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BMPER Promotes Epithelial-Mesenchymal Transition in the Developing Cardiac Cushions.

Dyer L, Lockyer P, Wu Y, Saha A, Cyr C, Moser M, Pi X, Patterson C - PLoS ONE (2015)

Bottom Line: In the BMPER-/- embryo, EMT is dysregulated in the atrioventricular and outflow tract cushions compared with their wild-type counterparts, as indicated by a significant increase of Sox9-positive cells during cushion formation.These results indicate that BMPER negatively regulates BMP-induced Smad and Sox9 activity during valve development.Together, these results identify BMPER as a regulator of BMP2-induced cardiac valve development and will contribute to our understanding of valvular defects.

View Article: PubMed Central - PubMed

Affiliation: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, United States of America.

ABSTRACT
Formation of the cardiac valves is an essential component of cardiovascular development. Consistent with the role of the bone morphogenetic protein (BMP) signaling pathway in cardiac valve formation, embryos that are deficient for the BMP regulator BMPER (BMP-binding endothelial regulator) display the cardiac valve anomaly mitral valve prolapse. However, how BMPER deficiency leads to this defect is unknown. Based on its expression pattern in the developing cardiac cushions, we hypothesized that BMPER regulates BMP2-mediated signaling, leading to fine-tuned epithelial-mesenchymal transition (EMT) and extracellular matrix deposition. In the BMPER-/- embryo, EMT is dysregulated in the atrioventricular and outflow tract cushions compared with their wild-type counterparts, as indicated by a significant increase of Sox9-positive cells during cushion formation. However, proliferation is not impaired in the developing BMPER-/- valves. In vitro data show that BMPER directly binds BMP2. In cultured endothelial cells, BMPER blocks BMP2-induced Smad activation in a dose-dependent manner. In addition, BMP2 increases the Sox9 protein level, and this increase is inhibited by co-treatment with BMPER. Consistently, in the BMPER-/- embryos, semi-quantitative analysis of Smad activation shows that the canonical BMP pathway is significantly more active in the atrioventricular cushions during EMT. These results indicate that BMPER negatively regulates BMP-induced Smad and Sox9 activity during valve development. Together, these results identify BMPER as a regulator of BMP2-induced cardiac valve development and will contribute to our understanding of valvular defects.

No MeSH data available.


Related in: MedlinePlus

BMPER inhibits BMP2-induced signaling in the developing cushions.(A) Recombinant BMPER and BMP2 proteins were combined as indicated and immunoprecipitated using an anti-BMPER antibody, an anti-BMP2 antibody, or the appropriate species-specific IgG antibody controls. As a loading control, all unbound proteins in the supernatant were run in the Input lanes (right lanes). The anti-BMP2 antibody recognized recombinant BMP2 and co-immunoprecipitated BMPER when both proteins were present. Similarly, the anti-BMPER antibody recognized recombinant BMPER and co-immunoprecipitated BMP2 when both proteins were present. (B) BMPER inhibits BMP2-induced Smad1,5,8 phosphorylation (pSmad) in cultured endothelial cells. MECs were treated with BMP2 and increasing doses of BMPER for 45 minutes. As expected, BMP2 treatment induced Smad phosphorylation (second lane). With increasing concentrations of BMPER, the pSmad levels were exponentially reduced (R2 = 0.98). Data are presented as the fold change compared with BMP2 treatment in the absence of BMPER treatment. Due to reduced signaling intensity when stripping and reprobing blots, β-actin was used as a loading control instead of total Smad. (C) BMP2 increases the Sox9 protein level in cultured endothelial cells. MECs were treated with 0.6 nM BMP2 for the indicated time periods. As expected, BMP2 treatment increased Sox9 protein levels. The arrow indicates the Sox9 protein band. N.S., not significant. *p<0.05, compared with cells without treatment. n = 3. (D) BMPER inhibits BMP2-induced Sox9 protein expression in cultured endothelial cells. MECs were treated with 0.6 nM BMP2 and 5 nM BMPER for 4 hours. BMPER co-treatment blocks BMP2-induced Sox9 protein expression. The arrow indicates the Sox9 protein band. N.S., not significant. *p<0.05, compared with control cells without treatment; #p<0.05, compared with cells with BMP2 treatment only. n = 3. (E) BMPER affects downstream Smad1/5/8 activity in the developing atrioventricular cushions. At E9.5, BMPER-/- atrioventricular cushions display reduced pSmad signals compared with their wild-type counterparts. However, by E10.5, the pSmad intensity increases in the BMPER-/- atrioventricular cushions compared with the wild-type counterparts. This increase is not maintained, with reduced pSmad intensity in the BMPER-/- cushions by E11.5. Fluorescence intensity is quantified on the right. (F) At E9.5, BMPER-/- outflow tract cushions display reduced pSmad compared with their wild-type counterparts. However, by E10.5, the pSmad intensity increases in the BMPER-/- outflow tract cushions compared with the wild-type counterparts. This increase is not maintained, with reduced pSmad intensity in the BMPER-/- cushions by E11.5. *p<0.05. Scale bar = 120 μm.
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pone.0139209.g004: BMPER inhibits BMP2-induced signaling in the developing cushions.(A) Recombinant BMPER and BMP2 proteins were combined as indicated and immunoprecipitated using an anti-BMPER antibody, an anti-BMP2 antibody, or the appropriate species-specific IgG antibody controls. As a loading control, all unbound proteins in the supernatant were run in the Input lanes (right lanes). The anti-BMP2 antibody recognized recombinant BMP2 and co-immunoprecipitated BMPER when both proteins were present. Similarly, the anti-BMPER antibody recognized recombinant BMPER and co-immunoprecipitated BMP2 when both proteins were present. (B) BMPER inhibits BMP2-induced Smad1,5,8 phosphorylation (pSmad) in cultured endothelial cells. MECs were treated with BMP2 and increasing doses of BMPER for 45 minutes. As expected, BMP2 treatment induced Smad phosphorylation (second lane). With increasing concentrations of BMPER, the pSmad levels were exponentially reduced (R2 = 0.98). Data are presented as the fold change compared with BMP2 treatment in the absence of BMPER treatment. Due to reduced signaling intensity when stripping and reprobing blots, β-actin was used as a loading control instead of total Smad. (C) BMP2 increases the Sox9 protein level in cultured endothelial cells. MECs were treated with 0.6 nM BMP2 for the indicated time periods. As expected, BMP2 treatment increased Sox9 protein levels. The arrow indicates the Sox9 protein band. N.S., not significant. *p<0.05, compared with cells without treatment. n = 3. (D) BMPER inhibits BMP2-induced Sox9 protein expression in cultured endothelial cells. MECs were treated with 0.6 nM BMP2 and 5 nM BMPER for 4 hours. BMPER co-treatment blocks BMP2-induced Sox9 protein expression. The arrow indicates the Sox9 protein band. N.S., not significant. *p<0.05, compared with control cells without treatment; #p<0.05, compared with cells with BMP2 treatment only. n = 3. (E) BMPER affects downstream Smad1/5/8 activity in the developing atrioventricular cushions. At E9.5, BMPER-/- atrioventricular cushions display reduced pSmad signals compared with their wild-type counterparts. However, by E10.5, the pSmad intensity increases in the BMPER-/- atrioventricular cushions compared with the wild-type counterparts. This increase is not maintained, with reduced pSmad intensity in the BMPER-/- cushions by E11.5. Fluorescence intensity is quantified on the right. (F) At E9.5, BMPER-/- outflow tract cushions display reduced pSmad compared with their wild-type counterparts. However, by E10.5, the pSmad intensity increases in the BMPER-/- outflow tract cushions compared with the wild-type counterparts. This increase is not maintained, with reduced pSmad intensity in the BMPER-/- cushions by E11.5. *p<0.05. Scale bar = 120 μm.

Mentions: Previous work has directly shown that BMPER interacts with BMP4 and indirectly shown that BMPER also interacts with BMP2 [14]. Because BMP2 is the major BMP involved in cardiac cushion development by inducing Sox9 [6], we tested the direct interaction of BMPER-BMP2 using co-immunoprecipitation with recombinant proteins BMPER and BMP2. As a positive control, the BMP2 antibody and the BMPER antibody both successfully immunoprecipitated BMP2 and BMPER, respectively (Fig 4A). In the presence of both proteins, both antibodies could immunoprecipitate both proteins (Fig 4A), thus indicating that BMPER and BMP2 directly interact in vitro.


BMPER Promotes Epithelial-Mesenchymal Transition in the Developing Cardiac Cushions.

Dyer L, Lockyer P, Wu Y, Saha A, Cyr C, Moser M, Pi X, Patterson C - PLoS ONE (2015)

BMPER inhibits BMP2-induced signaling in the developing cushions.(A) Recombinant BMPER and BMP2 proteins were combined as indicated and immunoprecipitated using an anti-BMPER antibody, an anti-BMP2 antibody, or the appropriate species-specific IgG antibody controls. As a loading control, all unbound proteins in the supernatant were run in the Input lanes (right lanes). The anti-BMP2 antibody recognized recombinant BMP2 and co-immunoprecipitated BMPER when both proteins were present. Similarly, the anti-BMPER antibody recognized recombinant BMPER and co-immunoprecipitated BMP2 when both proteins were present. (B) BMPER inhibits BMP2-induced Smad1,5,8 phosphorylation (pSmad) in cultured endothelial cells. MECs were treated with BMP2 and increasing doses of BMPER for 45 minutes. As expected, BMP2 treatment induced Smad phosphorylation (second lane). With increasing concentrations of BMPER, the pSmad levels were exponentially reduced (R2 = 0.98). Data are presented as the fold change compared with BMP2 treatment in the absence of BMPER treatment. Due to reduced signaling intensity when stripping and reprobing blots, β-actin was used as a loading control instead of total Smad. (C) BMP2 increases the Sox9 protein level in cultured endothelial cells. MECs were treated with 0.6 nM BMP2 for the indicated time periods. As expected, BMP2 treatment increased Sox9 protein levels. The arrow indicates the Sox9 protein band. N.S., not significant. *p<0.05, compared with cells without treatment. n = 3. (D) BMPER inhibits BMP2-induced Sox9 protein expression in cultured endothelial cells. MECs were treated with 0.6 nM BMP2 and 5 nM BMPER for 4 hours. BMPER co-treatment blocks BMP2-induced Sox9 protein expression. The arrow indicates the Sox9 protein band. N.S., not significant. *p<0.05, compared with control cells without treatment; #p<0.05, compared with cells with BMP2 treatment only. n = 3. (E) BMPER affects downstream Smad1/5/8 activity in the developing atrioventricular cushions. At E9.5, BMPER-/- atrioventricular cushions display reduced pSmad signals compared with their wild-type counterparts. However, by E10.5, the pSmad intensity increases in the BMPER-/- atrioventricular cushions compared with the wild-type counterparts. This increase is not maintained, with reduced pSmad intensity in the BMPER-/- cushions by E11.5. Fluorescence intensity is quantified on the right. (F) At E9.5, BMPER-/- outflow tract cushions display reduced pSmad compared with their wild-type counterparts. However, by E10.5, the pSmad intensity increases in the BMPER-/- outflow tract cushions compared with the wild-type counterparts. This increase is not maintained, with reduced pSmad intensity in the BMPER-/- cushions by E11.5. *p<0.05. Scale bar = 120 μm.
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pone.0139209.g004: BMPER inhibits BMP2-induced signaling in the developing cushions.(A) Recombinant BMPER and BMP2 proteins were combined as indicated and immunoprecipitated using an anti-BMPER antibody, an anti-BMP2 antibody, or the appropriate species-specific IgG antibody controls. As a loading control, all unbound proteins in the supernatant were run in the Input lanes (right lanes). The anti-BMP2 antibody recognized recombinant BMP2 and co-immunoprecipitated BMPER when both proteins were present. Similarly, the anti-BMPER antibody recognized recombinant BMPER and co-immunoprecipitated BMP2 when both proteins were present. (B) BMPER inhibits BMP2-induced Smad1,5,8 phosphorylation (pSmad) in cultured endothelial cells. MECs were treated with BMP2 and increasing doses of BMPER for 45 minutes. As expected, BMP2 treatment induced Smad phosphorylation (second lane). With increasing concentrations of BMPER, the pSmad levels were exponentially reduced (R2 = 0.98). Data are presented as the fold change compared with BMP2 treatment in the absence of BMPER treatment. Due to reduced signaling intensity when stripping and reprobing blots, β-actin was used as a loading control instead of total Smad. (C) BMP2 increases the Sox9 protein level in cultured endothelial cells. MECs were treated with 0.6 nM BMP2 for the indicated time periods. As expected, BMP2 treatment increased Sox9 protein levels. The arrow indicates the Sox9 protein band. N.S., not significant. *p<0.05, compared with cells without treatment. n = 3. (D) BMPER inhibits BMP2-induced Sox9 protein expression in cultured endothelial cells. MECs were treated with 0.6 nM BMP2 and 5 nM BMPER for 4 hours. BMPER co-treatment blocks BMP2-induced Sox9 protein expression. The arrow indicates the Sox9 protein band. N.S., not significant. *p<0.05, compared with control cells without treatment; #p<0.05, compared with cells with BMP2 treatment only. n = 3. (E) BMPER affects downstream Smad1/5/8 activity in the developing atrioventricular cushions. At E9.5, BMPER-/- atrioventricular cushions display reduced pSmad signals compared with their wild-type counterparts. However, by E10.5, the pSmad intensity increases in the BMPER-/- atrioventricular cushions compared with the wild-type counterparts. This increase is not maintained, with reduced pSmad intensity in the BMPER-/- cushions by E11.5. Fluorescence intensity is quantified on the right. (F) At E9.5, BMPER-/- outflow tract cushions display reduced pSmad compared with their wild-type counterparts. However, by E10.5, the pSmad intensity increases in the BMPER-/- outflow tract cushions compared with the wild-type counterparts. This increase is not maintained, with reduced pSmad intensity in the BMPER-/- cushions by E11.5. *p<0.05. Scale bar = 120 μm.
Mentions: Previous work has directly shown that BMPER interacts with BMP4 and indirectly shown that BMPER also interacts with BMP2 [14]. Because BMP2 is the major BMP involved in cardiac cushion development by inducing Sox9 [6], we tested the direct interaction of BMPER-BMP2 using co-immunoprecipitation with recombinant proteins BMPER and BMP2. As a positive control, the BMP2 antibody and the BMPER antibody both successfully immunoprecipitated BMP2 and BMPER, respectively (Fig 4A). In the presence of both proteins, both antibodies could immunoprecipitate both proteins (Fig 4A), thus indicating that BMPER and BMP2 directly interact in vitro.

Bottom Line: In the BMPER-/- embryo, EMT is dysregulated in the atrioventricular and outflow tract cushions compared with their wild-type counterparts, as indicated by a significant increase of Sox9-positive cells during cushion formation.These results indicate that BMPER negatively regulates BMP-induced Smad and Sox9 activity during valve development.Together, these results identify BMPER as a regulator of BMP2-induced cardiac valve development and will contribute to our understanding of valvular defects.

View Article: PubMed Central - PubMed

Affiliation: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, United States of America.

ABSTRACT
Formation of the cardiac valves is an essential component of cardiovascular development. Consistent with the role of the bone morphogenetic protein (BMP) signaling pathway in cardiac valve formation, embryos that are deficient for the BMP regulator BMPER (BMP-binding endothelial regulator) display the cardiac valve anomaly mitral valve prolapse. However, how BMPER deficiency leads to this defect is unknown. Based on its expression pattern in the developing cardiac cushions, we hypothesized that BMPER regulates BMP2-mediated signaling, leading to fine-tuned epithelial-mesenchymal transition (EMT) and extracellular matrix deposition. In the BMPER-/- embryo, EMT is dysregulated in the atrioventricular and outflow tract cushions compared with their wild-type counterparts, as indicated by a significant increase of Sox9-positive cells during cushion formation. However, proliferation is not impaired in the developing BMPER-/- valves. In vitro data show that BMPER directly binds BMP2. In cultured endothelial cells, BMPER blocks BMP2-induced Smad activation in a dose-dependent manner. In addition, BMP2 increases the Sox9 protein level, and this increase is inhibited by co-treatment with BMPER. Consistently, in the BMPER-/- embryos, semi-quantitative analysis of Smad activation shows that the canonical BMP pathway is significantly more active in the atrioventricular cushions during EMT. These results indicate that BMPER negatively regulates BMP-induced Smad and Sox9 activity during valve development. Together, these results identify BMPER as a regulator of BMP2-induced cardiac valve development and will contribute to our understanding of valvular defects.

No MeSH data available.


Related in: MedlinePlus