MMP-2 mediates local degradation and remodeling of collagen by annulus fibrosus cells of the intervertebral disc
© Rastogi et al.; licensee BioMed Central Ltd. 2013
Received: 19 June 2012
Accepted: 27 April 2013
Published: 27 April 2013
Degeneration of the intervertebral disc (IVD) is characterized by marked degradation and restructuring of the annulus fibrosus (AF). Although several matrix metalloproteinases (MMPs) have been found to be more prevalent in degenerate discs, their coordination and function within the context of the disease process are still not well understood. In this study, we sought to determine whether MMP-2 is associated with degenerative changes in the AF and to identify the manner by which AF cells use MMP-2.
Two established animal models of disc degeneration, static compression and transannular needle puncture of rodent caudal discs, were examined for MMP-2 immunopositivity. With lentiviral transduction of an shRNA expression cassette, we screened and identified an effective shRNA sequence for generating stable RNA interference to silence MMP-2 expression in primary rat AF cells. Gelatin films were used to compare gelatinase activity and spatial patterns of degradation between transduced cells, and both noninfected and nonsense shRNA controls. The functional significance of MMP-2 was determined by assessing the ability for cells to remodel collagen gels.
Both static compression and 18-g annular puncture of rodent caudal discs stimulated an increase in MMP-2 activity with concurrent lamellar disorganization in the AF, whereas 22-g and 26-g needle injuries did not. To investigate the functional role of MMP-2, we established lentivirus-mediated RNAi to induce stable knockdown of transcript levels by as much as 88%, and protein levels by as much as 95% over a 10-day period. Culturing transduced cells on gelatin films confirmed that MMP-2 is the primary functional gelatinase in AF cells, and that MMP-2 is used locally in regions immediately around AF cells. In collagen gels, transduced cells demonstrated an inability to remodel collagen matrices.
Our study indicates that increases in MMP-2 observed in human degenerate discs are mirrored in experimentally induced degenerative changes in rodent animal models. AF cells appear to use MMP-2 in a very directed fashion for local matrix degradation and collagen remodeling. This suggests that MMP-2 may have a functionally significant role in the etiology of degenerative disc disease and could be a potential therapeutic target.
KeywordsIntervertebral disc Matrix metalloproteinase Collagen Remodeling RNA interference
Various physiologic and pathophysiologic processes of connective tissues involve cell-mediated matrix-remodeling events, including tissue growth, repair, and degeneration. During remodeling, extracellular matrix (ECM) turnover rates are increased , causing shifts in tissue architecture and composition. Although a number of molecules are thought to regulate this process, matrix metalloproteinases (MMPs) are believed to be particularly important in ECM degradation [2–4]. In addition to being the key mediators of tissue remodeling, MMPs are also known to be involved in cell proliferation, migration, differentiation, angiogenesis, apoptosis, and host defense .
A notable example of chronic pathophysiologic remodeling occurs in the intervertebral disc (IVD) of the spine during degenerative disc disease (DDD). In the annulus fibrosus (AF), there is an acceleration of age-related matrix changes, which are thought to be caused by enhancement of catabolic processing of the ECM [3, 4, 6, 7]. Several MMPs have been shown to be expressed at elevated levels in the AF of diseased discs . One of these is MMP-2, a gelatinase that participates in the secondary breakdown of collagen during remodeling [2, 4, 8, 9]. Activation of MMP-2 above endogenous baseline levels has been found to be induced by mechanical stress [10, 11]. However, the functional roles of MMP-2 in disc health and degeneration remain unclear. An improved understanding of the molecular mechanisms involved in DDD would contribute to the development of interventional strategies  to mitigate degenerative processes.
In recent years, RNA interference (RNAi) has become a powerful and accessible tool for manipulating cellular function, particularly with the use of small RNAs for sequence-specific gene silencing [12, 13]. Although extensive studies are required to elucidate unanticipated effects, the capability of RNAi to induce both transient and stable silencing [12, 14] contribute to its promise as a technology for treating human disease.
We had previously observed that mechanical overloading of murine IVDs results in altered MMP-2 activation patterns [10, 15]. As a first step toward improving our understanding of MMPs in DDD, this study demonstrates that, in a rat annular-injury model of degenerative changes, discs similarly exhibit an upregulation of MMP-2. To investigate the functional implications of MMP-2, we then validated an approach to silence MMP-2 gene expression by using lentivirus-mediated RNAi in rat AF primary cell cultures. We found that MMP-2 silencing inhibited degradation of gelatin immediately surrounding cells and impaired the ability of cells to remodel collagen gels. To our knowledge, this is the first study to use small-hairpin RNAs (shRNAs) to achieve stable knockdown of MMP-2. Results suggest that MMP-2 is used locally by cells to drive changes in ECM structure and function.
Materials and methods
Sprague-Dawley rats (6 to 9 months old; Taconic Farms, Germantown, NY, USA) were subjected to annular puncture to induce degenerative changes , after approval from the Institutional Animal Care and Use Committee at the University of Maryland, College Park. Rats were anesthetized by using isoflurane, and the c6-7 motion segment was identified, marked, and confirmed by using fluoroscopy. A subcutaneous injection of analgesic (buprenorphine, 0.03 mg/0.1 ml) and an intramuscular injection of antibiotic (fluoroquinolone, 2.5 mg/0.1 ml) were given to the rat. The diameter of the tail at the puncture site was measured with calipers to determine the necessary depth of needle insertion. After establishing a sterile field and scrubbing with povidone/iodine (Betadine)-isopropyl alcohol, a cranial-caudal skin incision was made on the dorsal aspect of the tail in the proximity of c6-7. Soft tissues were parted to identify the c6-7 disc, and a sterile tapered hypodermic needle (26-g, 22-g, or 18-g) was marked to the depth corresponding to the radius of the tissue, and then carefully inserted by hand. Fluoroscopy was used to confirm proper position and depth. For sham surgeries, the skin incision was made, but discs were left uninjured. Incisions were then sutured closed. After surgery, rats were revived and returned to normal cage activity for 2 weeks, with daily observation for pain and distress. Rats were initially given an injection of analgesic (buprenorphine, 0.03 mg/0.1 ml), after which analgesic (buprenorphine at 0.05 mg/kg rat), antiinflammatory (Carprofen at 5 mg/kg rat), and antibiotic (cephalexin at 60 mg/kg rat) were administered, if necessary, in accordance with our pain/distress assessments and consultation with the veterinarian.
After 2 weeks, the rats were killed by carbon dioxide asphyxiation (6 to 8 L/min until loss of consciousness, followed by 12 L/min for 10 minutes) and bilateral thoracotomy. Motion segments were then dissected from tails, and then fixed in formalin, decalcified in a solution containing 10% (wt/vol) sodium citrate and 20% formic acid, and processed in graded ethanol and xylene baths before paraffin embedding (TP1020/EG1160; Leica Microsystems, Buffalo Grove, IL, USA). Paraffin blocks were sectioned at 6 μm by using a microtome (HM355; Microm/Thermo Fisher Scientific, Waltham, MA, USA). Slides containing serial sections close to the midsagittal regions of punctured or control discs were deparaffinized and rehydrated. Sections were blocked with goat serum for 1 hour, washed, and then incubated with primary antibody against MMP-2 (clone SPM346; Abcam, Cambridge, MA, USA) at either 0 (no Ab control), 1, or 10 μg/ml. A biotin-labeled goat anti-mouse IgG secondary antibody was used with horseradish peroxidase-conjugated streptavidin (Vectastain ABC; Vector Laboratories, Burlingame, CA, USA), and 3,3 -diaminobenzidine substrate to develop a color reaction. Each batch of 26-g, 22-g, 18-g, and nonpunctured discs was immunostained concurrently under identical conditions, and images were captured under identical exposure conditions to ensure valid qualitative comparisons of staining intensity.
Primary cell isolation and culture
Healthy AF tissues were freshly harvested under sterile conditions from Sprague-Dawley rat caudal discs immediately after euthanasia, performed as described earlier according to procedures approved by the Institutional Animal Care and Use Committee at the University of Maryland, College Park. Tissues were minced and digested overnight in culture media containing 3 mg/ml collagenase. Digests were then centrifuged, and cells were resuspended in fresh culture media and plated in tissue-culture flasks. Cells were cultured until passage 3 in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C with 5% CO2, with media changes 3 times per week.
In preliminary experiments, we found that rat AF cells express similar levels of types I and II collagen, sox9, and aggrecan during low-passage culture. Passage 3 cells were selected as a balance between adequate cell expansion and retention of cell phenotype.
Lentivirus construct preparation
Matrix metalloproteinase 2 (MMP-2) shRNA sequences
Top and bottom "stem-loop-stem" sequences, 5 to 3
AF cells were plated in six-well tissue-culture plates in complete media (DMEM, supplemented with 10% FBS and 1% penicillin-streptomycin; Gibco/Invitrogen, Carlsbad, CA, USA) at 50% confluence (150,000 cells/well). Each of the six viral constructs (shMMP2a-e, shNon) was added to one well each, at multiplicity of infection (MOI) of 0.1, along with 6 µg/ml Polybrene (Sigma, St. Louis, MO, USA) to facilitate transduction. Viral particles were removed the next day through aspiration of the culture medium, and replaced with complete culture medium. Blasticidin (Invitrogen) was added in appropriate samples to select for transduced cells the day after virus removal, at a concentration of 8 μg/ml, based on preliminary experiments of AF cell sensitivity to blasticidin. Media was changed every 3 days. In appropriate samples, each media change contained blasticidin. Each time point also included a corresponding noninfected control (plated cells not exposed to viral particles).
Three separate experiments were conducted to determine MMP2 expression for each of the five shMMP viral constructs. Experiment 1 was conducted to determine the mixed-population response. As such, cells were infected and harvested at 1, 4, 7, and 10 days after virus removal without selection. Experiment 2 examined the response of the pure transduced cell population. After infection, cells were harvested at 1, 4, 7, and 10 days under continuous blasticidin treatment. Experiment 3 assessed the stability of gene silencing by subjecting cells to a 10-day continuous blasticidin treatment, followed by culture in blasticidin-free complete media for an additional 1, 4, 7, and 10 days. At each time point, samples were collected in Tris-HCl, pH 7.5 to 8.0 by using a cell scraper. Each harvested sample was divided into three groups to measure (a) DNA content, (b) MMP-2 content, and (c) gene expression. Conditioned media was also harvested from samples for measurement of MMP-2 content. All samples were maintained at -80°C until use.
MMP-2 content was quantified by using a microplate-based activity assay (R&D Systems Inc., Minneapolis, MN, USA). One fourth (25%) of each harvested sample (see Cell transduction) was thawed, pulverized with a pestle, and then loaded into microplates along with conditioned media samples and standards. Protein levels of MMP-2 were obtained according to the manufacturer's protocol. Another one fourth (25%) of each harvested sample was subsequently thawed, subjected to five additional freeze/thaw cycles for cell lysis, and loaded into a microtiter plate along with standards generated from Lambda DNA. PicoGreen (Invitrogen) reagent was then added to all wells to measure DNA content. Absorbance (MMP-2) and fluorescence (DNA) measurements were made with a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA). All measured MMP-2 protein levels were normalized to DNA content from the corresponding sample, determined by using PicoGreen, to examine comparable protein knockdown for each population.
Sequences of primers used for RT-PCR analysis
Forward and reverse primers
GenBank accession no.
Length of product (bp)
A 5% (wt/vol) gelatin solution was pipetted onto glass slides and allowed to air dry. Once cooled, the slides were chemically crosslinked in 4% formalin for 1 hour at room temperature, washed extensively with sterile PBS, and then stored overnight in PBS at 4°C. The next day, the PBS was removed and slides were equilibrated in complete cell culture media for 30 minutes before cell seeding.
Separate populations of AF cells infected with the most effective of the five shRNA constructs against MMP2 (hereafter designated shMMP2), and the nonsense shRNA construct (shNon) were treated with blasticidin to obtain pure populations of transduced cells. Transduced cells (n = 4 for shMMP2 and shNon each) and noninfected control cells (n = 4) were seeded onto the films at densities of approximately 200,000 cells/film. The cell suspension was allowed to settle for 30 minutes before cell-culture media was added. Gelatin films without cells served as negative controls (n = 4). After 4 days in normal culture conditions, media was removed from the dish, and films were rinsed once with PBS, stained with 1% Ponceau S for 1 minute, and washed with DI water for 5 minutes on a shaker. Each replicate of gelatin films was stained in parallel and imaged immediately after staining under identical exposures to standardize comparisons. Extent of degradation of gelatin films was quantified by using Image J (National Institutes of Health, Bethesda, MD, USA). In brief, images were first segmented by thresholding to distinguish degraded (white) from nondegraded (red) regions. The areas of these regions were then used to calculate a percentage degradation equal to the ratio of degraded area to total area in each field of view. Statistical analysis (SPSS) was performed by using a Kruskal-Wallis test with Games-Howell post hoc analysis.
Three-dimensional type I collagen gels were made from rat-tail collagen (BD Biosciences, San Jose, CA, USA) at a concentration of 3 mg/ml by following the manufacturer's protocols. All components were prechilled and sterile. In brief, the collagen I stock solution was diluted in 10× PBS (Gibco/Invitrogen), sterile water, and 1N NaOH to achieve a final concentration of 3 mg/ml collagen in 1× PBS at pH 7.4. The collagen solution was kept on ice until use.
AF cells were infected with shMMP2 or shNon virus, as described previously. Two separate populations of cells were used for each of the two viruses, one of which was treated with blasticidin to obtain a pure population of only transduced cells, whereas the other was used as a mixed population of transduced and noninfected cells (n = 3 for each). Noninfected cells served as positive control (n = 3), with collagen gels without cells (n = 3) serving as negative control. Cells were trypsinized, pelleted, and resuspended in collagen solution at 500,000 cells/ml. The collagen-cell solution was pipetted into custom-made dog-bone-shaped molds (3 mm thick), fabricated to be 15 mm in width at the grips with a 5-mm-wide × 10-mm-long gauge region, placed in petri dishes. Polypropylene mesh (Small Parts, Inc., Logansport, IN, USA) with approximately 300-μm pore size was embedded in the collagen at each end of the mold for gripping. Collagen gel constructs were incubated for 90 minutes at 37°C to allow polymerization, and then placed in six-well plates in free-floating culture with complete media. Gels were cultured for 7 days, at which point, they were used either for mechanical testing to ascertain changes in material properties, or fixed for histology to evaluate structural changes.
Mechanical testing and histology
The short-term effective modulus (that is, as t → 0), E1 + E2; the long-term effective modulus (that is, as t →∞), E2; the viscous parameter, μ; and the stress at collagen-gel rupture were used for comparison among experimental groups. One-way analysis of variance with Tukey post hoc tests was performed (SPSS) to determine whether differences among groups were statistically significant (α = 0.05).
For histology, gels were fixed overnight in 10% buffered formalin. Samples were then dehydrated through sequential ethanol baths, cleared in xylene, and infiltrated with paraffin. Paraffin blocks were cut to obtain 6-μm sections, which were stained with Safranin-O/Fast green to evaluate changes in collagen structure.
Disruption of mechanical integrity of the AF enhances MMP-2 expression
MMP2 expression can be silenced in primary AF cells through stable RNA interference
To elucidate the role of MMP-2 in matrix organization, we set out to develop an experimental system for silencing MMP-2 in rat IVD cells to complement our in vivo studies. After constructing five lentiviral vectors for expressing shRNA to target MMP2, designated shMMP2a-e, and a nonsense control shNon (Table 1), we performed a preliminary screen to identify an effective silencing construct (data not shown). We examined the knockdown of MMP2 expression in (a) mixed (transduced and nontransduced) populations, (b) pure (blasticidin-selected) populations, and (c) long-term cultures of pure populations. All constructs, except shMMP2a, led to decreased mRNA and protein levels, as determined with qRT-PCR and MMP-2 activity assay, respectively. In Experiment 1, the mixture of transduced and nontransduced cells exhibited downregulation, peaking at 4 days after infection, but then progressively climbed higher through 10 days, presumably because of proliferation of nontransduced cells. In Experiments 2 and 3, downregulation remained more consistent over time. Overall, shMMP2c and d were more successful in knocking down gene expression compared with the other constructs, with up to 80% silencing observed for both. Protein levels for shMMP2b-e were all markedly lower than shNon controls. Based on the composite data, we selected shMMP2d (hereafter designated shMMP2) for use in subsequent experiments.
RNAi of MMP2 expression inhibits local matrix degradation in AF cells
Collagen remodeling is impaired in AF cells deficient in MMP-2
Despite the striking histologic differences in collagen microstructure observed in the shMMP2 group compared with both noninfected and shNon, MMP2 silencing did not correspondingly affect collagen gel properties on the larger scale. In terms of macroscopic appearance, blank gel construct size remained the same size through 7 days of culture, whereas gels embedded with noninfected AF cells contracted approximately 83% relative to their original size. Gels embedded with cells transduced with shMMP2 contracted to a lesser degree (18%) than did gels with cells transduced with shNon (31%).
This study demonstrated that MMP2 expression is stimulated in the AF of IVDs subjected to overload or puncture injury in rodent caudal discs, and strongly supports the notion that MMP-2 plays a significant functional role in the degenerative changes previously observed in both animal models [16, 19]. In particular, we showed that silencing MMP2 expression all but eliminated gelatinase activity in AF cells and profoundly compromised their ability to remodel collagen matrices. We also demonstrated that, similar to tumor invadopodia [20–23], MMP-2 preferentially functions in a localized region surrounding AF cells, rather than being secreted and acting at a distance. Contrary to our expectations, the impaired ability of AF cells to degrade and remodel their local microstructure did not translate to the macroscale characterization of the collagen-gel mechanical properties. The lack of clear trends indicates that bulk collagen-gel properties depend on more than just collagen microstructure. Others have similarly shown that collagen fiber alignment does not fully explain the direction-dependent material properties of collagen gels .
Various matrix-degrading enzymes have been found to be expressed at higher levels in degenerate human discs [2, 9, 25–29]. Although the biochemical aspects of MMP function have been elucidated, their specific functional roles in the multifaceted nature of the disorder are not well understood. For instance, it has been shown that MMPs can contribute not only to matrix degradation, but also to inflammatory conditions, innervation, and apoptosis [5, 30, 31] all of which have been observed in DDD. Although the precise coordination of the molecular events behind degenerative tissue remodeling remains unclear, demonstrating that MMP-2 is critical for localized matrix turnover by AF cells serves as an important step toward mechanistic understanding of DDD.
The observed effects in AF cells are consistent with the putative role of MMP-2 in other systems in vivo. Because gelatinases exhibit much greater enzymatic activity on denatured collagen than on native collagen , they are believed to work alongside other matrix-degrading enzymes in mediating radical changes in the ECM by removing bulk denatured collagen material (gelatin). Although MMP-2 is considered in most circles to function in the secondary processing of collagen, studies have shown its ability to cleave intact triple-helical collagen with some efficiency [5, 33]. It is interesting to speculate whether MMP-2 might also serve a collagenolytic capacity in our collagen gel preparations, because the inability for shMMP2-transduced cells to remodel the gels was so dramatic. Further studies manipulating collagenase activity are needed to investigate the relative collagenolytic and gelatinolytic roles that potentially underlie MMP-2 function.
Changes mediated by MMP-2 can be either physiologic or pathophysiologic in context. For instance, MMP-2 and -9 have been implicated not only in development and repair, but also in pathologies of the tracheal gland , renal system [34, 35], tumors [20, 30, 36], bone [37–39], and circulatory system [40, 41], among others. In the current study, knocking down MMP-2 both impaired the localized degradation of gelatin and inhibited AF cells from remodeling the collagen matrix. This was evident in the histologic structure and contraction of collagen gels. Despite the known association between gelatinase activity and collagen gel remodeling, until now, no direct evidence suggested that these effects are directly coupled in AF cells.
This ambiguity stems in part from a complex relation that has been observed to exist among ECM-related cues, actin cytoskeletal organization, and MMP-2 activation. It has long been observed that cells can cause contraction of native collagen gels through reorganization of collagen fibrillar structure . Generally, contraction of gels is accompanied by MMP-2 activation [43–47], which is thought to be linked with the suppression of stress fibers by collagen gel deformability [45, 46, 48]. Studies have also demonstrated that integrin binding is another critical factor in potentiating MMP-2 function [21, 22, 45, 49–52].
Although these previous studies demonstrate an outside-in phenomenon in the regulation of MMP-2 activation by the collagen microenvironment, our data suggest that effects may be bidirectional. We found that pure populations of MMP-2-deficient AF cells remained rounded in collagen gels and were unable to remodel them, although cells remained adherent to gelatin films over several days. Thus, it appears that MMP2 expression may have profound influence over actin-cytoskeleton-dependent collagen gel remodeling, but no effect on cell-collagen binding, presumably via β1 integrins. Furthermore, it suggests that collagen gel contraction is more directly influenced by cytoskeletal organization than integrin ligation, which may be a necessary, but not sufficient, condition.
In terms of IVD physiology, our findings support the pursuit of further in vivo investigation. Evidence from the literature points to a potentially important role of MMP-2 in degradative changes in the AF. Across various species and insult techniques, animal models of degenerative changes in the IVD have demonstrated an association of MMP-2 with gross morphologic changes in the AF [3, 10, 53], which parallels observations in human discs during development and pathogenesis [2, 27–29]. Consistent with these other studies, our data in mice and rats show that MMP-2 activity could increase relatively early in response to injurious load and annular puncture [16, 19], suggesting possible involvement in the initial phases of degradation.
Experiments using in vitro cell-culture models provide some insight into the potential functional significance of MMP-2. Silencing MMP2 in primary AF cells abolished gelatinolytic activity in the vicinity of cells, implicating MMP-2 as the primary physiologic gelatinase responsible for localized breakdown of gelatin but of little relevance in distant matrix degradation. This observation is consistent with previous reports in the literature by others, that negligible MMP-9 is expressed in nonherniated human IVDs . Collagen gel experiments underscore the functional significance of MMP-2 in structural reorganization of the ECM. Noninfected AF cells were able to modify the existing collagen scaffold into a more fibrous compact structure, resulting in gels that exhibited higher strength and lower viscosity, presumably due in part to the ability to remove denatured collagen. It also is possible that MMP-2 may contribute to the cells' ability to modify their biophysical environment through contraction, migration, and manipulation of collagen fibrils.
Several technical observations were also made during the course of this study. In our experiments, five shRNA sequences were tested for effectiveness in silencing MMP2 in rat AF cells. As expected, some constructs were more effective in silencing MMP2 than were others. It is known that siRNAs targeting different regions of the same gene vary markedly in their effectiveness , which depends on factors such as the secondary structure of the mRNA target, presence of the RNA-binding proteins, and thermodynamic stability of the duplex [14, 54]. In mixed-populations cultures, it is also possible that the transduced and nontransduced populations exhibit differences in proliferation rates, although we did not measure this directly. It was, therefore, not unexpected that the level of knockdown might change over time.
Treating cells with blasticidin to obtain a pure population of only transduced cells eliminated any potential masking effects by nontransduced cells. Therefore, it was expected that the pure population would exhibit higher levels of knockdown compared with a mixed population. Instead, we observed less suppression of transcript levels in Experiments 2 and 3 for all of our constructs. It is possible that the 10-day blasticidin treatment used to kill nontransduced cells in these experiments may nonspecifically alter expression of other genes, as antibiotics in culture could interfere with multiple metabolic processes . Despite the reduced silencing observed at the mRNA level, we continued to observe almost no protein expression.
Even though the shNon contained a scrambled sequence, some level of knockdown was observed in these cells as well. It is known that the interferon response may be triggered in the process of cell transfection with a viral vector [14, 54] and may cause overall changes in the gene-expression profile in cells as well . These factors together could lead to some level of gene knockdown observed in cells transduced with the shNon vector. In addition, some debate exists as to the specificity of sequences required for effective RNAi . Various bases in the scrambled nonsense sequence may be able to match the MMP2 mRNA found in cells causing some knockdown effect. Compared with the nonsense construct, however, shMMP2 was significantly more effective in reducing mRNA and protein levels. Therefore, the chosen sequence for shMMP2 was considered effective for use in future RNAi experiments.
Although more work is needed, elucidating the functional roles of degradative enzymes in degenerative remodeling of the disc ECM contributes to the identification of potential targets for gene therapy. RNAi technology offers the advantage of stable silencing of catabolic factors, which can be of therapeutic value in treating disc degeneration. To this extent, lentiviral vectors have provided an advancement in RNAi and offer the means to achieve significant levels of gene transfer both in vitro and in vivo . Various studies have used siRNAs for RNAi in IVD cells [58, 59], but to our knowledge, this study is the first to use shRNAs to ascertain the functional role of a molecule putatively involved in disc degeneration. MMP2 as a gene-therapy target has already been studied in vivo by using animal models as a strategy to treat tumors. In nude mice, siRNAs against MMP2 resulted in decreased tumor invasion, migration, and angiogenesis, with a 60% reduction in tumor size [60, 61]. This approach allows only transient downregulation of gene expression, making it difficult to adapt for therapeutic purposes. However, shRNA delivery can allow long-term studies to determine the functional role of genes relevant to intervertebral disc degeneration, such as MMP2.
Taken together, results from this study suggest that the increased presence of MMP-2 in animal models of disc degeneration may be associated with the degradative changes that are observed in the AF. Whether a similar relation might exist in degradative changes in human DDD remains to be determined. By using RNAi, we showed that MMP-2 functions as the primary gelatinase and is used by AF cells for degrading the local surrounding matrix. Notably, in the absence of MMP-2, collagen remodeling is significantly impaired, suggesting that it may play a significant role in collagen turnover and structural alterations in the AF. This mechanism could offer one avenue for therapeutic intervention against degenerative disc disease.
degenerative disc disease
Dulbecco Modified Eagle Medium
fetal bovine serum
reverse transcriptase polymerase chain reaction
The authors acknowledge financial support for this study from the University of Maryland, College Park, the National Institutes of Health (AR054051), and the National Science Foundation (CBET-0845754).
- Iatridis JC, MacLean JJ, Roughley PJ, Alini M: Effects of mechanical loading on intervertebral disc metabolism in vivo. J Bone Joint Surg Am. 2006, 41-46. 88 Suppl 2
- Rutges JP, Kummer JA, Oner FC, Verbout AJ, Castelein RJ, Roestenburg HJ, Dhert WJ, Creemers LB: Increased MMP-2 activity during intervertebral disc degeneration is correlated to MMP-14 levels. J Pathol. 2008, 214: 523-530. 10.1002/path.2317.View ArticlePubMedGoogle Scholar
- Salo J, Mackiewicz Z, Indahl A, Konttinen YT, Holm AK, Sukura A, Holm S: Plasmin-matrix metalloproteinase cascades in spinal response to an experimental disc lesion in pig. Spine. 2008, 33: 839-844. 10.1097/BRS.0b013e31816b1f1d.View ArticlePubMedGoogle Scholar
- Seguin CA, Pilliar RM, Madri JA, Kandel RA: TNF-alpha induces MMP2 gelatinase activity and MT1-MMP expression in an in vitro model of nucleus pulposus tissue degeneration. Spine. 2008, 33: 356-365. 10.1097/BRS.0b013e3181642a5e.View ArticlePubMedGoogle Scholar
- Nagase H, Woessner JF: Matrix metalloproteinases. J Biol Chem. 1999, 274: 21491-21494. 10.1074/jbc.274.31.21491.View ArticlePubMedGoogle Scholar
- Akeda K, An HS, Pichika R, Patel K, Muehleman C, Nakagawa K, Uchida A, Masuda K: The expression of NG2 proteoglycan in the human intervertebral disc. Spine. 2007, 32: 306-314. 10.1097/01.brs.0000254108.08507.04.View ArticlePubMedGoogle Scholar
- Levicoff EA, Kim JS, Sobajima S, Wallach CJ, Larson JW, Robbins PD, Xiao X, Juan L, Vadala G, Gilbertson LG, Kang JD: Safety assessment of intradiscal gene therapy II: effect of dosing and vector choice. Spine. 2008, 33: 1509-1516. 10.1097/BRS.0b013e318178866c. discussion 1517PubMed CentralView ArticlePubMedGoogle Scholar
- Shen B, Melrose J, Ghosh P, Taylor F: Induction of matrix metalloproteinase-2 and -3 activity in ovine nucleus pulposus cells grown in three-dimensional agarose gel culture by interleukin-1beta: a potential pathway of disc degeneration. Eur Spine J. 2003, 12: 66-75.PubMedGoogle Scholar
- Kozaci LD, Guner A, Oktay G, Guner G: Alterations in biochemical components of extracellular matrix in intervertebral disc herniation: role of MMP-2 and TIMP-2 in type II collagen loss. Cell Biochem Funct. 2006, 24: 431-436. 10.1002/cbf.1250.View ArticlePubMedGoogle Scholar
- Hsieh AH, Lotz JC: Prolonged spinal loading induces matrix metalloproteinase-2 activation in intervertebral discs. Spine. 2003, 28: 1781-1788. 10.1097/01.BRS.0000083282.82244.F3.View ArticlePubMedGoogle Scholar
- MacLean JJ, Roughley PJ, Monsey RD, Alini M, Iatridis JC: In vivo intervertebral disc remodeling: kinetics of mRNA expression in response to a single loading event. J Orthop Res. 2008, 26: 579-588. 10.1002/jor.20560.PubMed CentralView ArticlePubMedGoogle Scholar
- Aigner A: Delivery systems for the direct application of sirnas to induce rna interference (RNAi) in vivo. J Biomed Biotechnol. 2006, 2006: 71659-PubMed CentralView ArticlePubMedGoogle Scholar
- Wall NR, Shi Y: Small RNA: can RNA interference be exploited for therapy?. Lancet. 2003, 362: 1401-1403. 10.1016/S0140-6736(03)14637-5.View ArticlePubMedGoogle Scholar
- Dykxhoorn DM, Lieberman J: The silent revolution: RNA interference as basic biology, research tool, and therapeutic. Annu Rev Med. 2005, 56: 401-423. 10.1146/annurev.med.56.082103.104606.View ArticlePubMedGoogle Scholar
- Hsieh AH, Lotz JC: Time-course regulation of MMP-2 activity in loaded intervertebral discs. Presented at the International Society for the Study of the Lumbar Spine 30th Annual Meeting, 2003. 2003Google Scholar
- Hsieh AH, Hwang D, Ryan DA, Freeman AK, Kim H: Degenerative anular changes induced by puncture are associated with insufficiency of disc biomechanical function. Spine. 2009, 34: 998-1005. 10.1097/BRS.0b013e31819c09c4.View ArticlePubMedGoogle Scholar
- Primer3. [http://frodo.wi.mit.edu/]
- 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: 402-408. 10.1006/meth.2001.1262.View ArticlePubMedGoogle Scholar
- Lotz JC, Colliou OK, Chin JR, Duncan NA, Liebenberg E: Compression-induced degeneration of the intervertebral disc: an in vivo mouse model and finite-element study. Spine. 1998, 23: 2493-2506. 10.1097/00007632-199812010-00004.View ArticlePubMedGoogle Scholar
- Deryugina EI, Bourdon MA, Reisfeld RA, Strongin A: Remodeling of collagen matrix by human tumor cells requires activation and cell surface association of matrix metalloproteinase-2. Cancer Res. 1998, 58: 3743-3750.PubMedGoogle Scholar
- Deryugina EI, Ratnikov B, Monosov E, Postnova TI, DiScipio R, Smith JW, Strongin AY: MT1-MMP initiates activation of pro-MMP-2 and integrin alphavbeta3 promotes maturation of MMP-2 in breast carcinoma cells. Exp Cell Res. 2001, 263: 209-223. 10.1006/excr.2000.5118.View ArticlePubMedGoogle Scholar
- Silletti S, Kessler T, Goldberg J, Boger DL, Cheresh DA: Disruption of matrix metalloproteinase 2 binding to integrin alpha vbeta 3 by an organic molecule inhibits angiogenesis and tumor growth in vivo. Proc Natl Acad Sci USA. 2001, 98: 119-124.PubMed CentralPubMedGoogle Scholar
- Fujiwara A, Shibata E, Terashima H, Shishido A, Nishiki J, Yoshida K, Miyauchi K, Madachi A, Matsuura N: Evaluation of matrix metalloproteinase-2 (MMP-2) activity with film in situ zymography for improved cytological diagnosis of breast tumors. Breast Cancer. 2006, 13: 272-278. 10.2325/jbcs.13.272.View ArticlePubMedGoogle Scholar
- Thomopoulos S, Fomovsky GM, Chandran PL, Holmes JW: Collagen fiber alignment does not explain mechanical anisotropy in fibroblast populated collagen gels. J Biomech Eng. 2007, 129: 642-650. 10.1115/1.2768104.View ArticlePubMedGoogle Scholar
- Nemoto O, Yamagishi M, Yamada H, Kikuchi T, Takaishi H: Matrix metalloproteinase-3 production by human degenerated intervertebral disc. J Spinal Disord. 1997, 10: 493-498.View ArticlePubMedGoogle Scholar
- Pockert AJ, Richardson SM, Le Maitre CL, Lyon M, Deakin JA, Buttle DJ, Freemont AJ, Hoyland JA: Modified expression of the ADAMTS enzymes and tissue inhibitor of metalloproteinases 3 during human intervertebral disc degeneration. Arthritis Rheum. 2009, 60: 482-491. 10.1002/art.24291.View ArticlePubMedGoogle Scholar
- Rutges JP, Nikkels PG, Oner FC, Ottink KD, Verbout AJ, Castelein RJ, Creemers LB, Dhert WJ: The presence of extracellular matrix degrading metalloproteinases during fetal development of the intervertebral disc. Eur Spine J. 2010, 19: 1340-1346. 10.1007/s00586-010-1378-x.PubMed CentralView ArticlePubMedGoogle Scholar
- Weiler C, Nerlich AG, Zipperer J, Bachmeier BE, Boos N: 2002 SSE Award Competition in Basic Science: Expression of major matrix metalloproteinases is associated with intervertebral disc degradation and resorption. Eur Spine J. 2002, 11: 308-320. 10.1007/s00586-002-0472-0.PubMed CentralView ArticlePubMedGoogle Scholar
- Kang JD, Georgescu HI, McIntyre-Larkin L, Stefanovic-Racic M, Donaldson WF, Evans CH: Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2. Spine. 1996, 21: 271-277. 10.1097/00007632-199602010-00003.View ArticlePubMedGoogle Scholar
- Martin MD, Matrisian LM: The other side of MMPs: protective roles in tumor progression. Cancer Metastasis Rev. 2007, 26: 717-724. 10.1007/s10555-007-9089-4.View ArticlePubMedGoogle Scholar
- Page-McCaw A, Ewald AJ, Werb Z: Matrix metalloproteinases and the regulation of tissue remodelling. Nature Rev Mol Cell Biol. 2007, 8: 221-233. 10.1038/nrm2125.View ArticleGoogle Scholar
- Tournier JM, Polette M, Hinnrasky J, Beck J, Werb Z, Basbaum C: Expression of gelatinase A, a mediator of extracellular matrix remodeling, by tracheal gland serous cells in culture and in vivo. J Biol Chem. 1994, 269: 25454-25464.PubMedGoogle Scholar
- Aimes RT, Quigley JP: Matrix metalloproteinase-2 is an interstitial collagenase: inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4- and 1/4-length fragments. J Biol Chem. 1995, 270: 5872-5876. 10.1074/jbc.270.11.5872.View ArticlePubMedGoogle Scholar
- Lelongt B, Trugnan G, Murphy G, Ronco PM: Matrix metalloproteinases MMP2 and MMP9 are produced in early stages of kidney morphogenesis but only MMP9 is required for renal organogenesis in vitro. J Cell Biol. 1997, 136: 1363-1373. 10.1083/jcb.136.6.1363.PubMed CentralView ArticlePubMedGoogle Scholar
- Thrailkill KM, Clay Bunn R, Fowlkes JL: Matrix metalloproteinases: their potential role in the pathogenesis of diabetic nephropathy. Endocrine. 2009, 35: 1-10. 10.1007/s12020-008-9114-6.PubMed CentralView ArticlePubMedGoogle Scholar
- Bjorklund M, Koivunen E: Gelatinase-mediated migration and invasion of cancer cells. Biochim Biophys Acta. 2005, 1755: 37-69.PubMedGoogle Scholar
- Inoue K, Mikuni-Takagaki Y, Oikawa K, Itoh T, Inada M, Noguchi T, Park JS, Onodera T, Krane SM, Noda M, Itohara S: A crucial role for matrix metalloproteinase 2 in osteocytic canalicular formation and bone metabolism. J Biol Chem. 2006, 281: 33814-33824. 10.1074/jbc.M607290200.View ArticlePubMedGoogle Scholar
- Lieu S, Hansen E, Dedini R, Behonick D, Werb Z, Miclau T, Marcucio R, Colnot C: Impaired remodeling phase of fracture repair in the absence of matrix metalloproteinase-2. Dis Models Mech. 2011, 4: 203-211. 10.1242/dmm.006304.View ArticleGoogle Scholar
- Okada Y, Naka K, Kawamura K, Matsumoto T, Nakanishi I, Fujimoto N, Sato H, Seiki M: Localization of matrix metalloproteinase 9 (92-kilodalton gelatinase/type IV collagenase = gelatinase B) in osteoclasts: implications for bone resorption. Lab Invest. 1995, 72: 311-322.PubMedGoogle Scholar
- Galis ZS, Muszynski M, Sukhova GK, Simon-Morrissey E, Libby P: Enhanced expression of vascular matrix metalloproteinases induced in vitro by cytokines and in regions of human atherosclerotic lesions. Ann NY Acad Sci. 1995, 748: 501-507.View ArticlePubMedGoogle Scholar
- Johnson JL, Baker AH, Oka K, Chan L, Newby AC, Jackson CL, George SJ: Suppression of atherosclerotic plaque progression and instability by tissue inhibitor of metalloproteinase-2: involvement of macrophage migration and apoptosis. Circulation. 2006, 113: 2435-2444. 10.1161/CIRCULATIONAHA.106.613281.View ArticlePubMedGoogle Scholar
- Bell E, Ivarsson B, Merrill C: Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci USA. 1979, 76: 1274-1278. 10.1073/pnas.76.3.1274.PubMed CentralView ArticlePubMedGoogle Scholar
- Azzam HS, Thompson EW: Collagen-induced activation of the M(r) 72,000 type IV collagenase in normal and malignant human fibroblastoid cells. Cancer Res. 1992, 52: 4540-4544.PubMedGoogle Scholar
- Nguyen M, Arkell J, Jackson CJ: Three-dimensional collagen matrices induce delayed but sustained activation of gelatinase A in human endothelial cells via MT1-MMP. Int J Biochem Cell Biol. 2000, 32: 621-631. 10.1016/S1357-2725(00)00013-3.View ArticlePubMedGoogle Scholar
- Seltzer JL, Lee AY, Akers KT, Sudbeck B, Southon EA, Wayner EA, Eisen AZ: Activation of 72-kDa type IV collagenase/gelatinase by normal fibroblasts in collagen lattices is mediated by integrin receptors but is not related to lattice contraction. Exp Cell Res. 1994, 213: 365-374. 10.1006/excr.1994.1211.View ArticlePubMedGoogle Scholar
- Tomasek JJ, Halliday NL, Updike DL, Ahern-Moore JS, Vu TK, Liu RW, Howard EW: Gelatinase A activation is regulated by the organization of the polymerized actin cytoskeleton. J Biol Chem. 1997, 272: 7482-7487. 10.1074/jbc.272.11.7482.View ArticlePubMedGoogle Scholar
- Wang DR, Sato M, Li LN, Miura M, Kojima N, Senoo H: Stimulation of pro-MMP-2 production and activation by native form of extracellular type I collagen in cultured hepatic stellate cells. Cell Structure Function. 2003, 28: 505-513. 10.1247/csf.28.505.View ArticlePubMedGoogle Scholar
- Allenberg M, Weinstein T, Li I, Silverman M: Activation of procollagenase IV by cytochalasin D and concanavalin A in cultured rat mesangial cells: linkage to cytoskeletal reorganization. J Am Soc Nephrol. 1994, 4: 1760-1770.PubMedGoogle Scholar
- Ellerbroek SM, Wu YI, Overall CM, Stack MS: Functional interplay between type I collagen and cell surface matrix metalloproteinase activity. J Biol Chem. 2001, 276: 24833-24842. 10.1074/jbc.M005631200.View ArticlePubMedGoogle Scholar
- Giannelli G, Bergamini C, Fransvea E, Marinosci F, Quaranta V, Antonaci S: Human hepatocellular carcinoma (HCC) cells require both alpha3beta1 integrin and matrix metalloproteinases activity for migration and invasion. Lab Invest. 2001, 81: 613-627. 10.1038/labinvest.3780270.View ArticlePubMedGoogle Scholar
- Yan L, Moses MA, Huang S, Ingber DE: Adhesion-dependent control of matrix metalloproteinase-2 activation in human capillary endothelial cells. J Cell Sci. 2000, 113: 3979-3987.PubMedGoogle Scholar
- Zigrino P, Drescher C, Mauch C: Collagen-induced proMMP-2 activation by MT1-MMP in human dermal fibroblasts and the possible role of alpha2beta1 integrins. Eur J Cell Biol. 2001, 80: 68-77. 10.1078/0171-9335-00134.View ArticlePubMedGoogle Scholar
- Bergknut N, Rutges JP, Kranenburg HJ, Smolders LA, Hagman R, Smidt HJ, Lagerstedt AS, Voorhout G, Hazewinkel HH, Grinwis GC, Creemers LB, Meij BP, Dhert WJ: The dog as an animal model for intervertebral disc degeneration?. Spine. 2012, 37 (5): 351-358. 10.1097/BRS.0b013e31821e5665. 011, as originally listed), and the complete reference info is now provided],View ArticlePubMedGoogle Scholar
- Pushparaj PN, Aarthi JJ, Manikandan J, Kumar SD: siRNA, miRNA, and shRNA: in vivo applications. J Dent Res. 2008, 87: 992-1003. 10.1177/154405910808701109.View ArticlePubMedGoogle Scholar
- Kuhlmann I: The prophylactic use of antibiotics in cell culture. Cytotechnology. 1996, 19: 95-105. 10.1007/BF00749764.View ArticleGoogle Scholar
- Nagy V, Watzele M: FuGENE 6 Transfection Reagent: minimizing reagent-dependent side effects as analyzed by gene-expression profiling and cytotoxicity assays. Nature Methods. 2006, 3 (5): iii-v.View ArticleGoogle Scholar
- Sledz CA, Williams BR: RNA interference and double-stranded-RNA-activated pathways. Biochem Soc Trans. 2004, 32: 952-956. 10.1042/BST0320952.View ArticlePubMedGoogle Scholar
- Nishida K, Doita M, Takada T, Kakutani K, Miyamoto H, Shimomura T, Maeno K, Kurosaka M: Sustained transgene expression in intervertebral disc cells in vivo mediated by microbubble-enhanced ultrasound gene therapy. Spine. 2006, 31: 1415-1419. 10.1097/01.brs.0000219945.70675.dd.View ArticlePubMedGoogle Scholar
- Seki S, Asanuma-Abe Y, Masuda K, Kawaguchi Y, Asanuma K, Muehleman C, Iwai A, Kimura T: Effect of small interference RNA (siRNA) for ADAMTS5 on intervertebral disc degeneration in the rabbit anular needle-puncture model. Arthritis Res Ther. 2009, 11: R166-10.1186/ar2851.PubMed CentralView ArticlePubMedGoogle Scholar
- Tsung AJ, Kargiotis O, Chetty C, Lakka SS, Gujrati M, Spomar DG, Dinh DH, Rao JS: Downregulation of matrix metalloproteinase-2 (MMP-2) utilizing adenovirus-mediated transfer of small interfering RNA (siRNA) in a novel spinal metastatic melanoma model. Int J Oncol. 2008, 32: 557-564.PubMed CentralPubMedGoogle Scholar
- Sun Y, Liu M, Yang B, Li B, Lu J: Role of siRNA silencing of MMP-2 gene on invasion and growth of laryngeal squamous cell carcinoma. Eur Arch Otorhinolaryngol. 2008, 265: 1385-1391. 10.1007/s00405-008-0684-y.View ArticlePubMedGoogle Scholar
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