Aquaporin 1 and 5 expression decreases during human intervertebral disc degeneration: novel HIF-1-mediated regulation of aquaporins in NP cells (original) (raw)
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PLoS ONE, 2011
Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O 2 transport. It has been reported that this protein contributes to gas permeation (CO 2 , NO and O 2 ) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 59 proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a doseand time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl 2 ) and by overexpressing stabilized mutated forms of HIF-1a. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1a siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1a participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1a other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases.
Aquaporin 1 – a novel player in spinal cord injury
Journal of Neurochemistry, 2008
The role of water channel aquaporin 1 (AQP-1) in uninjured or injured spinal cords is unknown. AQP-1 is weakly expressed in neurons and gray matter astrocytes, and more so in white matter astrocytes in uninjured spinal cords, a novel finding. As reported before, AQP-1 is also present in ependymal cells, but most abundantly in small diameter sensory fibers of the dorsal horn. Rat contusion spinal cord injury (SCI) induced persistent and significant four-to eightfold increases in AQP-1 levels at the site of injury (T10) persisting up to 11 months post-contusion, a novel finding. Delayed AQP-1 increases were also found in cervical and lumbar segments, suggesting the spreading of AQP-1 changes over time after SCI. Given that the antioxidant melatonin significantly decreased SCI-induced AQP-1 increases and that hypoxia inducible factor-1α was increased in acutely and chronically injured spinal cords, we propose that chronic hypoxia contributes to persistent AQP-1 increases after SCI. Interestingly; AQP-1 levels were not affected by long-lasting hypertonicity that significantly increased astrocytic AQP-4, suggesting that the primary role of AQP-1 is not regulating isotonicity in spinal cords. Based on our results we propose possible novel roles for AQP-1 in the injured spinal cords: (i) in neuronal and astrocytic swelling, as AQP-1 was increased in all surviving neurons and reactive astrocytes after SCI and (ii) in the development of the neuropathic pain after SCI. We have shown that decreased AQP-1 in melatonin-treated SCI rats correlated with decreased AQP-1 immunolabeling in the dorsal horns sensory afferents, and with significantly decreased mechanical allodynia, suggesting a possible link between AQP-1 and chronic neuropathic pain after SCI.
Arthritis Research & Therapy, 2009
Introduction The aim of this study was to compare the effects of tumour necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β) on protease and catabolic cytokine and receptor gene expression in normal and degenerate human nucleus pulposus cells in alginate culture. Methods Cells isolated from normal and degenerate nucleus pulposus regions of human intervertebral discs were cultured in alginate pellets and stimulated by the addition of 10 ng/mL TNFα or IL-1β for 48 hours prior to RNA extraction. Quantitative realtime polymerase chain reaction was used to assess the effect of TNF-α or IL-β stimulation on the expression of matrix metalloproteinase (MMP)-3,-9 and-13, TNF-α, TNF receptor 1 (TNF-R1), TNF receptor 2 (TNF-R2), IL-1α, IL-1β, IL-1 receptor 1 (IL-1R1) and IL-1 receptor antagonist (IL-1Ra). Results MMP-3 and MMP-9 gene expressions were upregulated to a greater level by IL-1β than TNF-α. MMP-13 was upregulated by each cytokine to a similar extent. TNF-α and TNF-R2 expressions were upregulated by both TNF-α and IL-β, whereas TNF-R1 expression was not significantly affected by either cytokine. IL-1β and IL-1Ra expressions were significantly upregulated by TNF-α, whereas IL-1α and IL-1R1 were unchanged. Conclusions TNF-α does not induce MMP expression to the same degree as stimulation by IL-1β, but it does act to upregulate IL-1β expression as well as TNF-α and TNF-R2. The net result of this would be an increased inflammatory environment and accelerated degradation of the matrix. These results support the hypothesis that, while TNF-α may be an important initiating factor in matrix degeneration, IL-1β plays a greater role in established pathological degradation.
Molecular Brain Research, 2001
Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens [Cell 39 (1984) 49], that increase plasma membrane water permeability in secretory and absorptive cells. In the central nervous system (CNS), we detected the transcripts of AQP3, 5 and 8 in addition to the previously reported transcripts of AQP4 and 9 in astrocytes, of AQP3, 5 and 8 in neurons, of AQP8 in oligodendrocytes, and none of them in microglia using RNase protection assay and the reverse transcription-polymerase chain reaction (RT-PCR). Hypoxia evoked a marked decrease in the expression levels of AQP4, 5 and 9, but not of AQP3 and 8 mRNAs, and in astrocytes in vitro subsequent reoxygenation elicited the restoration of the expression of AQP4 and 9 to their basal levels. Interestingly, AQP5 showed a transient up-regulation (about 3-fold) and subsequent down-regulation of its expression within 20 h of reoxygenation after hypoxia. The changes in the profiles of AQP expression during hypoxia and reoxygenation were also observed by Western blot analysis. These results suggest that AQP5 may be one of the candidates for inducing the intracranial edema in the CNS after ischemia injury.
bioRxiv (Cold Spring Harbor Laboratory), 2023
Hypoxia-inducible factors (HIFs) are essential to the homeostasis of hypoxic tissues. Although HIF-2α, is expressed in nucleus pulposus (NP) cells, consequences of elevated HIF-2 activity on disc health remains unknown. We expressed HIF-2α with proline to alanine substitutions (P405A;P531A) in the Oxygen-dependent degradation domain (HIF-2αdPA) in the NP tissue using an inducible, nucleus pulposus-specific K19 CreERT allele to study HIF-2α function in the adult intervertebral disc. Expression of HIF-2α in NP impacted disc morphology, as evident from small but significantly higher scores of degeneration in NP of 24-month-old K19 CreERT ; HIF-2α dPA (K19-dPA) mice. Noteworthy, comparisons of grades within each genotype between 14 months and 24 months indicated that HIF-2α overexpression contributed to more pronounced changes than aging alone. The annulus fibrosus (AF) compartment in the 14month-old K19-dPA mice exhibited lower collagen turnover and Fourier transform-infrared (FTIR) spectroscopic imaging analyses showed changes in the biochemical composition of the
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.
Frontiers in Immunology, 2021
Oxidative stress (OS) irreversibly affects the pathogenesis of intervertebral disc degeneration (IDD). Certain non-coding RNAs act as competitive endogenous RNAs (ceRNAs) that regulate IDD progression. Analyzing the signatures of oxidative stress-related gene (OSRG) pairs and regulatory ceRNA mechanisms and immune-infiltration patterns associated with IDD may enable researchers to distinguish IDD and reveal the underlying mechanisms. In this study, OSRGs were downloaded and identified using the Gene Expression Omnibus database. Functional-enrichment analysis revealed the involvement of oxidative stress-related pathways and processes, and a ceRNA network was generated. Differentially expressed oxidative stress-related genes (De-OSRGs) were used to construct De-OSRG pairs, which were screened, and candidate De-OSRG pairs were identified. Immune cell-related gene pairs were selected via immune-infiltration analysis. A potential long non-coding RNA–microRNA–mRNA axis was determined, and...