Gene network dynamics controlling keratinocyte migration

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Hauke Busch¹, David Camacho-Trullio¹, Zbigniew Rogon¹, Kai Breuhahn², Peter Angel³, Roland Eils^1,4,5 & Axel Szabowski^3,5

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Synopsis

So far it is unclear how to translate large-scale 'omics' data into a coherent model of cellular regulation that allows one to simulate, predict and control cellular behavior. The functional components that lead to a cellular decision have been debated with respect to requiring a controlled interplay of protein signaling and gene regulation. Combining these complex processes into one model is inherently difficult: protein signaling pathways and gene regulation are feedback-entangled processes, yet occurring on different timescales in the range of minutes and hours, respectively. This makes it practically impossible to experimentally observe all required cellular components at a sufficiently high sampling rate.

Here, we propose a complexity reduction approach for the above problem based on a timescale separation of cellular events. The slaving principle states that the long-term macroscopic behavior of a system is governed by its slowest evolving variables (Haken, 2004).

Hence, the long-term phenotypic response of a cell can be expressed in terms of its slowest evolving functional elements. Postulating that a cell reaches a decision on a timescale of hours, its phenotype should be controlled by the slow protein turnover rates. Assuming a proportionality between protein and mRNA concentrations, information on cellular processes lasting hours or days should be reflected in the cellular gene expression dynamics, which are experimentally accessible by time-resolved microarray experiments.

We studied the mechanisms of keratinocyte cell migration in a heterologous co-culture combining primary human keratinocytes and genetically defined murine fibroblasts as an in vitro model for cell migration in the context of wound healing (Szabowski et al, 2000). The dermal-derived hepatocyte growth factor (HGF) is involved in the regulation of migration via the activation of the Met receptor, a proto-oncogene commonly mutated in metastasizing epithelial cells (Birchmeier et al., 2003). However, from a system control point of view, it is not known how the cell produces a sustained and context-dependent migratory response upon stimulation of Met.

We stimulated keratinocytes with HGF and performed DNA microarray experiments at 1, 2, 3, 4, 6 and 8 h after stimulation. We identified a putative set of genes responsible for the decision to migrate, by ranking the gene kinetics according to their mean and peak fold expression. Interestingly, the rank score distribution of genes displays a long tail at high rank scores (Figure 1B), likely indicating that only a small set of genes mediate the decision to migrate. Genes that are strongly regulated upon the migratory HGF stimulus show a weak or even a downregulated response upon FGF-7 stimulation, a cytokine mediating proliferation (Figure 1C). Subsequent filtering according to their biological function further narrowed down candidate genes for in silico modeling.

Figure 1

Identification of genes mediating HGF-induced cellular transition to migration. (A) Workflow of data analysis, inverse modeling and experimental validation. (B) Histogram of rank scores for all 20 188 measured genes after HGF stimulation (cf. Materials and methods). The ranks numbered 1–20 correspond to the 20 highest ranked genes (cf. gene list in (C)). (C) Top: mean and peak fold expression of the 20 top-ranked genes upon HGF stimulation. Bottom: expression of the same genes upon FGF-7 stimulation. Data points were normalized to the maximal mean and peak fold expression of the respective experiments (cf. Materials and methods).

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In the next step, a dynamic gene regulatory network was inferred from the kinetics of the identified genes using a continuous time recurrent neural network as the model function (Beer, 1995). In our model, all genes can interact, time-delayed, with each other, including themselves via a sigmoid activation function. The impact of the HGF-induced protein signaling on gene expression is taken into account via a time-varying input function for each gene. Gene expression kinetics was fitted using a genetic algorithm combined with a search for robust system solutions based on the maximal Lyapunov exponent evaluation (Rosenstein et al, 1993). Network parameters were obtained from averaging over 250 out of 5000 fittest and most robust solutions. For the complete workflow, see Figure 1A.

The inferred model allowed predictions on the gene network topology and its long-term dynamic behavior. Network topology was shown to be robust with increasing network size. Starting with the top-ranked genes and growing the network in size from three to nine genes, each time repeating the inverse modeling workflow, qualitatively conserved the topology of the smallest three-gene network. This core consists of the highest ranked genes considered for modeling, namely the transcription factor egr3, akap12 and ptgs2. Moreover, it could be shown that the regulatory effect of genes not in the core network is weak to negligible, as the external input and the higher ranked genes control their dynamics. Hence, a network of nine genes sufficed to include most genes having a regulatory effect on the cellular decision toward migration.

Model verification was performed experimentally on the protein level, with the assumption that (i) protein levels follow mRNA change over time and (ii) fast protein signaling events can be adiabatically approximated. We subsequently transformed the obtained gene regulatory network topology and dynamics to the level of signaling pathways to predict in silico and verify in vitro the order of events necessary to initiate, maintain and stop keratinocyte migration.

We showed that the transition to cell migration is controlled by the following time sequential events. (I) HGF triggers the activation of the proto-oncogene receptor Met, putting the cell into a responsive system state. (II) A consecutive permanent activation of the EGF receptor, starting within the next 7 h, initiates and sustains migration. (III) The protein kinase A signaling pathway enhances, delays or stops migration. (IV) The minimal EGF receptor signaling strength necessary to sustain cell migration is modulated via several proteins, whose genes are associated with tumor malignancy.

Time ordered sequential events control cellular decision of migration. (A) Schematic representation of events regulating the initiation, maintenance, modulation and stopping of cell migration. The migratory response depends on a certain threshold of gene activity, as indicated by the dashed line, and proceeds as long as no context information is provided via a stop signal or the sustained activation is depleted. (B) Summary scheme of the model for migration. A first pulse-like HGF stimulus is required to transform the cell into a sensitive state for migration. To initiate and sustain migration a second input preferably through the EGF-R signaling pathway is required. This second input can be modulated by a number of genes. Context information is integrated through the PKA pathway, which can stop migration at any point in time.

The data analysis and modeling provide a new way of obtaining a holistic view on the dynamic orchestration of various pathways and a broad degree of interdependency that controls cell migration. A summary of the biological results is depicted in (Figure 7A and B. It was found that only a few genes mediate the transition toward migration, a behavior reminiscent of phase transition in self-organized systems. From this point of view, our data analysis and modeling approach might be generally applicable to understand other cellular processes involving decision making as well.

Figure 7

Time ordered sequential events control cellular decision of migration. Schematic representation of events regulating the initiation, maintenance, modulation and stopping of cell migration. (A) The migratory response depends on a certain threshold of gene activity, as indicated by the dashed line, and proceeds as long as no context information is provided via a stop signal or the sustained activation is depleted. (B) Summary scheme of the model for migration. A first pulse-like HGF stimulus is required to transform the cell into a sensitive state for migration. To initiate and sustain migration, a second input preferably through the EGF-R signaling pathway is required. This second input can be modulated by a number of genes. Context information is integrated through the PKA pathway, which can stop migration at any point in time.

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