Glial fibrillary acidic protein transcription responses to transforming growth factor-beta1 and interleukin-1beta are mediated by a nuclear factor-1-like site in the near-upstream promoter.

Elevated expression of glial fibrillary acidic protein (GFAP) is associated with astrocyte activation during responses to injury in the CNS. Because transforming growth factor-beta1 (TGF-beta1) and interleukin-1beta (IL-1beta) are released during neural responses to injury and because these cytokines also modulate GFAP mRNA levels, it is of interest to define their role in GFAP transcription. The increases of GFAP mRNA in response to TGF-beta1 and decreases in response to IL-1beta were shown to be transcriptionally mediated in rat astrocytes transfected with a luciferase-reporter construct containing 1.9 kb of 5'-upstream rat genomic DNA. Constructs containing sequential deletions of the rat GFAP 5'-upstream promoter identified a short region proximal to the transcription start (-106 to -53 bp) that provides full responses to TGF-beta1 and IL-1beta. This region contains an unusual sequence motif with overlapping nuclear factor-1 (NF-1)- and nuclear factor-kappaB (NF-kappaB)-like binding sites and homology to known TGF-beta response elements. Mutagenesis (3-bp exchanges) in -70 to -68 bp blocked the induction of GFAP by TGF-beta1 and the repression by IL-1beta. Gel shift experiments showed that the DNA segment -85 to -63 bp was bound by a factor(s) in nuclear extracts from astrocytes. The concentrations of these DNA binding factors were increased by treatment of astrocytes with TGF-beta1 and decreased by IL-1beta. Binding of these nuclear factors was blocked by mutation of -70 to -68 bp. Despite homology to NF-1 or NF-kappaB binding sites in the GFAP promoter at segment -79 to -67 bp, anti-NF-kappaB or anti-NF-1 antibodies did not further retard the gel shift of the nuclear factors/DNA complex. Moreover, astrocytic nuclear proteins do not compete for the specific binding to NF-1 consensus sequence. Thus, nuclear factors from astrocytes that bind to the -85- to -63-bp promoter segment might be only distantly related to NF-1 or NF-kappaB. These findings are pertinent to the use of GFAP promoter constructs in transgenic animals, because cisacting elements in the GFAP promoter are sensitive to cytokines that may be elaborated in response to expression of transgene products.