CONTRIBUTION OF BOTH STAT AND SRF TCF TO C-FOS PROMOTER ACTIVATION BYGRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR/

Granulocyte-macrophage colony-stimulating factor (GMCSF) is a hematopo ietic growth factor that has been shown to support cell proliferation in murine fibroblasts engineered to stably express both chains of the human GM-CSF receptor (NIH-GMR). Because the proto-oncogene c-fos is b elieved to provide a link between short-term signals elicited at the m embrane and long-term cellular response, we chose to study the mechani sm of GM-CSF-dependent cell regulation using c-fos promoter activity a s a molecular marker in both NIH-GMR transfectants and in the CD34(+) cell line TF-1. The importance of c-fos and related AP-1 activity in G M-CSF signalling was suggested by a tight correlation between GMCSF-de pendent activation of the c-fos promoter and cell proliferation and by the inhibitory effect of a trans-dominant c-fos mutant on cell growth . To evaluate the contribution of the serum response factor (SRF) asso ciated with the ternary complex factor (TCF) and of STAT proteins to c -fos promoter activation in response to GM-CSF, the SRF binding site ( SRE) and/or the STAT binding site (SIE) were inactivated. In serum-fre e medium, both SRE and SIE are essential to c-fos promoter activation by GM-CSF in NIH-GMR transfectants and in TF-1 cells. No response to G M-CSF was observed when both sites were mutated. The nature of the STA T family member was further investigated by Western blots and DNA reta rdation assays using an SIE probe. Our data indicate that GM-CSF induc ed DNA binding of both STAT1 and STAT3 in NIH-GMR and mainly of STAT3 in TF-1 cells. STAT5 tyrosine phosphorylation was also observed in TF- 1 cells. Finally, expression of a dominant negative MAPK mutant, ERK19 2A, resulted in a decrease of both SRE- and SIE-dependent activation o f c-fos promoter by GM-CSF, suggesting that STAT1/3 are regulated not only by tyrosine kinases, but also partially by MAPK. (C) 1996 by The American Society of Hematology.