Chondrocyte viability decreases with cartilage injury, aging and disease (3
). In osteoarthritic cartilage there have been several reports of chondrocytes exhibiting classical signs of apoptosis (6
). Since chondrocytes are essential for maintaining the integrity of the cartilage extracellular matrix, thus enabling normal joint function, identifying the cellular mechanisms that control cell survival could be an important step in developing treatments to prevent cartilage loss.
Our results demonstrate for the first time that lamin A is upregulated in OA chondrocytes, where it inhibits mitochondrial function, lowers cellular ATP levels, promotes senescence, and induces apoptosis. Lamin A is induced by PGE2
, a mediator produced in significant amounts by OA chondrocytes (23
). Furthermore, our results highlight a potential role for lamin A in premature aging and (apoptosis) senescence of chondrocytes associated with osteoarthritis. Lamin A-mediated overexpression led to apoptosis, evidenced by mitochondrial depolarization, caspase activation, decreased cellular ATP and increased cytosolic cytochrome C. These effects were partially reversed following FTI treatment, suggesting that farneyslation of lamin A is necessary for induction of apoptosis. Recent studies have also shown that treatment with FTIs can reverse the nuclear morphology defects in cells expressing the truncated lamin A variant in HGPS (24
We have previously shown that increased production of PGE2
is associated with chondrocyte apoptosis (21
). Our current study provides strong supporting evidence that addition of PGE2
leads to increased lamin A expression, subsequently leading to change in mitochondrial function and cell death. PGE2
-induced expression of lamin A was confirmed at gene expression level (qPCR), by immunostaining (confocal microscopy), and by immunoblot analysis. PGE2
mediates catabolic effects in OA chondrocytes via EP4 receptor (20
), and the current study supports this observation, as we have shown that PGE2
, via the EP2/EP4 receptor, increased lamin A and not B1 lamin expression. In contrast, IL-1 was found to inhibit, rather than stimulate, lamin A expression in chondrocytes. This result was somewhat unexpected, since we have shown that IL-1β is known to stimulate PGE2
in chondrocytes and promote apoptosis (21
). These results suggest that lamin A expression is suppressed by downstream components of IL-1β signaling, independently of PGE2
; however, the extent to which lamin A is required for IL-1β induced apoptosis was not examined in this study.
The decreased level of lamin A in IL-1-stimulated chondrocytes may occur due to activation of multiple pathways: 1) IL-1-induced mitochondrial dysfunction may lead to NLRP3 inflammasome-mediated caspase activation which leads to lamin degradation (27
); 2) increased reactive oxygen or nitrogen species generated by IL-1 treatment activate caspases (28
); 3) lamins are substrate for IL-1-induced caspase activity; and 4) IL-1 has also been shown to induce carboxymethylation of lamin and alters subnuclear distribution and degradation (29
Mitochondria are involved in many cellular processes, and mitochondrial dysfunction has been associated with apoptosis, aging, and a number of pathological conditions including osteoarthritis (8
). Mitochondrial dysfunction due to disruption of mitochondrial membrane potential may lead to senescence and apoptosis of cells via a cascade of signals including changes in ATP levels, release of cytochrome C into the cytosol, and activation of caspase 3 (21
). Lamin A overexpression in chondrocytes effected the activation of these classical pathways, culminating in cell death by apoptosis. Similar observations have been reported in fibroblasts isolated from patients with LMNA
mutations, which demonstrate mitochondrial respiratory chain protein obliteration, and changes in mitochondrial membrane potential (32
Similarly, it has been shown that human skin fibroblasts isolated from lipodystrophy syndrome patients with LMNA
mutations have increased p16 and p21 expression, leading to senescence with increasing cellular passages (32
). Increased expression of cyclin-dependent kinase inhibitors (CDK inhibitors) p21 and p16 has been associated with inhibition of cell cycle arrest or senescence in many cell types (33
). Lamin A-overexpressing chondrocytes had elevated levels of p21 but not p16 at 24 h post transfection as compared to control or vector transfected cells. It is further noted that at onset of early phase of senescence, p21 expression is increased, and in the later phase p16 is increased dramatically, which is involved in the inhibition of Rb kinases leading to cell arrest (33
). Furthermore, Zhou et al (36
) have previously reported that knockdown of p16 by siRNA contributed to the recovery of chondrocytes with increased proliferation and overall increased repair capacity. In our studies, overexpression of lamin A increased p21 levels compared to transfected controls, accompanied by decreased proliferative capacity as determined by BrdU incorporation.
Thus, our data show that minor perturbations in the expression of lamin A led to cellular senescence, decreased cellular energy stores and apoptosis. These results further demonstrate that lamin A accumulation/metabolism is not limited to its role in the development of accelerated aging observed in laminopathy syndromes, but that overexpression of the normal lamin A protein in disease may also play a role in the premature aging of chondrocytes in osteoarthritis. It is of further interest to note that senescence (cell cycle arrest) may also lead to hypertrophic cell phenotype, decreased proliferation, decreased response to growth factors, dysregulated gene expression, and aging of cartilage tissue (8
). Since the molecular pathology of several laminopathies resembles an accelerated form of normal ageing, it is intriguing to draw a parallel between alteration in lamin A function, senescence and apoptosis in chondrocytes.