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

Kharait S, Hautaniemi S, Wu S, Iwabu A, Lauffenburger DA, Wells A - BMC Syst Biol (2007)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA. sok4@pitt.edu

ABSTRACT

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.

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Related in: MedlinePlus

Immunoblotting data for EGF treatment of 5 minutes (A) and 1 hour (B) across different fibronectin concentration of surfaces. Tissue culture plates were coated with different fibronectin (FN) concentrations. NR6WT cells were grown on these surfaces for 24 hours in complete growth medium and quiesced for another 24 hours in medium containing 0.5% dialyzed FBS. EGF was added for a period of 1 hour, cells washed once with PBS and lysed. Cell lysates were resolved using SDS-PAGE and immunoblotted using specific antibodies for various phosphorylated proteins. At least 5 replicates for each signaling protein were created for polynomial modeling. Actin served as a loading control.
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Figure 1: Immunoblotting data for EGF treatment of 5 minutes (A) and 1 hour (B) across different fibronectin concentration of surfaces. Tissue culture plates were coated with different fibronectin (FN) concentrations. NR6WT cells were grown on these surfaces for 24 hours in complete growth medium and quiesced for another 24 hours in medium containing 0.5% dialyzed FBS. EGF was added for a period of 1 hour, cells washed once with PBS and lysed. Cell lysates were resolved using SDS-PAGE and immunoblotted using specific antibodies for various phosphorylated proteins. At least 5 replicates for each signaling protein were created for polynomial modeling. Actin served as a loading control.

Mentions: Addition of EGF activated EGFR within minutes and this signal was transmitted downstream to signaling cascades measured (Figure 1A). Interestingly, the EGFR activation profile mirrored that of ERK within early time periods of EGF stimulation (5 minutes). ERK activation was robust immediately after addition of EGF compared to quiesced cells and remained significantly increased for over an hour (of EGF stimulus) with minimal change over different surface fibronectin concentrations (Figure 1B). Thus, ERK functioned like a 'switch' turned on dependent mainly on EGFR signaling. EGFR signaling also activated PLCγ and PKCδ, with their activation increasing linearly across increasing surface FN levels with resultant MLC activation downstream of PKCδ [13]. However, fibronectin does exert a significant influence on cell speed, as predicted [11], biphasic with surface adhesiveness (Figure 2A).


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

Kharait S, Hautaniemi S, Wu S, Iwabu A, Lauffenburger DA, Wells A - BMC Syst Biol (2007)

Immunoblotting data for EGF treatment of 5 minutes (A) and 1 hour (B) across different fibronectin concentration of surfaces. Tissue culture plates were coated with different fibronectin (FN) concentrations. NR6WT cells were grown on these surfaces for 24 hours in complete growth medium and quiesced for another 24 hours in medium containing 0.5% dialyzed FBS. EGF was added for a period of 1 hour, cells washed once with PBS and lysed. Cell lysates were resolved using SDS-PAGE and immunoblotted using specific antibodies for various phosphorylated proteins. At least 5 replicates for each signaling protein were created for polynomial modeling. Actin served as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1839898&req=5

Figure 1: Immunoblotting data for EGF treatment of 5 minutes (A) and 1 hour (B) across different fibronectin concentration of surfaces. Tissue culture plates were coated with different fibronectin (FN) concentrations. NR6WT cells were grown on these surfaces for 24 hours in complete growth medium and quiesced for another 24 hours in medium containing 0.5% dialyzed FBS. EGF was added for a period of 1 hour, cells washed once with PBS and lysed. Cell lysates were resolved using SDS-PAGE and immunoblotted using specific antibodies for various phosphorylated proteins. At least 5 replicates for each signaling protein were created for polynomial modeling. Actin served as a loading control.
Mentions: Addition of EGF activated EGFR within minutes and this signal was transmitted downstream to signaling cascades measured (Figure 1A). Interestingly, the EGFR activation profile mirrored that of ERK within early time periods of EGF stimulation (5 minutes). ERK activation was robust immediately after addition of EGF compared to quiesced cells and remained significantly increased for over an hour (of EGF stimulus) with minimal change over different surface fibronectin concentrations (Figure 1B). Thus, ERK functioned like a 'switch' turned on dependent mainly on EGFR signaling. EGFR signaling also activated PLCγ and PKCδ, with their activation increasing linearly across increasing surface FN levels with resultant MLC activation downstream of PKCδ [13]. However, fibronectin does exert a significant influence on cell speed, as predicted [11], biphasic with surface adhesiveness (Figure 2A).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA. sok4@pitt.edu

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus