Elsevier

The Spine Journal

Volume 9, Issue 8, August 2009, Pages 658-666
The Spine Journal

Basic Science
Senescence mechanisms of nucleus pulposus chondrocytes in human intervertebral discs

https://doi.org/10.1016/j.spinee.2009.04.018Get rights and content

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 (H2O2) content as a marker for an oxidative stress in the human NP specimens.

Methods

SA-β-gal expression, telomere length, telomerase activity, and H2O2 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 H2O2 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.

Introduction

Cellular senescence is a program activated by normal cells in response to various types of stress [1]. One important mechanism responsible for cellular senescence is the progressive telomere shortening and eventual telomere dysfunction that occur as a result of incomplete DNA replication (an end-replication problem) at the telomeres (“replicative senescence” or “intrinsic senescence”) [1], [2], [3], [4], [5], [6]. This end-replication problem can be resolved by a holoenzyme telomerase, which elongates the telomeric DNA in the 5′-to-3′ direction [6], [7], [8], [9], [10], [11]. In the absence of telomerase or when its expression levels are very low, the telomeric DNA progressively shortens with each round of cell division [12]. In addition to the replicative senescence, cellular senescence can also be induced in a rapid manner by a number of stresses that are independent of telomere shortening (“stress-induced premature senescence” or “stress or aberrant signaling-induced senescence”) [13], [14]. Such stresses include oxidative stress, DNA damage, oncogenic activity, and other metabolic perturbations [15].

Cellular senescence such as apoptosis can be viewed as a powerful tumor-suppressor mechanism that withdraws cells with irreparable DNA damages from the cell cycle [16], [17]. Therefore, the senescence signals, that is, a telomere-based one or a stress-based one, trigger a DNA damage response and this response shares a common signaling pathway that converges on either or both of the well-established two tumor-suppressor proteins, p53 (the p53-p21-pRB pathway) and pRB proteins (the p16-pRB pathway) [1], [14], [15], [18], [19], [20]. In the p53-p21-pRB pathway, senescence stimuli activate the p53, which then can induce senescence by activating pRB through p21, which is a transcriptional target of p53. This senescence can be reversed upon subsequent inactivation of p53. In the p16-pRB pathway, senescence stimuli induce p16, which activates pRB. Once the pRB pathway is engaged by p16, the senescence cannot be reversed by subsequent inactivation of p53, silencing of p16 or inactivation of pRB [18]. Although there appears to be overlap between the two pathways, the emerging consensus is that the p53-p21-pRB pathway mediates the senescence that is primarily because of telomere shortening and the p16-pRB pathway is thought to mediate premature senescence [1], [14], [20]. However, a population of growing cells suffers from a combination of various physiologic stresses that act simultaneously, and the relative importance of the p53-p21-pRB or p16-pRB pathway for the senescence response may differ depending on the tissue and the species of origin [1], [20]. Once cells have entered senescence, they are arrested in the G1 phase of the cell cycle and they display a characteristic morphology (vacuolated, flattened cells) and gene expression, including markers such as a senescence-associated β-galactosidase (SA-β-gal) [21], [22].

Degenerative changes of the intervertebral disc (IVD) occur as a natural part of aging [23]. Gruber et al. [24] and Roberts et al. [25] recently provided important insights regarding the close link between cellular senescence and disc degeneration based on the observations that SA-β-gal-positive disc cells increased with the increasing disc degeneration or they increased in specimens of herniated disc. However, the mechanism and signaling pathways involved in the senescence of the nucleus pulposus (NP) chondrocytes are unknown. We hypothesized that with increasing age and advancing disc degeneration, senescent NP chondrocytes might be increased or accumulated in the NP. To demonstrate the mechanisms involved in the senescence of the NP chondrocytes, we examined cell senescence markers (SA-β-gal, telomere length, telomerase activity, p53, p21, pRB, and p16) and the hydrogen peroxide (H2O2) content as a marker for an oxidative stress in the human NP specimens.

Section snippets

Materials and methods

Twenty-five patients (14 female and 11 male) who underwent open discectomy for symptomatic herniated NP were included in this study. After a thorough removal of the protruded or extruded NP fragments, the NP specimens remaining in the central part of the IVD were pooled, and then they were immediately preserved at −75°C. The NP specimens were grouped according to a grading system for IVD degeneration that was based on the preoperative magnetic resonance images [26]: there were 3 patients with

Results

The Kolmogorov-Smirnov test showed that among the variables (age, percentages of SA-β-gal-positive NP chondrocytes, telomere length, telomerase activity, and the H2O2 content), age and the percentage of SA-β-gal-positive NP chondrocytes showed a normal distribution.

Discussion

In general, senescent cells become unresponsive to mitogenic stimuli, yet they can remain viable for extended periods of time [21]. They also express elevated levels of the extracellular matrix-degrading proteases, collagenase, and the matrix metalloproteinase family members [20], [21]. In contrast, they express decreased levels of the matrix metalloproteinase inhibitor TIMP1 and decreased levels of extracellular matrix components such as elastin, laminin, and several forms of collagen [20],

Acknowledgments

This article in part has been published in the Asian Spine Journal (2008;2:1–8), which is currently circulated only in Korea, and is unavailable online.

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    FDA device/drug status: not applicable.

    Author disclosures: none.

    This study was supported in part by the Catholic Medical Research Foundation.

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