DLX5 regulates the osteogenic differentiation of spinal ligaments cells derived from ossification of the posterior longitudinal ligament patients via NOTCH signaling

Tao Tang; Zhengya Zhu; Zhongyuan He; Fuan Wang; Hongkun Chen; Shengkai Liu; Mingbin Zhan; Jianmin Wang; Wei Tian; Dafu Chen; Xinbao Wu; Xizhe Liu; Zhiyu Zhou; Shaoyu Liu

doi:10.1002/jsp2.1247

DLX5 regulates the osteogenic differentiation of spinal ligaments cells derived from ossification of the posterior longitudinal ligament patients via NOTCH signaling

JOR Spine. 2023 Jan 28;6(2):e1247. doi: 10.1002/jsp2.1247. eCollection 2023 Jun.

Authors

Tao Tang^{1

2}, Zhengya Zhu¹, Zhongyuan He^{1

2}, Fuan Wang^{1

2}, Hongkun Chen¹, Shengkai Liu², Mingbin Zhan², Jianmin Wang¹, Wei Tian³, Dafu Chen³, Xinbao Wu³, Xizhe Liu², Zhiyu Zhou^{1

2}, Shaoyu Liu^{1

2}

Affiliations

¹ Innovation Platform of Regeneration and Repair of Spinal Cord and Nerve Injury, Department of Orthopaedic Surgery, The Seventh Affiliated Hospital Sun Yat-sen University Shenzhen China.
² Guangdong Provincial Key Laboratory of Orthopedics and Traumatology, Orthopaedic Research Institute/Department of Spinal Surgery The First Affiliated Hospital of Sun Yat-sen University Guangzhou China.
³ Laboratory of Bone Tissue Engineering, Beijing Laboratory of Biomedical Materials Beijing Research Institute of Orthopaedics and Traumatology, Beijing Jishuitan Hospital Beijing China.

Abstract

Background: Ossification of the posterior longitudinal ligaments (OPLL) is common disorder characterized by heterotopic ossification of the spinal ligaments. Mechanical stimulation (MS) plays an important role in OPLL. DLX5 is an essential transcription factor required for osteoblast differentiation. However, the role of DLX5 during in OPLL is unclear. This study aims to investigate whether DLX5 is associated with OPLL progression under MS.

Methods: Stretch stimulation was applied to spinal ligaments cells derived from OPLL (OPLL cells) and non-OPLL (non-OPLL cells) patients. Expression of DLX5 and osteogenesis-related genes were determined by quantitative real-time polymerase chain reaction and Western blot. The osteogenic differentiation ability of the cells was measured using alkaline phosphatase (ALP) staining and alizarin red staining. The protein expression of DLX5 in the tissues and the nuclear translocation of NOTCH intracellular domain (NICD) was examined by immunofluorescence.

Results: Compared with non-OPLL cells, OPLL cells expressed higher levels of DLX5 in vitro and vivo (p < 0.01). Upregulated expression of DLX5 and osteogenesis-related genes (OSX, RUNX2, and OCN) were observed in OPLL cells induced with stretch stimulation and osteogenic medium, whereas there was no change in the non-OPLL cells (p < 0.01). Cytoplasmic NICD protein translocated from the cytoplasm to the nucleus inducing DLX5 under stretch stimulation, which was reduced by the NOTCH signaling inhibitors (DAPT) (p < 0.01).

Conclusions: These data suggest that DLX5 play a critical role in MS-induced progression of OPLL through NOTCH signaling, which provides a new insight into the pathogenesis of OPLL.

Keywords: DLX5; NOTCH signaling; OPLL; cyclic stretch; mechanical stimulation; osteogenic differentiation.