Neuregulin-1 Induces Expression of Egr-1 and Activates Acetylcholine Receptor Transcription Through an Egr-1-binding Site

https://doi.org/10.1016/j.jmb.2004.04.018Get rights and content

Abstract

Localization of acetylcholine receptors (AChRs) to neuromuscular synapses is mediated, in part, through selective transcription of AChR genes in myofiber synaptic nuclei. Neuregulin-1 (NRG-1) and its receptors, ErbBs, are concentrated at synaptic sites, and NRG-1 activates AChR synthesis in cultured muscle cells, suggesting that NRG-1-ErbB signaling functions to activate synapse-specific transcription. Previous studies have demonstrated that NRG-1-induced transcription is conferred by cis-acting elements located within 100 bp of 5′ flanking DNA from the AChR epsilon subunit gene, and that it requires a GABP binding site within this region. To determine whether additional regulatory elements have a role in NRG-1 responsiveness, we used transcriptional reporter assays in a muscle cell line, and we identified an element that is required for NRG-1-induced transcription (neuregulin response element, NRE). Proteins from myotube extracts bind the NRE and NRG-1 treatment of the cells stimulates this binding. The ability of NRG-1 to stimulate formation of a protein–DNA complex with the NRE requires induction of protein expression. The complex contains early growth response-1 (Egr-1), a member of the Egr family of transcription factors, because proteins in the complex bind specifically to an Egr consensus site, and formation of the complex is inhibited by antibodies to Egr-1. NRG-1 induces expression of Egr-1 in myotubes, which presumably is responsible for the ability of NRG-1 to stimulate protein binding to the NRE. These results suggest that NRG-1 signaling in myotubes involves induction of Egr-1 expression, which in turn serves to activate transcription of the AChR epsilon subunit gene.

Introduction

Shortly after motor axons contact developing myotubes, signals are exchanged between nerve and muscle that mediate the organization of a highly differentiated presynaptic nerve terminal and a highly specialized postsynaptic apparatus.1., 2. Acetylcholine receptors (AChRs) are among the proteins that become localized to the postsynaptic region in muscle, and their localization to this small patch of the myofiber membrane during development is a hallmark of the inductive events of synapse formation.

It appears that two distinct signaling pathways mediate postsynaptic differentiation, including clustering of AChRs. In one pathway, agrin, a motor neuron-derived ligand, activates muscle-specific kinase (MuSK), a receptor tyrosine kinase, to stimulate the post-translational reorganization of proteins, including AChRs, in the muscle cell membrane.3., 4. MuSK is also able to initiate some aspects of postsynaptic differentiation independently of agrin in muscle that is not innervated.5., 6. A second pathway, whose signal is not known, leads to enhanced transcription of AChR genes in myofiber nuclei that are situated at synaptic sites.

Synapse-specific transcription has been demonstrated in transgenic mice that carry gene fusions between regulatory regions of AChR subunit genes and reporter genes.7., 8., 9. These transgenes are transcribed at a higher rate in myofiber nuclei near the synaptic site than in nuclei elsewhere in the myofiber, suggesting that motor neurons supply a signal to myofibers that activates AChR transcription in synaptic nuclei. Synapse-specific transcription leads to accumulation of AChR mRNA at synaptic sites,10., 11., 12. resulting in increased AChR protein synthesis in the synaptic region of the myofiber. RNAs encoding other synaptic proteins, including acetylcholine esterase, MuSK, Rapsyn, S-laminin, N-CAM, utrophin, and the regulatory subunit of protein kinase A, are also concentrated at synaptic sites, implying that synapse-specific transcription may be a general and important mechanism for clustering proteins at developing and adult neuromuscular synapses.

Neuregulin-1 (NRG-1), a widely expressed growth and differentiation factor that is structurally related to EGF, is currently the best candidate for the signal that activates synapse-specific transcription.13., 14., 15. NRG-1 is synthesized by motor neurons and concentrated at synaptic sites;16., 17., 18. like the signal that activates synapse-specific gene expression, NRG-1 is present in the synaptic basal lamina.16., 18. The receptors for NRGs, ErbB3 and ErbB4, and the co-receptor ErbB2 are members of the EGF receptor family;19 both ErbB2 and ErbB4 are concentrated in the postsynaptic membrane at neuromuscular synapses.20., 21., 22., 23. NRG-1 activates AChR gene expression in cultured muscle cells,16., 17. and the same cis-acting region that confers NRG-1 responsiveness also confers synapse-specific transcription in transgenic mice.24., 25. Further, a 5 bp regulatory element within this cis-acting region is required both for NRG-1-induced and synapse-specific transcription.26., 27., 28., 29., 30.

Mouse genetic approaches have been used to address whether NRG-1-mediated signaling is indeed required for synapse-specific gene expression. Mice lacking NRG-1, ErbB2 or ErbB4 are not informative because they die, owing to defects in cardiac development, several days prior to neuromuscular synapse formation.31., 32., 33. Mice lacking ErbB2 only in skeletal muscle are viable and have a mild deficiency in synaptic transmission and fewer AChRs at their neuromuscular synapses, providing further evidence that NRG-1-ErbB signaling may indeed have the suspected role in synapse formation.34 Because AChRs are not completely absent in these mice, ErbB3 and ErbB4 might partially compensate for loss of ErbB2. Mice that are heterozygous for an allele of NRG-1 that deletes the Ig-like domain (NRGIg+/−) also have reduced AChRs and less efficient synaptic transmission.35 Motor neurons do not appear to be an essential source of NRG-1 for stimulating AChR transcription, however, because AChRs are expressed normally in mice lacking NRG-1 only in motor neurons.6 NRG-1 also is expressed by skeletal muscle cells,36 where it may function in an autocrine fashion due to the influence of the agrin-MuSK pathway, which is required for all aspects of neuromuscular synapse formation.37., 38. In support of this idea, agrin can induce clustering of muscle-derived NRG-1 and ErbB receptors and stimulate AChR transcription through activation of ErbBs.39

NRG-1-induced transcription is conferred by cis-acting elements located within 100 bp of 5′ flanking DNA from the AChR ε subunit gene.17., 29. Within this region, a consensus binding site for Ets proteins is required to respond to NRG-1.29 GABP, a heterodimer of GABPα, an Ets protein, and GABPβ, a non-Ets protein that enhances the DNA-binding activity of GABP, are the predominant proteins in myotube nuclear extracts that bind this element.28., 30. NRG-1 stimulation does not increase the binding of GABP to DNA. NRG-1-stiumlated phosphorylation of GABP causes changes to its quaternary structure and increases its transcriptional activity, suggesting that NRG-1 signaling stimulates transcription by increasing the transcriptional activity of GABP without affecting its DNA-binding activity.40

Transcriptional reporter assays using P-19 teratocarcinoma cells, a non-muscle cell line, led to identification of a different element, a CA-rich sequence, that is required for NRG-1-induced transcription of the AChR ε subunit gene.41 NRG-1 stimulates binding of proteins from P-19 cells and from several other non-muscle cell lines to this element, and Sp1 is among the proteins that bind this element. The level of Sp1 in these cells is not altered by NRG-1 stimulation, indicating that Sp1 is most likely modified in these cells to increase its capacity to bind DNA. These studies did not evaluate the role of the CA-rich element in skeletal muscle cells, where expression of the AChR ε subunit gene is restricted. Because different cell types might exhibit different complements of transcriptional regulatory proteins that respond to NRG-1, it was not clear what function, if any, this element would have in muscle cells. We characterized the role of the CA-rich element of AChR ε subunit gene in cultured muscle cells and provide evidence for a different mechanism that could mediate a transcriptional response to NRG-1, one involving the transcription factor early growth response-1 (Egr-1). We show that the CA-rich element is required for NRG-1-induced transcription in muscle cells and that NRG-1 stimulates binding of Egr-1, rather than Sp1, to this element. Further, we show that expression of Egr-1 is increased by NRG-1, suggesting that NRG-1 signaling involves induction of Egr-1 expression, which in turn serves to activate AChR transcription.

Section snippets

Identification of a neuregulin-1 response element in muscle cells

We used a transcriptional reporter assay to determine whether NRG-1-induced expression of the AChR ε subunit gene in skeletal muscle cells requires the CA-rich element. We generated AChR ε subunit (−228/+25)-human growth hormone (hGH) gene fusions using wild-type AChR gene regulatory sequences and using AChR sequences with various nucleotide substitution mutations within this element. We then stably transfected Sol8 muscle cells with these gene fusions and measured the amount of hGH expression

Discussion

Our results demonstrate that a binding-site for Egr proteins in the AChR ε subunit gene is required for transcriptional induction of the AChR gene by NRG-1. We show that Egr-1 binds this NRE, and that expression of Egr-1 is induced by NRG-1. These results suggest that NRG-1 signaling in muscle cells involves induction of Egr-1 expression, which in turn serves to activate AChR transcription.

Egr-1 expression is induced by many different stimuli, including various growth factors, conditions of

Cell culture

Sol8 myoblasts were grown on dishes coated with Matrigel (BD Biosciences) and fed with growth media (Dulbecco's modification of Eagle's medium (DMEM), 10% (v/v) fetal bovine serum, 0.5% (v/v) chick embryo extract, 50 μg/ml gentamycin). To induce differentiation into myotubes, cells were grown to confluency and the media was replaced with differentiation media (DMEM, 5% horse serum, 50 μg/ml gentamycin).

To prepare whole cell extracts, myotubes grown on 60 mm dishes that had been in

Acknowledgments

This work was supported by a research grant from the NIH (NS44924) to L.F.

References (66)

  • J Si et al.

    Induction of acetylcholine receptor gene expression by ARIA requires activation of mitogen-activated protein kinase

    J. Biol. Chem.

    (1996)
  • V.P Sukhatme et al.

    A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization

    Cell

    (1988)
  • P.R Mittelstadt et al.

    Role of Egr-2 in up-regulation of Fas ligand in normal T cells and aberrant double-negative lpr and gld T cells

    J. Biol. Chem.

    (1999)
  • S.F Yan et al.

    Hypoxia-associated induction of early growth response-1 gene expression

    J. Biol. Chem.

    (1999)
  • D.M Cohen et al.

    Urea inducibility of egr-1 in murine inner medullary collecting duct cells is mediated by the serum response element and adjacent Ets motifs

    J. Biol. Chem.

    (1996)
  • S.A Qureshi et al.

    v-Src activates mitogen-responsive transcription factor Egr-1 via serum response elements

    J. Biol. Chem.

    (1991)
  • C Sweeney et al.

    Growth factor-specific signaling pathway stimulation and gene expression mediated by ErbB receptors

    J. Biol. Chem.

    (2001)
  • J Si et al.

    Identification of an element required for acetylcholine receptor-inducing activity (ARIA)-induced expression of the acetylcholine receptor epsilon subunit gene

    J. Biol. Chem.

    (1997)
  • Y Li et al.

    Promoter elements and transcriptional control of the mouse acetylcholinesterase gene

    J. Biol. Chem.

    (1993)
  • R Marais et al.

    accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain

    Cell

    (1993)
  • R Higuchi

    Recombinant PCR

  • S.J Burden

    The formation of neuromuscular synapses

    Genes Dev.

    (1998)
  • J.R Sanes et al.

    Induction, assembly, maturation and maintenance of a postsynaptic apparatus

    Nature Rev. Neurosci.

    (2001)
  • U.J McMahan

    The agrin hypothesis

    Cold Spring Harb. Symp. Quant. Biol.

    (1990)
  • W Lin et al.

    Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse

    Nature

    (2001)
  • A Klarsfeld et al.

    An acetylcholine receptor alpha-subunit promoter conferring preferential synaptic expression in muscle of transgenic mice

    EMBO J.

    (1991)
  • J.R Sanes et al.

    Selective expression of an acetylcholine receptor-lacZ transgene in synaptic nuclei of adult muscle fibers

    Development

    (1991)
  • A.M Simon et al.

    Spatial restriction of AChR gene expression to subsynaptic nuclei

    Development

    (1992)
  • J.P Merlie et al.

    Concentration of acetylcholine receptor mRNA in synaptic regions of adult muscle fibres

    Nature

    (1985)
  • B Fontaine et al.

    Localization of nicotinic acetylcholine receptor alpha-subunit transcripts during myogenesis and motor endplate development in the chick

    J. Cell Biol.

    (1989)
  • K.L Carraway et al.

    Neuregulin-2, a new ligand of ErbB3/ErbB4-receptor tyrosine kinases

    Nature

    (1997)
  • H Chang et al.

    Ligands for ErbB-family receptors encoded by a neuregulin-like gene

    Nature

    (1997)
  • D.L Falls et al.

    ARIA, a protein that stimulates acetylcholine receptor synthesis, is a member of the neu ligand family

    Cell

    (1993)
  • Cited by (15)

    • Regulation of Gene expression at the neuromuscular Junction

      2020, Neuroscience Letters
      Citation Excerpt :

      Egr-3 mediates Neuregulin response in intrafusal muscle fibers and activates AChR expression ([101] and references therein). Neuregulins were also shown to activate Egr-1 expression in myotubes and an Egr binding site was identified in the AChR ε promoter [102]. Interestingly, Egr-1 was recently shown to negatively regulate Agrin expression [103].

    • The zinc finger protein Zfpm1 modulates ventricular trabeculation through Neuregulin-ErbB signalling

      2019, Developmental Biology
      Citation Excerpt :

      To examine whether upregulation in nrg1 expression could lead to enhancement of ErbB signalling, the phosphorylation level of MAPK and AKT were examined and increased phosphorylation of both proteins in Zfpm1-/- samples was observed (Fig. 5B). The expression of targets gene, junba, erg1 and erg2b (Fromm and Rhode, 2004; Sweeney et al., 2001), was explored and the stronger signal in Zfpm1-/- samples further confirmed arisen Neuregulin-ErbB signalling activity (Fig. 5C and data not shown). Other trabecular markers, including gja5a, also displayed disarrayed expression in Zfpm1-/- ventricles (Fig. 5C and data not shown).

    • Advanced age enhances the sepsis-induced up-regulation of the γ- and α7-nicotinic acetylcholine receptors in different parts of the skeletal muscles

      2016, Archives of Gerontology and Geriatrics
      Citation Excerpt :

      Neuregulin-1 could bind to muscle cell membrane receptors (ErbB2) to form a complex that phosphorylated the ras protein, which further activated the MAP kinase signaling pathway. The latter could lead to transient activation of c-JNK and c-fos and subsequent phosphorylation of the GABP α/β protein, which could bind to the N-box of the nAChR promoter and activate the transcription and expression of ε-nAChR (Fromm & Rhode, 2004; Kim et al., 2013; Si, Tanowitz, Won, & Mei, 1998). Therefore, advanced age and sepsis reduced the synthesis and release of neuregulin-1 from the motor neurons, which could reduce the inhibitory signals, thereby activating γ-nAChR and α7-nAChR expression.

    • Transmitting the message: intracellular mRNA localization

      2010, Current Opinion in Cell Biology
      Citation Excerpt :

      In multinucleate myofibers, transcripts δ-subunit and ɛ-subunit of the acetylcholine receptor (AChR) accumulate in the vicinity of synaptic myonuclei [23,24]. Local transcriptional control involves the activation of synaptic expression by the nerve-derived signal agrin and the trophic factor neuregulin, and concomitant expression in extrasynaptic nuclei by electrical activity [25–27] (Figure 1a). Another well-studied case of localized transcription in a syncytium is the Drosophila blastoderm embryo, where an intricate pattern of transcription factor gene expression leading to complex overlapping of localized patterns of mRNA is expressed in different subsets of a monolayer of nuclei.

    • How does an mRNA find its way? Intracellular localisation of transcripts

      2007, Seminars in Cell and Developmental Biology
    View all citing articles on Scopus
    View full text