Cyclic tensile stretch modulates proteoglycan production by intervertebral disc annulus fibrosus cells through production of nitrite oxide

Abstract

Degeneration of the intervertebral disc is the main pathophysiological process implicated in low back pain and is a prerequisite to disc herniation. Clinically, mechanical forces are important modulators of the degeneration, but the underlying molecular mechanism is not known and needs investigation to identify the biological target. The aim of this work was to study, at the molecular level, the effects of cyclic tensile stretch (CTS) on the production of proteoglycan by intervertebral disc annulus fibrosus cells since proteoglycans seem to be implicated in the dynamic process of intervertebral disc degeneration. Such cells of rabbit were cultured at high density on plates with a flexible bottom. CTS was applied with use of a pressure-operated instrument to deform the plates. With CTS at 1% elongation (1 Hz frequency), the level of (35)S-labeled neosynthesized proteoglycans that accumulated in the cellular pool or were secreted in the culture medium did not change, but at 5% elongation, the level was significantly reduced after 8 h of stimulation (30 and 21%, respectively) and further reduced at 24 h (43 and 41%, respectively). Introducing the protein synthesis inhibitor cycloheximide had no effect on this result. Neither aggrecan and biglycan expression nor proteoglycan physical properties were modified. The level of nitrite oxide production significantly increased by 3.5 times after 8 h of 5% elongation. Introducing the nitric oxide synthase (NOS) inhibitors N(G)-methyl-l-arginine or N-omega nitro-l-arginine diminished the effects of CTS on the production of nitrite oxide and proteoglycans. By contrast, introducing N-iminoethyl-l-lysine (a more specific inhibitor of inductible NOS [iNOS]) had little or no effect. Taken together, these results suggest that cNOS activation seems to be more implicated in the 5% CTS modulation of proteoglycan production than iNOS activation. These results suggest that CTS can help regulate the intervertebral disc matrix by decreasing proteoglycan production through a post-translational regulation involving nitrite oxide. This result could be of interest in the development of local therapeutic strategies aimed at controlling intervertebral disc degeneration.

Cyclic stretch-induced apoptosis in rat annulus fibrosus cells is mediated in part by endoplasmic reticulum stress through nitric oxide production

“…Excessive NO may directly disturb ER function and activate ER stress pathway [14,17,40]. Here, we demonstrated significant increase of NO production and iNOS mRNA in rat AF cells exposed to cyclic stretch, which was consistent with previous studies showing that NO were up-regulated by the stimulation of prolonged cyclic stretch in AF cells [2,33]. NO overproduction appears to occur before the onset of ER stress and subsequent apoptosis, thus it is conceivable that NO production is a potential trigger of ER stress observed in rat AF cells during cyclic stretch.…”

“…For instance, endoplasmic reticulum stress triggered by NO overproduction, leads to b-cell apoptosis via CHOP induction and caspase-12 activation [29]. Furthermore, cyclic stretch can induce NO overproduction [2,33] and even cause apoptosis [34] in AF cells. These data suggest that NO may participate in force-induced signals into apoptotic responses by endoplasmic reticulum stress.…”

The effects of dynamic loading on the intervertebral disc

“…Cell membrane stretch due to cell flattening activates ion channels and induces alterations in membrane potential and hence affects the cell metabolism [11,81]. The stretching of rabbit AF cells (5% elongation or 15% area change) has been shown to increase nitric oxide production, to decrease proteoglycan production and to increase apoptosis of AF cells [8,96,97]. However, a 2% cyclic strain applied on human AF cells was shown to increase aggrecan and decrease matrix metalloproteinase (MMP) gene expression [85].…”

The effects of cyclic tensile strain on the organisation and expression of cytoskeletal elements in bovine intervertebral disc cells: an in vitro study

“…These forces remain in the NP and dissipate into adjacent IVDs through the stiff vertebral body. Previous studies have shown that the strain resisted by the OAF is between 1 and 13% (Broberg, 1983;Klein et al, 1983;Ebara et al, 1996;Rannou et al, 2003).…”