Senescence mechanisms of nucleus pulposus chondrocytes in human intervertebral discs.
BACKGROUND
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.


OBJECTIVE
To demonstrate the mechanisms involved in the senescence of NP chondrocytes.


METHODS
Senescence-related markers were assessed in the surgically obtained human NP specimens.


METHODS
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.


METHODS
We examined cell senescence markers (senescence-associated beta-galactosidase [SA-beta-gal], telomere length, telomerase activity, p53, p21, pRB, and p16) and the hydrogen peroxide (H(2)O(2)) content as a marker for an oxidative stress in the human NP specimens.


METHODS
SA-beta-gal expression, telomere length, telomerase activity, and H(2)O(2) 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-beta-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 H(2)O(2) contents (p=.18). The NP specimens with Grade III or Grade IV degeneration showed significantly higher percentages of SA-beta-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-beta-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.
Kim
Ki-Won
KW
Department of Orthopedic Surgery, St. Mary's Hospital, The Catholic University of Korea, 62 Yoido-dong, Youngdeungpo-ku, Seoul, Republic of Korea. kiwonk62@yahoo.co.kr
Chung
Ha-Na
HN
Ha
Kee-Yong
KY
Lee
Jun-Seok
JS
Kim
Young-Yul
YY
D000328:Adult; D000368:Aged; D015153:Blotting, Western; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D019902:Chondrocytes / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007150:Immunohistochemistry; D007403:Intervertebral Disc / Q000378:metabolism* / Q000473:pathology; D007405:Intervertebral Disc Displacement / Q000378:metabolism / Q000473:pathology; D008297:Male; D008875:Middle Aged; D019098:Telomerase / Q000378:metabolism; D016615:Telomere / Q000378:metabolism / Q000473:pathology; D001616:beta-Galactosidase / Q000378:metabolism
D006861:Hydrogen Peroxide; D019098:Telomerase; D001616:beta-Galactosidase