Transcriptional profiling of the murine intervertebral disc and age-associated changes in the nucleus pulposus

ABSTRACT

Purpose/Aim: The intervertebral disc (IVD) is composed of cell types whose subtle phenotypic differences allow for the formation of distinct tissues. The role of the nucleus pulposus (NP) in the initiation and progression of IVD degeneration is well established; however, the genes and pathways associated with NP degeneration are poorly characterized.

Materials and Methods: Using a genetic strategy for IVD lineage-specific fluorescent reporter expression to isolate cells, gene expression and bioinformatic analysis was conducted on the murine NP at 2.5, 6, and 21 months-of-age and the annulus fibrosus (AF) at 2.5 and 6 months-of-age. A subset of differentially regulated genes was validated by qRT-PCR.

Results: Transcriptome analysis identified distinct profiles of NP and AF gene expression that were remarkably consistent at 2.5 and 6 months-of-age. Prg4, Cilp, Ibsp and Comp were increased >50-fold in the AF relative to NP. The most highly enriched NP genes included Dsc3 and Cdh6, members of the cadherin superfamily, and microRNAs mir218-1 and mir490. Changes in the NP between 2.5 and 6 months-of-age were associated with up-regulation of molecular functions linked to laminin and Bmp receptor binding (including up-regulation of Bmp5 & 7), with the most up-regulated genes being Mir703, Shh, and Sfrp5. NP degeneration was associated with molecular functions linked to alpha-actinin binding (including up-regulation of Ttn & Myot) and cytoskeletal protein binding, with the overall most up-regulated genes being Rnu3a, Snora2b and Mir669h.

Conclusions: This study provided insight into the phenotypes of NP and AF cells, and identified candidate pathways that may regulate degeneration.

KEYWORDS:

• Nucleus pulposus
• annulus fibrosus
• intervertebral disc
• transcriptional profiling
• bioinformatics

Acknowledgments

We thank David Carter and the London Regional Genomics Centre for expertise and guidance with microarrays and bioinformatic analyses using Partek Genomics Suite. We also thank Diana Quinonez for assistance with animal breeding and genotyping. We also thank Khader Zimmo for his contributions to qPCR primer design and optimization.

Author contributions

All authors were involved in drafting the article or revising it critically for content, and all authors approved the final version. Dr. Séguin had access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of its analysis.

Study conception and design: M.R.M., and C.A.S.

Acquisition of data: M.R.M., M.A.V., and N.A.T.

Analysis and interpretation of data: M.R.M., M.A.V., and C.A.S.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was funded by the Natural Sciences and Engineering Research Council of Canada [371546] to C.A.S. Infrastructure was provided in part by the Canada Foundation for Innovation-Leaders Opportunity Fund [25086 to C.A.S]. M.R.M. was supported by a Canadian Institutes of Health Research (CIHR) Doctoral Award. M.R.M., and N.A.T. were supported in part by the Joint Motion Program - A CIHR Training Program in Musculoskeletal Health Research and Leadership. M.A.V. is supported by a Collaborative Program in Musculoskeletal Health Research Transdisciplinary Training Award, an Ontario Graduate Scholarship, and a scholarship from The Arthritis Society. C.A.S. is the recipient of a CIHR New Investigator Award and an Early Researcher Award from the Ontario Ministry of Research and Innovation.