ATP promotes extracellular matrix biosynthesis of intervertebral disc cells

We have recently found a high accumulation of extracellular adenosine triphosphate (ATP) in the center of healthy porcine intervertebral discs (IVD). Since ATP is a powerful extracellular signaling molecule, extracellular ATP accumulation might regulate biological activities in the IVD. The objective of this study was therefore to investigate the effects of extracellular ATP on the extracellular matrix (ECM) biosynthesis of porcine IVD cells isolated from two distinct anatomical regions: the annulus fibrosus (AF) and nucleus pulposus (NP). ATP treatment significantly promotes ECM deposition and corresponding gene expression (aggrecan and type II collagen) by both cell types in three-dimensional agarose culture. A significant increase in ECM accumulation has been found in AF cells at a lower ATP treatment level (20 μM) compared with NP cells (100 μM), indicating that AF cells are more sensitive to extracellular ATP than NP cells. NP cells also exhibit higher ECM accumulation and intracellular ATP than AF cells under control and treatment conditions, suggesting that NP cells are intrinsically more metabolically active. Moreover, ATP treatment also augments the intracellular ATP level in NP and AF cells. Our findings suggest that extracellular ATP not only promotes ECM biosynthesis via a molecular pathway, but also increases energy supply to fuel that process.

[1]  R. E. Neuman,et al.  The determination of hydroxyproline. , 1950, The Journal of biological chemistry.

[2]  Mehran Kasra,et al.  Frequency response of pig intervertebral disc cells subjected to dynamic hydrostatic pressure , 2006, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[3]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[4]  E. Liechty,et al.  Exogenous adenine nucleotides replete endothelial cell adenosine triphosphate after oxidant injury by adenosine uptake. , 1990, The Journal of laboratory and clinical medicine.

[5]  L. Setton,et al.  Collagen gene expression and mechanical properties of intervertebral disc cell–alginate cultures , 2001, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[6]  Weiyong Gu,et al.  Energy metabolism of intervertebral disc under mechanical loading , 2013, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[7]  S. Honma,et al.  Synchronized ATP oscillations have a critical role in prechondrogenic condensation during chondrogenesis , 2012, Cell Death and Disease.

[8]  N. Boos,et al.  Biological treatment strategies for disc degeneration: potentials and shortcomings , 2007, European Spine Journal.

[9]  C. Boesch,et al.  Quantitative MR imaging of lumbar intervertebral disks and vertebral bodies: influence of diurnal water content variations. , 1993, Radiology.

[10]  Jeffrey C Lotz,et al.  Biological response of the intervertebral disc to dynamic loading. , 2004, Journal of biomechanics.

[11]  J. Estrela,et al.  Inhibition of cancer growth and selective glutathione depletion in Ehrlich tumour cells in vivo by extracellular ATP. , 1994, The Biochemical journal.

[12]  G. Lee,et al.  ATP release by mechanically loaded porcine chondrons in pellet culture. , 2000, Arthritis and rheumatism.

[13]  P. Robbins,et al.  Transporters of nucleotide sugars, ATP, and nucleotide sulfate in the endoplasmic reticulum and Golgi apparatus. , 1998, Annual review of biochemistry.

[14]  D. Buttle,et al.  Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. , 1986, Biochimica et biophysica acta.

[15]  D L Bader,et al.  Measurement of the deformation of isolated chondrocytes in agarose subjected to cyclic compression. , 1998, Medical engineering & physics.

[16]  John F. Bolton,et al.  Chondrocyte deformation within compressed agarose constructs at the cellular and sub-cellular levels. , 2000, Journal of biomechanics.

[17]  J. Urban,et al.  Intervertebral disk nutrition: a review of factors influencing concentrations of nutrients and metabolites. , 2011, The Orthopedic clinics of North America.

[18]  D. Bergel,et al.  Effect of static load on matrix synthesis rates in the intervertebral disc measured in vitro by a new perfusion technique , 1995, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[19]  J A Buckwalter,et al.  Aging and degeneration of the human intervertebral disc. , 1995, Spine.

[20]  Mauro Alini,et al.  Anabolic and catabolic mRNA levels of the intervertebral disc vary with the magnitude and frequency of in vivo dynamic compression , 2004, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[21]  P. Roughley.  Biology of Intervertebral Disc Aging and Degeneration: Involvement of the Extracellular Matrix , 2004, Spine.

[22]  G. Burnstock.  The past, present and future of purine nucleotides as signalling molecules , 1997, Neuropharmacology.

[23]  M. Knight,et al.  Purinergic pathway suppresses the release of .NO and stimulates proteoglycan synthesis in chondrocyte/agarose constructs subjected to dynamic compression. , 2006, Journal of cellular physiology.

[24]  R. Terkeltaub,et al.  Mediation of spontaneous knee osteoarthritis by progressive chondrocyte ATP depletion in Hartley guinea pigs. , 2004, Arthritis and rheumatism.

[25]  H. Kwon.  Extracellular ATP signaling via P2X(4) receptor and cAMP/PKA signaling mediate ATP oscillations essential for prechondrogenic condensation. , 2012, The Journal of endocrinology.

[26]  V. Morsch,et al.  Heparin and chondroitin sulfate inhibit adenine nucleotide hydrolysis in liver and kidney membrane enriched fractions. , 2001, The international journal of biochemistry & cell biology.

[27]  P. Hatton,et al.  Extracellular ATP and UTP stimulate cartilage proteoglycan and collagen accumulation in bovine articular chondrocyte pellet cultures. , 2000, Biochimica et biophysica acta.

[28]  A. Freemont,et al.  The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain. , 2008, Rheumatology.

[29]  Rocky S Tuan,et al.  Intervertebral disc cell response to dynamic compression is age and frequency dependent , 2009, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[30]  M. Adams,et al.  What is Intervertebral Disc Degeneration, and What Causes It? , 2006, Spine.

[31]  W. Gu,et al.  Mechanical loading affects the energy metabolism of intervertebral disc cells , 2011, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[32]  H E Gruber,et al.  Human intervertebral disc cells from the annulus: three-dimensional culture in agarose or alginate and responsiveness to TGF-beta1. , 1997, Experimental cell research.

[33]  S. Nicoll,et al.  The Effect of Serial Monolayer Passaging on the Collagen Expression Profile of Outer and Inner Anulus Fibrosus Cells , 2006, Spine.

[34]  S. Waldman,et al.  Harnessing the purinergic receptor pathway to develop functional engineered cartilage constructs. , 2010, Osteoarthritis and cartilage.

[35]  W C Hutton,et al.  The effect of hydrostatic pressure on intervertebral disc metabolism. , 1999, Spine.

[36]  E B Hunziker,et al.  Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture. , 1995, Journal of cell science.

[37]  L. Claes,et al.  Regulation of gene expression in intervertebral disc cells by low and high hydrostatic pressure , 2006, European Spine Journal.

[38]  L. Reed,et al.  Lipoic acid activation of the alpha-ketobutyrate oxidation system in cell-free extracts of Streptococcus faecalis. , 1955, Biochimica et biophysica acta.

[39]  H. Cheung,et al.  Difference in Energy Metabolism of Annulus Fibrosus and Nucleus Pulposus Cells of the Intervertebral Disc , 2011, Cellular and molecular bioengineering.

[40]  W. Halperin,et al.  Back pain prevalence in US industry and estimates of lost workdays. , 1999, American journal of public health.

[41]  S. Waldman,et al.  The Therapeutic Potential of Exogenous Adenosine Triphosphate (ATP) for Cartilage Tissue Engineering , 2012, Cartilage.

[42]  J. Urban,et al.  Nutrition of the Intervertebral Disc , 2004, Spine.

[43]  M. E. Adams,et al.  Degeneration and the chemical composition of the human lumbar intervertebral disc , 1987, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[44]  Bruce Caterson,et al.  Human intervertebral disc aggrecan inhibits nerve growth in vitro. , 2002, Arthritis and rheumatism.