Hypoxia‐inducible factor regulation of ANK expression in nucleus pulposus cells: Possible implications in controlling dystrophic mineralization in the intervertebral disc

Abstract

Objective. Since nucleus pulposus cells reside under conditions of hypoxia, we determined if the expression of ANK, a pyrophosphate transporter, is regulated by the hypoxia-inducible factor (HIF) proteins.Methods. Quantitative reverse transcriptionpolymerase chain reaction and Western blot analyses were used to measure ANK expression in nucleus pulposus cells from rats and humans. Transfections were performed to determine the effect of HIF-1/2 on ANK promoter activity.Results. ANK was expressed in embryonic and mature rat discs. Oxygen-dependent changes in ANK expression in nucleus pulposus cells were minimal. However, silencing of HIF-1␣ and HIF-2␣ resulted in increased ANK expression and up-regulation of promoter activity. HIF-mediated suppression of ANK was validated by measuring promoter activity in HIF-1␤-null embryonic fibroblasts. Under conditions of hypoxia, there was induction of promoter activity in the null cells as compared with the wild-type cells. Overexpression of HIF-1␣ and HIF-2␣ in nucleus pulposus cells resulted in a significant suppression of ANK promoter activity. Since the ANK promoter contains 2 hypoxiaresponsive elements (HREs), we performed site-directed mutagenesis and measured promoter activity. We found that HIF-1 can bind to either of the HREs and can suppress promoter activity; in contrast, HIF-2 was required to bind to both HREs in order to suppress activity. Finally, analysis of human nucleus pulposus tissue showed that while ANK was expressed in normal tissue, there was increased expression of ANK along with alkaline phosphatase in the degenerated state.Conclusion. Both HIF-1 and HIF-2 serve as negative regulators of ANK expression in the disc. We propose that baseline expression of ANK in the disc serves to prevent mineral formation under physiologic conditions.Within the spine, the vertebrae are separated by a complex tissue, the intervertebral disc. At the periphery of the disc, a ligamentous tissue, the anulus fibrosus, encloses the proteoglycan-rich nucleus pulposus. Although details of the ontology of cells of the adult nucleus pulposus are obscure, it is known that they are derived from the notochord (1), an embryonic tissue with a limited blood supply. The superior and inferior boundaries of the intervertebral disc are formed by the cartilage end plates. A limited number of blood vessels infiltrate the end plates and the outer anulus fibrosus but do not enter the nucleus pulposus (2,3). For this reason, there is considerable support for the view that the nucleus pulposus cells reside in a hypoxic environment (4)(5)(6). Surprisingly, while the disc contains both fibrous proteins and a hydrated extracellular matrix, calcified deposits are absent in the nucleus pulposus and the anulus fibrosus in healthy state.

In vitro extracellular matrix accumulation of nasal and articular chondrocytes for intervertebral disc repair

“…Importantly, the disc microenvironment is characterised by low oxygen (5%-1%) (Bartels et al, 1998), low glucose (<5mM) (Bibby et al, 2005) and low pH conditions (Bartels et al, conditions may be due to downregulation of ANK, a pyrophosphate transporter known to be involved in controlling mineralization, which is negatively regulated by both HIF1 and HIF2 (Skubutyte et al, 2010). Therefore, it is highly unlikely that the native disc environment could provide the necessary biochemical cues to promote osteogenesis, although this would need to be confirmed using animal models.…”

Molecular Biology and Interactions in Intervertebral Disc Development, Homeostasis, and Degeneration, with Emphasis on Future Therapies: A Systematic Review

“…As ANK expression is sensitive to oxygen tension, the observed high expression levels were surprising since the NP is avascular. High ANK expression was expected in the calcified zone of the endplate, which has a rich vascular supply (Skubutyte et al, 2010). Hypoxia Inducible Factors HIF-1 and HIF-2 have a role in regulating energy metabolism and matrix synthesis in the NP.…”

“…Also, within the hypoxic IVD, HIF-1 and HIF-2 expression is important for maintaining basal ANK expression. It is proposed that under normal physiological conditions, control of mineralization is facilitated by Ank, which helps transport the mineralization inhibitor PPi (Skubutyte et al, 2010). In addition to HIF proteins, TGF-β seems to influence ANK expression in NP cells, which increases in response to TGF-β treatment.…”

Reconstruction of an In Vitro Niche for the Transition from Intervertebral Disc Development to Nucleus Pulposus Regeneration

“…One unique trait of NPCs is the continued expression of HIF-1a under normoxic conditions, whereas, in other cell types, this isoform is quickly degraded [60]. Known to be involved in controlling mineralization in various tissues, expression of ANK, a pyrophosphate transporter, is negatively regulated by both HIF-1 and HIF-2 in the NP and is thought to prevent mineralization [61]. A decrease in ACAN expression is also observed upon HIF-1 inhibition, suggesting that the unique oxygen-independent stabilization of HIF-1a is an important metabolic adaptation for both glycolysis and ACAN expression [62].…”