A Somatic Cell Genetic System for Dissecting Hemopoietic Cytokine Signal Transduction

Rachael T Richardson,Robyn Starr,Leecia J.L Angus,Douglas J Hilton

doi:10.1074/jbc.m202189200

Rachael T Richardson, Robyn Starr + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m202189200

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Abstract

Somatic cell genetics has proven to be a powerful tool for the dissection of cytokine signal transduction pathways. Here we describe a system in which interleukin-6 (IL-6) signaling may be dissected using myeloid leukemic M1 cells. We utilized two properties of M1 cell differentiation to isolate IL-6-unresponsive mutants. First, M1 differentiation is associated with cessation of cell division. Second, differentiated M1 cells migrate rapidly and form dispersed colonies in agar. Mutant clones that are unresponsive to IL-6 are, therefore, large and compact as compared with clones derived from IL-6-responsive wild type M1 cells. Following spontaneous or chemically induced mutagenesis and selection in a high dose of IL-6, we isolated 27 M1 clones unresponsive to IL-6. Three harbored mutations that acted in a dominant manner, whereas 24 contained recessive mutations. gp130, an IL-6 receptor component, was affected in many mutant clones. We show that these clones display IL-6 and leukemia inhibitory factor receptors with reduced binding affinities and express gp130 at reduced levels. The IL-6-unresponsive phenotype of these mutant clones was fully rescued by the transfection of exogenous gp130 DNA. Therefore, this approach targets components of the IL-6 signaling pathway and may be suitable to study signaling from a variety of cytokines.

Highlights

Genetics has been successfully used to identify and analyze components of signal transduction pathways in a number of systems, including chemotaxis in bacteria [1], the response to the mating pheromone in yeast [2], the development of the Drosophila melanogaster visual system [3] and nematode vulva [4], and the increase in gene transcription in response to interferon (IFN)1 in mammalian somatic cells [5]
We demonstrate that M1 cells provide the basis for a somatic cell genetic approach to dissect the signal transduction pathways used by cytokines to stimulate macrophage differentiation
Selection of Spontaneously Occurring Cytokine-unresponsive M1 Cells—To determine the suitability of the M1 cell line as a somatic cell genetic tool, we first assessed the frequency of spontaneous cytokine unresponsiveness

Summary

Introduction

Genetics has been successfully used to identify and analyze components of signal transduction pathways in a number of systems, including chemotaxis in bacteria [1], the response to the mating pheromone in yeast [2], the development of the Drosophila melanogaster visual system [3] and nematode vulva [4], and the increase in gene transcription in response to interferon (IFN) in mammalian somatic cells [5]. We demonstrate that M1 cells provide the basis for a somatic cell genetic approach to dissect the signal transduction pathways used by cytokines to stimulate macrophage differentiation. Two spontaneous and 25 ICR191-induced IL-6-unresponsive clones of M1 cells were selected in agar based on their ability to form tightly packed colonies, rather than small clusters of dispersed cells, in the presence of high concentrations of this cytokine. These lines were at least 2 orders of magnitude less responsive to IL-6 than wild type M1 cells. Among the recessive cell lines, one group appears to lack functional gp130, consistent with the important role that this receptor component plays in IL-6 signal transduction

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