Hypoxia-inducible erythropoietin gene expression in human neuroblastoma cells

Abstract

Two human neuroblastoma (NB) cell lines, SH-SY5Y and Kelly, were found to express the gene for erythropoietin (EPO) in an oxygen (O 2 )-dependent manner. However, NB cells had maximal production of EPO with lower partial pressure of O 2 values than the well-characterized hepatoma cell line HepG2. This maximal EPO expression was preceded by accumulation of the O 2 -sensitive ␣ subunit of the heterodimeric transcription-factor complex hypoxia-inducible factor 1 (HIF-1). Western blot analysis revealed that the amount of the ␤ subunit of HIF-1, identical to aryl hydrocarbon receptor nuclear translocator 1 (ARNT1), and the homolog ARNT2 increased in nuclear extracts from SH-SY5Y cells exposed to anoxia. In neuronal cells, ARNT1 and ARNT2 can form a heterodimer with HIF-1␣, generating a functional HIF-1 complex. Using the hypoxia response element of the human EPO enhancer, we conducted electrophoretic mobility shift assays that showed accumulation and binding of HIF-1 complexes containing both ARNT1 and ARNT2 in NB cells. In addition to the HIF-1 complex, hepatocyte nuclear factor 4␣ (HNF4␣) was found to be indispensable for hypoxiainduced EPO gene expression in hepa- IntroductionHypoxia-inducible expression of the glycoprotein hormone erythropoietin (EPO) is part of the body's response to hypoxia, which includes up-regulation of oxygen (O 2 )-dependent genes involved in vascular tone and growth, metabolic adaptation, and O 2 delivery. 1,2 EPO is produced primarily by the kidneys and liver, but EPO gene expression has also been found in several other tissues, including brain tissue, 3 breast cancer cells, 4 female genital tract tissues, 5 and rat Sertoli cells. 6 EPO gene expression is regulated by the heterodimeric transcription-factor complex hypoxia-inducible factor 1 (HIF-1), which is composed of a 120-kDa O 2 -regulated ␣ subunit and a 91-to 94-kDa constitutively expressed ␤ subunit. 7 Under normoxic conditions, HIF-1␣ is posttranslationally hydroxylated at proline residues 402 and 564, 8 which tags the protein for ubiquitination by the E3 ubiquitin ligase complex containing the von Hippel-Lindau tumor-suppressor protein. 9,10 Subsequently, HIF-1␣ protein is rapidly degraded by the proteasome system. 11 Under hypoxic conditions, the ␣ subunit is stabilized because of the lack of proline hydroxylation and accumulates. Stabilized HIF-1␣ translocates into the nucleus and forms an HIF-1 complex with the almost ubiquitously expressed HIF-1␤ (identical to aryl hydrocarbon receptor nuclear translocator 1 [ARNT1]). The HIF-1 complex binds to hypoxia response elements (HREs) found in enhancers or promoters of hypoxia-inducible genes. 12 In addition to ARNT1, kidney and neuronal cells express the ARNT1 homolog ARNT2, which can form a functional HIF complex with HIF-1␣. 13 The HIF-1 binding site (HBS) in the 3Ј EPO enhancer is one of 3 sites that are important for hypoxia-induced EPO gene transcription. 14 The HBS is the most upstream element and is followed by a 4-base-pair (bp) CACA repeat and finally a direct repeat of ...

Induction of Apoptosis by Flavopiridol in Human Neuroblastoma Cells Is Enhanced under Hypoxia and Associated With N-myc Proto-oncogene Down-Regulation

“…Recent studies have documented an important role for hypoxia in neuroblastoma cell acquisition of an immature, angiogenic, and more aggressive neural-crest-like phenotype (14,15,17). Accordingly, xenografted hypoxia-pretreated neuroblastoma cells were reported to form palpable tumors early and to grow faster than grafted normoxia-treated cells (14).…”

“…HIF-1 transactivates the hypoxia-responsive element (HRE) present in the promoter or enhancer elements of many genes encoding angiogenic, metabolic and metastatic factors (10,12,13), inducing their expression and contributing to the acquisition of tumor cell aggressive phenotypic changes (8 -10). Accumulation of HIF-1␣/2␣ proteins was observed in vitro in advanced-stage, metastatic human neuroblastoma cell lines exposed to hypoxia and in the hypoxic areas of experimental neuroblastoma xenografts grown in mice (14,15). Recent findings have demonstrated that hypoxia has a profound impact on neuroblastoma aggressive behavior.…”

Erythropoietin and Oxidative Stress

“…Work performed in vivo with subjects exposed to hypoxia also demonstrates an increase in expression of EPO and EPOR mRNA following reduced oxygenation (Marti, et al, 1996). Furthermore, both primary neurons , Liu, et al, 2006 and neuronal cell lines (Stolze, et al, 2002) have been found to retain the capacity to express EPO in an oxygen-dependent manner. Although EPO is recognized as a critical modulator of erythropoiesis, a low concentration of red blood cells alone does not directly stimulate EPO production, but requires the presence of a diminished oxygen tension.…”

Endogenous Erythropoietin as Part of the Cytokine Network in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

“…Human Kelly neuroblastoma cells were cultured and stimulated with the HIF-inducer deferoxamine mesylate (DFX, Sigma) as reported (22), with or without recombinant human IFN-γ (HyCult Biotechnology, Uden, The Netherlands) or recombinant human TNF-α (R&D Systems, Minneapolis, MN, USA). After 24 h of incubation, EPO was measured in the supernatant by EnzymeLinked ImmunoSorbent Assay (ELISA) (Quantikine EPO, R&D Systems, sensitivity 0.6 mU/mL).…”

“…To this end, we used Kelly human neuroblastoma cells, known to produce significant amounts of EPO protein, detectable by ELISA (22). IFN-γ or TNF, alone or in combination, did not induce EPO production in Kelly cells (not shown).…”