Phenotypic characteristics of the nucleus pulposus: expression of hypoxia inducing factor-1, glucose transporter-1 and MMP-2.
Attempts to study the biology of the nucleus pulposus have been limited in scope due to the low rates of cell proliferation, difficulties in maintaining viable disc cells in culture and the absence of a clearly defined phenotype. The major objective of this communication is to construct a phenotypic signature for cells of the nucleus pulposus that is based on the hypothesis that in response to restriction on oxygen and nutrient flux, there is expression of HIF-1, GLUT-1 and MMP-2. Nucleus pulposus, as well as annulus fibrosus and cartilage of the vertebral end plates, was collected from rat spinal units. Western blot analysis and immunohistochemistry clearly showed that there was a significant level of expression of the HIF-1 beta isoform in the nucleus pulposus; HIF-1 beta was present at lower levels in cells of the annulus and the end plate. In contrast to HIF-1 beta, HIF-1 alpha was expressed only in the nucleus pulposus. This isoform was absent from both the cartilage end plate and annulus. We detected HIF-1 alpha immunohistochemically in the nucleus pulposus; however, the staining was light and diffuse. Cells of the nucleus pulposus expressed GLUT-1; in contrast, when probed by Western blot analysis the annulus and cartilage were negative for this protein. Western blot analysis also showed that in the nucleus pulposus the level of MMP-2 was high when compared to the adjacent tissues. We suggest that the differential expression of the two HIF isoforms, and GLUT-1 and MMP-2, provides a phenotypic signature that permits cells of the nucleus pulposus to be distinguished from neighboring tissues. Moreover, the presence of these isoforms provides evidence that cells of the disc respond to hypoxia and nutrient stress by upregulating stress-responsive genes.
Rajpurohit
Ramesh
R
Department of Orthopaedic Surgery, Jefferson Medical College, Thomas Jefferson University, 501 Curtis Building, 1015 Walnut St., Philadelphia, PA 19107, USA.
Risbud
Makarand V
MV
Ducheyne
Paul
P
Vresilovic
Edward J
EJ
Shapiro
Irving M
IM
D000818:Animals; D015415:Biomarkers; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000032:analysis / Q000096:biosynthesis*; D051272:Glucose Transporter Type 1; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007150:Immunohistochemistry; D007403:Intervertebral Disc / Q000737:chemistry / Q000378:metabolism*; D020778:Matrix Metalloproteinase 2 / Q000032:analysis / Q000096:biosynthesis*; D009004:Monosaccharide Transport Proteins / Q000032:analysis / Q000096:biosynthesis*; D009687:Nuclear Proteins / Q000032:analysis / Q000096:biosynthesis*; D010641:Phenotype; D051381:Rats; D014157:Transcription Factors
D015415:Biomarkers; D004268:DNA-Binding Proteins; D051272:Glucose Transporter Type 1; C497444:Hif1a protein, rat; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009004:Monosaccharide Transport Proteins; D009687:Nuclear Proteins; C495679:Slc2a1 protein, rat; D014157:Transcription Factors; D020778:Matrix Metalloproteinase 2