Structure of the EGF receptor transactivation circuit integrates multiple signals with cell context.

Transactivation of the epidermal growth factor receptor (EGFR) is thought to be a process by which a variety of cellular inputs can be integrated into a single signaling pathway through either stimulated proteolysis (shedding) of membrane-anchored EGFR ligands or by modification of the activity of the EGFR. As a first step towards building a predictive model of the EGFR transactivation circuit, we quantitatively defined how signals from multiple agonists were integrated both upstream and downstream of the EGFR to regulate extracellular signal regulated kinase (ERK) activity in human mammary epithelial cells. By using a "non-binding" reporter of ligand shedding, we found that transactivation triggers a positive feedback loop from ERK back to the EGFR such that ligand shedding drives EGFR-stimulated ERK that in turn drives further ligand shedding. Importantly, activated Ras and ERK levels were nearly linear functions of ligand shedding and the effect of multiple, sub-saturating inputs was additive. Simulations showed that ERK-mediated feedback through ligand shedding resulted in a stable steady-state level of activated ERK, but also showed that the extracellular environment can modulate the level of feedback. Our results suggest that the transactivation circuit acts as a context-dependent integrator and amplifier of multiple extracellular signals and that signal integration can effectively occur at multiple points in the EGFR pathway.

Three-dimensional culture conditions lead to decreased radiation induced cytotoxicity in human mammary epithelial cells.

For both targeted and non-targeted exposures, the cellular responses to ionizing radiation have predominantly been measured in two-dimensional monolayer cultures. Although convenient for biochemical analysis, the true interactions in vivo depend upon complex interactions between cells themselves and the surrounding extracellular matrix. This study directly compares the influence of culture conditions on radiation induced cytotoxicity following exposure to low-LET ionizing radiation. Using a three-dimensional (3D) human mammary epithelial tissue model, we have found a protective effect of 3D cell culture on cell survival after irradiation. The initial state of the cells (i.e., 2D versus 3D culture) at the time of irradiation does not alter survival, nor does the presence of extracellular matrix during and after exposure to dose, but long term culture in 3D which offers significant reduction in cytotoxicity at a given dose (e.g. approximately 4-fold increased survival at 5Gy). The cell cycle delay induced following exposure to 2 and 5Gy was almost identical between 2D and 3D culture conditions and cannot account for the observed differences in radiation responses. However the amount of apoptosis following radiation exposure is significantly decreased in 3D culture relative to the 2D monolayer after the same dose. A likely mechanism of the cytoprotective effect afforded by 3D culture conditions is the down regulation of radiation induced apoptosis in 3D structures.

Basic fibroblast growth factor regulates persistent ERK oscillations in premalignant but not malignant JB6 cells.

The regulation of extracellular signal-regulated kinase (ERK) oscillations in the context of wound healing and carcinogenesis have been investigated in premalignant and malignant JB6 mouse epidermal cells stimulated with basic fibroblast growth factor (bFGF) and 12-O-tetradecanoyl phorbol-13-acetate (TPA). In premalignant JB6 cells, bFGF stimulation (1) increases cellular phospho-ERK and phospho-c-Jun levels, (2) increases serum-dependent cell proliferation, (3) induces an apparent epithelial-to-mesenchymal transition, and (4) induces the persistent nuclear-cytosolic oscillation of an ERK1-green fluorescent protein (ERK1-GFP) chimera. In contrast, TPA induces persistent activation of ERK in the absence of oscillations and does not induce efficient migration. Treatment of malignant or transformed JB6 cells with bFGF is associated with a transient nuclear translocation of ERK1-GFP but not oscillations or efficient cell migration. Our data suggest that bFGF regulates ERK oscillations in premalignant but not malignant JB6 cells.

Rapid and sustained nuclear-cytoplasmic ERK oscillations induced by epidermal growth factor.

Although the ERK pathway has a central role in the response of cells to growth factors, its regulatory structure and dynamics are incompletely understood. To investigate ERK activation in real time, we expressed an ERK-GFP fusion protein in human mammary epithelial cells. On EGF stimulation, we observed sustained oscillations of the ERK-GFP fusion protein between the nucleus and cytoplasm with a periodicity of approximately 15 min. The oscillations were persistent (>45 cycles), independent of cell cycle phase, and were highly dependent on cell density, essentially disappearing at confluency. Oscillations occurred even at ligand doses that elicited very low levels of ERK phosphorylation, and could be detected biochemically in both transfected and nontransfected cells. Mathematical modeling revealed that negative feedback from phosphorylated ERK to the cascade input was necessary to match the robustness of the oscillation characteristics observed over a broad range of ligand concentrations. Our characterization of single-cell ERK dynamics provides a quantitative foundation for understanding the regulatory structure of this signaling cascade.

HER/ErbB receptor interactions and signaling patterns in human mammary epithelial cells.

BACKGROUND: Knowledge about signaling pathways is typically compiled based on data gathered using different cell lines. This approach implicitly assumes that the cell line dependence is not important. However, different cell lines do not always respond to a particular stimulus in the same way, and lack of coherent data collected from closely related cellular systems can be detrimental to the efforts to understand the regulation of biological processes. To address this issue, we created a clone library of human mammary epithelial (HME) cells that expresses different levels of HER2 and HER3 receptors in combination with endogenous EGFR/HER1. Using our clone library, we have quantified the receptor activation patterns and systematically tested the validity of the existing hypotheses about the interaction patterns between HER1-3 receptors. RESULTS: Our study identified HER2 as the dominant dimerization partner for both EGFR and HER3. Contrary to earlier suggestions, we find that lateral interactions with HER2 do not lead to strong transactivation between EGFR and HER3, i.e., EGFR activation and HER3 activation are only weakly linked in HME cells. We also find that observed weak transactivation is uni-directional where stimulation of EGFR leads to HER3 activation whereas HER3 stimulation does not activate the EGFR. Repeating our experiments at lower cell confluency established that cell confluency is not a major factor in the observed interaction patterns. We have also quantified the dependence of the kinetics of Erk and Akt activation on different HER receptors. We found that HER3 signaling makes the strongest contribution to Akt activation and that, stimulation of either EGFR or HER3 leads to significant Erk activation. CONCLUSION: Our study shows that clone cell libraries can be a powerful resource in systems biology research by making it possible to differentiate between various hypotheses in a consistent cellular background. Using our constructed clone library we profiled the cell signaling patterns to establish the role of HER2 in the crosstalk between EGFR and HER3 receptors in HME cells. Our results for HME cells show that the weak linkage between EGFR and HER3 pathways can lead to distinct downstream cellular signaling patterns in response to the ligands of these two receptors.

Regulation of the low-dose radiation paracrine-specific anchorage-independent growth response by annexin A2.

Here we identify the release of annexin A2 into the culture medium in response to low-dose X-radiation exposure and establish functional linkages to an established paracrine factor-mediated anchorage-independent growth response. Using a standard bicameral coculture model, we demonstrate that annexin A2 is secreted into the medium by irradiated cells (seeded in upper chamber) and is capable of binding to nonirradiated neighboring cells (seeded in lower chamber). The paracrine factor-mediated anchorage-independent growth response to low-dose X irradiation is reduced when irradiated annexin A2-silenced (shRNA) JB6 cells are co-cultured with nonirradiated cells relative to co-culture with irradiated annexin A2-competent vector control cells. Consistent with this observation, purified bovine annexin A2 tetramer induces anchorage-independent growth. These observations suggest that annexin A2 regulates, in part, the radiation paracrine factor-specific anchorage-independent growth response in JB6 cells.

Cellular dichotomy between anchorage-independent growth responses to bFGF and TPA reflects molecular switch in commitment to carcinogenesis.

We have investigated gene expression patterns underlying reversible and irreversible anchorage-independent growth (AIG) phenotypes to identify more sensitive markers of cell transformation for studies directed at interrogating carcinogenesis responses. In JB6 mouse epidermal cells, basic fibroblast growth factor (bFGF) induces an unusually efficient and reversible AIG response, relative to 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced AIG which is irreversible. The reversible and irreversible AIG phenotypes are characterized by largely nonoverlapping global gene expression profiles. However, a subset of differentially expressed genes were identified as common to reversible and irreversible AIG phenotypes, including genes regulated in a reciprocal fashion. Hepatic leukemia factor (HLF) and D-site albumin promoter-binding protein (DBP) were increased in both bFGF and TPA soft agar colonies and selected for functional validation. Ectopic expression of human HLF and DBP in JB6 cells resulted in a marked increase in TPA- and bFGF-regulated AIG responses. HLF and DBP expression were increased in soft agar colonies arising from JB6 cells exposed to gamma radiation and in a human basal cell carcinoma tumor tissue, relative to paired nontumor tissue. Subsequent biological network analysis suggests that many of the differentially expressed genes that are common to bFGF- and TPA-dependent AIG are regulated by c-Myc, SP-1, and HNF-4 transcription factors. Collectively, we have identified a potential molecular switch that mediates the transition from reversible to irreversible AIG.

An extensive survey of tyrosine phosphorylation revealing new sites in human mammary epithelial cells.

Protein tyrosine phosphorylation represents a central regulatory mechanism in cell signaling. Here, we present an extensive survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell (HMEC) line by applying antiphosphotyrosine peptide immunoaffinity purification coupled with high sensitivity capillary liquid chromatography tandem mass spectrometry. A total of 481 tyrosine phosphorylation sites (covered by 716 unique peptides) from 285 proteins were confidently identified in HMEC following the analysis of both the basal condition and acute stimulation with epidermal growth factor (EGF). The estimated false discovery rate was 1.0% as determined by searching against a scrambled database. Comparison of these data with existing literature showed significant agreement for previously reported sites. However, we observed 281 sites that were not previously reported for HMEC cultures and 29 of which have not been reported for any human cell or tissue system. The analysis showed that a majority of highly phosphorylated proteins were relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed, raising the possibility of more important functional roles for such highly phosphorylated pTyr sites. By mapping to major signaling networks, such as the EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which provides interesting targets for future hypothesis-driven and targeted quantitative studies involving tyrosine phosphorylation in HMEC or other human systems.

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.

Quantifying the effects of co-expressing EGFR and HER2 on HER activation and trafficking.

The human epidermal growth factor receptor (HER) system is an intricately regulated system that plays critical roles in development and tumorigenesis. Here, we apply integrated experimentation and modeling to analyze HER receptor activation in a panel of cell lines expressing endogenous levels of EGFR/HER1 and different levels of HER2. A mathematical model that includes the fundamental processes involved in receptor activation and trafficking was used to fit the experimental data, and values of the independent parameters for active receptor dimer formation affinities, trafficking rates and relative phosphorylation levels were estimated. Obtained parameter values quantitatively support the existing ideas on the effect of HER2 on EGFR dynamics, and enable us to predict the abundances of various phosphorylated receptor dimers in the cell lines.

EGF-receptor-mediated mammary epithelial cell migration is driven by sustained ERK signaling from autocrine stimulation.

EGF family ligands are synthesized as membrane-anchored precursors whose proteolytic release yields mature diffusible factors that can activate cell surface receptors in autocrine or paracrine mode. Expression of these ligands is altered in pathological states and in physiological processes, such as development and tissue regeneration. Despite the widely documented biological importance of autocrine EGF signaling, quantitative relationships between protease-mediated ligand release and consequent cell behavior have not been rigorously investigated. We thus explored the relationship between autocrine EGF release rates and cell behavioral responses along with activation of ERK, a key downstream signal, by expressing chimeric ligand precursors and modulating their proteolytic shedding using a metalloprotease inhibitor in human mammary epithelial cells. We found that ERK activation increased monotonically with increasing ligand release rate despite concomitant downregulation of EGF receptor levels. Cell migration speed was directly related to ligand release rate and proportional to steady-state phospho-ERK levels. Moreover, migration speed was significantly greater for autocrine stimulation compared with exogenous stimulation, even at comparable phospho-ERK levels. By contrast, cell proliferation rates were approximately equivalent at all ligand release rates and were similar regardless of whether the ligand was presented endogenously or exogenously. Thus, in our mammary epithelial cell system, migration and proliferation are differentially sensitive to the mode of EGF ligand presentation.

Cellular trafficking of phospholamban and formation of functional sarcoplasmic reticulum during myocyte differentiation.

Phospholamban (PLB) associates with the Ca(2+)-ATPase in sarcoplasmic reticulum (SR) membranes to permit the modulation of contraction in response to beta-adrenergic signaling. To understand how coordinated changes in the abundance and intracellular trafficking of PLB and the Ca(2+)-ATPase contribute to the maturation of functional muscle, we measured changes in abundance, location, and turnover of endogenous and tagged proteins in myoblasts and during their differentiation. We found that PLB is constitutively expressed in both myoblasts and differentiated myotubes, whereas abundance increases of the Ca(2+)-ATPase coincide with the formation of differentiated myotubes. We observed that PLB is primarily present in highly mobile vesicular structures outside the endoplasmic reticulum, irrespective of the expression of the Ca(2+)-ATPase, indicating that PLB targeting is regulated through vesicle trafficking. Moreover, using pulse-chase methods, we observed that in myoblasts, PLB is trafficked through directed transport through the Golgi to the plasma membrane before endosome-mediated internalization. The observed trafficking of PLB to the plasma membrane suggests an important role for PLB during muscle differentiation, which is distinct from its previously recognized role in the regulation of the Ca(2+)-ATPase.

High speed method for in situ multispectral image registration.

High speed data registration is required for the study of fluorescence resonance energy transfer in real time as well as fast dynamic intra- and inter-cellular signaling events. Multispectral confocal spinning disk microscopy provides a high resolution method for performing such real time live cell imaging. However, optical distortions and the physical misalignments introduced by the use of multiple acquisition cameras can obscure spatial information contained in the captured images. In this manuscript, we describe a multispectral method for real time image registration whereby the image from one camera is warped onto the image from a second camera via a polynomial correction. This method provides a real time pixel-for-pixel match between images obtained over physically distinct optical paths. Using an in situ calibration method, the polynomial is characterized by a set of coefficients, using a least squares solver. Error analysis demonstrates optimal performance results from the use of cubic polynomials. High-speed evaluation of the warp is then performed through forward differencing with fixed-point data types. Forward differencing is an iterative approach for evaluating polynomials on the condition that the function variable changes with constant steps. Image reconstruction errors are reduced through bilinear interpolation. The registration techniques described here allow for successful registration of multispectral images in real time (exceeding 15 frame/s) and have a broad applicability to imaging methods requiring pixel matching over multiple data channels.

Cholesterol dictates the freedom of EGF receptors and HER2 in the plane of the membrane.

The flow of information through the epidermal growth factor receptor (EGFR) is shaped by molecular interactions in the plasma membrane. The EGFR is associated with lipid rafts, but their role in modulating receptor mobility and subsequent interactions is unclear. To investigate the role of nanoscale rafts in EGFR dynamics, we used single-molecule fluorescence imaging to track individual receptors and their dimerization partner, human epidermal growth factor receptor 2 (HER2), in the membrane of human mammary epithelial cells. We found that the motion of both receptors was interrupted by dwellings within nanodomains. EGFR was significantly less mobile than HER2. This difference was likely due to F-actin because its depolymerization led to similar diffusion patterns between the EGFR and HER2. Manipulations of membrane cholesterol content dramatically altered the diffusion pattern of both receptors. Cholesterol depletion led to almost complete confinement of the receptors, whereas cholesterol enrichment extended the boundaries of the restricted areas. Interestingly, F-actin depolymerization partially restored receptor mobility in cholesterol-depleted membranes. Our observations suggest that membrane cholesterol provides a dynamic environment that facilitates the free motion of EGFR and HER2, possibly by modulating the dynamic state of F-actin. The association of the receptors with lipid rafts could therefore promote their rapid interactions only upon ligand stimulation.

The membrane-anchoring domain of epidermal growth factor receptor ligands dictates their ability to operate in juxtacrine mode.

All ligands of the epidermal growth factor (EGF) receptor (EGFR) are synthesized as membrane-anchored precursors. Previous work has suggested that some ligands, such as EGF, must be proteolytically released to be active, whereas others, such as heparin-binding EGF-like growth factor (HB-EGF) can function while still anchored to the membrane (i.e., juxtacrine signaling). To explore the structural basis for these differences in ligand activity, we engineered a series of membrane-anchored ligands in which the core, receptor-binding domain of EGF was combined with different domains of both EGF and HB-EGF. We found that ligands having the N-terminal extension of EGF could not bind to the EGFR, even when released from the membrane. Ligands lacking an N-terminal extension, but possessing the membrane-anchoring domain of EGF, still required proteolytic release for activity, whereas ligands with the membrane-anchoring domain of HB-EGF could elicit full biological activity while still membrane anchored. Ligands containing the HB-EGF membrane anchor, but lacking an N-terminal extension, activated EGFR during their transit through the Golgi apparatus. However, cell-mixing experiments and fluorescence resonance energy transfer studies showed that juxtacrine signaling typically occurred in trans at the cell surface, at points of cell-cell contact. Our data suggest that the membrane-anchoring domain of ligands selectively controls their ability to participate in juxtacrine signaling and thus, only a subclass of EGFR ligands can act in a juxtacrine mode.

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.

Quantitative analysis of HER2-mediated effects on HER2 and epidermal growth factor receptor endocytosis: distribution of homo- and heterodimers depends on relative HER2 levels.

Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR) family. Many cell types express multiple EGFR family members (including EGFR, HER2, HER3, and/or HER4) that interact to form an array of homo- and heterodimers. Differential trafficking of these receptors should strongly affect signaling through this system by changing substrate access and heterodimerization efficiency. Because of the complexity of these dynamic processes, we used a quantitative and computational model to understand their integrated operation. Parameters characterizing EGFR and HER2 interactions were determined using experimental data obtained from mammary epithelial cells constructed to express different levels of HER2, enabling us to estimate receptor-specific internalization rate constants and dimer uncoupling rate constants. Significant novel results obtained from this work are as follows: first, that EGFR homodimerization and EGFR/HER2 heterodimerization occur with comparable affinities; second, that EGFR/HER2 heterodimers traffic as single entities. Furthermore, model predictions of the relationship of HER2 expression levels to consequent distribution of EGFR homodimers and EGFR/HER2 heterodimers suggest that the levels of HER2 found on normal cells are barely at the threshold necessary to drive efficient heterodimerization. Thus, altering HER2 concentrations, either overall or local, could provide an effective mechanism for regulating EGFR/HER2 heterodimerization and may explain why HER2 overexpression found in some cancers has such a profound effect on cell physiology.

Coregulation of epidermal growth factor receptor/human epidermal growth factor receptor 2 (HER2) levels and locations: quantitative analysis of HER2 overexpression effects.

Elevated expression of human epidermal growth factor receptor 2 (HER2) is known to alter cell signaling and behavioral responses implicated in tumor progression. However, multiple diverse mechanisms may be involved in these overall effects, including signaling by HER2 itself, modulation of signaling by epidermal growth factor receptor (EGFR), and modification of trafficking dynamics for both EGFR and HER2. Because these processes are so tightly interrelated, the net effect of HER2 overexpression is difficult to reliably attribute to any single particular mechanism. To take an important first step toward dissecting the effects of HER2 overexpression on cell responses in terms of the various specific underlying mechanisms, we have developed and validated a quantitative model of the relevant trafficking processes. We then use our model for successful prediction of EGFR and HER2 level and location changes attributable to HER2 overexpression in 184A1 human mammary epithelial cells expressing a series of HER2 levels by retroviral infection. Model predictions are based on our independent experimental measurement of key trafficking parameters for both EGFR and HER2. In terms of trafficking processes, HER2 overexpression reduces the EGFR internalization rate constant and increases the fraction of EGFR recycled. Consequently, our model successfully predicts that HER2 increases the overall level of activated EGFR by both enhancing its recycling and reducing its internalization, but it increases activated EGFR localization at the cell surface almost solely by its reduction of internalization. Furthermore, the model also successfully predicts the effects of monoclonal antibody 2C4, which interferes with HER2/EGFR heterodimerization, on EGFR and HER2 levels and compartmental locations. We anticipate that this model should ultimately be useful in parsing the relative contributions of direct effects of HER2 via signaling vis-a-vis indirect effects of HER2 via modification of EGFR signaling.

Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library.

A nonimmune library of 10(9) human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow-cytometric sorting. The yeast library can be amplified 10(10)-fold without measurable loss of clonal diversity, allowing its effectively indefinite expansion. The expression, stability, and antigen-binding properties of >50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the usefulness of this approach for high-throughput antibody isolation for proteomics applications.