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Inhibition of SIRT1 impairs the accumulation and transcriptional activity of HIF-1α protein under hypoxic conditions.

Laemmle A, Lechleiter A, Roh V, Schwarz C, Portmann S, Furer C, Keogh A, Tschan MP, Candinas D, Vorburger SA, Stroka D - PLoS ONE (2012)

Bottom Line: Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2.Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes.In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α.

View Article: PubMed Central - PubMed

Affiliation: Clinic of Visceral Surgery and Medicine, Visceral and Transplantation Surgery, University Hospital Bern and University of Bern, Bern, Switzerland.

ABSTRACT
Sirtuins and hypoxia-inducible transcription factors (HIF) have well-established roles in regulating cellular responses to metabolic and oxidative stress. Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2. Here we investigated the role of SIRT1, a NAD+-dependent deacetylase, in the regulation of HIF-1 activity in hypoxic conditions. Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes. In hypoxic models in vitro and in in vivo models of systemic hypoxia and xenograft tumor growth, knockdown of SIRT1 protein with shRNA or inhibition of its activity with small molecule inhibitors impaired the accumulation of HIF-1α protein and the transcriptional increase of its target genes. In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α. Taken together, our data suggest that HIF-1α and SIRT1 proteins interact in HCC cells and that HIF-1α is a target of SIRT1 deacetylase activity. Moreover, SIRT1 is necessary for HIF-1α protein accumulation and activation of HIF-1 target genes under hypoxic conditions.

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SIRT1 and HIF-1α are simultaneously expressed in hypoxic cells.A. Hep3B, HepG2 and Huh-7 cells were exposed to (N) normoxia (21% O2) or (H) hypoxia (1% O2) for 8 hours. SIRT1 and HIF-1α proteins were detected by Western blot. An antibody against Sp1 was used as a control for equal loading. A representative blot of 3 independently performed experiments is shown. B. Total RNA was isolated from Hep3B cells exposed to 1% O2 for 16 hours. RT-qPCR was performed and relative fold increase of target gene mRNA was calculated by comparing ΔCt values of cells cultured at 21% O2 to ΔCt values of cells exposed to 1% O2. Relative mRNA was increased 10.7±5-fold for BNIP3, 21.8±4-fold for CA9 and 101±17-fold. Columns, mean values from 3 independent experiments. Bars, ±SD. C. Hep3B and HepG2 cells were exposed to (N) normoxia (21% O2) or (H) hypoxia (1% O2) for 12, 24 and 48 hours. SIRT1 and α-tubulin proteins were detected in total cell lysates by Western blot. A representative blot of 6 independently performed experiments is shown. D. Total RNA was isolated from Hep3B cells exposed to 1% O2 for 12, 24 and 48 hours and ΔCt values of SIRT1 mRNA was compared to cells cultured at 21% O2 by RT-qPCR. Columns, mean values from 6 independent experiments. Bars, ±SD. E. Total RNA was isolated from Hep3B cells exposed to 1% O2 and 0.1% O2 for 24 hours and ΔCt values of SIRT1 mRNA was compared to cells cultured at 21% O2 by RT-qPCR. Columns, mean values from 6 independent experiments. Bars, ±SD. 1%O2.
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pone-0033433-g001: SIRT1 and HIF-1α are simultaneously expressed in hypoxic cells.A. Hep3B, HepG2 and Huh-7 cells were exposed to (N) normoxia (21% O2) or (H) hypoxia (1% O2) for 8 hours. SIRT1 and HIF-1α proteins were detected by Western blot. An antibody against Sp1 was used as a control for equal loading. A representative blot of 3 independently performed experiments is shown. B. Total RNA was isolated from Hep3B cells exposed to 1% O2 for 16 hours. RT-qPCR was performed and relative fold increase of target gene mRNA was calculated by comparing ΔCt values of cells cultured at 21% O2 to ΔCt values of cells exposed to 1% O2. Relative mRNA was increased 10.7±5-fold for BNIP3, 21.8±4-fold for CA9 and 101±17-fold. Columns, mean values from 3 independent experiments. Bars, ±SD. C. Hep3B and HepG2 cells were exposed to (N) normoxia (21% O2) or (H) hypoxia (1% O2) for 12, 24 and 48 hours. SIRT1 and α-tubulin proteins were detected in total cell lysates by Western blot. A representative blot of 6 independently performed experiments is shown. D. Total RNA was isolated from Hep3B cells exposed to 1% O2 for 12, 24 and 48 hours and ΔCt values of SIRT1 mRNA was compared to cells cultured at 21% O2 by RT-qPCR. Columns, mean values from 6 independent experiments. Bars, ±SD. E. Total RNA was isolated from Hep3B cells exposed to 1% O2 and 0.1% O2 for 24 hours and ΔCt values of SIRT1 mRNA was compared to cells cultured at 21% O2 by RT-qPCR. Columns, mean values from 6 independent experiments. Bars, ±SD. 1%O2.

Mentions: HIF-1 is tightly regulated by oxygen availability and functions predominantly in hypoxic conditions. In order to demonstrate that SIRT1 is necessary for the accumulation of HIF-1α protein, we first verified that SIRT1 and HIF-1α proteins are co-expressed in hypoxic conditions. SIRT1 protein was strongly expressed in Hep3B, HepG2 and Huh7 HCC cells lines in normal culture conditions as well as in cells incubated at 1% O2 (Figure 1A). As predicted, HIF-1α protein was not detected in cells cultured at 21% O2 and was stabilized in cells incubated at 1% O2 (Figure 1A). Stabilization of HIF-1α protein led to the transcriptional increase of HIF-1 target genes BNIP3, CA9 as well as EPO, a target gene shared with HIF-2 (Figure 1B). To further verify that SIRT1 protein is not altered by hypoxia, HCC cells were exposed to 1% O2 for up to 48 hours. There was no change in SIRT1 protein or mRNA in cells exposed for 12, 24 or 48 hours to 1% O2 (Figure 1C & D). In addition, there was no significant change in SIRT1 mRNA in Hep3B cells exposed to more severe hypoxia of 0.1% O2 for 24 hours (Figure 1E). These data show that both SIRT1 and HIF-1α are present in cells under hypoxic conditions and HIF-1 is transcriptionally active. Moreover, unlike HIF-1α, SIRT1 expression is not tightly regulated by hypoxia in HCC cells.


Inhibition of SIRT1 impairs the accumulation and transcriptional activity of HIF-1α protein under hypoxic conditions.

Laemmle A, Lechleiter A, Roh V, Schwarz C, Portmann S, Furer C, Keogh A, Tschan MP, Candinas D, Vorburger SA, Stroka D - PLoS ONE (2012)

SIRT1 and HIF-1α are simultaneously expressed in hypoxic cells.A. Hep3B, HepG2 and Huh-7 cells were exposed to (N) normoxia (21% O2) or (H) hypoxia (1% O2) for 8 hours. SIRT1 and HIF-1α proteins were detected by Western blot. An antibody against Sp1 was used as a control for equal loading. A representative blot of 3 independently performed experiments is shown. B. Total RNA was isolated from Hep3B cells exposed to 1% O2 for 16 hours. RT-qPCR was performed and relative fold increase of target gene mRNA was calculated by comparing ΔCt values of cells cultured at 21% O2 to ΔCt values of cells exposed to 1% O2. Relative mRNA was increased 10.7±5-fold for BNIP3, 21.8±4-fold for CA9 and 101±17-fold. Columns, mean values from 3 independent experiments. Bars, ±SD. C. Hep3B and HepG2 cells were exposed to (N) normoxia (21% O2) or (H) hypoxia (1% O2) for 12, 24 and 48 hours. SIRT1 and α-tubulin proteins were detected in total cell lysates by Western blot. A representative blot of 6 independently performed experiments is shown. D. Total RNA was isolated from Hep3B cells exposed to 1% O2 for 12, 24 and 48 hours and ΔCt values of SIRT1 mRNA was compared to cells cultured at 21% O2 by RT-qPCR. Columns, mean values from 6 independent experiments. Bars, ±SD. E. Total RNA was isolated from Hep3B cells exposed to 1% O2 and 0.1% O2 for 24 hours and ΔCt values of SIRT1 mRNA was compared to cells cultured at 21% O2 by RT-qPCR. Columns, mean values from 6 independent experiments. Bars, ±SD. 1%O2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316573&req=5

pone-0033433-g001: SIRT1 and HIF-1α are simultaneously expressed in hypoxic cells.A. Hep3B, HepG2 and Huh-7 cells were exposed to (N) normoxia (21% O2) or (H) hypoxia (1% O2) for 8 hours. SIRT1 and HIF-1α proteins were detected by Western blot. An antibody against Sp1 was used as a control for equal loading. A representative blot of 3 independently performed experiments is shown. B. Total RNA was isolated from Hep3B cells exposed to 1% O2 for 16 hours. RT-qPCR was performed and relative fold increase of target gene mRNA was calculated by comparing ΔCt values of cells cultured at 21% O2 to ΔCt values of cells exposed to 1% O2. Relative mRNA was increased 10.7±5-fold for BNIP3, 21.8±4-fold for CA9 and 101±17-fold. Columns, mean values from 3 independent experiments. Bars, ±SD. C. Hep3B and HepG2 cells were exposed to (N) normoxia (21% O2) or (H) hypoxia (1% O2) for 12, 24 and 48 hours. SIRT1 and α-tubulin proteins were detected in total cell lysates by Western blot. A representative blot of 6 independently performed experiments is shown. D. Total RNA was isolated from Hep3B cells exposed to 1% O2 for 12, 24 and 48 hours and ΔCt values of SIRT1 mRNA was compared to cells cultured at 21% O2 by RT-qPCR. Columns, mean values from 6 independent experiments. Bars, ±SD. E. Total RNA was isolated from Hep3B cells exposed to 1% O2 and 0.1% O2 for 24 hours and ΔCt values of SIRT1 mRNA was compared to cells cultured at 21% O2 by RT-qPCR. Columns, mean values from 6 independent experiments. Bars, ±SD. 1%O2.
Mentions: HIF-1 is tightly regulated by oxygen availability and functions predominantly in hypoxic conditions. In order to demonstrate that SIRT1 is necessary for the accumulation of HIF-1α protein, we first verified that SIRT1 and HIF-1α proteins are co-expressed in hypoxic conditions. SIRT1 protein was strongly expressed in Hep3B, HepG2 and Huh7 HCC cells lines in normal culture conditions as well as in cells incubated at 1% O2 (Figure 1A). As predicted, HIF-1α protein was not detected in cells cultured at 21% O2 and was stabilized in cells incubated at 1% O2 (Figure 1A). Stabilization of HIF-1α protein led to the transcriptional increase of HIF-1 target genes BNIP3, CA9 as well as EPO, a target gene shared with HIF-2 (Figure 1B). To further verify that SIRT1 protein is not altered by hypoxia, HCC cells were exposed to 1% O2 for up to 48 hours. There was no change in SIRT1 protein or mRNA in cells exposed for 12, 24 or 48 hours to 1% O2 (Figure 1C & D). In addition, there was no significant change in SIRT1 mRNA in Hep3B cells exposed to more severe hypoxia of 0.1% O2 for 24 hours (Figure 1E). These data show that both SIRT1 and HIF-1α are present in cells under hypoxic conditions and HIF-1 is transcriptionally active. Moreover, unlike HIF-1α, SIRT1 expression is not tightly regulated by hypoxia in HCC cells.

Bottom Line: Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2.Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes.In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α.

View Article: PubMed Central - PubMed

Affiliation: Clinic of Visceral Surgery and Medicine, Visceral and Transplantation Surgery, University Hospital Bern and University of Bern, Bern, Switzerland.

ABSTRACT
Sirtuins and hypoxia-inducible transcription factors (HIF) have well-established roles in regulating cellular responses to metabolic and oxidative stress. Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2. Here we investigated the role of SIRT1, a NAD+-dependent deacetylase, in the regulation of HIF-1 activity in hypoxic conditions. Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes. In hypoxic models in vitro and in in vivo models of systemic hypoxia and xenograft tumor growth, knockdown of SIRT1 protein with shRNA or inhibition of its activity with small molecule inhibitors impaired the accumulation of HIF-1α protein and the transcriptional increase of its target genes. In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α. Taken together, our data suggest that HIF-1α and SIRT1 proteins interact in HCC cells and that HIF-1α is a target of SIRT1 deacetylase activity. Moreover, SIRT1 is necessary for HIF-1α protein accumulation and activation of HIF-1 target genes under hypoxic conditions.

Show MeSH
Related in: MedlinePlus