Inhibition of SIRT1 Impairs the Accumulation and Transcriptional Activity of HIF-1α Protein under Hypoxic Conditions

Alexander Laemmle,Antje Lechleiter,Stephan A Vorburger,Vincent Roh,Adrian Keogh,Deborah Stroka,Cynthia Furer,Mario P Tschan,Daniel Candinas,Christa Schwarz,Simone Portmann

doi:10.1371/journal.pone.0033433

Alexander Laemmle, Antje Lechleiter + Show 9 more

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https://doi.org/10.1371/journal.pone.0033433

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Journal: PLoS ONE	Publication Date: Mar 30, 2012
Citations: 194	License type: CC BY 4.0

Affiliation: University Hospital of Bern, University of Bern

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.

Highlights

Silent information regulator 2 (Sir2) was initially identified in Saccharomyces cerevisiae as the first member of the highly conserved sirtuin family of proteins [1]
SIRT1 protein was strongly expressed in Hep3B, HepG2 and Huh7 hepatocellular carcinoma (HCC) cells lines in normal culture conditions as well as in cells incubated at 1% O2 (Figure 1A)
Stabilization of hypoxia-inducible transcription factors (HIF)-1a protein led to the transcriptional increase of HIF-1 target genes BNIP3, carbonic anhydrase 9 (CA9) as well as EPO, a target gene shared with HIF-2 (Figure 1B)

Summary

Introduction

Silent information regulator 2 (Sir2) was initially identified in Saccharomyces cerevisiae as the first member of the highly conserved sirtuin family of proteins [1]. Sirtuin proteins are nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases. Their dependency on NAD+ suggests that sirtuin activity serves as a sensor of the cytosolic ratio of NAD+/NADH and directly links sirtuin activity to the metabolic and cellular energy state of a cell [2,3]. SIRT proteins have different subcellular localizations and described functions. SIRT1 regulate pathways in metabolism, inflammation and tumorigenesis and SIRT2 functions as a tubulin deacetylase [5]. SIRT3 and SIRT5 are localized in mitochondria and regulate metabolism and ammonia detoxification, respectively [6,7]. SIRT7 interacts with RNA polymerase I histones to promote Pol I-mediated rRNA transcription in the nucleolus [12]

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