A proximal E-box modulates NGF effects on rat PPT-A promoter activity in cultured dorsal root ganglia neurones

Abstract

The rat preprotachykinin A (rtPPTA) promoter fragment spanning -865+92, relative to the major transcriptional start, has previously been demonstrated to be nerve growth factor (NGF) responsive in primary cultures of rat dorsal root ganglion (DRG) neurones [Harrison, P.T., Dalziel, R.G., Ditchfield, N.A., Quinn, J.P., 1999. Neuronal-specific and nerve growth factor-inducible expression directed by the preprotachykinin-A promoter delivered by an adeno-associated virus vector. Neuroscience 94, 997-1003]. In this communication, we demonstrate that an E box element at -60, in part, regulates the activity of this rtPPT-A promoter fragment in DRG neurones in response to NGF. Differential regulation of the promoter is observed in the presence or absence of NGF when the E Box site is present. Under basal conditions binding of proteins to this -60 element may antagonise promoter activity. Hence, in the absence of NGF, mutation of the -60 E box increased reporter gene expression. Further, comparison of levels of reporter gene expression supported by both WT and mutated promoter indicate that in the presence of NGF the -60 E box element also plays a role as an activator domain. This represents a novel mechanism for NGF regulation of rtPPT-A. Similarly, an important role for this signalling pathway was observed in neonate rat DRG neuronal cultures, which require NGF for their survival, namely mutation of the -60 element resulted in higher levels of reporter gene expression.