Stat3 and G-CSF-Induced Myeloid Differentiation

Abstract

Granulocyte colony-stimulating factor (G-CSF) is the cytokine critical for directing neutrophilic granulocyte differentiation. Early G-CSF signaling events in myeloid cells involves activation of STATs, proteins that serve the dual function of signal transduction and activation of transcription, especially the activation of Stat3. A dominant-negative mutant construct of Stat3 inhibited G-CSF-mediated neutrophilic differentiation indicating that Stat3 is a essential component for driving the G-CSF-mediated differentiation program in myeloid cells. Three isoforms of Stat3 have been identified, aL (p92), β (p83) and γ (p72) each derived from a single gene. Stat3aL is the predominant isoform expressed in most cells. Stat3γ is derived from Stat3aL by alternative RNA splicing. Stat3β is derived from Stat3aL by limited proteolysis. Mapping of Stat3aL and Stat3β activation in M1 murine myeloid leukemia cells revealed that their optimal activation required G-CSFR constructs containing both Y704 and Y744. These amino acid residues has previously been demonstrated to be essential for G-CSF-induced differentiation in this cells. Phosphopeptide affinity and phosphopeptide inhibition studies indicate that Stat3aL and Stat3β are recruited to the G-CSF receptor complex through their interaction with the receptor at phosphotyrosines Y704 and Y744. Y744 is followed at the +3 position by Cys (C). This sequence YXXC, represents a novel motif implicated in the recruitment and activation of Stat3aL, Stat3β and Stat3γ by the hG-CSFR. Structurally, Stat3aL, Stat3β and Stat3γ differ from each other in their C-terminal transactivation domain. In the β isoform, the Stat3aL transactivation domain is replaced by 7 amino acid residues which enable Stat3β to interact with c-Jun. In the γ isoform, the Stat3aL transactivation domain is removed by limited proteolysis creating a dominant negative isoform. In immature human myeloid cells capable of differentiating into neutrophils in response to G-CSF, G-CSF did not activate Stat3aL; rather, it activated predominantly Stat3β. These findings combined with recent reports linking Stat3aL with proliferation and transformation suggest that the β isoform of Statβ may be more critical for G-CSF-mediated differentiation. Activation of Stat3γ occurred predominantly in terminally differentiated neutrophils suggesting that it may be part of a controlled proteolytic mechanism modulating pro-proliferative protein(s) in mature myeloid cells.

Key Words:

• Signal transducer and activator of transcription
• Granulocyte-colony
• stimulating factor receptor
• Stat3aL
• Stat3β