Department of Advanced Medicine for Spine and Spinal Cord Disorders, Hokkaido University Graduate School of Medicine, North-15, West-7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan
Department of Advanced Medicine for Spine and Spinal Cord Disorders, Hokkaido University Graduate School of Medicine, North-15, West-7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan. T: 81-11-706-5934; F: 81-11-706-6054.
Intervertebral disc degeneration is thought to be associated with genetic factors as well as excessive mechanical loading, altering the biomechanical properties of the intervertebral disc.1, 2 Although the precise molecular biological mechanism of disc degeneration is still unclear, previous studies have suggested that apoptosis or programmed cell death of disc cells may play one of the most important roles in disc degeneration.3–5
Apoptosis plays a central role in the homeostasis of all tissues during normal development and tissue turnover, and two major signaling pathways control the initiation of apoptosis in mammals. The extrinsic pathway involves engagement of cell-surface cell death receptors by ligands that belong to the tumor necrosis factor-receptor superfamily and consequent activation of caspase-8. The intrinsic pathway involving caspase-9 as the initiator originates from mitochondria. Stressed mitochondria release a set of molecules, including cytochrome c and Apaf-1, to form the apoptosome molecular cluster that activates caspase-9 and its downstream effector caspase-3. Moreover, crosstalk between the death receptor and mitochondrial pathways is mediated by caspase-8 cleavage of the protein Bid,1, 6 which contains a Bcl-2 homologous domain 3.
Park et al.5 examined human herniated lumbar disc tissues with the use of immunohistochemical staining and Western blot analysis to determine the presence of several proteins associated with apoptosis. They found that the proteins associated with the intrinsic pathway stained positively in all samples, and the expression of intrinsic pathway proteins was higher than the expression of extrinsic pathway (caspase-8) on Western blot analysis. The results of their study suggested that disc cells participate in the intrinsic pathway, which subsequently undergo apoptotic cell death through mitochondrial involvement. Among the intrinsic pathway, bcl-2 prevents or delays apoptotic induction by a large variety of stimuli in many cell types.7 Molecular intervention at the level of bcl-2 in the apoptotic pathway, therefore, has the potential to enhance cell survival.
There are several strategies to prevent apoptotic cell death. Gene therapeutic approaches have the potential to provide sustained and local endogenous synthesis of potential therapeutic proteins that undergo certain posttranslational processing. The purposes of this study were to investigate whether bcl-2 overexpression in rat intervertebral disc cells was effective in preventing serum starvation-induced apoptotic cell death and to discuss the potential advantages of this approach to provide a therapeutic benefit in regulating disc degeneration.
All animal procedures were performed under the guidance of our animal research committee. Rat nucleus pulposus cells were isolated as previously reported with modifications.8 Lumbar intervertebral discs from male Sprague–Dawley rats (age 13 weeks) were harvested immediately after they were killed. The gel-like nucleus pulposus was separated from the annulus fibrosus using a dissecting microscope and the tissue specimens were placed in a complete tissue culture medium consisting of Dulbecco's modified Eagle's medium (DMEM; Sigma–Aldrich, St. Louis, MO), supplemented with 10% fetal bovine serum (FBS; BioWhittaker, Inc., Walkersville, MD), 1% penicillin/streptomycin, and 1.25 µg/ml Fungizone (Invitrogen, Carlsbad, CA). Any dense annulus tissue or cartilaginous endplate tissue was carefully removed. After the specimens were centrifuged twice at 1,000 rpm for 3 min, they were then treated with DMEM supplemented with 0.25% collagenase in a shaking incubator at 37°C for 30 min and centrifuged twice at 1,000 rpm for 3 min. The cells released from the matrix were placed in 6-cm tissue culture dishes and incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide. When confluent, the cells were detached using 0.25% trypsin/EDTA (Invitrogen) solution and subcultured on 10-cm dishes.
Coding sequence of rat bcl-2 (NM_016993) was codon optimized for the expression in Rattus norvegicus and synthesized by GENEART (Regensberg, Germany) in an effort to enhance the protein expression.9 Repeat sequences, regions of very high (>80%) or very low (<30%) GC content, and other several negatively cis-acting motifs that might hamper expression in mammals were removed without alterations within the encoded protein.10 In addition, a Kozak consensus sequence was introduced to increase translational initiation11 and two stop codons were added to ensure efficient termination (Fig. 1). The synthesized gene, designed with BamHI/HindIII restriction sites, was cloned into a pGA4 vector. The resulting plasmid was purified and sequence verified. The insert was digested with restriction enzymes BamHI and HindIII, loaded onto an agarose gel and purified with the DNA gel extraction kit (Qiagen, Hilden, Germany). The purified fragment was subcloned into a pBApo-CMV vector cut with the same restriction enzymes. Finally, high-quality plasmid DNA, pBApo-CMV-bcl2, was isolated using PureYield™ Plasmid Midiprep System (Promega, Madison, WI) according to the manufacturer's instructions. A control plasmid vector that did not contain the bcl-2 gene was also generated. Transfection was carried out with 1 µg recombinant plasmids premixed with 3 µl TransIT-LT1 (Mirus Bio, Madison, WI) in 100 µl Opti-MEM (Invitrogen) using 6 cm dishes (3 × 105 cells), according to the manufacturer's instructions.
Forty-eight hours after transfection, cells were washed with phosphate-buffered saline (PBS; Invitrogen) followed by two washes with DMEM to remove any remaining culture medium and incubated in serum-deprived medium which consisted of DMEM supplemented with 1% penicillin/streptomycin and 1.25 µg/ml Fungizone (Invitrogen) at 37°C with 5% CO2. At various time points (6 and 48 h) after serum withdrawal, the cells were harvested and analyzed. Nucleus pulposus cells that had not undergone either transfection or serum starvation were used as untreated controls and untransfected serum-starved cells were described as “serum-starved only” cells.
All extraction procedures were performed at 4°C or on ice. Cells were lysed with 0.1 ml of ice-cold radio immunoprecipitation assay (RIPA) buffer (Pierce Biotechnology, Rockford, IL) containing protease inhibitor cocktail (Roche, Basel, Switzerland), which was added immediately before use. Cell lysates (10 µg protein/lane) were loaded and separated on 5–20% sodium dodecyl sulfate gradient gel and transferred to polyvinylidene difluoride membranes by electroblotting. After blocking with 5% nonfat milk containing 0.3% Tween-20 (Bio-Rad Laboratories, Hercules, CA) overnight at 4°C, the membranes were incubated overnight with antibodies to bcl-2 (1:200, catalog number, SC-7382; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C. The membranes were washed three times with Tris-buffered saline–Tween-20 and further incubated with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:2,000) for 1 h. The membrane was then exposed to an enhanced chemiluminescent system, and charge-coupled device (CCD) image analyzer was used to visualize immunoreactive bands. β-Actin (1:2,000, antibodies to β-actin was from Applied Biological Materials, Inc., BC, Canada) was used as an internal control for protein loading.
To investigate the effects of overexpression of Bcl-2 on the final pathways of apoptosis and on basic genes important for nucleus pulposus homeostasis, messenger RNA (mRNA) levels of caspase-3, type II collagen, and aggrecan were quantified by real-time RT-PCR. Total RNA extraction from nucleus pulposus cells was performed with the FastPure™ RNA Kit (TaKaRa Bio, Otsu, Japan). RNA was reverse transcribed into cDNA and real-time PCR was performed with SYBR PrimeScript® RT-PCR Kit (TaKaRa Bio). Primers for rat caspase-3, type II collagen, aggrecan, and glyceraldehyde phosphate dehydrogenase (GAPDH) were custom designed (Table 1) and synthesized. To confirm amplification specificity, the PCR products were subjected to a melting curve analysis.12 A positive standard curve for each primer was obtained by real-time PCR with serially diluted cDNA samples. The data were analyzed using the Thermal Cycler Dice® Real-Time System (TaKaRa Bio). Relative mRNA expression of the target genes per GAPDH was calculated using the comparative threshold cycle (Ct) method.12 The numerical value for caspase-3 mRNA was compared to an untreated control sample at 6 h and the values for type II collagen and aggrecan mRNA were compared to an untreated control samples at 48 h after serum withdrawal, respectively, and finally reported as a ratio (%) to control.
Table 1. Sequences of Primers for Real-Time Reverse Transcription-Polymerase Chain Reaction Analysis
GAPDH, glyceraldehyde phosphate dehydrogenase.
Type II collagen (alpha 1) (forward)
Type II collagen (alpha 1) (reverse)
Apoptotic cells were detected based on the principles of annexin V binding to translocated membrane phosphatidylserine (PS).13 Fluorescein isothiocyanate (FITC)-labeled annexin V was added to cultured cells and bound to exposed PS. FITC signals were detected by flow cytometry. Propidium iodide (PI) was added to cultured cells to identify the loss of integrity of the cell membrane. Annexin V-Fluorescein Staining Kit (Wako Pure Chemical Industries, Osaka, Japan) was used in this analysis of apoptosis. Cells from each treatment were detached using 0.25% trypsin/EDTA (Invitrogen) solution and centrifuged at 1,500 rpm for 5 min. After the cell pellets were centrifuged again with PBS, they were resuspended and incubated for 10 min in 100 µl binding buffer containing annexin V-FITC and PI according to the manufacturer's instructions. Apoptosis was monitored by a flow cytometer (COULTER EPICS XL Flow Cytometer; Beckman Coulter, Inc., Fullerton, CA). FITC−/PI−, FITC+/PI−, and FITC+/PI+ represented a viable cell condition (intact cell), first stage of apoptosis (early apoptotic cell), and completed apoptotic cell death (late apoptotic cell), respectively.4 In the present study, apoptotic cells included both early and late apoptotic cells.
All values in the text and figures were expressed as mean ± standard deviation of eight observations. ANOVA with subsequent use of Student–Newman–Keul's method was performed to determine the significance of the difference in multiple comparisons. p-Values of <0.05 were accepted as significant.
To examine the role of Bcl-2 protein induction by pBApo-CMV-bcl-2, pBApo-CMV-bcl-2 was transfected into rat nucleus pulposus cells. After 48 h post-transfection with pBApo-CMV (negative control plasmid vector) or pBApo-CMV-bcl-2, the cells were serum starved. Western blot analyses with an anti-Bcl-2 specific antibody demonstrated strong induction of Bcl-2 expression in cells transfected with the pBApo-CMV-bcl-2 plasmid, harvested at both 6 and 48 h after serum starvation. This result indicated that transfection of optimized Bcl-2 gene increased protein level of Bcl-2 in nucleus pulposus cells (Fig. 2).
Flow cytometric analyses demonstrated that serum withdrawal significantly increased apoptotic rates in the nucleus pulposus cells compared to untreated control cells in the early stage (6 h) after serum starvation. At 48 h after serum removal, further increases in apoptotic cells were observed in both serum-starved only and negative control plasmid vector groups compared to the untreated control group. Conversely, significant inhibition of apoptosis was seen in bcl-2-transfected group compared to the other serum-starved groups (Fig. 3).
To investigate the effects of bcl-2 on caspase-3, a key mediator of apoptosis, mRNA levels of caspase-3 were measured. At 6 h after serum removal, there was a significant increase in caspase-3 mRNA compared to untreated controls. Conversely, at 48 h after serum withdrawal in both serum-starved and control plasmid vector groups, there remained significantly higher caspase-3 mRNA levels compared to untreated control groups. However, caspase-3 mRNA was significantly reduced in cells transfected with bcl-2 compared to other serum-starved groups (Fig. 4).To investigate whether the cells overexpressing Bcl-2 alter the expression of basic genes important for nucleus pulposus homeostasis, mRNA levels of type II collagen and aggrecan were measured at 48 h after serum withdrawal. mRNA levels of type II collagen and aggrecan were significantly decreased in bcl-2-transfected and control plasmid vector groups compared to untreated controls. However, there remained significantly higher type II collagen and aggrecan mRNA levels in bcl-2-transfected groups compared to control plasmid vector groups (Fig. 5).
Some studies have demonstrated that efficient gene transfer into intervertebral disc cells can provide local, sustained endogenous synthesis of therapeutic proteins aimed at restoring the degenerated intervertebral discs.14, 15 Recently, gene therapy has proven its ability to modulate disc cell the biological processes. However, as Evans pointed out,16 successful gene transfer requires the presence of an adequate number of responding cells because disc degeneration is typically accompanied by the loss of disc cell cellularity. Therefore, inhibiting the active cell death of the endogenous cell population is required to restore cell numbers to make gene therapy practical.16 The present study evolved with the aim of regulating apoptosis to regulate the processes of disc degeneration.
There have been no reports regarding transfer of the bcl-2 gene into the intervertebral disc cells. In the current study, rat nucleus pulposus cells were successfully transfected with bcl-2, which effectively reduced apoptotic cell numbers that were induced by serum starvation. These new findings confirm our proof-of-principle to demonstrate that the regulation of apoptotic cell death is possible via exogenous bcl-2 overexpression in nucleus pulposus cells.
Although the apoptotic cascade is not yet fully understood, overexpression of bcl-2 has previously been shown to prevent the release of apoptotic induction factors and the subsequent activation of caspase-3.17 The apoptotic pathway has not been studied in the intervertebral disc cells. This study clearly revealed that in vitro overexpression of bcl-2 reduced caspase-3 mRNA levels in rat nucleus pulposus cells. Caspase-3, the most prominent of all effector caspases, is localized downstream in the caspase cascade and represents the main effector molecule of apoptosis and irreversibly executes programmed cell death. The results of this study indicated that bcl-2 was also effective in suppressing the later stages of programmed cell death in nucleus pulposus cell, which confirmed previous observations using other cells.7, 17
There have been many studies using genetically engineered virus vectors that have proved effective for gene transfer to various cells, including the intervertebral disc.14–16 For effective clinical treatment, we must avoid the high biological risk associated with severe viral immune reactions seen with the adenoviral vectors. Furthermore, there is a possibility of permanent integration and transgene expression when these genes are introduced using retroviral vectors. As bcl-2 is a proto-oncogene and is known to be highly expressed in many human tumors,18 the integrating viral vector approach may be associated with the long-term oncogenic potential of the treatment. The current study used nonviral vector that exclude many of the potential risks associated with viral gene transfer. These cationic lipid reagents deliver genetic material into a cell by fusing with the cell's phospholipid membrane and fail to integrate in the genome of target cells. As transgenic expression from a plasmid vector alone has a limited expression window, transfer of the bcl-2 gene may enhance anti-apoptotic cell survival with less risk, and increased clinical benefit.
In this study, mRNA levels of type II collagen and aggrecan were significantly higher in bcl-2-transfected groups compared to control plasmid groups at 48 h after serum withdrawal. On the other hand, in complete tissue culture medium conditions, there was no difference of type II collagen and aggrecan mRNA levels among untreated control, control plasmid, and bcl-2 groups (data not shown). These results indicate that bcl-2-transfected cells did not produce more type II collagen and aggrecan but just resisted the serum-starved condition. Hence, the overexpression of Bcl-2 did not alter or hamper the expression of basic genes important for nucleus pulposus homeostasis. When designing bcl-2 disc gene therapy, direct injection of bcl-2 genes may be conducted by in vivo strategies in a similar way that genes encoding growth factors have been injected into intervertebral discs in certain animal models.14, 16 As an alternate approach, optimized disc cells or progenitor cells such as bone marrow-derived stromal cells may also be directly introduced into the disc in an ex vivo situation. In this situation, bcl-2 gene transfer may be used as a promising candidate gene during the initial experimental stages as groundwork for successful cell therapy by preventing apoptotic disc cell death and enhancing cell viability and number. In a clinical setting, the most available source of cells is the tissues obtained in surgeries to treat disc herniation and degenerative disc disease. Because the degenerative disc exists in a harsh environment that is acidic, hypoxic, and poor in nutrients, beneficial transplants may have to be preconditioned by genetic manipulation in order to survive and restore normal matrix components under these conditions.16
Using rabbit models, several reports have documented that more than 1 year of transgene expression has been achieved with some recombinant viral vectors.14, 16 The immunologic isolation of the disc and the low rate of cell division within it presumably account for these findings.16 However, it is not yet clear that similar outcomes have been achieved in the degenerating disc. We observed transient exogenous bcl-2 overexpression that was sustained for 7 days under disc cell maintenance in complete tissue culture medium conditions (data not shown). However, in the degenerate human disc, there is a paucity of disc cells and most of the cells are necrotic that are unresponsive for repair with a gene therapy. The limitation of this study is that it was impossible to demonstrate that already advanced degenerative disc was treated by this method. In addition, conditions in the advanced degenerate disc may not be favorable for this approach, particularly if the nutritional pathway has been compromised by endplate calcification. In such case, the present strategies would be limited in their use to treat degenerative disc disease and would be replaced by other methods such as disc arthroplasty.
In conclusion, the present study was conducted in an attempt to inhibit active cell death to make subsequent gene therapy a more practical concept because disc degeneration is typically accompanied by the loss of disc cells numbers. This new approach to combat degenerative disc disease has not previously been documented and there have been no reports regarding the transfer of the bcl-2 gene into the intervertebral disc cells. We think that this study has important implications in the further development of suitable therapies for degenerative disc disease and provides valuable new information about a novel approach to treat or delay disc degeneration.
This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (17790991), the Uehara Memorial Foundation, and the Nakatomi Foundation.