Recovery of function in osteoarthritic chondrocytes induced by p16INK4a-specific siRNA in vitro

H. W. Zhou, S. Q. Lou and K. Zhang

Department of Orthopaedic Surgery, Third Hospital, Peking University, Beijing 100083, China.

Correspondence to: H. Zhou, Department of Orthopaedic Surgery, Third Hospital, Peking University, 38 Xueyuan Road, Beijing 100083, People's Republic of China. E-mail: mailto:doctorzhou6654{at}sina.com


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. To demonstrate the roles of p16INK4a in the senescence of human chondrocytes and the progression of osteoarthritis (OA).

Methods. Immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to examine p16INK4a expression in fetal, normal age-matched and OA cartilage, and Western blot was used in primary cultured chondrocytes from different origins. To explore a functional p16INK4a knockdown in OA chondrocytes, the primary cultured cells were treated with p16INK4a-specific small interfering ribonucleic acids (siRNAs). Expression of p16INK4a, p14ARF and p53 was observed by Western blot and RT-PCR. The phosphorylation status of pRb, senescence-associated ß-galactosidase (SA-ß-gal), cell G1/S transition and cell proliferation were studied by Western blot, histological staining, 3H-thymidine incorporation and cell counts respectively. Expression of the collagen I, collagen II and aggrecan genes was measured by semiquantitative RT-PCR. To establish the response of chondrocytes to cytokines, cells were treated with transforming growth factor-ß1 (TGF-ß1) or interleukin-1{alpha} (IL-1{alpha}) and examined for incorporation of 3H-thymidine, 3H-proline and 35S-sulphate respectively.

Results. A significant increase of p16INK4a was detected in OA chondrocytes compared with normal age-matched and fetal chondrocytes (P<0.01) in vivo and in vitro. Treated with p16INK4a-specific siRNAs, OA chondrocytes displayed a significant decrease in p16INK4a expression with an increase of phosphorylated pRb, but no alteration of p14ARF and p53 expression, followed by decreases of senescent features and increases in the expression of some chondrocyte-specific genes and overall repair capacity.

Conclusions. p16INK4a is instrumental in the senescence of human articular chondrocytes or OA. The reduction of p16INK4a by RNA interference (RNAi) contributed to the recovery of osteoarthritic chondrocytes, suggesting that p16INK4a may be a viable future therapeutic candidate.

KEY WORDS: p16INK4a, Senescence, Osteoarthritis, siRNA.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Age is a major risk factor for osteoarthritis (OA). Extracellular matrix (ECM) synthesis by chondrocytes decreases with age, and there is an age-dependent decline in the responsiveness of these cells to growth factors [13]. The proliferative potential of articular chondrocytes also decreases with age, and in common with other cell types, chondrocytes can only undergo a limited number of cell divisions in vitro [4]. Age-related alterations in the chondrocyte phenotype may therefore allow ECM degradation to predominate, resulting in OA-like changes.

Progressive telomere shortening has been proposed to be one critical determinant of senescence [57]. With each round of cell division and deoxyribonucleic acid (DNA) replication, the telomere DNA sequence is under-replicated, leading to progressive shortening of the telomere [8]. Eventually the shortened telomere may no longer be able to protect the end of the chromosome, and the unprotected chromosomal DNA end may release a senescence-inducing signal to the cell. Martin and Buckwalter [9] have recently reported that the average telomere length in cartilage chondrocytes decreases with age, which suggests that the replicative senescence induced by telomere shorting contributes to either the development or the progression of OA.

It is clear, however, that senescence is not simply the end result of shortened telomeres. Telomere shortening is not a consistent measure of the onset of senescence, as the rate of telomere erosion varies between cells during in vitro culture [6, 1014] and there is no consistent telomere length at which senescence predictably takes place [15]. In addition, the ectopic expression of hTERT in some cell types restores telomerase enzymatic function and telomere lengths but still does not allow these cells to by-pass senescence [16, 17]. Importantly, other recent findings suggest that normal human cells respond to certain types of DNA damage [1820], histone deacetylase inhibitors [21] and oncogenic forms of Ras or Raf [22, 23] by adopting a phenotype that closely resembles replicative senescence. These data suggest that even when shortened telomeres are restored by the ectopic expression of hTERT, additional signals activate or enforce the senescence programme.

The telomere-independent pathway activated by environmental and intrinsic stress is primarily associated with up-regulated expression of p16INK4a. The gene for p16INK4a is located at the INK4a/ARF locus, which yields two transcripts derived from alternative first exons, 1{alpha}, 1ß. p16INK4a is specified by the {alpha} transcript (containing exons 1{alpha}, 2 and 3), and ß transcript contains exons 1ß, 2 and 3 encoding p19ARF in the mouse and p14ARF in humans [24]. Both p16INK4a and p14ARF play an important role in regulating cell growth and senescence: p16INK4a competes with the activating D-type cyclins for association with CDK4 or CDK6, thereby preventing phosphorylation of pRb proteins that control G1 exit; However, p14ARF, due to its binding to and promotion of rapid degradation of MDM2 subsequently leading to p53 stabilization and accumulation, can trigger cell cycle arrest or apoptosis [24].

The activation of either the p16INK4a/pRB pathway or the p19ARF/p53 pathway in senescence appears to depend on the type and species of origin of the cell. Mouse embryo fibroblasts may preferentially rely on the p19ARF/p53 pathway [25], whereas human keratinocytes employ the p16INK4a/pRB pathway to enforce the senescence programme [16, 26]. In addition, p16INK4a loss seems to be more common than p14ARF in human tumour cells [24, 27], confirming a strong selection against this gene as human cells escape senescence. Therefore, we focused on p16INK4a and hypothesized that p16INK4a acts as a suppressor that mediates the senescence of chondrocytes, thus activating the onset of OA, even though there is no evidence that p14/p53 pathway can be excluded completely. Accordingly, we sought to examine the role of p16INK4a in this cell type, both in vitro and in vivo.

Immunohistochemistry and Western blot show that the p16INK4a gene is highly expressed in OA and age-matched chondrocytes compared with fetal chondrocytes in vivo and in vitro, and even more significantly in the former. Following this, we used small interfering RNAs (siRNAs), which are able to trigger RNA interference in mammalian somatic cells [28, 29], to inhibit the expression of p16INK4a in cultured chondrocytes. We found that p16INK4a transient expression silencing led to decreases of senescent features and increases in the expression of some chondrocyte-specific genes and overall repair capacity in comparison with mock-treated samples. To exclude interference of p14ARF by siRNA because of the overlap of coding regions with p16INK4a, we compared the expression of p14ARF and p53 within siRNA-treated, untreated OA chondrocyte and fetal chondrocytes, and found no evidence for p14ARF inactivation in our own study. Taken together, our results suggest that p16INK4a plays at least partially a critical role in chondrocyte senescence and that inhibition of p16INK4a could be a potential therapeutic strategy for blocking the senescence of articular chondrocytes for the prevention or treatment of OA.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Samples and grade
Thirty-two osteoarthritic cartilage samples were obtained from 26 patients (mean age 67.60 yr, range 65–80) undergoing total knee replacement for severe OA of the knee joints. Twenty age-matched control cartilage samples were collected from 18 trauma patients who underwent amputation (mean age 68.20 yr, range 63–81). To consistently define joint characteristics, all of the articular cartilages were graded macroscopically and scored microscopically as previously described [30]. Only cartilage samples of Collins grade 1 (no grade 0 was found) and a Mankin score of 3 were used as normal age-matched controls. All OA cartilages were Collins grade 3 or 4 and Mankin score 6. Additionally, fetal cartilage samples were obtained from six first-trimester fetuses after consent had been obtained from the parents as required under Chinese law.

Immunohistochemistry assay
5 µm sections were prepared as previously described [31]. Before staining, the sections were deparaffinized in xylene and rehydrated. The appropriate epitopes were recovered by pronase treatment [5 min incubation at 37°C in 100 µg/ml pronase in 0.1% phosphate-buffered slaine (PBS)]. Sections were then incubated overnight at 4°C with a monoclonal mouse anti-p16INK4a (IgG) diluted 1:100 (Santa Cruz), followed by incubation with a biotin-conjugated goat anti-mouse secondary antibody (Santa Cruz), labelling with streptavidin-conjugated horseradish peroxidase and staining with a DAB Kit (Santa Cruz). Finally, the sections were counterstained with haematine. As additional controls, the primary antibodies were neutralized by pre-absorption with the blocking peptide available (Santa Cruz), or the blots were probed with irrelevant mouse or goat IgG monoclonal antibodies. Secondary antibodies were also tested for non-specific binding.

Cells were examined by two independent observers under a Leica Q 550 CW microscope with a magnification of 400. The frequency of nuclear p16INK4a staining was determined by counting positive and negative chondrocytes over the entire focal area, which contained between 80 and 120 cells. Large variances were resolved by common review, and means of the percentage of p16INK4a-positive chondrocytes from six sections were calculated.

Chondrocyte cultures
For generation of the primary chondrocyte cultures, the cartilage tissue samples were minced into ~1 mm3 pieces, washed in DMEM/F12 (Gibco), and subjected to a 60 min 0.2% trypsin digestion followed by overnight digestion in 0.2% collagenase II (Gibco), DMEM/F12 and 10% fetal bovine serum (FBS). The resulting cell suspension was filtered through 100 µm nylon meshes, washed repeatedly with PBS and centrifuged at 250 g for 5 min. The average yield was (3.0 ± 0.3) x 106 chondrocytes. Cells were seeded into Costar 24-well plates (Costar, Corning, NY, USA) at a final density of 1 x 104 cells/cm2 as monolayers in DMEM/F12 supplemented with 10% FBS for 3 days under normal growth conditions (37°C and 5% CO2). Monolayers were then incubated in 1% FBS with or without the transfection complexes for 4 h as described below, after which they were processed for the various analyses.

siRNA template design
Ambion’s siRNA target design online tool was utilized to choose five sequences (Table 1) for targeting human p16INK4a mRNA [32]. BLASTN searches were conducted on all sequences to ensure gene specificity. All siRNA duplexes were synthesized with the Silencer siRNA construction kit (Ambion) according to the manufacturer’s protocol. The positive (anti-GAPDH siRNA) and negative control (scrambled siRNA) siRNAs were purchased from Ambion.


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TABLE 1. siRNA template sequences

 
siRNA transfection
Three days’ post-seeding, p16INK4a-specific siRNA was transfected into chondrocyte monolayers using the TransMessenger transfection reagent (Qiagen) according to the manufacturer’s protocol. After 4 h of transfection, the complexes were removed from the 24-well plates, cells were washed with PBS, and 500 µl fresh medium containing 10% FBS was added to the plates. After 1, 2, 3, 4 and 5 days, chondrocytes were harvested from three independent wells and used for the following analyses, each of which was performed at least three times. The positive and negative controls were analysed in parallel.

Selection of optimal siRNA and determination of transfection efficiency
Transfection efficiency was determined by Western blot and reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The starting amount for transfection of siRNA and the ratio of siRNA to TransMessenger transfection reagent was 0.8 µg and 1:5 respectively according to the manufacturer’s recommendations. To optimize the contribution of siRNA dose and siRNA/transfection reagent ratio to transfection efficiency, total amounts of siRNA were varied between 0.4 µg and 1.6 µg siRNA and siRNA/transfection reagent ratios were varied between 1:2.5 and 1:10 µg/µl.

mRNA expression analysis
Total mRNA was isolated directly from fresh cartilage as previously described [33] or isolated from cultured cells with the RNAqueousTM-4 PCR kit (Ambion), according to the manufacturer’s protocol. The specific primers used for RT-PCR are shown in Table 2. ß-Actin was amplified as an internal control for normalization. Aliquots of each RT sample were assessed for ß-actin expression following 21 PCR cycles. PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide staining.


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TABLE 2. Specific primers used for RT-PCR

 
Cytokine induction
On the third day after the 4 h siRNA treatment, the culture medium was replaced with DMEM/F12 containing 0.5% FCS. One day later, chondrocytes in three wells were incubated in 0.5% FCS with interleukin-1{alpha} (IL-1{alpha}) (40 U/ml) (Sigma) or transforming growth factor-ß1 (TGF-ß1) (10 ng/ml) (Sigma) for another 24 h, while chondrocytes in three additional wells were exposed to the same conditions without IL-1{alpha} or TGF-ß1. Cells were then processed for RNA extraction and the measurements described below. Control chondrocytes were treated and analysed in parallel.

Western blot analysis
Total proteins were extracted from the cultured chondrocyte monolayers on the indicated days, and Western blot analysis was performed as previously described [40]. The blots were immersed overnight at 4°C in 5% skimmed milk and subsequently incubated for another overnight at 4°C with the following primary antibodies: monoclonal mouse anti-p16INK4a (1:1500), anti-p53 (1:1000) and anti-GAPDH (1:1500), polyclonal goat anti-p14ARF (1:1000) and anti-phosphorylated pRb (1:150) as well as polyclonal rabbit anti-pRb (1:2000) followed by a goat anti-mouse or goat anti-rabbit or horse anti-goat secondary IgG (1/10,000) conjugated to horseradish peroxidase (HRP). Visualization of the immunocomplexes was conducted with the Luminol reagent kit according to the manufacturer’s specifications. The blots imaged by autoradiography were quantified by densitometry. The negative controls for all antibodies were performed as the immunohistochemistry assay described. All regents were from Santa Cruz, except for anti-GAPDH (Ambion).

Cell counts
Chondrocytes were trypsinized on the indicated days and counted with a haemocytometer. As the number of viable cells isolated from different donors was variable, we quantify cell proliferation by dividing the cells counted on the indicated day in culture by the number of cells alive right before transfection as a starting cell number. Chondrocyte viability was assessed by trypan blue exclusion (Sigma). Approximately 200 cells were evaluated in each sample under the haemacytometer. Viable cells were identified by their ability to exclude trypan blue.

DNA synthesis
Chondrocytes were treated with 1 µCi of 3H-thymidine (Amersham) per ml for the last 6 h of day 3 after siRNA treatment, or following 24 h of culturing in the presence of cytokines in the induction experiment. Cells were then rinsed twice in PBS, twice with ice-cold 5% trichloroacetic acid (TCA) and twice with 80% ethanol; the incorporated radioactivity of cell lysates was measured in a liquid scintillation counter [41].

DNA content
At the same time points as mentioned above, the DNA content was analysed with a Hoechst 33258 (Sigma) dye assay [42] measured by fluorophotometry, EX 360 nm–EM 460 nm. The results were expressed as relative fluorescence units (RFU).

Proteoglycan and collagen synthesis analysis
Proteoglycan and collagen synthesis were monitored as described in [43], with a slight modification. Briefly, for proteoglycan, 5 mCi/ml 35S-sulphate (Amersham) was added to each well on 3 days’ post-transfection, or following 24 h of culturing in the presence of cytokines in the induction experiment. The monolayers were incubated for a further 24 h. The labelled chondrocytes and the medium were harvested at the end of incubation. The cells were digested with 0.2% (w/v) papain in 0.1 M sodium acetate buffer for 24 h at 55°C. Proteoglycans in the media and cell digests were precipitated by the addition of an equal volume of 10% cetylpyridinium chloride in the presence of carriers (chondroitin sulphates, 100 mg/ml) for 1 h at 37°C. After centrifugation (10 000 g for 15 min, 37°C), the pellets were dissolved in 2 M MgCl2 and precipitated again with 5 vol. of ethanol at 4°C overnight.

For collagen synthesis, the cells were labelled with 20 µCi/ml 3H proline (Amersham) for 24 h. The media and corresponding pepsin digests of cell monolayers were pooled and adjusted to pH 3 with acetic acid and added to 100 mg/ml type I collagen from bovine skin, as the carrier. Collagen was precipitated by adding an equal volume of 4 M NaCl. The precipitate was dissolved in 0.5 ml 3% (v/v) acetic acid, incubated at 20°C for 2 h with 2 mg/ml pepsin (Sigma), and precipitated again.

All the radioactivities of the resulting pellets were determined in a liquid scintillation counter.

ß-Gal staining
siRNA treated or control chondrocytes in 24-well plates were washed twice in PBS 3 days’ post-treatment, followed by ß-galactosidase (ß-gal) staining according to Dimri et al. [44]. Positive staining was evident within 2 h and maximal by 16 h. Representative wells were photographed under an Olympus IX 70 microscope. Means of the percentage of ß-gal-positive chondrocytes of three wells of six separate experiments in each group were calculated.

Statistical analysis
The data are reported as mean ± standard error of the mean (SEM) of the indicated number of experiments. Values were assessed by one-way analysis of variance (ANOVA) and LSD post hoc multiple comparison tests, at a significance level of P<0.01.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
p16INK4a expression in articular cartilage
p16INK4a-immunopositive chondrocytes were detected in all layers of normal control and OA cartilage (data not shown). Sections in Fig. 1 show representative nuclear distribution of p16INK4a in chondrocytes from fetus, age-matched control and OA samples. In the OA cartilage there were significantly more p16INK4a stained chondrocytes compared with age-matched cartilage (P<0.01) and significantly fewer positive cells in the fetal control cartilage (P<0.01) (Table 3). To determine p16INK4a expression at the mRNA level, we directly isolated total RNA from the same samples and analysed p16INK4a mRNA expression by semiquantitative RT-PCR (Fig. 1) in parallel the changes in protein expression. We also used Western blot to analyse p16INK4a protein expression in primary cultured chondrocytes from fetus, age-matched control and OA donors (Fig. 2). The results indicated that the trend of p16INK4a expression in the primary cultures was consistent with the in vivo results, though there was some small variation from cultures examined 3 to 7 days post-seeding (data not shown). Thus, we concluded that primary cultured chondrocytes could be used as an in vitro model for the study of p16INK4a expression in human chondrocytes.



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FIG. 1. p16INK4a expression in human articular cartilage tissue. Upper panel: Representative serial sections of from OA (A), age-matched normal (B) and fetal human articular cartilage (C). Arrowhead indicates a representative immunopositive chondrocyte. Lower panel: Corresponding RT-PCR analysis for both p16INK4a and ß-actin; the graph shows the corresponding quantification (mean ± SEM from three independent experiments) normalized to those of ß-actin. Significant differences were observed between all paired data points: P<0.01.

 

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TABLE 3. Percentages of p16INK4a positive chondrocytes in each type of cartilage. Percentages of p16INK4a immunostained chondrocytes in six sections of each sample from three different donors are given as means (SEM). Significant differences were observed between all paired data points: P<0.01.

 


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FIG. 2. p16INK4a expression in primary cultured chondrocytes at 5 days post-seeding, from fetal, age-matched and OA subjects. Upper panel: Representative Western blot. Lower panel: Graphs show the corresponding mean ± SEM of three independent experiments. Significant differences were observed between all paired data points: P<0.01.

 
Optimization of the siRNA transfection
The transfection efficiency varied with sequence of siRNA, the total siRNA content for transfection and the siRNA/transfection reagent ratio. Of the five siRNAs tested, the maximal silencing of p16INK4a was achieved at 72 h post-transfection with siRNA465–485- and siRNA699–719-based RNA respectively with 0.8 µg siRNA and a transfection reagent/siRNA ratio of 1:5 (Fig. 3). The length of 72 h was found to be maximal for reversing p16INK4a activities later. This was not the case when either half or double the quantities and ratios were used in the study (data not shown). To determine the optimal time point for analysis, a time-course experiment was performed at multiple time points after transfection. Representative time-course data induced by siRNA465–485 are shown in Fig. 4. These optimal schemes were used in subsequent experiments. To further examine siRNA specificity, p16INK4a expression was detected in mock controls and no change was observed compared with test cells (Fig. 5). The positive control GAPDH-specific siRNA yielded the expected results, confirming the selectivity of the p16INK4a-specific siRNA and the viability of the system (Fig. 5).



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FIG. 3. Different RNAi activities induced by different siRNAs. Upper panel: Representative Western blot at a standardized representative time point of 72 h post-transfection. Graphs show the mean ± SEM of three independent experiments. *P<0.01 versus control. Lower panel: Corresponding RT-PCR analysis for both p16INK4a and ß-actin levels, and a graph showing p16INK4a mRNA levels relative to that of the untreated control.

 


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FIG. 4. siRNA silencing of p16INK4a over time: the viability of endogenous p16INK4a decreased by 24 h after transfection of siRNA and reached its maximum at 72 h, then gradually recovered with time. Upper panel: Representative Western blot of endogenous p16INK4a expression in OA chondrocytes at various time points following transfection with p16INK4a siRNA duplexes. Graphs show the mean ± SEM of three independent experiments. Lower panel: Corresponding RT-PCR analysis for both p16INK4a and ß-actin levels, and a graph showing p16INK4a mRNA levels relative to that of the untreated control. P<0.01 versus control.

 


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FIG. 5. p16INK4a and GAPDH expression at 72 h post-transfection. Upper panel: Transfection with two different p16INK4a-specific siRNAs significantly reduced expression of p16INK4a; similarly, the positive control, GAPDH-specific siRNA, led to significant reductions in GAPDH expression. Graphs show the mean ± SEM of three independent experiments. *P<0.01 versus control. Lower panel: Corresponding RT-PCR analysis for p16INK4a, GAPDH and ß-actin levels, and a graph showing p16INK4a and GAPDH mRNA levels relative to that of the untreated control. Within each test group, analyses of p16INK4a, GAPDH and ß-actin levels were performed on the same protein or RNA samples.

 
p14ARF and p53 expression is not altered in p16INK4a-specific siRNA-treated OA chondrocytes
Reduced expression of p14ARF, the alternative ß-transcript of the p16INK4a gene, seemed a likely candidate for such a change in the post-transfection chondrocytes. We therefore observed the level of p14ARF expression by Western blot and RT-PCR analysis in siRNA-treated chondrocytes over 120 h. All cells exhibited a similar expression pattern for p14ARF at various time points. The expression of p14ARF in OA chondrocytes treated by siRNA465–485-based RNA is shown in Fig. 6. We also investigated the status of p53, an established target of p14ARF, and found no marked differences (P>0.2) over the time course (Fig. 6). These provided compelling evidence that the p14ARF/p53 pathway was not interfered with by the selected siRNAs in our own study. One explanation could be that there are different second and/or tertiary structures of mRNA between p16INK4a and p14ARF, possibly due to different reading frames, which may participate the induction of distinct RNAi activities [45]. Whether the other three siRNAs in our study which have no effect on p16INK4a have an effect on p14ARF expression is currently being investigated.



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FIG. 6. No significant effect of siRNA on p14ARF and p53 expression was observed over time. Upper panel: Representative Western blot of endogenous p14ARF and p53 expression in OA chondrocytes at various time points following transfection with siRNA duplexes. Graphs show the mean ± SEM of three independent experiments. Lower panel: Corresponding RT-PCR analysis for p14ARF, p53 and ß-actin levels, and a graph showing p16INK4a mRNA levels relative to that of the untreated control. P<0.01 versus control. Within each test group, analyses of p14ARF, p53 and ß-actin levels were performed on the same protein or RNA samples.

 
Senescence-associated markers reveal recovery of OA chondrocytes by p16INK4a-specific siRNA
72 h post-treatment, most of the mock-transfected OA chondrocytes, including untreated (Fig. 7A), buffer-only and siRNAscrambled-treated samples (data not shown), showed gross enlargement, flattening and strongly positive SA-ß-gal staining, and no significant changes were observed within them. However, the siRNA-treated OA chondrocytes were characterized by refractive cytoplasm with long, thin fibrocyte-like projections and few cells showed positive SA-ß-gal staining (Fig. 7B), like fetal chondrocytes cultured for the same period, which showed no significant response to siRNA treatment (Figs. 7C, D).



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FIG. 7. Representative morphology and SA-ß-gal staining of chondrocytes at 72 h post-transfection: (A) OA untreated group; (B) OA siRNA group; (C) fetal untreated group; (D) fetal siRNA group. Chondrocytes were microphotographed at a magnification of 10 x 20. Diagram shows mean ± SEM percentage of SA-ß-gal-positive staining found in the various groups. *P<0.01 versus untreated OA, n = 6.

 
To determine the re-emergence of chondrocyte division in the absence of p16INK4a, cell proliferation was studied in OA and fetal chondrocytes in cultures. Since the percentage of viable chondrocytes freshly isolated from the different donors was different (fetal 91.95 ± 3.0% and OA 81.12 ± 7.63%), the average rate of proliferation of different groups was tested as in Materials and methods—siRNA-treated OA chondrocyte, 3.40; untreated OA chondrocytes, 1.68; siRNA-treated fetal chondrocytes, 3.75; untreated fetal chondrocytes, 3.48 at 72 h post-transfection. The statistical analysis shows that OA chondrocyte proliferation is affected by silencing of p16INK4a (Fig. 8) (P<0.01) and reaches a similar level to fetal chondrocytes (P>0.02), on which silencing of p16INK4a by siRNA has a limited influence (P>0.1). These were further confirmed with DNA synthesis assay using 3H-thymidine (Fig. 11A) (P<0.01) and Hoechst 33258 staining of DNA (Fig. 11B) (P<0.01). However, buffer and negative-control reagents have no obvious effect on either, and the similar results were revealed at other time points (data not shown). Taken together, the results suggest that silencing of p16INK4a by a p16INK4a-specific siRNA accelerated the entry of G0/G1-arrested OA chondrocytes into the S-phase of the cell cycle, thereby inducing the proliferation of previously senescent chondrocytes.



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FIG. 8. Effect of p16INK4a-specific siRNA on chondrocyte proliferation at 72 h post-transfection. Values represent mean ± SEM of six independent experiments. *P<0.01 versus untreated OA.

 


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FIG. 11. Rates of DNA, protein and proteoglycan synthesis and DNA content, in the presence and absence of cytokine on day 3 post-treatment with siRNA and mock reagents. From left to right within each group the bars represent cultures that did not receive cytokine treatment, followed by TGF-ß1 and IL-1{alpha} co-treated cultures in siRNA and control groups. Bars show mean ± SEM from triplicate labelled cultures. P<0.01 for siRNA-treated OA or fetal chondrocytes versus untreated OA controls. *P<0.01 for co-treated with TGF-ß1 or IL-1{alpha} versus no TGF-ß1 and IL-1{alpha} treated.

 
Since p16INK4a specifically binds to and inhibits the D-cyclin/CDK complexes that phosphorylate pRb, thus blocking G1 cell-cycle progression, the alteration of the phosphorylated form of pRb was also examined in our study. Western blot analysis with anti-pRb antibody showed two separate but closely migrating bands for all samples, the upper one representing the hyperphosphorylated pRb and the lower one the unphosphorylated pRb (Fig. 9A). The striking bands of phosphorylated pRb were observed in siRNA-treated OA chondrocytes and fetal controls. We also used a specific antibody for phospho-Rb to detect the alterations of the phosphorylated form and observed a similar level of phosphorylation in tested OA chondrocytes to fetal chondrocytes, which is considerably higher than that of mock-treated chondrocytes (P<0.01) (Fig. 8B). This was consistent with the amelioration of other senescence-associated markers examined above and provided compelling evidence that the p16INK4a/pRb pathway was functional in chondrocyte senescence.



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FIG. 9. Induction of Rb phosphorylation by p16INK4a silencing at a representative time point. pRbunphos: unphosphorylated form of the Rb protein. pRbphos: phosphorylated form of the Rb protein. Graphs show the mean ± SEM of three independent experiments. *P<0.01 versus control. Within each test group, analyses of pRbunphos, pRbphos levels were performed on the same protein samples.

 
Alteration of chondrocytic gene expression in OA chondrocytes by p16INK4a-specific RNAi
Phenotypic changes are a central feature of chondrocytes. Beside deactivation, phenotypic alterations represent the second potential reason for the anabolic failure of chondrocytes in OA cartilage. This is known from many studies of chondrocyte differentiation in vivo and in vitro, in particular in monolayer culture. Under these conditions, the chondrocytes stop expressing the cartilage-typical aggrecan and collagen type II, though they are still very active and synthesize collagen types I, III and V and are of typical spindle-like or stellate shape, so-called dedifferentiated chondrocytes. So, we performed expression analyses in test and control OA chondrocytes using the markers for chondrocyte differentiation: collagen I, collagen II and aggrecan. Semiquantitative RT-PCR readily permits parallel and direct comparisons of relative levels of expression among multiple samples in various normal or pathological conditions [46]. Accordingly, we used RT-PCR to detect the levels of collagen I, collagen II and aggrecan.

In siRNA-treated chondrocytes, the copy number of mRNA for collagen II and aggrecan was similar to that in fetal chondrocytes (P>0.01), exceeding that in mock-treated chondrocytes (P<0.01). In contrast, collagen I mRNA was expressed at similarly low levels within siRNA, mock-treated and fetal chondrocytes (Fig. 10.). These results verify that inhibition of p16INK4a recovered the expression of several chondrocytic genes on the mRNA level without detectable increases of type I collagen, which was beneficial to delay of dedifferentiation of chondrocytes, though dedifferentiation could not be avoided in our own study.



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FIG. 10. RT-PCR analysis for chondrocyte-specific mRNA expression 72 h post-transfection. Upper panels: collagen I, collagen II, aggrecan and ß-actin RT-PCR products. Lower panels: the corresponding quantitation (mean ± SEM from three independent experiments) normalized in relation to expression of ß-actin. *P<0.01 versus mock-treated controls. In each experiment, the total RNA in each group was extracted from the chondrocytes in four wells of a 24-well plate, and RT-PCR reactions were run in parallel.

 
The effect of p16INK4a-specific siRNA on the metabolic response of OA chondrocytes to cytokine stimulation
With increasing age or in OA pathological conditions, chondrocytes are less responsive to anabolic cytokines and much more sensitive to catabolic cytokines than young or normal chondrocytes, which suggests that older or degenerative articular cartilage is less able to repair and restore itself. To further explore the silencing effect of p16INK4a on the function of OA chondrocytes, chondrocytes from siRNA-treated and mock-treated OA groups as well as fetal samples were co-treated with TGF-ß1 or IL-1{alpha} for 24 h to demonstrate the specificity of the response to the cytokines. As shown in Fig. 11, TGF-ß1 induced significant increases (P<0.01) in 3H-proline (75.80%, 85.64%), 35S-sulphate (72.29%, 81.68%) and 3H-thymidine (82.17%, 91.04%) incorporation, and DNA content (46.86%, 50.66%) in siRNA-treated OA and fetal chondrocytes respectively compared with the mock-treated controls (<20% increase; P>0.05). Administration of IL-1{alpha} to the mock-treated cultures (untreated, buffer-only and negative control) resulted in significant (P<0.01) reductions of 3H-proline (45.73%, 45.62% and 48.68% respectively), 35S-sulphate (38.05%, 39.98% and 47.56% respectively) and 3H-thymidine (45.72%, 46.09% and 59.91% respectively) incorporation, and DNA content (26.12%, 23.33% and 28.36% respectively) as compared with non-significant (P>0.05) decreases of 3H-proline (11.92%, 10.86%), 35S-sulphate (12.55%, 7.34%), 3H-thymidine (15.16%, 10.18%) and DNA content (12.58%, 10.52%) respectively in siRNA-treated OA and fetal chondrocytes. These results suggest that siRNA-treated OA chondrocytes become more sensitive to TGF-ß1 and less sensitive to IL-1{alpha}, which is similar to the properties of fetal chondrocytes in our own study, than mock-treated chondrocytes. The rate of 3H-proline, 35S-sulphate and 3H-thymidine incorporation was normalized in relation to the cellular DNA content, which showed that this increased synthesis was not due to cell proliferation [47].


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Senescence is a feature of all mitotic cells studied to date, although the mechanisms that cause senescent growth arrest vary with cell lineage and type. It is clear that human cells undergo replicative senescence due to the acquisition of one or more critically short telomeres. However, recent findings suggest that telomere shortening is not the only inducer of the senescent phenotype. Senescence response may be a fail-safe programme for suppressing tumorigenesis against DNA damage or inappropriate mitogenic signals [22].

It is now generally accepted that OA lesions are often localized to weight-bearing cartilage or to sites of trauma. Mechanical compression and repetitive mechanical injury has been proposed as the critical signal for the initiation and progression of OA [4852]. All these abnormal biomechanical factors can stimulate the rate of chondrocyte turnover and result in DNA damage or/and free radical production, thereby altering the normal functional activities of chondrocytes. Telomere shortening observed in the study described by Martin and Buckwalter [9] was possibly at least partially a result of acceleration of chondrocyte turnover rather than a cause of OA, and the shortening could not rule out the possibility that other processes such as oxidative stress and damage to DNA may have induced senescence in OA before telomere erosion, even though exogenous expression of telomerase was able to increase the lifespan of OA chondrocytes in vitro [37]. Studies in various cell types have suggested that p16INK4a may accumulate in response to particular forms of stress, including cumulative oxidative damage [5355].

We reported here, for the first time, that the p16INK4a senescence gene was more highly expressed in older subjects than young subjects, and further that it was even more highly expressed in the pathological condition of OA. This may indicate that p16INK4a is physiologically involved in the processes of chondrocytic aging, and may be responsible, at least in part, for the senescence of chondrocytes seen in OA.

To further elucidate the role of p16INK4a in the biological activity of chondrocytes and in the overall metabolism of cartilage, siRNA technology was used to decrease p16INK4a expression in the present study. Although RNAi cannot replace a gene knockout experiment in which both alleles are deleted cleanly from the genome, it nevertheless produces hypomorphic mutants that are extremely useful for understanding gene functions, and also plays an important role in therapeutic applications for a number of diseases [56].

It has been postulated that the levels of siRNA knockdown depend on the efficiency of the transient transfections used to introduce the duplexes into the test cells. To monitor this, we examined p16INK4a protein level by Western blot and mRNA level by RT-PCR level and found that expression of p16INK4a was obviously less in siRNA-transfected cells than in mock-transfected cells on both levels. Both methods also indicated good knockdown by the positive control siRNAGAPDH, further indicating that the system worked correctly and with good efficiency. All data suggested that in our own study, the p16INK4a-specific siRNA was efficiently taken up by primary cultured human chondrocytes. In general, the transfection efficiency of plasmid DNA into primary cells is extremely low. Therefore, siRNA provides many advantages for biological studies and clinical applications.

Following the silencing of p16INK4a gene expression, OA chondrocytes showed increases in DNA synthesis and cell growth rate. By contrast, staining for SA-ß-gal, which is expressed by senescent cells, showed a significant decrease. Also, the p16INK4a-specific siRNA efficiently promoted the phosphorylation of pRb in siRNA-treated OA chondrocytes compared with mock-treated OA chondrocytes, a further observation that verified that p16INK4a functions as a key factor in the blockade of Rb protein phosphorylation in senescent chondrocytes. These facts suggested that p16INK4a silencing by siRNA led to increased phosphorylation of pRb, accelerated entry of G0/G1-arrested OA chondrocytes into the S-phase of the cell cycle and improved the senescent feature of OA chondrocytes.

Although p14ARF, an alternative reading frame protein encoded by the INK4a locus, seems also to be important for cell senescence, our data presented here, which demonstrated no evidence for p14ARF activation and inactivation, strongly suggest that the siRNAs used in our study selectively perturbed functions related to p16INK4a without directly altering p14ARF itself.

An increased understanding of differential mRNA expression could be important in our understanding of chondrocyte responses in situations leading to OA development. Accordingly, we examined mRNA levels for several relevant molecules (collagen I, collagen II and aggrecan) that play known roles in maintaining cartilage function. While not all-inclusive, the molecules assessed are representative of cellular activities known to influence cartilage function. Although it is difficult to avoid dedifferentiation of chondrocytes under monolayer culture, the results from our experiment indicated that p16INK4a silencing in OA chondrocytes is a potent stimulator of the synthesis of the two most abundant cartilage-specific matrix molecules, type II collagen and aggrecan, by chondrocytes. The parallel increases indicate a balanced up-regulation of cartilage-specific matrix synthesis, which is beneficial to keeping the chondrocytic phenotype as culture proliferation increases.

Irrespective of the initiating event, OA is caused by an imbalance between anabolic and catabolic processes, which results in a slow but progressive destruction of articular cartilage. These lesions have been attributed to a loss of tissue integrity due to metabolic and structural changes, and may reflect the altering ability of chondrocytes to respond to various stimuli and stresses in the aging organism. Chondrocyte activation and the induction of anabolic or catabolic responses are thought to be primarily the functions of cytokines and growth factors. Pro-inflammatory cytokine has been shown to contribute to the accelerated damage of articular tissue by inducing proteolytic enzymes, interfering with the activity of growth factor or decreasing the synthesis of key matrix components and amplifying the inflammatory response. In contrast, growth factors have the potential to act as mitogens to stimulate cell proliferation and stimulate production of ECM and protease inhibitors.

IL-1 and TGF-ß are considered to be the most important catabolic and anabolic factors respectively in joint diseases. IL-1 could stimulate production of matrix metalloproteinases (MMPs) and NO, and lead to proteoglycan (PG) breakdown and synthesis reduction. However, TGF-ß is known to enhance PG synthesis, decrease NO synthesis and counteract the effects of IL-1 on cartilage. Additional evidence revealed that the effect of TGF-ß on the anabolic pathway appeared to be more pronounced in young cartilage than old cartilage [5759]. By contrast, IL-1 appears to be more stimulatory on catabolic pathway in cartilage from old animals compared with young animals [57, 59]. One explanation is that old cartilage shows a very strong induction of NO, an important second mediator of IL-1, after IL-1 stimulation compared with younger cartilage. Meanwhile, old cartilage itself produces a significant amount of IL-1, which could disturb the TGF-ß signalling pathway, resulting in the loss of TGF-ß-mediated inhibition of NO synthesis [59]. In addition, TGF-ß has been reported both to inhibit and stimulate proliferation of articular chondrocytes in monolayer cultures [60, 61]. In these previous studies, TGF-ß enhanced the proliferation of actively dividing cells, a high percentage of which were in the S phase. In contrast, it inhibited proliferation of slowly dividing cells.

Interestingly, our results showed that silencing of p16INK4a by siRNA triggered a profound modification of the genetic response to anabolic and catabolic cytokines in human OA chondrocytes. For the cytokine induction, IL-1{alpha} and TGF-ß1 were introduced to all cultures. The results obtained in our own study indicated that in p16INK4a-specific siRNA-treated OA chondrocytes, TGF-ß1 can strikingly stimulate matrix synthesis; by contrast, mock-treated chondrocytes have limited ability to increase matrix synthesis via TGF-ß1. However, IL-1 inhibited the synthesis of PG, collagen and DNA in both test and control OA chondrocytes, and the effect appeared to be more pronounced in the controls. All effects of TGF-ß1 and IL-1 on siRNA-treated chondrocytes are similar to fetal chondrocytes. These are probably due to the facts that p16INK4a silencing promoted OA chondrocyte entry into S phase and division and the function of OA chondrocytes becomes close to that of younger cells.

All this suggests that silencing of p16INK4a decreases the response of OA chondrocytes to catabolic cytokines and increases the response to anabolic growth factor, consistent with the observations as stated above that young chondrocytes are more able to respond to TGF-ß than to IL-1, thus favouring stimulation of catabolic pathways in chondrocytes from old cartilage versus those of young cartilage. This modification may allow chondrocytes to adapt to the internal environment of OA joints, where various cytokines act on chondrocytes, and may thereby be potentially useful for possible future treatment of OA.

Many researchers have hypothesized [29, 62, 63] that the accumulation of p16INK4a mRNA with each population doubling in vitro may lead to cell senescence, since p16INK4a mRNA is extremely stable. However, in our own study, p16INK4a expression was clearly detected in the normal age-matched samples, all of which lacked clinical symptoms of OA. This suggests that chondrocytes may require sufficient amounts of nuclear p16INK4a to induce cell-cycle arrest and senescence leading to OA, and/or that another mechanism may exist in OA patients to further increase p16INK4a overexpression. This is supported by the observation that senescent OA chondrocytes showed even higher expression levels of p16INK4a when compared with chondrocytes from age-matched normal controls.

The major findings of the present study refer to the effects of transient exposure of monolayer-cultured chondrocytes to p16INK4a-specific siRNA. The inevitable dedifferentiation and short-term effects, although biologically important, may not be relevant to the situation in vivo, where long-term effects are of great importance. Thus, future studies will be required to examine the long-term situation, such as application of siRNA expression vectors, in vivo.

In conclusion, although the precise mechanism of action remains to be determined, it is likely that p16INK4a silencing in OA chondrocytes can help recover certain beneficial biological functions. Our results reversely support the notion that p16INK4a acts as a suppressor to mediate the senescence of chondrocytes, thereby activating the onset of OA. Furthermore, p16INK4a RNAi technology could provide many advantages for biological studies in the pathological process of osteoarthritis and a useful theoretical basis for future therapy of OA.


    Acknowledgments
 
We are grateful to Professor Tanjun Tong for excellent technical assistance.

The authors have declared no conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 2 August 2003; revised version accepted 5 December 2003.



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