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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

AQPs 1 and 5 are expressed in healthy rat discSections of rat intervertebral disc were stained with either H&E (A, B) or antibodies against AQP1 (H, I) or AQP5 (E, F). Note both AQPs are expressed in NP and AF tissues (arrows). In NP cells, prominent plasma membrane localization was seen. G, Western blot of AQPs from three rats demonstrates expression of 29 kDa AQP1 and AQP5 protein in NP tissues. Immunofluorescent detection of AQP1 and AQP5 protein in cultured NP and AF cells (H-K). L and M, Comparable levels of AQP 1 and 5 mRNA expression in rat NP (L) and AF (M) tissue samples was seen. Scale bar in H-K is 100 μm.
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Figure 2: AQPs 1 and 5 are expressed in healthy rat discSections of rat intervertebral disc were stained with either H&E (A, B) or antibodies against AQP1 (H, I) or AQP5 (E, F). Note both AQPs are expressed in NP and AF tissues (arrows). In NP cells, prominent plasma membrane localization was seen. G, Western blot of AQPs from three rats demonstrates expression of 29 kDa AQP1 and AQP5 protein in NP tissues. Immunofluorescent detection of AQP1 and AQP5 protein in cultured NP and AF cells (H-K). L and M, Comparable levels of AQP 1 and 5 mRNA expression in rat NP (L) and AF (M) tissue samples was seen. Scale bar in H-K is 100 μm.

Mentions: Since AQP expression was observed to be sensitive to disc degeneration, it was of interest to study their expression and regulation in native NP tissue. For this purpose, sections of NP and AF from rat intervertebral discs were first stained with antibody to detect either AQP1 (Fig. 2C and 2D) or AQP5 (Fig. 2E and 2F) localization. Additional sections were counterstained with H&E for assessment of general tissue morphology (Fig. 2A and 2B). Both AQP1 and AQP5 protein were detected in NP and AF tissues, with AQP1 showing more robust plasma membrane expression than AQP5 in NP sections. Protein expression of AQPs was further assessed in rat NP tissue and cultured NP and AF cells with Western blot analysis and immunofluorescence microscopy, respectively. As shown in Fig. 2G, both AQPs are expressed in freshly isolated NP tissue from three rats as evidenced by specific bands present at 29 kDa. Cultured NP and AF cells (Fig. 2H-2K) also expressed both AQPs. AQP mRNA expression was measured for both AQPs in NP (Fig. 2L) and AF (Fig. 2M) tissue isolated from three rats. All experimental data demonstrate a trend of similar expression of both AQPs 1 and 5 in NP cells and tissue.


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)

AQPs 1 and 5 are expressed in healthy rat discSections of rat intervertebral disc were stained with either H&E (A, B) or antibodies against AQP1 (H, I) or AQP5 (E, F). Note both AQPs are expressed in NP and AF tissues (arrows). In NP cells, prominent plasma membrane localization was seen. G, Western blot of AQPs from three rats demonstrates expression of 29 kDa AQP1 and AQP5 protein in NP tissues. Immunofluorescent detection of AQP1 and AQP5 protein in cultured NP and AF cells (H-K). L and M, Comparable levels of AQP 1 and 5 mRNA expression in rat NP (L) and AF (M) tissue samples was seen. Scale bar in H-K is 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: AQPs 1 and 5 are expressed in healthy rat discSections of rat intervertebral disc were stained with either H&E (A, B) or antibodies against AQP1 (H, I) or AQP5 (E, F). Note both AQPs are expressed in NP and AF tissues (arrows). In NP cells, prominent plasma membrane localization was seen. G, Western blot of AQPs from three rats demonstrates expression of 29 kDa AQP1 and AQP5 protein in NP tissues. Immunofluorescent detection of AQP1 and AQP5 protein in cultured NP and AF cells (H-K). L and M, Comparable levels of AQP 1 and 5 mRNA expression in rat NP (L) and AF (M) tissue samples was seen. Scale bar in H-K is 100 μm.
Mentions: Since AQP expression was observed to be sensitive to disc degeneration, it was of interest to study their expression and regulation in native NP tissue. For this purpose, sections of NP and AF from rat intervertebral discs were first stained with antibody to detect either AQP1 (Fig. 2C and 2D) or AQP5 (Fig. 2E and 2F) localization. Additional sections were counterstained with H&E for assessment of general tissue morphology (Fig. 2A and 2B). Both AQP1 and AQP5 protein were detected in NP and AF tissues, with AQP1 showing more robust plasma membrane expression than AQP5 in NP sections. Protein expression of AQPs was further assessed in rat NP tissue and cultured NP and AF cells with Western blot analysis and immunofluorescence microscopy, respectively. As shown in Fig. 2G, both AQPs are expressed in freshly isolated NP tissue from three rats as evidenced by specific bands present at 29 kDa. Cultured NP and AF cells (Fig. 2H-2K) also expressed both AQPs. AQP mRNA expression was measured for both AQPs in NP (Fig. 2L) and AF (Fig. 2M) tissue isolated from three rats. All experimental data demonstrate a trend of similar expression of both AQPs 1 and 5 in NP cells and tissue.

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