Basic ScienceSenescence mechanisms of nucleus pulposus chondrocytes in human intervertebral discs
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.