GM-CSF-induced internal autocrine proliferation occurs in a compartment outside of the endoplasmic reticulum

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been observed to be produced by myeloid leukemias, suggesting the capacity of these cells to support their growth via autocrine mechanisms. To investigate whether GM-CSF can function through an internal autocrine loop, we tested the proliferation of the GM-CSF-dependent line, FDCP2-1D, following transduction with the wild-type (WT) GM-CSF gene and a GM-CSF gene modified for intracellular retention in the endoplasmic reticulum (ER) by the addition of sequences encoding the amino acids SEKDEL on the carboxyl terminus. Four retroviruses were constructed to express either the WT or the modified GM-CSF genes under transcriptional regulation of a retroviral long terminal repeat (LTR) or a simian virus 40 (SV40)-promoter. The SEKDEL-modified gene under LTR control produced a biologically active protein, but despite evidence of relative intracellular retention, all FDCP2-1D clones transduced with this virus (L-GM/KDEL-PD) were shown to secrete GM-CSF. However, when a virus expressing the SEKDEL-modified gene transcriptionally regulated by the relatively weaker SV40 promoter (LN-SV40-GM) was used, clones could be established that did not secrete GM-CSF. These clones, containing intracellular GM-CSF without secretion following transduction with the LN-SV40-GM/KDEL virus, were tested for growth independence and were not viable when exogenous GM-CSF was withdrawn, suggesting that binding of GM-CSF to its intracellular receptor either did not occur in the ER or was unable to induce a proliferative signal. In contrast, transduced clones secreting WT GM-CSF regulated by the SV40 promoter (LN-SV40-GM) were shown to be factor independent, and GM-CSF-blocking antibodies minimally affected this autocrine-induced growth, implicating intracellular function of the cytokine. The importance of internal GM-CSF production in the autocrine proliferation of this clone was documented by providing an antisense GM-CSF oligonucleotide to the culture, which resulted in inhibition of proliferation. In addition, suramin, an agent that dissociates cytokines from their receptors, minimally affected the proliferative rate of clones expressing the WT GM-CSF gene, while proliferation of untransduced FDCP2-1D cells sustained by exogenous GM-CSF was blocked by suramin. These studies provide evidence that 1) secretion is not required for stimulation by GM-CSF, and 2) the internal retention of GM-CSF by SEKDEL modification is insufficient to induce autocrine growth, suggesting that GM-CSF can interact functionally with its receptor without extracellular release at a location other than the ER.