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NFAT1 Deficiency Causes Chondrocyte Dysfunction and Ectopic Ossification in Murine Intervertebral joints
Yi Feng1, Qinghua Lu1, Brian M. Egan1, Kenneth D. Brandt1, Jinxi Wang1
1
University of Kansas Medical Center, Kansas City, KS, USA
Disclosures: The authors declare that they have no competing interests.
INTRODUCTION: Our previous studies demonstrated that adult mice with deletion of NFAT1 transcription factor displayed osteoarthritis (OA) in
synovial joints, including cartilage fibrillation, synovial reaction, osteophyte formation, and subchondral bone changes [1]. The present study aimed to
investigate whether NFAT1 deficiency affects cellular function, tissue metabolism, and development of intervertebral disc degeneration (IDD) in mice.
METHODS: Nfat1-/- and wild-type mice (both sexes) were used for various analyses at 2, 6, 12, and 18 months of age. All animal procedures were
performed with the approval of the Institutional Animal Care and Use Committee (IACUC) in compliance with all federal and state laws and regulations.
Lumbar intervertebral discs (IVDs) were isolated for quantitative gene expression analysis by qRT-PCR. Lumbar spines were processed for histochemistry
and immunohistochemistry (IHC). Whole-body radiographs were taken to assess radiographic changes in non-synovial intervertebral joints and appendicular
synovial joints. Degenerative changes in disc height and disc bulge were quantitatively analyzed by micromeasurements. Quantitative data (4 animal per
strain/time point for qRT-PCR and 6 animals per strain/time point for other analyses) were presented as the mean ± 95% confidence intervals. The difference
between means from two groups was analyzed by Student’s t-test; the difference between means for three or more groups was assessed by one-way ANOVA.
A p-value of less than 0.05 was considered statistically significant.
RESULTS: Gene expression analyses demonstrated no significant differences in expression of anabolic and catabolic genes between wild-type and Nfat1-/mice at 2 and 6 months. The expression levels of anabolic genes Acan, Col2a1, and Sox9 were significantly decreased in Nfat1-/- IVDs at 12 and 18 months,
compared to wild-type IVDs. However, no significant differences in expression of major catabolic genes were detected between wild-type and Nfat1-/- IVDs
at 12 and 18 months (Figure1). Histochemistry and IHC revealed age-dependent reduction of safranin-O stained proteoglycans in the Nfat1-/- nucleus
pulposus, but not in the growth plate-like cartilaginous tissue (Figure 2A) and reduced collagen-2 protein expression in the Nfat1-/- endplates (Figure 2B).
Ectopic ossification was detected in the Nfat1-/- endplates. However, no significant differences in degenerative structural changes (e.g. reduced disc height,
disc bulge, and spondylophytes) were observed between wild-type and Nfat1-/- intervertebral joints at any age groups. No gender differences were observed.
DISCUSSION: NFAT1 deficiency causes chondrocyte dysfunction and ectopic ossification in the endplates; it also compromises the synthesis of aggrecan
in the nucleus pulposus, but not in the growth plate-like cartilaginous tissue. However, Nfat1 deficiency does not cause typical osteoarthritic changes (e.g.
loss of cartilage, osteophytes, and subchondral bone sclerosis) or accelerate degenerative structural changes in murine intervertebral joints which lack
synovium. The data suggest that synovial reaction plays an important role in the development of NFAT1 deficiency-induced OA in the synovial joints.
SIGNIFICANCE: The results from this study has identified the role NFAT1 in maintaining the homeostasis of specific tissues (endplate and nucleus
pulposus) of murine intervertebral joints. The data also highlight the importance of synovial reaction for the initiation and progression of osteoarthritis.
REFERENCES: 1. Wang, J. et al. J Pathol 2009; 219:163-72.
ACKNOWLEDGEMENTS: Research reported in this article was supported in part by the NIH/NIAMS Award Number R01 AR059088 (to J. Wang
Figure 1. qRT-PCR analyses of
anabolic and catabolic gene
expression in IVDs of wild-type
and Nfat1-1- mice at 12 and 18
months. Figure 2. (A) Safranin-O stained
proteoglycans (red) are severely
reduced in the nucleus pulposus
of Nfat1-1- mice, with ectopic
ossification spots in the end plate
(white arrows) at 18 months of
age. (B) IHC reveals focal
reduction of collagen-2 protein
expression in the endplate of an
18-month-old Nfat1-1- mouse. ORS 2017 Annual Meeting Poster No.1864