Skeletal dysplasias comprise a heterogeneous group of over 450 bone and cartilage diseases with an overall birth incidence of 1 in 5000 (Krakow and Rimoin, 2010; Nemec et al., 2012; Ngo et al., 2018; Orioli et al., 1986; Superti-Furga and Unger, 2007). Mutations in transient receptor potential vanilloid 4 (TRPV4), a non-selective cation channel, can lead to varying degrees of skeletal dysplasia, including moderate autosomal-dominant brachyolmia and severe metatropic dysplasia (Andreucci et al., 2011; Kang, 2012). For example, a V620I substitution (exon 12, G858A) in TRPV4 is responsible for moderate brachyolmia, which exhibits short stature, scoliosis, and delayed development of deformed bones (Kang et al., 2012; Rock et al., 2008; Kang, 2012). These features, albeit more severe, are also present in metatropic dysplasia. Metatropic dysplasia can be caused by a TRPV4 T89I substitution (exon 2, C366T) and leads to joint contractures, disproportionate measurements, and, in severe cases, neonatal death due to small chest size and cardiopulmonary compromise (Camacho et al., 2010; Kang et al., 2012; Kang, 2012). Both V620I and T89I TRPV4 mutations are considered gain-of-function variants (Leddy et al., 2014b; Loukin et al., 2011). Given the essential role of TRPV4 during chondrogenesis (Muramatsu et al., 2007; Willard et al., 2021) and cartilage homeostasis (O’Conor et al., 2014), it is hypothesized that TRPV4 mutations may affect endochondral ossification during skeletal development.

Endochondral ossification is a process by which bone tissue is created from a cartilage template (Breeland et al., 2021; Camacho et al., 2010; Krakow and Rimoin, 2010; Rimoin et al., 2007). During this process, chondrocytes transition from maintaining the homeostasis of cartilage, regulated by transcription factor SRY-box containing gene 9 (SOX9) (Breeland et al., 2021; Nishimura et al., 2012b; Prein and Beier, 2019; Sophia Fox et al., 2009), to hypertrophy. Hypertrophy is driven by runt-related transcription factor 2 (RUNX2) and bone morphogenic protein (BMP) signaling (Breeland et al., 2021; Nishimura et al., 2012b; Prein and Beier, 2019) and leads to chondrocyte apoptosis or differentiation into osteoblasts to form bone (Breeland et al., 2021; Nishimura et al., 2012b; Prein and Beier, 2019). However, how TRPV4 and its signaling cascades regulate endochondral ossification remains to be determined.

The activation of TRPV4 increases SOX9 expression (Muramatsu et al., 2007) and prevents chondrocyte hypertrophy and endochondral ossification (Amano et al., 2009; Hattori et al., 2010; Lui et al., 2019; Nishimura et al., 2012a; Nishimura et al., 2012b). One study found that overexpressing wildtype (WT) Trpv4 in mouse embryos increased intracellular calcium (Ca²⁺) concentration and delayed bone mineralization (Weinstein et al., 2014), a potential link between intracellular Ca²⁺, such as with gain-of-function TRPV4 mutations, and delayed endochondral ossification. Our previous study also observed increased expression of follistatin (FST), a potent BMP inhibitor, and delayed hypertrophy in porcine chondrocytes overexpressing human V620I- and T89I-TRPV4 (Leddy et al., 2014a; Leddy et al., 2014b). While previous studies have greatly increased our knowledge of the influence of TRPV4 mutations on chondrogenesis and hypertrophy, most of them often involved animal models (Leddy et al., 2014b; Weinstein et al., 2014) or cells (Camacho et al., 2010; Krakow and Rimoin, 2010; Leddy et al., 2014b; Loukin et al., 2011; Rock et al., 2008) overexpressing mutant TRPV4. Therefore, these approaches may not completely recapitulate the effect of TRPV4 mutations on human chondrogenesis.

Human-induced pluripotent stem cells (hiPSCs), derived from adult somatic cells (Takahashi et al., 2007), offer a system for modeling human disease to study the effect of mutations throughout differentiation (Adkar et al., 2017; Lee et al., 2021). In fact, two studies have used patient-derived hiPSCs with TRPV4 mutations to study lethal and non-lethal metatropic dysplasia-causing variants I604M (Saitta et al., 2014) and L619F (Nonaka et al., 2019), respectively. However, patient samples are often challenging to procure due to the rarity of skeletal dysplasias. In this regard, CRISPR-Cas9 technology allows the creation of hiPSC lines harboring various mutations along with isogenic controls (i.e., WT).

The goal of this study was to elucidate the molecular mechanisms underlying how two TRPV4 gain-of-function mutations lead to strikingly distinct severities of skeletal dysplasias (i.e., moderate brachyolmia vs. lethal metatropic dysplasia). To achieve this goal, we generated CRISPR-Cas9 gene-edited hiPSC lines bearing either the V620I or T89I TRPV4 mutation, and their isogenic WT control, to delineate the effects of TRPV4 mutations on chondrogenesis and hypertrophy using RNA sequencing and transcriptomic analysis. We further examined the effects of the mutations on channel function and matrix production and properties. We hypothesized the V620I and T89I TRPV4 mutations would enhance chondrogenesis with distinct degrees of altered hypertrophy. This study will improve our understanding of the role of TRPV4 in chondrocyte homeostasis and maturation and lay the foundation for treatment and prevention of TRPV4-mediated dysplasias.

To elucidate the detailed molecular mechanisms underlying the distinct severity of skeletal dysplasias caused by two TRPV4 mutations (moderate brachyolmia-causing V620I vs. severe metatropic dysplasia-causing T89I), we used CRISPR-Cas9 gene editing to generate hiPSC-derived chondrocytes bearing either V620I or T89I mutation. We observed that day-28 chondrocytes exhibited differences in channel function and gene expression between the mutants and WT control. Differences in transcriptomic profiles between V620I and T89I and from WT became more apparent with maturation following 4 additional weeks of culture with TGFβ3 or hypertrophic differentiation with BMP4 treatment. Of note, WT was significantly more sensitive to BMP4-induced hypertrophy. At the transcriptomic and proteomic levels, TRPV4 mutations inhibited chondrocyte hypertrophy, particularly with the T89I mutation, whereas V620I exhibited a milder phenotype, consistent with the clinical presentation of these two conditions. Our results suggest that skeletal dysplasias may be, at least in part, resulting from improper chondrocyte hypertrophy downstream of altered TRPV4 function. Furthermore, with our genome-wide RNA sequencing analysis, we also identified several putative genes that may be responsible for these dysregulated pathways in human chondrocytes bearing V620I or T89I TRPV4 mutations.

Our findings are generally consistent with previous non-human models of V620I and T89I mutations. Two other models that have studied the V620I and T89I mutations include X. laevis oocytes injected with rat TRPV4 cRNA (Loukin et al., 2011) or primary porcine chondrocytes transfected with human mutant TRPV4 (Leddy et al., 2014b). Both reports and our current study investigated the baseline currents of the mutant TRPV4 compared to WT. Here, we used patch clamping and observed high basal currents in V620I with a significant decrease when TRPV4 was inhibited. However, this characteristic was trending, but not significant, in T89I, despite both V620I and T89I being reported as gain-of-function mutations (Camacho et al., 2010; Rock et al., 2008). Both the X. laevis oocyte and porcine chondrocyte models confirmed high basal currents through V620I-TRPV4 (Leddy et al., 2014b; Loukin et al., 2011). Interestingly, X. laevis oocytes, but not the humanized porcine chondrocytes, showed an increase in basal Ca²⁺ signaling through T89I (Leddy et al., 2014b; Loukin et al., 2011). Furthermore, our results were consistent with a summary of TRPV4 channelopathies reporting an increase in conductivity in V620I but no change in T89I (Kang, 2012). Interestingly, V620I also had increased expression of PRKCA, the gene encoding for protein kinase C alpha. Phosphorylation by PKC has been shown to alter TRPV4 activation (Cao et al., 2018) and therefore may play a role in the altered signaling with these mutations. In future experiments, we will further investigate PKC and PKA phosphorylation of TRPV4 and the effects on channel activity in these mutations. The conflicting basal current results could be due to differences in phosphorylation or the species of the TRPV4, but this was not the case regarding channel activation. As mentioned, the hiPSC-derived chondrocytes with V620I and T89I TRPV4 had reduced currents and Ca²⁺ signaling in response to chemical agonist GSK101. However, our previous study showed the porcine chondrocytes with mutant human TRPV4 had increased peak Ca²⁺ signaling in response to hypotonic changes (Leddy et al., 2014b). This discrepancy could be due to the mode of activation of TRPV4 (i.e., osmotic vs. chemical agonist). In contrast, the oocytes with mutant rat TRPV4 had lower currents in response to both hypotonic and chemical (GSK101) TRPV4 activation compared to WT-TRPV4, consistent with our findings. It can be speculated that there is decreased sensitivity to the antagonist because the mutated hiPSC-derived chondrocytes are compensating for the increased basal activity by reducing the number of TRPV4 channels or other ion channels and signaling transducers as shown with the RNAseq data and associated GO terms. The increased basal currents and decreased channel sensitivity to TRPV4 agonist GSK101 with mutated TRPV4 are also likely resulting from an increased open probability of TRPV4 making the channels less likely to be activated by a chemical agonist (Loukin et al., 2011). The obvious differences in both resting and activated states confirm functional differences with TRPV4 mutations that may ultimately lead to changes in downstream signaling of the channel, which alter joint development and result in skeletal dysplasias.

It was hypothesized, in the porcine chondrocyte study, that the increased Ca²⁺ signaling due to the V620I and T89I TRPV4 mutations increased FST expression that inhibited BMP signaling and hypertrophy (Leddy et al., 2014a; Leddy et al., 2014b). Surprisingly, we found no differences in FST expression in mutant hiPSC-derived chondrocytes compared to WT. However, our previous study used non-human cells, which could alter the effects of the human TRPV4 mutations and downstream gene expression. Another previous hypothesis made was that the altered TRPV4 signaling increased SOX9 expression, a known regulator of resting and proliferating chondrocytes up-regulated by TRPV4 activation (Muramatsu et al., 2007), thus decreasing hypertrophy (Rock et al., 2008). SOX9-knockin mice exhibit a dwarfism phenotype (Amano et al., 2009), and SOX9 overexpression inhibits hypertrophy and endochondral ossification (Hattori et al., 2010; Lui et al., 2019), likely via parathyroid hormone-related protein (PTHrP) (Amano et al., 2009; Nishimura et al., 2012b). However, PTHrP was not strongly regulated in our data set. Furthermore, our RT-qPCR revealed that only V602I significantly up-regulated SOX9, and the RNAseq data showed that SOX9 had a smaller fold change compared to other chondrogenic genes, such as GDF5, COL6A1, COL6A3, and COMP. In fact, these genes, which were up-regulated in V620I- and T89I-hiPSC-derived chondrocytes, have a pro-chondrogenic but anti-hypertrophic phenotype (Caron et al., 2020; Chu et al., 2017; Hecht and Sage, 2006). Therefore, these results suggest additional and alternative pathways to FST and SOX9 that are responsible for the V620I and T89I skeletal dysplasias.

Our results are generally consistent with previous reports on the effects of other TRPV4 mutations such as lethal and non-lethal metatropic dysplasia-causing I604M (Saitta et al., 2014) and L619F (Nonaka et al., 2019). The data also reveal potential differences in the effects of these varying TRPV4 mutations on cell electrophysiology or differentiation. For example, we saw an increase in SOX9 expression in V620I, while no change in T89I. Gain-of-function mutation L619F also increased SOX9 expression (Nonaka et al., 2019), while I604M, which has been reported to not alter conductivity like T89I (Kang, 2012), decreased SOX9 expression (Saitta et al., 2014). I604M also decreased COL2A1, COL10A1, and RUNX2 expression consistent with our T89I results (Saitta et al., 2014). Intriguingly, the L619F mutation was reported to increase Ca²⁺ signaling with activation via a TRPV4 agonist (Nonaka et al., 2019). However, we observed that V620I and T89I had significantly reduced Ca²⁺ signaling compared to WT in response to chemical agonist GSK101, as confirmed by both confocal imaging and patch clamping. These results highlight that TRPV4 mutations have heterogeneous effects on downstream signaling pathways and thus lead to diverse disease phenotypes, despite similar classification of these mutations as ‘gain-of-function’. It is also important to note that in previous studies, chondrogenic differentiation of iPSCs (Saitta et al., 2014) or dental pulp cells (Nonaka et al., 2019) were performed in short-term micromass culture, and not long-term pellet culture as in our study, potentially leading to different levels of chondrogenesis and maturation of the cells.

Our transcriptomic analysis showed significant changes in various HOX family genes due to TRPV4 mutations, suggesting a potential role of these genes in maintaining the immature, chondrogenic phenotype in the mutated lines. At both days 28 and 56, the top 25 up-regulated genes in the V620I and T89I lines included genes from the anterior HOX family (Iimura and Pourquié, 2007; Seifert et al., 2015). The high expression of anterior HOX genes indicates that the mutants are maintaining the chondrocytes in an early developmental stage with axial patterning. At days 28 and 56, HOXA2, HOXA3, and HOXA4 were in the top up-regulated genes, with HOXA4 having the largest fold change. Interestingly, gain-of-function mutations or overexpression of HOXA2, HOXA3, and HOXA4 impair chondrogenesis, limit skeletal development, decrease endochondral ossification regulators, and delay mineralization in animal models (Creuzet et al., 2002; Deprez et al., 2013; Kanzler et al., 1998; Li and Cao, 2006; Massip et al., 2007; Seifert et al., 2015). HOXA5 was also highly up-regulated at both days 28 and 56, and mutations in this gene showed disordered patterning of limb bud development (Pineault and Wellik, 2014). Finally, the rib and spine phenotypes associated with brachyolmia and metatropic dysplasia could be contributed to the altered expression of HOXA4 to HOXA7 as it has been shown that these genes are associated with rib and spine patterning, and alterations in expression have led to defects (Chen et al., 1998; Wellik, 2009). The only up-regulated posterior HOX genes were HOXC8 and HOXD8 at day 56 (Iimura and Pourquié, 2007; Seifert et al., 2015). The absence of posterior HOX9, HOX11, and HOX13, which are associated with limb development and hypertrophic RUNX2/3 expression (Pineault and Wellik, 2014; Qu et al., 2020), may be at least partially responsible for the improper development in skeletal dysplasias. Interestingly, many links have been identified between HOX genes and TGFβ3-family signaling, specifically through SMAD proteins, both within skeletal development and other processes (e.g., murine lung development) (Li and Cao, 2006; Li and Cao, 2003; Volpe et al., 2013).

In fact, TRPV4 and TGF-β signaling have recently been shown to interact, with effects specific to the order in which they occur (Nims et al., 2021; O’Conor et al., 2014; Woods et al., 2021). Consistent with previous finding with hiPSCs housing the I604M TRPV4 mutations (Saitta et al., 2014), the altered TRPV4 activity in our hiPSC-derived chondrocytes could be altering their response to the TGFβ3 and BMP4 treatments. Furthermore, the V620I and T89I mutations increased expression of HTRA1, which has been shown to bind to and alter the response to members of the TGFβ family (Polur et al., 2010). Furthermore, TGFβ3 and TWIST, which is downstream of TGFβ3 signaling, were both up-regulated in TRPV4-mutated hiPSC-derived chondrocytes. It has been reported that TGFB3 expression and signaling prevent osteoblastogenesis of mesenchymal stem cells (Nishimura et al., 2012a; Nishimura et al., 2012b), while TWIST inhibits hypertrophy regulators RUNX2 and FGFR2 (Michigami, 2014; Miraoui and Marie, 2010). Therefore, another mechanism of hypertrophic dysregulation with these mutations could be altered response to TGFβ family signaling.

In fact, in response to treatment with BMP4, a member of the TGFβ family, there was increased expression of GSTA1, which produces the antioxidant glutathione (Chen et al., 2008; Hayes et al., 2005), in WT but not in mutants. BMP4 treatment of T89I-mutated chondrocytes significantly increased expression of another antioxidant catalase (CAT) compared to its TGFβ3 control group; however, TRPV4-mutated chondrocytes without BMP4 treatment had significantly lower CAT expression. This may potentially indicate an association between antioxidants, which remove reactive oxygen species (ROS; e.g., H₂O₂), and chondrocyte maturation. While one study observed that chondrocyte maturation is associated with decreasing catalase (Morita et al., 2007), this is inconsistent with other findings. Another report stated hypoxia, which increases ROS, inhibits hypertrophic differentiation and endochondral ossification (Leijten et al., 2012). Many others found that ROS prevent endochondral ossification, potentially via inhibition of the hedgehog pathways (Atashi et al., 2015; Chen et al., 2008; Fragonas et al., 1998). Interestingly, IHH also had the lowest expression level in our T89I mutant chondrocytes. These findings suggest that decreased expression of CAT and GSTA1 in TRPV4 mutants may also be involved in dysregulating endochondral ossification in these cells.

GSTA1 is one of many genes with significantly lower expression in mutated chondrocytes compared to WT in response to BMP4 treatment including ALPL, AMELX, IFITM5, IBSP, and MEPE. These genes play important roles in bone development. For example, IBSP is downstream of RUNX2, a primary transcription factor of hypertrophic differentiation and osteoblast differentiation (Komori, 2018). Further, MEPE negatively (Lu et al., 2004; Staines et al., 2012) and IFITM5 (Hanagata et al., 2011; Moffatt et al., 2008) and ALPL (Millán, 2013; Strzelecka- Kiliszek et al., 2018) positively regulate bone mineralization during skeletogenesis, respectively. Mutations in these genes also lead to bone mineralization diseases such as rickets (Lu et al., 2004; Staines et al., 2012), osteogenesis imperfecta (Hanagata, 2016), and hypophosphatasia with deformed long bones (Taillandier et al., 2015). Not only did we observe significantly lower gene expression of ALPL in TRPV4-mutated chondrocytes treated with BMP4; we also demonstrated that ALPL protein production is negatively associated with disease severity. Our results indicate that not only do the mutated cells have an altered hypertrophic response to BMP4, but there is a connection between these genes, particularly ALPL, or tissue-nonspecific alkaline phosphatase, and delayed endochondral ossification in chondrocytes bearing V620I or T89I mutations. However, how these genes and their transcription are associated with TRPV4 function and mutations still warrants further investigation.

The gene expression and protein production of ALPL, as well as IHH, in response to BMP4 treatment was not only significantly lower in mutants compared to WT, but it was also significantly lower for the severe T89I mutation compared to the moderate V620I mutation. This is one of the many targets showing the different levels of genetic and protein production between the two mutations that may be responsible for the distinct severity and phenotype of the corresponding diseases. Furthermore, several other genes were also detected to be uniquely up- or down-regulated compared to WT in one mutated line but not the other. Interestingly, there were more uniquely expressed genes between the two mutated lines at day 56, suggesting the separation of transcriptomic profiles in V620I and T89I occurs at later time points of chondrocyte maturity. Among those unique to severe T89I include down-regulation of IBSP, a positive mineralization regulator, at day 28, and up-regulation of negative regulator MEPE at day 56. These unique genes were also not associated with many of the same biological processes as WT and V620I, especially those regarding endochondral ossification, when treated with BMP4. This, in conjunction with the high number of unique DEGs, represents a potential inhibition of hypertrophy, particularly in response to BMP4 treatment, with the T89I mutation leading to severe metatropic dysplasia. V620I also had unique differences from both WT and T89I. The decrease in mechanical properties with increased basal current of the V620I mutant was unexpected since TRPV4 activation was previously shown to increase matrix production and properties (O’Conor et al., 2014). Furthermore, genes uniquely up-regulated in V620I were associated with interferon type I (IFNβ). IFNβ has been reported to decrease inflammatory markers and matrix degradation (Hu et al., 2005; Palmer et al., 2004; van Holten et al., 2004; Zhao et al., 2014), despite the decrease in moduli observed in the day-42 V620I chondrogenic pellets. Interestingly, a study comparing bone marrow-derived MSCs from healthy and systemic lupus erythematous patients found that IFNβ-inhibited osteogenesis via suppression of RUNX2 and other osteogenic genes (Gao et al., 2020). Highlighting a potential, unique regulator of the delayed hypertrophy in V620I leading to brachyolmia.

Here, we present multiple putative genes and pathways that could be involved in delaying, and potentially inhibiting, chondrocyte hypertrophy in V620I- and T89I-TRPV4 mutants. It should be noted, however, that this study has some potential limitations. It is well-recognized that Wnt/β-catenin signaling plays an important role in chondrocyte hypertrophy (Hou et al., 2019; Huang et al., 2018; Michigami, 2014). After 56 days of differentiation, we observed increased expression of β-catenin-coding gene CTNNB1 in T89I-mutated chondrocytes highlighting this pathway could be playing a role in the inhibited hypertrophy. However, we may be preventing some hypertrophy since our chondrogenic protocol uses a pan-Wnt inhibitor to prevent off-target differentiation and promote a homogenous chondrocyte population (Wu et al., 2021). Nevertheless, our WT chondrocytes, but not TRPV4 mutants, exhibited hypertrophic differentiation with BMP4 treatment, suggesting that DEGs/pathways detected in our sequencing analysis are still robust. Since this study focuses on TRPV4 gain-of-function mutations, future studies could fully or partially inhibit TRPV4 signaling to determine if that would increase similarity between the mutant and WT lines at various stages of chondrogenic and hypertrophic differentiation. Additionally, this study only activated TRPV4 using the pharmacological activator GSK101. Other future experiments could activate the channel osmotically or with mechanical loading to investigate additional differences in TRPV4 function leading to skeletal dysplasias during development.

In summary, our study found that dysregulated skeletal development in the V620I- and T89I-TRPV4 dysplasias is likely due, at least in part, to delayed and inhibited chondrocyte hypertrophy. The gain-of-function mutations may lead to increased HOX gene expression, altered TGFβ signaling, decreased hypertrophic and biomineralization gene expression (e.g., ALPL, AMELX, IFITM5, IBSP, and MEPE), and genes regulating hedgehog pathways and ROS accumulation (e.g., GSTA1 and CAT). Our findings lay a foundation for the development of therapeutics for these diseases and provide significant insights into the regulation of endochondral ossification via TRPV4.

All RNAseq data files generated and reported in this study are available on GEO (accession number GSE225446,https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE225446).

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Author details

1. Amanda R Dicks

   1. Department of Biomedical Engineering, Washington University in St. Louis, St Louis, United States
   2. Department of Orthopedic Surgery, Washington University School of Medicine, St. Louis, St Louis, United States
   3. Shriners Hospitals for Children - St. Louis, St. Louis, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6036-280X

2. Grigory I Maksaev

   Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, St Louis, United States

   Contribution

   Data curation, Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

3. Zainab Harissa

   1. Department of Biomedical Engineering, Washington University in St. Louis, St Louis, United States
   2. Department of Orthopedic Surgery, Washington University School of Medicine, St. Louis, St Louis, United States
   3. Shriners Hospitals for Children - St. Louis, St. Louis, United States

   Contribution

   Data curation, Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

4. Alireza Savadipour

   1. Department of Orthopedic Surgery, Washington University School of Medicine, St. Louis, St Louis, United States
   2. Shriners Hospitals for Children - St. Louis, St. Louis, United States
   3. Department of Mechanical Engineering and Material Science, Washington University in St. Louis, St. Louis, United States

   Contribution

   Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

5. Ruhang Tang

   1. Department of Orthopedic Surgery, Washington University School of Medicine, St. Louis, St Louis, United States
   2. Shriners Hospitals for Children - St. Louis, St. Louis, United States

   Contribution

   Data curation, Formal analysis, Investigation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

6. Nancy Steward

   1. Department of Orthopedic Surgery, Washington University School of Medicine, St. Louis, St Louis, United States
   2. Shriners Hospitals for Children - St. Louis, St. Louis, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

7. Wolfgang Liedtke

   1. Department of Neurology, Duke University School of Medicine, Durham, United States
   2. Department of Molecular Pathobiology - NYU College of Dentistry, New York, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing – review and editing

   Competing interests

   Patents on TRPV4 inhibitors have been licensed to TRPblue (US Patents 9,701,675; 10,329,265; and 11,014,896). Dr. Liedtke is an executive employee of Regeneron Pharmaceuticals (Tarrytown NY).

8. Colin G Nichols

   Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, St Louis, United States

   Contribution

   Conceptualization, Supervision, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4929-2134

9. Chia-Lung Wu

   Department of Orthopaedics and Rehabilitation, Center for Musculoskeletal Research, University of Rochester, Rochester, United States

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   Additional information

   co-senior author

   "This ORCID iD identifies the author of this article:" 0000-0001-9598-7036

10. Farshid Guilak

    1. Department of Orthopedic Surgery, Washington University School of Medicine, St. Louis, St Louis, United States
    2. Shriners Hospitals for Children - St. Louis, St. Louis, United States

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Writing – review and editing

    For correspondence

    guilak@wustl.edu

    Competing interests

    Patents on TRPV4 inhibitors licensed to TRPblue Inc (US Patents 9,701,675; 10,329,265; and 11,014,896)

    Additional information

    co-senior author

    "This ORCID iD identifies the author of this article:" 0000-0001-7380-0330

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