Poster Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Expression of Caspase Recruitment Domain-Containing Protein 14 (CARD14) in Human Nucleus Pulposus: Implication for Intervertebral Disc Degeneration (#53)

Koki Kawaguchi 1 , Koji Akeda 1 , Yusuke Togo 1 , Kento Watanabe 1 , Norihiko Takagemi 1 , Tatsuhiko Fujiwara 1 , Akihiro Sudo 1
  1. Orthopaedic Surgery, Mie University Graduate School of Medicine, Edobashi, Tsu, Japan

INTRODUCTION: A previous study evaluated the association between DNA methylation and intervertebral disc (IVD) degeneration by genome-wide association analysis of human nucleus pulposus (NP) tissues and identified 220 differentially methylated loci (DMLs) associated with human IVD degeneration. Caspase recruitment domain-containing protein 14 (CARD14), one of the DMLs, have been identified as the essential causative genes of psoriasis, and its mutation causes activation of NF-κB. Psoriatic arthritis develops as a complication of psoriasis and causes low back pain. However, the expression of CARD14 in IVD remains unknown. The purpose of this study was (1) to examine the expression of CARD14 in human NP cells, and (2) to evaluate the expression of CARD14 and NF-κB in human NP tissues in early and advanced stages of degeneration.

METHODS: Human NP cells obtained during spine surgeries were isolated from IVD tissues (Pfirrmann’s classification: grades 2-4, n=5), and the cells were cultured in a monolayer. To examine the effect of proinflammatory cytokines on the expression of CARD14, human NP cells were cultured in the presence of interleukin-1β (IL-1β; 0.1, 1, and 10 ng/ml). mRNA expression of CARD14 was quantified by real-time PCR analysis. Protein expression was evaluated by Western blot and immunohistochemical techniques. Human NP tissues were divided into the early degeneration (ED) group (Pfirrmann’s grade 2-3, n=15) or advanced degeneration (AD) group (Pfirrmann’s grade 4, n=14), and immunopositive or negative cells were manually counted. Immunopositive cells were classified as slightly positive (1+) or strongly positive (2+) according to the intensity of staining. Furthermore, using the same tissue samples, double immunofluorescence staining for CARD14 and NF-κB (p65) was performed, and the number of positive cells for each was counted.

RESULTS: 1. IL-1β increased the mRNA expression of CARD14 in a dose-dependent manner. A single band against CARD14 (113 kDa) was identified using Western blot analysis.

  1. The percentage of immunopositive cells in the AD group (76.7±17.4%) was significantly higher than that in the ED group (41.0±13.9%, P<0.05). The percentage of immunopositive cells and 2+ positive cells in the Pfirrmann grade 4 group was significantly higher than those in the Pfirrmann grade 2 and 3 groups (P<0.05).
  2. The percentage of p65-immunopositive cells in the AD group (69.4±4.9%) was significantly higher than that in the ED group (50.0±10.0%, P<0.05). The p65 immunopositive rate in CARD14-positive cells in the AD group (90.8±4.0%) was significantly higher than that in the ED group (83.7±4.0%, P<0.05).

DISCUSSION: The results of this study showed that human NP cells constitutively express GARD14 at both mRNA and protein levels. CARD14 expression in NP cells was shown to be stimulated by inflammatory stimuli, and its expression was upregulated in human NP tissues at the advanced stage of degeneration. Since CARD14 is associated with inflammatory activation, the enhanced expression of CARD14 in degenerated IVDs might be associated with the process of tissue degeneration. 6554ab75477cb-ISSLS2024Figure.jpg