Abstract
Abstract 3618
Poster Board III-554
The potential influence over cytokine activity by soluble receptors and the precise cytokine specificity they display suggests that soluble cytokine receptors may play a significant role in the response of cells to cytokine-mediated signaling. GM-CSF biology is mediated by binding to a heterodimeric receptor consisting of a major binding subunit (GMRα) and a major signaling subunit (β). GM-CSF Receptor can signal an astonishing variety of cellular functions, including protection from apoptosis, differentiation, maturation, activation and homing. GM-CSF Receptor α (GMRα) exists in both trans-membrane (tmGMRα) and soluble isoforms (sGMRα). sGMRα is believed to arise exclusively through alternative splicing of the GMRα gene product which removes the exon encoding the transmembrane domain. The splicing is such that the amino terminal 317 residues of sGMRα are homologous to the extracellular domain of tmGMRα, however, the deletion and subsequent frameshift replaces the transmembrane and cytoplasmic domains of tmGMRα with a unique 16 amino acid tail on sGMRα. It has been demonstrated that normal human monocytes constitutively secrete sGMRα, and sGMRα has been implicated in the biology of acute myeloid leukemias and the tumor-nerve interactions evoking bone cancer pain.
sGMRα has been shown to bind directly to GM-CSF. We hypothesized that recombinant sGMRα(r-sGMRα) competitively inhibits the biological properties of GM-CSF. Using a baculoviral expression system, we produced N-terminal-His-tagged r-sGMRα. We exposed the GM-CSF and IL-3 dependent myeloid cell line, Mo7e, to varying concentrations of r-sGMRα and found that r-sGMRα directly inhibits GM-CSF dependent cell survival and proliferation in a dose-dependent manner. We found that r-sGMRα was toxic to Mo7e cells at concentrations as low as the Kd of r-sGMRα for GM-CSF (3nM). The finding that lower concentrations of GM-CSF resulted in lower Mo7e stimulation, but not toxicity, indicated that the mechanism of r-sGMRα toxicity was unlikely to simply be a reduction in effective GM-CSF concentration.
To test whether the r-sGMRα toxicity seen in Mo7e cells was a result of GM-CSF neutralization or a result of signaling inhibition, we demonstrated that GM-CSF neutralizing antibody is cytotoxic during GM-CSF withdrawal, but not cytotoxic in the presence of IL-3, whereas r-sGMRα was cytotoxic under both conditions. Previous kinetic studies have demonstrated a time-dependent strengthening of the GMRα : β : GM-CSF complex; forming discrete “loose” and “tight” binding forms over minutes. Our studies indicate that neither pre-incubation of r-sGMRα with GM-CSF (6 hours) nor pre-incubation of cells with GM-CSF (prior to r-sGMRα addition) diminishes r-sGMRα's ability to inhibit GM-CSF-dependent cell survival and proliferation. We also determined that both r-sGMRα and GM-CSF degradation is independent of the other in vitro.
To determine if r-sGMRα mediated cell death resulted from mechanisms other than GM-CSF signaling inhibition, we also tested additional myeloid cell lines that do not require exogenous cytokines for survival, including K562, (which does not express GMRα) as well as KG-1 and HL-60 cells (which both co-express GMRα and GMRb). While r-sGMRα does not inhibit survival or proliferation of KG-1 cells, r-sGMRα does inhibit proliferation of both HL-60 and K562 cells, but only at concentrations tenfold higher than the Kd (30nM). This inhibition was independent of GM-CSF's presence. These data indicate that soluble receptor may have biological effects beyond competition for ligand with the membrane bound receptor, and beyond the modification of GM-CSF half-life.
The ability of r-sGMRα to inhibit IL-3 dependent survival in Mo7e cells, but not K562 cells, supports the notion that r-sGMRα may be directly inducing cytotoxicity by interacting with surface components that are likely part of the GM-CSF Receptor complex. Collectively, our data represent the first evidence demonstrating the modulation of GM-CSF signaling in human myeloid neoplasms by r-sGMRα and further illustrates the potentially significant role of soluble receptors in the response of cells to cytokine-mediated signaling.
Disclosures:
No relevant conflicts of interest to declare.
G-CSFR Ubiquitination Critically Regulates Myeloid Cell Survival and Proliferation
