Glial fibrillary acidic protein transcription responses to transforming growth factor-beta 1 and interleukin-1 beta 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 t ransforming growth factor-beta 1 (TGF-beta 1) and interteukin-1 beta (IL-1 beta) are released during neural responses to injury and because these cyto kines also modulate GFAP mRNA levels, it is of interest to define their rol e in GFAP transcription. The increases of GFAP mRNA in response to TGF-beta 1 and decreases in response to IL-1 beta were shown to be transcriptionall y mediated in rat astrocytes transfected with a luciferase-reporter constru ct containing 1.9 kb of 5'-up-stream rat genomic DNA. Constructs containing sequential deletions of the rat GFAP 5'-up-stream promoter identified a sh ort region proximal to the transcription start (-106 to -53 bp) that provid es full responses to TGF-beta 1 and IL-1 beta. This region contains an unus ual sequence motif with overlapping nuclear factor-1 (NF-1)- and nuclear fa ctor-kappa B (NF-kappa B)-like binding sites and homology to known TGF-beta response elements. Mutagenesis (3-bp exchanges) in -70 to -68 bp blocked t he induction of GFAP by TGF-beta 1 and the repression by IL-1 beta. Gel shi ft experiments showed that the DNA segment -85 to -63 bp was bound by a fac tor(s) in nuclear extracts from astrocytes, The concentrations of these DNA binding factors were increased by treatment of astrocytes with TGF-beta 1 and decreased by IL-1 beta. Binding of these nuclear factors was blocked by mutation of -70 to -68 bp. Despite homology to NF-1 or NF-kappa B binding sites in the GFAP promoter at segment -79 to -67 bp, anti-NF-kappa B or ant i-NF-1 antibodies did not further retard the gel shift of the nuclear facto rs/DNA complex. Moreover, astrocytic nuclear proteins do not compete for th e specific binding to NF-1 consensus sequence. Thus, nuclear factors from a strocytes that bind to the -85- to -63-bp promoter seg ment might be only d istantly related to NF-I or NF-kappa B. These findings are pertinent to the use of GFAP promoter constructs in transgenic animals, because cis-acting elements in the GFAP promoter are sensitive to cytokines that may be elabor ated in response to expression of transgene products.