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A novel pathway for MuSK to induce key genes in neuromuscular synapse formation.

Lacazette E, Le Calvez S, Gajendran N, Brenner HR - J. Cell Biol. (2003)

Bottom Line: Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes.The same pathways are used to regulate synaptic expression of AChR epsilon.We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChR epsilon. We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

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GABP binds to the N-box of a nsk2/musk promoter fragment and activates it. (A) Binding activity of wild-type nuclear extracts to the N-box present in the probe. The specificity of the signal is eliminated by competition with an unlabeled wild-type probe (WTcomp), whereas an unlabeled mutant competitor (MUTcomp) leaves the signal unaffected. (B) Sequences of the oligonucleotides used as probes or competitors. (C) Unlabeled oligonucleotide containing the N-box motif of the AChRɛ subunit promoter (ɛ-comp) competes with N-box of musk for binding of nuclear extract. (D) The N-box binding activity produced by nuclear extracts from C2C12 cells (lane 1) is supershifted (bold arrow) by antibodies directed against GABPα (lane 2) and GABPβ (lane 3). (E) Nuclear extracts of transiently transfected HEK293 cells expressing GABPα-myc and GABPβ-myc (lanes 1 and 2) or only GABPα-myc (lanes 3 and 4) or GABPβ-myc (lanes 5 and 6) possess an N-box binding activity (arrow), which is supershifted (bold arrow) in the presence of an anti-myc antibody (Ab, lanes 2, 4, and 6). The second band seen in the shift may originate from binding activity specific for HEK293 cells. (F) Agrin-induced activation of the 2.1-kb nsk2/musk promoter fragment in C2C12 myotubes is blocked by overexpression of GABPβDN. ***P < 0.001 in two-tailed t test. Bars ± SEM. Note that for better resolution of the agrin-specific induction, luciferase activities in parallel control cultures (not depicted) are subtracted.
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fig4: GABP binds to the N-box of a nsk2/musk promoter fragment and activates it. (A) Binding activity of wild-type nuclear extracts to the N-box present in the probe. The specificity of the signal is eliminated by competition with an unlabeled wild-type probe (WTcomp), whereas an unlabeled mutant competitor (MUTcomp) leaves the signal unaffected. (B) Sequences of the oligonucleotides used as probes or competitors. (C) Unlabeled oligonucleotide containing the N-box motif of the AChRɛ subunit promoter (ɛ-comp) competes with N-box of musk for binding of nuclear extract. (D) The N-box binding activity produced by nuclear extracts from C2C12 cells (lane 1) is supershifted (bold arrow) by antibodies directed against GABPα (lane 2) and GABPβ (lane 3). (E) Nuclear extracts of transiently transfected HEK293 cells expressing GABPα-myc and GABPβ-myc (lanes 1 and 2) or only GABPα-myc (lanes 3 and 4) or GABPβ-myc (lanes 5 and 6) possess an N-box binding activity (arrow), which is supershifted (bold arrow) in the presence of an anti-myc antibody (Ab, lanes 2, 4, and 6). The second band seen in the shift may originate from binding activity specific for HEK293 cells. (F) Agrin-induced activation of the 2.1-kb nsk2/musk promoter fragment in C2C12 myotubes is blocked by overexpression of GABPβDN. ***P < 0.001 in two-tailed t test. Bars ± SEM. Note that for better resolution of the agrin-specific induction, luciferase activities in parallel control cultures (not depicted) are subtracted.

Mentions: In cultured myotubes, musk, like AChR, is also activated by NRG-1 (Ip et al., 2000). The NRG-dependent activation of AChR genes depends on the binding of active transcription factor GABP to the N-box (Koike et al., 1995; Duclert et al., 1996; Fromm and Burden, 1998; Schaeffer et al., 1998). Therefore, we next tested in mobility shift assays whether the transcription factor complex GABP, composed of GABPα- and β subunits (Batchelor et al., 1998), binds to the N-box of the nsk2/musk promoter fragment, using a radiolabeled 24-base fragment of the first intron containing the N-box motif as a probe. Exposure of the probe to nuclear extracts from C2C12 myotubes revealed binding activity in the extract (Fig. 4 A). Adding nonlabeled probe eliminated the detectable shifted complex, indicating specificity of binding. Integrity of the N-box was necessary, since the nonradiolabeled probe with the N-box mutated was ineffective in competing for binding of the nuclear extract (Fig. 4, A and C). We next examined whether addition of a 24-nucleotide fragment of the rat AChRɛ subunit promoter, which also contains an N-box, ɛ-comp (Fig. 4 B), could compete for binding activity from C2C12 nuclear extracts. Indeed, a 25-fold molar excess of ɛ-comp prevented the retardation of the labeled N-box probe from nsk2/musk that had been produced by the addition of C2C12 nuclear extract (Fig. 4 C). Furthermore, a supershift was produced by binding of anti-GABPα or anti-GABPβ antibodies (Fig. 4 D). Likewise, when the labeled probe was exposed to an extract from HEK293 cells transiently expressing myc-tagged GABPα and GABPβ, respectively, supershifts were observed in the presence of anti-myc antibody (Fig. 4 E). These data combined indicate that the N-box motif of the MuSK promoter specifically binds GABP.


A novel pathway for MuSK to induce key genes in neuromuscular synapse formation.

Lacazette E, Le Calvez S, Gajendran N, Brenner HR - J. Cell Biol. (2003)

GABP binds to the N-box of a nsk2/musk promoter fragment and activates it. (A) Binding activity of wild-type nuclear extracts to the N-box present in the probe. The specificity of the signal is eliminated by competition with an unlabeled wild-type probe (WTcomp), whereas an unlabeled mutant competitor (MUTcomp) leaves the signal unaffected. (B) Sequences of the oligonucleotides used as probes or competitors. (C) Unlabeled oligonucleotide containing the N-box motif of the AChRɛ subunit promoter (ɛ-comp) competes with N-box of musk for binding of nuclear extract. (D) The N-box binding activity produced by nuclear extracts from C2C12 cells (lane 1) is supershifted (bold arrow) by antibodies directed against GABPα (lane 2) and GABPβ (lane 3). (E) Nuclear extracts of transiently transfected HEK293 cells expressing GABPα-myc and GABPβ-myc (lanes 1 and 2) or only GABPα-myc (lanes 3 and 4) or GABPβ-myc (lanes 5 and 6) possess an N-box binding activity (arrow), which is supershifted (bold arrow) in the presence of an anti-myc antibody (Ab, lanes 2, 4, and 6). The second band seen in the shift may originate from binding activity specific for HEK293 cells. (F) Agrin-induced activation of the 2.1-kb nsk2/musk promoter fragment in C2C12 myotubes is blocked by overexpression of GABPβDN. ***P < 0.001 in two-tailed t test. Bars ± SEM. Note that for better resolution of the agrin-specific induction, luciferase activities in parallel control cultures (not depicted) are subtracted.
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fig4: GABP binds to the N-box of a nsk2/musk promoter fragment and activates it. (A) Binding activity of wild-type nuclear extracts to the N-box present in the probe. The specificity of the signal is eliminated by competition with an unlabeled wild-type probe (WTcomp), whereas an unlabeled mutant competitor (MUTcomp) leaves the signal unaffected. (B) Sequences of the oligonucleotides used as probes or competitors. (C) Unlabeled oligonucleotide containing the N-box motif of the AChRɛ subunit promoter (ɛ-comp) competes with N-box of musk for binding of nuclear extract. (D) The N-box binding activity produced by nuclear extracts from C2C12 cells (lane 1) is supershifted (bold arrow) by antibodies directed against GABPα (lane 2) and GABPβ (lane 3). (E) Nuclear extracts of transiently transfected HEK293 cells expressing GABPα-myc and GABPβ-myc (lanes 1 and 2) or only GABPα-myc (lanes 3 and 4) or GABPβ-myc (lanes 5 and 6) possess an N-box binding activity (arrow), which is supershifted (bold arrow) in the presence of an anti-myc antibody (Ab, lanes 2, 4, and 6). The second band seen in the shift may originate from binding activity specific for HEK293 cells. (F) Agrin-induced activation of the 2.1-kb nsk2/musk promoter fragment in C2C12 myotubes is blocked by overexpression of GABPβDN. ***P < 0.001 in two-tailed t test. Bars ± SEM. Note that for better resolution of the agrin-specific induction, luciferase activities in parallel control cultures (not depicted) are subtracted.
Mentions: In cultured myotubes, musk, like AChR, is also activated by NRG-1 (Ip et al., 2000). The NRG-dependent activation of AChR genes depends on the binding of active transcription factor GABP to the N-box (Koike et al., 1995; Duclert et al., 1996; Fromm and Burden, 1998; Schaeffer et al., 1998). Therefore, we next tested in mobility shift assays whether the transcription factor complex GABP, composed of GABPα- and β subunits (Batchelor et al., 1998), binds to the N-box of the nsk2/musk promoter fragment, using a radiolabeled 24-base fragment of the first intron containing the N-box motif as a probe. Exposure of the probe to nuclear extracts from C2C12 myotubes revealed binding activity in the extract (Fig. 4 A). Adding nonlabeled probe eliminated the detectable shifted complex, indicating specificity of binding. Integrity of the N-box was necessary, since the nonradiolabeled probe with the N-box mutated was ineffective in competing for binding of the nuclear extract (Fig. 4, A and C). We next examined whether addition of a 24-nucleotide fragment of the rat AChRɛ subunit promoter, which also contains an N-box, ɛ-comp (Fig. 4 B), could compete for binding activity from C2C12 nuclear extracts. Indeed, a 25-fold molar excess of ɛ-comp prevented the retardation of the labeled N-box probe from nsk2/musk that had been produced by the addition of C2C12 nuclear extract (Fig. 4 C). Furthermore, a supershift was produced by binding of anti-GABPα or anti-GABPβ antibodies (Fig. 4 D). Likewise, when the labeled probe was exposed to an extract from HEK293 cells transiently expressing myc-tagged GABPα and GABPβ, respectively, supershifts were observed in the presence of anti-myc antibody (Fig. 4 E). These data combined indicate that the N-box motif of the MuSK promoter specifically binds GABP.

Bottom Line: Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes.The same pathways are used to regulate synaptic expression of AChR epsilon.We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChR epsilon. We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

Show MeSH
Related in: MedlinePlus