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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Objective

Protein kinase Cδ (PKCδ) activation has been shown to be a principal rate-limiting step in matrix-degrading enzyme production in human articular chondrocytes. The aim of this study was to assess the role of the PKC pathways, specifically PKCδ, in intervertebral disc tissue homeostasis.

Methods

Using in vitro, ex vivo, and in vivo techniques, we evaluated the pathophysiologic role of the PKCδ pathway by examining 1) proteoglycan deposition, 2) matrix-degrading enzyme production and activity, 3) downstream signaling pathways regulated by PKCδ, and 4) the effect on in vivo models of disc degeneration in genetically engineered PKCδ-knockout mice.

Results

Studies of pathway-specific inhibitors revealed a vital role of the PKCδ/MAPK (ERK, p38, JNK) axis and NF-κB in disc homeostasis. Accordingly, in an in vivo model of disc injury, PKCδ-knockout mice were markedly resistant to disc degeneration.

Conclusion

Suppression of the PKCδ pathway may be beneficial in the prevention and/or treatment of disc degeneration. The results of this study provide evidence for a potential therapeutic role of pathway-specific inhibitors of the PKCδ cascade in the future.

Low back pain is a common clinical symptom that has a significant impact on the aging population. Although the etiology of back pain is multifactorial, 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, biologic treatments capable of inhibiting ECM degradation have been considered, and clinical trials of emerging techniques for spine and joint cartilage preservation and repair are under way (4, 5).

Previous studies have also shown a destructive role of matrix metalloproteinases (e.g., MMP-1, MMP-3, MMP-13, and MMP-14) and aggrecanases (ADAMTS-4 and ADAMTS-5) in disc degeneration (6–8). Therefore, antagonizing these proteases may potentially retard degeneration and preserve disc tissue over time. Previously, our group demonstrated the critical catabolic role of protein kinase Cδ (PKCδ) in the up-regulation of MMP-13 after stimulation with fibronectin fragments or phorbol myristate acetate (PMA) (9) as well as interleukin-1 (IL-1) and fibroblast growth factor 2 (FGF-2) (6) in human adult articular chondrocytes. PKCδ is an upstream regulator of the MAPK (ERK, JNK, p38) and NF-κB signaling cascades and was the only PKC isoform associated with MMP-13 induction after FGF-2 stimulation in adult human articular chondrocytes (6). More recently, silencing of PKC gene expression in human chondrocytes revealed that PKCδ is involved in collagenase induction by IL-1 (10). Based on these results and others for articular chondrocytes (6, 9), we hypothesized that the PKCδ pathway plays a pivotal role in degeneration of the IVD. The aim of the present study was to evaluate the pathophysiologic role of the PKCδ pathway in IVD homeostasis, using in vitro, ex vivo, and in vivo animal models. Our results provide important new information on spine disc metabolism mediated by the PKCδ pathway.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

In vitro studies.

Alginate bead culture (long-term studies).

IVD tissue was harvested from the coccygeal discs of 15–18–month-old bovine animals, and disc cells were isolated from the NP, digested, and captured in alginate for 21 days to assess accumulated PG production by dimethylmethylene blue assay, as previously described (11, 12). Bone morphogenetic protein 7 (BMP-7) (100 ng/ml; a generous gift from Stryker Biotech) was used as a positive control.

Monolayer cell culture (short-term studies).

NP cells isolated from either bovine or human discs (provided by the Gift of Hope Organ & Tissue Donor Network) were cultured in serum-free monolayer and treated with pathway-specific inhibitors, including inhibitors of PKCδ (PKCδi, rottlerin; 4 μM), PKCα/β (Gö6976; 10 μM), PKCζ (H-Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu-OH; 10 μM), and PKCε (H-Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr-OH; 10 μM [Calbiochem]) (6). Inhibitors of MAPK and NF-κB (helenalin) were purchased from either Calbiochem or Tocris.

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 human immunodeficiency virus TAT gene product (YGRKKRRQRRR: amino acids 47–57 of TAT) by crosslinking 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 amounts of total protein in the conditioned medium were measured by protein assay (Pierce) and loaded on 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis gels, transferred, and blotted using anti–MMP-1, anti–MMP-13, anti–MMP-3, anti–phospo-specific NF-κB p65, and MAPKs (p38 and ERK) purchased from R&D Systems, and ADAMTS-4 and ADAMTS-5 purchased from Chemicon, as previously described (15).

Real-time quantitative polymerase chain reaction (PCR).

Total RNA extraction using TRIzol reagent (Invitrogen) and reverse transcription using a ThermoScript RT-PCR system (Invitrogen) were performed according to the manufacturers' instructions. Real-time PCR and relative gene induction analysis were performed using a MyiQ Real-Time PCR Detection System (Bio-Rad). The sets of primer sequences, optimized PCR conditions, and NCBI reference numbers are available from the corresponding author.

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 SDS–polyacrylamide gel containing 1 mg/ml gelatin. After electrophoresis, the gel was washed to remove SDS, using 50 mM 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 R250.

Active MMP-13 enzyme-linked immunosorbent assay (ELISA).

The activity of MMP-13 was assessed with an InviLISA Human Act MMP13 system (Protealmmum), using conditioned media of human NP cells according to the manufacturer's instructions (standard sandwich ELISA). A highly specific monoclonal antibody for the activated form of human MMP-13 permits specific detection of the active form of MMP-13, with a sensitivity of 7 pg/ml.

Ex vivo analysis of an organ culture model with intradiscal injection.

NZW rabbits (2.5–3 kg, mixed male and female) received 1.3 ml of heparin intravenously while under general anesthesia. After the heparin circulated for 5 minutes, the rabbits were killed with a lethal dose of pentobarbital to permit dissection of lumbar motion segments, followed by intradiscal injection en bloc with IL-1 (10 ng/ml) in the presence or absence of PKCδi (4 μM) (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 studies.

Eight PKCδ-knockout (PKCδ−/−) mice and 12 wild-type (WT) mice were used in disc degeneration experiments (4–5–month-old mice with a C57BL/6 genetic background, weight >25 gm). In vivo disc degeneration was induced by either intradiscal injection of IL-1 (100 ng/disc at L4/L5; PeproTech) or tail disc needle puncture under fluoroscopic guidance using a 26-gauge needle in both WT and knockout mice. For lumbar disc exposure, mice were placed in the supine position with the neck hyperextended, and anesthesia (1.5% isoflurane in oxygen) was administered via a face mask at a rate of 1 liter/minute. The abdominal hair was shaved, and the abdomen was thoroughly scrubbed with alcohol and a topical antiseptic solution (chlorhexadine gluconate) and draped in a sterile manner. After the adequacy of anesthesia was confirmed, a midline, ventral abdominal incision ∼2 cm in length was made with a scalpel with a #15 blade. The 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 sutures, and all skin margins were closed with wound clips, which were removed 7 days after surgery. For 2 days following surgery, the mice received buprenorphine 0.1 mg/kg subcutaneously twice daily for analgesia. Recovery was closely monitored for 2 days following surgery to ensure that no acute postoperative complications occurred.

Histologic assessment.

Discs obtained either ex vivo (rabbit discs) or in vivo (8 PKCδ−/− mice and 12 WT mice) were fixed in 4% paraformaldehyde in phosphate buffered saline, decalcified, embedded in paraffin, and sectioned for histologic assessments. Sagittal sections (5-μm thickness) of each IVD were stained with Safranin O. An investigator grouped the slides in an unblinded manner and randomly numbered them; these groups were then graded in a blinded manner by 2 different investigators (H-JI and XL). For ex vivo samples, a relative grade from 0 to 4 was assigned, where 0 = no staining (PG loss) and 4 = the most intense stain (normal disc), based on Safranin O staining. Grading for in vivo samples was performed according to a previously described grading system (total 12 points) (20; additional information is available from the corresponding author). Two independent examinations were performed, and the repeatability of grading on the 2 occasions was determined using Cohen's kappa statistics.

For immunostaining of matrix-degrading proteases, sections were incubated with 20 μg/ml of proteinase K for 30 minutes at 37°C for antigen retrieval. Endogenous peroxidase activity was blocked using 3% H2O2 and then by using blocking solution containing 0.1% horse serum, followed by overnight incubation with anti–MMP-13 (R&D Systems) or anti–ADAMTS-5 (Chemicon) antibodies at 4°C and visualization using a Vectastain kit (Vector) and a Nikon SMZ1000 stereomicroscope (model 3.2.0l; Diagnostic Instruments). For histometric analysis, we selected outer NP sections from the sagittal plane of the lumbar discs. The percentage of immunopositive chondrocytes was calculated under a fixed measuring frame (400 μm × 140 μm) as follows: (immunopositive cell number/total cell number) × 100.

Micro–computed tomography (micro-CT) imaging analyses.

Structural alterations of discs and end-plate architecture were evaluated by micro-CT scanning, as previously described (21). Freshly dissected lumbar motion segments were immediately fixed in 10% formalin followed by micro-CT imaging analyses at the Rush University Imaging Core Facility using a desktop micro-CT system (μCT 40; Scanco Medical). Sagittal views of the lumbar discs (L4/5 and L5/6) were scanned in a 10-mm region of the intact rat vertebral column at high resolution (20-mm tube, 10-μm resolution, 55 kVP, 145 μA, 300 msec 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 3-dimensional renderings (additional information is available from the corresponding author).

Statistical analysis.

The significance of differences among means of data for radiograph measurements was analyzed by repeated-measures analysis of variance and Fisher's protected least significant difference post hoc test. All data were expressed as the mean ± SEM. Statistical analysis was performed using StatView version 5.0 (SPSS). Cohen's kappa value was calculated using an internet-based program (http://department.obg. cuhk.edu.hk/researchsupport/Cohen_Kappa_data.asp). P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Effect of PKCδ pathway blockade on PMA- and IL-1–mediated PG loss in bovine NP cells.

To determine the biologic impact of the PKCδ pathway on IVD homeostasis, bovine NP cells were cultured in alginate with PMA (0.5 μM), a potent nonspecific PKC activator, in the presence or absence of the pharmacologic inhibitor of PKCδ, δV1-1 (2 μM), for 21 days. Treatment with PMA significantly suppressed PG accumulation by ∼35% compared with control (untreated), and this effect was reversed in the presence of PKCδi (Figure 1A). Similarly, depletion of PG by IL-1 (1 ng/ml), an activator of PKCδ (6), was reversed by PKCδi (Figure 1B). Interestingly, blocking the PKCδ pathway not only rescued the catabolic response of either IL-1 or PMA but also enhanced the anabolic response mediated by BMP-7. Of note, treatment of NP cells with PMA, IL-1, or PKCδi at the concentrations used in the studies had no significant impact on cell viability throughout the culture period (21 days), as determined by Live/Dead cell assay (calcein AM) (Figure 1C). Our data suggest that activation of the PKCδ pathway was negatively involved in disc homeostasis by suppressing PG accumulation in bovine disc cells, and that blocking this pathway not only rescued catabolism but also enhanced BMP-7–mediated IVD anabolism.

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Figure 1. Effect of protein kinase Cδ inhibitor (PKCδi; rottlerin) on proteoglycan (PG) accumulation and cell proliferation in bovine nucleus pulposus cells cultured in alginate for 21 days followed by dimethylmethylene blue assays for accumulated PG content. A and B, Incubation with phorbol myristate acetate (PMA) (A) or interleukin-1 (IL-1) (B) suppressed PG deposition, and the addition of PKCδi reversed this effect. Blocking the PKCδ pathway not only rescued the catabolic response of either IL-1 or PMA but also enhanced the anabolic response mediated by bone morphogenetic protein 7 (BMP-7). C, Cell viability was assessed weekly, and each treatment demonstrated >95% cell viability throughout the long-term culture period (21 days). Values are the mean ± SEM.

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Role of PKCδi in reversing catabolic and antianabolic gene expression modulated by IL-1 in bovine disc cells.

In order to understand the molecular mechanisms by which PKCδ inhibition rescues PG loss, we evaluated the modulation of ECM-associated gene expression in the presence of PKCδi after stimulation with IL-1 (Figure 2). Cells in monolayers were cultured in the presence of IL-1 with or without PKCδi. The conditioned media or cells were subjected to real-time PCR to analyze messenger RNA levels of matrix-degrading enzymes (MMP-1, MMP-3, MMP-13, MMP-14, ADAMTS-4, and ADAMTS-5), aggrecan, and type II collagen. Stimulation of cells with IL-1 markedly induced the expression of multiple proteases. In the presence of PKCδi, however, stimulation was significantly suppressed, in a dose-dependent manner (Figure 2A). The nonspecific PKC inhibitor bisindolylmaleimide I (PKCi), which blocks the activity of multiple PKC isoforms, was included as a positive control. Interestingly, PKCδi not only rescued the IL-1–induced suppression of aggrecan and type II collagen but also significantly enhanced the expression of aggrecan when combined with BMP-7 (P < 0.05) (Figure 2B). The combination of PKCδi and BMP-7 had a significant effect on the expression of type II collagen (P < 0.01), while BMP-7 itself had no effect on type II collagen (Figure 2C).

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Figure 2. Effects of pharmacologic inhibition of PKCδ on IL-1–modulated expression of matrix-degrading enzymes (A) and matrix-associated genes (B and C). For the assessment of aggrecan and type II collagen induction, BMP-7 was used as a positive control. Values are the mean ± SEM. MMP-1 = matrix metalloproteinase 1 (see Figure 1 for other definitions).

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PKCδ pathway–driven activation of NF-κB and MAPK and IL-1–mediated up-regulation of matrix-degrading enzyme production in bovine NP cells.

Activation of the PKCδ/MAPK axis and NF-κB pathways has been reported to be responsible for IL-1–mediated catabolism in both articular cartilage (6, 15) and IVD tissue (11). Here, we showed that IL-1 exerted similar activity in the spine via activation of the PKCδ pathway, and that inhibition of this pathway by PKCδi attenuated IL-1–mediated catabolic effects. Stimulation of cells with IL-1 rapidly activated PKCδ within 5 minutes (Figure 3A), similar to results obtained previously in human articular chondrocytes (6). Rapid activation of the MAPK (p38, ERK) and NF-κB pathways was also observed and was sustained for >60 minutes, as represented by phosphorylation (Figure 3A). Blocking the PKCδ pathway using PKCδi significantly reduced the upstream signaling of IL-1–induced phosphorylation of MAPK and NF-κB, suggesting that the MAPK and NF-κB pathways are regulated by PKCδ in bovine NP cells (Figure 3B).

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Figure 3. Signaling cascades mediated by IL-1 in bovine intervertebral disc cells. A and B, IL-1 rapidly activated the PKCδ pathway and the ERK, p38 MAPK, and NF-κB pathways (A), and this activation was attenuated in the presence of PKCδi (B). C and D, Cartilage-degrading enzyme production in bovine nucleus pulposus cells cultured in monolayer was assessed using pharmacologic inhibitors of PKC isoforms (PKCζi, PKCεi, PKCα/βi, and PKCδi) (C) and inhibitors of the MAPK and NF-κB pathways (NF-κBi, JNKi, p38i, and ERKi) (D). E, Left panel, The correlation between enzymatic activity and production levels of matrix metalloproteinase 13 (MMP-13) and MMP-1 was assessed using zymography. Coincubation of cells with the PKCδ-specific inhibitory peptide (PKCδi p; δV1-1) or PKCδi significantly attenuated catabolic enzyme activity. Right panel, Results of an enzyme-linked immunosorbent assay that specifically detected the activated form of MMP-13 showed that inhibition of PKCδ attenuated the MMP-13 enzyme activity stimulated by IL-1. Cont = control (see Figure 1 for other definitions).

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IL-1 activates multiple isoforms of PKC that lead to the activation of MAPK and NF-κB, which are 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–mediated induction of 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 blot analyses using conditioned medium for secreted catabolic enzyme production (MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5). Among the PKC isoforms, PKCδ induced the greatest inhibition of MMP and ADAMTS enzyme production (Figure 3C). More specifically, the presence of PKCδi abolished the production of MMP-13, MMP-1, and ADAMTS-4 and attenuated the production of 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 NF-κB pathway appeared to play the most significant role in matrix-degrading enzyme production, because the addition of a pharmacologic inhibitor of NF-κB (NF-κBi; 10 μM) potently diminished IL-1–induced enzyme production (Figure 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 is regulated by similar signaling mechanisms involving the NF-κB pathway but not the MAPK pathway. The catabolic gene expression levels in the presence of PKC isoforms are summarized in Table 1.

Table 1. Summary of the signaling pathways responsible for the expression of cartilage-degrading enzymes in disc nucleus pulposus cells stimulated with interleukin-1*
Target geneMMP-13MMP-3MMP-1ADAMTS-4ADAMTS-5
  • *

    MMP-13 = matrix metalloproteinase 13; PKC = protein kinase C; +++++ or ++++ = great effect; ++ or + = moderate effect; +/− or −/− = slight or no effect on target gene expression.

PKC     
 PKCδ+++++++++++++++++++++++
 PKCα/β++/−−/−+++++/−
 PKCε−/−−/−−/−−/−−/−
 PKCζ−/−−/−−/−−/−−/−
MAPK ERK+++++++++++++++
 p38++++++/−++++++/−
 JNK++++++++++++++++
NF-κB++++++++++++++++++++++

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 revealed that the production levels of MMP-13 and MMP-1 were correlated with enzymatic activity, as reflected by the digested clear zone on zymography (Figure 3E, left panel). The co-incubation of cells with either PKCδi (4 μM) or δV1-1 (5 μM) significantly attenuated catabolic enzyme activity, as assessed by zymography. Specific MMP-13 enzyme activity was further assessed by ELISA that specifically detected the activated form of MMP-13 (Figure 3E, right panel), demonstrating that inhibition of PKCδ attenuated MMP-13 enzyme activity stimulated by IL-1.

Effect of PKCδi on the catabolic actions mediated by IL-1 in the ex vivo rabbit disc organ culture model.

Given the in vitro results, we sought to elucidate potential physiologic effects of PKCδ inhibition in an ex vivo organ culture model. Rabbit discs were dissected and intradiscally coinjected with IL-1 (10 ng/ml) and PKCδi (4 μM). After 14 days of ex vivo culture, the discs were harvested, decalcified, and subjected to Safranin O staining for PG content (Figures 4A–C). A set of discs was analyzed for cell viability by Live/Dead cell assay (calcein AM). Green (live) and dead (red) cells were counted, and the percentage of live cells was calculated under a fixed measuring frame (2.5 mm2); for each treatment group, cell viability was >90–94%. Based on blinded, semiquantitative histologic analyses using a relative grading system based on Safranin O staining, we observed the following histologic changes: compared with control (grade 4, n = 12) (Figure 4A), PG content was markedly reduced following intradiscal injection of IL-1β (grade 0 or 1; n = 6) (Figure 4B), and this reduction was rescued by coinjection with PKCδi (grade 3 or 4; n = 6) (Figure 4C). The overall histologic scores are shown in Figure 4D. This simplified grading system was designed to test a pharmacologic compound with reliability of grading on 2 occasions by 2 different investigators (Cohen's κ < 0.05). The intraobserver and interobserver correlation coefficients for grading by the 2 observers were 0.9 and 0.89, respectively, revealing excellent interobserver reliability.

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Figure 4. Effect of PKCδi on PG accumulation in a rabbit disc ex vivo organ culture model. A–C, Representative images showing that injection with phosphate buffered saline revealed maintenance of abundant PG deposition in rabbit nucleus pulposus tissue after 14 days of ex vivo organ culture (A), intradiscal injection with IL-1 demonstrated catabolic suppression of PG content after 14 days (B), and injection with a combination of IL-1 plus PKCδi (4 μM) rescued PG loss compared with injection with IL-1 alone (C). D, Overall mean ± SEM histologic scores. See Figure 1 for definitions.

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Protection against disc degeneration in PKCδ−/− mice after challenge with intradiscal injection of IL-1 in vivo.

Our in vitro and ex vivo organ culture studies were further corroborated by an in vivo model comparing PKCδ−/− mice (n = 8) and WT mice (n = 12). To evaluate any potential disc abnormality that might affect the results of disc degeneration, we carefully studied the phenotypes of PKCδ−/− and WT mice for the past 6 years in terms of the sizes between and within sexes, fertility, tendency to obesity, and behavior in general, including eating habits and aggressiveness (fighting pattern). We did not observe any noticeable difference between PKCδ−/− and WT mice, with the exception that female mice were slightly smaller than male mice, although the difference was not significant (data not shown). In the current study, we also failed to observe any morphologic difference in discs between WT and PKCδ−/− mice (Figures 5A and B). We further evaluated potential structural abnormalities in disc end plates that might confound our results, via micro-CT imaging analyses, and failed to detect any significant structural differences in disc end plates (additional information is available from the corresponding author).

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Figure 5. A, Representative images of the lumbar discs of wild-type (WT) and protein kinase Cδ (PKCδ)–knockout (KO) mice following intradiscal injection of interleukin-1 and Safranin O staining. B, Representative images of the lumbar discs of WT and PKCδ−/− mice following tail disc needle puncture and Safranin O staining. C and D, Representative results of immunohistochemical analysis and Western blotting after intradiscal injection of IL-1 in WT and PKCδ−/− mice, using anti–matrix metalloproteinase 13 (anti–MMP-13) (C) and anti–ADAMTS-5 antibodies (D). E, Results of active MMP-13 enzyme-linked immunosorbent assay (ELISA). Bars show the mean ± SEM.

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Considering the similar phenotypes between WT and PKCδ−/− mice, our results were quite striking, demonstrating that PKCδ−/− mice were completely protected against disc degeneration, with control-like (intact) integrity of the disc structure even after challenge with IL-1 (Figure 5A). In contrast, the lumbar discs of WT mice were severely altered, with depleted NP, similar to results observed in the in vivo rabbit disc puncture model after challenge with IL-1. We observed similar results using a tail disc puncture model (Figure 5B). These reproducible results from both disc injury models were assessed by a previously described histologic grading system (12 points) (20), with statistical significance (P < 0.05). Importantly, our results from immunohistochemical analysis and Western blotting demonstrated significantly reduced (or the absence of) MMP-13 and ADAMTS-5 in PKCδ−/− mice (5–12% immunopositive) compared with WT mice (75–88% immunopositive; P < 0.01) after intradiscal injection of IL-1β (Figures 5C and D, respectively). Similarly, our ELISAs also revealed that PKCδ ablation may be sufficient to abrogate MMP-13 protein expression induced by IL-1β (see Figure 5E). Taken together, these findings reveal the significant potential for PKCδi to suppress disc damage–induced production of multiple cartilage-degrading enzymes and prevent disc degeneration in vivo.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Here, we revealed a critical pathophysiologic 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/NF-κB axis was activated in NP cells, leading to PG loss and production of multiple matrix-degrading enzymes. The results from the current study corroborate our previous findings from articular cartilage, because PKCδ mediated potent catabolic and antianabolic effects in in vitro and ex vivo experiments. Our results were further supported by ablation of the PKCδ gene in mice 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 was markedly antagonized by introduction of siPKCδ in human articular chondrocytes (Im HJ: unpublished observations), which supports recently published data (6). Collectively, our data suggest an important biologic role of PKCδ in ECM homeostasis in joints.

Importantly, the effect of PKCδi on pathway-specific inhibition of the PKCδ pathway has been questioned in the literature. For example, it has been suggested that PKCδi may exert its anti-PKCδ effects not via direct inhibition of this enzyme but rather via uncoupling of associated cellular processes resulting in energy (i.e., ATP) depletion and therefore may nonspecifically inhibit different PKC isoforms (24, 25). However, our studies using the PKCδ-specific peptide inhibitor δV1-1 (13) suggest that the anticatabolic effects induced by the PKCδ-specific peptide inhibitor are comparable with results obtained using PKCδi, suggesting that PKCδi does in fact exert anticatabolic effects. Inhibition of PKCδ, using either a pharmacologic inhibitor, PKCδi, or a PKCδ-specific inhibitory peptide, significantly reduced PG depletion and down-regulated the production and activity of matrix-degrading enzymes in the discs of all the species tested in the current studies, including bovine animals, rabbits, and humans. Nevertheless, the specific function of PKCδi remains unknown, 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 biologic 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 protected mice from disc degeneration, favoring an anabolic and anticatabolic disc environment. In contrast, when the identical experimental procedures were used, discs from WT mice were severely degenerated by both needle puncture and intradiscal injection of IL-1, which provides in vivo corroboration of our in vitro and ex vivo studies demonstrating a pathogenic association of the PKCδ pathway in disc homeostasis. Our data further reveal that the potent resistance to disc 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 ADAMTS-5. Although this model is still an injury-induced model of disc degeneration, 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 potential therapeutic benefit of PKCδi, it is necessary to recognize certain limitations of this study before the findings are translated to a clinical setting. First, the majority of our studies assessed the effects of IL-1 (or PMA) on NP tissue alone without assessing the effects on the anulus fibrosus; therefore, further studies are necessary to determine whether our results can be corroborated throughout all of the structural components of the disc.

Second, assuming a therapeutic benefit of PKCδ inhibition in the prevention of degenerative disc 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 treatment on lung barrier function both in vitro and in vivo (26). The PKCδ inhibitor 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. Although PKCδ inhibition may be beneficial in the spine, its use may have detrimental effects 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 with systemic administration.

Third, PKCδ inhibitor therapy may be beneficial only when the cellular components are available (e.g., during the early and intermediate degenerative stages of disease). At the end stages of degeneration (during which there is a lack of cellular components), PKCδ inhibitor therapy may be limited and may need to be combined with cell-based therapy or other tissue-engineering techniques. Finally, given that our results were not achieved using human tissue, the proper dosing, route of administration, and concentration would need to be elucidated for translation to a clinical setting in the future.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

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

Study conception and design. Ellman, Kim, Li, Chen, Nakayama, Im.

Acquisition of data. Kim, Kroin, Li, Yan, Nakayama, Liu, Morgan, Im.

Analysis and interpretation of data. Ellman, Kim, An, Li, Chen, Yan, Buechter, Liu, Morgan, Im.

ROLE OF THE STUDY SPONSOR

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Synthes was not involved in the study design, data collection, data analysis, or writing of the manuscript. Synthes approved the content of the submitted manuscript and has agreed to submit the manuscript for publication, although publication was not contingent on approval by Synthes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

We would like to thank the tissue donors and their family members, Drs. Cs-Szabo and Margulis (Rush University Medical Center), and the Gift of Hope Organ & Tissue Donor Network for providing human lumbar disc tissue samples. We also thank Dr. Mochly-Rosen at Stanford for her kind gift of δV1-1 for the studies.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES
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