Abstract
Introduction
Aquaporins are a family of transmembrane water channels that aid in osmoregulation, a critical function in the proteoglycan-rich nucleus pulposus. Previous reports have demonstrated expression of aquaporins in the intervertebral disc.1,2 Hypoxia, a defining feature of the nucleus pulposus environment has been shown to regulate expression of aquaporin1 (AQP1) and aquaporin 5 (AQP5).3-6 In some cases, this regulation has been shown to occur through HIF-1α and to depend on hypoxia responsive elements (HREs). The goal of this study was to examine if hypoxia and HIF-1α play a role in regulation and function of aquaporin1 (AQP1) and aquaporin5 (AQP5) in nucleus pulposus (NP) cells of the intervertebral disc.
Materials and Methods
Quantitative reverse transcription-polymerase chain reaction, Western blot, and immunohistochemistry were used to measure AQP1 and AQP5 expression in nucleus pulposus (NP) and annulus fibrosus (AF) cells and tissues. Transfections were used to determine the role of HIF-1α on AQP1 and AQP5 promoter activity in normoxia and hypoxia. The JASPAR database was used to identify two putative hypoxia response elements (HREs) in the promoter of each aquaporin. HIF-1α levels were modulated with treatment by DMOG or shRNAs. Expression of aquaporins in human tissue samples was evaluated using immunohistochemistry and polymerase chain reaction.
Results
We found that both AQP1 and AQP5 were expressed in tissues and cells of human and rat discs. To determine whether expression of the aquaporins was regulated by hypoxia, we treated NP cells with hypoxia (1% O2) for 8 to 72 hours. Treatment had no effect on promoter activity, mRNA expression, or protein expression of either aquaporin. Using the JASPAR database, we identified two HREs in the promoter of each aquaporin. Mutation of the HREs in either AQP1 or AQO5 had no suppressive effect on the basal activity of the promoters. We then investigated whether AQP1 and AQP5 were regulated in a HIF1α-dependent manner. Accumulation of HIF-1α by DMOG did not affect expression of either aquaporin. However, suppression of HIF-1α by lentiviral delivery of shRNA significantly decreased both mRNA and protein expression of AQP1 and AQP5. In addition, we have found changes in expression of these aquaporins in human tissue samples from patients with varying degrees of intervertebral disc degeneration.
Conclusion
These results indicate that, under hypoxic conditions, HIF-1α maintains basal expression of both AQP1 and AQP5 in the NP; however, this regulation is independent of HIF binding to the identified HREs in their promoters. Analysis of human tissue samples suggests that aquaporin expression may be linked to health of the intervertebral disc.
None declared
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