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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Arthritis Rheum. Author manuscript; available in PMC Jun 1, 2013.
Published in final edited form as:
PMCID: PMC3348367
NIHMSID: NIHMS347791

Perturbation of nuclear lamin A causes cell death in chondrocytes

Abstract

Objective

Mutations in LMNA encoding the A-type lamins cause several diseases including those with features of premature aging and skeletal abnormalities. We therefore examined the expression of lamin A in cartilage from humans with osteoarthritis (OA) and studied the effects of its overexpression on chondrocyte senescence and apoptosis.

Methods

Human chondrocyte-like cells (SW1353) were utilized in this study. RNA isolated from human OA and non-OA cartilages were used for profiling mRNA expression using Affymetrix microarray. Effects of lamin A overexpression on mitochondrial function and apoptosis were examined by assessing mitochondrial membrane potential, ATP levels, cytochrome C release and TUNEL assay. Western blots were performed for protein expression.

Results

Lamin A expression was markedly elevated in OA cartilage samples compared with non-OA controls. Western analysis confirmed elevated levels of lamin A expression in OA compared to non-OA cartilage. IL-1β treatment inhibited, whereas PGE2 caused a marked increase in lamin A accumulation. These effects of exogenous PGE2 on lamin A expression were mediated via EP2/4 receptor. Transfected chondrocytes that expressed lamin A displayed markers of early senescence/apoptosis.

Conclusion

Our results suggest that lamin A is upregulated in OA chondrocytes, and increased nuclear accumulation of lamin A in response to catabolic stress may account for the premature aging phenotype and apoptosis of chondrocytes in OA.

Keywords: lamin, senescence, mitochondria, chondrocytes, osteoarthritis

INTRODUCTION

Osteoarthritis (OA) is an age-related disease characterized by progressive loss of articular cartilage, new bone formation and, often, synovial proliferation. Over the past decade, there have been significant developments in the scientific understanding of OA, including the identification of a variety of mediators and signaling pathways that contribute to cartilage and bone remodeling (1;2). However, the molecular mechanisms that play a role in the initiation and perpetuation of arthritis are not clear.

Central to OA is the altered, catabolic phenotype of the articular chondrocyte, the single cellular component in cartilage. Therefore, understanding dysregulation of chondrocyte function, change in phenotype and altered extracellular matrix (ECM) interactions in osteoarthritis are major foci of investigation. OA chondrocytes undergo a series of complex changes, including hypertrophy, proliferation, catabolic alteration and, ultimately, death. These characteristic changes result in loss of proteoglycan and collagens due to increased production of matrix metalloproteinases (MMPs), and aggrecanases (ADAMTSs).

Since age is the risk factor most strongly correlated with OA, understanding age-related changes of cartilage is essential. With age, chondrocytes undergo decrease in mitotic and synthetic activity, exhibit decreased responsiveness to anabolic growth factors, and synthesize smaller and less uniform large aggregating proteoglycans and fewer functional link proteins (3). Age also appears to be an independent factor that predisposes articular chondrocytes to apoptosis, since the expression levels of specific pro-apoptotic genes (Fas, FasL, caspase-8, and p53) are higher in aged cartilage (4;5).

Relating the normal aging process to the molecular and biochemical events that lead to chondrocyte senescence in OA is therefore under intense investigation. A number of authors (610) have suggested that inflammatory mediators associated with OA may promote “premature” chondrocyte senescence, leading to progressive degeneration of cartilage. In particular, increasing evidence suggests that oxidative stress due to the production of reactive oxygen and nitrogen species are particularly implicated in chondrocyte senescence and cartilage aging (11).

In an effort to gain insights into the accelerated aging phenomena observed in OA, we asked whether lamin A, a nuclear envelope protein that has been implicated in progeria, could play a role in premature senescence of OA chondrocytes. Lamins are intermediate filament proteins, which form the nuclear lamina, a meshwork of intermediate filaments on the inner nuclear envelope membrane. In humans, three genes encode nuclear lamins. LMNA encodes the A-type lamins, consisting of lamin A and lamin C, the major somatic cell isoforms. The lamins provide the physical scaffolding and structural support for the nucleus and an anchor for various proteins, some of which interact with DNA. The lamins may use both direct and indirect interactions with chromatin to affect gene transcription, nuclear organization, transport of material in and out of the nucleus, cell cycle regulation and cell differentiation (12;13) Mutations in LMNA lead to inherited diseases collectively called laminopathies (14). One of these diseases is the Hutchinson-Gilford progeria syndrome (HGPS), in which the LMNA mutation leads to a defect in prelamin A processing, resulting in accumulation of a truncated, permanently farnesylated lamin A variant. This leads to accelerated aging of mesenchymal tissues and development of bone and joint abnormalities at young ages (15). Furthermore, the A-type lamins play an important role in cell responses to mechanical force (16). For these reasons, we examined the potential role of lamin A in OA. We report that lamin A is upregulated in OA cartilage, and provide evidence that increased expression causes mitochondrial dysfunction, ATP depletion and chondrocyte apoptosis.

METHODS

Reagents

All media and FBS were purchased from Life Technologies (Gaithersburg, MD). IL-1β was purchased from PeproTech (Rocky Hill, NJ) and ELISA kits from either R&D Systems (Active Caspase 3 kit), or Active motif (Cytochrome C kit). Other chemicals, EP2 receptor antagonist (AH6809), EP4 receptor antagonist (AH23848) and Chemiluminescent ATP determination kits were purchased from Sigma-Aldrich (St. Louis, MO). Mitochondrial JC-1 dye was purchased from Molecular Probes (Eugene, OR). The antibodies for western analysis were obtained from various sources including lamin A (Abcam), lamin B1 antibody, p16 and p21 (Santa Cruz Biotechnology), β-actin, catalase antibodies (Sigma).

Vectors

Complementary DNA constructs encoding lamin A and the R482Q lamin A variant have been described previously (17). The heterozygous LMNA mutation leading to the R482Q substitution in the C-terminal domain of lamins A and C causes Dunnigan-type familial partial lipodystrophy. We used R482Q constructs as a positive control because, in OA cartilage or in isolated OA chondrocytes, 4'-6-diamidino-2-phenylindole (DAPI) staining did not reveal any gross change in nuclear morphology, and overexpression of R482Q does not cause nucleoplasmic foci in contrast to other variants. However, overexpression of other variants of lamin A causes strong nuclear morphological changes.

Procurement of human cartilage

Human cartilage was obtained from the knees of patients with the diagnosis of advanced OA (age: approximately 50–85 yr and 85% female) who were undergoing knee replacement surgery, and from non-arthritic knees (normal controls: age 50–88 yr and 50% female) under the guidelines of the Institutional Review Board (IRB) of New York University School of Medicine for use of surgically discarded human tissues. Non-arthritic knee cartilage was obtained from National Disease Research Interchange (NDRI, Philadelphia, PA, USA). OA patients were free of steroidal/non-steroidal anti-inflammatory drugs for at least 2 weeks before surgery. All specimens were examined by the authors and confirmed to have gross evidence of OA (i.e., thinning of cartilage, focal eburnation and erosion and reduced proteoglycan content indicated by Safranin O staining). All specimens were histologically confirmed as OA by the pathologists at NYU Hospital for Joint Diseases.

RNA isolation from OA cartilage

Cartilage was milledinto fine powder in Freezer/Mill 6800 (CE) and total RNA was isolated as described previously (18).

Cells and cell culture

The SW 1353 cell line was isolated from a primary grade II chondrosarcoma of the right humerus obtained from a 72 year old female Caucasian (ATCC, Manassas, VA). SW1353 cells were cultured in 10 mL DMEM containing 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2 in air. The cells were subcultured at split ratios of 1:3–1:4 using trypsin-EDTA. The medium was changed every 3 days.

OA chondrocyte monolayer culture

Briefly, OA cartilage slices were minced finely and digested with collagenase for 12–16 h in Ham’s F12 medium (with 5% FBS), as described previously (19). The cell suspension was used to establish cultures in T175 flasks. Within 2–3 days of harvest, primary chondrocytes were replated at 80% confluence in 100 mm tissue culture dishes or 24-well plates for experiments.

Interleukin-1β (IL-1β) and prostaglandin E2 (PGE2) treatment

For all studies, the cultures were adapted to serum-free conditions with overnight incubation in Ham’s F12 medium containing 0.2% endotoxin-free human albumin. Cultures were then either left untreated or incubated with IL-1β (10 ng/ml) or PGE2 (10 μM) for the remainder of the experiment. EP2 receptor antagonist (AH6809), EP4 receptor antagonist (AH23848) were used at 10μM concentration to block PGE2 activity based on our previous studies (20).

Western analysis

Proteins were extracted from monolayer chondrocytes using M-PER reagent (Pierce, Rockland, IL). Protein concentrations were estimated using BCA reagent (Pierce) and 20–100 μg total proteins were electrophoresed on a SDS-PAGE gel using the Bio-Rad electrophoresis system. Proteins were transferred to nitrocellulose (1 h at 100 V) and blots probed with a mouse anti-lamin A monoclonal antibody (Abcam) and α-tubulin/β-actin/catalase monoclonal antibody to correct for variations in sample loading. Blots were developed with an ECL detection kit, and were quantitated by scanning and determining pixel counts using Image J 1.43 (NIH). Raw intensity data of lamin A were normalized to the internal protein control catalase/β-actin and plotted in bar graphs.

Transfection of human chondrocytes

The cells were grown in DMEM complete medium (10% FBS) in 24-well plates with 200,000 cells/well for 4–6 h before transfection. The cells were transferred to OPTI-MEM medium 1–3 h before transfection. The transfection with Trans-LT1 reagent was carried out according to the manufacturer’s protocol (Mirus Bio LLC).

Proliferation assay

Bromodeoxyuridine (BrdU) uptake was used to compare the proliferation between control and chondrocytes overexpressing lamin A using BrdU cell proliferation kit (EMD Biosciences). After 24 or 48 h post transfection, the cells were incubated at 37°C overnight with BrdU, as per the manufacturer’s protocol. The cells were then washed with growth medium and fixed with formalin, and BrdU incorporation was measured spectrophotometrically at 450–540 nm. The extent of proliferation was expressed as % BrdU uptake as compared to control.

Chemiluminescence ATP determination

The levels of intracellular ATP were determined by using the Bioluminescent Somatic Cell Assay Kit (Sigma-Aldrich) according to the manufacturer’s protocol and as reported previously (21). The amount of ATP released was measured as nmoles/106 cells.

Active caspase 3 assay

Apoptosis of cells as a result of lamin A overexpression was determined quantitatively by measuring active caspase 3 in cell lysates using an ELISA kit (R&D Systems) according to the manufacturer’s protocol. Active caspase 3 was measured and expressed as ng/mg total protein. Caspase 3-specific inhibitor (Ac-DEVD-CHO) and caspase-negative inhibitor (Z-FA-FMK) were obtained from BD Biosciences.

Mitochondrial staining

The mitochondrial membrane potential changes were observed using the fluorescent dye JC-1 (Molecular Probes) as reported previously (21). Cells were visualized using a florescence microscope (IX71, Olympus, software: IPlab).

Measurement of cytochrome C

Monolayer cells were harvested 24 h after treatment, and mitochondrial and cytosolic fractions were isolated using the Mitochondrial Fractionation Kit (Active Motif) according to the manufacturer’s instructions. Cytochrome C levels (μg/mg protein) were determined from purified fractions using an ELISA (Active Motif).

TUNEL staining

TdT terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) was used to evaluate apoptosis. SW1353 cells were seeded at a density of 5 × 105 cells/well into 6-well microplates. The following day, cells were transfected with lamin A and control vector (pCDNA4) using TransIT-LT reagent (Mirus Bio LLC, Madison, WI) as described in the methods. The cells were fixed with 4% paraformaldehyde and processed for the TUNEL assay using an in situ cell death detection kit (Click-iT), as recommended by the supplier (Invitrogen, Carlsbad, CA).

Immunofluorescence microscopy

Cells were washed twice with PBS and fixed with 500 μL of IC Fixation buffer containing paraformaldehyde (eBiosciences) for 15 min at room temperature. After fixation, the cells were serially diluted with PBS and washed six times. Cells were permeabilized using 0.25% Triton X-100 in PBS for 5 min at room temperature. After permeabilization, the cells were washed twice with 500 μL of PBS and incubated in blocking solution (10% BSA in PBS) for 30 minutes at 37°C. The cells were then incubated with primary antibody (either anti-FLAG M2 monoclonal antibody or anti-lamin A antibody, Abcam) at a dilution of 1:500 (anti-FLAG) or 1:100 (anti-lamin) for 1 h at 37°C. The cells were then washed three times with PBS (5 min each) and incubated further with secondary antibody (anti-mouse FITC-labeled antibody, Sigma-Aldrich) (1:100 dilution) for 1 h at 37°C. Post secondary antibody incubation, the cells were washed three times with PBS and then viewed using a fluorescence microscope (IX71, Olympus, software: IPlab).

Labeling and hybridization of microarrays

Five micrograms of total RNA were used for double-stranded (ds) cDNA synthesis using Gibco BRL superscript choice system. Purified ds cDNA was used for synthesis of biotin-labeled cRNA using ENZO BioArray High yield RNA transcript labeling kit (Affymetrix). The cRNA was purified using Qiagen RNeasy kit and was fragmented at 95°C for 35 min for target preparation. This was hybridized against both Human U95Av2 and U133A arrays as suggested by manufacturer (Affymetrix), and expression was normalized as described previously (18).

Real Time PCR (QPCR)

Total RNA (1 microgram) was primed using oligo (dT) 18 primers and cDNA synthesized using the Clontech cDNA synthesis kit following the manufacturer’s directions (Clontech, Mountain View, CA, USA). Predesigned TaqMan primer sets were purchased from Applied Biosystems. Real-time PCR reactions were run on the ABI Prism 7300 sequence detection system (Applied Biosystems, Foster City, CA, USA) and relative expression levels were calculated using the comparative CT method (22). Briefly, in the comparative CT method (CT value = the number of PCR cycles that elapse before the threshold is reached for the target nucleic acid), arithmetic formulas are used to calculate relative expression levels compared with normal (non-diseased) samples. The amount of target, normalized to an endogenous housekeeping gene [glyceraldehyde-3 phosphate dehydrogenase (GAPDH)] and relative to the non-OA control samples, is then calculated by the formula:

equation M1

The equation thus represents the normalized expression of the target gene in the individual patient, relative to the normalized expression of the mean of the non-OA control patients.

Statistical analysis

All the experiments were done three to six times, and each condition was done in triplicate. Data are expressed as mean ± SD. Unpaired, two-tailed t test was performed to analyze significance using GraphPad prism software (ver. 4.0).

RESULTS

Expression of lamins in cartilage

We investigated relative levels of mRNAs for lamins in normal and OA cartilage by gene expression profiling. Comparison of RNA from pooled samples of OA and normal cartilage using U95Av2 and U133A Affymetrix microarray revealed a significant ~2-fold upregulation of A-type lamin mRNA in diseased tissue (p <0.009 and 0.005; Figure 1A). Expression levels of lamin B1 and B2 mRNAs were equivalent to non-OA controls (p = 0.8 and 0.1, respectively) (data not shown). We further validated the elevated expression of lamin A (not B type) mRNA in OA (p=0.03) as compared to non-OA individual cartilage samples by quantitative PCR (Figure 1B). Lamin A upregulation in OA cartilage was also confirmed at the protein level; immunoblot of cartilage protein extracts using a lamin A-specific monoclonal antibody revealed a protein with an apparent molecular mass of 70–72 kDa (Figure 1C). The mean lamin A levels in five OA cartilage extracts compared to four non-OA cartilage samples were determined by Image J (NIH) analyses (normalized to the catalase control) (Figure 1C). In all OA samples, elevated expression of lamin A was observed compared to non-OA controls (p=0.0012). Immunohistochemical staining of tissue sections of OA cartilage revealed uniform staining of lamin A throughout the depth of cartilage from the articular surface to the deep zone. Staining was also found in both lesional and non-lesional areas (data not shown).

Figure 1
Lamin expression is elevated in osteoarthritis (OA) cartilage. A) Gene expression (microarray) analysis of lamins: pooled RNA samples from non-OA (N) and OA cartilage specimens were used for microarray analysis using U95Av2 and U133A microarray chips. ...

PGE2 increases lamin A expression through the EP2/4 receptors

There have been no reports of factors that induce lamin A expression in mammalian cells. In order to identify potential inducers of lamin A expression in human articular chondrocytes, we studied the effect of inflammatory mediators, PGE2 and IL-1β. OA chondrocytes, when cultured in the presence of PGE2 (10 μM) for 24 h, stained more strongly than unstimulated cells following immunofluorescence labeling of lamin A. Conversely, 10 ng/ml IL-1β treatment for 24 h decreased lamin A expression (Figure 2A). We also confirmed the increased expression of lamin A in PGE2- and IL-1 treated cells by immunoblot. Preincubation of IL-1-treated chondrocytes with the cyclooxygenase-2 (COX-2) selective inhibitor, celecoxib, had an additive inhibitory effect on lamin A protein expression (Figure 2B). To further elucidate the role of endogenous PGE2 in the regulation of lamin A expression by chondrocytes, primary cells were cultured in the presence of celecoxib. Chondrocytes treated with celecoxib expressed less lamin A compared to untreated cells (Figure 2B and C). In contrast, lamin B1 levels were not affected by addition of PGE2,IL-1β or celecoxib (Figures 2B and C).

Figure 2
Prostaglandin E2 (PGE2) increases lamin A expression in human OA chondrocytes. Human OA chondrocyte monolayer cultures were incubated in serum- free Ham’s F12 medium either in the presence or absence of PGE2/IL-1β. A) Expression of lamin ...

To further investigate the mechanism of PGE2-dependent induction of lamin A, we stimulated chondrocytes with PGE2 following blockade of the PGE2 receptors EP2/4. Blocking the EP2/4 with receptor-specific antagonists (EP2 receptor antagonist AH6809; EP4 receptor antagonist AH23848) at 10 μM concentration neutralized the effect of PGE2; this was accompanied by downregulation of lamin A at the protein levels, detectable by immunoblot (Figure 2C). These findings suggest that PGE2 significantly increases lamin A expression through the EP2/4 receptors. However, blocking of EP2/4 receptors in unstimulated conditions did not affect lamin A or lamin B1 expression (data not shown).

Lamin A overexpression disrupts chondrocyte proliferation and viability

To elucidate the role of lamin A in cellular aging, we studied the proliferative capacity using incorporation of bromodeoxyuridine (BrdU) 24 and 48 h following overexpression of wild type lamin A and its variant, R482Q, in chondrocytes. Transient overexpression of lamin A in these cells increased its mRNA by 6- to 10-fold as compared to control vector-transfected cells (Figure 3A). The increased expression of lamin A in these experiments was also confirmed by immunoblot (Figure 3A).

Figure 3
Overexpression of lamin A induces expression of senescence markers p21 and p16 in chondrocytes. Chondrocytes were transfected with lamin A constructs (wild type and variant R482Q), and were analyzed 24 h post transfection. A) Relative lamin A expression ...

We observed a significant decrease in proliferation of cells overexpressing lamin A (by 52% at 24 h and 47% at 48 h, as compared to control vector-transfected cells) (p <0.001). Overexpression of the lamin A R482Q variant had a similar inhibitory effect on proliferation of chondrocytes (by 81% at 24 h, and 52% at 48 h) (p <0.001), suggesting that both wild type lamin A and the variant had similar effects on chondrocyte proliferation. Furthermore, overexpression of lamin A also decreased cell viability, as measured by decreased total LDH activity from 14.05 ± 1.9 to 7.35 ± 0.41 U/mg protein (p <0.02) as compared to control vector-transfected cells.

Increased expression of senescence markers

To determine whether decreased cellular proliferation was accompanied by increased cellular senescence following lamin A overexpression, we measured expression of regulatory proteins p21 (WAF1) and Cyclin-dependent kinase inhibitor 2A (p16 ink4A). In senescent cells, expression of these inhibitors is induced. As shown in Figure 3B, cells transiently overexpressing lamin A and the R482Q variant showed increased expression of p21, while cells transfected with vector alone express barely detectable amounts of p21 by immunoblot. Overexpression of lamin A results in significant increase in the protein expression of p21 in wild type and lamin A mutants by 2- to 3-fold. However, unlike p21, p16 levels were found to decrease following lamin A overexpression in chondrocytes.

Lamin A overexpression increased cytosolic cytochrome C accumulation

Mitochondrial depolarization leads to a redistribution of cytochrome C within the cytosolic and mitochondrial fractions. We investigated the role of lamin A in this process by measuring cytochrome C levels in cytosol and mitochondrial fractions. Transient expression of lamin A in chondrocytes (Figure 4A) increased the levels of cytochrome C in the cytosol (from 3.76 ± 0.4 to 6.04 ± 1.4 ng/mg protein) (p <0.5), and this was accompanied by a concomitant decrease in the mitochondrial fraction (from 14.4 ± 7.9 to 10.6 ± 5.9 ng/mg protein). IL-1β, a known inducer of apoptosis in cultured chondrocytes, also caused a redistribution of cytochrome C levels and was included as a positive control. In the same experiment we also assessed ATP levels: overexpression of lamin A in chondrocytes resulted in 40–50% decrease in cellular ATP levels, 24 h post transfection (vector control 11.3 ± 0.89 pmol/106 cells; lamin A, 5.99 ± 0.41) (p <0.02). This redistribution of cytochrome C indicates that lamin A overexpression leads to breakdown of mitochondrial membrane integrity and reduced cellular energy levels.

Figure 4
Dysregulated mitochondrial functions in wild type lamin A-overexpressing chondrocytes. Human chondrocytes (SW1353) were transfected with lamin A or vector control, and mitochondrial functions and apoptosis (by TUNEL staining) were determined 24–48 ...

Overexpression of lamin A in human chondrocytes leads to caspase 3 activation

We tested whether overexpression of wild type lamin A in chondrocytes can lead to increased levels of caspase 3, a known mediator of chondrocyte apoptosis.. Overexpression of lamin A increased caspase 3 levels by 2- to 3-fold (2.7 ± 0.7 μg/mg protein) (p <0.01) over basal levels (0.5 ± 0.09 μg/mg protein) within 24 h (Figure 4B). This effect was inhibited by preincubation with AC-DEVD-CHO, a caspase 3-specific inhibitor (Figure 4B). In the same experiments, the compound Z-FA-FMK (a peptide that has no effect on caspases) did not inhibit caspase activation and had no effect on lamin A-induced caspase 3. Lamin A overexpression did not change nuclear morphology in human chondrocytes.

Overexpression of lamin A in human chondrocytes leads to apoptosis

In parallel studies, we also confirmed overexpression lamin A induced DNA fragmentation as an alternative indicator of apoptosis. TUNEL assay detects apoptosis-mediated DNA fragmentation. Overexpression of lamin A induced significant DNA fragmentation at 48 h post-transfection in comparison to control vector transfected chondrocytes, as indicated by the green fluorescence in the TUNEL assay (Fig. 4C). As expected, IL-1β (10 ng/ml) treatment promoted apoptosis, indicated by positive TUNEL staining, in cultured chondrocytes.

Protein farnesyltransferase inhibitor (FTI) reverses lamin A-mediated cellular defects

Since overexpression of lamin A slows growth of chondrocytes, we tested the effect of FTI on the cellular defects observed in cells expressing wild type lamin A. Culturing transfected chondrocytes overexpressing lamin A with FTI prevented lamin A-induced mitochondrial membrane depolarization indicated by increased red fluorescence of healthy mitochondria following staining with JC-1 dye (Figure 5A). This was accompanied by a moderate decrease in the leak of cytochrome C from the mitochondria to the cytoplasm, by 7% compared to untreated lamin A-overexpressing cells (data not shown).

Figure 5
Dysregulated mitochondrial function induced in response to lamin A overexpression is mitigated by farnesyltransferase inhibitor (FTI) pretreatment in chondrocytes. Chondrocytes (SW1353) were pre-incubated with FTI (10 μM) 24 h prior to lamin A ...

Similarly, addition of FTI to cells overexpressing lamin A decreased active caspase 3 levels (from 2048.0±732 to 349±273 ng/mg protein; p <0.03) (Figure 5B) and increased cellular ATP to near control values (from 6.69±0.55 to 9.32±0.29 nmoles/106 cells; p=0.02) (Figure 5C). These data suggest that the farnesylation step during lamin synthesis is in part necessary for lamin A-mediated induction of chondrocyte apoptosis.

DISCUSSION

Chondrocyte viability decreases with cartilage injury, aging and disease (3;6;8). In osteoarthritic cartilage there have been several reports of chondrocytes exhibiting classical signs of apoptosis (6;8). 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 (2426).

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;30;31). 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 (3335). 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 (3335). 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.

Supplementary Material

Supp Table S1 & Fig S1-S4

Acknowledgments

This work was supported in part by grants from the National Institutes of Health to Dr. Abramson (AR054817 and AR07176, with salary support to Drs. Ben-Artzi and Palmer) and Dr. Worman (AR048997).

The authors thank Ann Rupel for assistance in preparation of the manuscript and figures. The authors also wish to thank Ms. Jyoti Patel for processing, storage and maintenance of primary chondrocyte cells. The authors would like to thank Mandar Dave for performing ATP, caspase and mitochondrial staining assays.

Footnotes

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Attur had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Ben-Artzi, Attur and Abramson.

Acquisition of data. Attur, Ben-Artzi, Yang and Al-Mussawir.

Analysis and interpretation of data. Attur, Ben-Artzi, Palmer, Worman and Abramson.

References

1. Wieland HA, Michaelis M, Kirschbaum BJ, Rudolphi KA. Osteoarthritis - an untreatable disease? Nat Rev Drug Discov. 2005;4:331–44. [PubMed]
2. Lories RJ. Joint homeostasis, restoration, and remodeling in osteoarthritis. Best Pract Res Clin Rheumatol. 2008;22:209–20. [PubMed]
3. Martin JA, Buckwalter JA. Aging, articular cartilage chondrocyte senescence and osteoarthritis. Biogerontology. 2002;3:257–64. [PubMed]
4. Todd Allen R, Robertson CM, Harwood FL, Sasho T, Williams SK, Pomerleau AC, et al. Characterization of mature vs aged rabbit articular cartilage: analysis of cell density, apoptosis-related gene expression and mechanisms controlling chondrocyte apoptosis. Osteoarthritis Cartilage. 2004;12:917–23. [PubMed]
5. Robertson CM, Pennock AT, Harwood FL, Pomerleau AC, Allen RT, Amiel D. Characterization of pro-apoptotic and matrix-degradative gene expression following induction of osteoarthritis in mature and aged rabbits. Osteoarthritis Cartilage. 2006;14:471–6. [PubMed]
6. Aigner T, Kim HA, Roach HI. Apoptosis in osteoarthritis. Rheum Dis Clin North Am. 2004;30:639–53. [PubMed]
7. Buckwalter JA, Martin JA. Osteoarthritis. Adv Drug Deliv Rev. 2006;58:150–67. [PubMed]
8. Loeser RF. Aging and osteoarthritis: the role of chondrocyte senescence and aging changes in the cartilage matrix. Osteoarthritis Cartilage. 2009;17:971–9. [PMC free article] [PubMed]
9. Blanco FJ, Lopez-Armada MJ, Maneiro E. Mitochondrial dysfunction in osteoarthritis. Mitochondrion. 2004;4:715–28. [PubMed]
10. Del Carlo M, Jr, Loeser RF. Cell death in osteoarthritis. Curr Rheumatol Rep. 2008;10:37–42. [PubMed]
11. Loeser RF., Jr Aging cartilage and osteoarthritis--what's the link? Sci Aging Knowledge Environ. 2004;2004:e31. [PubMed]
12. Shimi T, Pfleghaar K, Kojima S, Pack CG, Solovei I, Goldman AE, et al. The A- and B-type nuclear lamin networks: microdomains involved in chromatin organization and transcription. Genes Dev. 2008;22:3409–21. [PubMed]
13. Dechat T, Pfleghaar K, Sengupta K, Shimi T, Shumaker DK, Solimando L, et al. Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin. Genes Dev. 2008;22:832–53. [PubMed]
14. Worman HJ, Fong LG, Muchir A, Young SG. Laminopathies and the long strange trip from basic cell biology to therapy. J Clin Invest. 2009;119:1825–36. [PMC free article] [PubMed]
15. Merideth MA, Gordon LB, Clauss S, Sachdev V, Smith AC, Perry MB, et al. Phenotype and course of Hutchinson-Gilford progeria syndrome. N Engl J Med. 2008;358:592–604. [PMC free article] [PubMed]
16. Lammerding J, Schulze PC, Takahashi T, Kozlov S, Sullivan T, Kamm RD, et al. Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction. J Clin Invest. 2004;113:370–8. [PMC free article] [PubMed]
17. Ostlund C, Bonne G, Schwartz K, Worman HJ. Properties of lamin A mutants found in Emery-Dreifuss muscular dystrophy, cardiomyopathy and Dunnigan-type partial lipodystrophy. J Cell Sci. 2001;114:4435–45. [PubMed]
18. Attur MG, Palmer GD, Al-Mussawir HE, Dave M, Teixeira CC, Rifkin DB, et al. F-spondin, a neuroregulatory protein, is up-regulated in osteoarthritis and regulates cartilage metabolism via TGF-beta activation. FASEB J. 2009;23:79–89. [PubMed]
19. Attur MG, Dave M, Cipolletta C, Kang P, Goldring MB, Patel IR, et al. Reversal of autocrine and paracrine effects of interleukin 1 (IL-1) in human arthritis by type II IL-1 decoy receptor. Potential for pharmacological intervention. J Biol Chem. 2000;275:40307–15. [PubMed]
20. Attur M, Al-Mussawir HE, Patel J, Kitay A, Dave M, Palmer G, et al. Prostaglandin E2 exerts catabolic effects in osteoarthritis cartilage: evidence for signaling via the EP4 receptor. J Immunol. 2008;181:5082–8. [PubMed]
21. Dave M, Attur M, Palmer G, Al-Mussawir HE, Kennish L, Patel J, et al. The antioxidant resveratrol protects against chondrocyte apoptosis via effects on mitochondrial polarization and ATP production. Arthritis Rheum. 2008;58:2786–97. [PubMed]
22. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–8. [PubMed]
23. Amin AR, Attur M, Patel RN, Thakker GD, Marshall PJ, Rediske J, et al. Superinduction of cyclooxygenase-2 activity in human osteoarthritis-affected cartilage. Influence of nitric oxide. J Clin Invest. 1997;99:1231–7. [PMC free article] [PubMed]
24. Capell BC, Erdos MR, Madigan JP, Fiordalisi JJ, Varga R, Conneely KN, et al. Inhibiting farnesylation of progerin prevents the characteristic nuclear blebbing of Hutchinson-Gilford progeria syndrome. Proc Natl Acad Sci U S A. 2005;102:12879–84. [PubMed]
25. Glynn MW, Glover TW. Incomplete processing of mutant lamin A in Hutchinson-Gilford progeria leads to nuclear abnormalities, which are reversed by farnesyltransferase inhibition. Hum Mol Genet. 2005;14:2959–69. [PubMed]
26. Mallampalli MP, Huyer G, Bendale P, Gelb MH, Michaelis S. Inhibiting farnesylation reverses the nuclear morphology defect in a HeLa cell model for Hutchinson-Gilford progeria syndrome. Proc Natl Acad Sci U S A. 2005;102:14416–21. [PubMed]
27. Zhou R, Yazdi AS, Menu P, Tschopp J. A role for mitochondria in NLRP3 inflammasome activation. Nature. 2011;469:221–5. [PubMed]
28. Kim J, Xu M, Xo R, Mates A, Wilson GL, Pearsall AW, et al. Mitochondrial DNA damage is involved in apoptosis caused by pro-inflammatory cytokines in human OA chondrocytes. Osteoarthritis Cartilage. 2010;18:424–32. [PubMed]
29. Veluthakal R, Amin R, Kowluru A. Interleukin-1 beta induces posttranslational carboxymethylation and alterations in subnuclear distribution of lamin B in insulin-secreting RINm5F cells. Am J Physiol Cell Physiol. 2004;287:C1152–C1162. [PubMed]
30. Kuhn K, D'Lima DD, Hashimoto S, Lotz M. Cell death in cartilage. Osteoarthritis Cartilage. 2004;12:1–16. [PubMed]
31. Kim HA, Blanco FJ. Cell death and apoptosis in osteoarthritic cartilage. Curr Drug Targets. 2007;8:333–45. [PubMed]
32. Caron M, Auclair M, Donadille B, Bereziat V, Guerci B, Laville M, et al. Human lipodystrophies linked to mutations in A-type lamins and to HIV protease inhibitor therapy are both associated with prelamin A accumulation, oxidative stress and premature cellular senescence. Cell Death Differ. 2007;14:1759–67. [PubMed]
33. Alcorta DA, Xiong Y, Phelps D, Hannon G, Beach D, Barrett JC. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc Natl Acad Sci U S A. 1996;93:13742–7. [PubMed]
34. Brookes S, Rowe J, Gutierrez Del AA, Bond J, Peters G. Contribution of p16(INK4a) to replicative senescence of human fibroblasts. Exp Cell Res. 2004;298:549–59. [PubMed]
35. McConnell BB, Gregory FJ, Stott FJ, Hara E, Peters G. Induced expression of p16(INK4a) inhibits both. Mol Cell Biol. 1999;19:1981–9. [PMC free article] [PubMed]
36. Zhou HW, Lou SQ, Zhang K. Recovery of function in osteoarthritic chondrocytes induced by p16INK4a-specific siRNA in vitro. Rheumatology (Oxford) 2004;43:555–68. [PubMed]