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Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice.

Sengle G, Carlberg V, Tufa SF, Charbonneau NL, Smaldone S, Carlson EJ, Ramirez F, Keene DR, Sakai LY - PLoS Genet. (2015)

Bottom Line: These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy.In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development.New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, Oregon Health & Science University, Portland, Oregon, United States of America; Shriners Hospital for Children, Portland, Oregon, United States of America.

ABSTRACT
Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.

No MeSH data available.


Related in: MedlinePlus

In vitro BMP bioactivity assay using C2C12 cells.(A) Comparison of Fbn1 and Fbn2 mRNA levels during myoblast to myotube differentiation. (B) BMP activity measured with and without RNAi of Fbn2 during myoblast to myotube differentiation. (C) Domain structure of fibrillin-2 and recombinant polypeptide rF86. (D) Interaction of fibrillin-2 with the prodomain of BMP-7 complex confers latency to the growth factor. Top left: cartoon depicting BMP-7 complex, a growth factor dimer non-covalently associated with two processed prodomains. Top right: cartoon of the assay: BMP-7 complex was coupled via prodomain interactions either through a mAb directed against the N-terminal His6-tag of the BMP-7 prodomain or an N-terminal fibrillin-2 peptide (rF86) which was captured by mAb689. C2C12 cells were then seeded. Below: quantitation of coupled amounts of immobilized BMP-7 complex determined by comparison with standards of known concentration. (E) qPCR expression levels of Id3 from treated and untreated wells. Error bars indicate mean ± SD. Each experiment was performed in triplicates.
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pgen.1005340.g008: In vitro BMP bioactivity assay using C2C12 cells.(A) Comparison of Fbn1 and Fbn2 mRNA levels during myoblast to myotube differentiation. (B) BMP activity measured with and without RNAi of Fbn2 during myoblast to myotube differentiation. (C) Domain structure of fibrillin-2 and recombinant polypeptide rF86. (D) Interaction of fibrillin-2 with the prodomain of BMP-7 complex confers latency to the growth factor. Top left: cartoon depicting BMP-7 complex, a growth factor dimer non-covalently associated with two processed prodomains. Top right: cartoon of the assay: BMP-7 complex was coupled via prodomain interactions either through a mAb directed against the N-terminal His6-tag of the BMP-7 prodomain or an N-terminal fibrillin-2 peptide (rF86) which was captured by mAb689. C2C12 cells were then seeded. Below: quantitation of coupled amounts of immobilized BMP-7 complex determined by comparison with standards of known concentration. (E) qPCR expression levels of Id3 from treated and untreated wells. Error bars indicate mean ± SD. Each experiment was performed in triplicates.

Mentions: To gain further insight into the molecular mechanisms by which fibrillin-2 might control BMP activity, we performed in vitro experiments. First, we measured the mRNA expression levels of Fbn1 and Fbn2 during mouse C2C12 myoblast to myotube differentiation. Fbn2 mRNA was 15-fold increased 2 days after initiation of differentiation when compared to levels at the initiation start, while Fbn1 mRNA expression was increased only 2.5-fold (Fig 8A). In a second experiment, we performed the C2C12 myoblast to myotube differentiation assay while Fbn2 expression was knocked down by RNAi. Two days after initiation of differentiation the cell culture supernatant was removed and added to C2C12BRA cells, which harbor the BMP-inducible plasmid BRE-luc and serve as reporter cells for BMP bioactivity [28]. Supernatant from cells subjected to Fbn2 RNAi contained 4 times more BMP bioactivity than supernatant from control cells (Fig 8B). These results were consistent with in vivo observations of increased BMP activity in Fbn2 forelimb muscle. In addition, the in vitro results indicate that, in the process of muscle maturation, fibrillin-2 performs a much more predominant role than fibrillin-1.


Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice.

Sengle G, Carlberg V, Tufa SF, Charbonneau NL, Smaldone S, Carlson EJ, Ramirez F, Keene DR, Sakai LY - PLoS Genet. (2015)

In vitro BMP bioactivity assay using C2C12 cells.(A) Comparison of Fbn1 and Fbn2 mRNA levels during myoblast to myotube differentiation. (B) BMP activity measured with and without RNAi of Fbn2 during myoblast to myotube differentiation. (C) Domain structure of fibrillin-2 and recombinant polypeptide rF86. (D) Interaction of fibrillin-2 with the prodomain of BMP-7 complex confers latency to the growth factor. Top left: cartoon depicting BMP-7 complex, a growth factor dimer non-covalently associated with two processed prodomains. Top right: cartoon of the assay: BMP-7 complex was coupled via prodomain interactions either through a mAb directed against the N-terminal His6-tag of the BMP-7 prodomain or an N-terminal fibrillin-2 peptide (rF86) which was captured by mAb689. C2C12 cells were then seeded. Below: quantitation of coupled amounts of immobilized BMP-7 complex determined by comparison with standards of known concentration. (E) qPCR expression levels of Id3 from treated and untreated wells. Error bars indicate mean ± SD. Each experiment was performed in triplicates.
© Copyright Policy
Related In: Results  -  Collection

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pgen.1005340.g008: In vitro BMP bioactivity assay using C2C12 cells.(A) Comparison of Fbn1 and Fbn2 mRNA levels during myoblast to myotube differentiation. (B) BMP activity measured with and without RNAi of Fbn2 during myoblast to myotube differentiation. (C) Domain structure of fibrillin-2 and recombinant polypeptide rF86. (D) Interaction of fibrillin-2 with the prodomain of BMP-7 complex confers latency to the growth factor. Top left: cartoon depicting BMP-7 complex, a growth factor dimer non-covalently associated with two processed prodomains. Top right: cartoon of the assay: BMP-7 complex was coupled via prodomain interactions either through a mAb directed against the N-terminal His6-tag of the BMP-7 prodomain or an N-terminal fibrillin-2 peptide (rF86) which was captured by mAb689. C2C12 cells were then seeded. Below: quantitation of coupled amounts of immobilized BMP-7 complex determined by comparison with standards of known concentration. (E) qPCR expression levels of Id3 from treated and untreated wells. Error bars indicate mean ± SD. Each experiment was performed in triplicates.
Mentions: To gain further insight into the molecular mechanisms by which fibrillin-2 might control BMP activity, we performed in vitro experiments. First, we measured the mRNA expression levels of Fbn1 and Fbn2 during mouse C2C12 myoblast to myotube differentiation. Fbn2 mRNA was 15-fold increased 2 days after initiation of differentiation when compared to levels at the initiation start, while Fbn1 mRNA expression was increased only 2.5-fold (Fig 8A). In a second experiment, we performed the C2C12 myoblast to myotube differentiation assay while Fbn2 expression was knocked down by RNAi. Two days after initiation of differentiation the cell culture supernatant was removed and added to C2C12BRA cells, which harbor the BMP-inducible plasmid BRE-luc and serve as reporter cells for BMP bioactivity [28]. Supernatant from cells subjected to Fbn2 RNAi contained 4 times more BMP bioactivity than supernatant from control cells (Fig 8B). These results were consistent with in vivo observations of increased BMP activity in Fbn2 forelimb muscle. In addition, the in vitro results indicate that, in the process of muscle maturation, fibrillin-2 performs a much more predominant role than fibrillin-1.

Bottom Line: These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy.In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development.New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, Oregon Health & Science University, Portland, Oregon, United States of America; Shriners Hospital for Children, Portland, Oregon, United States of America.

ABSTRACT
Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.

No MeSH data available.


Related in: MedlinePlus