Progressive IVD degeneration induced by WBV was associated with increased expression of Il-1β within the IVD that preceded Mmp and Adamts gene induction. Moreover, WBV-induced IVD degeneration is not reversed following cessation of vibration.
Mice lacking equilibrative nucleoside transporter 1 (ENT1
−/−) demonstrate progressive calcification of spinal tissues including the annulus fibrosus (AF) of the intervertebral disc (IVD). We previously established ENT1 as the primary nucleoside transporter in the AF and demonstrated dysregulation of biomineralization pathways. To identify cellular pathways altered by loss of ENT1, we conducted microarray analysis of AF tissue from wild‐type (WT) and ENT1
−/− mice before calcification (2 months of age) and associated with calcification (6 months of age). Bioinformatic analyses identified cell cycle dysregulation in ENT1
−/− AF tissues and implicated the E2f family of transcription factors as potential effectors. Quantitative polymerase chain reaction analysis confirmed increased expression of multiple E2f transcription factors and E2f interacting proteins (
Rb1 and Cdk2) in ENT1
−/− AF cells compared with WT at 6 months of age. At this time point, ENT1
−/− AF tissues showed increased JNK MAPK pathway activation, CDK1, minichromosome maintenance complex component 5 (Mcm5), and proliferating cell nuclear antigen (PCNA) protein expression, and PCNA‐positive proliferating cells compared with WT controls. The current study demonstrates that loss of ENT1‐mediated adenosine transport leads to increased cell proliferation in the AF of the IVD.
The comprehensiveness of data collected by "omics" modalities has demonstrated the ability to drastically transform our understanding of the molecular mechanisms of chronic, complex diseases such as musculoskeletal pathologies, how biomarkers are identified, and how therapeutic targets are developed. Standardization of protocols will enable comparisons between findings reported by multiple research groups and move the application of these technologies forward. Herein, we describe a protocol for parallel proteomic and metabolomic analysis of mouse intervertebral disc (IVD) tissues, building from the combined expertise of our collaborative team. This protocol covers dissection of murine IVD tissues, sample isolation, and data analysis for both proteomics and metabolomics applications. The protocol presented below was optimized to maximize the utility of a mouse model for "omics" applications, accounting for the challenges associated with the small starting quantity of sample due to small tissue size as well as the extracellular matrix-rich nature of the tissue.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.