Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced 02 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. We (8,9).The molecular mechanisms that mediate genetic responses to hypoxia have been extensively investigated for the EPO gene, which encodes a growth factor that regulates erythropoiesis and, thus, blood 02-carrying capacity (1, 4). Cis-acting DNA sequences required for transcriptional activation in response to hypoxia were identified in the EPO 3' flanking region, and a trans-acting factor that binds to the enhancer, hypoxia-inducible factor 1 (HIF-1), fulfilled criteria for a physiological regulator of EPO transcription: inducers of EPO expression (1% 02, CoC12, and desferrioxamine) also induced HIF-1 DNA-binding activity with similar kinetics, inhibitors of EPO expression (actinomycin D, cycloheximide, and 2-aminopurine) blocked induction of HIF-1 activity, and mutations in the EPO 3' flanking region that eliminated HIF-1 binding also eliminated enhancer function (4). These results also support the hypothesis that 02 tension is sensed by a hemoprotein (10) and suggest that a signal transduction pathway requiring ongoing transcription, translation, and protein phosphorylation participates in the induction of HIF-1 DNA-binding activity and EPO transcription in hypoxic cells (4 Glycerol gradient sedimentation, UV crosslinking, and methylation interference studies suggested that HIF-1 was a heterodimer consisting of the 120-kDa HIF-la subunit complexed with a 91-to 94-kDa HIF-13 subunit and that both subunits contacted DNA in the major groove (11,13,14). Here we show that HIF-la and HIF-13 are basic-helix-loop-helix (bHLH)-PAS proteins whose expression is regulated by cellular 02 tension.t MATERIALS AND METHODSProtein Microsequence Analysis. Purified HIF-1 subunits were fractionated by SDS/PAGE, and the 120-and 94-kDa polypeptides were transferred to poly(vinylidene difluoride) membranes and individually digested with trypsin in situ, and peptides were fractionated by reverse-phase HPLC (13). Protein microsequence analysis was performed at the Wistar Protein Microchemistry Laboratory (Philadelphia) (15).cDNA Library Construction and Screening. Poly(A)+ RNA was isolated from Hep 3B human hepatoma cells cultured for 16 hr at 37°C in a chamber flushed with 1% 02/5% C02/94% N2. cDNA was synthesized by using oligo(dT) and random hexamer primers, and bacteriophage libraries were constructed in Agtl 1 and Uni-ZAP XR (Stratagene). cDNA libraries were screened with 32P-labeled cDNA fragments by plaque hybridization (16).PCR. Degenerate oligonucleotide primers were designed by using codon preference rules (17). aF1 (5'-ATCGGATCCAT-CACIGARCTSATGGGITATA-3') was based upon the amino terminus of HIF-la peptide 87-1 and used as a forward primer. Two nested reverse primers, aRl (5'-ATTAAGCTTT-TGGTSAGGTGGTCISWGTC-3') and aR2 (5'-ATTAAGCT-TGCATGGTAGTAYTCATAGAT-3'), were based upon the carboxyl terminus of peptide ...

We have identified a 50-nucleotide enhancer from the human erythropoietin gene 3'-flanking sequence which can mediate a sevenfold transcriptional induction in response to hypoxia when cloned 3' to a simian virus 40 promoter-chloramphenicol acetyltransferase reporter gene and transiently expressed in Hep3B cells. Nucleotides (nt) Erythropoietin (EPO) is the glycoprotein hormone which regulates mammalian erythrocyte production and, as a result, tissue oxygen delivery. EPO RNA levels increase several hundredfold in rodent liver and kidney in response to hypoxia (8,23,24) or anemia (3,4,18). Human EPO RNA levels show similar increases in transgenic mouse liver and kidney (17,25,26,28). Hypoxia also induces EPO RNA expression in Hep3B human hepatoma cells (10, 12), demonstrating that the same cell type can sense hypoxia and respond by increasing EPO RNA levels. The 50-fold increase in steady-state EPO RNA in hypoxic Hep3B cells requires new protein synthesis and is accounted for by an approximately 10-fold increase in the rate of transcription, with the remaining increase due to posttranscriptional mechanisms (10, 11). Nuclear extracts prepared from hypoxic Hep3B cells support a higher level of EPO gene transcription in vitro than extracts from nonhypoxic cells (5).We have previously demonstrated that a 256-nucleotide (nt) EPO gene 3'-flanking sequence element functions as a hypoxia-inducible enhancer when cloned 3' to a simian virus 40 (SV40) early-region promoter-chloramphenicol acetyltransferase (CAT) reporter gene and transiently expressed in Hep3B cells (27 We have now performed functional studies and binding assays to further characterize the hypoxia-inducible enhancer. These studies indicate that hypoxia-inducible EPO gene transcription is mediated by a 50-nt element that contains at least three different transcription factor binding sites. One of these sites binds a factor in Hep3B nuclear extracts which is induced by hypoxia. MATERIALS AND METHODSPlasmid constructs. The pSVcat reporter plasmid (pCATPromoter; Promega) contained SV40 early-region promoter (excluding the 72-bp repeat enhancer), bacterial CAT coding sequence, SV40 splice site, and polyadenylation signal.

CUTL1, also known as CDP (CCAAT Displacement Protein), Cut, or Cux-1, is a homeodomain transcription factor known to play an essential role in development and cell cycle progression. Previously, we identified CUTL1 as modulator of cell motility and invasiveness. Here we report that protein kinase A (PKA), known to inhibit tumor progression in various tumor types, directly phosphorylates CUTL1 at serine 1215 in NIH3T3 fibroblasts. The PKA-induced phosphorylation results in decreased DNA binding affinity of CUTL1 and diminished CUTL1-mediated cell cycle progression and cell motility. Furthermore, the expression of several CUTL1 target genes involved in proliferation and migration, such as DNA polymerase A and DKK2, was modulated by PKA-induced phosphorylation. These data identify CUTL1 as a novel target of PKA through which this protein kinase can modulate tumor cell motility and tumor progression. Protein kinase A (PKA)2 belongs to the family of serine/threonine kinases whose activity is implicated in the regulation of a wide variety of cellular functions, such as cell metabolism, secretion, proliferation, differentiation, and neoplastic transformation (1). PKA consists of two separate subunits, the catalytic (C) and regulatory (R) subunits that interact to form an inactive holoenzyme complex (2). Activation of PKA is achieved by binding of the second messenger cAMP to the R subunit, which consequently induces a conformational change in the R subunit and leads to the dissociation of the holoenzyme into its constituent subunits (1, 2). The free active C subunit is then able to phosphorylate a variety of cytoplasmic and nuclear protein substrates, including transcription factors (1).cAMP and protein kinase A have been shown to play a major role in the modulation of tumor progression. However, these effects appear to be dependent on the cellular context (3). cAMP is known to have antiproliferative effects by antagonizing ERK activation via PKA in a variety of cell systems including fibroblasts, glial cells, and lymphocytes (4). Furthermore, cAMP has been shown to inhibit cell motility and invasiveness of several epithelial cancers (5, 6). In several neuronal, endocrine, and other epithelial cells, however, cAMP is able to stimulate growth by activating ERKs (7-9).CUTL1, also known as CDP (CCAAT displacement protein), belongs to a family of homeobox transcription factors involved in the regulation of cell growth and differentiation (10). It is evolutionarily conserved and contains four DNA binding domains, three of which are known as Cut repeats and one as a Cut homeodomain (11).CUTL1 has been described as a transcriptional activator as well as a transcriptional repressor. Its activity has been associated with cellular proliferation and cell cycle progression (12-14) as well as modulation of genes involved in terminal differentiation (15, 16). Accordingly, knockout studies with the murine homologue Cux-1 revealed reduced growth, retarded differentiation of the lung epithelia, hair follicle defects, reduced male ferti...