The goal of the study was to investigate bone morphogenetic protein 2 (BMP-2) and transforming growth factor β (TGF-β) control of the expression of β1,3-glucuronosyl transferase 1 (GlcAT-1), an important regulator of chondroitin sulfate synthesis in cells of the nucleus pulposus. Treatment with both growth factors resulted in induction of GlcAT-1 expression and promoter activity. Deletion analysis indicated that promoter constructs lacking AP1 and TonE sites were unresponsive to growth factor treatment. Experiments using dominant-negative proteins showed that these transcription factors along with Sp1 were required for induction of GlcAT-1 promoter activity. Moreover, when either AP1 or TonE binding sites were mutated, induction was suppressed. Both BMP-2 and TGF-β increased c-Jun and TonEBP expression and phosphorylation of transactivation domains. We investigated the role of the mitogen-activated protein kinase (MAPK) signaling pathway following growth factor treatment; a robust and transient activation of ERK1/2, p38, and JNK was noted. Treatment with MAPK inhibitors blocked BMP-2- and TGF-β-induced AP1 reporter function, GlcAT-1 expression, and GAG accumulation. We found that DN-ERK1 but not DN-ERK2 resulted in suppression of growth factor–mediated induction of GlcAT-1 promoter activity; we also showed that p38δ was important in GlcAT-1 activation. Results of these studies demonstrate that BMP-2 and TGF-β regulate GlcAT-1 expression in nucleus pulposus cells through a signaling network comprising MAPK, AP1, Sp1, and TonEBP. It is concluded that by controlling both GAG and aggrecan synthesis, these growth factors positively influence disk cell function. © 2010 American Society for Bone and Mineral Research.
Background:The regulation of PHD expression and function under inflammatory conditions in the nucleus pulposus is unknown. Results: Expression of PHD3 is regulated by TNF-␣ and IL-1. PHD3 controls TNF-␣ activity by modulating NF-B signaling. Conclusion: PHD3 promotes the catabolic effects of TNF-␣ on nucleus pulposus cells. Significance: PHD3 may play an important role in pathogenesis of disc disease.
Objectives of this study were to investigate whether AQP1 and AQP5 expression is altered during intervertebral disc degeneration and if hypoxia and HIF-1 regulate their expression in NP cells. AQP expression was measured in human tissues from different degenerative grades; regulation by hypoxia and HIF-1 was studied using promoter analysis and gain- and loss-of-function experiments. We show that both AQPs are expressed in the disc and that mRNA and protein levels decline with human disease severity. Bioinformatic analyses of AQP promoters showed multiple evolutionarily conserved HREs. Surprisingly, hypoxia failed to induce promoter activity or expression of either AQP. While genomic chromatin immunoprecipitation showed limited binding of HIF-1α to conserved HREs, their mutation did not suppress promoter activities. Stable HIF-1α suppression significantly decreased mRNA and protein levels of both AQPs, but HIF-1α failed to induce AQP levels following accumulation. Together, our results demonstrate that AQP1 and AQP5 expression is sensitive to human disc degeneration and that HIF-1α uniquely maintains basal expression of both AQPs in NP cells, independent of oxemic tension and HIF-1 binding to promoter HREs. Diminished HIF-1 activity during degeneration may suppress AQP levels in NP cells, compromising their ability to respond to extracellular osmolarity changes.
Objective. To determine whether hypoxia and hypoxia-inducible factor (HIF) proteins regulate expression of -1,3-glucuronyltransferase 1 (GlcAT-1), a key enzyme in glycosaminoglycan synthesis in nucleus pulposus cells.Methods. Real-time reverse transcriptasepolymerase chain reaction and Western blotting were used to measure GlcAT-1 expression. Transfections were performed to determine the effect of HIF-1␣ and HIF-2␣ on GlcAT-1 promoter activity.Results. Under hypoxic conditions there was an increase in GlcAT-1 expression; a significant increase in promoter activity was seen both in nucleus pulposus cells and in N1511 chondrocytes. We investigated whether HIF controlled GlcAT-1 expression. Suppression of HIF-1␣ and HIF-2␣ induced GlcAT-1 promoter activity and expression only in nucleus pulposus cells. Transfection with CA-HIF-1␣ as well as with CA-HIF-2␣ suppressed GlcAT-1 promoter activity only in nucleus pulposus cells, suggesting a cell type-specific regulation. Site-directed mutagenesis and deletion constructs were used to further confirm the suppressive effect of HIFs on GlcAT-1 promoter function in nucleus pulposus cells. Although it was evident that interaction of HIF with hypoxia-responsive elements resulted in suppression of basal promoter activity, it was not necessary for transcriptional suppression. This result suggested both a direct and an indirect mode of regulation, possibly through recruitment of a HIF-dependent repressor. Finally, we showed that hypoxic expression of GlcAT-1 was also partially dependent on MAPK signaling.Conclusion. These studies demonstrate that hypoxia regulates GlcAT-1 expression through a signaling network comprising both activator and suppressor molecules, and that this regulation is unique to nucleus pulposus cells.The intervertebral disc is a specialized tissue that permits rotation as well as flexure and extension of the human spine. It consists of an outer ligamentous anulus fibrosus that encloses the gel-like nucleus pulposus. The superior and inferior boundaries of the intervertebral disc are formed by the cartilage end plates. Blood vessels infiltrate the superficial region of the end plates and the outer third of the anulus fibrosus, but they do not enter the nucleus pulposus (1,2). In line with the avascular nature of this tissue there is a robust and constitutive expression of both hypoxia-inducible factor 1␣ (HIF-1␣) and HIF-2␣, suggesting that the nucleus pulposus cells reside in a hypoxic environment (3-5).The hypoxic cells in the nucleus pulposus secrete a complex extracellular matrix that contains the hydrophilic proteoglycan aggrecan substituted with many glycosaminoglycan (GAG) chains (6). The major GAG of the nucleus pulposus is chondroitin sulfate. Structurally, this molecule is a heteropolysaccharide containing repeating units of N-acetylgalactosamine linked to glucuSupported by the NIH (grants R01-AR-050087 and R01-AR-055655).
The objective of our study was to examine the regulation of hypoxic expression of Hsp70 in nucleus pulposus cells and to determine if Hsp70 promoted HIF-1α degradation. Rat nucleus pulposus cells were maintained in culture in either 21% or 1% oxygen. To determine the regulation of Hsp70 expression by Tonicity enhancer binding protein (TonEBP) and HIF-1/2, loss- and gain-of-function experiments and mutational analysis of Hsp70 promoter were performed. Hypoxia increased Hsp70 expression in nucleus pulposus cells. Noteworthy, hypoxia increased TonEBP transactivation and mutation of TonE motifs blocked hypoxic induction of Hsp70 promoter. In contrast, mutation of HRE motifs coupled with loss of function experiments suggested that HIF-1 and HIF-2 suppressed Hsp70 promoter activity and transcription. Interestingly, HIF-α interferes with TonEBP function and suppresses inductive effect of TonEBP on Hsp70 promoter. In terms of Hsp70 function, when treated with Hsp70 transcriptional inhibitor, KNK437, there was an increase in HIF-1α protein stability and transcriptional activity. Likewise, when Hsp70 was overexpressed, the stability of HIF-1α and its transcriptional activity decreased. Hsp70 interacted with HIF-1α under hypoxic conditions and evidenced increased binding when treated with MG132, a proteasomal inhibitor. These results suggest that Hsp70 may promote HIF-1α degradation through proteasomal pathway in nucleus pulposus cells. In hypoxic and hyperosmolar nucleus pulposus cells, Hsp70, TonEBP and HIFs form a regulatory loop. We propose that the positive regulation by TonEBP and negative regulation of Hsp70 by HIF-1 and HIF-2 may serve to maintain Hsp70 levels in these cells, whereas Hsp70 may function in controlling HIF-1α homeostasis.
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