Granulocyte-Macrophage Colony-stimulating Factor Signals for Increased Glucose Transport via Phosphatidylinositol 3-Kinase- and Hydrogen Peroxide-dependent Mechanisms

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates cellular glucose uptake by decreasing the apparent K m for substrate transport through facilitative glucose transporters on the plasma membrane. Little is known about this signal transduction pathway and the role of the ␣ subunit of the GM-CSF receptor (␣GMR) in modulating transporter activity. We examined the function of phosphatidylinositol 3-kinase (PI 3-kinase) in GM-CSF-stimulated glucose uptake and found that PI 3-kinase inhibitors, wortmannin and LY294002, completely blocked the GM-CSF-dependent increase of glucose uptake in Xenopus oocytes expressing the low affinity ␣GMR and in human cells expressing the high affinity ␣␤GMR complex. We identified a Src homology 3 domain-binding motif in ␣GMR at residues 358 -361 as a potential interaction site for the PI 3-kinase regulatory subunit, p85. Physical evidence for p85 binding to ␣GMR was obtained by co-immunoprecipitation with antibodies to ␣GMR and p85, and an ␣GMR mutant with alteration of the Src homology 3 binding domain lost the ability to bind p85. Experiments with a construct eliminating most of the intracellular portion of ␣GMR showed a 50% reduction in GM-CSF-stimulated glucose uptake with residual activity blocked by wortmannin. Searching for a proximally generated diffusible factor capable of activating PI 3-kinase, we identified hydrogen peroxide (H 2 O 2 ), generated by ligand or antibody binding to ␣GMR, as the initiating factor. Catalase treatment abrogated GM-CSF-or anti-␣GMR antibody-stimulated glucose uptake in ␣GMR-expressing oocytes, and H 2 O 2 activated PI 3-kinase and led to some stimulation of glucose uptake in uninjected oocytes. Human myeloid cell lines and primary explant human lymphocytes expressing high affinity GM-CSF receptors responded to ␣GMR antibody with increased glucose uptake. These results identify the early events in the stimulation of glucose uptake by GM-CSF as involving local H 2 O 2 generation and requiring PI 3-kinase activation. Our findings also provide a mechanistic explanation for signaling through the isolated ␣ subunit of the GM-CSF receptor.

GM-CSF Enhances Macrophage Glycolytic Activity In Vitro and Improves Detection of Inflammation In Vivo

“…GM-CSF acts to mobilize peripheral-blood progenitor cells, resulting in shorter durations of neutropenia in patients receiving induction chemotherapy for hematologic malignancies (25). Further, it is widely appreciated that GM-CSF increases the hematopoietic 18 F-FDG signal and that the heightened signal may persist for weeks after the last dose (16,(26)(27)(28)(29)(30)(31). The findings of this study provide further understanding of the mechanisms underlying this observation.…”

Alternative modes of GM-CSF receptor activation revealed using activated mutants of the common β-subunit

“…The regulatory subunit of PI3K, p85, has been previously shown to associate with the GMR-␣ subunit cytoplasmic domain, an interaction that is dependent upon the SH3 domain of p85. 19 In addition, IK␤ has been shown to directly interact with the GMR-␣ in a yeast-2-hybrid screen. 20 With a view to identify the signaling components that are present in the GMR-␣:FI⌬ complex, we used several approaches to identify proteins binding to the GMR-␣-subunit cytoplasmic domain.…”

“…16 The membrane-proximal region, and in particular, the SH3 binding site/PROX-like (SBP) motif, 17 is essential for GM-CSF-induced activation of JAK2. 18 Some signaling molecules have been shown to associate with the GMR-␣ cytoplasmic domain, including the p85 regulatory subunit of PI3K, 19 and IK␤ 20 ; however, the nature and the role of these interactions remain poorly characterized. Nevertheless, it is interesting to note that although GMR-␣ is essential for GM-CSF receptor activation, it does not signal by itself, highlighting the significance of the GMR-␣ interaction with ␤ c .…”

A role for extracellular matrix binding receptors in regulating hematopoietic growth factor signaling

“…GM-CSF initiates its function by binding to its heterodimeric receptor present on myeloid progenitors and mature monocytes, neutrophils, eosinophils, basophils, and dendritic cells (3)(4)(5)(6). Distinct GM-CSF-activated signaling pathways (7)(8)(9) are critical in regulating the proliferation, differentiation, and maturation of myeloid cells and stimulating macrophage proliferation (10,11). In addition, GM-CSF primes the respiratory burst and enhances the effector function of mature granulocytes and mononuclear phagocytes.…”

“…The ␣ subunit consists of a cytoplasmic domain of limited size, whereas the ␤ subunit consists of a more extensive intracellular domain with multiple tyrosine residues that are targets for phosphorylation by tyrosine kinases including Janus kinase (JAK), to result in the recruitment of Src homology 2 (SH2)-containing proteins to this receptor subunit (14). After ligand binding and the assembly of the high affinity GM-CSF receptor, tyrosine kinases such as JAK2 are recruited to the intracellular domain of the ␤ receptor to activate the JAK͞signal transducer and activator of transcription (STAT), ras͞mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3-kinase) pathways, leading to increases in the proliferation, maturation, survival, and activation of myeloid and monocyte lineages (1,(7)(8)(9)13). Because a subset of GM-CSF receptors preexists as heterodimers in the absence of ligand binding (14), mechanisms are likely opera- tive that prevent the activation of this receptor in the absence of tissue injury.…”