Cascade transactivation of growth factor receptors in early human hematopoiesis

Highly purified progenitors (including erythroid [BFU-E], granulo- monocytic [CFU-GM], multipotent [CFU-GEMM] progenitors, as well as multipotent progenitors with self-renewal capacity [CFU-B]) express high-affinity growth factor receptors (GFRs), with prevalent interleukin-3 receptors (IL-3Rs) (2,700/cell), a > or = 10-fold lower number of IL-6Rs (145/cell) and granulocyte-macrophage colony- stimulating factor receptors (GM-CSFRs) (300/cell), and a barely detectable level of erythropoietin (Ep) receptors (75/cell). Hematopoietic growth factor (HGF) dosages inducing peak clonogenetic effects are associated with partial/subtotal occupancy of the homologous HGF receptor (HGFR). Cross-reactivity between GFRs and heterologous GFs (including IL-6, IL-3, GM-CSF, Ep, and the kit ligand [KL]) was explored by competition experiments on purified progenitors with radiolabeled and excess cold HGFs at +4 degrees C. No cross- reaction was observed between IL-6R, IL-3R, EpR, and the heterologous GFs, whereas the GM-CSFR showed cross-reactivity with IL-3 and, to a lesser extent, KL. Modulation of GFRs was examined after 18 or 40 hours of incubation with GF(s) at 37 degrees C, followed by ligand-binding assay at 20 degrees C. IL-6, IL-3, GM-CSF, and Ep induce a marked down- modulation of their own receptors. Interestingly, each GF induces the transactivation of the R(s) for the “distal” GF(s): (1) IL-6 induces transactivation of IL-3R, but not of GM-CSFR/EpR; (2) IL-3 causes a rapid upmodulation of GM-CSFR/EpR (“pure” progenitors treated with IL-3 show upmodulation of GM-CSFR alpha-chain mRNA by reverse transcriptase- polymerase chain reaction); whereas (3) GM-CSF induces the transactivation of the EpR. This chain upmodulation of HGFRs may underlie the synergistic interactions between the HGFs in clonogenetic culture. It is emphasized that KL does not induce upmodulation of the other GFRs. Finally, Ep, GM-CSF, and IL-3 do not modulate the expression of the “proximal” HGFRs (ie, GM-CSFR/IL-3R/IL-6R, IL-3R/IL- 6R, and IL-6R, respectively). These results allow insight into the cellular basis of hematopoiesis, ie, the complex and coordinate interactions between HGFs and their receptors. They are compatible with a model of cascade transactivation via upmodulation of GFRs in the initial key steps of hematopoietic differentiation, whereby the action of each GF enhances the effect of the distal GF(s) by a multistep chain- potentiation mechanism.