Increased HIF-2α Activity in the Nucleus Pulposus Causes Intervertebral Disc Degeneration in the Aging Mouse Spine (original) (raw)
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Journal of Biological Chemistry, 2013
Background: The relationship between connective tissue growth factor (CCN2) and hypoxia-inducible factor (HIF)-1 in hypoxic nucleus pulposus is unknown. Results: HIF-1␣ suppresses CCN2 expression, whereas CCN2 represses basal HIF-1␣ levels and transcriptional activity. Conclusion: In nucleus pulposus, HIF-1␣ and CCN2 form a negative regulatory circuit. Significance: Tight control of CCN2 and HIF-1 activities may play a role in disc homeostasis.
Arthritis and Rheumatism, 2010
Objective-Since nucleus pulposus cells reside in hypoxia, we determined if expression of ANK, a pyrophosphate transporter, is regulated by the HIF proteins. Methods-Quantitative RT-PCR and Western blot were used to measure ANK expression in nucleus pulposus cells. Transfections were performed to determine the effect of HIF-1/-2 on ANK promoter activity. Results-ANK was expressed in embryonic and mature rat disc. Oxygen dependent changes in ANK expression in nucleus pulposus cells were minimal. However, silencing of HIF-1α and HIF-2α resulted in increased ANK expression and upregulation of promoter activity. HIF mediated suppression of ANK was validated by measuring promoter activity in HIF-1β null embryonic fibroblasts. Compared with wild type cells, in hypoxia, there was induction of promoter activity in the null cells. We overexpressed HIF-1α and HIF-2α in nucleus pulposus cells and noted a significant suppression in ANK promoter activity. Since the ANK promoter contains two hypoxia response elements (HRE), we performed site-directed mutagenesis and measured promoter activity. We found that HIF-1 can bind to either of the HRE and suppress promoter activity. In contrast, HIF-2 was required to bind to both HRE to suppress activity. Finally, analysis of human nucleus pulposus tissue showed that while ANK was expressed in normal tissue, there was increased expression of ANK along with alkaline phosphatase in the degenerate state. Conclusion-Both HIF-1 and HIF-2 serve as negative regulators of ANK expression in the disc. We propose that baseline ANK expression in the disc serves to prevent mineral formation under physiological conditions.
AJP: Cell Physiology, 2007
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)-1α, a transcription factor that responds to oxygen tension and regulates glycolysis. The goal of the present study was to examine the importance of HIF-1α 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-1α protein levels and mRNA stability were similar at 20 and 2% O2; there was a small, but significant increase in HIF-1α transactivation domain activity in hypoxia. With respect to HIF-1α 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 cel...
European Cells and Materials, 2015
Aged and degenerated intervertebral discs are characterised by a significant increase in the number of senescent cells, which may be associated with the deterioration of this tissue due to their catabolic phenotype. On the other hand, carboxymethyl-lysine has been found to be accumulated with ageing in the proteins of the disc, evidencing the existence of oxidative stress in this tissue. Accordingly, here we investigated the effect of oxidative stress on the physiology of human nucleus pulposus cells. Hydrogen peroxide (H 2 O 2) at subcytotoxic concentrations transiently increased the intracellular levels of reactive oxygen species, activated the p38 MAPK, ERKs, JNKs and Akt signalling pathways and induced the nuclear translocation of NF-κΒ and Nrf2. It also provoked DNA damage and triggered a DNA repair response by activating the ATM-Chk2-p53-p21 WAF1-pRb pathway, ultimately resulting in a G1 cell cycle delay and the decrease of cells' proliferation. Prolonged exposure to H 2 O 2 led to premature cellular senescence, as characterised by the inhibition of proliferation, the enhanced senescence-associated β galactosidase staining and the over-expression of known molecular markers, without though a significant decrease in the chromosome telomere length. H 2 O 2-senescent cells were found to possess a catabolic phenotype, mainly characterised by the up-regulation of extracellular matrixdegrading enzymes (MMP-1,-2,-9 and ADAMTS-5) and the down-regulation of their inhibitors (TIMPs), as well as of several proteoglycans, including aggrecan, the major component of the nucleus pulposus. The senescent phenotype could be reversed by N-acetyl-L-cysteine, supporting the use of antioxidants for the improvement of disc physiology and the deceleration of disc degeneration.
Arthritis & Rheumatism, 2010
Objective-Since nucleus pulposus cells reside in hypoxia, we determined if expression of ANK, a pyrophosphate transporter, is regulated by the HIF proteins. Methods-Quantitative RT-PCR and Western blot were used to measure ANK expression in nucleus pulposus cells. Transfections were performed to determine the effect of HIF-1/-2 on ANK promoter activity. Results-ANK was expressed in embryonic and mature rat disc. Oxygen dependent changes in ANK expression in nucleus pulposus cells were minimal. However, silencing of HIF-1α and HIF-2α resulted in increased ANK expression and upregulation of promoter activity. HIF mediated suppression of ANK was validated by measuring promoter activity in HIF-1β null embryonic fibroblasts. Compared with wild type cells, in hypoxia, there was induction of promoter activity in the null cells. We overexpressed HIF-1α and HIF-2α in nucleus pulposus cells and noted a significant suppression in ANK promoter activity. Since the ANK promoter contains two hypoxia response elements (HRE), we performed site-directed mutagenesis and measured promoter activity. We found that HIF-1 can bind to either of the HRE and suppress promoter activity. In contrast, HIF-2 was required to bind to both HRE to suppress activity. Finally, analysis of human nucleus pulposus tissue showed that while ANK was expressed in normal tissue, there was increased expression of ANK along with alkaline phosphatase in the degenerate state. Conclusion-Both HIF-1 and HIF-2 serve as negative regulators of ANK expression in the disc. We propose that baseline ANK expression in the disc serves to prevent mineral formation under physiological conditions.
Cell and Tissue Research, 2002
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-1β isoform in the nucleus pulposus; HIF-1β was present at lower levels in cells of the annulus and the end plate. In contrast to HIF-1β, HIF-1α was expressed only in the nucleus pulposus. This isoform was absent from both the cartilage end plate and annulus. We detected HIF-1α 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.
American Journal of Pathology, 2021
Endoplasmic reticulum (ER) stress is shown to promote nucleus pulposus (NP) cell apoptosis and intervertebral disc degeneration. However, little is known about ER stress regulation by the hypoxic disc microenvironment and its contribution to extracellular matrix homeostasis. NP cells were cultured under hypoxia (1% partial pressure of oxygen) to assess ER stress status, and gain-of-function and loss-of-function approaches were used to assess the role of hypoxia-inducible factor (HIF)-1a in this pathway. In addition, the contribution of ER stress induction on the NP cell secretome was assessed by a nontargeted quantitative proteomic analysis by sequential windowed data independent acquisition of the total high-resolution mass spectraemass spectrometry. NP cells exhibited a lower ER stress burden under hypoxia. Knockdown of HIF-1a increased C/EBP homologous protein, protein kinase RNAlike endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) levels, whereas HIF-1a stabilization decreased the expression of ER stress markers Ddit3, Hsp5a, Atf6, and Eif2a. Interestingly, ER stress inducers tunicamycin and thapsigargin induced HIF-1a activity under hypoxia while promoting the unfolded protein response. NP cell secretome analysis demonstrated an impact of ER stress induction on extracellular matrix secretion, with decreases in collagens and cell adhesionerelated proteins. Moreover, analysis of transcriptomic data of NP tissues from aged mice and degenerated human discs showed higher levels of unfolded protein response markers and decreased levels of matrix components. Our study shows, for the first time, that hypoxia and HIF-1a attenuate ER stress responses in NP cells, and ER stress promotes inefficient extracellular matrix secretion under hypoxia.
Journal of Orthopaedic Research, 2013
Oxidative damage is a well-established driver of aging. Evidence of oxidative stress exists in aged and degenerated discs, but it is unclear how it affects disc metabolism. In this study, we first determined whether oxidative stress negatively impacts disc matrix metabolism using disc organotypic and cell cultures. Mouse disc organotypic culture grown at atmospheric oxygen (20% O 2 ) exhibited perturbed disc matrix homeostasis, including reduced proteoglycan synthesis and enhanced expression of matrix metalloproteinases, compared to discs grown at low oxygen levels (5% O 2 ). Human disc cells grown at 20% O 2 showed increased levels of mitochondrial-derived superoxide anions and perturbed matrix homeostasis. Treatment of disc cells with the mitochondria-targeted reactive oxygen species (ROS) scavenger XJB-5-131 blunted the adverse effects caused by 20% O 2 . Importantly, we demonstrated that treatment of accelerated aging Ercc1 À/D mice, previously established to be a useful in vivo model to study age-related intervertebral disc degeneration (IDD), also resulted in improved disc total glycosaminoglycan content and proteoglycan synthesis. This demonstrates that mitochondrial-derived ROS contributes to age-associated IDD in Ercc1 À/D mice. Collectively, these data provide strong experimental evidence that mitochondrial-derived ROS play a causal role in driving changes linked to aging-related IDD and a potentially important role for radical scavengers in preventing IDD. ß
Study Design. NF-κ B activity was pharmacologically and genetically blocked in an accelerated aging mouse model to mitigate age-related disc degenerative changes. Objective. To study the mediatory role of NF-κ B-signaling pathway in age-dependent intervertebral disc degeneration. Summary of Background Data. Aging is a major contributor to intervertebral disc degeneration (IDD), but the molecular mechanism behind this process is poorly understood. NF-κ B is a family of transcription factors that play a central role in mediating cellular response to damage, stress, and infl ammation. Growing evidence implicates chronic NF-κ B activation as a culprit in many aging-related diseases, but its role in aging-related IDD has not been adequately explored. We studied the effects of NF-κ B inhibition on IDD, using a DNA repair-defi cient mouse model of accelerated aging ( Ercc1 − /∆ mice) previously been reported to exhibit age-related IDD.