Journal of Molecular Biology
Neuregulin-1 Induces Expression of Egr-1 and Activates Acetylcholine Receptor Transcription Through an Egr-1-binding Site
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.
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2010, Current Opinion in Cell BiologyCitation 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
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