Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived chondrocytes
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Evaluation Summary:
TRPV4 is an ion channel protein and mutations in TRPV4 gene resulted in different types of skeletal defects. In this study, the authors created two types of TRPV4 mutations (mild V620I and lethal T89I mutations) in human iPS cells through CRISPR-Cas9 gene editing. They identified key molecules potentially involved in TRPV4 mutation-induced changes in chondrocyte activities and concluded that the inhibition of chondrocyte hypertrophy induced by the mutations may cause the bone diseases.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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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.
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Author Response:
Reviewer #1 (Public Review):
Dicks et al. in this study characterized electrophysiological properties of mutant and wild-type hiPSC-chondrocytes and the expression of chondrocyte-associated markers during chondrogenic differentiation of the cells, and analyzed the differential expression of global transcriptome between the different chondrocyte groups. They demonstrated TRPV4 mutation-induced changes in calcium signaling, mechanical property of matrix, and transcriptome of hiPSC-chondrocytes and concluded that the V620I and T89I mutations of TRPV4 in chondrocytes delay or inhibit hypertrophy, which may be a potential cause of skeletal dysplasias.
This study applied a gene-editing tool to creating mutant hiPSCs as a human cell model of the disease in culture to study TRPV4 mutation-induced alteration in cellular …
Author Response:
Reviewer #1 (Public Review):
Dicks et al. in this study characterized electrophysiological properties of mutant and wild-type hiPSC-chondrocytes and the expression of chondrocyte-associated markers during chondrogenic differentiation of the cells, and analyzed the differential expression of global transcriptome between the different chondrocyte groups. They demonstrated TRPV4 mutation-induced changes in calcium signaling, mechanical property of matrix, and transcriptome of hiPSC-chondrocytes and concluded that the V620I and T89I mutations of TRPV4 in chondrocytes delay or inhibit hypertrophy, which may be a potential cause of skeletal dysplasias.
This study applied a gene-editing tool to creating mutant hiPSCs as a human cell model of the disease in culture to study TRPV4 mutation-induced alteration in cellular activities and molecular regulation. Establishing such an hiPSC model for disease study is novel and considered a major strength. Other strengths of this report include adequate background information, solid data analysis, and well-referenced discussions. The iPSC model established in this study could potentially be used to study pathogenic mechanisms of the diseases and identify molecular targets involved in regulating the mechanisms for the development of disease treatments.
However, there are two weaknesses identified in this current report, which are described below.
- Through comparison, differences in biological response and activities between mutant and wild-type hiPSC-chondrocytes were shown, and molecules and mechanisms of interest were identified as potential regulators involved in the mutation-induced changes. However, critical experiments such as gain- and loss-of-function assays to determine whether and how some or all of the identified molecules or mechanisms (HOXs, TGFB, biomineralization genes …) are regulated by the mutations to alter chondrocyte activities are missing. These experiments are needed to strengthen their conclusions. The discussions about the identified molecules and mechanisms with cited references are inadequate as a support for the conclusions.
We agree with the reviewer that gain- and loss-of-function experiments would be critical for identifying whether the proposed mechanisms are in fact responsible for the differences caused by the TRPV4 mutations and the disease phenotypes. However, these experiments are out of the scope of this study, and we plan to investigate each of these mechanisms in future studies. In the meantime, we have added additional citations to the discussion to further support these conclusions.
- The data currently presented in Figures 1, 5 and 6 are insufficient to justify the claims regarding mutation-induced changes of TRPV4, chondrocyte hypertrophy, and expression levels of the identified molecules.
To further support the conclusions in Figures 1, 5, and 6, we have added additional data. As suggested, we investigated the role of TRPV4 phosphorylation on channel function and activity. We found V620I had increased expression of PRKCA, the gene encoding for protein kinase C alpha. These data indicate that TRPV4 phosphorylation may be responsible for the increased basal calcium signaling through V620I TRPV4.
We then performed western blots to investigate production of hypertrophic proteins to validate the gene expression and support the claims that V620I and T89I had delayed hypertrophy in response to BMP4 treatment. Indeed, BMP4 treatment increased ALPL, COL10A1, IHH, and RUNX2 gene and protein expression compared to TGFβ3 controls, and this response was more prominent in WT than mutant lines. These data have been added to the paper to support our conclusions (Fig. 4 – Fig. S1B, Fig. 5, Fig. 5 – Fig. S1).
Reviewer #2 (Public Review):
In this manuscript, Dicks et al. generated two human iPSC lines with TRPV4 mutations (mild V620I or lethal T89I) using a CRISPR-Cas9 approach and examined their channel function and differentiation abilities into chondrocytes. While their initial goal is to elucidate the detailed molecular mechanisms underlying how these two mutations lead to strikingly distinct severities of skeletal dysplasias, most of their data found that these two mutations behave in a similar manner. The minor differences they found are: 1) increased basal currents in V620I cells; 2) reduced mechanical properties of cartilage matrix in V620I chondrocytes; 3) some differences in DEGs of RNA-seq data. They also stated that "The severe T89I mutation inhibits chondrocyte hypertrophy more than moderate V620I 298 mutation" (page 16). However, no substantiated data were provided to support this conclusion. While a serial of RNA-seq experiments were performed to explore the underlying mechanism, they were not followed by validation experiments to pinpoint the exact pathways or molecular mechanisms. Thus, although using CRISPR-Cas9 and iPSCs are novel and potentially important, this manuscript is overall descriptive with limited mechanistic information.
We thank the reviewer for the summary of the paper. We have further investigated the differences between WT and the two mutant lines to add to the RNA-seq experiments. As suggested by another reviewer, we looked at protein kinase gene expression, which may be altering TRPV4 phosphorylation and ultimately changes in channel activation. This expression data is consistent with the basal calcium differences we saw, and we believe these warrant further investigation in a follow-up study regarding biochemical changes to the channel structure and activation.
We also further validated the differences in BMP4-induced hypertrophy by looking at protein production. BMP4 not only increased hypertrophic proteins COL10A1, ALPL, IHH, and RUNX2, but we saw much larger increases in WT compared to mutants. Further, ALPL production was increased in the moderate V620I mutation compared to the severe T89I mutation, indicating a potential player in the differences in disease severity caused by the two mutants.
Finally, we investigated the DEGs between V620I and T89I to highlight the differences between the two mutations. We believe this study has served as a foundation for identifying potential mechanisms leading to the disease phenotypes of moderate and severe skeletal dysplasias. In future studies, we hope to validate these mechanisms.
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Evaluation Summary:
TRPV4 is an ion channel protein and mutations in TRPV4 gene resulted in different types of skeletal defects. In this study, the authors created two types of TRPV4 mutations (mild V620I and lethal T89I mutations) in human iPS cells through CRISPR-Cas9 gene editing. They identified key molecules potentially involved in TRPV4 mutation-induced changes in chondrocyte activities and concluded that the inhibition of chondrocyte hypertrophy induced by the mutations may cause the bone diseases.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Dicks et al. in this study characterized electrophysiological properties of mutant and wild-type hiPSC-chondrocytes and the expression of chondrocyte-associated markers during chondrogenic differentiation of the cells, and analyzed the differential expression of global transcriptome between the different chondrocyte groups. They demonstrated TRPV4 mutation-induced changes in calcium signaling, mechanical property of matrix, and transcriptome of hiPSC-chondrocytes and concluded that the V620I and T89I mutations of TRPV4 in chondrocytes delay or inhibit hypertrophy, which may be a potential cause of skeletal dysplasias.
This study applied a gene-editing tool to creating mutant hiPSCs as a human cell model of the disease in culture to study TRPV4 mutation-induced alteration in cellular activities and molecular …
Reviewer #1 (Public Review):
Dicks et al. in this study characterized electrophysiological properties of mutant and wild-type hiPSC-chondrocytes and the expression of chondrocyte-associated markers during chondrogenic differentiation of the cells, and analyzed the differential expression of global transcriptome between the different chondrocyte groups. They demonstrated TRPV4 mutation-induced changes in calcium signaling, mechanical property of matrix, and transcriptome of hiPSC-chondrocytes and concluded that the V620I and T89I mutations of TRPV4 in chondrocytes delay or inhibit hypertrophy, which may be a potential cause of skeletal dysplasias.
This study applied a gene-editing tool to creating mutant hiPSCs as a human cell model of the disease in culture to study TRPV4 mutation-induced alteration in cellular activities and molecular regulation. Establishing such an hiPSC model for disease study is novel and considered a major strength. Other strengths of this report include adequate background information, solid data analysis, and well-referenced discussions. The iPSC model established in this study could potentially be used to study pathogenic mechanisms of the diseases and identify molecular targets involved in regulating the mechanisms for the development of disease treatments.
However, there are two weaknesses identified in this current report, which are described below.
1. Through comparison, differences in biological response and activities between mutant and wild-type hiPSC-chondrocytes were shown, and molecules and mechanisms of interest were identified as potential regulators involved in the mutation-induced changes. However, critical experiments such as gain- and loss-of-function assays to determine whether and how some or all of the identified molecules or mechanisms (HOXs, TGFB, biomineralization genes ...) are regulated by the mutations to alter chondrocyte activities are missing. These experiments are needed to strengthen their conclusions. The discussions about the identified molecules and mechanisms with cited references are inadequate as a support for the conclusions.
2. The data currently presented in Figures 1, 5 and 6 are insufficient to justify the claims regarding mutation-induced changes of TRPV4, chondrocyte hypertrophy, and expression levels of the identified molecules. -
Reviewer #2 (Public Review):
In this manuscript, Dicks et al. generated two human iPSC lines with TRPV4 mutations (mild V620I or lethal T89I) using a CRISPR-Cas9 approach and examined their channel function and differentiation abilities into chondrocytes. While their initial goal is to elucidate the detailed molecular mechanisms underlying how these two mutations lead to strikingly distinct severities of skeletal dysplasias, most of their data found that these two mutations behave in a similar manner. The minor differences they found are: 1) increased basal currents in V620I cells; 2) reduced mechanical properties of cartilage matrix in V620I chondrocytes; 3) some differences in DEGs of RNA-seq data. They also stated that "The severe T89I mutation inhibits chondrocyte hypertrophy more than moderate V620I 298 mutation" (page 16). …
Reviewer #2 (Public Review):
In this manuscript, Dicks et al. generated two human iPSC lines with TRPV4 mutations (mild V620I or lethal T89I) using a CRISPR-Cas9 approach and examined their channel function and differentiation abilities into chondrocytes. While their initial goal is to elucidate the detailed molecular mechanisms underlying how these two mutations lead to strikingly distinct severities of skeletal dysplasias, most of their data found that these two mutations behave in a similar manner. The minor differences they found are: 1) increased basal currents in V620I cells; 2) reduced mechanical properties of cartilage matrix in V620I chondrocytes; 3) some differences in DEGs of RNA-seq data. They also stated that "The severe T89I mutation inhibits chondrocyte hypertrophy more than moderate V620I 298 mutation" (page 16). However, no substantiated data were provided to support this conclusion. While a serial of RNA-seq experiments were performed to explore the underlying mechanism, they were not followed by validation experiments to pinpoint the exact pathways or molecular mechanisms. Thus, although using CRISPR-Cas9 and iPSCs are novel and potentially important, this manuscript is overall descriptive with limited mechanistic information.
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Reviewer #3 (Public Review):
TRPV4 is an ion channel protein and mutations in TRPV4 gene resulted in different types of skeletal defects. In this study, the authors created two types of TRPV4 mutations (mild V620I and lethal T89I mutations) in human iPS cells through CRISPR-Cas9 gene editing. They found that mutations of TRPV4 accelerated calcium signaling and reduced response of calcium signaling to TRPV4 agonist. In addition, the V620I mutation led to decreased mechanical properties of cartilage matrix later in chondrogenesis. TRPV4 mutations also upregulated HOX genes and downregulated antioxidant genes, such as CAT and GSTA1, through entire chondrogenesis process. BMP4-induced chondrocyte hypertrophy was also inhibited in TRPV4 mutant cells. This study provided novel information about the functions of TRPV4 in chondrogenesis and …
Reviewer #3 (Public Review):
TRPV4 is an ion channel protein and mutations in TRPV4 gene resulted in different types of skeletal defects. In this study, the authors created two types of TRPV4 mutations (mild V620I and lethal T89I mutations) in human iPS cells through CRISPR-Cas9 gene editing. They found that mutations of TRPV4 accelerated calcium signaling and reduced response of calcium signaling to TRPV4 agonist. In addition, the V620I mutation led to decreased mechanical properties of cartilage matrix later in chondrogenesis. TRPV4 mutations also upregulated HOX genes and downregulated antioxidant genes, such as CAT and GSTA1, through entire chondrogenesis process. BMP4-induced chondrocyte hypertrophy was also inhibited in TRPV4 mutant cells. This study provided novel information about the functions of TRPV4 in chondrogenesis and help us understand the skeletal dysplasia observed in patients with TRPV4 mutations.
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