Background
The social and economic burden of low back pain continues to escalate due mainly to changes in lifestyle and demographic profile, with such pain now the leading cause of disability worldwide [
1]. Although many different pathophysiological causes might trigger low back pain, it has been demonstrated that intervertebral disc (IVD) degeneration plays a primary underlying role. By connecting the neighboring vertebrae of the spinal column and allowing slight movement flexibility of the spine, IVDs act to absorb axial compressive forces and facilitate load transmission. IVDs comprise an annulus fibrosus surrounding the central nucleus pulposus, the fibrocartilage, and endplates.
Decades of research into IVDs have revealed many secrets of structure, function, and molecular mechanisms; however, how these large structures can survive and function even under the most difficult physiological conditions remains an enigma. The lumbar spine carries considerable forces and has no dedicated blood supply, thus the vertebral endplate plays an important role in balancing the contradictory functions of permeability for nutrients to diffuse between disc cells and capillaries in the adjacent vertebra and vascularized tissues with sufficient strength to prevent damage or fracture. In addition, endplates function to absorb and separate significant pressure from the spine’s mechanical burden and prevent the nucleus pulposus from bulging into the adjacent vertebrae and are essential for disc metabolism.
Some studies have suggested that IVD degeneration is closely correlated with the state of the vertebral endplates [
2,
3], which often show significant morphological changes with aging-related IVD degenerations [
4]. The biochemical changes in endplates have been reported extensively, from normal to different degenerative conditions [
4]. Ariga et al. showed that increased apoptosis in the cartilaginous endplate with age resulted in markedly decreased cell density and destruction of the cartilaginous endplate [
5], followed by the structure of the cartilaginous endplate beginning to disappear. Multiple independent factors can cause the initiation and progression of degeneration through endplate changes (reviewed in [
6‐
8]). The process is a chain of biochemical, cellular, structural, and functional changes in the endplates, with mechanical stress, nutrient supply, osmotic and ionic environments, hormones, cytokines, growth factors, and matrix molecules all reported to affect disc cell degeneration, and many other pathological causes still to be explored.
Receptor interacting serine/threonine kinase 1 (RIPK1) is involved in Toll-like receptor (TLR), tumor necrosis factor (TNF), interferon, and interleukin (IL)1α signaling pathways [
9‐
14]. Several studies found that activated RIPK1 can associate with RIPK3 to induce mixed lineage kinase domain like pseudokinase (MLKL)-dependent necroptosis and production of inflammatory cytokines or recruit Fas-associated protein with death domain (FADD) and activate caspase-8 to induce apoptosis following DNA damage or TLR signaling. In addition, it participates in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation independent of its kinase activity. Abnormal activities of RIPK1 have been indicated in several disease processes, such as ischemic injuries, chronic and acute inflammatory diseases, axonal degeneration, neutrophilic dermatosis, autoinflammatory and autoimmune pathology, and cancers [
14]. Due to RIPK1′s role in regulating necroptosis and apoptosis, it has gained interests as a treatment target for the RIPK1-dependent diseases already mentioned. In addition, the cumulative effects of various functions performed by RIPK1 may collaboratively contribute to molecular pathologies of autoimmune, degenerative, and inflammatory diseases.
This study sought to identify potential molecular pathogenic markers of IVD degeneration through building signaling networks based on known pathways important in IVD degeneration and to screen against the network using clinical specimens to reveal the underlying mechanisms of such molecules.
Materials and methods
Antibodies and other reagents
The following antibodies were used in this study: RIPK1 (Cell Signaling Technology, Danvers, MA), p-IKKα/βSer176/Ser180 (Thermo Fisher Scientific, Waltham, MA), p-JNKThr183 (Abcam, Cambridge, MA), p-IKKα/βSer176/180, p-p38Thr180/Tyr182, β-actin, and cleaved caspase 3 (Cell Signaling Technology). Other reagents purchased are as follows: 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) (X-Gal), zVAD (N-benzyloxycarbonyl-Val-Ala-Asp (O-Me) fluoromethyl ketone), and necrostatin-1 (Nec1) from Sigma-Aldrich (St. Louis, MO), cycloheximide (CHX) from Santa Cruz Biotech (Dallas, Texas), collagenase II from Thermo Fisher Scientific, BCA Protein Assay Kit from Beyotime Biotechnology (Shanghai, China), HiScript Q Select RT SuperMix for qPCR and AceQ qPCR Probe Master Mix from Vazyme Biotech (Nanjing, China), and all remaining reagents from Sigma-Aldrich unless otherwise specified.
Building a signaling network representing intervertebral disc diseases with pathology-associated proteins
Reported pathology-associated proteins in IVD diseases were used as signaling nodes [
1,
6‐
8]. These molecules were input into the meta-search engine of protein–protein interaction database String [
15], organized, and analyzed as previously described [
16]. Interactions identified by experiment, database, neighborhood, gene fusion, co-expression, and co-occurrence were included in the search. A confidence score of 0.15 was used and only direct protein–protein interactions were counted. Due to the limited amounts of mRNA extracted from patient specimens, 70 genes with the highest confidence and interaction scores were maintained in the signaling network for later experiments.
Quantification of mRNA expression in patients’ specimens and primary chondrocyte cells using quantitative real-time PCR (qRT-PCR)
Conditions of normal and degenerative endplates were confirmed using nuclear magnetic resonance (NMR) analysis, the Modic scoring system, and the Pfriimann disc degeneration grading system. These endplates were removed from a patient’s spine during surgery then rapidly frozen using liquid nitrogen. Total RNAs were extracted using a RNeasy Plus Micro Kit (Qiagen) according to the manufacturer’s instructions, and fold changes were calculated based on mRNA expression levels of the degenerated versus normal endplates, with change thresholds set at 0.8 and 1.2 to pick hits. Primary chondrocyte cells were plated in 6-well dishes, treated, and then harvested. Total RNAs were extracted in TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions for qRT-PCR experiments, carried out as previously described [
17] using the LightCycler 96 (Roche Diagnostics, Rotkreuz, Switzerland). Primers used are listed in Additional file
1: Table S1. The cycling conditions were: 95 °C for 5 min, 40 cycles of 95 °C for 10 s and 60 °C for 30 s, and then 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. The fluorescence was measured during 40 cycles of the 60 °C step. Relative mRNA expression was analyzed using the 2
−ΔΔCt method.
RIPK1 knockdown was achieved through viral transduction in primary chondrocyte cells using lentiviral transduction particles for shRNAs. The sequences for the short hairpin RNAs for
RIPK1 (shRIPK1) are listed in Additional file
2: Table S2. The shRIPK1s were cloned into the vector pTripz, characterized, and then sequenced. Lentiviral vector packaging and lentiviral transduction were carried out as described previously [
18], and shRNA expression was inducted in the presence of doxycycline.
Overexpression of RIPK1 in primary chondrocyte cells
Full-length cDNA encoding
RIPK1 (NM_001359997.1) was amplified from the
Mus musculus fibroblast cell line NIH/3T3 (ATCC, USA) using the following primers: 5′-GCTCTAGAGCCACCATGCAACCAGACATGTCCTTGGACA-3′ (
Xbal) and 5′-TAGGATCCGCTCTGGCTGGCACGAATCAAGTGG-3′ (
BamHI). These cDNAs were then cloned into the vector pCDH-EF1-MCS-T2A-Puro (Addgene, Cambridge, MA), characterized, and sequenced. Lentiviral vector packaging and lentiviral transduction were carried out as described previously [
18].
Isolation, culture, and identification of primary chondrocyte cells
Cartilage was obtained from the hind knees of 6- to 10-day-old ICR mice or from IVD degeneration models at the indicated times. After incision of the fiber annulus and removal of the nucleus pulposus with a blade under a dissecting microscope, the translucent endplate cartilage was exposed. The cartilages are shallow, dish-like structures, thin in the center and thick at the boundary. They were minced into 1 mm3 pieces with ophthalmic scissors, washed three times with phosphate-buffered saline (PBS) containing 1000 U/mL penicillin and streptomycin 1 mg/L, and then collected aseptically on a sterile bench. Chondrocytes were obtained by digestion with collagenase II, then the cells were washed with PBS and cultured in petri dishes in a humidified incubator containing 5% CO2 and 10% O2 at 37 °C. Chondrocytes were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and streptomycin 0.1 mg/L). The cells were trypsinized and replated for a few times to purify chondrocytes. Primary chondrocyte cells were identified using Toluidine blue staining. Cells were fixed in 4% formaldehyde and stained with Toluidine blue for microscope observation.
Cellular senescence assays
Senescence β-galactosidase Staining Kits (Cell Signaling Technology) were used for SA-β-gal staining according to the manufacturer’s instructions. Cells were cultured in petri dishes at 37 °C in a humidifier incubator containing 5% CO2 and 10% O2. Briefly, primary chondrocyte cells were fixed with 2% formaldehyde and 0.2% glutaraldehyde, and then incubated with X-gal staining solution. Cells were visualized and imaged with a Nikon Eclipse Ni-U microscope (Nikon, Tokyo, Japan). The percentages of positively stained cells were calculated based on three independent experiments. Chondrocyte senescence was induced using IL-1β at 10 ng/mL for 48 h.
Animal model of IVD degeneration and histological staining
A total of 18 ICR mouse, aged 6 weeks, were used for caudal vertebra degeneration. Mice were raised in two groups randomly. To develop degeneration, mice received a surgical procedure. Briefly, the mice were anesthetized with 0.3 mL of 0.6% pentobarbital sodium intraperitoneally. The levels between the sixth and seventh, seventh and eighth, and eighth and ninth coccygeal vertebrae were identified under surgical microscope. Induction of degeneration was performed by percutaneous puncture with a 1-mL syringe needle. The needle was introduced until it reached the nucleus pulposus, when it was turned 360° and maintained in the same position for 2 min. The mice were raised in double cages where animals can run through two cages.
The samples were collected at 0, 1, and 3 months after the puncture, and then the animals were executed by neck dislocation. The samples were removed and decalcified in 15% EDTA for a week, then the samples were stored in 10% formaldehyde for 48–72 h, before water flushing for 4 h followed by paraffin embedding. Finally, paraffin sections (3 µm in thickness) were stained with hematoxylin and eosin (H&E) by a standard procedure and imaged at 40–200× magnification (Nikon).
Apoptosis assays
Primary chondrocyte cells were plated at 6 × 10
5 cells/dish in 6-well plates and cultured for either 1 week after Lentiviral infection or 48 h of drug treatment. Cells were treated and subjected to apoptosis kit reagents as previously described [
17,
19]. Cisplatin (5 μM) was used in this assay as a positive control for methodology. The data were analyzed using FlowJo software (Ashland, OR, USA).
Western blot assays
The standard procedures used in this study are described previously [
16,
19,
20]. Each experiment was independently performed at least three times. Briefly, cartilage cells were lysed in Mammalian Protein Extraction Reagent (MPER™, Thermo Fisher Scientific) and lysis buffer was supplemented with Halt™ Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) and Complete™ Mini Protease Inhibitor Cocktail (Roche Diagnostics). BCA Protein Assay Kits were used to determine protein concentrations. Proteins were resolved on 4–12% gradient SDS-PAGE gels (Bio-Rad, Hercules, CA) and transferred to PVDF membrane (EMD Millipore, Temecula, CA). Membranes were incubated with primary antibody, followed by HRP-conjugated secondary antibody (Cell Signaling Technology) and signal detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). Immunoblots were quantified using Image J as described. Each experiment was independently performed at least for three times.
Cytokine assay
Cytokines were measured using the Bio-Plex Pro mouse cytokine 23-plex assay (Bio-Rad) according to the manufacturer’s instructions for the Luminex 200 instrument. Where a value was above or below the reference range, it was assigned the value of the highest or lowest standard, respectively. Lysates were made by homogenizing organs in ice-cold protein DISC lysis buffer (Roche, 30 mM Tris–HCl (pH 7.0), 120 mM NaCl, 10% Glycerol, 1% Triton X-100, complete protease inhibitors) followed by protein level normalization using a BCA assay (Thermo Scientific).
Statistical analysis
Data are reported as mean ± SD, unless otherwise noted. Significance was analyzed by one-way ANOVA using GraphPad Prism version 5.00 (GraphPad, San Diego, CA, USA), unless otherwise specified [
19]. *
P < 0.05; **
P < 0.01; ***
P < 0.001.
Discussion
IVD degeneration is a complicated issue involving a myriad of factors. Pathological changes have been recognized and classified as ‘degeneration’ as early as in the second decade of life. Recent studies have focused on understanding the molecular and genetic aspects of disc degeneration to diagnosis the degeneration early, identify the optimal time to start therapeutic intervention, and halt or slow down the degenerative process. Nonetheless, apoptosis or programmed cell death appears to correlate with age-related degeneration, with a higher percentage of apoptosis present in older people [
23]. Identifying the molecular causes or capturing early events in apoptosis during disc degeneration could revolutionize current treatment of back pain. To this end, the Fas receptor is expressed shortly after the onset of disc degeneration [
24], while a high mechanical load, decreased production of important matrix proteins (such as type II collagen and aggrecan), and increased production of degradative, inflammatory, and catabolic molecules (such as TNF, ILs, MMPs, cathepsin, aggrecanase, lysozyme, nitric oxide, and free radicals) are also implicated causally [
25].
Since IVD degeneration is a complex disease and there are no prominent therapeutic agents identified to target pathological molecules, the signaling network based on systems biology and protein–protein interaction database analysis might offer new opportunities to implicate targets and biomarkers for IVD degeneration. The signaling network built by us was based on identified pathological molecules as well as proteins not yet implicated in IVD degeneration, but that show interactions with the known pathological molecules in IVD degeneration disease. Since the cells on patient endplates are very limited, we only had enough for qRT-PCR screening to look for differentially expressed mRNAs of the network proteins in degenerated versus normal specimens. Our screening identified ESR1, a known regulator in multiple degenerative and aging diseases such as Alzheimer’s [
26,
27], radiographic hip osteoarthritis [
28], aging macula disorder [
29], and cancers [
30]. More importantly, ESR1 has been implicated to play a role in the bone metabolism of osteoporosis, osteocytes, osteoclasts, osteoblasts, immune cells, and other cells to maintain bone mineral density with varieties of mechanisms [
21]. Therefore, it was not surprising to detect abnormal expression of ESR1 in patients with IVD disease and it proved that our signaling network and qRT-PCR screening was effective, informative, and identified potential molecules. Another strong hit, PPP5C, inhibits cell growth when it is knocked down in several cell types. We also investigated the strong hit of RIPK1, a mediator of necroptosis, apoptosis, and inflammation [
14]. Multiple RIPK1 deficiency studies have shown persuasive evidence that RIPK1 suppresses FADD/caspase-8-dependent apoptosis in some cell types, and RIPK3/MLKL-dependent necroptosis in others [
9‐
12,
31‐
33]. We showed herein that short-term RIPK1 knockdown can increase inflammatory cytokines, while long-term RIPK1 knockdown led to apoptosis in primary chondrocyte cells. We further demonstrated that long-term RIPK1 knockdown triggered apoptosis through the cleaved caspase 3 pathway while downregulating NF-κB and MAPKs cascades and decreasing inflammation and cell survival. Our capture of both short- and long-term results of RIPK1 knockdown relatively represent the complex and lengthy degeneration process, which in turn seems to be associated with inflammation and infection. Possibly, the impact of apoptosis is more predominant after inflammation and infection to worsen the IVD degeneration, and in animal disease models, the mRNA expression of RIPK1 was lower in degeneration models after 3 months, while that of TNF was significantly higher.
Several caspase proteins have been implicated in the apoptosis of disc degeneration and thus proposed as therapeutic targets [
34‐
36]. The apoptosis we observed is also dependent on caspase as shown by zVAD blockage, and zVAD significantly inhibited apoptosis induced by THF and CHX in primary chondrocyte cells cultured from the animal models. These findings further confirmed that the apoptosis is caspase-mediated and RIPK1 dependent. Our studies suggest that inhibitors of caspase and overexpression of RIPK1 could have therapeutic potential in halting or delaying degeneration in IVD diseases or reversing the IL-1β induced senescence, and thus for developing RIPK1 agitators with low toxicity for the treatment and/or delay of IVD degeneration.
A great number of biomolecular therapies, gene and interfering RNA therapies have been broadly investigated in IVD degeneration. Injection of various growth factors, such as BMPs, EGF, TGF-βs, has shown promising results in delaying degeneration [
37]. Viral and non-viral gene delivery of genes, such as Sox-9, OP-1, TIMP-1, and BMP-2, dramatically increased disc height, gene expression, and matrix molecules [
37,
38]. Despite harsh environment where these biological molecules are injected and further improvement are required for clinical application, these studies have provided encouraging results about delaying apoptosis and long-term promotion of regeneration. Both natural and synthetic materials can provide favorable scaffolds for tissue engineering and bioactive agent delivery. Natural materials have the advantages such as low toxicity, similarity to native tissue, and easy large-scale production, and synthetic materials are highly reproducible and their mechanical and physicochemical properties can be finely adjusted. All such development will surely contribute to combating IVD degeneration with gene therapy strategies, e.g. gene therapy with
RIPK1, in the near future.
Authors’ contributions
XQ, ZLu, and DC designed and performed experiments, XQ and MZ analyzed the data, CZ participated in the patients’ surgery, XQ, ZLiu, and JL drafted the manuscript. All authors read and approved the final manuscript.