Involvement of TRPV4 genetic mutations in regulation of intracellular calcium level and bone and cartilage abnormalities

Abstract

Continuous remodeling and reshaping of the bones are essential for repairing micro-damaged bones and maintaining calcium balance in the body. The transient receptor potential (TRP) superfamily of genes includes calcium channels that serve as a sensory system for the musculoskeletal system. One such channel is Transient Receptor Potential Vanilloid family member 4 gene (TRPV4), which is a non-selective cation channel capable of allowing calcium ions (Ca2+) to pass through. TRPV4 functions as both a mechanosensory and osmosensory channel, contributing to the development of various musculoskeletal tissues. During the formation of joints, TRPV4 is involved in chondrocyte proliferation and joint shaping. It also plays a crucial role in bone and cartilage development, osmoregulation, pain sensation, and apoptosis. Considering the crucial functions of TRPV4 in the development of bone and cartilage, it is not surprising that mutations in this mechanosensor channel can result in various skeletal abnormalities. Over 50 mutations in TRPV4 gene have been identified, leading to channelopathies affecting the skeletal system, ranging from dwarfism to prenatal death, skeletal dysplasia, and arthropathies. While the location of the mutation is likely related to the specific phenotype observed, little is currently known about the underlying mechanisms. Therefore, the objective of this project was to investigate the disease mechanisms caused by TRPV4 mutations that disrupt calcium regulation in multiple tissues, particularly chondrocytes. This will be accomplished through experiments involving HEK and chondrocyte cells, as well as mouse models, as TRPV4 mutations result in abnormal bone and cartilage development in human skeletal dysplasias. Scottish fold cats show a dominantly inherited osteochondrodysplasia involving malformation in the distal forelimbs, distal hindlimbs and tail, and progressive joint destruction. Using genome mapping techniques, our collaborators identified a p.V342F mutation (c.1024G>T) in TRPV4 as the underlying cause of the Scottish fold cat phenotype. Functional analysis in stably transfected HEK293 cells indicated that the TRPV4 V342F variant was weakly expressed on the cell surface in comparison to TRPV4 WT and therefore the maximum response to a synthetic agonist, GSK1016709A, was decreased. A higher basal activity and an increased response to hypotonic conditions were detected in mutant TRPV4 channels.

So far, researchers have created TRPV4 knockout (KO) mouse models and transgenic mouse models, where mutated genes are overexpressed alongside a wild-type (WT) background. We have characterised a TRPV4 V620A mouse model, which was generated by ENU mutagenesis, and is the first knock-in TRPV4 mutant mouse. Our findings revealed significant changes in spine curvature, vertebral angle, tibia and femur length, as well as several trabecular bone parameters in TRPV4 mutant mice compared to WT mice as well as TRPV4 KO mice. The phenotype observed in the TRPV4 V620A mouse is milder compared to the more severe phenotype of the TRPV4 V620I transgenic mouse. Moreover, the TRPV4 V620A mouse exhibits a less pronounced phenotype than the human TRPV4 V620I condition. In contrast to the findings in the transgenic model (V620I mutant), where skeletal malformations were observed in the mutant model compared to WT mice, the TRPV4 V620A mice in this study exhibited no significant alterations when compared to WT mice. Experiments on the stimulation of the TRPV4 WT, TRPV4 KO, TRPV4V620A/+, and TRPV4V620A/V620A chondrocytes indicated less calcium influx in response to GSK101 stimulation in homozygous mutant chondrocytes compared to WT and heterozygous chondrocytes. Although TRPV4V620A/+, and TRPV4V620A/V620A chondrocytes showed higher constitutive intracellular calcium than WT and KO, after normalization, all mutant and WT chondrocytes and even KO showed a normal peak in response to hypotonicity. However, increased level of [Ca2+]i remained lower in heterozygous and even much lower in homozygous and KO compared to WT, but this level in TRPV4V620A/+ cells reached the same level as WT in sustained phase over the time course. TRPV4 KO and homozygous mutant chondrocytes did not respond to hypotonicity stimulation, while the heterozygous mutant TRPV4 has a reduced response compared to WT. The V620A heterozygous variant showed no significant effect on the response of PAR1 and 2 channels when stimulated with PAR1 and PAR2 agonists. Experiments to explore the mechanism by which V620I and V620A variants in TRPV4 could disrupt ion channel function in HEK293 cells indicated that TRPV4 WT transfected HEK cells as well as mutant channels responded to agonist GSK101 but non-transfected cells did not respond to the treatment. However, the maximum response to GSK101 was low in both mutant TRPV4 than WT. Same as chondrocytes, in which TRPV4 WT, TRPV4V620A/V620A and TRPV4V620A/+ indicated proper response to GSK101 stimulation, HEK cells transfected with TRPV4 WT, TRPV4 p.620A and TRPV4 p.620I had favorable response towards GSK101 stimulation. HEK cells transfected with TRPV4 p.V620I had a lower maximum response as well as decreased sustained [Ca2+]i level compared to cells expressing WT, subsequent to the

adjustment of the leading basal action. This shows that the mutant V620I channels have a low hypotonicity sensitivity than the WT channel. However, cells transfected with TRPV4 p.V620A did not respond to hypotonicity stimulation. The results indicated that PAR1 peak responses in both TRPV4 p.V620A and TRPV4 p.V620A expressing HEK cells. However, the peak response to PAR1 agonist treatment was a little lower in the TRPV4 p.V620A cells compared to the TRPV4 p.V620A expressing cells. The peak responses were typical in TRPV4 WT expressing HEK cells. Same pattern of baseline and peak responses were observed when cells were treated with PAR2 agonist treatment. Therefore, PAR2 stimulates the activation of TRPV4, allowing an influx of calcium. In conclusion, this research aimed to investigate the role of TRPV4 genetic mutations on the calcium metabolism in different models that might be associated with bone and cartilage abnormalities. Our data on the effect of TRPV4 620A variant on the pathologic presentations of the mouse indicated that this variant was associated with larger thoracolumbar kyphosis and cervicothoracic lordosis, but shorter tibia and femur length, suggest abnormalities during endochondral ossification. Likewise, the larger bone volume fraction and the smaller trabecular number of the trabecular bone, as well as the smaller trabecular separation of the femoral head in the TRPV4V620A/+ and TRPV4V620A/V620A mice compared to WT group, confirmed the hypothesis that this variant has harmful effects on bone development. In addition, TRPV4 620A variant in mouse chondrocytes was associated with an impaired response to the synthetic agonist triggering and hypotonicity stimulation. Putting these observations together, TRPV4 V620A variant is associated with impaired channel function that is probably mirrored in raising pathologic bone and cartilage presentations. On the other hand, the behaviors of HEK cells transfected with TRPV4 620A and TRPV4 620I were not similar to each other. As a consequence, our results on Trpv4 620A variant in mice partly resemble the phenotype of the V620I variant in human Brachyolmia.