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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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Agrin uses two pathways for the activation of the 2.1-kb nsk2/musk promoter fragment in cultured myotubes. (A) In C2C12 myotubes, activation of p1.6luc-N by Agrin is fully blocked by HER2KM and HER4KM, dominant-negative mutants of human ErbB2 and ErbB4, respectively, whereas wild-type HER2 mimics the induction by Agrin. (B) Same as in A but blockade by dominant-negative JNK, JNK-APF; conversely, overexpression of MKK7D, a constitutively active kinase activating JNK, activates the fragment in the absence of Agrin. (C) Same as in B but with an AChRɛ promoter fragment (pLCF216ɛ). Note similar activation patterns of nsk2/musk and AChRɛ promoter fragments. (D) Constitutively active Rac, RacV12, activates p1.6luc-N; induction by Rac is completely blocked by GABPβDN. (E) With ErbB signaling blocked, p1.6luc-N is activated more strongly by agrin than by saturating (2 nM) NRG-1, and responses are fully blocked by dominant- negative Rac, RacN17. Constitutively active Rac, RacV12, strongly activates the promoter in spite of blockade of ErbB signaling. All experiments were performed on C2C12 cells transfected with p1.6luc-N. (F) Agrin does not discernibly increase ErbB2 levels in C2C12 myotubes. Experiments performed as in Fig. 3 with plasmids transfected as indicated. Responses are normalized to control values in the left-hand columns, and nonspecific background luciferase activities as observed in myotubes on agrin-free substrate are subtracted (C). *P < 0.05, **P < 0.01, and ***P < 0.001, respectively, in two-tailed t test. Bars ± SEM.
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fig5: Agrin uses two pathways for the activation of the 2.1-kb nsk2/musk promoter fragment in cultured myotubes. (A) In C2C12 myotubes, activation of p1.6luc-N by Agrin is fully blocked by HER2KM and HER4KM, dominant-negative mutants of human ErbB2 and ErbB4, respectively, whereas wild-type HER2 mimics the induction by Agrin. (B) Same as in A but blockade by dominant-negative JNK, JNK-APF; conversely, overexpression of MKK7D, a constitutively active kinase activating JNK, activates the fragment in the absence of Agrin. (C) Same as in B but with an AChRɛ promoter fragment (pLCF216ɛ). Note similar activation patterns of nsk2/musk and AChRɛ promoter fragments. (D) Constitutively active Rac, RacV12, activates p1.6luc-N; induction by Rac is completely blocked by GABPβDN. (E) With ErbB signaling blocked, p1.6luc-N is activated more strongly by agrin than by saturating (2 nM) NRG-1, and responses are fully blocked by dominant- negative Rac, RacN17. Constitutively active Rac, RacV12, strongly activates the promoter in spite of blockade of ErbB signaling. All experiments were performed on C2C12 cells transfected with p1.6luc-N. (F) Agrin does not discernibly increase ErbB2 levels in C2C12 myotubes. Experiments performed as in Fig. 3 with plasmids transfected as indicated. Responses are normalized to control values in the left-hand columns, and nonspecific background luciferase activities as observed in myotubes on agrin-free substrate are subtracted (C). *P < 0.05, **P < 0.01, and ***P < 0.001, respectively, in two-tailed t test. Bars ± SEM.

Mentions: In cultured myotubes, NRG-1/ErbB-induced activation of AChRδ and ɛ is mediated by ERK and JNK MAPKs (Tansey et al., 1996; Altiok et al., 1997; Si et al., 1999). Therefore, we examined whether nsk2/musk was induced indirectly by Agrin, i.e., via the organization of an NRG-1/ErbB pathway with NRG derived from the myotubes, as we had observed previously for the induction of AChRɛ by Agrin (Meier et al., 1998b). As predicted for such a mechanism, activation of p1.6luc-N by mini-Agrin in C2C12 myotubes was abolished by overexpression of pHER2KM or pHER4KM, dominant-negative mutants of ErbB2 and ErbB4 (Fig. 5 A). Activation of nsk2/musk by Agrin was also blocked by overexpression of a dominant-negative mutant of JNK, pJNK-APF (Fig. 5 B). Conversely, a constitutively active mutant of MKK7, pMKK7D, which activates JNK, induced nsk2/musk in the absence of Agrin. A promoter–reporter construct of AChRɛ (Jones et al., 1996) was regulated similarly (Fig. 5 C). These data suggest that Agrin organizes a secondary NRG-1/ErbB pathway, which in turn activates musk and AChRɛ via ERK and JNK.


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)

Agrin uses two pathways for the activation of the 2.1-kb nsk2/musk promoter fragment in cultured myotubes. (A) In C2C12 myotubes, activation of p1.6luc-N by Agrin is fully blocked by HER2KM and HER4KM, dominant-negative mutants of human ErbB2 and ErbB4, respectively, whereas wild-type HER2 mimics the induction by Agrin. (B) Same as in A but blockade by dominant-negative JNK, JNK-APF; conversely, overexpression of MKK7D, a constitutively active kinase activating JNK, activates the fragment in the absence of Agrin. (C) Same as in B but with an AChRɛ promoter fragment (pLCF216ɛ). Note similar activation patterns of nsk2/musk and AChRɛ promoter fragments. (D) Constitutively active Rac, RacV12, activates p1.6luc-N; induction by Rac is completely blocked by GABPβDN. (E) With ErbB signaling blocked, p1.6luc-N is activated more strongly by agrin than by saturating (2 nM) NRG-1, and responses are fully blocked by dominant- negative Rac, RacN17. Constitutively active Rac, RacV12, strongly activates the promoter in spite of blockade of ErbB signaling. All experiments were performed on C2C12 cells transfected with p1.6luc-N. (F) Agrin does not discernibly increase ErbB2 levels in C2C12 myotubes. Experiments performed as in Fig. 3 with plasmids transfected as indicated. Responses are normalized to control values in the left-hand columns, and nonspecific background luciferase activities as observed in myotubes on agrin-free substrate are subtracted (C). *P < 0.05, **P < 0.01, and ***P < 0.001, respectively, in two-tailed t test. Bars ± SEM.
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fig5: Agrin uses two pathways for the activation of the 2.1-kb nsk2/musk promoter fragment in cultured myotubes. (A) In C2C12 myotubes, activation of p1.6luc-N by Agrin is fully blocked by HER2KM and HER4KM, dominant-negative mutants of human ErbB2 and ErbB4, respectively, whereas wild-type HER2 mimics the induction by Agrin. (B) Same as in A but blockade by dominant-negative JNK, JNK-APF; conversely, overexpression of MKK7D, a constitutively active kinase activating JNK, activates the fragment in the absence of Agrin. (C) Same as in B but with an AChRɛ promoter fragment (pLCF216ɛ). Note similar activation patterns of nsk2/musk and AChRɛ promoter fragments. (D) Constitutively active Rac, RacV12, activates p1.6luc-N; induction by Rac is completely blocked by GABPβDN. (E) With ErbB signaling blocked, p1.6luc-N is activated more strongly by agrin than by saturating (2 nM) NRG-1, and responses are fully blocked by dominant- negative Rac, RacN17. Constitutively active Rac, RacV12, strongly activates the promoter in spite of blockade of ErbB signaling. All experiments were performed on C2C12 cells transfected with p1.6luc-N. (F) Agrin does not discernibly increase ErbB2 levels in C2C12 myotubes. Experiments performed as in Fig. 3 with plasmids transfected as indicated. Responses are normalized to control values in the left-hand columns, and nonspecific background luciferase activities as observed in myotubes on agrin-free substrate are subtracted (C). *P < 0.05, **P < 0.01, and ***P < 0.001, respectively, in two-tailed t test. Bars ± SEM.
Mentions: In cultured myotubes, NRG-1/ErbB-induced activation of AChRδ and ɛ is mediated by ERK and JNK MAPKs (Tansey et al., 1996; Altiok et al., 1997; Si et al., 1999). Therefore, we examined whether nsk2/musk was induced indirectly by Agrin, i.e., via the organization of an NRG-1/ErbB pathway with NRG derived from the myotubes, as we had observed previously for the induction of AChRɛ by Agrin (Meier et al., 1998b). As predicted for such a mechanism, activation of p1.6luc-N by mini-Agrin in C2C12 myotubes was abolished by overexpression of pHER2KM or pHER4KM, dominant-negative mutants of ErbB2 and ErbB4 (Fig. 5 A). Activation of nsk2/musk by Agrin was also blocked by overexpression of a dominant-negative mutant of JNK, pJNK-APF (Fig. 5 B). Conversely, a constitutively active mutant of MKK7, pMKK7D, which activates JNK, induced nsk2/musk in the absence of Agrin. A promoter–reporter construct of AChRɛ (Jones et al., 1996) was regulated similarly (Fig. 5 C). These data suggest that Agrin organizes a secondary NRG-1/ErbB pathway, which in turn activates musk and AChRɛ via ERK and JNK.

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