Intervertebral disc (IVD) degeneration is the leading cause of disability with no disease-modifying treatment. IVD degeneration is associated with instable mechanical loading in the spine, but little is known about how mechanical stress regulates nucleus notochordal (NC) cells to maintain IVD homeostasis. Here we report that mechanical stress can result in excessive integrin αvβ6-mediated activation of transforming growth factor beta (TGFβ), decreased NC cell vacuoles, and increased matrix proteoglycan production, and results in degenerative disc disease (DDD). Knockout of TGFβ type II receptor (TβRII) or integrin αv in the NC cells inhibited functional activity of postnatal NC cells and also resulted in DDD under mechanical loading. Administration of RGD peptide, TGFβ, and αvβ6-neutralizing antibodies attenuated IVD degeneration. Thus, integrin-mediated activation of TGFβ plays a critical role in mechanical signaling transduction to regulate IVD cell function and homeostasis. Manipulation of this signaling pathway may be a potential therapeutic target to modify DDD.
Degenerative disc disease (DDD) remains a common musculoskeletal disorder that brings an enormous socioeconomic burden.1,
Dysfunction of nucleus pulposus (NP) cells is the key in the onset of intervertebral disc (IVD) degeneration.1,6,
Temporal–spatial activation of latent matrix transforming growth factor beta (TGFβ) has been shown to modulate chondrocyte anabolic activity in articular cartilage, maintain bone homeostasis during bone remodeling, and help with tissue repair.18,19 The αv integrins in combination with -β6, -β5, and -β8 have been shown to mediate the activation of TGFβ.20,
In this study, we systematically investigated the role of mechanical stress on the functional transition of NC cells and IVD homeostasis. Utilizing multiple rodent models, we found that mechanical stress resulted in integrin αvβ6-mediated activation of TGFβ. Abnormal stress resulted in excessive TGFβ signaling and accelerated NC cells functional transition. Administration of RGD peptide and neutralizing antibodies against TGFβ and αvβ6 attenuated these changes. On the other hand, conditional knockout of TβRII or αv also impeded NC cells’ transition and caused IVD degeneration by mechanical stimuli. Thus, precise integrin-induced activation of TGFβ is required to maintain IVD cell function and homeostasis.
Materials and methods
Lumbar spine instability mouse model
C57BL/6J (male, 8-week old) mice were purchased from Charles River, Wilmington, MA, USA. After anesthetized with ketamine and xylazine, they were operated by resection of the lumbar 3th–lumbar 5th (L3–L5) spinous processes along with the supraspinous and interspinous ligaments to induce instability of lumbar spine.28,29 Sham operations were carried out only by detachment of the posterior paravertebral muscles from the L3–L5 vertebrae. The operated mice were intraperitoneally injected with either TβRI inhibitor (SB-505124, Sigma-Aldrich, St Louis, MO, USA) at a dose of 1 mg·kg−1 (SB group) or the equivalent volume of vehicle (dimethyl sulfoxide; Veh group) once every 2 days. Mice (8-week old) were killed at 0, 1, 2, 4, and 8 weeks after the surgery (n=6 per group).
Caudal spine instability mouse model
The instability of caudal spine was induced by fully depth annular stab and NP removal of the caudal 7th–8th (C7–8) IVD.30 The adjacent C8–9 IVDs were chosen for observation at 4 weeks post surgery (n=6 per group). The treatments were the same as those described in the first model.
Rat caudal IVD compression model
Twelve-week-old male Sprague–sDawley rats (Charles River) were attached at caudal vertebrae with a loading device.31,
Noto-cre (CD1 background mouse expressing Cre recombinase driven by Noto promoter) mouse strain was obtained from the lab of Cheryle A. Séguin.10 TgfβRIIflox/flox mouse strain (C57BL/6J background mouse homozygous for TgfβRII flox allele) was obtained from the lab of H.L. Moses.35 Notocre::TgfβRIIflox/+ mice were generated by crossing heterozygote Noto-cre strain with homozygote TgfβRIIflox/flox strain. TgfβRII−/− and TgfβRI+/+ (wild-type (WT) littermates) offspring were generated by crossing two Notocre::TgfβRIIflox/+ strains (Notocre::TgfβRIIflox/flox mice that are TgfβRII conditional deletion in Noto lineage cells are referred to as “TgfβRII−/−”. Notocre::TgfβRII+/+ mice that are heterozygous for Noto-cre are referred to as “TgfβRII+/+” in the text, n=6 per group).
The genotype of the mice was determined by PCR analyses of genomic DNA isolated from mouse tails using the following primers: Noto-directed cre forward, 5′- ATACCGGCAGATCATGCAAGC-3′, and reverse, 5′- ATGCACATATGCAACCCACA-3′.10 The loxP TgfβRII allele was identified with the primers forward, 5′- TAAACAAGGTCCGGAGCCCA-3′, and reverse, 5′- ACTTCTGCAAGAGGTCCCCT-3′.36
αvflox/flox mice mouse strain was obtained from the lab of Andrew Burich, Benaroya Research Institute at Virginia Mason. Notocre::αvflox/flox mice that are αv conditional deletion in Noto lineage cells are referred to as “αv−/−”. Notocre:: αv+/+ mice that are heterozygous for Noto-cre are referred to as “αv+/+” in the text (n=3 per group). The loxP αv allele was identified with the primers forward, 5′- TTCAGGACGGCACAAAGACCGTTG-3′, and reverse, 5′- CACAAATCAAGGATGACCAAACTGAG-3′.
C57BL/6J mice of the following ages: 16.0-day pregnant, 3 days, 1 weeks, 1 month, 2 months, 3 months and 4 months were purchased from Charles River (n=6 per time point).
All animals (rat and mouse) were maintained in the Animal Facility of the Johns Hopkins University School of Medicine. The experimental protocols for both species were reviewed and approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University, Baltimore, MD, USA.
IVD ex vivo compression model
The L1–L5 lumbar IVDs were removed under sterile conditions from 4-week-old C57BL/6J mice. The collected IVDs were organ-cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 1% penicillin–streptomycin (MediaTech, Dallas, TX, USA) and pretreated with one of the followings: 2 ng·mL−1 of recombinant mouse TGFβ1 (7666-MB-005, R&D, Minneapolis, Minnesota, USA), 5.0 μg·ml−1 of 1D11, 2 μmol·L−1 of RGD peptide (3498, Tocris Bioscience, Burlington, NC, USA), 100 μg·mL−1 of anti-αVβ6-neutralizing antibody (ab77906, Abcam, Cambridge, MA, USA) or Veh (13C4, R&D Systems) overnight. Compression loads of 0 or 1.0 MPa were applied by vertically placing weights on the IVDs after pretreatment. Loading duration was 24 h (n=6 per group).37
We determined the sample size based on power analysis via website http://www.biomath.info (for two groups, 80% power, 5% significance, two-sided), or via G*Power (for more than two groups, 80% power, 5% significance). For animal model and TgfβRII knockout studies, each experiment was conducted twice: one pilot experiment in three samples and two independent experiments of six samples. For αv knockout mice, three independent experiments with six samples were conducted.
Micro Computed Tomography (μCT)
The lower thoracic and whole lumbar spine from mice were dissected, fixed in 10% buffered formalin for 48 h, transferred into phosphate-buffered saline, and then examined by high-resolution μCT (Skyscan1172). The ribs on the lower thoracic were included for the identification of L4–L5 IVD localization. Images were reconstructed and analyzed using NRecon v1.6 and CTAn v1.9 (Skyscan company, San Jose, CA, USA), respectively. Three-dimensional model visualization software, CTVol v2.0 (Skyscan company, San Jose, CA, USA), was used to analyze parameters of the L4–L5 IVD with half height of L4 and L5 vertebrae. The scanner was set at a voltage of 49 kVp, a current of 200 μA, and a resolution of 6.8 μm per pixel to measure the IVD and endplate (EP). A resolution 16.8 μm of per pixel was set for the whole L5 vertebral body measurement. Coronal images of the L4–L5 IVD were used to perform three-dimensional histomorphometric analyses of IVD. IVD volume was defined by the region of interest to cover the whole invisible space between L4 and L5 vertebrae. Parameter: TV (total tissue volume) was used for three-dimensional structural analysis.
Histochemistry, immunohistochemistry, and histomorphometry
The specimens were fixed in 10% buffered formalin for 48 h, decalcified in 10% EDTA (pH 7.4) for 14 days, dehydrated, and embedded in paraffin. Four-micrometer-thick coronal-oriented sections of the L4–L5, C7–9, or C8–10 spine were processed for safranin O and fast green staining. Tartrate-resistant acid phosphatase staining was performed using a standard protocol (Sigma-Aldrich). Immunostaining was performed using a standard protocol. Sections were incubated with primary antibodies to mouse aggrecan (1:200, AB1031, Millipore, Billerica, MA, USA), CCN2 (1:400, ab6992, Abcam), pSmad2/3 (1:200, sc-11769, Santa Cruz, Dallas, TX, USA), integrin αVβ6 (1:100, bs-5791R-Biotin, Bioss, Woburn, MA, USA), integrin αVβ3 (1:100, bs-1310R, Bioss), integrin αVβ5 (1:100, bs-1356R, Bioss), integrin β8 (1:300, ab80673, Abcam), integrin αV (Santa Cruz, 1:100, sc-6617-R), integrin β6 (Santa Cruz, 1:100, sc-6632), and TGFβRII (1:100, sc-400, Santa Cruz) at 4 °C overnight. For immunohistochemical staining, a horseradish peroxidase-streptavidin detection system (Dako, Carpinteria, CA, USA) was subsequently used to detect the immunoactivity, followed by counterstaining with hematoxylin (Sigma-Aldrich). For immunofluorescent assay, the slides were incubated with secondary antibodies conjugated with fluorescence at room temperature for 1 h while avoiding light. Morphometric study was performed by an image autoanalysis system (Olympus DP71, SEG Biological Microscopes, Center Valley, Pennsylvania, USA). Triplicates of each sample were used for staining.
Quantitative histomorphometric analysis
Quantitative histomorphometric analysis was conducted in a blinded manner with Image-Pro Plus Software version 6.0 (Media Cybernetics Inc, Rockville, Maryland, USA). EP and IVD scores were obtained as previously described.1,38 The percentage of pSmad2/3-positive cells was obtained by counting the number of positive staining cells to the number of total cells in the NP region. The expression of integrin αVβ6 was calculated by the sum of integrated optical density in the NP region. The area of CCN2-positive staining was calculated in the whole L4–L5 IVD in lumbar spine instability (LSI) mice (2-month old). In other experiments, the area of CCN2-positive staining was calculated only in the NP region. The percentage of Acan-positive staining was calculated by counting the positive staining area of region of interest that covers all cells in NP.
Quantitative reverse transcription-PCR
Total RNA was extracted from NP tissue of IVD in ex vivo assay using TRIzol reagent (Sigma-Aldrich) according to the manufacturer’s instruction. The yield and purity of RNA were estimated spectrophotometrically using theA260/A280 ratio. Two micrograms of RNA was reverse-transcribed into complementary DNA using the SuperScript first-strand synthesis system (Invitrogen). One microliter of complementary DNA was subjected to quantitative reverse transcription-PCR amplification using SYBR GREENPCR Master Mix (Promega, Madison, WI, USA) and sequence-specific primers for Acan: 5′- CAGATGGCACCCTCCGATAC-3′and 5′- GACACACCTCGGAAGCAGAA-3′. The value of gene expression was normalized relative to the mouse GAPDH: 5′- AATGTGTCCGTCGTGGATCTGA-3′ and 5′- AGTGTAGCCCAAGATGCCCTTC-3′. PCR reactions were performed in triplicates. The data were analyzed using the 2−ΔΔCT method.
Western blot analyses were conducted on the protein extraction from NP tissue in ex vivo assay. The protein extraction was centrifuged, and the concentration of supernatants was evaluated by DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA), then the proteins were separated by SDS-polyacrylamide gel electrophoresis and blotted on a polyvinylidene fluoride membrane (Bio-Rad Laboratories). After incubation in specific antibodies, proteins were detected using an enhanced chemiluminescence kit (Amersham Biosciences, Pittsburgh, PA, USA). The target protein concentrations were examined by antibodies recognizing mouse pSmad2 (1:1 000, 3101, Cell Signaling Technology Inc., Danvers, MA, USA), Smad2 (1:1 000, 3103, Cell Signaling Technology Inc.), integrin αV (1:500, sc-6617-R, Santa Cruz), and GAPDH (1:1 000, 8884, Cell Signaling Technology Inc.).
The data were expressed as mean±s.d., and statistical significance was determined using a Student’s t-test in time point or genetic mice comparison, or one-way analysis of variance followed by a post hoc Least-Significance-Difference (LSD) test (homogeneity of variance) or a Tukey’s test (heterogeneity of variance) in treatment or ex vivo assay comparison. The level of significance was defined as P<0.05. All data analyses were performed using SPSS 15.0 analysis software (SPSS Inc, Chicago, Illinois, USA).
Activation of TGFβ associates with reduced NC cell vacuoles and increased extracellular proteoglycan in response to mechanical stress
The IVD height increased in mice from birth through 1 month, and then remained stable before decreasing around 4 months. The NC cells’ sphere-shaped vacuole observed at birth changed to a spindle-like shape by 4 months (Figure 1a and c). Extracellular aggrecan secretion was stimulated on day 3 after birth and accumulated around the NC cells to generate IVD extracellular space adjacent to endplates from day 7 (Figure 1b). To investigate that these changes were associated with mechanical stress, we employed a LSI mouse model by removing spinous processes and posterior supraspinous and interspinous ligaments (Figure 1d).28,29 The effects of mechanical stress on NC cells were analyzed by immunostaining of the IVD sections collected from 2-month-old LSI mice, in which the IVD space is peaked. The vacuole sizes of NC cells gradually decreased beginning at 2 weeks post surgery in LSI, whereas in the sham-operated mice NC size decreased at 4 weeks with Safranin O staining (Figure 1e). Significant IVD degeneration was observed in LSI mice relative to sham-operated mice, as shown by IVD score beginning as early as 1 week post surgery1,38 (Figure 1g). Mechanical stress has been shown to activate latent TGFβ.20,21,39,40 Immunostaining revealed that phosphorylated Smad2/3-positive cells (pSmad2/3+) in the NP were significantly increased 1, 2, and 4 weeks post surgery in the LSI mice relative to the sham controls (Figure 1f and h). Further, the levels of CCN2, a TGFβ downstream factor that upregulates the synthesis of matrix proteins in IVDs,26,27,41,
Integrin αvβ6 induces TGFβ activation in response to mechanical stress to regulate NC cell function
We then investigated the mechanism of mechanical stress-induced activation of latent TGFβ. The αvβ integrins are one known mechanism that mediates cell-induced conformational change of TGFβ latent complex to release active TGFβ.21,
To determine a causal relationship, active TGFβ, RGD peptide, neutralizing antibodies against TGFβ orαvβ6, or vehicle were applied to an ex vivo IVD compression loading model.37 Immunostaining demonstrated that RGD peptide, antibodies against TGFβ (1D11), or αvβ6 all inhibited stress-induced phosphorylation of Smad2/3 in NC cells (Figure 3i and k). The result was confirmed by western blot analysis (Figure 3l). Importantly, the morphology of NC cell vacuoles was altered in the active TGFβ and vehicle-compression treatment groups (Figure 3j, second and third row), whereas the sphere shape of vacuoles was maintained in the RGD, TGFβ (1D11), and αvβ6 antibody groups (Figure 3j, bottom three rows) relative to the zero compression vehicle group (Figure 3j, top row). In contrast, the extracellular CCN2 and aggrecan were significantly stimulated in the active TGFβ group and compression group treated with vehicle (Figure 4a and b, rows 2 and 3; Figure 4c and d) but inhibited in the RGD and TGFβ antibody groups (Figure 4a and b, rows 4 and 5). Messenger RNA expression of aggrecan was similarly inhibited by RGD and TGFβ antibody (Figure 4e).
Conditional knockout of TGFβ type II receptor prohibits functional transition of NC cells
To confirm the role of TGFβ in control of NC cell function in vivo, notochord-originated cell-specific TgfbrII knockout mice (TgfbrII−/−) were generated by crossing floxed TgfbrII mice with Noto-Cre mice. TβRII- and pSmad2/3-positive NC cells were almost undetectable in immunostaining of IVD sections of TgfbrII−/− mice (Figure 5a–d), validating our mouse model. Immunostaining demonstrated that CCN2 and aggrecan were much lower in the NPs of TgfbrII−/− mice relative to WT TgfbrII+/+ littermates (Figure 5e–h), further confirming CCN2 and aggrecan as downstream signaling targets. Safranin O staining of IVD sections showed that NC cells have much larger vacuoles with less proteoglycan in 4-week-old TgfbrII−/− mice relative to their WT TgfbrII+/+ littermates (Figure 5i). Furthermore, the IVD volume was significantly increased in the TgfbrII−/− mice in comparison to their WT TgfbrII+/+ littermates in μCT analysis (Figure 5j). These genetic data demonstrate that TGFβ signaling in NC cells affects vacuole size for IVD morphology.
Conditional knockout of integrin αv impedes postnatal functional transition of NC cells
We then examined the role of integrin αv in activation of TGFβ in notochord cells as αv chain is the core to mediate the activation of TGFβ. Floxed αv mice were crossed with Noto-Cre mice to generate αv knockout mice (αv−/−). Immunostaining of IVD sections validated that αv expression was diminished specifically in NC cells, whereas the expression remained unchanged in AF and EP cells in αv−/−mice relative to their WT αv+/+ littermates (Figure 6a). Integrin β6 expression was unaffected in the αv−/− NC cells (Figure 6b). The reduction of αv in the αv−/− NC cells was further confirmed by western blot analysis (Figure 6k). As expected, αvβ6- and pSmad2/3-positive NC cells were significantly reduced in αv−/−mice relative to WT αv+/+ littermates (Figure 6c, d, g and h). The contents of CCN2 and aggrecan were also much lower in the NPs of αv−/− mice relative to WT αv+/+ littermates (Figure 6e, f, i and j). Similar to TgfbrII−/−mice, NC cells had much larger vacuoles with less proteoglycan in the αv−/− mice relative to their WT littermates (Figure 6m). The αv−/− and TgfbrII−/− mice demonstrate that disruption of TGFβ signaling can delay NC morphologic transition. Therefore, to evaluate whether the morphologic change of vacuoles was sufficient to prevent mechanically induced IVD degeneration, we further subjected αv−/− and TgfbrII−/− mice to LSI or sham surgery (Figure 1d). Safranin O staining showed that IVD was significantly degenerated in both LSI αv−/− and TgfbrII−/− mice relative their sham-operated controls (Figure 6n), and demonstrated that complete loss of NC cell TGFβ signaling can also be as detrimental as excessive TGFβ signaling.
Inhibition of excess activation of TGFβ attenuates IVD degeneration
We examined whether inhibition of excessive active TGFβ is beneficial for IVD degeneration. TGFβ type I receptor (TβRI) inhibitor (1 mg·kg−1) was intraperitoneally injected in the LSI mice post surgery. Degeneration of IVD was attenuated as assessed by IVD score (Figure 7a). The vacuolar morphology was preserved with the TβRI inhibitor treatment relative to sham-operated versus LSI-operated with vehicle treatment (Figure 7d). As expected, pSmad2/3+ cells were reduced by TβRI inhibitor (Figure 7b and e). Expression of aggrecan and CCN2 was maintained at similar levels of sham-operated mice as opposed to the elevation of both seen in the LSI-operated vehicle-treated group (Figure 7c, f and g).
In parallel, we administrated the TGFβ-neutralizing antibody directly into the IVD in a rat caudal IVD compression model.31,
The initiation of DDD by NP dysfunction is a hot topic in the spinal disease field. NP cells originate from the notochord. During development, NC cells regulate the spinal morphogenesis by control of intracellular vacuoles evenly distributed in the NP to generate intervertebral space.6,10,49,50 After birth, IVDs gradually change their content with a reduction of intracellular vacuoles of NC cells corresponding to increase extracellular proteoglycans.15 Loss of vacuolar NC cells and their morphologic and functional transition are thought as early degenerative changes of IVD.12 However, the mechanism of NC cells transition is not well understood. In this study, we revealed that integrin αvβ6-induced activation of TGFβ controls the functional transition of NC cells in IVDs in multiple rodent models. Active TGFβ reduced intracellular vacuoles of NC cells, but increased extracellular proteoglycans, thereby shifting the burden of mechanical loads from the cells to the extracellular environment.
Mechanical loads are one of the most important etiologies of DDD.50 In normal physiological situations, appropriate mechanical loading produced by body weight and muscle force is necessary to activate TGFβ in the NP to modulate anabolic activity of NC cells and maintain IVD homeostasis, similar to its role in homeostasis of the bone and cartilage.40,51,
Central to this mechanosignal transduction is the activation of TGFβ by integrin αvβ6. Several of integrin α and β chains have been found in IVD involving in cell–matrix interaction.25,54 Integrin α5β1 has recently been shown to be associated with mechanotransduction in IVD cells.55 Mechanical stress has been shown to induce cell cytoskeleton changes, leading to integrin αvβ6 binding to the latent TGFβ complex for its conformational change to allow active TGFβ to bind to its receptor.45 Similarly, we found it is integrin αvβ6 that correlates to active TGFβ signaling pathway and regulates NC cells function by mechanical stimili. TGFβ signaling activates transcription and secretion of CCN2, which in turn binds to CCN2 receptor to stimulate a cascade of changes in extracellular proteoglycan expression.56 Aggrecan, the major proteoglycan in IVD, exerts osmotic pressure to resist compressive loads.57 However, aggrecan also affects the osmotic pressure in the IVD. Besides shifting water from outside the IVD, our study suggests that aggrecan likely also results in a shifting of water from intracellular to extracelluar matrix and leads to NC cells morphologic transition by dehydration.
TGFβ is recognized as an anabolic factor in IVD and has been found to prevent DDD.58 However, high levels of TGFβ1 have also been observed in the IVDs from DDD patients.39,40,59,
DDD is a polygenic disease exacerbated by many different factors.2,16,50,64,
We thank the following colleagues for their technical support: Xin Xu, Wenlong Liu, Gehua Zhen, Shouan Zhu, Xiaokai Wang, Changjun Li, Hui Xie and Liang Xie.
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