Abstract
Objective
PKCδ activation was found to be a principal rate-limiting step in matrix-degrading enzyme production in human articular chondrocytes. However, the role of the PKC pathways, specifically PKCδ, has not yet been assessed in intervertebral disc tissue homeostasis.
Methods
Using in vitro, ex vivo, and in vivo techniques, we evaluated the pathophysiological role of the PKCδ pathway by examining (i) proteoglycan deposition; (ii) matrix-degrading enzyme production and activity; (iii) downstream signaling pathways regulated by PKCδ; and (iv) the effect on in vivo models of disc degeneration in genetically-engineered PKCδ knockout mice.
Results
Pathway-specific inhibitor studies reveal a vital role of PKCδ-MAPK (ERK, p38, JNK) axis and NFκB in disc homeostasis. Accordingly, PKCδ knockout mice are markedly resistant to disc degeneration in a disc injury model in vivo.
Conclusion
Suppression of the PKCδ pathway may be beneficial in the prevention and/or treatment of disc degeneration, and these findings provide evidence for the potential therapeutic role of pathway-specific inhibitors of the PKCδ cascade in the future.
Keywords: PKCδ, MMP-13, intervertebral disc degeneration, signaling pathways, MAPK, Intervertebral disc, MMPs, ADAMTS
Introduction
Low back pain (LBP) is a common clinical problem that has a significant impact on today’s aging population. While the etiology of back pain is multi-factorial, it has been associated with intervertebral disc (IVD) degeneration (1, 2). It has been suggested that the degenerative process begins in the nucleus pulposus (NP) of the IVD and is associated with progressive loss of proteoglycans (PGs) from the extracellular matrix (ECM) (3). Recently, biological treatments capable of inhibiting ECM degradation have been considered, and clinical trials for spine and joint cartilage preservation and repair are underway (4, 5).
Studies have also shown a destructive role of matrix metalloproteinases (e.g. MMP-1, MMP-3, MMP-13, MMP-14) and aggrecanases (ADAMTS-4 and -5) in disc degeneration (6-8). Therefore, antagonizing these proteases may potentially retard degeneration and preserve disc tissue over time. Previously, we demonstrated the critical catabolic role of PKCδ in the upregulation of MMP-13 after stimulation with fibronectin fragment (Fn-F) or phorbol-12-myristate 13-acetate (PMA)(9) as well as IL-1 and FGF-2(6) in human adult articular chondrocytes. PKCδ is an upstream regulator of the MAPKs (ERK, JNK, p38) and NFκB signaling cascades, and was the only PKC isoform associated with MMP-13 induction after FGF-2 stimulation in human adult articular chondrocytes (6). More recently, silencing of PKC gene expression in human chondrocytes reveals that PKCδ is involved in collagenase induction by IL-1 (10) Based on these results and others from articular chondrocytes (6, 9) we hypothesize that the PKCδ pathway plays a pivotal role in degeneration of the IVD. The aim of the present study is to evaluate the pathophysiological role of the PKCδ pathway in IVD homeostasis using in vitro, ex vivo and in vivo animal model approaches. Our results provide important new information on spine disc metabolism mediated by the PKCδ pathway.
Materials & Methods
In vitro
Alginate bead culture (long-term studies)
IVD tissue was harvested from bovine coccygeal discs (15-18 months old) and disc cells were isolated from the NP, digested, and captured in alginate for 21 days to assess accumulated PG production by dimethylmethylene blue (DMMB) assay as previously described (11, 12). BMP7 (100ng/ml, Stryker Biotech, Hopkinton, MA) was used as a positive control for PG production.
Monolayer cell culture (short-term studies)
NP cells isolated from either bovine or human discs (obtained from Gift of Hope Tissue Donor Network) were cultured in serum-free monolayer and treated with pathway-specific inhibitors, including inhibitors of PKCδ (rottlerin, 4μM), PKCα/β [Gö6976, 10μM], PKCζ (H-Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu-OH, 10μM), and PKCε (H-Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr-OH, 10μM, Calbiochem, Gibbstown, NJ)(6). Inhibitors of MAPK and NFκB (Helenalin) were purchased from either Calbiochem or Toris Bioscience (Ellisville, MO, USA).
Synthesis of a PKCδ peptide inhibitor
PKCδ was selectively inhibited using the δV1-1 peptide antagonist (13) that consists of a peptide derived from the first unique region (V1) of PKCδ (SFNSYELGSL: amino acids 8-17 of PKCδ) coupled to a membrane permeant peptide sequence in the HIV TAT gene product (YGRKKRRQRRR: amino acids 47-57 of TAT) by cross-linking an N-terminal Cys-Cys bond to the membrane-permeable TAT peptide, as previously described (14). The peptides were a gift from the Mochly-Rosen laboratory at Stanford University.
Western blotting
Equal amount of total protein in the conditioned medium was measured by protein assay (Pierce, Rockford, IL) and loaded on 10% SDS-PAGE gels, transferred, and blotted using anti-MMP-1, MMP-13 (R&D System, Minneapolis, MN), MMP-3, anti-phospo-specific anti-NFκB (p65), and MAP kinases (p38 and ERK) purchased from R&D (Minneapolis, MN), and ADAMTS-4, ADAMTS-5 purchased from Chemicon (Massachusetts, MA), as previously described (15).
Quantitative real-time PCR
Total RNA extraction using the Trizol reagent (Invitrogen, Carlsbad, CA) and reverse transcription using ThermoScript TM RT-PCR system (Invitrogen, Carlsbad, CA) were performed by provided manufacture’s instruction. Real time PCR and relative gene induction analysis were performed using MyiQ real-Time PCR Detection System (Bio-Rad, Hercules, CA). The sets of primer sequence, optimized PCR conditions, and NCBI reference numbers are supplied in the Supplemental Methods (Table 1).
Protease activity assessments
Zymography
To analyze MMP activity, concentrated human NP media were mixed with sample buffer without reducing agent or boiling, and loaded onto 1mg/mL gelatin-containing SDS-polyacrylamide gel. After electrophoresis, the gel was washed to remove SDS by 50mM Tris-HCl (pH 7.5) containing 2.5% Triton X-100 for 1 hour at room temperature, allowing the reactivation of MMPs. Enzyme activities were revealed by staining with Coomassie Brilliant Blue R-250. Active MMP13 ELISA: Activity of MMP-13 was assessed by Active MMP-13 ELISA kit (Protealmmune, InviLISA human ACT MMP-13, Cupertino, CA 95014) using conditioned media of human NP cells by following the manufacturer’s instruction (standard sandwich ELISA). A highly specific monoclonal antibody for the activated form of human MMP-13 permits to detect specifically active form of MMP-13 at the sensitivity of 7pg/mL.
Ex vivo analysis via an intradiscal injection organ culture model
New Zealand white rabbits (2.5-3kg, mixed male and female) were given 1.3 ml of heparin intravenously under general anesthesia. After the heparin circulated for five minutes, the rabbits were euthanized with a lethal dose of pentobarbital to permit dissection of lumbar motion segments followed by intradiscal injection en bloc with IL-1 (10ng/mL) in the presence or absence of PKCδ inhibitor (PKCδi, 4μM)(16) Discs (n=6 per treatment) were then separated and maintained individually in organ culture for 14 days in complete medium, as previously described (17-19).
In vivo
PKCδ−/− (knockout, KO, n=8) and wild type (WT, n=12) mice were used in disc degeneration experiments (4-5 months old mice with C57B1/6 genetic background, weight >25g). In vivo disc degeneration was induced by either intradiscal injection of IL-1 (100ng/disc at L4/L5, Peprotech, Rocky Hill, NJ), or tail disc needle puncture under fluoroscopic guidance using a 26-gauge needle in both WT and KO mice. For lumbar disc exposure, mice were positioned supine with the neck hyperextended and anesthesia (1.5% isoflurane in oxygen) was administered via a facemask at a rate of 1 L/minute. The abdominal hair was shaved, thoroughly scrubbed with alcohol and a topical antiseptic solution (chlorhexadine gluconate), and draped in sterile fashion. After confirming adequate anesthesia, a midline, ventral abdominal incision approximately 2 cm in length was made with a #15 blade scalpel. Abdominal viscera were gently retracted to allow visualization and access to the spine and lumbar disc space. Needle puncture or intradiscal injection was then performed as indicated in the study protocol. The muscles were then closed with 4-0 vicryl suture, and all skin margins were closed with wound clips, which were removed 7 days post-surgery. For two days following surgery, the mice received buprenorphine 0.1mg/kg subcutaneously twice daily for analgesia. Recovery was closely monitored for two days following surgery to ensure there were no acute postoperative complications.
Histological assessment
Discs either from ex vivo (rabbit discs) or in vivo [mouse, PKCδ KO (n=8) and WT (n=12)] were fixed in 4% paraformaldehyde in PBS, decalcified, embedded in paraffin and sectioned for histological assessments. Sagittal sections (5μM thickness) of each IVD were stained with Safranin-O. An unblinded investigator grouped the slides and randomly numbered them; these groups were then graded by two different blinded investigators (H-JI and XL). For ex vivo samples, a relative grade was assigned from 0-4, where 0 means no staining (PG loss) and 4 means the most intense stain (normal disc) based on Safranin-O. Grading for in vivo samples were performed by following the grading system described previously (total 12 points, Supplemental Method Table 2)(20). Two independent examinations were performed, and the repeatability of grading on the two occasions was determined using Cohen kappa statistics.
For immunostaining of matrix-degrading proteases, sections were incubated with 20μg/mL proteinase K for 30 min at 37°C for antigen retrieval. The endogenous peroxidase activity was blocked, using 3% H2O2 and followed by blocking solution containing 0.1% horse serum and overnight incubation with anti-MMP13 (R&D, Minneapolis, MN) or anti-ADAMTS5 (Chemicon, Massachusetts, MA) antibodies at 4°C followed by visualization using Vectastain Kit (Vector Laboratories) and Nikon SMZ1000 (Model #3.2.0, Diagnostic Instrument, Inc. Sterling Heights, MI). For histometric analysis, we selected outer NP sections from the sagittal plane of the lumbar discs. The % of immuno-positive chondrocytes was calculated under a fixed measuring frame (400μm×140μm) (immune-positive cell number/total cell number × 100).
μCT imaging analyses
Structural alterations of discs and end-plate architecture were evaluated by μCT scanning, as performed previously (21). Freshly dissected lumbar motion segments were immediately fixed in 10% formalin followed by μCT imaging analyses in the Rush Imaging Core Facility using a Scanco Model 40 Desktop μCT. Sagittal orientation (L4/L5-vertebrate-L5/L6) of the lumbar discs was scanned in a 10mm region of the intact rat vertebral column at high resolution (20mm tube, 10μm resolution, 55kVP, 145μA, 300ms integration time). Each specimen was scanned in air with the x-ray beam oriented perpendicular to the long axis of the vertebral column. The imaging threshold was set at 270 for the creation of 3D renderings (Supplemental Data Figure 1).
Data Analyses
The significance of differences among means of data on radiograph measurements was analyzed by analysis of variance (ANOVA) for repeated measurements and Fisher PLSD as a post hoc test. All data were expressed as the mean ± standard error. Statistical analysis was performed using the Statview (Version 5.0, SPSS, Chicago, IL) program package. The Cohen kappa value was calculated using the internet-based program (http://department.obg.cuhk.edu.hk/researchsupport/Cohen_Kappa_data.asp). P<0.05 was defined as significant for all tests.
Results
Blockade of the PKCδ pathway rescues PMA- and IL-1-mediated PG loss in bovine NP cells
To determine the biological impact of the PKCδ pathway on IVD homeostasis bovine NP cells were cultured in alginate with PMA (0.5μM), a potent non-specific PKC activator, in the presence or absence of the pharmacological inhibitor of PKCδ (δV1-1, 2μM) for 21 days. The treatment with PMA significantly suppressed PG accumulation (p<0.05) by approximately 35% compared to control (untreated), and this effect was completely rescued in the presence of PKCδi, Rottlerin (Fig.1A). Similarly, depletion of PG by IL-1 (1 ng/mL), an activator of PKCδ (6) was reversed by rottlerin (Fig.1B). Interestingly, blocking the PKCδ pathway not only rescues the catabolic response of either IL-1 or PMA, but also it enhances the anabolic response mediated by BMP7. Of note, treatment of NP cells with PMA, IL-1 or the δPKC inhibitor, rottlerin, showed no significant impact on cellular viability at the concentrations used in the studies as shown by Live and Dead cell assay (Calcin AM) throughout the culture period (21 days) (Fig.1C). Our data suggest that activation of the PKCδ pathway is negatively involved in disc homeostasis by suppressing PG accumulation in bovine disc cells, and blocking this pathway not only rescues catabolism but also enhances BMP7-mediated IVD anabolism.
Figure 1.
Effect of PKCδi, rottlerin, on PG accumulation and cell proliferation in bovine NP cells cultured in alginate for 21 days followed by DMMB assays for accumulated PG content. Incubation with PMA (0.5 μM) (A) or IL-1 (1 ng/mL) (C) suppresses PG deposition, and addition of PKCδi, rottlerin, (2 μM) reverses this effect above the control levels (non-treatment). Furthermore, by co-treatment with BMP7 (100 ng/mL), PKCδI, rottlerin, significantly (p<0.05) enhances BMP7-induced PG; B) The PMA-mediated increase in cell proliferation is negated by addition of the PKCδ inhibitor.
Pharmacological inhibitor of PKCδ reverses catabolic and anti-anabolic gene expression modulated by IL-1 in bovine disc cells
In order to understand the molecular mechanisms by which PKCδ inhibitor rescues PG loss, we determined the modulation of ECM-associated gene expression in the presence of rottlerin after stimulation with IL-1 (Fig.2). Cells in monolayers were cultured in the presence of IL-1 with or without the PKCδ inhibitor, rottlerin. The conditioned media or cells were subjected to real-time PCR to analyze mRNA levels of matrix-degrading enzymes (MMP-1, -3, -13, -14, ADAMTS-4 and -5), aggrecan, and collagen type II (COLII). Stimulation of cells with IL-1 markedly induced expression of multiple proteases. In the presence of PKCδ inhibitor, however, these stimulations were significantly suppressed in a dose-dependent manner [Fig.2A (a)–(f)]. Non-specific PKC inhibitor, Bisindolylmaleimide I (PKCi), which blocks activity of multiple PKC isoforms, was included as a positive control. Interestingly, the PKCδ inhibitor, rottlerin, not only rescues IL-1-supressed aggrecan and COLII, but also significantly enhances expression of aggrecan with BMP7 (Fig.2B; p<0.05). More dramatic results were obtained in the expression levels of COLII by the combination of the rottlerin and BMP7 (p<0.01) while BMP7 itself has no effect on COLII (Fig.2C).
Figure 2.
Effects of pharmacological inhibitor of PKCδ on IL-1 (10 ng/mL)-modulated expression of matrix-degrading enzymes [A; (a)-(f)] and matrix-associated gene in which BMP7 was used as a positive control (B &C).
PKCδ pathway-driven activation of NFκB and MAPK is responsible for the IL-1-mediated upregulation of matrix-degrading enzyme production in bovine NP cells
The activation of the PKCδ-MAPK axis and NFκB pathways has been reported to be responsible for IL-1-mediated catabolism in both articular cartilage (6, 15) and IVD tissue (11). Here, we show that IL-1 exerts similar activity in the spine via activation of the PKCδ pathway, and inhibition of this pathway by PKCδ inhibitor attenuates IL-1-mediated catabolic effects. Stimulation of cells with IL-1 rapidly activates PKCδ within 5 minutes [Fig.3A (a)], similar to results obtained previously in human articular chondrocytes (6). Rapid activation of the MAPK (p38, ERK) and NFκB pathway is also observed and is sustained for >60 min as represented by phosphorylation [Fig.3A (b)]. Blocking the PKCδ pathway (rottlerin) significantly reduces the IL-1-induced phosphorylation of MAPK and NFκB, suggesting that both MAPK and NFκB pathways are regulated by PKCδ as an upstream regulator in bovine NP cells (Fig.3B).
Figure 3.
Signaling cascades mediated by IL-1 in bovine IVD cells. IL-1 rapidly activates PKCδ [A (a)], and ERK, p38 MAPKs and NFκB pathways [A (b)] which were attenuated in the presence of PKCδi, rottlerin (B); Pharmacological inhibitors of PKC isoforms (C); and inhibitors of MAPKs, NFκB pathways were tested on cartilage degrading enzyme production in bovine NP cells cultured in monolayer. (D); PKCδ specific inhibitory peptide (δV1-1) tested by zymogram, cartilage-degrading enzyme production (E, left panel) and MMP-13 activity assessed by Active MMP-13 ELISA (E, right panel) using human NP cells in monolayers.
IL-1 activates multiple isoforms of PKC that lead to the activation of MAPK and NFκB, key signaling cascades associated with protease expression (6, 15, 22, 23). To determine which of these signaling cascades, if any, are essential for IL-1-induced matrix-degrading enzymes, cells were stimulated in the presence of IL-1 with or without inhibitors of PKC isoforms such as α/β, δ, ε, and ζ, for 24 hours followed by western blotting analyses using conditioned medium for secreted catabolic enzyme production (MMP-1, -3, -13, ADAMTS-4 and -5). Among PKC isoforms, PKCδ inhibition demonstrates the greatest inhibition of MMPs and ADAMTS enzyme production (Fig.3C). More specifically, the presence of rottlerin bolished the production of MMP-13, MMP-1, and ADAMTS-4, and attenuated MMP-3 and ADAMTS-5. In contrast, inhibitors of other PKC isoforms (PKCα/βi, ζi, and εi) failed or only partially suppressed the production of these enzymes. Of the PKC-mediated downstream signaling cascades, the NFκB pathway appears to play the most significant role in matrix-degrading enzyme production as addition of a pharmacological inhibitor of NFκB (NFκBi, 10μM) potently diminishes IL-1-induced enzyme production (Fig.3D). Interestingly, we observed almost identical expression patterns between MMP-3 and ADAMTS-5 in our inhibitor studies, suggesting that production of MMP-3 and ADAMTS-5 are regulated by similar signaling mechanisms involving NFκB, but not MAPK pathways. The catabolic gene expression levels in the presence of PKC isoforms are collectively summarized in Table 1.
Table 1.
Summary of signaling pathways responsible for the expression of cartilage-degrading enzymes in disc NP cells. [+++++ or ++++, great effect; ++ or + moderate effect; +/− or −/− slight or no effect on target gene expression]
| Stimulation cells with IL-1 | ||||||
|---|---|---|---|---|---|---|
| PKC | PKCδ | +++++ | ++++ | +++++ | +++++ | ++++ |
| PKCα/β | + | +/− | −/− | ++++ | +/− | |
| PKCε | −/− | −/− | −/− | −/− | −/− | |
| PKCζ | −/− | −/− | −/− | −/− | −/− | |
| MAPK | Erk | +++++ | + | ++++ | ++++ | + |
| P38 | +++++ | +/− | + | ++++ | +/− | |
| JNK | +++++ | + | ++++ | ++++ | ++ | |
| NFκB | +++++ | +++++ | ++++ | ++++ | ++++ | |
 |
 |
 |
 |
 |
||
| Target gene | MMP-13 | MMP-3 | MMP-1 | ADAMTS-4 | ADAMTS-5 | |
Despite challenges with IL-1, we failed to detect activated forms of MMPs in bovine disc cells, perhaps due to the sensitivity threshold of antibody affinity (e.g, reduced antibody affinity to bovine cells). Thus, we tested human disc NP cells for MMP production and enzyme activity. Our results reveal that the production levels of MMP-13 and -1 are correlated with enzymatic activity reflected by the digested clear zone on zymography (Fig.3E; left upper panel). The co-incubation of cells with either chemical PKCδi (rottlerin 4μM) or PKCδ-specific inhibitor peptide (δV1-1; 1 or 5μM) significantly attenuates catabolic enzyme activity assessed by zymography. Specific MMP-13 enzyme activity was further assessed by ELISA that specifically detects the activated form of MMP-13 (Fig.3E; right panel), demonstrating that inhibition of PKCδ attenuates MMP-13 enzyme activity that is stimulated by IL-1.
PKCδ inhibitor antagonizes the catabolic actions mediated by IL-1 in ex vivo rabbit disc organ culture model
Given the in vitro results obtained above, we sought to elucidate potential physiologic effects of PKCδ inhibition in an ex vivo organ culture model. Rabbit discs were dissected and intradiscally co-injected with IL-1 (10ng/mL) and rottlerin, 4μM. After 14 days of ex vivo culture, discs were harvested, decalcified, and subjected to safranin O staining for PG content (Fig.4A [A]–[C]). A set of discs were analyzed for cell viability by Live and Dead cell assay (Calcin AM). Green (live) and dead (red) cells were counted, and the % of live cells were calculated under a fixed measuring frame (2.5mm2), and we identified >90-94% cell viability for each treatment group. Based on blinded, semi-quantitative histological analyses using a relative grading system based on Safranin-O, we noticed a typical histologic change: compared to PG content in the control level [A; grade 4; n=12], intradiscal injection with IL-1β markedly reduced PG content, as represented by color intensity [B; grade 0 or 1; n=6], and this was significantly rescued by co-injection with rottlerin (4μM) [C; grade 3 or 4; p<0.05; n=6]. Overall grading scores are summarized in Fig.4D. This simplified blinded grading system was designed to test a pharmacological compound with reliability of grading on two occasions by two different investigators (Cohen kappa statistics: <0.05). The intra- and interobserver reliability correlation coefficients of grading by the 2 observers were κ =0.9 and 0.89, respectively, revealing excellent inter-observer reliability.
Figure 4.
Effect of PKCδi, rottlerin, on PG accumulation in a rabbit disc ex vivo organ culture model. (A) Control – injection with Phosphate-Buffered Saline reveals maintenance of abundant PG deposition in rabbit NP tissue after 14 days of ex vivo organ culture. (B) Intradiscal injection with IL-1 (10 ng/ml) demonstrates catabolic suppression of PG content after 14 days. (C) Injection with a combination of IL-1 + PKCδI (rottlerin, 4 μM) illustrates rescued PG loss compared to injection with (B) IL-1 alone.
Disc degeneration is protected in PKCδ knockout (PKCδ−/−) mice after challenged with intradiscal injection of IL-1 in vivo
Our in vitro and ex vivo organ culture studies are further corroborated by an in vivo model comparing PKCδ−/− (n=8) versus WT (n=12) mice. To evaluate any potential abnormality in discs that might affect the results of disc degeneration, we carefully studied the phenotypes of KO and WT mice for the past 6 years in terms of the sizes between and within genders, fertility, tendency to obesity and behavior in general, including eating habits and aggressiveness (fighting pattern). We did not observe any noticeable difference between PKCδ KO and WT mice, with the exception that female mice are slightly smaller than male mice, which is not statistically significant (data not shown). In the current study, we also failed to notice any morphological difference in discs between control groups of WT and KO mice [Fig.5 (A)–(D); control WT versus control KO]. We further evaluated potential structural abnormalities in disc endplates that might confound our results via the use of μCT imaging analyses, and failed to detect any significant structural differences in disc endplates (Supplemental Data Fig.1)
Figure 5.
(A) Intradiscal injection of IL-1 comparing PKCδ KO and WT mice followed by safranin-O staining, (B) Tail disc needle puncture of KO and WT mice followed by safranin-O staining for PG content. Immunohistochemistry was performed on disc level L4/L5 that was intradiscally injected with IL-1, using (C) anti-MMP13 and (D) anti-ADAMTS5 antibodies comparing PKCδ WT and PKCδ KO mice tissues.
Considering the similar phenotypes between WT and KO mice, our results are quite striking, demonstrating that PKCδ KO mice are completely protected from disc degeneration [Fig.5A; L4/L5] with control-like (intact; L5/L6) integrity of disc structure even after challenge with IL-1. In contrast, the lumbar discs (L4/L5) from WT mice were severely altered with depleted NP, similar to results found in an in vivo rabbit disc puncture model28 after challenge with IL-1. We observed similar remarkable results using a tail disc puncture model [Fig.5B]. These reproducible results from both disc injury models were assessed by a previously described histological grading system (12 points)(20) with statistical significance (p<0.05). Importantly, our results from immunohistochemistry and western blotting analyses demonstrate significantly reduced (or absence of) MMP-13 and ADAMTS5 in PKCδ KO (5-12% immune-positive) compared to WT mice (75-88% immune-positive, p<0.01) after intradiscal injection of IL-1β (Fig.5C & D, respectively). Taken together, these findings reveal the significant potential for the pharmacological inhibitor of the PKCδ pathway to suppress disc damage-induced production of multiple cartilage-degrading enzymes, and prevent disc degeneration in vivo.
Discussion
We reveal a critical pathophysiological role of the PKCδ pathway on IVD homeostasis in in vitro, ex vivo and in vivo studies, suggesting that inhibition of PKCδ may protect discs from degeneration over time. After treatment with IL-1, the PKCδ-MAPK-NFκB axis is activated in NP cells, leading to PG loss and production of multiple matrix-degrading enzymes. The results from this current study corroborate our previous findings from articular cartilage, as PKCδ mediates potent catabolic and anti-anabolic effects using in vitro and ex vivo experimental approaches. Our results were further supported by ablation of the PKCδ gene in mouse in vivo.
Further, in experiments using small interfering RNA (siRNA) targeting PKCδ (siPKCδ) we observed that FGF-2 or IL-1β-induced MMP-13 production is markedly antagonized by introduction of siPKCδ in human articular chondrocytes (unpublished data), which supports recently published data (6). Collectively, our data suggest an important biological role of PKCδ on ECM homeostasis in joints.
Importantly, the effect of rottlerin (PKCδi) on pathway-specific inhibition of the PKCδ pathway has been questioned in the literature. For example, it has been suggested that rottlerin may exert its anti-PKCδ effects not via direct inhibition of this enzyme, but rather via uncoupling of associated cellular processes resulting in energy (ie ATP) depletion, and therefore may non-specifically inhibit different PKC isoforms (24, 25). However, our studies using the PKCδ-specific peptide inhibitor, δV1-1 (13) suggest that the anti-catabolic effects shown by the PKCδ-specific peptide inhibitor are comparable to those results obtained by the use of rottlerin, suggesting that rottlerin does in fact exert anti-catabolic effects. Administration with a PKCδi using either a pharmacological inhibitor, rottlerin, or PKCδ-specific inhibitory peptide, significantly reduces PG depletion, downregulates production and activity of matrix-degrading enzymes in discs of all different species we tested in the current studies, including bovine, rabbit and human. Nevertheless, the specific function of rottlerin remains unknown and contradictory, and future studies are warranted to truly characterize it as a pathway-“specific” inhibitor of the PKCδ pathway. It is often difficult to extrapolate in vitro and ex vivo data to a clinical setting; however, in vivo results provide a greater understanding of the complexity with which biological factors and cytokines work together in the metabolic system to maintain or disrupt homeostasis. In our genetically engineered mouse model in vivo, we observed striking results in which ablation of PKCδ completely protects mice from disc degeneration, favoring an anabolic and anti-catabolic disc environment. On the other hand, discs from WT mice with the identical experimental procedures are severely degenerated by both disc injury models (needle puncture and intradiscal injection of IL-1) which provide in vivo corroboration of our in vitro and ex vivo studies demonstrating pathogenic association of the PKCδ pathway in disc homeostasis. Our data further reveal that the potent resistance to degeneration in PKCδ−/− mice is, at least in part, due to the reduction and/or absence of key matrix proteases such as MMP-13 and ADAMTS5. Although this model is still an injury-induced degeneration model and it may not reflect the precise events in human cases, these in vivo findings in PKCδ−/− mice provide essential evidence for the potential clinical utility of a PKCδ inhibitor in the prevention of disc degeneration.
Despite the exciting potential therapeutic benefit of PKCδi, it is necessary to recognize certain limitations of this study before translation to a clinical setting. First, the majority of our studies assessed effects of IL-1 (or PMA) on NP tissue alone without its effects on the AF, so further studies are necessary to determine if our results are corroborated throughout the entire structural components of disc. Second, assuming a therapeutic benefit of PKCδ inhibition in the prevention of disc degeneration diseases in the spine, further studies are warranted to determine the systemic effects of PKCδ inhibition on other organ systems throughout the body, as well as optimal routes of administration. For example, recent studies have elucidated harmful effects of PKCδi, rottlerin, treatment on lung barrier function both in vitro and in vivo (26). The PKCδ ihibitor, rottlerin, increased lung barrier dysfunction in pulmonary endothelial cell monolayers and caused pulmonary edema in rats, suggesting that the PKCδ pathway may also play a beneficial role in maintaining lung barrier function. While PKCδ inhibition may be beneficial in the spine, its use may be detrimental in other organ systems and further studies are necessary to gain a better understanding of these different effects. Logically, localized injection of a PKCδ inhibitor would seem to offer greater health benefits with reduced risk compared to systemic administration. Third, a PKCδ inhibitor therapy may be beneficial only when the cellular components are available (e.g., the early- and intermediate-degenerative stages of disease progress). At end-stages of degeneration (lack of cellular components), a PKCδ inhibitor therapy may be limited and may need combination with cell-based therapy or other tissue-engineering techniques. Finally, given our results were not found in human tissue, the proper dosing, route of administration, and concentration would need to be elucidated for translation to a clinical setting in the future.
Supplementary Material
ACKNOWLEDGMENTS
The current studies were supported by Synthes. We would like to thank the tissue donors and the family members, Drs. Cs-Szabo and Margulis, and the Gift of Hope Organ and Tissue Donor Network for human lumbar disc tissue samples. We also thank Stryker Biotech for their generous gift providing BMP7 and Dr. Mochly-Rosen at Stanford for her kind gift of δPKC inhibitor, δV1-1 for the studies.
Footnotes
Drs. Ellman and Kim are co-first authors.
References
- 1.Mooney V. The classification of low back pain. Ann Med. 1989;21(5):321–5. doi: 10.3109/07853898909149215. [DOI] [PubMed] [Google Scholar]
- 2.Freemont TJ, LeMaitre C, Watkins A, Hoyland JA. Degeneration of intervertebral discs: current understanding of cellular and molecular events, and implications for novel therapies. Expert Rev Mol Med. 2001;2001:1–10. doi: 10.1017/S1462399401002885. [DOI] [PubMed] [Google Scholar]
- 3.Buckwalter JA, Kuettner KE, Thonar EJ. Age-related changes in articular cartilage proteoglycans: electron microscopic studies. J Orthop Res. 1985;3(3):251–7. doi: 10.1002/jor.1100030301. [DOI] [PubMed] [Google Scholar]
- 4.An H, Boden SD, Kang J, Sandhu HS, Abdu W, Weinstein J. Summary statement: emerging techniques for treatment of degenerative lumbar disc disease. Spine. 2003;28(15 Suppl):S24–5. doi: 10.1097/01.BRS.0000076894.33269.19. [DOI] [PubMed] [Google Scholar]
- 5.An HS, Thonar EJ, Masuda K. Biological repair of intervertebral disc. Spine. 2003;28(15 Suppl):S86–92. doi: 10.1097/01.BRS.0000076904.99434.40. [DOI] [PubMed] [Google Scholar]
- 6.Im HJ, Muddasani P, Natarajan V, Schmid TM, Block JA, Davis F, et al. Basic fibroblast growth factor stimulates matrix metalloproteinase-13 via the molecular cross-talk between the mitogen-activated protein kinases and protein kinase Cdelta pathways in human adult articular chondrocytes. J Biol Chem. 2007;282(15):11110–21. doi: 10.1074/jbc.M609040200. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Le Maitre CL, Freemont AJ, Hoyland JA. Localization of degradative enzymes and their inhibitors in the degenerate human intervertebral disc. J Pathol. 2004;204(1):47–54. doi: 10.1002/path.1608. [DOI] [PubMed] [Google Scholar]
- 8.Salminen HJ, Saamanen AM, Vankemmelbeke MN, Auho PK, Perala MP, Vuorio EI. Differential expression patterns of matrix metalloproteinases and their inhibitors during development of osteoarthritis in a transgenic mouse model. Ann Rheum Dis. 2002;61(7):591–7. doi: 10.1136/ard.61.7.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Loeser RF, Forsyth CB, Samarel AM, Im HJ. Fibronectin fragment activation of proline-rich tyrosine kinase PYK2 mediates integrin signals regulating collagenase-3 expression by human chondrocytes through a protein kinase C-dependent pathway. J Biol Chem. 2003;278(27):24577–85. doi: 10.1074/jbc.M304530200. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Litherland GJ, Elias MS, Hui W, Macdonald CD, Catterall JB, Barter MJ, et al. Protein kinase C isoforms zeta and iota mediate collagenase expression and cartilage destruction via STAT3- and ERK-dependent c-fos induction. J Biol Chem. 285(29):22414–25. doi: 10.1074/jbc.M110.120121. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Li X, An HS, Ellman M, Phillips F, Thonar EJ, Park DK, et al. Action of fibroblasti growth factor-2 on the intervertebral disc. Arthritis Res Ther. 2008;10(2):R48. doi: 10.1186/ar2407. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Li X, Phillips FM, An HS, Ellman M, Thonar EJ, Wu W, et al. The action of resveratrol, a phytoestrogen found in grapes, on the intervertebral disc. Spine (Phila Pa 1976) 2008;33(24):2586–95. doi: 10.1097/BRS.0b013e3181883883. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Chen L, Hahn H, Wu G, Chen CH, Liron T, Schechtman D, et al. Opposing cardioprotective actions and parallel hypertrophic effects of delta PKC and epsilon PKC. Proc Natl Acad Sci U S A. 2001;98(20):11114–9. doi: 10.1073/pnas.191369098. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Chen L, Wright LR, Chen CH, Oliver SF, Wender PA, Mochly-Rosen D. Molecular transporters for peptides: delivery of a cardioprotective epsilonPKC agonist peptide into cells and intact ischemic heart using a transport system, R(7) Chem Biol. 2001;8(12):1123–9. doi: 10.1016/s1074-5521(01)00076-x. [DOI] [PubMed] [Google Scholar]
- 15.Muddasani P, Norman JC, Ellman M, van Wijnen AJ, Im HJ. Basic fibroblast growth factor activates the MAPK and NFkappaB pathways that converge on Elk-1 to control production of matrix metalloproteinase-13 by human adult articular chondrocytes. J Biol Chem. 2007;282(43):31409–21. doi: 10.1074/jbc.M706508200. [DOI] [PubMed] [Google Scholar]
- 16.Ellman MBLX, An HS, Park DK, Davis F, Im HJ. The Role of the PKC delta pathway in the Intervertebral Disc. Ortho Trans. 2008;33:1414. [Google Scholar]
- 17.Haschtmann D, Stoyanov JV, Ettinger L, Nolte LP, Ferguson SJ. Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system. Spine. 2006;31(25):2918–25. doi: 10.1097/01.brs.0000247954.69438.ae. [DOI] [PubMed] [Google Scholar]
- 18.Chiba K, Andersson GB, Masuda K, Momohara S, Williams JM, Thonar EJ. A new culture system to study the metabolism of the intervertebral disc in vitro. Spine. 1998;23(17):1821–7. doi: 10.1097/00007632-199809010-00002. discussion 8. [DOI] [PubMed] [Google Scholar]
- 19.Risbud MV, Sittinger M. Tissue engineering: advances in in vitro cartilage generation. Trends Biotechnol. 2002;20(8):351–6. doi: 10.1016/s0167-7799(02)02016-4. [DOI] [PubMed] [Google Scholar]
- 20.Masuda K, Aota Y, Muehleman C, Imai Y, Okuma M, Thonar EJ, et al. A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine (Phila Pa 1976) 2005;30(1):5–14. doi: 10.1097/01.brs.0000148152.04401.20. [DOI] [PubMed] [Google Scholar]
- 21.Kim J, Kroin JS, Buvanendran A, Li X, van Wijnen AJ, Tuman KJ, Im H-J. Characterization of a new animal model for evaluation and treatment of back pain due to lumbar facet joint osteoarthritis. Arthritis Rheum. 2011 doi: 10.1002/art.30487. In press. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Liacini A, Sylvester J, Li WQ, Zafarullah M. Inhibition of interleukin-1-stimulated MAP kinases, activating protein-1 (AP-1) and nuclear factor kappa B (NF-kappa B) transcription factors down-regulates matrix metalloproteinase gene expression in articular chondrocytes. Matrix Biol. 2002;21(3):251–62. doi: 10.1016/s0945-053x(02)00007-0. [DOI] [PubMed] [Google Scholar]
- 23.Mengshol JA, Vincenti MP, Coon CI, Barchowsky A, Brinckerhoff CE. Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor kappaB: differential regulation of collagenase 1 and collagenase 3. Arthritis Rheum. 2000;43(4):801–11. doi: 10.1002/1529-0131(200004)43:4<801::AID-ANR10>3.0.CO;2-4. [DOI] [PubMed] [Google Scholar]
- 24.Klarl BA, Lang PA, Kempe DS, Niemoeller OM, Akel A, Sobiesiak M, et al. Protein kinase C mediates erythrocyte “programmed cell death” following glucose depletion. Am J Physiol Cell Physiol. 2006;290(1):C244–53. doi: 10.1152/ajpcell.00283.2005. [DOI] [PubMed] [Google Scholar]
- 25.Tapia JA, Jensen RT, Garcia-Marin LJ. Rottlerin inhibits stimulated enzymatic secretion and several intracellular signaling transduction pathways in pancreatic acinar cells by a non-PKC-delta-dependent mechanism. Biochim Biophys Acta. 2006;1763(1):25–38. doi: 10.1016/j.bbamcr.2005.10.007. [DOI] [PubMed] [Google Scholar]
- 26.Klinger JR, Murray JD, Casserly B, Alvarez DF, King JA, An SS, et al. Rottlerin causes pulmonary edema in vivo: a possible role for PKCdelta. J Appl Physiol. 2007;103(6):2084–94. doi: 10.1152/japplphysiol.00695.2007. [DOI] [PubMed] [Google Scholar]
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