Research ArticleMUSCLE DEVELOPMENT

MuSK is a BMP co-receptor that shapes BMP responses and calcium signaling in muscle cells

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Science Signaling  06 Sep 2016:
Vol. 9, Issue 444, pp. ra87
DOI: 10.1126/scisignal.aaf0890
  • Fig. 1 MuSK ectodomain binds to BMP4.

    (A) Schematic representation of the MuSK ectodomain fusion protein used in the reporter and the solution-binding assays. The fusion protein contains the three immunoglobulin (Ig)–like domains (Ig1, Ig2, and Ig3) and the cysteine-rich domain (CRD) of the extracellular portion of MuSK fused to a His-tag at the C terminus. (B) BMP4 depletion. Soluble BMP4 was co-incubated with His-tagged MuSK ectodomain or a control protein [His-tagged tobacco etch virus (TEV) protease] followed by precipitation with nickel beads. Residual soluble BMP4 activity was measured using the C2C12BRA reporter cell line. The average value of untreated cells was set as 100% activity. Data are means ± SD from three independent experiments with eight replicates in each [n = 3; **P < 0.01; ns, not significant versus BMP4-only treatment, Bonferroni-adjusted one-way analysis of variance (ANOVA)]. (C) BMP4 coprecipitates with the MuSK ectodomain. The amount of BMP4 in eluates from the bead pellets was analyzed by ELISA. Data are representative of three independent experiments. Values indicate the average from eight replicates and are means ± SD (***P < 0.0001, unpaired, two-sided Student’s t test). RU, response units. (D) SPR binding analysis of MuSK with BMP4. Representative SPR profiles are shown for various concentrations of MuSK binding to BMP4. Sensorgrams were normalized for MuSK binding to a mock-coupled flow cell. The black lines show the experimental measurements of a twofold serial dilution over the concentration range 2 mM to 1.96 nM of each sensorgram, and the red lines correspond to global fits of the data to a 1:1 model using a heterogeneous surface model with the program EVILFIT. (E) Schematics of Fc-fusion of full length (FL) and Ig3-lacking (ΔIg3) MuSK ectodomain proteins used in (F). (F) The MuSK Ig3 domain is required for BMP4 binding. Immobilized FL or ΔIg3 MuSK ectodomain Fc-fusions were incubated with BMP4 (0 to 200 nM). Data are means ± SD from three independent experiments with four technical replicates in each (n = 3).

  • Fig. 2 MuSK influences BMP4-induced expression of a subset of genes in myoblasts.

    (A) Wild-type H-2Kb-tsA58 and MuSK−/− myoblasts were serum-deprived for 4 hours and then treated with BMP4. Microarray analysis identified differentially expressed genes upon BMP4 treatment in both genotypes. The number of genes up-regulated in response to BMP4 in wild-type and MuSK−/− myoblasts are grouped into a Venn diagram as wild type only, shared, and MuSK−/− only. Data represent the averages of three independent biological replicates. FDR-corrected P < 0.05 and ±1.5-fold change were used as selection criteria to identify genes differentially expressed between samples. (B to E) Validation of microarray results for four genes. Transcript abundances for MuSK-independent expression of Fabp7 (B) and MuSK-regulated expression of Ptgs2 (C), Ptger4 (D), and Rgs4 (E) were measured by qRT-PCR. Data are means ± SD from three biological replicate experiments (***P < 0.0001; **P < 0.01; *P < 0.05; ns, not significant versus untreated conditions, Bonferroni-adjusted one-way ANOVA).

  • Fig. 3 MuSK influences BMP4-induced expression of a subset of genes in myotubes.

    (A) Wild-type H-2Kb-tsA58 and MuSK−/− myotube cultures were treated with BMP4. RNA was isolated and subjected to transcriptomic analysis. The number of genes up-regulated in response to BMP4 responses for up-regulated genes in wild-type and MuSK−/− myotubes are grouped in a Venn diagram as wild type only, shared, and MuSK−/− only. Data represent the averages of three independent biological replicates. FDR-corrected P < 0.05 and ±1.5-fold change were used as selection criteria to identify genes differentially expressed between samples. (B) Venn diagram showing the number of transcripts that increased in abundance in response to BMP4 in a MuSK-dependent manner in myoblasts and myotubes. (C) Validation of microarray results. Wild-type H-2Kb-tsA58 and MuSK−/− myotubes were treated with BMP4, and the abundance of Myh15 and Car3 transcripts was analyzed in myoblasts and myotubes by qRT-PCR. Data are means ± SD from five biological replicate experiments (**P < 0.01; *P < 0.05; ns, not significant versus untreated condition, one-way ANOVA with Bonferroni correction). (D) MuSK, Myh15, and Car3 expression in soleus muscles compared to extensor digitorum longus (EDL) muscles. The abundance of MuSK, Myh15, and Car3 transcripts was analyzed by qRT-PCR. Data are means ± SD from five different animals (***P < 0.0001; **P < 0.01, unpaired, two-sided Student’s t test).

  • Fig. 4 MuSK regulation of BMP signaling is independent of MuSK kinase activity.

    (A) Wild-type H-2Kb-tsA58 myotubes were treated with BMP4 (25 ng/ml) or with agrin for the indicated times. MuSK was then immunoprecipitated, and the tyrosine kinase activation was assessed by Western blotting with a phosphotyrosine-specific antibody (upper panel). The blots were stripped and reprobed with a MuSK-specific antibody to assess total MuSK (bottom panel). Blots are representative of three independent experiments. IP, immunoprecipitation; IB, immunoblot. (B) MuSK−/− myoblasts or MuSK−/− myoblasts transgenically expressing wild-type, kinase-dead (K608A), or Y553F MuSK were treated with BMP4 (3.25 ng/ml for 2.5 hours), and Rgs4 transcript abundance was measured by qRT-PCR. Data are means ± SD from three biological replicate experiments (*P < 0.05, unpaired, two-sided Student’s t test).

  • Fig. 5 MuSK is a BMP co-receptor and regulates the canonical BMP4 pathway.

    (A) Wild-type H-2Kb-tsA58 and MuSK−/− myoblasts were treated with BMP4 at the indicated concentrations, and total and phosphorylated SMAD1/5/8 (pSMAD1/5/8) were then assessed by Western blotting (left panel). The abundance of pSMAD1/5/8 in Western blots was normalized to total SMAD1/5/8 for each condition. Fold change in pSMAD1/5/8 was calculated and plotted as the ratio of BMP4-treated conditions to the untreated controls (right panel). Data are means ± SEM from three biological replicate experiments and their independent Western blots. (B) Wild-type H-2Kb-tsA58 and MuSK−/− myoblasts were treated with BMP4 (3.25 ng/ml) for 2 hours. Id1 transcript abundance was quantified by qRT-PCR. Data are means ± SD from three biological replicates (**P < 0.01, one-way ANOVA with Bonferroni correction). (C) Serum-deprived wild-type H-2Kb-tsA58 myoblasts were treated with BMP4 in the presence or absence of the BMP type I receptor inhibitor LDN-193189, and Rgs4 transcript abundance was quantified by qRT-PCR. Data are means ± SD from three biological replicate experiments (**P < 0.01, one-way ANOVA with Bonferroni correction). (D) Serum-deprived wild-type H-2Kb-tsA58 myoblasts were treated with BMP4 in the presence or absence of LDN-193189, and Id1 transcript abundance was measured by qRT-PCR. Data are means ± SD from three biological replicate experiments (**P < 0.01, one-way ANOVA with Bonferroni correction). (E) Immobilized His-tagged MuSK ectodomain was incubated with the indicated concentrations of recombinant purified Fc-fusion versions of ALK2, ALK3, ALK4, ALK6, BMPRII, ActRIIB, and TROY. Bound receptors were detected with horseradish peroxidase (HRP)–conjugated antibodies recognizing human and mouse IgG. Data are representative of three independent experiments. Values indicate the average from four replicates and are means ± SD. (F) Pooled detergent extracts of cultured H-2Kb-tsA58 myotubes were divided into equal volumes and incubated with antibodies recognizing ALK3 or ALK6 or with normal IgG and immunoprecipitated. Immunoprecipitates were immunoblotted to show MuSK or ALK3 and ALK6 as indicated. A nonspecific band (n.s.) in the MuSK immunoblots is indicated. Data are representative of three biological replicate experiments. HC, heavy chain.

  • Fig. 6 BMP4 treatment inhibits carbachol-induced Ca2+ responses in wild-type H-2Kb-tsA58 myoblasts in an RGS4-dependent manner.

    (A) Pseudochrome Fluo-4 fluorescence images of H-2Kb-tsA58 myoblasts stimulated with carbachol in the absence (top) or presence (bottom) of BMP4. The images were acquired during baseline (I), peak carbachol response (II), before ionomycin (IM) addition (III), and at peak IM response (IV). Scale bar, 100 μm. The representative average of normalized Ca2+ imaging responses (Fnorm) after a 4-hour BMP4 treatment (right). (B) Pseudochrome Fluo-4 fluorescence images of wild-type myoblasts treated with the RGS4 inhibitor 11b and stimulated with carbachol in the absence (top) or presence (bottom) of BMP4 treatment. The images were acquired during baseline (I), peak carbachol response (II), before IM addition (III), and at peak IM response (IV) (left). Scale bar, 100 μm. The representative average of normalized Ca2+ imaging responses (Fnorm) after BMP4 treatment in the presence of the RGS4 inhibitor 11b (right). (C) The normalized peak fluorescence of carbachol-induced Ca2+ responses (Fnorm,max) was significantly decreased by BMP4 treatment only in the absence of the RGS4 inhibitor 11b. Data are means ± SD from seven independent experiments for each condition (**P < 0.01, one-way ANOVA with Bonferroni correction).

  • Fig. 7 Model for MuSK regulation of BMP4 signaling.

    MuSK binds to BMPs and the BMP receptors ALK3 and ALK6 and regulates the transcriptional output of BMP signaling in muscle cells. MuSK regulation of BMP signaling is cell type–specific because BMP induced the expression of different sets of genes in myoblasts and myotubes in a manner that depended on MuSK. The kinase activity of MuSK, which is required for proper NMJ formation, is dispensable for MuSK-mediated regulation of BMP signaling. JM, juxtamembrane; TK, tyrosine kinase; CRD/Fz, CRD/frizzled-like domain.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/444/ra87/DC1

    Fig. S1. SPR binding analysis of MuSK with BMP2 and BMP7.

    Fig. S2. Soluble MuSK ectodomain inhibits BMP4 activity.

    Fig. S3. Equivalent binding of biglycan to MuSK ectodomain either containing (FL) or lacking (ΔIg3) the third Ig domain.

    Fig. S4. BMP4 treatment stimulates Rgs4 expression in wild-type but not MuSK−/− myoblasts.

    Fig. S5. MuSK favors the transcription of signaling- and transcription-related genes.

    Fig. S6. Abundant phosphorylated SMAD1/5/8 in cytosolic granules in wild-type but not MuSK−/− myoblasts.

    Table S1. Association and dissociation rate constants and overall Kd values for the interaction of MuSK with BMP2, BMP4, and BMP7, as determined by SPR.

    Table S2. Transcripts induced by BMP4 only in wild-type myoblasts.

    Table S3. Transcripts induced by BMP4 in both wild-type and MuSK−/− myoblasts.

    Table S4. Transcripts induced by BMP4 only in MuSK−/− myoblasts.

    Table S5. Transcripts induced by BMP4 only in wild-type myotubes.

    Table S6. Transcripts induced by BMP4 in both wild-type and MuSK−/− myotubes.

    Table S7. Transcripts induced by BMP4 only in MuSK−/− myotubes.

    Table S8. Transcripts induced by BMP4 in a MuSK-dependent manner in both myoblasts and myotubes.

    Table S9. Transcripts induced by BMP4 in a MuSK-dependent manner only in myoblasts.

    Table S10. Transcripts induced by BMP4 in a MuSK-dependent manner only in myotubes.

  • Supplementary Materials for:

    MuSK is a BMP co-receptor that shapes BMP responses and calcium signaling in muscle cells

    Atilgan Yilmaz, Chandramohan Kattamuri, Rana N. Ozdeslik, Carolyn Schmiedel, Sarah Mentzer, Christoph Schorl, Elena Oancea, Thomas B. Thompson, Justin R. Fallon*

    *Corresponding author. Email: justin_fallon{at}brown.edu

    This PDF file includes:

    • Fig. S1. SPR binding analysis of MuSK with BMP2 and BMP7.
    • Fig. S2. Soluble MuSK ectodomain inhibits BMP4 activity.
    • Fig. S3. Equivalent binding of biglycan to MuSK ectodomain either containing (FL) or lacking (ΔIg3) the third Ig domain.
    • Fig. S4. BMP4 treatment stimulates Rgs4 expression in wild-type but not MuSK−/− myoblasts.
    • Fig. S5. MuSK favors the transcription of signaling- and transcription-related genes.
    • Fig. S6. Abundant phosphorylated SMAD1/5/8 in cytosolic granules in wild-type but not MuSK−/− myoblasts.
    • Table S1. Association and dissociation rate constants and overall Kd values for the interaction of MuSK with BMP2, BMP4, and BMP7, as determined by SPR.
    • Table S2. Transcripts induced by BMP4 only in wild-type myoblasts.
    • Table S3. Transcripts induced by BMP4 in both wild-type and MuSK−/− myoblasts.
    • Table S4. Transcripts induced by BMP4 only in MuSK−/− myoblasts.
    • Table S5. Transcripts induced by BMP4 only in wild-type myotubes.
    • Table S6. Transcripts induced by BMP4 in both wild-type and MuSK−/− myotubes.
    • Table S7. Transcripts induced by BMP4 only in MuSK−/− myotubes.
    • Table S8. Transcripts induced by BMP4 in a MuSK-dependent manner in both myoblasts and myotubes.
    • Table S9. Transcripts induced by BMP4 in a MuSK-dependent manner only in myoblasts.
    • Table S10. Transcripts induced by BMP4 in a MuSK-dependent manner only in myotubes.

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    Citation: A. Yilmaz, C. Kattamuri, R. N. Ozdeslik, C. Schmiedel, S. Mentzer, C. Schorl, E. Oancea, T. B. Thompson, J. R. Fallon, MuSK is a BMP co-receptor that shapes BMP responses and calcium signaling in muscle cells. Sci. Signal. 9, ra87 (2016).

    © 2016 American Association for the Advancement of Science

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