Studies of nucleotides of growth-plate cartilage: evidence linking changes in cellular metabolism with cartilage calcification

Shapiro, Irving M.; Golub, Ellis E.; May, Mary; Rabinowitz, Joseph L.

doi:10.1007/bf01122899

Cited by 29 publications

(14 citation statements)

References 21 publications

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“…We found that in normoxia, basal concentrations of ATP in nucleus pulposus cells were between 20 and 25 nM/mg protein. These values are comparable to levels reported for articular chondrocytes (18), another cell type that uses glycolysis to generate energy (29,30). In the presence of 2-DG, a potent inhibitor of glycolysis, ATP generation was suppressed almost 80%.…”

Section: Discussionsupporting

confidence: 71%

“…hypoxia; hypoxia-inducible factor-1␣ THE LOCAL OXYGEN TENSION promotes cellular differentiation and regulates tissue energy metabolism. When the oxygen tension is low, there is almost complete reliance on glycolysis to generate ATP and reducing equivalents (12,29,30). In specialized tissues such as the intervertebral disk, the mechanism by which cells conserve energy has received little attention.…”

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confidence: 99%

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Normoxic stabilization of HIF-1α drives glycolytic metabolism and regulates aggrecan gene expression in nucleus pulposus cells of the rat intervertebral disk

Agrawal¹,

Guttapalli²,

Narayan³

et al. 2007

American Journal of Physiology-Cell Physiology

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Agrawal A, Guttapalli A, Narayan S, Albert TJ, Shapiro IM, Risbud MV. Normoxic stabilization of HIF-1␣ drives glycolytic metabolism and regulates aggrecan gene expression in nucleus pulposus cells of the rat intervertebral disk. Am J Physiol Cell Physiol 293: C621-C631, 2007. First published April 18, 2007; doi:10.1152/ajpcell.00538.2006.-The nucleus pulposus is an aggrecan-rich, avascular tissue that permits the intervertebral disk to resist compressive loads. In the disk, nucleus pulposus cells express hypoxia-inducible factor (HIF)-1␣, a transcription factor that responds to oxygen tension and regulates glycolysis. The goal of the present study was to examine the importance of HIF-1␣ in rat nucleus pulposus cells and to probe the function of this transcription factor in terms of regulating aggrecan gene expression. We found that HIF-1␣ protein levels and mRNA stability were similar at 20 and 2% O2; there was a small, but significant increase in HIF-1␣ transactivation domain activity in hypoxia. With respect to HIF-1␣ target genes GAPDH, GLUT-1, and GLUT-3, mRNA and protein levels were independent of the oxygen tension. Other than a modest increase in 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase reporter activity, the oxemic state did not change GAPDH, GLUT-1, and GLUT-3 promoter activities. Treatment of cells with 2-deoxyglucose (2-DG), a glycolytic inhibitor, resulted in a significant suppression in ATP synthesis in normoxia, whereas treatment with mitochondrial inhibitors did not affect ATP production and cell viability. However, measurement of the rate of fatty acid oxidation indicated that these cells contained functioning mitochondria. Finally, we showed that when HIF-1␣ was suppressed, irrespective of the oxemic state, there was a partial loss of aggrecan expression and promoter activity. Moreover, when cells were treated with 2-DG, there was inhibition in aggrecan promoter activity. Results of this study indicate that oxygen-independent stabilization of HIF-1␣ in nucleus pulposus cells is a metabolic adaptation that drives glycolysis and aggrecan expression. hypoxia; hypoxia-inducible factor-1␣ THE LOCAL OXYGEN TENSION promotes cellular differentiation and regulates tissue energy metabolism. When the oxygen tension is low, there is almost complete reliance on glycolysis to generate ATP and reducing equivalents (12,29,30). In specialized tissues such as the intervertebral disk, the mechanism by which cells conserve energy has received little attention. Nonetheless, it is known that in the intervertebral disk, the nucleus pulposus has no direct vascular supply (8,24,33), and ATP is probably generated by anaerobic glycolysis (1, 10). The dependence on glycolysis reflects the low oxygen tension in the disk, which has been reported to be low as low as 2%, and the observation that modulation in the oxygen supply can cause vertebral defects (15). Regulation of glycolysis is mediated by hypoxia-inducible factor (HIF)-1␣, a transcription factor that responds to the local oxygen tension, and has been show...

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Section: Discussionsupporting

confidence: 71%

mentioning

confidence: 99%

Normoxic stabilization of HIF-1α drives glycolytic metabolism and regulates aggrecan gene expression in nucleus pulposus cells of the rat intervertebral disk

Agrawal¹,

Guttapalli²,

Narayan³

et al. 2007

American Journal of Physiology-Cell Physiology

Self Cite

160

188

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“…Wuthier [20] showed that nucleotide concentrations in hypertrophic zone were greater than in proliferating zone. Recently, Shapiro et al [21], using HPLC measurement, concluded that the concentration of various nucleotides in cartilage was low (6-8 n tool/rag dry weight). The ATP and ADP concentrations were estimated to be much greater than AMR Assuming a 60% water content in cartilage, ATP concentration can be calculated to be about 4 ~M, according to the data of Shapiro et al [21].…”

Section: Discussionmentioning

confidence: 98%

“…Recently, Shapiro et al [21], using HPLC measurement, concluded that the concentration of various nucleotides in cartilage was low (6-8 n tool/rag dry weight). The ATP and ADP concentrations were estimated to be much greater than AMR Assuming a 60% water content in cartilage, ATP concentration can be calculated to be about 4 ~M, according to the data of Shapiro et al [21]. If ATP is accessible to pyrophosphohydrolase and is held at this level in vivo, then the enzyme should be at least partially active because its Km was estimated to be 10 txM [9].…”

Section: Discussionmentioning

confidence: 98%

The deposition of calcium pyrophosphate and phosphate by matrix vesicles isolated from fetal bovine epiphyseal cartilage

Hsu

Anderson

1984

Calcif Tissue Int

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Since calcium (Ca) deposition by isolated fetal bovine matrix vesicles is selectively supported by nucleoside triphosphate, and since the Ca deposits appear to be amorphous by transmission electron microscopy, attempts were made to study further the nature of these Ca deposits. Calcification of isolated matrix vesicles was allowed to occur in a calcifying medium in which either inorganic phosphate (Pi) or [gamma-P]ATP was labeled with 32P. 32P in Ca P (pyrophosphate) deposits were analyzed by a Dowex 1 X 10 anion exchange chromatography. The results of the analysis indicate that the (32P) radioactivity was mainly associated with Pi when Pi in the calcifying media was labeled with 32P. In contrast, 32P was found to be associated with inorganic pyrophosphate (PPi) when [gamma-32P]ATP was used. Using a specific enzyme coupling assay for PPi, the presence of PPi in the Ca deposits was demonstrated. The amounts of Pi and PPi in the Ca deposits initiated by fetal calf matrix vesicles were found to be approximately equal. To exclude the possibility that the major part of PPi of Ca P deposit existed as adsorbed form, the deposition was performed under the conditions in which Pi was omitted from calcifying medium. The results of these experiments showed that substantial amount of PPi and Ca deposits remained the same and was not correlated to the amount of Pi in these deposits. In contrast, Pi of CaP was decreased if Pi was omitted from the calcifying medium. Thus, it appears that the major portion of PPi exists as mineral rather than adsorbed form.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…When the oxygen tension is low, there is almost complete reliance on glycolysis to generate ATP and reducing equivalents (1)(2)(3). In specialized tissues such as the intervertebral disc, the mechanism by which cells conserve energy has received little attention.…”

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confidence: 99%

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

Agrawal¹,

Gajghate²,

Smith³

et al. 2008

Arthritis & Rheumatism

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Objective. To determine whether nucleus pulposus cells of the intervertebral disc express hypoxiainducible factor 2␣ (HIF-2␣), and to assess the role of HIF-1 and HIF-2 in controlling cited2 and vascular endothelial growth factor (VEGF) expression.Methods. Rat cells were cultured under normoxic (21% O 2 ) or hypoxic (2% O 2 ) conditions, and expression and promoter activity of HIF-2 target genes were evaluated. Gain-or loss-of-function experiments were performed to investigate the contribution of HIF isoforms to cited2 activity as well as the role of cited2 in regulating VEGF expression.Results. We found that HIF-2␣ protein was expressed in vivo and that protein and messenger RNA expression were similar under both normoxic and hypoxic conditions. However, there was a significant increase in HIF-2␣ transactivation under hypoxic conditions. With respect to functional activity, unlike the case in most other tissues, HIF-2 failed to increase the transcriptional activities of superoxide dismutase 2 and frataxin, 2 common target genes involved in radical dismutation. However, under hypoxic conditions, HIF-2 preferentially regulated the expression and promoter activity of cited2, a p300 binding protein. When HIF-2␣ or HIF-1␣ was suppressed, cited2 promoter activity was inhibited. Finally, we showed that forced expression or suppression of cited2 resulted in corresponding changes in expression of VEGF, a common target gene for HIF-1 and HIF-2 in the nucleus pulposus cells.Conclusion. Results of this study indicate that in nucleus pulposus cells, HIF-2 and HIF-1 modulate their own transcriptional activity through cited2. We suggest that the 2 arms of the regulatory circuit serve to maintain survival activities and inhibit angiogenesis in the healthy disc.

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Studies of nucleotides of growth-plate cartilage: evidence linking changes in cellular metabolism with cartilage calcification

Cited by 29 publications

References 21 publications

Normoxic stabilization of HIF-1α drives glycolytic metabolism and regulates aggrecan gene expression in nucleus pulposus cells of the rat intervertebral disk

Normoxic stabilization of HIF-1α drives glycolytic metabolism and regulates aggrecan gene expression in nucleus pulposus cells of the rat intervertebral disk

The deposition of calcium pyrophosphate and phosphate by matrix vesicles isolated from fetal bovine epiphyseal cartilage

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

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