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Published on: December 31, 2013
Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived
Amanda R Dicks1,2,3, Grigory I Maksaev4, Zainab Harissa1,2,3
1Department of Biomedical Engineering, Washington University in St. Louis, St Louis, United States.
Abstract:
Mutations in the TRPV4 ion channel can lead to a range of skeletal dysplasias. However, the mechanisms by which TRPV4 mutations lead to distinct disease severity remain unknown. Here, we use CRISPR-Cas9-edited human-induced pluripotent stem cells (hiPSCs) harboring either the mild V620I or lethal T89I mutations to elucidate the differential effects on channel function and chondrogenic differentiation. We found that hiPSC-derived chondrocytes with the V620I mutation exhibited increased basal currents through TRPV4. However, both mutations showed more rapid calcium signaling with a reduced overall magnitude in response to TRPV4 agonist GSK1016790A compared to wildtype (WT). There were no differences in overall cartilaginous matrix production, but the V620I mutation resulted in reduced mechanical properties of cartilage matrix later in chondrogenesis. mRNA sequencing revealed that both mutations up-regulated several anterior HOX genes and down-regulated antioxidant genes CAT and GSTA1 throughout chondrogenesis. BMP4 treatment up-regulated several essential hypertrophic genes in WT chondrocytes; however, this hypertrophic maturation response was inhibited in mutant chondrocytes. These results indicate that the TRPV4 mutations alter BMP signaling in chondrocytes and prevent proper chondrocyte hypertrophy, as a potential mechanism for dysfunctional skeletal development. Our findings provide potential therapeutic targets for developing treatments for TRPV4-mediated skeletal dysplasias.
Insights
Mutations in the TRPV4 ion channel cause skeletal dysplasias. This study reveals how TRPV4 mutations disrupt chondrocyte function and bone development, offering potential therapeutic targets for these conditions.
Area of Science:
- Genetics
- Cell Biology
- Developmental Biology
Background:
- Mutations in the TRPV4 ion channel are linked to various skeletal dysplasias.
- The precise mechanisms underlying differential disease severity in TRPV4-related skeletal disorders are not fully understood.
Purpose of the Study:
- To investigate the functional consequences of specific TRPV4 mutations (V620I and T89I) on chondrogenic differentiation and skeletal development.
- To elucidate the molecular mechanisms by which these mutations impact chondrocyte function and signaling pathways.
Main Methods:
- CRISPR-Cas9 gene editing was used to create human-induced pluripotent stem cells (hiPSCs) with V620I or T89I TRPV4 mutations.
- Electrophysiology, calcium imaging, mRNA sequencing, and biochemical assays were employed to analyze channel function and cellular responses.
- Chondrogenic differentiation and BMP4-induced hypertrophic maturation were assessed in wildtype and mutant hiPSC-derived chondrocytes.
Main Results:
- Both V620I and T89I TRPV4 mutations altered calcium signaling dynamics in response to agonists.
- The V620I mutation impaired the mechanical properties of cartilage matrix later in chondrogenesis.
- Mutations led to altered expression of HOX genes and antioxidant genes, and importantly, inhibited BMP4-induced chondrocyte hypertrophy.
Conclusions:
- TRPV4 mutations disrupt chondrocyte differentiation and hypertrophy, potentially through altered BMP signaling pathways.
- These findings highlight a mechanism for dysfunctional skeletal development in TRPV4-mediated skeletal dysplasias.
- The study identifies potential therapeutic targets for treating these genetic disorders.
