Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells

IntroductionNutrient deprivation is a likely contributor to intervertebral disc (IVD) degeneration. Silent mating type information regulator 2 homolog 1 (SIRT1) protects cells against limited nutrition by modulation of apoptosis and autophagy. However, little evidence exists regarding the extent to which SIRT1 affects IVD cells. Therefore, we conducted an in vitro study using human IVD nucleus pulposus (NP) cells.MethodsThirty-two IVD specimens were obtained from patients who underwent surgical intervention and were categorized based on Pfirrmann IVD degeneration grades. Cells were isolated from the NP and cultured in the presence of recombinant human SIRT1 (rhSIRT1) under different serum conditions, including 10 % (v/v) fetal bovine serum (FBS) as normal nutrition (N) and 1 % (v/v) FBS as low nutrition (LN). 3-Methyladenine (3-MA) was used to inhibit autophagy. Autophagic activity was assessed by measuring the absorbance of monodansylcadaverine and immunostaining and Western blotting for light chain 3 and p62/SQSTM1. Apoptosis and pathway analyses were performed by flow cytometry and Western blotting.ResultsCells cultured under LN conditions decreased in number and exhibited enhanced autophagy compared with the N condition. Medium supplementation with rhSIRT1 inhibited this decrease in cell number and induced an additional increase in autophagic activity (P < 0.05), whereas the combined use of rhSIRT1 and 3-MA resulted in drastic decreases in cell number and autophagy (P < 0.05). The incidence of apoptotic cell death increased under the LN condition, which was decreased by rhSIRT1 (P < 0.05) but increased further by a combination of rhSIRT1 and 3-MA (P < 0.05). Under LN conditions, NP cells showed a decrease in antiapoptotic Bcl-2 and an increase in proapoptotic Bax, cleaved caspase 3, and cleaved caspase 9, indicating apoptosis induction via the mitochondrial pathway. These changes were suppressed by rhSIRT1 but elevated further by rhSIRT1 with 3-MA, suggesting an effect of rhSIRT1-induced autophagy on apoptosis inhibition. Furthermore, the observed autophagy and apoptosis were more remarkable in cells from IVDs of Pfirrmann grade IV than in those from IVDs of Pfirrmann grade II.ConclusionsSIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human IVD NP cells, suggesting that rhSIRT1 may be a potent treatment agent for human degenerative IVD disease.

[1]  F. Alt,et al.  Developmental defects and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient mice , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[2]  H. Gruber,et al.  Anti-Apoptotic Effects of IGF-1 and PDGF on Human Intervertebral Disc Cells In Vitro , 2000, Spine.

[3]  H E Gruber,et al.  Analysis of Aging and Degeneration of the Human Intervertebral Disc: Comparison of Surgical Specimens With Normal Controls , 1998, Spine.

[4]  I. Park,et al.  Anti-apoptotic effects of caspase inhibitors on rat intervertebral disc cells. , 2006, The Journal of bone and joint surgery. American volume.

[5]  G. Kroemer,et al.  The apoptosis/autophagy paradox: autophagic vacuolization before apoptotic death , 2005, Journal of Cell Science.

[6]  Jong-Beom Park,et al.  Expression of Fas Receptor on Disc Cells in Herniated Lumbar Disc Tissue , 2001, Spine.

[7]  Ralph A. Nixon,et al.  Extensive Involvement of Autophagy in Alzheimer Disease: An Immuno-Electron Microscopy Study , 2005, Journal of neuropathology and experimental neurology.

[8]  S. Roberts,et al.  Transport Properties of the Human Cartilage Endplate in Relation to Its Composition and Calcification , 1996, Spine.

[9]  K. Wada,et al.  Aberrant Interaction between Parkinson Disease-associated Mutant UCH-L1 and the Lysosomal Receptor for Chaperone-mediated Autophagy* , 2008, Journal of Biological Chemistry.

[10]  M. Kurosaka,et al.  Expression of silent mating type information regulator 2 homolog 1 and its role in human intervertebral disc cell homeostasis , 2011, Arthritis research & therapy.

[11]  N Bogduk,et al.  The lumbar disc and low back pain. , 1991, Neurosurgery clinics of North America.

[12]  R. Chen,et al.  Silent information regulator, Sirtuin 1, and age‐related diseases , 2009, Geriatrics & gerontology international.

[13]  A. Minami,et al.  Regulation of apoptosis in nucleus pulposus cells by optimized exogenous Bcl‐2 overexpression , 2010, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[14]  I. Amelio,et al.  Cell death pathology: cross-talk with autophagy and its clinical implications. , 2011, Biochemical and biophysical research communications.

[15]  John Y-J Shyy,et al.  Intervertebral disc degeneration: the role of the mitochondrial pathway in annulus fibrosus cell apoptosis induced by overload. , 2004, The American journal of pathology.

[16]  A. Apel,et al.  Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy. , 2008, Cancer research.

[17]  D. Rubinsztein,et al.  Plasma membrane contributes to the formation of pre-autophagosomal structures , 2010, Nature Cell Biology.

[18]  T. Matsushita,et al.  SIRT1 regulation of apoptosis of human chondrocytes. , 2009, Arthritis and rheumatism.

[19]  J. Katz,et al.  Lumbar disc disorders and low-back pain: socioeconomic factors and consequences. , 2006, The Journal of bone and joint surgery. American volume.

[20]  Kevin Bray,et al.  Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. , 2006, Cancer cell.

[21]  S. Roberts,et al.  The influence of serum, glucose and oxygen on intervertebral disc cell growth in vitro: implications for degenerative disc disease , 2008, Arthritis research & therapy.

[22]  K. Riew,et al.  Mitochondrial involvement in fas-mediated apoptosis of human lumbar disc cells. , 2005, The Journal of bone and joint surgery. American volume.

[23]  Myriam Gorospe,et al.  Calorie Restriction Promotes Mammalian Cell Survival by Inducing the SIRT1 Deacetylase , 2004, Science.

[24]  G. Andersson.  Epidemiological features of chronic low-back pain , 1999, The Lancet.

[25]  T. Matsushita,et al.  Autophagy modulates osteoarthritis-related gene expression in human chondrocytes. , 2012, Arthritis and rheumatism.

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

[27]  T. Matsushita,et al.  Notochordal cell disappearance and modes of apoptotic cell death in a rat tail static compression-induced disc degeneration model , 2014, Arthritis Research & Therapy.

[28]  Nicholas E. Bruns,et al.  A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy , 2008, Proceedings of the National Academy of Sciences.

[29]  A. Salminen,et al.  SIRT1: regulation of longevity via autophagy. , 2009, Cellular signalling.

[30]  C. Pfirrmann,et al.  Magnetic Resonance Classification of Lumbar Intervertebral Disc Degeneration , 2001, Spine.

[31]  R. Weinberg,et al.  hSIR2SIRT1 Functions as an NAD-Dependent p53 Deacetylase , 2001, Cell.

[32]  A. Minami,et al.  Caspase 3 as a therapeutic target for regulation of intervertebral disc degeneration in rabbits. , 2011, Arthritis and rheumatism.

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

[34]  Guido Kroemer,et al.  Autophagy and the integrated stress response. , 2010, Molecular cell.

[35]  L. Dai,et al.  Autophagy in rat annulus fibrosus cells: evidence and possible implications , 2011, Arthritis research & therapy.

[36]  S. A. Shirazi-Adl,et al.  Nutrient supply and intervertebral disc metabolism. , 2006, The Journal of bone and joint surgery. American volume.

[37]  S. Roberts,et al.  The influence of nutrient supply and cell density on the growth and survival of intervertebral disc cells in 3D culture. , 2011, European cells & materials.

[38]  T. Matsushita,et al.  Disruption of Sirt1 in chondrocytes causes accelerated progression of osteoarthritis under mechanical stress and during ageing in mice , 2013, Annals of the rheumatic diseases.

[39]  Delin Chen,et al.  Negative Control of p53 by Sir2α Promotes Cell Survival under Stress , 2001, Cell.

[40]  T. Matsushita,et al.  The overexpression of SIRT1 inhibited osteoarthritic gene expression changes induced by interleukin‐1β in human chondrocytes , 2013, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.