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Regulation of colony-stimulating factor 1-induced proliferation by heterotrimeric Gi2 proteins
1Laboratoire d'Oncogénèse Immunohématologique, Institut de Biologie des Hôpitaux de Nantes, France.
Abstract:
Receptors for hematopoietic cytokines possess intrinsic tyrosine-kinases or are associated with tyrosine-kinases; interactions between metabolic pathways activated by tyrosine-kinases and heterotrimeric G proteins are suspected, but not yet proven. To investigate whether alteration of G protein function affects signal transduction of hematopoietic cytokines, we expressed mutant Gi2 proteins in BAC 1.2F5 cells, a murine macrophage cell line that is dependent from monocyte-macrophage colony-stimulating factor (CSF 1) for its proliferation. Mutations made in alpha subunits constitutively activate (alpha i2-Q205L) or inactivate (alpha i2-G204A) Gi2 heterotrimers. We show that expression of alpha i2-Q205L in BAC 1.2F5 cells does not induce independence from CSF 1, but reduces the cells' requirement in CSF 1, shortens the length of the G1 phase and the cell doubling time in response to CSF 1, and protects cells from death by apoptosis induced by CSF 1 withdrawal, exposure to H2O2 or heat shock, but not mitoxantrone. More importantly, expression of alpha i2-G204A, a dominant negative mutant, inhibits BAC 1.2F5 cell proliferation in response to CSF 1, increases the length of the G1 phase and the cell doubling time, and accelerates apoptotic cell death after withdrawal of CSF 1, exposure to H2O2 or heat shock. We conclude that the metabolic pathways regulated by Gi2 proteins and CSF 1 tyrosine-kinase receptors converge on a common effector necessary for the regulation of macrophage survival and proliferation.
Insights
Altering Gi2 protein function impacts macrophage response to colony-stimulating factor 1 (CSF 1). Constitutively active Gi2 reduces CSF 1 dependence and enhances survival, while inactive Gi2 inhibits proliferation and accelerates apoptosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- Hematopoietic cytokine receptors often have intrinsic or associated tyrosine-kinases.
- Interactions between tyrosine-kinase pathways and heterotrimeric G proteins are hypothesized but unproven.
- Macrophage proliferation is regulated by colony-stimulating factor 1 (CSF 1).
Purpose of the Study:
- To investigate if altered G protein function affects signal transduction of hematopoietic cytokines.
- To determine the role of Gi2 proteins in CSF 1 signaling pathways.
- To elucidate the convergence of Gi2 protein pathways and CSF 1 tyrosine-kinase receptors.
Main Methods:
- Expression of constitutively active (alpha i2-Q205L) and dominant-negative (alpha i2-G204A) Gi2 proteins in BAC 1.2F5 murine macrophage cells.
- Assessment of cell proliferation, G1 phase length, cell doubling time, and apoptosis.
- Evaluation of cellular response to CSF 1 withdrawal, hydrogen peroxide (H2O2), heat shock, and mitoxantrone.
Main Results:
- Constitutively active alpha i2-Q205L reduced CSF 1 requirement, shortened G1 phase and doubling time, and conferred resistance to apoptosis from CSF 1 withdrawal, H2O2, and heat shock.
- Dominant-negative alpha i2-G204A inhibited CSF 1-induced proliferation, increased G1 phase and doubling time, and accelerated apoptosis under stress conditions.
- Mitoxantrone-induced apoptosis was unaffected by Gi2 protein mutations.
Conclusions:
- Gi2 protein pathways and CSF 1 tyrosine-kinase receptors converge on a common effector.
- This convergence regulates macrophage survival and proliferation.
- Gi2 proteins play a crucial role in mediating CSF 1 signaling in macrophages.