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Combining engineering analyses and mathematical modeling with intervention and detection methodologies at the molecular level will allow manipulation of intracellular signal transduction pathways, and therefore rational control of functional processes central to medicine and biotechnology. We have formulated a simple mathematical model of a key signaling pathway required for regulated migration of fibroblasts and other cell types: activation of the intracellular enzyme phospholipase C (PLC) mediated by epidermal growth factor receptor (EGFR) and a multitude of other transmembrane receptors. One of the interesting features of this pathway is that the substrate of PLC, the lipid phosphatidylinositol (4,5)-bisphosphate (PIP(2)), is turned over quite rapidly and must be constantly resupplied to the plasma membrane by a known transfer mechanism. The model, which accounts for regulation of PIP(2) concentration, is sufficiently detailed to explain unique quantitative features of recent experimental data. We find that competitive pathways that deplete PIP(2) from the membrane, as well as receptor-mediated enhancement of PIP(2) supply, must be significant for agreement between model and experiment. Importantly, the mechanistic nature of the model also allowed us to predict the efficacy of various molecular intervention strategies, including overexpression of wild-type and variant proteins in the pathway as well as treatment with specific drug inhibitors. For many parameter conditions the intuitive strategy of targeting the enzyme itself is actually predicted to be relatively inefficient, with a novel and potentially useful alternative being disruption of the reactant supply mechanism.

Ligand-conjugated polymer (polyplex) gene delivery vectors have strong potential as targeted, in vivo gene transfer vehicles; however, they are currently limited by low delivery efficiency. A number of barriers to polyplex-mediated delivery have been previously identified, including receptor binding, internalization, endosomal escape, and nuclear localization. However, based on understanding of viral gene delivery systems, yet another potential barrier may exist; a limited ability to unpackage the plasmid DNA cargo following localization to the nucleus. We have developed a model system that employs a cationic polymer linked to epidermal growth factor (EGF) as a ligand to target delivery of plasmid DNA encoding the green fluorescent protein to mouse fibroblasts bearing the EGF receptor. Using fluorescence microscopy to simultaneously trace both the plasmid and polymer during gene delivery in combination with an in vitro transcription assay, we provide evidence that plasmid unpackaging can indeed be a limiting step for gene expression for sufficiently large polymer constructs. Short-term expression is significantly enhanced by using short polycations that dissociate from DNA more rapidly both in vitro and in vivo. Finally, we describe a thermodynamic model that supports these data by showing that shorter polycations can have a higher probability of dissociating from DNA. This work demonstrates that vector unpackaging should be added to the list of barriers to receptor-mediated polyplex gene delivery, thus providing an additional design principle for targeted synthetic delivery vehicles.

For effective migration, cells must establish an asymmetry in cell/substratum biophysical interactions permitting cellular protrusive and contractile motive forces to produce net cell body translocation; often this is superficially manifested as a polarized cell shape. This change is most easily noted for epithelial cells, which typically undergo a mesenchymal transition prior to rapid motility, and for hematopoietic cells, which must transition from non-adherent to adherent states. These two situations entail dramatic changes that also involve cell-cell contact and differentiation-related changes, and thus introduce confounding events and signals in defining control elements. Hence, a simpler biochemical and biophysical model system may be useful for gaining fundamental insights into the underlying mechanisms. Fortunately, even relatively "uniform" fibroblasts also undergo an initial shape change to commence locomotion. Investigators have recently begun to probe underlying signals that contribute to the reorganization of the actin cytoskeleton. We describe here a model for fibroblast shape changes involved in epidermal growth factor (EGF) stimulation of motility, focusing on signals through EGF receptor (EGFR) -mediated pathways influencing cytoskeletal organization and cell/substratum adhesion. We present new data addressing specifically phospholipase C-gamma (PLCgamma) pathway activation of actin-modifying proteins, including gelsolin, that contributes to these changes and promotes cell migration by increasing the fraction of cells in a motility-permissive morphology and the time spent in such a state.

Transforming growth factor alpha (TGF alpha) has been reported to be a more potent agonist when compared to epidermal growth factor (EGF) in several systems while acting via their common receptor, the epidermal growth factor receptor (EGFR). It has been postulated that this increased potency is mediated by the increased recycling of EGFR upon activation by TGF alpha as against receptor activation by EGF. The authors test this hypothesis by simultaneously measuring mitogenesis and the dynamics of surface receptor number in response to these ligands in NR6 mouse fibroblasts expressing the EGFR. The data demonstrates that increased receptor recycling due to endosomal dissociation of TGF alpha can indeed realise an increased mitogenic potency relative to EGF under appropriate cellular and experimental conditions (i.e. situations in which the increase in the number of occupied receptors due to receptor sparing by TGF alpha represents additional mitogenic signalling capacity). However, this difference in receptor trafficking does not uniquely determine the relative potencies of these ligands since TGF alpha is a less potent mitogen compared to EGF when experimental conditions are dominated by the effects of ligand trafficking on growth factor availability. Thus, the relative potencies of these growth factors are determined in a given context by the relative importance of ligand and receptor trafficking effects which determine the availability of these signalling components. These results are consistent with a suggested model of hormone responsiveness which favours dissociative ligands (such as TGF alpha) in receptor-limited situations and non-dissociative ligands (such as EGF) in the case of ligand limitation.

We have developed an experimental system for testing mathematical model predictions concerning escape of autocrine ligands into the extracellular bulk medium. This system employs anti-receptor blocking antibodies against the epidermal growth factor receptor (EGFR)/transforming growth factor alpha (TGFalpha) receptor/ligand pair. TGFalpha was expressed under the control of a tetracycline-repressed promoter, together with a constitutively expressed human EGFR in B82 mouse fibroblast cells. This expression system allowed us to vary TGFalpha synthesis rates over a roughly 300-fold range by adjusting tetracycline concentration. TGFalpha accumulation in the extracellular bulk medium was then measured as a function of cell density, TGFalpha synthesis rate, and anti-EGFR blocking antibody concentration. Consistent with model predictions, amounts of ligand in the medium on a per cell basis were found to diminish as cell density was increased but with reduced dependence on cell density at higher ligand synthesis rates. Similarly consistent with model predictions, higher ligand synthesis rates also decreased the effect of anti-receptor blocking antibodies. Our investigation has established that we can successfully analyze and understand autocrine ligand secretion behavior from the basis of our theoretical model.

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) elicit quantitatively different cell proliferation responses even though they act via a common receptor, the epidermal growth factor receptor (EGFR). We hypothesized that differential cellular trafficking of available ligand is responsible for the different mitogenic responses elicited by EGF and TGF alpha. Mitogenesis and ligand depletion were determined simultaneously in NR6 mouse fibroblasts expressing either wild-type (WT) or internalization-deficient cytoplasmic domain-truncated (c'973) EGFR. Thus we could determine the effects of both ligand-induced and low level constitutive ligand/receptor processing. For a given initial amount of growth factor, TGF alpha is a weaker stimulus than EGF in cells expressing either form of the EGFR. This difference in the mitogenic potencies correlates with increased depletion of TGF alpha observed during the growth assays. When this difference in ligand depletion is accounted for, or minimized, EGF and TGF alpha elicit quantitatively similar growth responses. Therefore, the relative mitogenic potencies of EGF and TGF alpha depend on ligand availability, as determined by the cellular trafficking of these ligands in conjunction with environmental circumstances. Interestingly, our data demonstrate that TGF alpha can be a less potent mitogenic stimulus than EGF under conditions where ligand availability is limited. Further, in our assays, differences in ligand processing are sufficient to explain the different mitogenic potencies of these growth factors in either of the receptor trafficking scenarios. Our results suggest a model of regulation of hormone responsiveness which favors dissociative ligands (such as TGF alpha) in receptor-limited situations and non-dissociative ligands (such as EGF) in the face of high receptor levels.

We describe experiments comparing the proliferation responses to epidermal growth factor (EGF) by NR6 fibroblasts expressing genetically engineered epidermal growth factor receptors (EGFRs). These cells present either wild-type (WT) EGFR or a cytoplasmic domain-truncated (c'973) EGFR that exhibits a decreased ligand-induced internalization rate constant. In two distinct in vitro proliferation assays, with or without medium replenishment, we measured the specific cell proliferation rate constants and EGF depletion kinetics for both WT and c'973 cells. When EGF depletion is minimized by replenishment, the EGF concentration dependencies of the two cell types are similar, whereas when EGF depletion is not prevented, maximal proliferation of WT cells requires an initial EGF concentration that is approximately 10x that required by c'973 cells. However, when EGF depletion is accounted for, the dependencies of growth rate for the two cell types on the current EGF concentration in both assays are essentially identical. Our results demonstrate that diminished depletion of EGF from the extracellular medium is a major reason for increased mitogenic sensitivity to EGF by cells possessing internalization-deficient receptors.

Intracellular trafficking of the epidermal growth factor receptor (EGF-R) is regulated by receptor occupancy. To investigate this, we developed an assay to study endosomal sorting under steady-state conditions. Using a cell line transfected with EGF-R variants, we found that the fraction of internalized EGF.EGF-R complexes sorted to lysosomes was a function of the number of intracellular complexes and required sequences in the cytoplasmic domain of the receptor. As the number of intracellular occupied wild-type receptors increased from 3 x 10(2) to 2 x 10(5)/cell, the fraction of internalized EGF that was degraded dropped from 70 to 20%. Transforming growth factor-alpha, which dissociates from the EGF-R at endosomal pH, was degraded to a uniform extent of approximately 50% at all intracellular ligand concentrations. EGF internalized by receptors lacking a cytoplasmic domain (c'647) was degraded to an extent of only 5-10% independent of the number of intracellular complexes. Mutant receptors truncated either at residues 1022 or 973 displayed sorting patterns intermediate between wild-type and c'647 receptors. Despite large differences in their internalization rates, the fractional sorting patterns of c'1022 and c'973 receptors were indistinguishable. Receptor tyrosine kinase activity appeared to have a small effect on sorting pattern, but only in the context of full-length receptors. Our results indicate that the default pathway of internalized receptors is rapidly recycling and that lysosomal targeting of occupied EGF-R is due to endosomal retention that is both specific and saturable. In addition, internalization and endosomal retention of EGF-R appear to be mediated by distinct structural elements.

Little is known about the regulation of EGF receptor (EGF-R) trafficking following endocytosis. We investigated this by using a series of EGF-R with altered cytoplasmic tails and comparing their ability to undergo recycling and lysosomal targeting in both the occupied and empty state. We found that 2-3% of empty EGF-R are internalized each minute, but rapidly recycle (t1/2 approximately 5 min). This constitutive internalization and recycling of empty receptors was independent of cytoplasmic receptor sequences. Occupied EGF-R, in contrast, displayed a much slower rate of recycling (t1/2 between 10-23 min) due to retention within recycling endosomes. Endosomal retention of different EGF-R correlated with lysosomal targeting of EGF. Intrinsic receptor tyrosine kinase activity had no discernible effect on postendocytic trafficking of EGF. Although sequences within the cytoplasmic tail of the EGF-R appear to be required for occupancy-dependent endosomal retention, they are distinct from those required for ligand-induced endocytosis. Our studies indicate that intracellular trafficking of the EGF-R is regulated by endosomal components that preferentially recognize occupied receptors. Down-regulation of the EGF-R thus involves two distinct regulatory processes: one at the level of internalization and one at the level of recycling.

Occupancy-induced down-regulation of cell surface epidermal growth factor (EGF) receptors attenuates signal transduction. To define mechanisms through which down-regulation of this class of growth factor receptors occurs, we have investigated the relative roles of ligand-induced internalization and recycling in this process. Occupied, kinase-active EGF receptors were internalized through a high affinity, saturable endocytic system at rates up to 10-fold faster than empty receptors. In contrast, full length EGF receptors lacking tyrosine kinase activity underwent internalization at a rate independent of occupancy. This "kinase-independent" internalization rate appeared to reflect constitutive receptor internalization since it was similar to the internalization rate of both receptors lacking a cytoplasmic domain and of antibodies bound to empty receptors. EGF internalized by either kinase-active or kinase-inactive receptors was efficiently recycled and was found within endosomes containing recycling transferrin receptors. However, targeting of internalized receptors to lysosomes did not require receptor kinase activity. All receptors that displayed ligand-induced internalization also underwent down-regulation, indicating that the proximal cause of down-regulation is occupancy-induced endocytosis. Tyrosine kinase activity greatly enhances this process by stabilizing receptor association with the endocytic apparatus.