Loss of ENT1 increases cell proliferation in the annulus fibrosus of the intervertebral disc

Abstract

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.

Chronic stress and antidepressant treatment alter purine metabolism and beta oxidation within mouse brain and serum

“…Since tissues from multiple mice were pooled, we are unable to correlate molecular profiles with individual animal behavior. Pooling tissue is an approach used by other groups to perform parallel analyses on limited starting tissues 52 . As the sensitivity of OMIC analyses improves, studies of tissues from individual animals, or even specific cell-types, should be performed to overcome the limitations of the present work.…”

Genetic murine models of spinal development and degeneration provide valuable insights into intervertebral disc pathobiology

“…Mice lacking ENT1, a model of diffuse idiopathic skeletal hyperostosis, show reduced expression of ANK, ENPP1, and ALPL in discs, increased AF cell proliferation, and progressive ectopic calcification of fibrous connective tissues along the spine (Warraich et al, 2013). Analyses of AF tissue isolated from ENT1 knockout mice has further revealed that the observed increased cell proliferation is associated with an upregulation of E2F transcription factors, the cell cycle regulators RB1 and CDK2, and stimulation of the JNK/MAPK pathway (Veras et al, 2019). Notably, the importance of genetic background in the ectopic calcification process has been recently demonstrated by the study of LG/J inbred mice, which show an agedependent increase in dystrophic mineralisation of discs (this mouse strain will be discussed later in the review) (Novais et al, 2020).…”

Protocol for parallel proteomic and metabolomic analysis of mouse intervertebral disc tissues

“…We have explored the effect of mechanical loading on the IVD in mice [42][43][44] and used transgenic mice to study IVD development, 45 disc degeneration, 16,46 and diffuse idiopathic skeletal hyperostosis (DISH). [47][48][49][50] Important insights have likewise been provided by others using mouse models to study disc development, [51][52][53] inflammation, 54 IVD degeneration, 10,13,[55][56][57][58] calcification, 59,60 and scoliosis. [61][62][63] The multitude of available mouse models of spine pathologies allows for global molecular comparisons to uncover novel biological insights.…”

“…Proteomics has also been used in mice for global characterization of the healthy IVD, 73 to examine the response to mechanical loading, 42 to characterize different mouse strains, 55 and to investigate ectopic calcification in the IVD. 48 To date, there have been no metabolomic studies of murine IVD tissues, and the analysis of human IVD tissues is limited to a single unbiased metabolite screen using high-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR), 74 which is much less sensitive than MS based metabolomics. 75 Despite the limitations to the starting quantity of sample associated with the small size of the mouse, it is possible to gain novel insights into mechanisms, biomarkers and therapeutic targets of IVD pathologies using optimized protocols for proteomic and metabolomic analyses.…”

“…86 These studies led to the development of the current protocol for label-free quantitative proteomics of murine IVD tissue. 48 Our group has also used metabolomics to develop biomarkers of chronic kidney disease, 87 muscle response to exercise in diabetes, 88 kidney function, 89 and characterize the association of the microbiome to atherosclerosis. 90 The metabolomics methods used previously to assess kidney and muscle tissues required minimal adaptation for use with murine IVD tissue.…”