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.

Figure 1

Expression of Notch receptors and ligands, as well as Notch target gene Hes1, in rat intervertebral discs. A, Transverse sections of disc tissue from mature rats were treated with anti–Notch-4 antibodies, and expression of Notch-4 was detected in cells of the nucleus pulposus (NP) and annulus fibrosus (AF) (arrows). Original magnification × 20. B, Expression of Notch-4, Jagged-1, and Hes1 was assessed in NP and AF tissue by Western blotting; anti–β-tubulin was used as a positive control. C, Expression of select Notch ligands (Jagged1 [J1], Jagged2 [J2], Delta1, and Delta3) and Notch receptors (N1–N4) was assessed in AF tissue by reverse transcription–polymerase chain reaction (RT-PCR). D, Messenger RNA expression of the 4 Notch receptors was assessed in primary cells of the AF (left) and NP (right) by real-time RT-PCR. Values for Notch2–Notch4 are expressed relative to that of Notch1 and are the mean ± SEM results from 3 independent experiments. * = P < 0.05. NS = not significant. E, AF and NP cells were assessed by immunofluorescence analysis for the expression of Notch3, Notch4, and Jagged1, as well as cleaved Notch1 (Notch1–intracellular domain [N1-ICD]). Original magnification × 20.

Figure 2

Hypoxic regulation of Notch receptor expression in rat disc cells. A–D, AF and NP cells were cultured under conditions of hypoxia (Hx) for 8 or 24 hours, or under normoxic (Nx) conditions, and the response to hypoxia was assessed by real-time reverse transcription–polymerase chain reaction analysis of mRNA expression for Notch receptors Notch1 (A), Notch2 (B), Notch3 (C), and Notch4 (D). Values are the mean ± SEM results from 3 independent experiments. * = P < 0.05. E and F, Notch-4 protein expression under conditions of hypoxia, or under normoxic conditions, was assessed in AF and NP cells by immunofluorescence analysis (after 24 hours of hypoxia) (E) and Western blotting (after 8 and 24 hours of hypoxia) (F). Anti–β-tubulin was used as a positive control in Western blots. Original magnification × 20. See Figure 1 for other definitions.

Figure 3

Hypoxic regulation of Notch ligand expression in rat disc cells. A and B, AF and NP cells were cultured under conditions of hypoxia (Hx) for 8 or 24 hours, or under normoxic conditions (Nx), and the response to hypoxia was assessed by real-time RT-PCR analysis of Jagged1 (A) and Jagged2 (B) mRNA expression. C and D, Jagged-1 protein expression under conditions of hypoxia, or under normoxic conditions, was assessed in AF and NP cells by Western blotting (after 8 and 24 hours) (C) and immunofluorescence analysis (after 24 hours) (D). Anti–β-tubulin was used as a positive control in Western blots. Original magnification × 20. E and F, The effect of hypoxia on Hes1 gene expression and promoter activity was assessed in AF and NP cells cultured for 8 or 24 hours under conditions of hypoxia, or under normoxic conditions, using real-time RT-PCR analysis of Hes1 mRNA expression (E) and Western blotting of Hes-1 protein expression, with anti–β-tubulin used as a positive control (F). G, AF cells were cotransfected with a Hes1 reporter (−194/+160 bp) along with increasing doses of Notch1–intracellular domain (N-ICD), and Hes1 reporter activity was measured under normoxic and hypoxic conditions. Values in A, B, E, and G are the mean ± SEM results from 3 independent experiments. * = P < 0.05. See Figure 1 for other definitions.

Figure 4

Effect of hypoxia on Notch signaling activity. A and B, The 12xCSL reporter (A) or CBF1 reporter containing a wild-type (W) or mutant (M) RBPJκ motif (B) was transfected into rat annulus fibrosus cells along with pRL-TK vector. Cells were cultured for 24 hours under normoxic conditions (21% O₂) (Nx) or hypoxic conditions (1% O₂) (Hx), and some cells were treated with γ-secretase inhibitor L685458 (4 μM), and luciferase reporter activity was measured. C and D, Rat annulus fibrosus cells were cotransfected with wild-type (WT) (C) or mutant (MT) (D) CBF1 reporters along with increasing doses of Notch1-ICD, and reporter activity was measured under normoxic and hypoxic conditions. Values are the mean ± SEM results from 3 independent experiments. * = P < 0.05. See Figure 1 for other definitions.

Figure 5

Role of Notch signaling in controlling AF cell proliferation. A, AF cells were cultured under normoxia (Nx) or hypoxia (Hx), with or without γ-secretase inhibitor L685458 (4 μM), for 24–48 hours, and cell proliferation was measured by MTT assay. B, The cell cycle of AF cells treated with or without L685458 was determined by flow cytometry, with results expressed as the percentage of AF cells in G0/G1 phase or S phase. Values in A and B are the mean ± SEM results from 3 independent experiments. * = P < 0.05. C–E, Messenger RNA expression of Notch receptors Notch1 and Notch4 (C), Notch ligands Jagged1 and Jagged2 (D), and Notch target gene Hes1 (E) was assessed by real-time RT-PCR in multiple human AF tissue samples in varying degrees of degeneration (Thompson grades 2–5 [G2–G5]) compared with normal controls (N) (controls n = 2, G2 n = 5, G3 n = 4, G4 n = 3, G5 n = 3). The mean expression of mRNA in normal control samples was set at 1.0 (horizontal line). See Figure 1 for other definitions.

Figure 6

Role of Notch signaling in controlling NP cell proliferation. A, NP cells were cultured under conditions of normoxia (Nx) or hypoxia (Hx), with or without γ-secretase inhibitor L685458 (4 μM), for 24–48 hours, and cell proliferation was measured by MTT assay. Values are the mean ± SEM results from 3 independent experiments. * = P < 0.05. B–D, Messenger RNA expression of Notch receptors Notch1 and Notch4 (B), Notch ligands Jagged1 and Jagged2 (C), and Notch target gene Hes1 (D) was assessed by real-time RT-PCR in multiple human NP tissue samples in varying degrees of degeneration (Thompson grades 2–5 [G2–G5]) compared with normal controls (N) (controls n = 1, G2 n = 3, G3 n = 4, G4 n = 7, G5 n = 4). The mean expression of mRNA in normal control samples was set at 1.0 (horizontal line). See Figure 1 for other definitions.