Decision tree modeling predicts effects of inhibiting contractility signaling on cell motility.

BACKGROUND: Computational models of cell signaling networks typically are aimed at capturing dynamics of molecular components to derive quantitative insights from prior experimental data, and to make predictions concerning altered dynamics under different conditions. However, signaling network models have rarely been used to predict how cell phenotypic behaviors result from the integrated operation of these networks. We recently developed a decision tree model for how EGF-induced fibroblast cell motility across two-dimensional fibronectin-coated surfaces depends on the integrated activation status of five key signaling nodes, including a proximal regulator of transcellular contractile force generation, MLC (myosin light chain) [Hautaniemi et al, Bioinformatics 21: 2027 {2005}], but we have not previously attempted predictions of new experimental effects from this model. RESULTS: In this new work, we construct an improved decision tree model for the combined influence of EGF and fibronectin on fibroblast cell migration based on a wider spectrum of experimental protein signaling and cell motility measurements, and directly test a significant and non-intuitive a priori prediction for the outcome of a targeted molecular intervention into the signaling network: that partially reducing activation of MLC would increase cell motility on moderately adhesive surfaces. This prediction was indeed confirmed experimentally: partial inhibition of the activating MLC kinase (MLCK) upstream using the pharmacologic agent ML-7 resulted in increased motility of NR6 fibroblasts. We further extended this exciting finding by showing that partial reduction of MLC activation similarly enhanced the transmigration of the human breast carcinoma cell line MDA-213 through a Matrigel barrier. CONCLUSION: These findings specifically highlight a central regulatory role for transcellular contractility in governing cell motility, while at the same time demonstrating the value of a decision tree approach to a systems "signal-response" model in discerning non-intuitive behavior arising from integrated operation a cell signaling network.

Motility signaled from the EGF receptor and related systems.

Cell motility is now recognized as central to many biological processes. Growth factors, such as those that activate the epidermal growth factor receptor (EGFR), drive biochemically and biologically distinct subsets of migration critical for (neo)organogenesis and tumor invasion. Thus, modulation of these events requires an understanding of the controls of EGFR-mediated motility. Deconstruction of motility into its component events enables this deeper insight. Herein we describe methods that measure the overall motility and its parameters as well as the biophysical processes extension, de-adhesion/retraction, and contraction.

Epidermal growth factor induces fibroblast contractility and motility via a protein kinase C delta-dependent pathway.

Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKCdelta is the most strongly phosphorylated isoform, and the preferential PKCdelta inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKCdelta is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKCdelta activation, whereas lack of PLCgamma signaling resulted in delayed PKCdelta activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKCdelta is required, a dominant-negative PKCdelta construct or RNAi-mediated PKCdelta depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKCdelta activation. All of the interventions that blocked PKCdelta activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKCdelta activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKCdelta to promote the acute phase of EGF-induced MLC activation.

Concomitant expression of heparin-binding epidermal growth factor-like growth factor mRNA and basic fibroblast growth factor mRNA in myocardial infarction in rats.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is mitogenic and chemotactic for many cell types. HB-EGF is induced in pathological states which require cell mitogenesis and proliferation, including angiogenesis, and has been reported to interact functionally with basic fibroblast growth factor (bFGF). To test our hypothesis that HB-EGF mRNA expression is increased in myocardial infarction, we used Northern hybridization in rats to investigate the expression of HB-EGF and EGF receptor mRNAs expression in the infarct zone compared to the expression of bFGF and FGF receptor mRNAs. We also performed in situ hybridization to identify the cells responsible for HB-EGF mRNA production. HB-EGF mRNA rapidly increased after ligation (mean +/- SE, 5.6+/-0.23-fold increase at 6 hours compared to the preligation heart levels) and reached a maximum level (9.1+/-0.42-fold increase) around 12 hours. HB-EGF mRNA then gradually decreased on day 1 (5.8+/-1.0-fold increase), day 2 (3.2+/-0.94-fold increase) and day 3 (1.9+/-0.33-fold increase) after ligation. Parallel changes in bFGF mRNA expression were observed (6, 12 hours, days 1, 2 and 3; 3.6+/-0.42-, 5.3+/-0.12-, 2.3+/-0.12-, 1.7+/-0.03- and 0.95+/-0.03-fold increase, respectively). EGF receptor (ErbB-1) mRNA was gradually increased on day 2 (2.4+/-0.53-fold increase), day 7(4.0+/-0.61-fold increase) and day 14 (7.0+/-0.61-fold increase). Similarly, FGF receptor (FGF receptor-1) mRNA was gradually increased (days 2,7 and 14; 1.3+/-0.13-, 1.5+/-0.17- and 2.3+/-0.15-fold increase, respectively). Reperfusion after a 2-hour ligation (too late to salvage myocytes) enhanced HB-EGF (12 hours, 16.8+/-1.8-fold increase) and bFGF (12 hours, 10.4+/-1.1-fold increase) mRNA expression. The cells responsible for the increased production of HB-EGF mRNA were shown by in situ hybridization to be surviving myocytes located in the infarct peripheral zone around infarct necrotizing tissue. In conclusion, our results demonstrated a rapid increase in HB-EGF mRNA expression concomitant with an increase in bFGF mRNA expression, suggesting that HB-EGF and bFGF might play some role in the course of pathological changes in the infarct in the early inflammatory phase. Reperfusion at times too late to salvage myocytes accelerated sequential changes in the expression of both HB-EGF and bFGF mRNAs.