NF-κB Signaling Pathway in Controlling Intervertebral Disk Cell Response to Inflammatory and Mechanical Stressors

Abstract

Background: Intervertebral disk degeneration (IDD) has a greater than 90% lifetime incidence and is one of the leading causes of chronic back pain in the United States. Despite the high societal cost of IDD, there is limited understanding of the biological effects of mechanical overloading on further degeneration. The transcription factor NF-κB (nuclear factor κB) has been implicated as a key mediator of disk cell response to inflammatory and mechanical stresses and represents a potential control point.

Objective: The study objective was to measure the effect of NF-κB signaling pathway inhibition on annulus fibrosus (AF) cell matrix synthesis and gene expression under conditions of combined inflammatory and mechanical stimulation.

Methods: Annulus fibrosus cells were harvested from rabbit intervertebral disks and grown in vitro on flexible plates. The cells were exposed to inflammatory and mechanical stimulation for 24 hours with and without NF-κB inhibition. Nuclear translocation of NF-κB was measured via immunofluorescent staining. Intervertebral disk cell homeostasis was assessed via inflammatory, anabolic, and catabolic gene expression and via matrix synthetic ability.

Results: NF-κB nuclear translocation in response to interleukin-1 beta (IL-1β) was reversed with exposure to NF-κB inhibition. NF-κB inhibition decreased matrix metalloproteinase-3, inducible nitric oxide synthase, and cyclooxygenase-2 gene expression and prostaglandin E2 production response to combined inflammatory and mechanical stimulation. Proteoglycan and collagen synthesis were decreased by combined stimulation, but this effect was not reversed by NF-κB inhibition.

Limitations: In vitro modeling of conditions within the disk may not fully reflect the response that AF cells have in native matrix.

Conclusions: NF-κB signaling mediates catabolic and inflammatory responses to inflammatory and mechanical stimulation but does not mediate the decrease in matrix synthesis under combined harmful stimulation. Identification of key control points in the cellular responses to inflammatory and mechanical stimuli will facilitate rational design of exercise-based therapies and facilitate synergistic treatments of novel biochemical treatments with rehabilitation regimens.

Figure 1.

Representative images of nuclear factor κB (NF-κB) staining for all conditions after 24 hours of cyclic tensile strain showing increased nuclear translocation of NF-κB after interleukin-1 beta (IL-1β) stimulation with and without stretching. Movement of NF-κB into the nucleus induced by IL-1β was diminished by ACHP preconditioning.

Figure 2.

Nuclear-to-cytoplasmic ratio of nuclear factor κB (NF-κB) in response to stretching and proinflammatory stimulation at 24 hours demonstrates a strong response to inflammatory (interleukin-1 beta [IL-1β]) signaling, but no further increase with the addition of mechanical stimulation. Three independent experiments were run, and a minimum of 3 cell images from each experiment were used. * Significantly greater than control (P<.05). ^† P<.05, N=6. A=ACHP, IL=IL-1β, S=stretch, CI=confidence interval.

Figure 3.

Relative change in messenger RNA (mRNA) gene expression of matrix metalloproteinase-3 (MMP-3), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) as measured by real-time polymerase chain reaction shows increased matrix breakdown and inflammatory response with interleukin-1 beta (IL-1β) stimulation with and without stretching, which is reversed by addition of ACHP. Matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) show more modest changes in response to inflammatory and mechanical stimulation, although the increase in TIMP-1 in response to IL-1β was not reversed by the addition of ACHP. All values represent mean±95% confidence interval of n=3 samples. * Significantly greater than control (P<.05). ^† P<.05. A=ACHP, IL=IL-1β, S=stretch.

Figure 4.

Relative prostaglandin E₂ (PGE₂) levels at 24 hours (n=8) in response to interleukin-1 beta (IL-1β), stretching, and nuclear factor κB (NF-κB) inhibition demonstrates synergistic effect of inflammatory and mechanical stimulation to elicit an inflammatory response from annulus fibrosus cells. A=ACHP, IL=IL-1β, S=stretch, CI-confidence interval. * Significantly greater than control (P<.05). ^†P<.05.

Figure 5.

Proteoglycan (PG) and total collagen synthesis normalized to noninflammatory nonstretched controls (n=3, mean±95% confidence interval) after 24 hours of strain measured by ³⁵S (20-μCi/mL media) and ³H-proline (10-μCi/mL media) incorporation via pulse labeling shows increased extracellular matrix synthesis with short-duration stretching, which is reversed by nuclear factor κB (NF-κB) inhibition, and decreased synthesis with 24-hour stretching, which is unaffected by NF-κB inhibition.

Figure 6.

Summary of annulus fibrosus response to inflammatory and mechanical stimulation. Mechanical stimulation increased all effects of inflammatory stimulation. Nuclear factor κB (NF-κB) inhibition reversed NF-κB activation and relative gene expression (RGE) response to both inflammatory and combined stimulation but did not reverse the decrease in matrix synthesis caused by inflammatory and combined stimulation. MMP-1=matrix metalloproteinase-1, MMP-3=matrix metalloproteinase-3, TIMP-1=tissue inhibitor of matrix metalloproteinase-1, iNOS=inducible nitric oxide synthase, COX-2=cyclooxygenase-2, PGE₂= prostaglandin E₂, PG=proteoglycan.