Nucleus pulposus cells express HIF-1α under normoxic culture conditions: A metabolic adaptation to the intervertebral disc microenvironment

Abstract

Nucleus pulposus (NP) cells of the intervertebral disc reside in an environment that has a limited vascular supply and generate energy through anaerobic glycolysis. The goal of the present study was to examine the expression and regulation of HIF-1alpha, a transcription factor that regulates oxidative metabolism in nucleus pulposus cells. Nucleus pulposus cells were isolated from rat, human, and sheep disc and maintained at either 21% or 2% oxygen for various time periods. Cells were also treated with desferrioxamine (Dfx), a compound that mimics the effects of hypoxia (Hx). Expression and function of HIF-1alpha were assessed by immunofluorescence microscopy, Western blot analysis, gel shift assays, and luciferase reporter assays. In normoxia (Nx), rat, sheep, and human nucleus pulposus cells consistently expressed the HIF-1alpha subunit. Unlike other skeletal cells, when maintained under low oxygen tension, the nucleus pulposus cells exhibited a minimal induction in HIF-1alpha protein levels. Electromobility shift assays confirmed the functional binding of normoxic HIF-1alpha protein to its putative DNA binding motif. A dual luciferase reporter assay showed increased HIF-1alpha transcriptional activity under hypoxia compared to normoxic level, although this induction was small when compared to HeLa and other cell types. These results indicate that normoxic stabilization of HIF-1alpha is a metabolic adaptation of nucleus pulposus cells to a unique oxygen-limited microenvironment. The study confirmed that HIF-1alpha can be used as a phenotypic marker of nucleus pulposus cells.

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

“…Rat and human NP cells were isolated using a method previously reported by Risbud et al 7 Human NP cells were isolated from surgically discarded, magnetic resonance imaging-graded tissue samples. Collection of animal and human tissues for cell isolation was performed as per approved protocols by Jefferson's IACUC and IRB, respectively.…”

“…[2][3][4][5] NP cells adapt to this hypoxic niche by modulating a key transcription factor, HIF1A (hypoxia inducible factor 1 a subunit), to promote cell survival, matrix synthesis, and to regulate glycolytic metabolism. [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.…”

Cited2 modulates hypoxia‐inducible factor–dependent expression of vascular endothelial growth factor in nucleus pulposus cells of the rat intervertebral disc

“…Regulation of glycolysis is mediated by hypoxia-inducible factor 1␣ (HIF-1␣), a key transcription factor that is expressed by the nucleus pulposus cells (8). Unlike other skeletal cells such as chondrocytes, in the nucleus pulposus there is normoxic stabilization of this otherwise oxygen-labile protein (6,9). Moreover, despite an absence of HIF-1␣ protein induction in hypoxia, an increase in its transcriptional activity was seen (6,9).…”

“…Unlike other skeletal cells such as chondrocytes, in the nucleus pulposus there is normoxic stabilization of this otherwise oxygen-labile protein (6,9). Moreover, despite an absence of HIF-1␣ protein induction in hypoxia, an increase in its transcriptional activity was seen (6,9). In a recent study of nucleus pulposus cells, Agrawal et al demonstrated that this transcription factor controlled both glycolysis and matrix synthesis (6).…”

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

“…Isolation of NP Cells, Cell Treatments, and Hypoxic CultureRat and human NP cells were isolated and characterized as reported earlier (5). T/C-28, a human chondrocyte line, was kindly provided by Dr. Mary Goldring (Hospital for Special Surgery, Weill Cornell Medical College, New York) (24).…”