Hypoxia activates the notch signaling pathway in cells of the intervertebral disc: Implications in degenerative disc disease

Abstract

Objective To investigate whether hypoxia regulates Notch signaling, and whether Notch plays a role in intervertebral disc cell proliferation. Methods Reverse transcription–polymerase chain reaction and Western blotting were used to measure expression of Notch signaling components in intervertebral disc tissue from mature rats and from human discs. Transfections were performed to determine the effects of hypoxia and Notch on target gene activity. Results Cells of the nucleus pulposus and annulus fibrosus of rat disc tissue expressed components of the Notch signaling pathway. Expression of Notch-2 was higher than that of the other Notch receptors in both the nucleus pulposus and annulus fibrosus. In both tissues, hypoxia increased Notch1 and Notch4 messenger RNA (mRNA) expression. In the annulus fibrosus, mRNA expression of the Notch ligand Jagged1 was induced by hypoxia, while Jagged2 mRNA expression was highly sensitive to hypoxia in both tissues. A Notch signaling inhibitor, L685458, blocked hypoxic induction of the activity of the Notch-responsive luciferase reporters 12xCSL and CBF1. Expression of the Notch target gene Hes1 was induced by hypoxia, while coexpression with the Notch–intracellular domain increased Hes1 promoter activity. Moreover, inhibition of Notch signaling blocked disc cell proliferation. Analysis of human disc tissue showed that there was increased expression of Notch signaling proteins in degenerated discs. Conclusion In intervertebral disc cells, hypoxia promotes expression of Notch signaling proteins. Notch signaling is an important process in the maintenance of disc cell proliferation, and thus offers a therapeutic target for the restoration of cell numbers during degenerative disc disease.

Notch1 controls development of the extravillous trophoblast lineage in the human placenta

“…By binding of NICD to HIF-1α, Notch signaling synergizes with hypoxia to keep cells in an undifferentiated state (58,59). Hence, we speculated that a lowoxygen environment could support mRNA expression and protein stability of Notch1, as shown in other cell types (59)(60)(61), and thereby maintain the proliferative capacity of CCT precursors. Indeed, the present data indicate that hypoxia increases Notch1 as well as activity of a canonical Notch reporter.…”

FIH-1-Mint3 Axis Does Not Control HIF-1α Transcriptional Activity in Nucleus Pulposus Cells

“…Of note, the in vitro K m of FIH-1 for Notch1 is much lower (ϳ250-fold) than it is for HIF-1␣ (42), although in vivo analyses suggest that hydroxylation of ankyrin repeat domain proteins and HIF-1␣ occurs within the same range (43). It is noteworthy that Notch1 and Notch2 are highly expressed in the NP, and their expression is hypoxia-sensitive (44). Importantly, our data suggest that both Notch1-and Notch2-ICD can rescue FIH-1-mediated suppression of HIF-1␣ and HIF-2␣ transactivation, raising the possibility that the high Notch expression in the NP may serve as a preferential substrate and outcompete HIF-1␣ for binding with FIH-1, contributing to the seeming lack of regulation of HIF-1 signaling by FIH-1.…”

Mesenchymal Stem Cell in the Intervertebral Disc

“…Various animal models have suggested the promising potential of mesenchymal stem cell (MSC) implantation to arrest IVD degeneration or even partially regenerate the disc [21,37]. However, there are two major issues in MSC therapies: first, most studies are focused on the exogenous stem cells but the limitation is their potential immunogenicity.…”

“…However, there are two major issues in MSC therapies: first, most studies are focused on the exogenous stem cells but the limitation is their potential immunogenicity. MSCs have indeed been shown to halt degeneration processes but are rarely able to completely regenerate the degenerative disc as the disc degeneration often continues after a certain period [37]. Besides, the therapy is invasive and therefore may potentially lead to complications such as infection and discitis.…”