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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 co-immunoprecipitate.A. Hep3B cells were treated with 125 µM DMOG for 5 hours. Cytoplasmic and nuclear protein fractions were prepared. Twenty-five µg nuclear proteins and 50 µg cytoplasmic proteins were analyzed for SIRT1 and HIF-1α by Western blot. Sp1 was used as a nuclear fraction marker and α-tubulin for the cytoplasmic fraction. A representative blot of 3 independently performed experiments is shown. B. Cytoplasmic proteins (1250 µg) prepared in (A) were immunoprecipitated with a rabbit polyclonal SIRT1 antibody, chicken polyclonal HIF-1α antibody or a rabbit IgG1 control antibody. Immunoprecipitated proteins were subjected to Western blot analysis. A representative blot of 3 independently performed experiments is shown. C. SIRT1 inhibition leads to increased acetylation of HIF-1α. Hep3B cells were infected with lentiviral vectors containing shRNA against SIRT1 (shSIRT1_1958 or shSIRT1_3206) or scrambled shRNA (SHC002) as a negative control. Seven days post-infection, cells were incubated under hypoxia for 5 hours in the presence of 10 µM MG132. Whole cell lysates (2 mg) were immunoprecipitated with a mouse monoclonal HIF-1α antibody (2 µg) and precipitates were analyzed by Western blotting with a rabbit polyclonal anti-acetyl-lysine and a polyclonal chicken anti-HIF-1α antibody. Whole cell lysates (WCL: 100 µg) were used as input controls and were analyzed for SIRT1 and α-tubulin. A representative blot of 2 independently performed experiments is shown.
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pone-0033433-g004: SIRT1 and HIF-1α co-immunoprecipitate.A. Hep3B cells were treated with 125 µM DMOG for 5 hours. Cytoplasmic and nuclear protein fractions were prepared. Twenty-five µg nuclear proteins and 50 µg cytoplasmic proteins were analyzed for SIRT1 and HIF-1α by Western blot. Sp1 was used as a nuclear fraction marker and α-tubulin for the cytoplasmic fraction. A representative blot of 3 independently performed experiments is shown. B. Cytoplasmic proteins (1250 µg) prepared in (A) were immunoprecipitated with a rabbit polyclonal SIRT1 antibody, chicken polyclonal HIF-1α antibody or a rabbit IgG1 control antibody. Immunoprecipitated proteins were subjected to Western blot analysis. A representative blot of 3 independently performed experiments is shown. C. SIRT1 inhibition leads to increased acetylation of HIF-1α. Hep3B cells were infected with lentiviral vectors containing shRNA against SIRT1 (shSIRT1_1958 or shSIRT1_3206) or scrambled shRNA (SHC002) as a negative control. Seven days post-infection, cells were incubated under hypoxia for 5 hours in the presence of 10 µM MG132. Whole cell lysates (2 mg) were immunoprecipitated with a mouse monoclonal HIF-1α antibody (2 µg) and precipitates were analyzed by Western blotting with a rabbit polyclonal anti-acetyl-lysine and a polyclonal chicken anti-HIF-1α antibody. Whole cell lysates (WCL: 100 µg) were used as input controls and were analyzed for SIRT1 and α-tubulin. A representative blot of 2 independently performed experiments is shown.

Mentions: We next questioned whether HIF-1α is a target of SIRT1 deacetylase activity. There is contradictory literature on whether there is a direct interaction between HIF-1α and SIRT1 [27], [28]. Therefore, we tested if endogenous proteins co-immunoprecipitate in the cells used in this study. Hep3B cells were treated with DMOG for 5 hours to stabilize HIF-1α under normoxic conditions. SIRT1 protein was detected in the cytoplasmic and nuclear fraction. A strong HIF-1α signal was found in the nuclear fraction, and a lower signal was detected in the cytoplasmic fraction (Figure 4A). Using endogenously expressed cytoplasmic proteins, we observed that HIF-1α immunoprecipitated with extracts for SIRT1, and likewise, SIRT1 is detected in extracts immunoprecipitated for HIF-1α (Figure 4B). In summary, our data are in agreement with others, and suggest that SIRT1 and HIF-1α proteins physically interact [27], [39]. From this data we hypothesize that SIRT1 may exert its effects on HIF-1α protein stability through a physical interaction.


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 co-immunoprecipitate.A. Hep3B cells were treated with 125 µM DMOG for 5 hours. Cytoplasmic and nuclear protein fractions were prepared. Twenty-five µg nuclear proteins and 50 µg cytoplasmic proteins were analyzed for SIRT1 and HIF-1α by Western blot. Sp1 was used as a nuclear fraction marker and α-tubulin for the cytoplasmic fraction. A representative blot of 3 independently performed experiments is shown. B. Cytoplasmic proteins (1250 µg) prepared in (A) were immunoprecipitated with a rabbit polyclonal SIRT1 antibody, chicken polyclonal HIF-1α antibody or a rabbit IgG1 control antibody. Immunoprecipitated proteins were subjected to Western blot analysis. A representative blot of 3 independently performed experiments is shown. C. SIRT1 inhibition leads to increased acetylation of HIF-1α. Hep3B cells were infected with lentiviral vectors containing shRNA against SIRT1 (shSIRT1_1958 or shSIRT1_3206) or scrambled shRNA (SHC002) as a negative control. Seven days post-infection, cells were incubated under hypoxia for 5 hours in the presence of 10 µM MG132. Whole cell lysates (2 mg) were immunoprecipitated with a mouse monoclonal HIF-1α antibody (2 µg) and precipitates were analyzed by Western blotting with a rabbit polyclonal anti-acetyl-lysine and a polyclonal chicken anti-HIF-1α antibody. Whole cell lysates (WCL: 100 µg) were used as input controls and were analyzed for SIRT1 and α-tubulin. A representative blot of 2 independently performed experiments is shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316573&req=5

pone-0033433-g004: SIRT1 and HIF-1α co-immunoprecipitate.A. Hep3B cells were treated with 125 µM DMOG for 5 hours. Cytoplasmic and nuclear protein fractions were prepared. Twenty-five µg nuclear proteins and 50 µg cytoplasmic proteins were analyzed for SIRT1 and HIF-1α by Western blot. Sp1 was used as a nuclear fraction marker and α-tubulin for the cytoplasmic fraction. A representative blot of 3 independently performed experiments is shown. B. Cytoplasmic proteins (1250 µg) prepared in (A) were immunoprecipitated with a rabbit polyclonal SIRT1 antibody, chicken polyclonal HIF-1α antibody or a rabbit IgG1 control antibody. Immunoprecipitated proteins were subjected to Western blot analysis. A representative blot of 3 independently performed experiments is shown. C. SIRT1 inhibition leads to increased acetylation of HIF-1α. Hep3B cells were infected with lentiviral vectors containing shRNA against SIRT1 (shSIRT1_1958 or shSIRT1_3206) or scrambled shRNA (SHC002) as a negative control. Seven days post-infection, cells were incubated under hypoxia for 5 hours in the presence of 10 µM MG132. Whole cell lysates (2 mg) were immunoprecipitated with a mouse monoclonal HIF-1α antibody (2 µg) and precipitates were analyzed by Western blotting with a rabbit polyclonal anti-acetyl-lysine and a polyclonal chicken anti-HIF-1α antibody. Whole cell lysates (WCL: 100 µg) were used as input controls and were analyzed for SIRT1 and α-tubulin. A representative blot of 2 independently performed experiments is shown.
Mentions: We next questioned whether HIF-1α is a target of SIRT1 deacetylase activity. There is contradictory literature on whether there is a direct interaction between HIF-1α and SIRT1 [27], [28]. Therefore, we tested if endogenous proteins co-immunoprecipitate in the cells used in this study. Hep3B cells were treated with DMOG for 5 hours to stabilize HIF-1α under normoxic conditions. SIRT1 protein was detected in the cytoplasmic and nuclear fraction. A strong HIF-1α signal was found in the nuclear fraction, and a lower signal was detected in the cytoplasmic fraction (Figure 4A). Using endogenously expressed cytoplasmic proteins, we observed that HIF-1α immunoprecipitated with extracts for SIRT1, and likewise, SIRT1 is detected in extracts immunoprecipitated for HIF-1α (Figure 4B). In summary, our data are in agreement with others, and suggest that SIRT1 and HIF-1α proteins physically interact [27], [39]. From this data we hypothesize that SIRT1 may exert its effects on HIF-1α protein stability through a physical interaction.

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