Aging aggravates intervertebral disc degeneration by regulating transcription factors toward chondrogenesis

Abstract

Osterix is a critical transcription factor of mesenchymal stem cell fate, where its loss or loss of Wnt signaling diverts differentiation to a chondrocytic lineage. Intervertebral disc (IVD) degeneration activates the differentiation of prehypertrophic chondrocyte-like cells and inactivates Wnt signaling, but its interactive role with osterix is unclear. First, compared to young-adult (5 mo), mechanical compression of old (18 mo) IVD induced greater IVD degeneration. Aging (5 vs 12 mo) and/or compression reduced the transcription of osterix and notochordal marker T by 40-75%. Compression elevated the transcription of hypertrophic chondrocyte marker MMP13 and pre-osterix transcription factor RUNX2, but less so in 12 mo IVD. Next, using an Ai9/td reporter and immunohistochemical staining, annulus fibrosus and nucleus pulposus cells of young-adult IVD expressed osterix, but aging and compression reduced its expression. Lastly, in vivo LRP5-deficiency in osterix-expressing cells inactivated Wnt signaling in the nucleus pulposus by 95%, degenerated the IVD to levels similar to aging and compression, reduced the biomechanical properties by 45-70%, and reduced the transcription of osterix, notochordal markers and chondrocytic markers by 60-80%. Overall, these data indicate that age-related inactivation of Wnt signaling in osterix-expressing cells may limit regeneration by depleting the progenitors and attenuating the expansion of chondrocyte-like cells.

Keywords: Wnt/β-catenin/LRPs; biomechanics; genetic animal models; osterix.

Figure 1.

Mouse intervertebral disc (IVD) degeneration was scored histologically on a 0–14 scale. Individual scores are noted for each representative image. (A) Mouse intervertebral disc (IVD) degeneration increased with aging in the lumbar region. (B) Mechanical injury by tail compression (n=5, age) or puncture (n=3) induce IVD degeneration in 5 mo and 18 mo mice. (C) Quantification of the tail IVD degeneration. The box plots show the median score (line), interquartile value and the whiskers are the min/max values. Scale bar: 100 μm. *: p<0.05.

Figure 2.

QPCR of 5 mo (n=5) and 12 mo (n=6) IVD subjected to tail compression. The relative gene expression in each loaded and control intervertebral disc was determined by normalizing to housekeeping gene IPO8 (CT value of 30) and then normalized to the control intervertebral disc. Mean+SD. *: Main effect of loading, #: main effect of aging, p<0.05. ~:0<0.1

Figure 3.

(A) Immunohistochemistry staining for osterix and counterstained with Safranin-O of tail and lumbar IVD. (A’) Magnification of the lumbar IVD. (B) Osterix staining of the NP of 5 mo IVD subjected to tail compression (n=3). (C) Magnification of the NP and rotated by 90° clockwise. Solid brown arrows denote cells with small cell nuclei (relative to cell size) stained with a high-intensity of osterix expression (Dark), empty brown arrows denote cells with large cell nuclei stained with a low-intensity of osterix expression (Light) and white arrows denote cells with cell nuclei stained with no osterix expression (No Stain). (D) Fraction of the NP cell population stained dark, light or not stained for osterix. Mean+SD. Scale bar for A, B: 100 μm; A’:25 μm, and C: 12.5 μm. CB: cortical bone, CEP: cartilage endplate, GP: growth plate, NP: nucleus pulposus, OAF: outer annulus fibrosus, TB: trabecular bone. *: p<0.05.

Figure 4.

(A) Merged DAPI (blue) and immunofluorescence of osterix expression of tail IVD from 5 mo and 12 mo OsxCreERT2/tdT mice. Immunofluorescence of osterix expression of (B) IVD from 5 mo and 12 mo OsxCreERT2/td mice. (C) IVD of 5 and 12 mo td mice without OsxCreERT2. AF: annulus fibrosus, CB: cortical bone, CEP: cartilage endplate, NP: nucleus pulposus, OAF: outer annulus fibrosus, TB: trabecular bone. Scale bar: 100 μm.

Figure 5.

From WT (n=6) and LRP5 cKO (n=6) mice, (A) LacZ staining for WNT signaling (blue arrow head), quantification of WNT signaling, Safranin-O/Fast green staining and histological scoring of (A-D) lumbar and (E-H) tail IVD. Mean+SD. Scale bar: 100 μm. *: p<0.05.

Figure 6.

(A) Force/Displacement curves of control (WT, n=5) and LRP5 cKO (n=5) tail IVD. (B) Stiffness in compression, neutral zone and tension of WT and LRP5 cKO IVD. Mean+SD. *: p<0.05.

Figure 7.

QPCR of (A) lumbar and (B) tail IVD from WT (n=5) and LRP5 cKO (n=5) mice. (C) LacZ staining for WNT signaling (blue arrow) and immunohistochemical staining for osterix (brown arrow) from WT and LRP5 cKO tail IVD. Cells stained for both are brown with blue lining. Mean+SD. Scale bar: 50 μm *: p<0.05.

Figure 8.

(A) Proposed role between chondrocyte-like (CLC) and notochordal (NC1) cells during optimal regeneration, suboptimal regeneration and IVD degeneration. NC1 cells have high WNT signaling and no osterix. IVD degeneration is characterized by a loss (X’s) of both cells. (B) During suboptimal regeneration, progenitor cells with high osterix expression gain WNT signaling to become NC2 and finally become CLC by losing osterix and WNT signaling. Aging limits differentiation of progenitors to CLC through NC2 and promotes it directly, which is suboptimal because fewer NC cells become involved.