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High-throughput screening for modulators of ACVR1 transcription: discovery of potential therapeutics for fibrodysplasia ossificans progressiva

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ABSTRACT

The ACVR1 gene encodes a type I receptor of bone morphogenetic proteins (BMPs). Activating mutations in ACVR1 are responsible for fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by congenital toe malformation and progressive heterotopic endochondral ossification leading to severe and cumulative disability. Until now, no therapy has been available to prevent soft-tissue swelling (flare-ups) that trigger the ossification process. With the aim of finding a new therapeutic strategy for FOP, we developed a high-throughput screening (HTS) assay to identify inhibitors of ACVR1 gene expression among drugs already approved for the therapy of other diseases. The screening, based on an ACVR1 promoter assay, was followed by an in vitro and in vivo test to validate and characterize candidate molecules. Among compounds that modulate the ACVR1 promoter activity, we selected the one showing the highest inhibitory effect, dipyridamole, a drug that is currently used as a platelet anti-aggregant. The inhibitory effect was detectable on ACVR1 gene expression, on the whole Smad-dependent BMP signaling pathway, and on chondrogenic and osteogenic differentiation processes by in vitro cellular assays. Moreover, dipyridamole reduced the process of heterotopic bone formation in vivo. Our drug repositioning strategy has led to the identification of dipyridamole as a possible therapeutic tool for the treatment of FOP. Furthermore, our study has also defined a pipeline of assays that will be useful for the evaluation of other pharmacological inhibitors of heterotopic ossification.

No MeSH data available.


Effect of Dipy on the BMP-mediated signaling pathway. (A) Luciferase activity measured in ATDC5 BRE-Luc cells treated with the indicated doses of Dipy and activated with [50 ng/ml] BMP2. The ratio of luciferase (Luc)/fluorescence (Fluo) was normalized to that obtained with DMSO (value 100). Bar graph represents the mean and s.d. of three independent experiments. §P<0.001. (B) Effect of Dipy on the expression level of Id1, Id2 and Id3 BMP target genes in native ATDC5 cells. Values were normalized on the β2M reference gene (relative quantification by the ΔCt method: ratio reference/target=2ΔCt). Bars represent the mean and s.d. of three independent experiments. ns, non-significant; *P<0.05, #P<0.01, §P<0.001. (C,D) Effect of Dipy on the activation of the Smad-dependent pathway. ATDC5 (C) and C2C12 (D) cells were treated with Dipy and activated with [200 ng/ml] BMP2 for 1 h.
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DMM023929F2: Effect of Dipy on the BMP-mediated signaling pathway. (A) Luciferase activity measured in ATDC5 BRE-Luc cells treated with the indicated doses of Dipy and activated with [50 ng/ml] BMP2. The ratio of luciferase (Luc)/fluorescence (Fluo) was normalized to that obtained with DMSO (value 100). Bar graph represents the mean and s.d. of three independent experiments. §P<0.001. (B) Effect of Dipy on the expression level of Id1, Id2 and Id3 BMP target genes in native ATDC5 cells. Values were normalized on the β2M reference gene (relative quantification by the ΔCt method: ratio reference/target=2ΔCt). Bars represent the mean and s.d. of three independent experiments. ns, non-significant; *P<0.05, #P<0.01, §P<0.001. (C,D) Effect of Dipy on the activation of the Smad-dependent pathway. ATDC5 (C) and C2C12 (D) cells were treated with Dipy and activated with [200 ng/ml] BMP2 for 1 h.

Mentions: In order to test the effect of Dipy on the activation state of the Smad-dependent BMP signaling pathway, we generated ATDC5 clones stably expressing the luciferase reporter gene under the control of a minimal promoter carrying a BMP-responsive element (BRE-Luc) isolated from Id1, a well-known BMP target gene (Monteiro et al., 2008). Cells were treated with Dipy in the presence of BMP2 for 6 h. As reported in Fig. 2A, Dipy weakened the amplitude of the activation induced by BMP2 in a dose-dependent manner. Consistently, we found a downregulation in the mRNA expression of native Id1, Id2 and Id3 target genes, as assessed by RT-qPCR in ATDC5 cells (Fig. 2B), and a significant reduction in the phosphorylation state of the Smad1/5 proteins both in ATDC5 and C2C12 cells (Fig. 2C,D and Table S2 for immunoblot densitometric analysis).Fig. 2.


High-throughput screening for modulators of ACVR1 transcription: discovery of potential therapeutics for fibrodysplasia ossificans progressiva
Effect of Dipy on the BMP-mediated signaling pathway. (A) Luciferase activity measured in ATDC5 BRE-Luc cells treated with the indicated doses of Dipy and activated with [50 ng/ml] BMP2. The ratio of luciferase (Luc)/fluorescence (Fluo) was normalized to that obtained with DMSO (value 100). Bar graph represents the mean and s.d. of three independent experiments. §P<0.001. (B) Effect of Dipy on the expression level of Id1, Id2 and Id3 BMP target genes in native ATDC5 cells. Values were normalized on the β2M reference gene (relative quantification by the ΔCt method: ratio reference/target=2ΔCt). Bars represent the mean and s.d. of three independent experiments. ns, non-significant; *P<0.05, #P<0.01, §P<0.001. (C,D) Effect of Dipy on the activation of the Smad-dependent pathway. ATDC5 (C) and C2C12 (D) cells were treated with Dipy and activated with [200 ng/ml] BMP2 for 1 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4920148&req=5

DMM023929F2: Effect of Dipy on the BMP-mediated signaling pathway. (A) Luciferase activity measured in ATDC5 BRE-Luc cells treated with the indicated doses of Dipy and activated with [50 ng/ml] BMP2. The ratio of luciferase (Luc)/fluorescence (Fluo) was normalized to that obtained with DMSO (value 100). Bar graph represents the mean and s.d. of three independent experiments. §P<0.001. (B) Effect of Dipy on the expression level of Id1, Id2 and Id3 BMP target genes in native ATDC5 cells. Values were normalized on the β2M reference gene (relative quantification by the ΔCt method: ratio reference/target=2ΔCt). Bars represent the mean and s.d. of three independent experiments. ns, non-significant; *P<0.05, #P<0.01, §P<0.001. (C,D) Effect of Dipy on the activation of the Smad-dependent pathway. ATDC5 (C) and C2C12 (D) cells were treated with Dipy and activated with [200 ng/ml] BMP2 for 1 h.
Mentions: In order to test the effect of Dipy on the activation state of the Smad-dependent BMP signaling pathway, we generated ATDC5 clones stably expressing the luciferase reporter gene under the control of a minimal promoter carrying a BMP-responsive element (BRE-Luc) isolated from Id1, a well-known BMP target gene (Monteiro et al., 2008). Cells were treated with Dipy in the presence of BMP2 for 6 h. As reported in Fig. 2A, Dipy weakened the amplitude of the activation induced by BMP2 in a dose-dependent manner. Consistently, we found a downregulation in the mRNA expression of native Id1, Id2 and Id3 target genes, as assessed by RT-qPCR in ATDC5 cells (Fig. 2B), and a significant reduction in the phosphorylation state of the Smad1/5 proteins both in ATDC5 and C2C12 cells (Fig. 2C,D and Table S2 for immunoblot densitometric analysis).Fig. 2.

View Article: PubMed Central - PubMed

ABSTRACT

The ACVR1 gene encodes a type I receptor of bone morphogenetic proteins (BMPs). Activating mutations in ACVR1 are responsible for fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by congenital toe malformation and progressive heterotopic endochondral ossification leading to severe and cumulative disability. Until now, no therapy has been available to prevent soft-tissue swelling (flare-ups) that trigger the ossification process. With the aim of finding a new therapeutic strategy for FOP, we developed a high-throughput screening (HTS) assay to identify inhibitors of ACVR1 gene expression among drugs already approved for the therapy of other diseases. The screening, based on an ACVR1 promoter assay, was followed by an in vitro and in vivo test to validate and characterize candidate molecules. Among compounds that modulate the ACVR1 promoter activity, we selected the one showing the highest inhibitory effect, dipyridamole, a drug that is currently used as a platelet anti-aggregant. The inhibitory effect was detectable on ACVR1 gene expression, on the whole Smad-dependent BMP signaling pathway, and on chondrogenic and osteogenic differentiation processes by in vitro cellular assays. Moreover, dipyridamole reduced the process of heterotopic bone formation in vivo. Our drug repositioning strategy has led to the identification of dipyridamole as a possible therapeutic tool for the treatment of FOP. Furthermore, our study has also defined a pipeline of assays that will be useful for the evaluation of other pharmacological inhibitors of heterotopic ossification.

No MeSH data available.