Hypoxia Induces c-fos Transcription via a Mitogen-activated Protein Kinase-dependent Pathway

Hypoxia is a pathophysiological condition that occurs during injury, ischemia, and stroke. It is characterized by a decrease of reactive oxygen intermediates and a change of the intracellular redox level. In tumors hypoxia is regarded as a trigger for enhanced growth and metastasis. Here we report that in HeLa cells, hypoxic conditions induce the transcriptional activation of c-fos transcription via the serum response element. Mutations in the binding site for the ternary complex factor Elk-1 and the serum response factor abolished this induction, indicating that a ternary complex at the serum response element is necessary for the induction of the c-fos gene under hypoxia. The transcription factor Elk-1 was covalently modified by phosphorylation in response to hypoxia. Furthermore this hyperphosphorylation of Elk-1, the activation of mitogen-activated protein kinase (MAPK), and the induction of c-fos transcripts were blocked by PD98059, a specific inhibitor of mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase kinase 1. Anin vitro kinase assay with Elk-1 as substrate showed that MAPK is activated under hypoxia. The activation of MAPK corresponds temporally with the phosphorylation and activation of Elk-1. Thus, a decrease of the intracellular reactive oxygen intermediate level by hypoxia induces c-fos via the MAPK pathway. These results suggest that the intracellular redox levels may be directly coupled to tumor growth, invasion, and metastasis via Elk-1-dependent induction of c-Fos controlled genes. Hypoxia is a pathophysiological condition that occurs during injury, ischemia, and stroke. It is characterized by a decrease of reactive oxygen intermediates and a change of the intracellular redox level. In tumors hypoxia is regarded as a trigger for enhanced growth and metastasis. Here we report that in HeLa cells, hypoxic conditions induce the transcriptional activation of c-fos transcription via the serum response element. Mutations in the binding site for the ternary complex factor Elk-1 and the serum response factor abolished this induction, indicating that a ternary complex at the serum response element is necessary for the induction of the c-fos gene under hypoxia. The transcription factor Elk-1 was covalently modified by phosphorylation in response to hypoxia. Furthermore this hyperphosphorylation of Elk-1, the activation of mitogen-activated protein kinase (MAPK), and the induction of c-fos transcripts were blocked by PD98059, a specific inhibitor of mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase kinase 1. Anin vitro kinase assay with Elk-1 as substrate showed that MAPK is activated under hypoxia. The activation of MAPK corresponds temporally with the phosphorylation and activation of Elk-1. Thus, a decrease of the intracellular reactive oxygen intermediate level by hypoxia induces c-fos via the MAPK pathway. These results suggest that the intracellular redox levels may be directly coupled to tumor growth, invasion, and metastasis via Elk-1-dependent induction of c-Fos controlled genes. The activation of c-fos by mitogens and changes of the intracellular redox level is mainly mediated by the serum response element (SRE). 1The abbreviations used are: SRE, serum response element; AP-1, activator protein 1; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated protein kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase kinase; PMA, phorbol 12-myristate 13-acetate; ROI, reactive oxygen intermediate; SRF, serum response factor; TCF, ternary complex factor; SAP, stress-activated protein. The SRE is a regulatory element found in many growth factor-regulated promotors that directs the rapid induction of gene expression (for review see Ref. 1Treisman R. Semin. Cancer Biol. 1990; 1: 47-58PubMed Google Scholar). The best studied SRE is that of the c-fos gene. Two kinds of transcription factors are required for SRE activity: the ubiquitous transcription serum response factor (SRF) (2Norman C. Runswick M. Pollock R. Treisman R. Cell. 1988; 55: 989-1003Abstract Full Text PDF PubMed Scopus (709) Google Scholar) and the ternary complex factors (TCFs), which form a ternary complex with the SRF. The human TCFs include Elk-1, SAP-1, and SAP-2 and constitute a subfamily within the Ets family of transcription factors (for review see Ref. 3Treisman R. Curr. Opin. Genet. Dev. 1994; 4: 96-101Crossref PubMed Scopus (622) Google Scholar). These proteins need SRF to bind tightly to the SRE (4Shaw P.E. Schroter H. Nordheim A. Cell. 1989; 56: 563-572Abstract Full Text PDF PubMed Scopus (346) Google Scholar). The N-terminal domains of Elk-1 and SAP-1 mediate DNA contact and ternary complex formation. The transactivation domain at the C terminus contains several conserved MAPK phosphorylation sites. Growth factor-stimulated activation of the MAPK pathway results in phosphorylation of the Elk-1 C terminus (5Hill C.S. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1198) Google Scholar), which then cooperates with the SRF C-terminal activation domain to activate transcription (6Hill C.S. Marais R. John S. Wynne J. Dalton S. Treisman R. Cell. 1993; 73: 395-406Abstract Full Text PDF PubMed Scopus (329) Google Scholar, 7Johansen F.E. Prywes R. Mol. Cell. Biol. 1993; 13: 4640-4647Crossref PubMed Scopus (112) Google Scholar). The transcription factor AP-1 is composed of members of the Fos family (c-Fos, Fos-B, Fra-1, and Fra-2) and the Jun family (c-Jun, JunB, and JunD) that form restricted homo- or heterodimers (for review see Ref.8Angel P.E. Herrlich P.A. The FOS and JUN Families of Transcription Factors. CRC Press, Boca Raton1994Google Scholar). With the exception of c-Jun homodimers, AP-1 is predominantly induced at a transcriptional level by novel synthesis of its subunits. This induction is controlled by cis-acting SRE elements and AP-1 binding sites in the promotors of several AP-1 genes. A large number of mitogenic and proinflammatory signals lead to activation of AP-1. Likewise, changes in the cellular redox status were reported to activate AP-1 (9Devary Y. Gottlieb R.A. Lau L.F. Karin M. Mol. Cell. Biol. 1991; 11: 2804-2811Crossref PubMed Scopus (603) Google Scholar, 10Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Crossref PubMed Scopus (3430) Google Scholar). Changes of the cellular redox status are directly coupled to the intracellular level of ROIs. For example, hypoxia and antioxidants reduce the intracellular level of ROIs. A pathophysiological role for reduced levels of ROIs is evident in tumor initiation, growth, and metastasis. We have previously reported that treatment of cells with antioxidants such as pyrolidinedithiocarbamate or N-acetyl-l-cysteine (11Meyer M. Schreck R. Baeuerle P.A. EMBO J. 1993; 12: 2005-2015Crossref PubMed Scopus (1271) Google Scholar), as well as overexpression of thioredoxin (12Schenk H. Klein M. Erdbrugger W. Droge W. Schulze-Osthoff K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1672-1676Crossref PubMed Scopus (644) Google Scholar), strongly activates AP-1. Recently we and others could show that hypoxia, a physiological correlate to antioxidants, also activates AP-1 (13Yao K.-S. Xanthoudakis S. Curran T. O'Dwyer P.J. Mol. Cell. Biol. 1994; 14: 5997-6003Crossref PubMed Scopus (240) Google Scholar, 14Rupec R.A. Baeuerle P.A. Eur. J. Biochem. 1995; 234: 632-640Crossref PubMed Scopus (161) Google Scholar) and that this depends on novel gene transcription. The activation of AP-1 by hypoxia prompted us to investigate the transcription factors responsible for AP-1 gene induction. The c-Fos protein is a component of the hypoxia-induced AP-1 complex (14Rupec R.A. Baeuerle P.A. Eur. J. Biochem. 1995; 234: 632-640Crossref PubMed Scopus (161) Google Scholar). The major cis-acting element responsible for the induction of c-fos transcription in response to a variety of mitogens is the SRE (15Treisman R. Trends Biochem. Sci. 1992; 17: 423-426Abstract Full Text PDF PubMed Scopus (353) Google Scholar, 16Janknecht R. Cahill M.A. Nordheim A. Carcinogenesis. 1995; 16: 443-450Crossref PubMed Scopus (112) Google Scholar). Here we show that hypoxia induces c-fostranscription via the SRE. Both the binding site for the ternary complex factor as well as the binding site for SRF are required for this induction. Hypoxia rapidly induces a hyperphosphorylated ternary complex at the c-fos SRE, which contains the ternary complex factor Elk-1. Furthermore, the activation of MAPK ERK2 but not JNK/SAPK parallels the activation of Elk-1. We therefore conclude that the activation of Elk-1 by the MAPK pathway represents an important link between hypoxia-induced cellular redox imbalance and the activation of the transcription factor AP-1 via c-fos transcription. The human cervical carcinoma cell line HeLa (ATCC, CCL2) was cultured as described in Ref. 14Rupec R.A. Baeuerle P.A. Eur. J. Biochem. 1995; 234: 632-640Crossref PubMed Scopus (161) Google Scholar. Hypoxic conditions and PMA stimulation were applied as described in Ref. 14Rupec R.A. Baeuerle P.A. Eur. J. Biochem. 1995; 234: 632-640Crossref PubMed Scopus (161) Google Scholar. Preparation of cell extracts cells were cultivated in the absence of serum. For transfections cells were kept in 10% fetal calf serum (Sigma) 14 h before stimulation. In the experiments using PD98059 2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one (New England Biolabs Inc.), cells were incubated with 10 μmPD98059 for 1 h prior to hypoxia. 3 × 105 cells/60-mm dish were serum-deprived overnight in Dulbecco's modified Eagle's medium and treated with hypoxic medium for the indicated time as described in Ref. 14Rupec R.A. Baeuerle P.A. Eur. J. Biochem. 1995; 234: 632-640Crossref PubMed Scopus (161) Google Scholar. The hypoxic medium was aspirated after the treatment, and the culture dish with the adherent cells was immediately frozen on liquid nitrogen. Cells were lysed in whole cell extract buffer during thawing (17Baccarini M. Sabatini D.M. App H. Rapp U.R. Stanley E.R. EMBO J. 1990; 9: 3649-3657Crossref PubMed Scopus (87) Google Scholar). EMSA binding reactions were performed as described in Refs. 18Zinck R. Hipskind R.A. Pingoud V. Nordheim A. EMBO J. 1993; 12: 2377-2387Crossref PubMed Scopus (143) Google Scholar and 19Müller J.M. Cahill M.A. Rupec R.A. Baeuerle P.A. Nordheim A. Eur. J. Biochem. 1997; 244: 45-52Crossref PubMed Scopus (99) Google Scholar). For Fig. 3, 6 μl of Elk-1 antibody (Santa Cruz Biotechnology Inc.) or a control antibody (α-glutathione S-transferase, Santa Cruz Biotechnology Inc.) was added to 0.5 μg of cell extract and incubated for 40 min on ice. Phosphatase inhibitors present in the extracts used in Fig. 3 B were removed. To achieve this whole cell extracts were microdialyzed on VS filters (0.025 μm; Millipore) against phosphatase buffer (18Zinck R. Hipskind R.A. Pingoud V. Nordheim A. EMBO J. 1993; 12: 2377-2387Crossref PubMed Scopus (143) Google Scholar). Phosphatase assays were performed as described (18Zinck R. Hipskind R.A. Pingoud V. Nordheim A. EMBO J. 1993; 12: 2377-2387Crossref PubMed Scopus (143) Google Scholar). Gels were vacuum-dried and exposed to Kodak XAR5 films at −80 °C for 12–48 h. HeLa cells (2 × 106 cells) were treated as described (14Rupec R.A. Baeuerle P.A. Eur. J. Biochem. 1995; 234: 632-640Crossref PubMed Scopus (161) Google Scholar). 56 μg of total cellular extract were immunoprecipitated with a phospho-MAPK antibody, washed, and incubated with a glutathione S-transferase-Elk-1 fusion protein as substrate. The reaction was separated by SDS gel electrophoresis, blotted, and incubated with an antibody directed against phosphorylated Ser383 of Elk-1 according to the instructions of the manufacturer (New England Biolabs Inc.). HeLa cells (1 × 106/100 mm dish) were treated with hypoxic medium for 15–90 min. Total cellular RNA was isolated as described in Ref. 20Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar. Separation of 10 μg total RNA/lane on a formaldehyde-agarose gel, blotting, hybridization, and washing steps were performed as described (21Zinck R. Cahill M.A. Kracht M. Sachsenmaier C. Hipskind R.A. Nordheim A. Mol. Cell. Biol. 1995; 15: 4930-4938Crossref PubMed Scopus (238) Google Scholar), except that hybridization was performed in Rapidhyb buffer (Amersham Corp.) with a 32P-labeled c-foscDNA probe. RNA integrity and equal amount were confirmed using UV shadowing and methylene blue staining of the membrane (not shown). Transient transfections were performed essentially as described (19Müller J.M. Cahill M.A. Rupec R.A. Baeuerle P.A. Nordheim A. Eur. J. Biochem. 1997; 244: 45-52Crossref PubMed Scopus (99) Google Scholar, 22Janknecht R. Ernst W.H. Pingoud V. Nordheim A. EMBO J. 1993; 12: 5097-5104Crossref PubMed Scopus (508) Google Scholar). The reporter plasmids pSRE2-tk80-luc (22Janknecht R. Ernst W.H. Pingoud V. Nordheim A. EMBO J. 1993; 12: 5097-5104Crossref PubMed Scopus (508) Google Scholar), pEL2-tk80 (ΔTCF), and pm2-tk80 (ΔSRE) were gifts of A. Nordheim. pEL2-tk80 and pm2-tk80 were identical to pSRE2-tk80-luc except they contained mutations in the SRE that drastically reduce the DNA binding of TCF/Elk-1 and SRF, respectively (4Shaw P.E. Schroter H. Nordheim A. Cell. 1989; 56: 563-572Abstract Full Text PDF PubMed Scopus (346) Google Scholar). Briefly 105 HeLa cells were plated in 60-mm dishes and transfected after changing the medium the next day using the calcium phosphate method. 16 h later the cells were supplied with fresh medium. The medium was aspirated after 16 h, and the cells were supplied with hypoxic medium and incubated in the hypoxic chamber. Control cells were transfected in parallel and cultivated under normal oxygen pressure. After 6 h cells were harvested and luciferase assays were performed as described (19Müller J.M. Cahill M.A. Rupec R.A. Baeuerle P.A. Nordheim A. Eur. J. Biochem. 1997; 244: 45-52Crossref PubMed Scopus (99) Google Scholar). Luciferase-generated light units were normalized to protein contents and are shown as relative luciferase activation versusuntreated cells. The means of four experiments performed in duplicate are shown. PMA stimulation was used as a control in the experiments and gave approximately double the stimulation of the hypoxia treatment (not shown). To test c-fos gene activation under hypoxia, HeLa cells were subjected to hypoxia. A rapid and transient increase of c-fos transcripts was observed (Fig. 1 A). c-fos mRNA became detectable as as min hypoxia (Fig. 1 induction was observed after min of stimulation c-fos mRNA was after min of stimulation have observed for the induction of the by treatment with mitogens (15Treisman R. Trends Biochem. Sci. 1992; 17: 423-426Abstract Full Text PDF PubMed Scopus (353) Google Scholar, 16Janknecht R. Cahill M.A. Nordheim A. Carcinogenesis. 1995; 16: 443-450Crossref PubMed Scopus (112) Google Scholar). We therefore the c-fos SRE is responsible for the induction under hypoxia. Transient experiments using a luciferase reporter by c-fos SRE binding elements showed an induction hypoxic stimulation (Fig. 1 mutations in the binding sites for the ternary complex factor or the serum factor we the of the binding sites for this induction. These mutations binding of the and SRF, respectively (4Shaw P.E. Schroter H. Nordheim A. Cell. 1989; 56: 563-572Abstract Full Text PDF PubMed Scopus (346) Google Scholar). induction could be using the and (Fig. 1 and These show that the binding site for the ternary complex factor as well as the binding site for the serum response factor are required for the induction of the c-fos gene during hypoxia. We studied the of reduced oxygen on the phosphorylation status of in HeLa cells. hyperphosphorylation be in by a of the (18Zinck R. Hipskind R.A. Pingoud V. Nordheim A. EMBO J. 1993; 12: 2377-2387Crossref PubMed Scopus (143) Google Scholar, 22Janknecht R. Ernst W.H. Pingoud V. Nordheim A. EMBO J. 1993; 12: 5097-5104Crossref PubMed Scopus (508) Google Scholar, R. Wynne J. Treisman R. Cell. 1993; 73: Full Text PDF PubMed Scopus Google Scholar). Cells were treated with hypoxia between min and 6 h (Fig. A). of cells with the PMA was used as a control (Fig. was to of the cells. Hypoxia rapidly induced a complex on the c-fos SRE as by EMSA Fig. A). This complex was in its mobility that induced stimulation with PMA, which is to lead to hyperphosphorylated TCFs R. Cahill M.A. Nordheim A. Carcinogenesis. 1995; 16: 443-450Crossref PubMed Scopus (112) Google Scholar) (Fig. with The ternary complex was observed within min Fig. a at and after 40 min (Fig. Fig. B that the observed complex is a ternary complex that depends on the of the SRE binding the SRF and extracts TCFs (4Shaw P.E. Schroter H. Nordheim A. Cell. 1989; 56: 563-572Abstract Full Text PDF PubMed Scopus (346) Google Scholar). SRF extract the SRF form this ternary complex with the c-fos SRE. of the modified induced under hypoxia in HeLa cells was by the of a specific antibody against Elk-1 to the DNA binding reaction (Fig. 3 We conclude that Elk-1 or with previously reported that Elk-1 the of HeLa cell R.A. Nordheim A. 1991; PubMed Scopus Google Scholar). To show that hyperphosphorylation of the TCFs is the of the a phosphatase treatment was of the cell extracts with phosphatase prior to abolished complex (Fig. 3 indicating that the of the hypoxia was by hyperphosphorylation of the TCFs In to the activation of Elk-1 by hypoxic activation was observed in gel of cells to oxygen not shown). We next the phosphorylation of TCF/Elk-1 under hypoxic conditions was to activation of of cells with PD98059, a specific inhibitor of in a of complex (Fig. and 3 with and This that is necessary for the phosphorylation of TCF/Elk-1 during hypoxia. In a hyperphosphorylated form of ERK2 could be after JNK/SAPK was not not shown). To investigate this hyperphosphorylated ERK2 with activation of MAPK, an in vitro assay was After of MAPK, Elk-1 was used as blotted, and by a specific Elk-1 be after min of hypoxia (Fig. indicating This after min of hypoxia (Fig. 3, The form of MAPK temporally corresponds to the of the hyperphosphorylated complex in EMSA (Fig. and Furthermore, the inhibitor PD98059 blocked activation of MAPK (Fig. and of the phosphorylated complex To test the induction of c-fos transcripts was also by the inhibitor PD98059, a was The induction of c-fos transcripts was blocked as well as the of the phosphorylated complex (Fig. and These results show that the MAPK pathway is responsible for the activation of TCF/Elk-1 and the induction of hypoxic The response of to hypoxia is a complex and Hypoxic cells in tumors show a reduced and R.A. J. 1988; PubMed Scopus Google Scholar, S. Biochem. PubMed Scopus Google Scholar, K. 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