The Proinflammatory Cytokine Interleukin 1B and Hypoxia Cooperatively Induce the Expression of Adrenomedullin in Ovarian Carcinoma Cells through Hypoxia Inducible Factor 1 Activation Stilla Frede, Patricia Freitag, Teresa Otto, Christina Heilmaier, and Joachim Fandrey Institut fu ¨r Physiologie, Universita ¨t Duisburg-Essen, Essen, Germany Abstract Adrenomedullin (ADM) is a potent hypotensive peptide produced by macrophages and endothelial cells during ischemia and sepsis. The molecular mechanisms that control ADM gene expression in tumor cells are still poorly defined. It is known, however, that hypoxia potently increases ADM expression by activation of the transcription factor complex hypoxia inducible factor 1 (HIF-1). Proinflammatory cytokines produced by tumor invading macrophages likewise activate expression of ADM. Herein, we show that apart from hypoxia, the proinflammatory cytokine interleukin 1B (IL-1B) induced the expression of ADM mRNA through activation of HIF-1 under normoxic conditions and enhanced the hypoxia- induced expression in the human ovarian carcinoma cell line OVCAR-3. IL-1B significantly increased accumulation and nuclear translocation of HIF-1A under normoxic conditions and amplified hypoxic HIF-1 activation. IL-1B treatment affected neither HIF-1A mRNA levels nor the hydroxylation status of HIF-1A and, thus, stability of the protein. Instead cycloheximide effectively prevented the increase in HIF-1A protein, indicating a stimulatory effect of IL-1B on HIF-1A translation. Finally, treatment of HIF-1A with short interfering RNA revealed a significant role for HIF-1 in the IL-1B– dependent stimulation of ADM expression. (Cancer Res 2005; 65(11): 4690-7) Introduction Rapid growth of solid tumors often results in the development of a poorly nourished and hypoxic microenvironment (1). Under these conditions, a wide variety of tumor- and host-derived factors are expressed promoting tumor growth, degradation of extracellular matrix, vascularization, angiogenesis, and metastasis (2). However, the molecular mechanism and the relative importance of specific factors and microenvironmental conditions for these processes are not well characterized. Adrenomedullin (ADM), a hypotensive peptide, was originally isolated from pheochromocytoma (3). ADM acts as a modulator of vascular tone, vascular homeostasis, and cell growth (4–6). ADM is expressed in a variety of tissues and cell types under different conditions. Elevated blood ADM concentrations have been described in heart failure and myocardial ischemia (7), sepsis (8), and endotoxic shock (9). A link between hypoxic or ischemic conditions and enhanced ADM expression was suggested by the observation that hypoxia increased the expression of ADM in different tumor cell lines (10). Analysis of the regulatory parts of the ADM (ADM) gene showed at least 20 putative binding sites for the transcription factor complex hypoxia inducible factor 1 (HIF-1). HIF-1 is the key regulator of hypoxia-inducible genes like erythropoietin, vascular endothelial growth factor (VEGF), as well as a growing number of glycolytic and metabolic enzymes (11). The heterodimeric HIF-1 is composed of an oxygen-sensitive HIF-1a and a constitutive HIF-1h subunit, which both belong to the family of basic helix-loop-helix and PAS domain proteins (12, 13). HIF-1 activity is primarily regulated by the abundance and activity of the a subunit. HIF-1a is degraded under normoxic conditions, which is initiated by hydroxylation of the proline residues 402 and 564 by oxygen-dependent proline hydroxylases (14). Binding of the von Hippel-Lindau tumor supressor gene product (pVHL) to hydroxy- proline HIF-1a (OH-HIF-1a) targets the subunit for proteasomal degradation (15). In addition, hydroxylation of the asparagine residue 803 within the COOH-terminal transactivating domain of human HIF-1a by the O 2 -sensitive asparagyl-hydroxylase factor inhibiting HIF-1 regulates the interaction of HIF-1a with transcriptional coactivators like CBP/p300 (16). Under hypoxic conditions, HIF-1a is stabilized, translocates into the nucleus where it dimerizes with HIF-1h, and transactivates genes contain- ing hypoxia-response elements within their promoter or enhancer. Despite intensive studies, understanding of the nonhypoxic regulation of HIF-1 is still limited. Bacterial lipopolysaccharides as well as proinflammatory cytokines, such as interleukin 1h (IL-1h) and tumor necrosis factor a (TNFa), are able to increase HIF-1a protein and enhance the HIF-1 DNA binding (17, 18). For different cell types it was proposed that mitogen-activated protein kinase (MAPK) signaling pathways and activation of nuclear factor-nB (NF-nB) are involved in cytokine-induced HIF-1 stabili- zation (19, 20). Interestingly, proinflammatory cytokines and lipopolysaccharides inhibit the hypoxic induction of the classically HIF-1–regulated gene erythropoietin despite their HIF-activating effects (21). In contrast to erythropoietin, ADM expression is induced both by hypoxia and by lipopolysaccharides but the role of HIF-1 for both stimuli has not been resolved (22). In human ovarian carcinoma, a significant correlation exists between expression levels of ADM and clinical outcome of the patients (23). Tumors expressing high levels of ADM are associated with a very low survival time. ADM seems to enhance the proliferation of ovarian carcinoma but stimuli that lead to a dramatic increase in ADM expression in certain types of tumors are not well defined. Hypoxic foci within the tumors as well as high levels of inflammatory cytokines secreted by tumor-associated macrophages have been found in different types of tumors (24). Requests for reprints: Joachim Fandrey, Institut fu ¨r Physiologie, Universita ¨t Duisburg-Essen, Hufelandstrasse 55, 45147 Essen, Germany. Phone: 49-201-723-4600; Fax: 49-201-723-4648; E-mail: joachim.fandrey@uni-essen.de. I2005 American Association for Cancer Research. Cancer Res 2005; 65: (11). June 1, 2005 4690 www.aacrjournals.org Research Article Research. on November 29, 2015. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from