Neuron Restrictive Silencer Factor NRSF/REST Is a Transcriptional Repressor of Neuropilin-1 and Diminishes the Ability of Semaphorin 3A to Inhibit Keratinocyte Migration

Peter Kurschat,Diane Bielenberg,Mireille Rossignol-Tallandier,Andreas Stahl,Michael Klagsbrun

doi:10.1074/jbc.m507860200

Abstract

Neuropilin-1 (NRP1) is expressed by endothelial cells and neurons and serves as a receptor for both vascular endothelial growth factor (VEGF), an angiogenesis factor, and semaphorin 3A (Sema3A), a mediator of axonal guidance. We show here that NRP1 is also expressed in keratinocytes in vitro and in vivo. However, nothing has been reported about the regulation or function of keratinocyte NRP1. Using NRP1 promoter constructs in HaCaT cells, a keratinocyte cell line, we could demonstrate that a neuron restrictive silencer element (NRSE) was implicated in transcriptional repression of the NRP1 gene. Electrophoretic mobility shift assays demonstrated that the neuron restrictive silencer factor (NRSF) binds to NRSE. Overexpression of NRSF in HaCaT cells decreased NRP1 RNA and protein, whereas a dominant negative NRSF increased NRP1. Furthermore, the histone deacetylase inhibitor trichostatin A, an inhibitor of NRSF silencing activity, also increased NRP1 levels. NRP2 expression was not affected. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) strongly up-regulated NRP1 expression, concomitant with down-regulation of NRSF. Other keratinocyte mitogens such as keratinocyte growth factor (KGF) had no effect. To address function, HaCaT cells were exposed to two NRP1 ligands, VEGF165 and Sema3A. Neither had an effect on proliferation, whereas Sema3A, but not VEGF165, inhibited cell migration. Down-regulation of NRP1 by NRSF overexpression reduced Sema3A activity. It was concluded that NRSF is a transcription factor that silences NRP1 expression and thereby diminishes the Sema3A mediated inhibition of HaCaT keratinocyte migration.

Highlights

Neuropilins (NRPs)2 are 130-kDa type I transmembrane receptors that mediate neuronal guidance, angiogenesis, and the immune response [1, 2]
Neuropilin-1 Is Expressed in Keratinocytes in Vivo and in Vitro—Previous analysis by in situ hybridization showed that NRP1 is expressed by epidermal keratinocytes in human skin [25]
Because NRPs depend on either plexins or VEGFRs to transduce the signal into the cell, the expression of these molecules in HaCaT cells was investigated

Summary

To whom correspondence should be addressed

T cells [3]. NRPs bind members of the vascular endothelial growth factor (VEGF) family, including VEGF-A, VEGF-B, VEGF-C, and placenta growth factor PlGF-2 [1, 3]. Overexpression of the transcription factor Prox-1 down-regulates NRP1 expression in the transition from vascular to lymphatic endothelial cells [20]. Whereas NRP1 function and binding of VEGF and semaphorins has been studied extensively in endothelial cells and neurons [1, 3], the regulation of NRP1 gene expression and the response to NRP1 ligands in keratinocytes has not been documented. The strong up-regulation of NRP1 by EGF or the heparin-binding HB-EGF in HaCaT cells is accompanied by a down-regulation of NRSF RNA and protein levels. This is the first report to show that keratinocyte NRP1 is biologically functional. In keratinocytes, NRP1 is functional as a Sema3A receptor and that NRP1 expression is negatively regulated by NRSF

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION