Peroxynitrite induces gene expression in intervertebral disc cells.
METHODS
In vitro stimulation of human intervertebral disc (IVD) cells.


OBJECTIVE
To investigate the oxidative/nitrosative effects of peroxynitrite on human nucleus pulposus (NP) cells.


BACKGROUND
Peroxynitrite is an important tissue-damaging species generated at sites of inflammation and degeneration. The aim of this study was to examine the effects of oxidative/nitrosative stress caused by peroxynitrite and the peroxynitrite donor SIN-1 in human NP cells.


METHODS
Degenerated human IVD tissue was analyzed for nitrosylation by immunofluorescence. In addition, human NP cells were isolated from IVDs, expanded and stimulated either with peroxynitrite itself or a stable peroxynitrite donor (SIN-1). Nitrosylation, accumulation of intracellular reactive oxygen species, NF-kappaB nuclear translocation, and cell viability were analyzed by fluorescence. Gene expression of TNF-alpha, IL-1beta, IL-6, IL-8, and IL-10 was quantified by real-time (RT)-PCR.


RESULTS
Degenerated IVD tissue showed strong nitrosylation, especially in the NP. Isolated human NP cells showed a strong signal for nitrosylation and intracellular reactive oxygen species on stimulation with peroxynitrite or SIN-1. NF-kappaB/p65 sustained nuclear translocation of NF-kappaB/p65 and stimulation of IL-1beta, IL-6, and IL-8 expression was noted on treatment of cells with SIN-1.


CONCLUSIONS
This study provides evidence that peroxynitrite may play a role in disc degeneration and discogenic back pain development by an increased synthesis of proinflammatory cytokines. Nuclear translocation of NF-kappaB was identified as the potential underlying pathway. Therefore, neutralizing peroxynitrite and its derivatives (e.g., via the use of antioxidants) may be a novel treatment option for discogenic back pain.
Poveda
Lucy
L
Spine Research Group, Competence Centre for Applied Biotechnology and Molecular Medicine, University of Zurich, Zurich, Switzerland.
Hottiger
Michael
M
Boos
Norbert
N
Wuertz
Karin
K
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000293:Adolescent; D000328:Adult; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D007150:Immunohistochemistry; D016753:Interleukin-10 / Q000235:genetics; D053583:Interleukin-1beta / Q000235:genetics; D015850:Interleukin-6 / Q000235:genetics; D016209:Interleukin-8 / Q000235:genetics; D007403:Intervertebral Disc / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007405:Intervertebral Disc Displacement / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008297:Male; D008875:Middle Aged; D008981:Molsidomine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000378:metabolism / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051996:Transcription Factor RelA / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D053583:Interleukin-1beta; D015850:Interleukin-6; D016209:Interleukin-8; D020030:Nitric Oxide Donors; D017382:Reactive Oxygen Species; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; C002385:linsidomine; D008981:Molsidomine