Limits...
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.

Show MeSH

Related in: MedlinePlus

SIRT1 inhibition represses HIF-1 transcriptional activity and HIF-1α protein.A. Hep3B cells were co-transfected with luciferase reporter carrying multiple HREs and a renilla luciferase control plasmid. Twenty-four hours after the transfection, cells were treated with 25, 50 and 100 µM sirtinol and exposed to hypoxia for 24 hours. Dual luciferase activities were measured and firefly values were normalized by renilla values. Luciferase activity was reduced from 35-fold of DMSO-treated controls to 32-fold with 25 µM sirtinol, p = 0.2611; to 23-fold with 50 µM, *p = 0.0225 and to 10.5-fold with 100 µM ***p = 0.0009. Columns, mean of triplicates from one representative experiment (n = 3, independent experiments) Bars, ±SD. B. Hep3B cells were either treated with 100 µM sirtinol or an equivalent concentration of DMSO for 4 hours and then exposed to 1% O2 for 16 hours. Fold increase was calculated from ΔCt values of hypoxic cells to average ΔCt values of cells cultured at 21% O2 in DMSO. BNIP3 mRNA was reduced from 14±1.5 to 3.4±1-fold (***p = 0.0005), CA9 mRNA from 21±3.5 to 3.8±0.8-fold (**p = 0.0011) and EPO from 100±11.5 to 6.1±1.6-fold (**p = 0.0012) Columns, mean of 3 independent experiments; bars, ±SD. C. Hep3B cells were infected with lentiviral vectors containing shRNA sequences that target SIRT1 (shSIRT1_1958) or with a scrambled control (SHC002). Five days after transduction cells were exposed to hypoxia for 12 hours. The relative fold increase of mRNA in hypoxic cells was calculated compared to normoxic controls. CA9 mRNA reduced from 27- to 5.5-fold (**p = 0.008) and EPO from 120- to 18-fold (**p = 0.004). Columns, mean of 3 independent experiments; bars, ±SD. D. Hep3B cells were treated with 0, 25, 50 and 100 µM sirtinol or an equivalent concentration of DMSO (D) for 16 hours and then exposed to 21% O2 (N) or 1% O2 (H) for 4 hours. Whole cell lysates were analyzed by Western blot using antibodies against HIF-1α. α-tubulin was used as a loading control. A representative blot of 6 independently performed experiments is shown. E. Hep3B cells were infected with lentiviral vectors containing shRNAs targeting SIRT1 (shSIRT1_1958 and shSIRT1_3206) or with a scrambled negative control (SHC002). Five days after transduction, cells were exposed to hypoxia for 4 hours and SIRT1 and HIF-1α were analyzed by Western blot. Representative blot of 3 independently performed experiments. F–G. Hep3B cells were treated with 100 µM sirtinol for 16, 2 and 0 hours before and 2 hours after the exposure to hypoxia for a total of 4 hours. HIF-1α expression was analyzed by Western blot and by RT-qPCR. H. RCC4 VHL+/+ and RCC4 VHL−/− cells were pretreated with 100 µM sirtinol or DMSO for 2 or 16 hours, followed by 4 hours exposure to 21% O2 (N) or 1% O2 (H). Whole cell lysates were analyzed for HIF-1α by Western blot.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3316573&req=5

pone-0033433-g002: SIRT1 inhibition represses HIF-1 transcriptional activity and HIF-1α protein.A. Hep3B cells were co-transfected with luciferase reporter carrying multiple HREs and a renilla luciferase control plasmid. Twenty-four hours after the transfection, cells were treated with 25, 50 and 100 µM sirtinol and exposed to hypoxia for 24 hours. Dual luciferase activities were measured and firefly values were normalized by renilla values. Luciferase activity was reduced from 35-fold of DMSO-treated controls to 32-fold with 25 µM sirtinol, p = 0.2611; to 23-fold with 50 µM, *p = 0.0225 and to 10.5-fold with 100 µM ***p = 0.0009. Columns, mean of triplicates from one representative experiment (n = 3, independent experiments) Bars, ±SD. B. Hep3B cells were either treated with 100 µM sirtinol or an equivalent concentration of DMSO for 4 hours and then exposed to 1% O2 for 16 hours. Fold increase was calculated from ΔCt values of hypoxic cells to average ΔCt values of cells cultured at 21% O2 in DMSO. BNIP3 mRNA was reduced from 14±1.5 to 3.4±1-fold (***p = 0.0005), CA9 mRNA from 21±3.5 to 3.8±0.8-fold (**p = 0.0011) and EPO from 100±11.5 to 6.1±1.6-fold (**p = 0.0012) Columns, mean of 3 independent experiments; bars, ±SD. C. Hep3B cells were infected with lentiviral vectors containing shRNA sequences that target SIRT1 (shSIRT1_1958) or with a scrambled control (SHC002). Five days after transduction cells were exposed to hypoxia for 12 hours. The relative fold increase of mRNA in hypoxic cells was calculated compared to normoxic controls. CA9 mRNA reduced from 27- to 5.5-fold (**p = 0.008) and EPO from 120- to 18-fold (**p = 0.004). Columns, mean of 3 independent experiments; bars, ±SD. D. Hep3B cells were treated with 0, 25, 50 and 100 µM sirtinol or an equivalent concentration of DMSO (D) for 16 hours and then exposed to 21% O2 (N) or 1% O2 (H) for 4 hours. Whole cell lysates were analyzed by Western blot using antibodies against HIF-1α. α-tubulin was used as a loading control. A representative blot of 6 independently performed experiments is shown. E. Hep3B cells were infected with lentiviral vectors containing shRNAs targeting SIRT1 (shSIRT1_1958 and shSIRT1_3206) or with a scrambled negative control (SHC002). Five days after transduction, cells were exposed to hypoxia for 4 hours and SIRT1 and HIF-1α were analyzed by Western blot. Representative blot of 3 independently performed experiments. F–G. Hep3B cells were treated with 100 µM sirtinol for 16, 2 and 0 hours before and 2 hours after the exposure to hypoxia for a total of 4 hours. HIF-1α expression was analyzed by Western blot and by RT-qPCR. H. RCC4 VHL+/+ and RCC4 VHL−/− cells were pretreated with 100 µM sirtinol or DMSO for 2 or 16 hours, followed by 4 hours exposure to 21% O2 (N) or 1% O2 (H). Whole cell lysates were analyzed for HIF-1α by Western blot.

Mentions: As demonstrated above, we observed a simultaneous expression of both SIRT1 and HIF-1α protein under hypoxic conditions. A recent report by Lim et al. has suggested that SIRT1 negatively regulates HIF-1 activity by suppressing the transcriptional regulation of its target genes [27]. Following the model proposed by their data, inhibition of SIRT1 should provoke an exaggerated HIF-mediated response. Therefore, we tested the effect of inhibiting SIRT1 on HIF-1 activity using sirtinol, a cell permeable specific inhibitor of SIRT deacetylase activity [41]. The IC50 value of sirtinol is 70 µM for SIRT1 activity; hence concentrations of 25, 50 and 100 µM were used in this study. First, we confirmed the functionality of sirtinol on SIRT1 deacetylase activity in HepG2 cells. HepG2 cells have abundant SIRT1 protein, as well as an intact p53 response to DNA damage. The acetylation of lysine 382 of p53 is modulated by SIRT1 [21]. We demonstrated that sirtinol had no detectable effect on the lysine 382 acetylation in the absence of DNA damage, whereas, sirtinol produced a time-dependent increase of acetylated p53 in cells treated with the DNA damaging agent, doxorubicin (Figure S1). These data confirm that sirtinol is an efficient inhibitor of SIRT1 activity in vitro. We next investigated the effect of inhibiting SIRT1 activity on HIF-mediated transcriptional activation. Cells were transfected with a HRE reporter construct that contains multiple HRE sites and is specifically induced by HIF proteins and thus represents a direct measurement of HIF activation [42]. Twenty-four hours after transfection, cells were treated with increasing doses of sirtinol and incubated 24 hours at 1% O2. Hypoxia increased reporter activity 38-fold and there was no significant change in cells treated with DMSO at a concentration equal to the amount needed to treat cells with 100 µM sirtinol. There was a dose-dependent decrease of HIF-1 transcriptional activity in cells treated with sirtinol. A significant reduction of reporter activity was observed with 50 µM and 100 µM sirtinol (Figure 2A). We next verified that inhibition of SIRT1 activity alters the transcriptional activity of HIF-1 target genes. Pretreatment with 100 µM sirtinol significantly reduced the hypoxic induction of specific HIF-1 targets BNIP3 and CA9 mRNA as well as EPO mRNA (Figure 2B). Although, sirtinol specifically inhibits SIRT1, it also affects other members of the sirtuin family such as SIRT2 with an IC50 value of 40 µM [41]. Therefore to verify that the effect of sirtinol on repressing HIF-mediated transcriptional activity is at least in part due to the inhibition of SIRT1, cells were infected with lentiviruses carrying shRNA sequences targeting SIRT1. Targeted disruption of SIRT1 with clone shSIRT1_1958 led to a nearly complete knockdown whereas, shSIRT1_3206 resulted in a partial knockdown of SIRT1 protein compared to parental and SHC002 controls (Figure 2E). Cells infected with lentiviruses expressing clone shSIRT1_1958 significantly reduced the hypoxic induction of CA9 and EPO mRNA (Figure 2C). These data demonstrate that inhibition of SIRT1 activity with a small molecule inhibitor and a genetic knockdown leads to a strong decrease of HIF-1-mediated transcriptional activity.


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 inhibition represses HIF-1 transcriptional activity and HIF-1α protein.A. Hep3B cells were co-transfected with luciferase reporter carrying multiple HREs and a renilla luciferase control plasmid. Twenty-four hours after the transfection, cells were treated with 25, 50 and 100 µM sirtinol and exposed to hypoxia for 24 hours. Dual luciferase activities were measured and firefly values were normalized by renilla values. Luciferase activity was reduced from 35-fold of DMSO-treated controls to 32-fold with 25 µM sirtinol, p = 0.2611; to 23-fold with 50 µM, *p = 0.0225 and to 10.5-fold with 100 µM ***p = 0.0009. Columns, mean of triplicates from one representative experiment (n = 3, independent experiments) Bars, ±SD. B. Hep3B cells were either treated with 100 µM sirtinol or an equivalent concentration of DMSO for 4 hours and then exposed to 1% O2 for 16 hours. Fold increase was calculated from ΔCt values of hypoxic cells to average ΔCt values of cells cultured at 21% O2 in DMSO. BNIP3 mRNA was reduced from 14±1.5 to 3.4±1-fold (***p = 0.0005), CA9 mRNA from 21±3.5 to 3.8±0.8-fold (**p = 0.0011) and EPO from 100±11.5 to 6.1±1.6-fold (**p = 0.0012) Columns, mean of 3 independent experiments; bars, ±SD. C. Hep3B cells were infected with lentiviral vectors containing shRNA sequences that target SIRT1 (shSIRT1_1958) or with a scrambled control (SHC002). Five days after transduction cells were exposed to hypoxia for 12 hours. The relative fold increase of mRNA in hypoxic cells was calculated compared to normoxic controls. CA9 mRNA reduced from 27- to 5.5-fold (**p = 0.008) and EPO from 120- to 18-fold (**p = 0.004). Columns, mean of 3 independent experiments; bars, ±SD. D. Hep3B cells were treated with 0, 25, 50 and 100 µM sirtinol or an equivalent concentration of DMSO (D) for 16 hours and then exposed to 21% O2 (N) or 1% O2 (H) for 4 hours. Whole cell lysates were analyzed by Western blot using antibodies against HIF-1α. α-tubulin was used as a loading control. A representative blot of 6 independently performed experiments is shown. E. Hep3B cells were infected with lentiviral vectors containing shRNAs targeting SIRT1 (shSIRT1_1958 and shSIRT1_3206) or with a scrambled negative control (SHC002). Five days after transduction, cells were exposed to hypoxia for 4 hours and SIRT1 and HIF-1α were analyzed by Western blot. Representative blot of 3 independently performed experiments. F–G. Hep3B cells were treated with 100 µM sirtinol for 16, 2 and 0 hours before and 2 hours after the exposure to hypoxia for a total of 4 hours. HIF-1α expression was analyzed by Western blot and by RT-qPCR. H. RCC4 VHL+/+ and RCC4 VHL−/− cells were pretreated with 100 µM sirtinol or DMSO for 2 or 16 hours, followed by 4 hours exposure to 21% O2 (N) or 1% O2 (H). Whole cell lysates were analyzed for HIF-1α by Western blot.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316573&req=5

pone-0033433-g002: SIRT1 inhibition represses HIF-1 transcriptional activity and HIF-1α protein.A. Hep3B cells were co-transfected with luciferase reporter carrying multiple HREs and a renilla luciferase control plasmid. Twenty-four hours after the transfection, cells were treated with 25, 50 and 100 µM sirtinol and exposed to hypoxia for 24 hours. Dual luciferase activities were measured and firefly values were normalized by renilla values. Luciferase activity was reduced from 35-fold of DMSO-treated controls to 32-fold with 25 µM sirtinol, p = 0.2611; to 23-fold with 50 µM, *p = 0.0225 and to 10.5-fold with 100 µM ***p = 0.0009. Columns, mean of triplicates from one representative experiment (n = 3, independent experiments) Bars, ±SD. B. Hep3B cells were either treated with 100 µM sirtinol or an equivalent concentration of DMSO for 4 hours and then exposed to 1% O2 for 16 hours. Fold increase was calculated from ΔCt values of hypoxic cells to average ΔCt values of cells cultured at 21% O2 in DMSO. BNIP3 mRNA was reduced from 14±1.5 to 3.4±1-fold (***p = 0.0005), CA9 mRNA from 21±3.5 to 3.8±0.8-fold (**p = 0.0011) and EPO from 100±11.5 to 6.1±1.6-fold (**p = 0.0012) Columns, mean of 3 independent experiments; bars, ±SD. C. Hep3B cells were infected with lentiviral vectors containing shRNA sequences that target SIRT1 (shSIRT1_1958) or with a scrambled control (SHC002). Five days after transduction cells were exposed to hypoxia for 12 hours. The relative fold increase of mRNA in hypoxic cells was calculated compared to normoxic controls. CA9 mRNA reduced from 27- to 5.5-fold (**p = 0.008) and EPO from 120- to 18-fold (**p = 0.004). Columns, mean of 3 independent experiments; bars, ±SD. D. Hep3B cells were treated with 0, 25, 50 and 100 µM sirtinol or an equivalent concentration of DMSO (D) for 16 hours and then exposed to 21% O2 (N) or 1% O2 (H) for 4 hours. Whole cell lysates were analyzed by Western blot using antibodies against HIF-1α. α-tubulin was used as a loading control. A representative blot of 6 independently performed experiments is shown. E. Hep3B cells were infected with lentiviral vectors containing shRNAs targeting SIRT1 (shSIRT1_1958 and shSIRT1_3206) or with a scrambled negative control (SHC002). Five days after transduction, cells were exposed to hypoxia for 4 hours and SIRT1 and HIF-1α were analyzed by Western blot. Representative blot of 3 independently performed experiments. F–G. Hep3B cells were treated with 100 µM sirtinol for 16, 2 and 0 hours before and 2 hours after the exposure to hypoxia for a total of 4 hours. HIF-1α expression was analyzed by Western blot and by RT-qPCR. H. RCC4 VHL+/+ and RCC4 VHL−/− cells were pretreated with 100 µM sirtinol or DMSO for 2 or 16 hours, followed by 4 hours exposure to 21% O2 (N) or 1% O2 (H). Whole cell lysates were analyzed for HIF-1α by Western blot.
Mentions: As demonstrated above, we observed a simultaneous expression of both SIRT1 and HIF-1α protein under hypoxic conditions. A recent report by Lim et al. has suggested that SIRT1 negatively regulates HIF-1 activity by suppressing the transcriptional regulation of its target genes [27]. Following the model proposed by their data, inhibition of SIRT1 should provoke an exaggerated HIF-mediated response. Therefore, we tested the effect of inhibiting SIRT1 on HIF-1 activity using sirtinol, a cell permeable specific inhibitor of SIRT deacetylase activity [41]. The IC50 value of sirtinol is 70 µM for SIRT1 activity; hence concentrations of 25, 50 and 100 µM were used in this study. First, we confirmed the functionality of sirtinol on SIRT1 deacetylase activity in HepG2 cells. HepG2 cells have abundant SIRT1 protein, as well as an intact p53 response to DNA damage. The acetylation of lysine 382 of p53 is modulated by SIRT1 [21]. We demonstrated that sirtinol had no detectable effect on the lysine 382 acetylation in the absence of DNA damage, whereas, sirtinol produced a time-dependent increase of acetylated p53 in cells treated with the DNA damaging agent, doxorubicin (Figure S1). These data confirm that sirtinol is an efficient inhibitor of SIRT1 activity in vitro. We next investigated the effect of inhibiting SIRT1 activity on HIF-mediated transcriptional activation. Cells were transfected with a HRE reporter construct that contains multiple HRE sites and is specifically induced by HIF proteins and thus represents a direct measurement of HIF activation [42]. Twenty-four hours after transfection, cells were treated with increasing doses of sirtinol and incubated 24 hours at 1% O2. Hypoxia increased reporter activity 38-fold and there was no significant change in cells treated with DMSO at a concentration equal to the amount needed to treat cells with 100 µM sirtinol. There was a dose-dependent decrease of HIF-1 transcriptional activity in cells treated with sirtinol. A significant reduction of reporter activity was observed with 50 µM and 100 µM sirtinol (Figure 2A). We next verified that inhibition of SIRT1 activity alters the transcriptional activity of HIF-1 target genes. Pretreatment with 100 µM sirtinol significantly reduced the hypoxic induction of specific HIF-1 targets BNIP3 and CA9 mRNA as well as EPO mRNA (Figure 2B). Although, sirtinol specifically inhibits SIRT1, it also affects other members of the sirtuin family such as SIRT2 with an IC50 value of 40 µM [41]. Therefore to verify that the effect of sirtinol on repressing HIF-mediated transcriptional activity is at least in part due to the inhibition of SIRT1, cells were infected with lentiviruses carrying shRNA sequences targeting SIRT1. Targeted disruption of SIRT1 with clone shSIRT1_1958 led to a nearly complete knockdown whereas, shSIRT1_3206 resulted in a partial knockdown of SIRT1 protein compared to parental and SHC002 controls (Figure 2E). Cells infected with lentiviruses expressing clone shSIRT1_1958 significantly reduced the hypoxic induction of CA9 and EPO mRNA (Figure 2C). These data demonstrate that inhibition of SIRT1 activity with a small molecule inhibitor and a genetic knockdown leads to a strong decrease of HIF-1-mediated transcriptional activity.

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