Senescence mechanisms of nucleus pulposus chondrocytes in human intervertebral discs

• Published in: The spine journal Vol. 9; no. 8; pp. 658 - 666
• Main Authors: Kim, Ki-Won, MD, Chung, Ha-Na, BS, Ha, Kee-Yong, MD, Lee, Jun-Seok, MD, Kim, Young-Yul, MD
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2009
• Subjects: Adult; Aged; beta-Galactosidase - metabolism; Blotting, Western; Cells, Cultured; Cellular Senescence - physiology; Chondrocytes; Chondrocytes - metabolism; Chondrocytes - pathology; Disc degeneration; Female; Humans; Hydrogen Peroxide - metabolism; Immunohistochemistry; Intervertebral disc; Intervertebral Disc - metabolism; Intervertebral Disc - pathology; Intervertebral Disc Displacement - metabolism; Intervertebral Disc Displacement - pathology; Male; Middle Aged; Nucleus pulposus; Orthopedics; Senescence; Telomerase - metabolism; Telomere - metabolism; Telomere - pathology; Senescence; Nucleus pulposus; Disc degeneration; Intervertebral disc; Chondrocytes
• ISSN: 1529-9430; 1878-1632
• DOI: 10.1016/j.spinee.2009.04.018

Abstract Background context The population of senescent disc cells has been shown to increase in degenerated or herniated discs. However, the mechanism and signaling pathway involved in the senescence of nucleus pulposus (NP) chondrocytes are unknown. Purpose To demonstrate the mechanisms involved in the senescence of NP chondrocytes. Study design/setting Senescence-related markers were assessed in the surgically obtained human NP specimens. Patient sample NP specimens remaining in the central region of the intervertebral disc were obtained from 25 patients (mean: 49 years, range: 20–75 years) undergoing discectomy. Based on the preoperative magnetic resonance images, there were 3 patients with Grade II degeneration, 17 patients with Grade III degeneration, and 5 patients with Grade IV degeneration. Outcome measures We examined cell senescence markers (senescence-associated β-galactosidase [SA-β-gal], telomere length, telomerase activity, p53, p21, pRB, and p16) and the hydrogen peroxide (H2 O2 ) content as a marker for an oxidative stress in the human NP specimens. Methods SA-β-gal expression, telomere length, telomerase activity, and H2 O2 content as well as their relationships with age and degeneration grades were analyzed. For the mechanism involved in the senescence of NP chondrocytes, expressions of p53, p21, pRB, and p16 in these cells were assessed with immunohistochemistry and Western blotting. Results The percentages of SA-β-gal-positive NP chondrocytes increased with age (r=.82, p<.001), whereas the telomere length and telomerase activity declined (r=−.41, p=.045; r=−.52, p=.008, respectively) However, there was no significant correlation between age and H2 O2 contents (p=.18). The NP specimens with Grade III or Grade IV degeneration showed significantly higher percentages of SA-β-gal-positive NP chondrocytes than those with Grade II degeneration (p=.01 and p=.025, respectively). Immunohistochemistry showed that the senescent NP chondrocytes in all the specimens expressed p53, p21, and pRB, but a few NP chondrocytes in only two specimens expressed p16. Western blotting showed that the expressions of p53, p21, and pRB displayed a corresponding pattern, that is, a strong p53 expression led to strong p21 and pRB expressions and vice versa. Conclusions Our in vivo study demonstrated that senescent NP chondrocytes increased or accumulated in the NP with increasing age and advancing disc degeneration. The NP chondrocytes in the aging discs exhibited characteristic senescent features such as an increased SA-β-gal expression, shortened telomeres, and decreased telomerase activity. We further demonstrated that the telomere-based p53-p21-pRB pathway, rather than the stress-based p16-pRB pathway, plays a more important role in the senescence of NP chondrocytes in an in vivo condition. Our results suggest that prevention or reversal of the senescence of NP chondrocytes can be a novel therapeutic target for human disc degeneration.