Expression of silent mating type information regulator 2 homolog 1 and its role in human intervertebral disc cell homeostasis

Abstract

Introduction: Intervertebral disc tissue homeostasis is modulated by a variety of molecules. Silent mating type information regulator 2 homolog 1 (SIRT1) plays a key role in various physiological processes. The aim of the present study was to verify the expression of SIRT1 and determine SIRT1 function in human intervertebral disc cell homeostasis.

Methods: Human nucleus pulposus (NP) cells were obtained from 24 surgical patients (mean age: 39.4 years) and monolayer-cultured. SIRT1 expression was investigated using RT-PCR analysis and immunohistochemical staining. Quantitative real-time RT-PCR was performed to detect mRNA expression of SIRT1 and other genes: aggrecan, collagen type 2 and Sox9. The effect of SIRT1 on the extracellular matrix metabolism of NP cells was examined using recombinant human SIRT1 protein and a protein delivery reagent. Cell number and proliferation activity were measured following SIRT1 treatment. To reveal the deacetylation potential of transfected recombinant human SIRT1, western blotting for acetylated p53 was utilized. R-phycoerythrin was used for the negative control.

Results: SIRT1 expression was confirmed at both mRNA and protein levels in almost all NP cells. Real-time RT-PCR analysis showed SIRT1 mRNA expression significantly increased with donor age (P <0.05, ρ = 0.492). Pfirrmann grade 3 discs showed significantly higher SIRT1 mRNA expression than other grades. SIRT1 treatment significantly reduced aggrecan, Sox9 and collagen type 2 mRNA expression in a dose-dependent manner in all disease classes and disc degeneration grades. Proliferation activity was decreased by SIRT1 treatment in lumbar spinal stenosis and lumbar disc herniation, Pfirrmann grade 3 and grade 4 discs. In contrast, it was significantly upregulated in idiopathic scoliosis, Pfirrmann grade 2 discs. The negative control protein did not affect extracellular matrix metabolism or proliferation activity.

Conclusions: We demonstrate for the first time that SIRT1 is expressed by human NP cells. SIRT1 expression was significantly elevated in an early degeneration stage. SIRT1 affected both extracellular matrix metabolism and proliferation activity; the effect of SIRT1 was altered according to disease class and disc degeneration grade. SIRT1 appears to play a key role in homeostasis during the human intervertebral disc degeneration process.

Figure 1

SIRT1 mRNA and protein expression in nucleus pulposus cells. (a)Silent mating type information regulator 2 homolog 1 (SIRT1) mRNA expression in human nucleus pulposus (NP) cells. Results of RT-PCR on NP cells derived from disc tissues of patients with lumbar spinal stenosis, lumbar disc herniation and idiopathic scoliosis are shown. (b) SIRT1 protein distribution in NP cells. Immunohistochemical staining of NP cells for β-actin (A) and SIRT1 (B) (images are merged in C). Bars = 15 μm. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 2

Correlation between SIRT1 mRNA expression, donor age, and Pfirrmann degeneration grade. (a) Correlation between mRNA expression of silent mating type information regulator 2 homolog 1 (SIRT1) and donor age in nucleus pulposus cells derived from the disc tissues of 19 patients with lumbar spinal stenosis, lumbar disc herniation, or idiopathic scoliosis. (b) Relationship of SIRT1 mRNA expression with the Pfirrmann intervertebral disc degeneration grade from magnetic resonance imaging (MRI). *P <0.05. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 3

Recombinant human SIRT1 transfection into nucleus pulposus cells. Transfection of recombinant human silent mating type information regulator 2 homolog 1 (rhSIRT1) labeled with HiLyte Fluor™ 555 by Pro-DeliverIN™ into human nucleus pulposus (NP) cells. (a) Immunohistochemical staining for SIRT1, β-actin and the nucleus (4',6-diamidino-2-phenylindole). Red fluorescence shows labeled rhSIRT1 and green fluorescence shows β-actin. Arrows show labeled rhSIRT1. Of the NP cells, 37.9% were transfected with labeled rhSIRT1. Bars = 20 μm. (b) Western blot analysis for acetylated p53 and total p53. Western blot analysis confirmed that transfected SIRT1 deacetylated p53.

Figure 4

Effect of exogenous SIRT1 on the extracellular matrix metabolism of nucleus pulposus cells. mRNA expression of (a) aggrecan, (b) Sox9 and (c) collagen type 2 following treatment with recombinant human silent mating type information regulator 2 homolog 1 (rhSIRT1). Data were obtained from 11 patients (six lumbar spinal stenosis and lumbar disc herniation patients, and five idiopathic scoliosis patients). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 5

Effect of SIRT1 on the number of nucleus pulposus cells. The number of human nucleus pulposus cells following treatment with recombinant human silent mating type information regulator 2 homolog 1 (rhSIRT1). (a) Total samples (n = 11). (b) Lumbar spinal stenosis and lumbar disc herniation (n = 6). (c) Idiopathic scoliosis (n = 5).

Figure 6

Effect of SIRT1 on the proliferation activity of nucleus pulposus cells. The normalized proliferation activity following treatment with recombinant human silent mating type information regulator 2 homolog 1 (rhSIRT1). (a) Total samples (n = 11). (b) Lumbar spinal stenosis and lumbar disc herniation (n = 6). (c) Idiopathic scoliosis (n = 5).