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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

HIF-1α maintains basal AQP expression in NP cellsA and B, Western blot (A) and corresponding densitometric analysis (B) show that treatment of rat NP cells with DMOG (1mM) has no effect on levels of AQP1 and AQP5, as expected accumulation of HIF-1α protein by DMOG is evident. (C), Suppression of HIF-1α expression by co-transfection with Si-HIF-1α does not significantly change activities of AQP1 and AQP5 promoters in comparison to rat NP cell transfected with control siRNA. (D), YFP expression demonstrates efficient transduction of human NP cells infected with lentivirus encoding both HIF-1α shRNA and YFP. (E), Stable Silencing of HIF-1α by lentiviral Sh-HIF-1α causes strong decrease in basal levels of both AQP 1 and AQP5 mRNA in human NP cells. As expected, HIF-1α mRNA levels were significantly lower in silenced cells compared to cells that received control shRNA. F and G, Western blot (F) and corresponding densitometric analysis (G) shows that HIF-1α silencing in human NP cells results in robust decrease in both AQP 1 and AQP5 protein levels.
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Figure 6: HIF-1α maintains basal AQP expression in NP cellsA and B, Western blot (A) and corresponding densitometric analysis (B) show that treatment of rat NP cells with DMOG (1mM) has no effect on levels of AQP1 and AQP5, as expected accumulation of HIF-1α protein by DMOG is evident. (C), Suppression of HIF-1α expression by co-transfection with Si-HIF-1α does not significantly change activities of AQP1 and AQP5 promoters in comparison to rat NP cell transfected with control siRNA. (D), YFP expression demonstrates efficient transduction of human NP cells infected with lentivirus encoding both HIF-1α shRNA and YFP. (E), Stable Silencing of HIF-1α by lentiviral Sh-HIF-1α causes strong decrease in basal levels of both AQP 1 and AQP5 mRNA in human NP cells. As expected, HIF-1α mRNA levels were significantly lower in silenced cells compared to cells that received control shRNA. F and G, Western blot (F) and corresponding densitometric analysis (G) shows that HIF-1α silencing in human NP cells results in robust decrease in both AQP 1 and AQP5 protein levels.

Mentions: The effect of HIF-1 on AQP expression in rat NP cells was further investigated using gain- and loss-of-function studies. To block prolyl hydroxylase activity and accumulate HIF-1α protein, cells were treated with 1 mM DMOG. Western blot and corresponding densitometric analysis (Fig. 6A and 6B) show that, while DMOG treatment led to accumulation of HIF-1α protein, expression levels of AQP1 and AQP5 remained unchanged. Next, rat NP cells were co-transfected with the wild-type reporter constructs of either the AQP1 or the AQP5 promoter along with siHIF-1α or a control siRNA. Results clearly showed that suppression of HIF-1 by siHIF-1α did not affect promoter activity of either wild-type AQP promoter construct (Fig. 6C). To assess effect of stable HIF-1 suppression on AQP expression, human NP cells were transduced by lentivirus co-expressing shHIF-1α and YFP. As shown in Fig. 6D, YFP signal indicated that lentiviral delivery resulted in high infection rates and a robust transgene expression in NP cells. As expected, shHIF-1α significantly decreased mRNA expression of HIF-1α compared to sh-control (Fig. 6E-6G). Importantly, silencing of HIF-1α resulted in dramatic decrease in mRNA expression of both AQP1 and AQP5 in human NP cells (Fig. 6E). Moreover, HIF-1α knockdown resulted in significant reduction in protein levels of both AQP1 and AQP5 (Fig. 6G).


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)

HIF-1α maintains basal AQP expression in NP cellsA and B, Western blot (A) and corresponding densitometric analysis (B) show that treatment of rat NP cells with DMOG (1mM) has no effect on levels of AQP1 and AQP5, as expected accumulation of HIF-1α protein by DMOG is evident. (C), Suppression of HIF-1α expression by co-transfection with Si-HIF-1α does not significantly change activities of AQP1 and AQP5 promoters in comparison to rat NP cell transfected with control siRNA. (D), YFP expression demonstrates efficient transduction of human NP cells infected with lentivirus encoding both HIF-1α shRNA and YFP. (E), Stable Silencing of HIF-1α by lentiviral Sh-HIF-1α causes strong decrease in basal levels of both AQP 1 and AQP5 mRNA in human NP cells. As expected, HIF-1α mRNA levels were significantly lower in silenced cells compared to cells that received control shRNA. F and G, Western blot (F) and corresponding densitometric analysis (G) shows that HIF-1α silencing in human NP cells results in robust decrease in both AQP 1 and AQP5 protein levels.
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Related In: Results  -  Collection

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Show All Figures
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Figure 6: HIF-1α maintains basal AQP expression in NP cellsA and B, Western blot (A) and corresponding densitometric analysis (B) show that treatment of rat NP cells with DMOG (1mM) has no effect on levels of AQP1 and AQP5, as expected accumulation of HIF-1α protein by DMOG is evident. (C), Suppression of HIF-1α expression by co-transfection with Si-HIF-1α does not significantly change activities of AQP1 and AQP5 promoters in comparison to rat NP cell transfected with control siRNA. (D), YFP expression demonstrates efficient transduction of human NP cells infected with lentivirus encoding both HIF-1α shRNA and YFP. (E), Stable Silencing of HIF-1α by lentiviral Sh-HIF-1α causes strong decrease in basal levels of both AQP 1 and AQP5 mRNA in human NP cells. As expected, HIF-1α mRNA levels were significantly lower in silenced cells compared to cells that received control shRNA. F and G, Western blot (F) and corresponding densitometric analysis (G) shows that HIF-1α silencing in human NP cells results in robust decrease in both AQP 1 and AQP5 protein levels.
Mentions: The effect of HIF-1 on AQP expression in rat NP cells was further investigated using gain- and loss-of-function studies. To block prolyl hydroxylase activity and accumulate HIF-1α protein, cells were treated with 1 mM DMOG. Western blot and corresponding densitometric analysis (Fig. 6A and 6B) show that, while DMOG treatment led to accumulation of HIF-1α protein, expression levels of AQP1 and AQP5 remained unchanged. Next, rat NP cells were co-transfected with the wild-type reporter constructs of either the AQP1 or the AQP5 promoter along with siHIF-1α or a control siRNA. Results clearly showed that suppression of HIF-1 by siHIF-1α did not affect promoter activity of either wild-type AQP promoter construct (Fig. 6C). To assess effect of stable HIF-1 suppression on AQP expression, human NP cells were transduced by lentivirus co-expressing shHIF-1α and YFP. As shown in Fig. 6D, YFP signal indicated that lentiviral delivery resulted in high infection rates and a robust transgene expression in NP cells. As expected, shHIF-1α significantly decreased mRNA expression of HIF-1α compared to sh-control (Fig. 6E-6G). Importantly, silencing of HIF-1α resulted in dramatic decrease in mRNA expression of both AQP1 and AQP5 in human NP cells (Fig. 6E). Moreover, HIF-1α knockdown resulted in significant reduction in protein levels of both AQP1 and AQP5 (Fig. 6G).

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