FEBS Letters 584 (2010) 4366–4372 journal homepage: www.FEBSLetters.org Hypoxia-inducible factor 1-mediated regulation of PPP1R3C promotes glycogen accumulation in human MCF-7 cells under hypoxia ⇑ Guo-Min Shen, Feng-Lin Zhang, Xiao-Ling Liu, Jun-Wu Zhang National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China article info abstract Article history: Hundreds of genes can be regulated by hypoxia-inducible factor 1 (HIF1) under hypoxia. Here we Received 30 July 2010 demonstrated a HIF1-mediated induction of protein phosphatase 1, regulatory subunit 3C gene Revised 10 September 2010 (PPP1R3C) in human MCF7 cells under hypoxia. By mutation analysis we confirmed the presence Accepted 24 September 2010 of a functional hypoxia response element that is located 229 bp upstream from the PPP1R3C gene. Available online 1 October 2010 PPP1R3C induction correlates with a significant glycogen accumulation in MCF7 cells under hypoxia. Edited by Judit Ovádi Knockdown of either HIF1a or PPP1R3C attenuated hypoxia-induced glycogen accumulation signif- icantly. Knockdown of HIF2a reduced hypoxia-induced glycogen accumulation slightly (but not sig- nificantly). Our results demonstrated that HIF1 promotes glycogen accumulation through regulating Keywords: Hypoxia-inducible factor PPP1R3C expression under hypoxia, which revealed a novel metabolic adaptation of cells to hypoxia. Protein phosphatase 1, regulatory subunit Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 3C Glycogen accumulation Hypoxia Human MCF7 cell 1. Introduction PHDs), which uses O2 and a-ketoglutarate as substrates in a reac- tion that generates CO2 and succinate as byproducts [5–8]. Pro- The response to hypoxia is certainly the most well-understood lylhydroxylated HIFa is bound by the von Hippel–Lindau tumor aspect of hypoxia-inducible factor (HIF)’s ability to regulate metab- suppressor protein, which recruits an E3-ubiquitin ligase that tar- olism [1]. HIF is a heterodimeric protein that is composed of a con- gets HIFa for proteasomal degradation. In addition, a specific aspa- stitutively expressed beta subunit (HIFb, also called Aryl receptor raginyl residue in the transactivation domain is hydroxylated in nuclear translocator, ARNT) and an O2-regulated alpha subunit well-oxygenated cells by factor inhibiting HIF (FIH), which blocks (HIFa) [2,3]. There are three HIFa proteins in higher metazoans. the binding of the coactivators p300 and CCAAT-binding protein HIF1a and HIF2a have been the most extensively studied. They (CBP) [9–11]. Under hypoxic conditions, hydroxylation is inhibited have a similar domain architecture, bind to the same core motif and HIFa rapidly accumulates, dimerizes with HIFb, binds to the and also are regulated in a similar manner. HIF3a is an inhibitor core DNA binding sequence 50-RCGTG-30 (R is purine A or G) in tar- of HIF1 that may be involved in feedback regulation because its get genes, recruits co-activators and activates transcription. expression is transcriptionally regulated by HIF1 [4]. In well-oxy- The hundreds of genes can be induced by HIF1 under hypoxia. genated cells, HIFa is hydroxylated at one (or both) of two highly The largest functional group of genes consistently regulated by conserved prolyl residues by a family of dioxygenases (EGL nine HIF1 in a number of cell types are associated with glucose metab- homologs, EGLNs, also called prolyl hydroxylase domain proteins, olism [1]. The glucose metabolism in cells under hypoxia is signif- icantly different from that under normoxia. Glucose uptake significantly increases under hypoxia, primarily in a HIF-depen- Abbreviations: HRE, hypoxia response element; HIF, hypoxia-inducible factor; PPP1R3C, protein phosphatase 1, regulatory subunit 3C; DFOM, deferoxamine dent manner. HIF1 induces the transcription of solute carrier fam- mesylate salt; PHD2, prolyl hydroxylase domain protein 2; VHL, von Hippel–Lindau ily 2, member 1 (SLC2A1) and SLC2A3, which encode the glucose tumor suppressor protein; PTG, protein targeting to glycogen; GYS1, glycogen transporter 1 (GLUT1) and GLUT3, respectively [12,13]. HIF1 can synthase 1 ⇑ also activate the transcription of hexokinase gene 1 (HK1) and Corresponding author at: National Laboratory of Medical Molecular Biology, HK2 [14]. Intracellular glucose is quickly phosphorylated to glu- Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China. Fax: +86 10 65240529. cose-6-phosphate by hexokinase. The charged glucose molecule E-mail address: [email protected] (J.-W. Zhang). cannot easily pass back through the plasma membrane and is 0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.09.040 G.-M. Shen et al. / FEBS Letters 584 (2010) 4366–4372 4367 trapped within the cell. Glucose-6-phosphate has several possible assessed using the Dual-Luciferase Reporter Assay System metabolic fates, such as, as a structural component in the synthesis (Promega). of glycoproteins, metabolized in the pentose shunt to generate ri- bose, used to synthesize glycogen or, predominantly, broken down 2.5. RNA isolation and PCR analysis to pyruvate in glycolysis [1]. Although mechanism of regulating glycolysis under hypoxia has Cells were dissolved in TRIZOL reagent (Invitrogen) and total been well defined, the effect of hypoxia on other glucose metabolic RNA was extracted according to the manufacturer’s instructions. fates is yet to be addressed. Now, some reports have described the Total RNA was converted into cDNA using the M-MLV reverse- glycogen accumulation in cells under hypoxia conditions [15–18]. transcriptional system (Invitrogen) in the presence of oligo(dT)18. Pescador et al. demonstrated that hypoxia promotes glycogen The cDNA was used for conventional PCR or quantitative real-time accumulation through HIF1-mediated induction of glycogen syn- PCR (qrt-PCR) with specific gene primers: PPP1R3C forward: thase 1 [18]. They also showed that hypoxia upregulates the 50-GCGTTGTGTTTGCTGACTCC, reverse: 50-CGGTTGAAGGCTGAGG- expression of UTP: glucose-1-phosphate urydylyltransferase GAAAT-30; b-actin forward: 50-CTGGCACCACACCTTCTACA-30, reverse: (UGP2) and 1,4-a glucan branching enzyme (GBE1), in a cell-type 50-AGCACAGCCTGGATAGCAAC-30;HIF1a forward: 50-AGGTGGATAT specific manner [18]. Does hypoxia induce other genes that pro- GTCTGGGTTG-30, reverse: 50-AAGGACACATTCTGTTT GTTG-30;HIF2a mote glycogen accumulation? Some reports showed that protein forward: 50-GTCTCTCCACCCCATGTCTC-30, reverse: 50-GGTTCTTCAT phosphatase 1, regulatory subunit 3C (PPP1R3C) could be induced CCGTTTCCAC-30. SYBR green PCR mix (Transgen) was used for the by hypoxia [19,20], and Ortiz-Barahona et al. predicted that real-time PCR. The relative abundance of PPP1R3C transcript was PPP1R3C is a new HIF-targeted gene [21]. PPP1R3C was also called quantified using the comparative Ct method with b-actin as an protein targeting to glycogen (PTG) and regulated glycogen metab- internal control. olism [22]. Here we demonstrated that hypoxia leads to glycogen accumulation in human MCF7 cells through HIF1-PPP1R3C path- 2.6. Chromatin immunoprecipitation (ChIP) way, a new HIF-dependent pathway of glycogen accumulation in cells under hypoxia conditions. MCF7 cells were plated into 15 cm plate and grown to about 70% confluence. Then the cells were exposed to hypoxia for 24 h 2. Materials and methods and ChIP was performed as previous described [18]. The precipi- tated DNA was amplified by PCR using the following primers: P1: 2.1. Cell culture and hypoxic exposures 50-ACCCGCAGTGACCCACAAAG-30, P2: 50-CGCGGGGAGGTTCTGGG- ACT-30, P3: 50-TTGAATCTGGCAACCGAGAGG-30, P4: 50-CGAGTCTT MCF7 cells were grown in Dulbecco’s modified Eagle’s medium TGGGACATTTGTGC-30. For a positive control, the PDK1 promoter (DMEM) (Gibco) supplemented with 10% FBS and 100 units/ml region that contains a known HIF1-binding site was amplified penicillin, 100 lg/ml streptomycin. Normoxic cells were main- using the forward primer, 50-CGCCCTGTCCTTGAGCC-30 and the re- 0 0 tained at 37 °C in a 5% CO2 and 95% air incubator. The cells were verse primer, 5 -CGGTATGGAGCGTCCCCT-3 [26]. exposed to hypoxia in three gas incubator (YCP-50S) in 5% CO2, 94% N2 and 1% O2 at 37 °C. 2.7. Cell transfection with small interference RNA (siRNA) 2.2. Plasmid constructs One day before transfection, MCF7 cells were plated into 6-well plates. The cells were grown to 50% confluence and then transfec- The nucleotide sequences encoding HIF1a-CA5 [23], HIF1a-DN ted with 25 nM (final concentration) of siGENOME non-targeting [23] and HIF2a-DPA [24] were amplified by PCR and cloned into siRNA2, human HIF1A siGENOME SMART pool, or human HIF2A FLAG-taged pcDNA6-V5-HisB vector (Invitrogen), respectively. siGENOME SMART pool, or human PPP1R3C siGENOME SMART Promoter region of PPP1R3C were amplified from human genomic pool (Dharmacon) using Dharma FECT1 transfection reagent DNA by PCR and cloned into the luciferase reporter vector pGL3- according to the manufacturer’s instructions (Dharmacon). After promoter (Promega). As a positive control, the nucleotide sequence 24 h incubation at normoxia, the