Novel gain-of-function mutation of TRPV4 associated with accelerated chondrogenic differentiation of dental pulp stem cells derived from a patient with metatropic dysplasia

研究成果: ジャーナルへの寄稿記事

抄録

Metatropic dysplasia is a congenital skeletal dysplasia characterized by severe platyspondyly, dumbbell-like deformity of long tubular bones, and progressive kyphoscoliosis with growth. It is caused by mutations in the gene TRPV4, encoding the transient receptor potential vanilloid 4, which acts as a calcium channel. Many heterozygous single base mutations of this gene have been associated with the disorder, showing autosomal dominant inheritance. Although abnormal endochondral ossification has been observed by histological examination of bone in a patient with lethal metatropic dysplasia, the etiology of the disorder remains largely unresolved. As dental pulp stem cells (DPSCs) are mesenchymal stem cells that differentiate into bone lineage cells, DPSCs derived from patients with congenital skeletal dysplasia might be useful as a disease-specific cellular model for etiological investigation. The purpose of this study was to clarify the pathological association between TRPV4 mutation and chondrocyte differentiation by analyzing DPSCs from a patient with non-lethal metatropic dysplasia. We identified a novel heterozygous single base mutation, c.1855C>T in TRPV4. This was predicted to be a missense mutation, p.L619F, in putative transmembrane segment 5. The mutation was repaired by CRISPR/Cas9 system to obtain isogenic control DPSCs for further analysis. The expression of stem cell markers and fibroblast-like morphology were comparable between patient-derived mutant and control DPSCs, although expression of TRPV4 was lower in mutant DPSCs than control DPSCs. Despite the lower TRPV4 expression in mutant DPSCs, the intracellular Ca2+ level was comparable at the basal level between mutant and control DPSCs, while its level was markedly higher following stimulation with 4α-phorbol 12,13-didecanoate (4αPDD), a specific agonist for TRPV4, in mutant DPSCs than in control DPSCs. In the presence of 4αPDD, we observed accelerated early chondrocyte differentiation and upregulated mRNA expression of SRY-box 9 (SOX9) in mutant DPSCs. Our findings suggested that the novel missense mutation c.1855C>T of TRPV4 was a gain-of-function mutation leading to enhanced intracellular Ca2+ level, which was associated with accelerated chondrocyte differentiation and SOX9 upregulation. Our results also suggest that patient-derived DPSCs can be a useful disease-specific cellular model for elucidating the pathological mechanism of metatropic dysplasia.

元の言語英語
記事番号100648
ジャーナルBiochemistry and Biophysics Reports
19
DOI
出版物ステータス出版済み - 9 1 2019

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Dental Pulp
Stem cells
Pulp
Stem Cells
Mutation
Chondrocytes
Bone
Missense Mutation
Metatropic dwarfism
Bone and Bones
Clustered Regularly Interspaced Short Palindromic Repeats
TRPV Cation Channels
Gene encoding
Calcium Channels
Mesenchymal Stromal Cells
Fibroblasts
Osteogenesis
Genes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

これを引用

@article{13f40c4a1f1440e4b4a14fd1bbf241bb,
title = "Novel gain-of-function mutation of TRPV4 associated with accelerated chondrogenic differentiation of dental pulp stem cells derived from a patient with metatropic dysplasia",
abstract = "Metatropic dysplasia is a congenital skeletal dysplasia characterized by severe platyspondyly, dumbbell-like deformity of long tubular bones, and progressive kyphoscoliosis with growth. It is caused by mutations in the gene TRPV4, encoding the transient receptor potential vanilloid 4, which acts as a calcium channel. Many heterozygous single base mutations of this gene have been associated with the disorder, showing autosomal dominant inheritance. Although abnormal endochondral ossification has been observed by histological examination of bone in a patient with lethal metatropic dysplasia, the etiology of the disorder remains largely unresolved. As dental pulp stem cells (DPSCs) are mesenchymal stem cells that differentiate into bone lineage cells, DPSCs derived from patients with congenital skeletal dysplasia might be useful as a disease-specific cellular model for etiological investigation. The purpose of this study was to clarify the pathological association between TRPV4 mutation and chondrocyte differentiation by analyzing DPSCs from a patient with non-lethal metatropic dysplasia. We identified a novel heterozygous single base mutation, c.1855C>T in TRPV4. This was predicted to be a missense mutation, p.L619F, in putative transmembrane segment 5. The mutation was repaired by CRISPR/Cas9 system to obtain isogenic control DPSCs for further analysis. The expression of stem cell markers and fibroblast-like morphology were comparable between patient-derived mutant and control DPSCs, although expression of TRPV4 was lower in mutant DPSCs than control DPSCs. Despite the lower TRPV4 expression in mutant DPSCs, the intracellular Ca2+ level was comparable at the basal level between mutant and control DPSCs, while its level was markedly higher following stimulation with 4α-phorbol 12,13-didecanoate (4αPDD), a specific agonist for TRPV4, in mutant DPSCs than in control DPSCs. In the presence of 4αPDD, we observed accelerated early chondrocyte differentiation and upregulated mRNA expression of SRY-box 9 (SOX9) in mutant DPSCs. Our findings suggested that the novel missense mutation c.1855C>T of TRPV4 was a gain-of-function mutation leading to enhanced intracellular Ca2+ level, which was associated with accelerated chondrocyte differentiation and SOX9 upregulation. Our results also suggest that patient-derived DPSCs can be a useful disease-specific cellular model for elucidating the pathological mechanism of metatropic dysplasia.",
author = "Kentaro Nonaka and Xu Han and Hiroki Kato and Hiroshi Sato and Haruyoshi Yamaza and Yuta Hirofuji and Keiji Masuda",
year = "2019",
month = "9",
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doi = "10.1016/j.bbrep.2019.100648",
language = "English",
volume = "19",
journal = "Biochemistry and Biophysics Reports",
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T1 - Novel gain-of-function mutation of TRPV4 associated with accelerated chondrogenic differentiation of dental pulp stem cells derived from a patient with metatropic dysplasia

AU - Nonaka, Kentaro

AU - Han, Xu

AU - Kato, Hiroki

AU - Sato, Hiroshi

AU - Yamaza, Haruyoshi

AU - Hirofuji, Yuta

AU - Masuda, Keiji

PY - 2019/9/1

Y1 - 2019/9/1

N2 - Metatropic dysplasia is a congenital skeletal dysplasia characterized by severe platyspondyly, dumbbell-like deformity of long tubular bones, and progressive kyphoscoliosis with growth. It is caused by mutations in the gene TRPV4, encoding the transient receptor potential vanilloid 4, which acts as a calcium channel. Many heterozygous single base mutations of this gene have been associated with the disorder, showing autosomal dominant inheritance. Although abnormal endochondral ossification has been observed by histological examination of bone in a patient with lethal metatropic dysplasia, the etiology of the disorder remains largely unresolved. As dental pulp stem cells (DPSCs) are mesenchymal stem cells that differentiate into bone lineage cells, DPSCs derived from patients with congenital skeletal dysplasia might be useful as a disease-specific cellular model for etiological investigation. The purpose of this study was to clarify the pathological association between TRPV4 mutation and chondrocyte differentiation by analyzing DPSCs from a patient with non-lethal metatropic dysplasia. We identified a novel heterozygous single base mutation, c.1855C>T in TRPV4. This was predicted to be a missense mutation, p.L619F, in putative transmembrane segment 5. The mutation was repaired by CRISPR/Cas9 system to obtain isogenic control DPSCs for further analysis. The expression of stem cell markers and fibroblast-like morphology were comparable between patient-derived mutant and control DPSCs, although expression of TRPV4 was lower in mutant DPSCs than control DPSCs. Despite the lower TRPV4 expression in mutant DPSCs, the intracellular Ca2+ level was comparable at the basal level between mutant and control DPSCs, while its level was markedly higher following stimulation with 4α-phorbol 12,13-didecanoate (4αPDD), a specific agonist for TRPV4, in mutant DPSCs than in control DPSCs. In the presence of 4αPDD, we observed accelerated early chondrocyte differentiation and upregulated mRNA expression of SRY-box 9 (SOX9) in mutant DPSCs. Our findings suggested that the novel missense mutation c.1855C>T of TRPV4 was a gain-of-function mutation leading to enhanced intracellular Ca2+ level, which was associated with accelerated chondrocyte differentiation and SOX9 upregulation. Our results also suggest that patient-derived DPSCs can be a useful disease-specific cellular model for elucidating the pathological mechanism of metatropic dysplasia.

AB - Metatropic dysplasia is a congenital skeletal dysplasia characterized by severe platyspondyly, dumbbell-like deformity of long tubular bones, and progressive kyphoscoliosis with growth. It is caused by mutations in the gene TRPV4, encoding the transient receptor potential vanilloid 4, which acts as a calcium channel. Many heterozygous single base mutations of this gene have been associated with the disorder, showing autosomal dominant inheritance. Although abnormal endochondral ossification has been observed by histological examination of bone in a patient with lethal metatropic dysplasia, the etiology of the disorder remains largely unresolved. As dental pulp stem cells (DPSCs) are mesenchymal stem cells that differentiate into bone lineage cells, DPSCs derived from patients with congenital skeletal dysplasia might be useful as a disease-specific cellular model for etiological investigation. The purpose of this study was to clarify the pathological association between TRPV4 mutation and chondrocyte differentiation by analyzing DPSCs from a patient with non-lethal metatropic dysplasia. We identified a novel heterozygous single base mutation, c.1855C>T in TRPV4. This was predicted to be a missense mutation, p.L619F, in putative transmembrane segment 5. The mutation was repaired by CRISPR/Cas9 system to obtain isogenic control DPSCs for further analysis. The expression of stem cell markers and fibroblast-like morphology were comparable between patient-derived mutant and control DPSCs, although expression of TRPV4 was lower in mutant DPSCs than control DPSCs. Despite the lower TRPV4 expression in mutant DPSCs, the intracellular Ca2+ level was comparable at the basal level between mutant and control DPSCs, while its level was markedly higher following stimulation with 4α-phorbol 12,13-didecanoate (4αPDD), a specific agonist for TRPV4, in mutant DPSCs than in control DPSCs. In the presence of 4αPDD, we observed accelerated early chondrocyte differentiation and upregulated mRNA expression of SRY-box 9 (SOX9) in mutant DPSCs. Our findings suggested that the novel missense mutation c.1855C>T of TRPV4 was a gain-of-function mutation leading to enhanced intracellular Ca2+ level, which was associated with accelerated chondrocyte differentiation and SOX9 upregulation. Our results also suggest that patient-derived DPSCs can be a useful disease-specific cellular model for elucidating the pathological mechanism of metatropic dysplasia.

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