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Aquaporin 1 and 5 expression decreases during human intervertebral disc degeneration: Novel HIF-1-mediated regulation of aquaporins in NP cells.

Johnson ZI, Gogate SS, Day R, Binch A, Markova DZ, Chiverton N, Cole A, Conner M, Shapiro IM, Le Maitre CL, Risbud MV - Oncotarget (2015)

Bottom Line: Bioinformatic analyses of AQP promoters showed multiple evolutionarily conserved HREs.While genomic chromatin immunoprecipitation showed limited binding of HIF-1α to conserved HREs, their mutation did not suppress promoter activities.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, PA, USA.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Expression of AQPs 1 and 5 is refractory to hypoxia in NP cellsA and B, AQP1 (A) and AQP5 (B) mRNA levels in rat NP cells cultured in normoxia (NX, 21% O2) or hypoxia (HX, 1% O2) for 8-72 h. mRNA expression showed no significant change under hypoxia. C-F, Western blot and corresponding Densitometric analysis shows that protein expression of AQP1 (C, E) and AQP5 (D, F) in rat NP cells is refractory to hypoxic conditions. G and H, Immunofluorescent detection of AQP1 and AQP5 in NP cells following 24 h in normoxia (NX) or hypoxia (HX). N-Cadherin is used as a marker for plasma membrane staining. Scale bar in G and H is 100 μm.
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Figure 5: Expression of AQPs 1 and 5 is refractory to hypoxia in NP cellsA and B, AQP1 (A) and AQP5 (B) mRNA levels in rat NP cells cultured in normoxia (NX, 21% O2) or hypoxia (HX, 1% O2) for 8-72 h. mRNA expression showed no significant change under hypoxia. C-F, Western blot and corresponding Densitometric analysis shows that protein expression of AQP1 (C, E) and AQP5 (D, F) in rat NP cells is refractory to hypoxic conditions. G and H, Immunofluorescent detection of AQP1 and AQP5 in NP cells following 24 h in normoxia (NX) or hypoxia (HX). N-Cadherin is used as a marker for plasma membrane staining. Scale bar in G and H is 100 μm.

Mentions: To test the effect of hypoxia on expression levels of AQP1 and AQP5, rat NP cells were cultured in 1% O2 for 8 to 72 h and mRNA and protein levels were measured. As shown in Fig. 5A and 5B, hypoxic culture did not significantly affect mRNA expression of either AQP in NP cells. Western blots and corresponding densitometric analyses confirm that hypoxia does not affect protein expression of either AQP1 (Fig. 5C and 5E) or AQP5 (Fig. 5D and 5F). To investigate possible changes in protein localization in response to hypoxia, cells were incubated in hypoxia for 24 h before immunofluorescent staining with antibodies directed against either AQP1 or AQP5 along with N-Cadherin. Fig. 5G-5H shows that expression and localization of both AQPs remain unaffected by hypoxia.


Aquaporin 1 and 5 expression decreases during human intervertebral disc degeneration: Novel HIF-1-mediated regulation of aquaporins in NP cells.

Johnson ZI, Gogate SS, Day R, Binch A, Markova DZ, Chiverton N, Cole A, Conner M, Shapiro IM, Le Maitre CL, Risbud MV - Oncotarget (2015)

Expression of AQPs 1 and 5 is refractory to hypoxia in NP cellsA and B, AQP1 (A) and AQP5 (B) mRNA levels in rat NP cells cultured in normoxia (NX, 21% O2) or hypoxia (HX, 1% O2) for 8-72 h. mRNA expression showed no significant change under hypoxia. C-F, Western blot and corresponding Densitometric analysis shows that protein expression of AQP1 (C, E) and AQP5 (D, F) in rat NP cells is refractory to hypoxic conditions. G and H, Immunofluorescent detection of AQP1 and AQP5 in NP cells following 24 h in normoxia (NX) or hypoxia (HX). N-Cadherin is used as a marker for plasma membrane staining. Scale bar in G and H is 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4494915&req=5

Figure 5: Expression of AQPs 1 and 5 is refractory to hypoxia in NP cellsA and B, AQP1 (A) and AQP5 (B) mRNA levels in rat NP cells cultured in normoxia (NX, 21% O2) or hypoxia (HX, 1% O2) for 8-72 h. mRNA expression showed no significant change under hypoxia. C-F, Western blot and corresponding Densitometric analysis shows that protein expression of AQP1 (C, E) and AQP5 (D, F) in rat NP cells is refractory to hypoxic conditions. G and H, Immunofluorescent detection of AQP1 and AQP5 in NP cells following 24 h in normoxia (NX) or hypoxia (HX). N-Cadherin is used as a marker for plasma membrane staining. Scale bar in G and H is 100 μm.
Mentions: To test the effect of hypoxia on expression levels of AQP1 and AQP5, rat NP cells were cultured in 1% O2 for 8 to 72 h and mRNA and protein levels were measured. As shown in Fig. 5A and 5B, hypoxic culture did not significantly affect mRNA expression of either AQP in NP cells. Western blots and corresponding densitometric analyses confirm that hypoxia does not affect protein expression of either AQP1 (Fig. 5C and 5E) or AQP5 (Fig. 5D and 5F). To investigate possible changes in protein localization in response to hypoxia, cells were incubated in hypoxia for 24 h before immunofluorescent staining with antibodies directed against either AQP1 or AQP5 along with N-Cadherin. Fig. 5G-5H shows that expression and localization of both AQPs remain unaffected by hypoxia.

Bottom Line: Bioinformatic analyses of AQP promoters showed multiple evolutionarily conserved HREs.While genomic chromatin immunoprecipitation showed limited binding of HIF-1α to conserved HREs, their mutation did not suppress promoter activities.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, PA, USA.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus