Regulation of low affinity neurotrophin receptor (p75NTR) by early growth response (Egr) transcriptional regulators

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Abstract

The low affinity neurotrophin receptor p75NTR is a multifunctional receptor with important roles in neurotrophin signaling, axon outgrowth, and oligodendroglia and neuron survival. It is transcriptionally regulated with spatial and temporal precision during nervous system development, injury and regeneration. Very little is known about how p75NTR expression is dynamically regulated but it is likely to influence how p75NTR signals in particular cellular contexts. Here, we identify the early growth response (Egr) transcriptional regulators, Egr1 and Egr3, as direct modulators of p75NTR gene expression. Egr1 and Egr3 bind and transactivate the p75NTR promoter in vitro and in vivo, using distinct response elements on the p75NTR promoter. Consistent with these results, p75NTR expression is greatly diminished in muscle spindle stretch receptors and in peripheral nerve Schwann cells in Egr gene deficient mice. Taken together, the results elucidate a novel mechanism whereby Egr proteins can directly modulate p75NTR expression and signaling in vivo.

Introduction

The low affinity neurotrophin receptor (p75NTR) is a member of the tumor necrosis factor (TNF) superfamily of transmembrane glycoprotein receptors with important roles in regulating axon growth and neuron/oligodendrocyte survival during nervous system development and repair (Barker, 2004, Nykjaer et al., 2005, Reichardt, 2006). For example, p75NTR binds neurotrophins with relatively low affinity (Chao, 1994, Chao and Hempstead, 1995), but when it is coexpressed with tropomyosin-related kinase (Trk) transmembrane tyrosine kinase receptors it substantially increases the affinity and specificity of the neurotrophins NGF, BDNF/NT-4 and NT-3 for TrkA, TrkB and TrkC respectively, to mediate neuron survival and differentiation through p75NTR/Trk signaling (Benedetti et al., 1993, Davies et al., 1993). Similarly, p75NTR can complex with Sortilin, a member of the Vps10p-domain receptor family, and bind pro-neurotrophin ligands such as pro-NGF to facilitate oligodendrocyte and neuron apoptosis in vitro and in vivo (Beattie et al., 2002, Harrington et al., 2004, Nykjaer et al., 2004). Through yet additional coreceptor interactions, p75NTR can complex with NogoR and Lingo-1 receptors to bind myelin based growth inhibitors such as Nogo, myelin associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG) that modulate RhoA GTPase signaling and the axon cytoskeleton during development and regeneration (Mi et al., 2004, Wang et al., 2002, Wong et al., 2002, Yamashita et al., 1999).

The mechanisms by which p75NTR mediates such diverse cellular responses are not well understood but may be partially explained by the cellular and developmental context in which it is expressed. p75NTR expression is transcriptionally regulated by a variety of stimuli including growth factor signaling (such as NGF) during development (Kuruvilla et al., 2004, Ma et al., 1992), by central and peripheral nervous system injury (Beattie et al., 2002, Kokaia et al., 1998, Rende et al., 1993), and by disease processes such as ischemia, epilepsy, Alzheimer's disease and amyotrophic lateral sclerosis (Kokaia et al., 1998, Lowry et al., 2001, Mufson and Kordower, 1992, Roux et al., 1999) to name a few. Thus, depending upon the particular co-receptors that are also expressed and/or regulated in a cellular/disease context, modulation of p75NTR expression could be expected to influence how particular cells respond to normal or abnormal stimuli. Despite the fact that well-coordinated regulation of p75NTR expression is likely to have an important role in diversifying its signaling functions, very little is known about how it is regulated and what particular transcriptional mechanisms may influence its expression.

Early growth response (Egr) proteins are inducible transcriptional regulators that have an important role in regulating gene expression in response to a variety of extracellular stimuli that influence cellular growth, differentiation, response to injury and reaction to chronic nervous system diseases (for review see Beckmann and Wilce, 1997, O'Donovan et al., 1999). Egr proteins (Egr1–4) are encoded by four separate genes that contain highly homologous DNA-binding domains. They bind to moderately well-conserved Egr response elements (ERE) in target gene promoters to modulate their expression by imprecisely defined mechanisms. Mice lacking functional Egr genes have a wide range of developmental abnormalities, including infertility (Lee et al., 1996, Tourtellotte et al., 1999, Tourtellotte et al., 2000), hindbrain morphogenesis defects (Schneider-Maunoury et al., 1993, Swiatek and Gridley, 1993), impaired peripheral nerve myelination (Topilko et al., 1994), impaired muscle stretch receptor morphogenesis (Tourtellotte et al., 2001, Tourtellotte and Milbrandt, 1998), sympathetic nervous system abnormalities (Eldredge and Tourtellotte, 2004) and defects in learning and memory (Jones et al., 2001, Li et al., 2007). Despite the wide range of abnormalities in Egr gene deficient mice, only a small number of target genes directly regulated by Egr proteins in vivo have been identified.

The p75NTR promoter lacks consensus TATA and CAAT sequences, but contains multiple GC-rich motifs, suggesting functional roles for transcriptional regulators which bind GC-rich sequences, such as Egr transcription factors (Poukka et al., 1996). Egr proteins are rapidly induced by stimuli that also induce p75NTR expression, and enforced expression of Egr1 (Nikam et al., 1995) and Egr3 (Albert et al., 2005) upregulates p75NTR expression in vitro. Considering the high degree of association between the expression of Egr proteins and p75NTR in developmental and reactive states such as nervous system injury and repair, these results suggest that Egr proteins may have an important role in regulating p75NTR expression. However, whether p75NTR is subject to direct regulation by Egr proteins, through which regulatory domains Egr proteins may bind and transactivate it, and whether Egr proteins regulate it in vivo have not been clearly defined. In this study, we show that Egr1 and Egr3 directly bind specific regulatory elements in the p75NTR promoter in vivo and are capable of inducing p75NTR gene expression. Moreover, in muscle spindle stretch receptors and peripheral nerves, where p75NTR expression has important functions in spindle morphogenesis and peripheral nerve myelination, respectively, p75NTR expression is deregulated in Egr gene-deficient mice. These results demonstrate a physiologically relevant role for Egr proteins in regulating p75NTR gene expression and define a highly dynamic regulatory mechanism for modulating p75NTR expression during development, injury and regeneration.

Section snippets

Enforced expression of Egr1 or Egr3 induces p75NTR expression in multiple cell types

The p75NTR gene was identified as a potential target of Egr3 regulation from a microarray analysis intended to characterize the target genes regulated by Egr3 during muscle stretch receptor development (Albert et al., 2005). The microarray result was confirmed by comparing p75NTR expression in primary murine myotubes infected with adenoviruses expressing either wild type (Egr3) or a transcriptionally inactive c-terminal truncation of Egr3 (Egr3Tr). p75NTR expression was upregulated 14.2-fold by

Discussion

Egr transcriptional regulators are well suited to dynamically modulate gene expression during development, injury and regeneration. Egr genes encode transiently expressed and relatively unstable transcriptional regulatory proteins that modulate target gene expression in a restricted temporal and spatial context. Here, we showed that the low affinity neurotrophin receptor p75NTR, a multi-functional receptor highly regulated during development, injury and regeneration, is directly regulated by

Animals

Egr1-deficient and Egr3-deficient mice were generated and genotyped as previously described (Lee et al., 1996, Tourtellotte and Milbrandt, 1998). Egr1-deficient mice were backcrossed 10 generations to C57BL/6J mice and Egr3-deficient mice were backcrossed 6 generations to C57BL/6J mice. p75NTR exonIII-deficient mice were obtained from Jackson laboratories on a C57BL/6 genetic background (Lee et al., 1992) and genotyped by PCR using primers: 5′-TGTTACGTTCTCTGACGTGGTGAG-3′,

Acknowledgments

We thank L. Eldredge and J. Carter for helpful discussion and comments on the manuscript. This study was supported by The National Institutes of Health (NS046468 and NS040748).

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