Intervertebral disc degeneration (IVDD) is one of the most common causes of lower back pain. Research has confirmed that the level of degeneration in patients with IVDD is positively correlated with the presence of calcification at the end-plates,1 which are located between the IVD and their respective vertebral bodies and function to supply nutrients to the annulus fibrosus and the nucleus pulposus.2 It has been suggested that end-plate calcification may prevent the passage of nutrients from the bloodstream to the IVD, which in turn causes a failure of the end-plate to maintain the nucleus pulposus and annulus fibrosus, thereby accelerating the progression of IVDD.
The ectonucleotide pyrophosphatase/phosphodiesterase (ENPP)-1 gene is a major contributor to cartilage calcification. Previous studies have demonstrated that ENPP-1 is an ectonucleotide triphosphatase that resides within the cell membrane and hydrolyzes extracellular nucleoside triphosphates into monophosphate and extracellular inorganic pyrophosphate (ePPi).3,4 ENPP-1 deficiency leads to excessive bone reconstruction and calcification. Additional accumulation of ePPi in the extracellular milieu also results from its reduced degradation in the pericellular matrix.5 In a previous study, we found that cyclic mechanical tension (CMT) can induce calcification in end-plate chondrocytes and results in the downregulation of ENPP-1 expression. However, there has been no other research on the regulation of ENPP-1 expression through CMT.
Transforming growth factor beta 1 (TGF-β1) plays important roles in cell proliferation, differentiation, apoptosis, and extracellular matrix synthesis, and some studies have demonstrated the involvement of TGF-β1 in calcium pyrophosphate dihydrate crystal deposition in cartilage. TGF-β1 elevates the production of ePPi in chondrocytes,6,7 presumably via the expression of ENPP-1.8–10 Previously, using real-time PCR, we showed that both ENPP-1 and TGF-β1 expressions were increased.11 However, the precise relationship between TGF-β1 and ENPP-1 is still unknown.
Numerous studies have outlined the role of mitogen-activated protein kinases (MAPKs) in mediating multiple biological processes in the cell.12 Extracellular signal-regulated kinases (ERK) 1/2, members of the MAPK family, are serine/threonine protein kinases widely expressed in eukaryotic cells that are crucial for mechanical signal transduction.13–15 However, there is little evidence that CMT regulates the expression of ENPP-1 through a TGF-β1/ERK1/2 pathway.
Thus, in this study, we applied CMT using different elongation stimuli over a timeframe of 20, 40, and 60 minutes to observe changes in the expression of ENPP-1. We also sought to decipher the TGF-β1-mediated regulation of ENPP-1 expression by identifying the relevant signaling pathway and to assess the role of ERK1/2 signaling in this pathway activation after CMT.
Chondrocyte isolation and culture
Primary chondrocytes were isolated from the lumbar spine end-plate cartilage of Sprague-Dawley (SD) rats (160-180 g). Cartilage from the L1-L5 end-plates was carefully removed from the vertebrae and minced into small pieces (<0.03 mm3). Samples were sequentially digested with 0.25% trypsin (Sigma, St. Louis, MO, USA) at 37°C for 20 minutes and by 0.02% collagenase (Sigma) at 37°C for 5 hours. Chondrocytes were washed twice with phosphate-buffered saline (PBS) and cultured on Petri dishes at 37°C at 5% CO2. Monolayer cultures were maintained in growth medium comprising Dulbecco's modified Eagle's medium/ F-12 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin, and 50 mg/ml streptomycin (all from Hyclone, Logan, UT, USA). Cells from the second passage were used for experiments.
Application of cyclic strain
End-plate chondrocytes were plated at a density of 1×105 cells/cm2 in 2 ml of growth medium in 6-well flexible silicone rubber BioFlexTM plates coated with collagen type I (Flexcell International Corporation, Hillsborough, NC, USA). The cells were cultured for 48 hours to allow them to attach and reach 80%-90% confluence, at which time the growth medium was refreshed, and mechanical strain was applied. A cyclic mechanical strain with 0.5 Hz sinusoidal curve at 3%, 6%, and 9% elongation was applied with an FX-4000TTM Flexercell® Tension PlusTM unit (Flexcell International Corporation). The end-plate chondrocytes were stimulated with 3%, 6%, and 9% elongation for 20, 40, and 60 minutes. The cultures were incubated in a humidified atmosphere at 37°C and 5% CO2 during stimulation. Cells were harvested immediately after the CMT stimulation.
Real-time reverse transcription PCR (RT-PCR)
Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. After a reverse transcription reaction, RT-PCR was performed with a Roche LightCycler 480 system using SYBR®Premix Ex TaqTM (Takara, Dalian, China) according to the manufacturer's instructions. The conditions for RT-PCR were as follows: denaturation at 95°C for 10 seconds; 40 cycles at 95°C for 10 seconds and 60°C for 30 seconds. A dissociation stage was added to the end of the amplification procedure. There was no nonspecific amplification, as determined by the dissociation curve. GAPDH was used as an internal control. Data were analyzed using the Ct comparison method and expressed as fold-change compared with control. Each sample was analyzed in triplicate. The primer sequences are shown in Table 1.
Cells were lysed on ice for 30 minutes in a lysis buffer containing 50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with protease inhibitors (10 μg/ml leupeptin, 10 μg/ml pepstatin A, and 10 μg/ml aprotinin). For Western blotting analysis, 20 μg of sample was resolved by 12% SDS-PAGE and electro-transferred onto nitrocellulose membranes (Whatman, Piscataway, NJ, USA). The primary antibodies used were anti-ERK1/2 (rabbit monoclonal anti-p44/42 MAPK; Cell Signaling Technology, Inc., Danvers, MA, USA); anti-p38 (rabbit monoclonal anti-p38 MAPK; Cell Signaling Technology, Inc.); anti-SAPK/JNK (rabbit monoclonal anti-SAPK/JNK; Cell Signaling Technology, Inc.); anti-phospho-p38 (rabbit monoclonal anti-phospho-p38 MAPK Thr180/Tyr182; Cell Signaling Technology, Inc.), anti-phospho-ERK1/2 (rabbit monoclonal anti-phospho p44/42 MAPK Thr202/Tyr204; Cell Signaling Technology, Inc.); anti-phospho-SAPK/ JNK (rabbit monoclonal anti-phospho-SAPK/JNK Thr183/ Tyr185; Cell Signaling Technology, Inc.); and anti-ENPP-1 (rabbit polyclonal anti-ENPP-1; Cell Signaling Technology, Inc.). All antibodies were used at a dilution of 1:1000. For normalization of protein loading, a β-actin antibody (Sigma) was used at 1:5000 dilution. Infrared-labeled secondary goat-anti-rabbit IRDye 800 antibody (Li-Cor Biosciences, Lincoln, NE, USA) was used as a secondary antibody, and the bound complex was detected using the Odyssey Infrared Imaging System (Li-Cor Biosciences). The images were analyzed using the Odyssey Application Software, version 1.2 (Li-Cor Biosciences) to obtain the integrated intensities. For the kinase assays, end-plate chondrocytes were pretreated with 50 μmol/L of a specific inhibitor of ERK1/2 (U0126; Calbiochem, San Diego, CA, USA) with CMT stimulation for 40 minutes.
Silencing experiments with small interfering RNA
siRNA sequences (designed by Shanghai GenePharma Co. Ltd, Shanghai, China) were used at a final concentration of 50 nmol/L: TGF-β1 sense 5'-CAGCUGUACAUUGACUUUATT-3' and antisense 5'-UAAAGUCAAU GUACAGCUGTT-3'. Transfection of TGF-β1 siRNA was performed after CTS stimulation for 40 minutes (0.5 Hz, 3% elongation). Briefly, siRNA and lipofectamine 2000 were diluted separately in serumfree medium, and then the diluted Lipofectamine was added to the siRNA. After 20 minutes incubation at room temperature, cells were washed with PBS and incubated for 4 hours with the siRNA/Lipofectamine mix. The cells were then incubated with media containing 10% FBS for 48 hours. Cells were then harvested and ENPP-1 mRNA expression was used as a negative control to check for the specificity of siRNA effects.
SPSS 11.0 statistical software (SPSS Inc. Chicago, IL, USA) was used for statistical analysis. Results are presented as the mean ± standard deviation (SD) of at least three independent experiments. Comparisons of overall differences between groups were made using analysis of variance (ANOVA), and the control group and treatment groups were compared by the least significant difference (LSD) method. A P value of less than 0.05 was considered statistically significant.
ENPP-1 expression following CMT elongation
We applied three levels of CMT elongation (3%, 6%, and 9%) to test whether the different levels of stimuli had an effect on ENPP-1 gene expression in end-plate chondrocytes. RT-PCR was used to detect the expression changes in ENPP-1 mRNA after 3% (Figure 1A), 6% (Figure 1B), and 9% (Figure 1C) elongation after 20, 40, and 60 minutes. The 3% elongation group showed an increase in the expression of ENPP-1 after 20 minutes (P=0.0087) and 40 minutes (P=0.0057) of stimulation, with a larger increase observed in the 40 minutes group. Western blotting analysis (Figure 1D) also confirmed an upregulation of ENPP-1 protein expression in the 20 and 40 minutes groups. Thus, we chose 3% elongation for 40 minutes to induce CMT in subsequent experiments.
Contribution of TGF-β1 to the regulation of ENPP-1 expression by cyclic mechanical strain
Both RT-PCR and Western blotting indicated that TGF-β1 induces ENPP-1 expression after 3% CMT (Figure 2A and 2B). We used TGF-β1 siRNA and scramble siRNA to investigate the role of TGF-β1 in the induction of ENPP-1 expression (Figure 2C and 2D). TGF-β1 siRNA effectively reduced ENPP-1 mRNA and protein expression. These results indicate that the expression of ENPP-1 may be caused by the activity of endogenous TGF-β1.
CMT-stimulated phosphorylation of MAPKs in endplate chondrocytes
As shown in Figure 3, the phosphorylation of ERK1/2 was activated 40 minutes after 3% CMT, but not in non-stimulated cells. The phosphorylation of ERK1/2, but not that of p38 MAPK or JNK, increased after CMT. Thus, CMT activates the ERK1/2 pathway in end-plate chondrocytes.
Contribution of the ERK1/2 pathway to TGF-β1-regulated ENPP-1 expression
The level of phospho-ERK was strongly reduced in cells transfected with TGF-β1 siRNA as compared with chondrocytes transfected with a scrambled siRNA (Figure 4A). Following treatment with U0126, the expression of ENPP-1 mRNA and protein was markedly decreased in cells stimulated with 3% CMT (Figure 4B and 4C), providing further evidence of the role of ERK1/2 signaling in CMT-induced ENPP-1 expression.
IVD calcification is a complication resulting from and further promoting the process of IVDD.16,17 We considered that mechanical strain could be a cause of calcification,18 and demonstrated this principle in our previous study.19 Some studies indicate that humans are generally exposed to approximately 1000 μstrain under physiological conditions and no more than 4000 μstrain during vigorous activity. In terms of our elongation measurements, 1000 μstrain is approximately equal to 1% elongation; therefore, 6% and 9% elongation strains are not suitable strain levels in humans.20,21 We previously showed a decrease in ENPP-1 expression with higher elongation strain that resulted in calcification in end-plates;11 however, that study was designed to first clarify whether there was a relationship between mechanical strain and ENPP-1. Therefore, for the current study, we choose 3% mechanical strain, as it is an acceptable strain threshold for humans. Indeed, after several tests at various strain levels, we determined that 3% elongation for 40 minutes resulted in the largest increase in the expression of ENPP-1 mRNA.
Studies have shown that the application of mechanical strain can produce collagenous proteins and fibronectin,22,23 which cause an increase in the expression of TGF-β1.24,25 In our current study, we demonstrated that the expression levels of TGF-β1 and ENPP-1 were increased by a 3% elongation CMT for 40 minutes and that ENPP-1 expression was higher in cells exposed to TGF-β1 than that in control cells. We speculate that ENPP-1 gene expression is mainly dependent on endogenous TGF-β1 regulation. In rat end-plate cells exposed to 5 or 10 ng/ml of TGF-β1 for 24 hours and 3% elongation CMT for 40 minutes, we observed that the expression of ENPP-1 was increased at the mRNA and protein levels. We used siRNA to investigate the contribution of TGF-β1 in ENPP-1 expression. TGF-β1 siRNA effectively reduced ENPP-1 expression at the mRNA and protein levels, indicating that the CMT-induced increase in ENPP-1 expression may be caused by endogenous TGF-β1.
MAPKs mediate multiple biological processes. The MAPK family is known to regulate the expression of the ankylosis, progressive homolog (ANKH) gene5,26 and is a central transducer of mechanical forces to biological responses in cartilage.27 When we studied the regulation of ENPP-1 expression by TGF-β1, we demonstrated that ERK1/2 and p38 MAPK were activated in our cell culture system. Other studies have reported that the contribution of p38 MAPK to TGF-β1 signaling in cartilage is greatly influenced by the species and the cell culture system.28 It is known that TGF-β1 induces ERK1/2 phosphorylation.5 We observed that ERK1/2 was activated under our experimental conditions; however, the phosphorylation of p38 MAPK and JNK remained unchanged. ENPP-1 expression was decreased when cells were exposed to U0126, a specific ERK phosphorylation inhibitor. Therefore, we surmised that TGF-β1 induced the phosphorylation of ERK1/2 after CMT stimulation.
In conclusion, the results of the current study demonstrate that the CMT-induced increase in the expression of ENPP-1 in end-plate chondrocytes is dependent on the induction of TGF-β1 by ERK1/2 signaling. This information may provide a new approach to treat IVDD caused by mechanical tension.
1. Xu HG, Chen XW, Wang H, Lu LM, Liu P, Xia LZ. Correlation between chondrocyte apoptosis of vertebral cartilage endplate and degeneration of intervertebral disc. Natl Med J China (Chin) 2008; 88: 194-197.
2. Urban JP, Smith S, Fairbank JC. Nutrition of the intervertebral disc. Spine 2004; 29: 2700-2709.
3. Howell DS, Martel-Pelletier J, Pelletier JP, Morales S, Muniz O. NTP pyrophosphohydrolase in human chondrocalcinotic and osteoarthritic cartilage: further studies on histologic and subcellular distribution. Arthritis Rheum 1984; 27: 193-199.
4. Ryan LM, Wortmann RL, Karas B, Lynch MP, McCarty DJ. Pyrophosphohydrolase activity and inorganic pyrophosphate content of cultured human skin fibroblasts. Elevated levels in some patients with calcium pyrophosphate dehydrate deposition disease. J Clin Invest 1986; 77: 1689-1693.
5. Cailotto F, Bianchi A, Sebillaud S, Venkatesan N, Moulin D, Jouzeau JY, et al. Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal regulated kinase pathways in chondrocytes. Arthritis Res Ther 2007; 9: R122.
6. Ryan LM, Rosenthal AK. Metabolism of extracellular pyrophosphate. Curr Opin Rheumatol 2003; 15: 311-314.
7. Rosenthal AK, Gohr CM, Henry LA, Le M. Participation of transglutaminase in the activation of latent transforming growth factor beta1 in aging articular cartilage. Arthritis Rheum 2000; 43: 1729-1733.
8. Terkeltaub RA. Inorganic pyrophosphate generation and disposition in pathophysiology. Am J Physiol Cell Physiol 2001; 281: C1-C11.
9. Lotz M, Rosen F, McCabe G, Quach J, Blanco F, Dudler J, et al. Interleukin 1 beta suppresses transforming growth factor-induced inorganic pyrophosphate (ppi) production and expression of the ppi-generating enzyme PC-1 in human chondrocytes. Proc Natl Acad Sci U S A 1995; 92: 10364-10368.
10. Johnson K, Vaingankar S, Chen Y, Moffa A, Goldring MB, Sano K, et al. Differential mechanisms of inorganic pyrophosphate production by plasma cell membrane glycoprotein-1 (pc-1) and B10 in chondrocytes. Arthritis Rheum 1999; 42: 1986-1997.
11. Xu HG, Li ZR, Wang H, Liu P, Xiang SN, Wang CD, et al. Intermittent cyclic mechanical tension
-induced down-regulation of ectonucleotide pyrophosphatase phosphodiesterase 1 gene expression is mainly dependent on TGF-β1 in end-plate chondrocytes. Orthop Surg 2013; 5: 40-45.
12. Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature 2001; 410: 37-40.
13. Chaturvedi LS, Marsh HM, Basson MD. Src and focal adhesion kinase mediate mechanical strain-induced proliferation and ERK1/2 phosphorylation in human H441 pulmonary epithelial cells. Am J Physiol Cell Physiol 2007; 292: C1701-C1713.
14. Ryan JA, Eisner EA, DuRaine G, You Z, Reddi AH. Mechanical compression of articular cartilage induces chondrocyte proliferation and inhibits proteoglycan synthesis by activation of the ERK pathway: implications for tissue engineering and regenerative medicine. J Tissue Eng Regen Med 2009; 3: 107-116.
15. Han Y, Pan JS, Chen DP, Mao XY, Qi YX, Yan ZQ. Proliferation of human periodontal ligament cells promoted by cyclic strain via ERK signaling pathway. J Med Biomech 2009; 24: 211-215.
16. Melrose J, Burkhardt D, Taylor TK, Dillon CT, Read R, Cake M. Calcification in the ovine intervertebral disc: a model of hydroxyapatite deposition disease. Eur Spine J 2009; 18: 479-489.
17. Xu HG, Chen XH, Ding GZ, Wang H, Wang LT, Chen XW. Effect of pcDNA3.1-vascular endothelial growth factor 165 recombined vector on vascular buds in rabbit vertebral cartilage endplate. Chin Med J 2012; 125: 4055-4060.
18. Bian Q, Liang QQ, Wan C, Hou W, Li CG, Zhao YJ, et al. Prolonged upright posture induces calcified hypertrophy in the cartilage endplate in rat lumbar spine. Spine (Phila Pa 1976) 2011; 36: 2011-2020.
19. Xu HG, Zhang XH, Wang H, Liu P, Wang LT, Zuo CJ, et al. Intermittent cyclic mechanical tension
induced calcification and down-regulation of ANK gene expression of endplate chondrocytes. Spine 2012; 37: 1192-1197.
20. Fermor B, Gundle R, Evans M, Emerton M, Pocock A, Murray D. Primary human osteoblast proliferation and prostaglandin E2 release in response to mechanical strain in vitro
. Bone 1998; 22: 637-643.
21. Burr DB, Milgrom C. In vivo
measurement of human tibial strains during vigorous activity. Bone 1996; 18: 405-410.
22. Harris RC, Haralson MA, Badr KF. Continuous stretchrelaxation in culture alters rat mesangial cell morphology, growth characteristics and metabolic activity. Lab Invest 1992; 66: 548-554.
23. Hirakata M, Kaname S, Chung UG, Joki N, Hori Y, Noda M, et al. Tyrosine kinase dependent expression of TGF-beta induced by stretch in mesangial cells. Kidney Int 1997; 51: 1028-1036.
24. Yasuda T, Kondo S, Homma T, Harris RC. Regulation of extracellular matrix by mechanical stress in rat glomerular mesangial cells. J Clin Invest 1996; 98: 1991-2000.
25. Riser BL, Cortes P, Heilig C, Grondin J, Ladson-Wofford S, Patterson D, et al. Cyclin stretching force selectively up regulates transforming growth factor-beta isoforms in cultured rat mesangial cells. Am J Pathol 1996; 148: 1915-1923.
26. Sohn P, Crowley M, Slattery E, Serra R. Developmental and TGF-beta-mediated regulation of ANK mRNA expression in cartilage and bone. Osteoarthritis Cartilage 2002; 10: 482-490.
27. Fitzgerald JB, Jin M, Chai DH, Siparsky P, Fanning P, Grodzinsky AJ. Shear- and compression-induced chondrocyte transcription requires MAPK activation in cartilage explants. J Biol Chem 2008; 283: 6735-6743.
28. Badger AM, Roshak AK, Cook MN, Newman-Tarr TM, Swift BA, Carlson K, et al. Differential effects of SB 24 242235, a selective P38 mitogen-activated protein kinase inhibitor on IL-1 treated bovine and human cartilage/chondrocyte cultures. Osteoarthritis Cartilage 2000; 8: 434-443.