Oral Presentation 51st International Society for the Study of the Lumbar Spine Annual Meeting 2025

Mechanosensitive TRPV4 controls intervertebral disc development in human and mice (115223)

Jiachen Lin 1 , Bowen Lv 1 , Mengrui Wu 1 , Yue Wang 1
  1. The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, ZHEJIANG, China

INTRODUCTION

Recently, genes that participate in mechanosensing and transduction have been reported to mediate skeletal development. TRPV4 (Transient Receptor Potential Cation Channel Subfamily V Member 4) is a subunit of a mechanosensitive cation channel which expresses in multiple mammalian organs. Mutations in TRPV4 genes may lead to a spectrum of congenital skeletal defects. Here we found 3 pedigrees carrying TRPV4 mutations with remarkable intervertebral disc malformations. We next used genetic modification in mice to recapitulate human dysmorphic disc and investigated the underlying cellular mechanisms.

METHODS

Written consent was obtained from each participant. Genetic sequencing was performed to identify the causative mutation in patients. CRISPR/Cas9 editing was applied to introduce conditionally-expressing Trpv4R594H mutant in mice. Specifically, the Trpv4R594H was activated in osteochondral cell lineage using Col2-cre line (conditional knock-in, CKI). Skeletal staining and micro-CT were used to evaluate gross appearance and bone maturation in the mouse spine. Immunostaining assays of histological sections were performed to examine cell events and gene expression in the disc. We further utilized single-cell RNA sequencing and bulk RNA sequencing on spine samples collected from P1, P3, P7, P14 littermates to trace cell populations and decipher molecular changes in detail.

RESULTS

From 3 families, we identified 3 de novo TRPV4 gene mutations (c.2146G>A[p.Ala716Thr], c.1781G>A[p.Arg594His], and c.958C>G[p.Arg320Gly]). Distinctive clinical features, including deformed endplates and annulus fibrosus, enlarged nucleus pulposus, and flat vertebral bodies (Figure A). The postnatal Col2-Cre;Trpv4R594H CKI mice demonstrated similar dysmorphic phenotypes, as delayed ossification and vertebral malformations were noted on skeletal staining and microCT images (Figure B, C). The level of osteoblastic activation and mineralization were not affected, though the organization of hypertrophic chondrocytes in endplate was disrupted (data not shown). Further histological study revealed more severe defects in chondrogenic lineage in the CKI mice. After birth, CKI mice did not form annulus fibrosus structure, and the cartilaginous endplates were poorly-shaped (Figure D). The fiber arrangement was disrupted along with the retention of extracellular matrix in the disc. Moreover, increased cell apoptosis and decreased cell proliferation were recorded using TUNEL and BrDU assays in the disc (data not shown). Besides, the synthesis of type I collagen was significantly suppressed, while the distribution of type II collagen was markedly altered (Figure E), suggesting the metabolism of the chondrogenic lineage was severely impaired. Lastly, we identify the differential genes were enriched in Hippo-YAP signaling and PI3K/AKT signaling pathways in chondrocyte lineage (data not shown), which gave rise to further investigation on the underlying molecular mechanisms.

DISCUSSION

The mechanosensitive TRPV4 determines the fate of osteochondral progenitors and thus controls the development of naïve discs. Over-activation of TRPV misled multiple biological process of osteochondral progenitors in spine region, including endochondral ossification, calcium influx, and cell apoptosis. Specifically, the chondrogenic lineage was affected with significant changes in cell proliferation, differentiation, biosynthesis and metabolism, suggesting TRPV4 plays an essential role in mechanotransduction and modulation of the chondrogenic lineage. 

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