Role of N-cadherin in mechano-mediated cell fate control in intervertebral disc homeostasis

Abstract

Intervertebral discs (IVDs) form the primary connective tissue between two vertebrae and are crucial for spinal motion and load absorption. IVD degeneration (IDD) may lead to pain in the neck or lower back and is implicated in disability, worldwide. The etiology of IDD remains unclear but is thought to be multifactorial; aging, genetics, and environmental factors have been implicated. Mechanics is an environmental factor affecting IVD maturation and degeneration. Nucleus pulposus (NP) plays an essential role in the mechanical function of IVD in responding to environmental stimuli. Primitive NP is derived directly from the notochord and maintains the notochordal-like phenotype. In comparison, mature human NP is fibrocartilaginous and shows a predominant population of chondrocyte-like and fibroblastic cells. N-cadherin is a cell protein that modulates cell-cell adhesion junctions and mediates mechanotransduction through downstream cytosolic signalling cascades. N-cadherin is highly expressed in the notochordal-like NP cells during early IVD development. Thus, we hypothesized the function of N-cadherin in regulating NP cell phenotype, and therefore, IVD homeostasis through the modulation of the mechanosensing machinery. Johns Hopkins Chordoma Line 7 (JHC7) is an in vitro model for notochordal cells. We generated an N-cadherin deficient JHC7 line and performed single-cell RNA sequencing using a monolayer culture system and mechanical compression by alginate encapsulation to test our hypothesis.

Single-cell RNA sequencing defined the distinct phenotypes in WT versus mutant JHC7 cells. Trajectory analysis indicated that N-cadherin deficiency could drive the transition of these cells from notochordal- to MSC-, smooth muscle-, and chondrocyte-like phenotypes. The mutant cells showed matrix enrichment and an overrepresentation of the TGF-beta and focal adhesion pathways by GO and KEGG analyses, respectively. The transcription factor (TFs), namely SOX4 and SOX9 related to chondrogenesis, and TEAD1, a downstream protein in the focal adhesion cascade, were activated in mutant cells. These results suggested that the focal adhesion pathway may play a role in chondrogenic transition in JHC7 by controlling the downstream TFs in the absence of N-cadherin. We verified chondrogenic transition in the cellular mechanical loading model, evidenced by a significantly high expression of COL2A1 and ACAN in mutants. The RGD-binding integrin abundantly expressed in the mutant, indicated a shift in the mechanical sensor in the absence of N-cadherin. Mutant cells treated with TGF-beta showed higher levels of SOX9, COL2A1, and ACAN expression relative to the WT, indicating that the TGF-beta pathway could elevate the expression of the cellular matrix proteins, thereby facilitating the cells’ chondrogenic transition.

In conclusion, using the notochordal cell model, JHC7, we elucidated the role of N-cadherin mechanotransduction in regulating NP cell fate. The phenotypic transition of JHC7 cells was dependent on the presence of N-cadherin. Loss of N-cadherin promoted the shift in the mechanical sensor toward RGD-binding integrin, thus triggering integrin-mediated focal adhesion and stimulating the TGF-beta signalling pathway for elevating chondrogenesis upon supplementation with TGF-beta. Taken together, the deficiency of N-cadherin in human NP resulted in the mature chondrocyte-like phenotype with high matrix content, which was mediated by N-cadherin/integrin mechanosensing shift and the activation of the TGF-beta signalling pathway.