title: Nucleus pulposus cells express HIF-1 alpha 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.
authors.affiliation: Department of Orthopaedic Surgery and Graduate Program in Tissue Engineering and Regenerative Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, 19107.
authors.forename: Makarand V
authors.initials: MV
authors.lastname: Risbud
authors.forename: Asha
authors.initials: A
authors.lastname: Guttapalli
authors.forename: David G
authors.initials: DG
authors.lastname: Stokes
authors.forename: David
authors.initials: D
authors.lastname: Hawkins
authors.forename: Keith G
authors.initials: KG
authors.lastname: Danielson
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Schaer
authors.forename: Todd J
authors.initials: TJ
authors.lastname: Albert
authors.forename: Irving M
authors.initials: IM
authors.lastname: Shapiro
mesh_terms: D000222:Adaptation, Physiological; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D007403:Intervertebral Disc / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism; D008297:Male; D010100:Oxygen / Q000378:metabolism*; D010641:Phenotype; D051381:Rats; D017208:Rats, Wistar
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010100:Oxygen