Loss of HIF-1α in the Notochord Results in Cell Death and Complete Disappearance of the Nucleus Pulposus

Abstract

The intervertebral disc (IVD) is one of the largest avascular organs in vertebrates. The nucleus pulposus (NP), a highly hydrated and proteoglycan-enriched tissue, forms the inner portion of the IVD. The NP is surrounded by a multi-lamellar fibrocartilaginous structure, the annulus fibrosus (AF). This structure is covered superior and inferior side by cartilaginous endplates (CEP). The NP is a unique tissue within the IVD as it results from the differentiation of notochordal cells, whereas, AF and CEP derive from the sclerotome. The hypoxia inducible factor-1α (HIF-1α) is expressed in NP cells but its function in NP development and homeostasis is largely unknown. We thus conditionally deleted HIF-1α in notochordal cells and investigated how loss of this transcription factor impacts NP formation and homeostasis at E15.5, birth, 1 and 4 months of age, respectively. Histological analysis, cell lineage studies, and TUNEL assay were performed. Morphologic changes of the mutant NP cells were identified as early as E15.5, followed, postnatally, by the progressive disappearance and replacement of the NP with a novel tissue that resembles fibrocartilage. Notably, lineage studies and TUNEL assay unequivocally proved that NP cells did not transdifferentiate into chondrocyte-like cells but they rather underwent massive cell death, and were completely replaced by a cell population belonging to a lineage distinct from the notochordal one. Finally, to evaluate the functional consequences of HIF-1α deletion in the NP, biomechanical testing of mutant IVD was performed. Loss of the NP in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress. These findings are similar to the changes usually observed during human IVD degeneration. Our study thus demonstrates that HIF-1α is essential for NP development and homeostasis, and it raises the intriguing possibility that this transcription factor could be involved in IVD degeneration in humans.

Hypoxia promotes noncanonical autophagy in nucleus pulposus cells independent of MTOR and HIF1A signaling

“…14 NP cells in both hif1a mutant and wild-type mice showed similar levels and patterns of LC3 puncta, indicating that autophagy was not controlled by HIF1A. Although NP-specific knockout of Hif1a results in cell death by birth, 14 cell lineage and viability of NP cells at E15.5 is not altered, allowing avoidance of confounding variables that affect cell survival in assessment of the autophagic process. 14 Taken together, our results clearly support the premise that HIF1A is not involved in autophagy modulation in NP cells.…”

“…14 We therefore investigated if HIF1A controls hypoxic induction of autophagy in NP cells possibly through a BNIP3-and BNIP3L-independent mechanism. NP cells were transduced with a lentivirus co-expressing YFP along with ShCTR or ShHIF1A to stably suppress HIF1A expression.…”

“…14 Briefly, Foxa2-Cre knock-in male mice were bred with homozygous Hif1a floxed (Hif1a f/f ) females, in order to obtain Foxa2-Cre positive heterozygous floxed (Foxa2-Cre;Hif1a f/C ) male mice. These newly generated males were crossed with female mice homozygous for the floxed Hif1a allele to generate Foxa2-Cre;Hif1a f/f mutant mice, Foxa2-Cre;Hif1a f/C and Hif1a f/f mice were used as controls.…”

“…[6][7][8][9][10][11][12][13] A recent study using NP-specific conditional Hif1a knockout mice also shows that HIF1A is required for postnatal NP cell survival in vivo. 14 Autophagy is a highly conserved cellular process where organelles and cytosolic proteins are encapsulated within target of rapamycin [serine/threonine kinase]) resulting in increased autophagy. 21 In a nutrient-abundant status, active MTOR complex 1 (MTORC1) phosphorylates ULK1 (unc51-like autophagy activating kinase 1) and disrupts association between ULK1 and AMPK.…”

HIF-1α and growth plate development: what we really know

“…32 As for the growth plate, HIF-1a is crucial for development of the nucleus pulposus as well, as loss of HIF-1a results in its complete disappearance. 33 All in all, these findings indicate that HIF-1a has a critical and non-redundant role in the development of avascular and hypoxic tissues. However, a series of important questions remain yet unresolved.…”