nucleotides were eluted and separated by TLC. Ras activity as measured by GTP:GDP ratio was increased by EGF, but not by PRL. Additionally, PRL in combination with EGF abolished the ability of EGF to induce Ras activity. Those studies suggest that PRL alters the EGF signaling pathway upstream of Ras. Because Ras activation by EGF involves EGF-stimulated association of EGF-R with Grb2, the EGF-R was immunoprecipitated and a Western blot was probed for Grb2. As expected we found that EGF stimulated an association of EGF-R with Grb2, PRL, however, blocked this association. When we looked at the ability of Shc to associate with the EGF-R, we found that PRL and EGF had little effect on this association. The studies demonstrate that PRL either directly or indirectly inhibits the ability of EGF to induce EGF-R association with Grb2, to activate Ras, and to activate and phosphorylate MAPK.

Photoreactive (caged) cyclic AMP analogs induce DNA synthesis in mammary epithelial cells.

Dimethoxy-2-nitrobenzyl adenosine-3',5' cyclic monophosphate (DMNB) is a metabolically active, photolabile cyclic AMP analog that yields free cyclic AMP upon UV hydrolysis. The analog is useful in that it permits short term, transient elevations of intracellular cyclic AMP. Addition of DMNB (1-10 microM) to mouse mammary epithelial cells, followed by UV irradiation of cells, caused a significant increase in DNA synthesis over that observed with controls, UV irradiation alone or DMNB alone. In subsequent studies, DMNB exhibited a modest, but statistically significant, interaction with epidermal growth factor in promoting DNA synthesis. Effects of DMNB were observed if DNA synthesis was measured as either 3H-thymidine incorporation into DNA or as percent S-phase cells. These results indicate that previously observed effects of agents such as cholera toxin and phosphodiesterase resistant cyclic AMP analogs on mammary epithelial proliferation can be mimicked, at least in part, by a short term pulse of cyclic AMP.

Binding of epidermal growth factor and transforming growth factor-alpha in mammalian preimplantation embryos.

Preimplantation embryos of the pig (Days 11 to 15), cow (Days 14 to 16), sheep (Day 14) and pony (Day 16) bind epidermal growth factor (EGF) specifically. Binding was not detected in embryos of the rabbit at Day 5 or 6 or the hamster at Day 3. Transforming growth factor-alpha displaced [(125)I] EGF in pig, cow and pony embryos almost as much as unlabeled EGF. The binding affinities of EGF ranged from 12 to 233 pM in pig and cow embryos. The range of species and binding features indicate that the EGF family may play a significant role in mammalian preimplantation development.

Control of mammary epithelial cell DNA synthesis by epidermal growth factor, cholera toxin, and IGF-1: specific inhibitory effect of prolactin on EGF-stimulated cell growth.

Epidermal growth factor (EGF) stimulates mouse mammary cell proliferation in vivo, but its maximal level in the mammary gland is at midlactation, when little mammary growth takes place. The present studies use a normal murine mammary gland epithelial cell line (NMuMG) to determine the effects of the mammary mitogens EGF, cholera toxin (CT), and insulin-like growth factor-1 (IGF-1) on cell proliferation and how cell growth is altered by addition of the lactogenic hormone prolactin (PRL). EGF and CT stimulated over a fourfold increase of DNA synthesis in NMuMG cells when compared to basal levels. Only a twofold stimulation of DNA synthesis was observed when cells were treated with IGF-1. There was a slight increase in the percentage of cells in S-phase when these agents were added in combination with each other. Physiological levels of PRL had no significant effect on CT- or IGF-1-induced DNA synthesis but reduced EGF-stimulated cell proliferation to basal levels. Furthermore, PRL reduced the percentage of cells in S-phase to IGF-1- and CT-induced levels in the presence of EGF. Interestingly, PRL increased cellular levels of EGF mRNA after 2 h of treatment, which is similar to the response of mouse mammary glands cultured in lactogenic hormones. We conclude that even though PRL can increase the amount of EGF mRNA in mammary epithelial cells, it also eliminates EGF-induced mitogenesis.

Epidermal growth factor modulates cholera toxin induced mammary gland development.

Ovariectomized mice were either sham operated or sialoadenectomized and injected daily for 18 days with saline, estradiol + progesterone, cholera toxin or estradiol + progesterone+cholera toxin. Mammary development score and DNA were increased by estradiol + progesterone, but not by cholera toxin alone. In combination with estradiol + progesterone, cholera toxin increased mammary development score and mammary DNA. Sialoadenectomy reduced the ability of estradiol, progesterone and cholera toxin to induce mammary development. In other experiments, mice were primed with estradiol + progesterone for 10 days, and mammary tissue removed for in vitro culture with various combinations of insulin, aldosterone, cholera toxin and epidermal growth factor. In combination with insulin and aldosterone, cholera toxin increased mammary development in vitro. Sialoadenectomy reduced the ability of cholera toxin to induce mammary development in vitro. The effect of sialoadenectomy on mammary development was alleviated by adding epidermal growth factor to culture medium. Biochemical studies indicated that sialoadenectomy reduced the ability of estrogen and progesterone to induce cyclic AMP dependent protein kinase levels in mammary tissue, and also the ability of cholera toxin to induce accumulation of cyclic AMP in tissues. These effects of sialoadenectomy were reversed by addition of EGF to culture media.