EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 35, No. 5, 350-357, October 2003 Hypoxia activates signal transducers and activators of transcription 5 (STAT5) and increases its binding activity to the GAS element in mammary epithelial cells Youn-Hee Joung1*, Jong-Hwan Park1* cules then form homo- or heterodimers through Taekyu Park2, Chang-Soo Lee3 SH2-mediated interaction and translocate into the Oun Hyun Kim4, Sang-Kyu Ye5 nucleus to activate the transcription of various γ Un Mok Yang6 , Kwang Jeon Lee7 target genes. STAT5 recognizes the interferon- and Young Mok Yang1,8 activated site TTCNNNGAA (GAS sequence) in the promoter region of the β-casein gene. Except for 1 prolactin-dependent β-casein production in mam- Department of Premedical Science mary gland cells, the biological consequences of College of Medicine and Bio-Food and Drug Research Center STAT5a activation in various systems are not 2Bio-Food and Drug Research Center clear. Here we showed that STAT5a was phos- 3 phorylated 10 min after desferrioxamine (DFO) Department of Biomedical Science College of Natural Science and Bio-Food treatment, and reached a maximum induction at 4 and Drug Research Center h in mammary epithelial cells (HC11) and trans- 4Department of Animal Science fected COS-7 cells. Under hypoxic conditions (2% College of Natural Science O 2), a maximal phosphorylation of STAT5a was Konkuk University, Chungju 380-701, Korea observed within 6 h. EMSA (electrophoretic mobili- 5Department of Pharmacology ty shift assay) showed that DFO or hypoxia en- College of Medicine, Seoul National University hanced the binding activities of STAT5a DNA to Seoul 110-799, Korea β-casein gene promoter in mammary epithelial 6Woosung Feed Co., LTD. cells (HC11) and transfected COS-7 cells. These 62-1, Ojeong-dong, Daeduk-gu results showed that DFO or hypoxia induces ty- Daejeon 306-785, Korea rosine phosphorylation of STAT5a and also in- 7Department of Dairy Science creases the binding activity of STAT5a DNA in College of Animal Husbandry mammary epithelial cells. Our data suggest that Konkuk University, Seoul 143-701, Korea the STAT5 may act as a mediator in hypoxia- 8Corresponding Author: Tel, 82-43-840-3754; mediated gene expression. Fax, 82-43-851-9329; E-mail, [email protected] *These authors contributed equally to this work. Keywords: binding activity; desferrioxamine; hypoxia; interferon-γ activated site; STAT5a; tyrosine phosphor- ylation Accepted 25 July 2003 Abbreviations: DFO, desferrioxamine; EMSA, electrophoretic mo- bility shift assay; EPO, erythropoietin; GAS, interferon-γ activated Introduction site; HIF-1, hypoxia-inducible factor-1; PRL, prolactin; ROS, re- active oxygen species; STAT, signal transducers and activators Hypoxia initiates transcription of a number of gene of transcription products that help to sustain the supply of O2 to tissues and to enhance cell survival during severe O2 deprivation. Gene products that augment O2 supply Abstract at the tissue level include erythropoietin (Epo), which increases the proliferation of erythrocytes, tyrosine STATs (signal transducers and activators of trans- hydroxylase, which is necessary for the synthesis of cription) are proteins with dual functions: signal the neurotransmitter dopamine in the carotid bodies, transducers in the cytoplasm and transcriptional and the angiogenic factor VEGF (vascular endothelial activators in the nucleus. STAT proteins act as cell growth factor), which stimulates growth of new transcription factors activated by phosphorylation capillaries (Chandel et al., 2000; Koh et al., 2002). on its tyrosine residues upon stimulation by vari- Oxygen homeostasis is primarily controlled by a cel- ous cytokines. The phosphorylated STAT mole- lular oxygen-sensing transcription factor, hypoxia-indu- STAT5 activated by hypoxia 351 cible factor 1 (HIF-1), which induces the transcription binding protein in tissue extracts from lactating mam- of more than 40 proteins, including VEGF (Choi et mary gland (Schmitt-Ney et al., 1991; Wakao et al., al., 2003). Desferrioxamine (DFO), an iron chelator, 1994). Molecular cloning and extensive molecular has been shown to activate HIF-1 in vitro, with kine- biological analysis of different tissues at various tics similar to those associated with hypoxia, and to stages of differentiation revealed that STAT5a was increase expression of HIF-1 target genes, including not only expressed in mammary epithelial cells Epo (Wang and Semenza, 1993). Like hypoxia, DFO (HC11) and activated by prolactin, but that many stabilizes HIF-1α subunits, and it has been suggested hormones, growth factors, and cytokines use STAT5a that the O2-sensing mechanism might involve O2- as a signal transducer (Groner, 2002). dependent radical production by a local Fenton re- Here we report that DFO or hypoxia activates action (Ren et al., 2000). As we have shown pre- STAT5 and its binding activities to the GAS se- viously, desferrioxamine, mimics hypoxia and a similar quence in the β-casein gene promoter in mammary oxygen sensor in the hypoxia regulation induces epithelial cells and transfected COS-7 cells. hypoxic condition (Tazuke et al., 1998; Park et al., 2000; Park et al., 2001). The analysis of hypoxia- inducible factor-1 (HIF-1) and STAT (signal trans- Materials and Methods ducers and activators of transcription) seems a priori less justified because these transcription factors were Materials initially associated with the response to lowered oxy- Dulbecco's modified Eagle's medium (DMEM) and gen tension HIF-1 (Choi et al., 2003) and to cytokines RPMI 1640 were purchased from Gibco-BRL (Grand STAT5 (Ihle, 1996). But, more recently, both the JAK Island, NY). Fetal calf serum (FCS), insulin, epidermal (janus kinase)-STAT pathway (Simon et al., 1998) growth factor (EGF), prolactin (PRL) and desferrioxa- and HIF-1 (Chandel et al., 2000) have been shown mine (DFO) were obtained from Sigma Chemical Co. to be activated by reactive oxygen species (ROS) (St. Louis, MO). FuGene 6 transfection reagent was (Tacchini et al., 2002). Accumulating evidence sup- purchased from Roche (Basel, Switzerland). Anti- ports a critical role for oxidative stress in the path- STAT5a and STAT3 antibodies were obtained from ogenesis of atherosclerosis, cancer, and other human Santa Cruz Biotechnology (Santa Cruz, CA). Horsera- diseases (Halliwell, 1989). High levels of reactive dish peroxidase-conjugated donkey anti-rabbit IgG, oxygen species damage DNA and inactivate proteins, enhanced chemiluminescence (ECL) detection kit and resulting in chronic cellular dysfunction (Shacter et al., [γ-32P]ATP were purchased from Amersham Phar- 1998). Many cell types have also harnessed ROS, macia Biotech. (Piscataway, NJ). RestoreTM Western albeit in lower concentrations, as intracellular signaling Blot Stripping Buffer was purchased from Pierce molecules to mediate growth factor and cytokine (Rockford, IL). Anti-phospho-STAT5a/b (Y694/Y699) responses (Sundaresan et al., 1995). Modulation of antibody was obtained from Upstate Biotechnology growth responses by ROS has been demonstrated in (Lake Placid, NY). The oligonucleotide electrophoretic a number of cell types, including vascular smooth mobility shift assay (EMSA) kit was purchased from muscle cells (VSMCs) (Madamanchi et al., 2001). The Promega (Madison, WI). The oligonucleotide probe signal transducer and activator of transcription (STAT) (5'-AGATTTCTAGGAATTCAAATC-3') for EMSA analy- factors were originally described as growth factor- and sis was synthesized from Bioneer (Daejeon, Korea). interferon-inducible DNA binding complexes (Levy et al., 1988). Subsequently, the STAT factors have been shown to be induced by a wide variety of growth fac- Expression vector tors and cytokines (Ihle, 1996). These factors parti- The expression vector for mouse STAT5a (pMX- cipate in the regulation of many genes, including the STAT5a; kindly provided by Dr. Koichi Ikuta, Kyoto c-fos protooncogene, caspases, and the cell cycle University, Japan) was constructed as previously des- regulator, which can also respond to oxidative stress cribed (Onishi et al., 1996). cDNA for mouse STAT5a (Kumar et al., 1997). The STAT factors are unique, was inserted into the EcoR I and Not I sites of the in that they are phosphorylated on tyrosine residues pMX vector. in response to a variety of growth factors and cyto- kines (Silvennoinen et al., 1993; Shin et al., 2002). Cell culture On phosphorylation, the STATs dimerize via SH2- phosphotyrosine interactions and become competent HC11, mouse mammary epithelial cells, were grown to bind DNA (Shuai et al., 1994). Before phospho- to confluency in RPMI 1640 medium containing 10% rylation the STATs are found in the cytoplasm and fetal bovine serum, insulin (5 µg/ml), and epidermal translocate to the nucleus upon activation (Simon et growth factor (10 ng/ml). COS-7 cells were cultured al., 1998). STAT5a was first identified as a DNA in Dulbecco's modified essential medium containing 352 Exp. Mol. Med. Vol. 35(5), 350-357, 2003 10% fetal bovine serum, 2 mM glutamine, and 100 Electrophoretic mobility shift assay o U/ml penicillin and streptomycin at 37 C in 5% CO2. STAT5a DNA binding activity was detected using an At the initiation of each experiment, the cells were 5 electrophoretic mobility shift assay (EMSA), in which resuspended in the medium at a density of 2.5×10 a labeled double-stranded DNA sequence was used cells/ml. For hypoxic conditions, the cells were placed as a DNA probe to bind active STAT5a protein in in airtight chambers (NuAire, Plymouth,