Hypoxia-inducible factor 1-mediated inhibition of peroxisome proliferator-activated receptor alpha expression during hypoxia.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone-binding proteins that regulate transcriptional responses to peroxisome proliferators and structurally diverse fatty acids. PPARs have been implicated in a wide variety of functions, including lipid homeostasis and inflammatory responses. In this study, we examined the expression of PPAR-alpha in response to ambient hypoxia. Initial studies using microarray analysis of intestinal epithelial mRNA revealed that hypoxia rapidly down-regulates PPAR-alpha mRNA and protein in epithelial cells in vitro and in vivo. Subsequent studies revealed that the PPAR-alpha gene bears a DNA consensus motif for the transcription factor hypoxia-inducible factor 1 (HIF-1). EMSA analysis revealed that ambient hypoxia induces HIF-1alpha binding to the HIF-1 consensus domain of PPAR-alpha in parallel to HIF-1 nuclear accumulation, and antisense depletion of HIF-1alpha resulted in a loss of PPAR-alpha down-regulation. The PPAR-alpha ligand pirinixic acid (WY14643) functionally promoted IFN-gamma-induced ICAM-1 expression in normoxic epithelia, and this response was lost in cells pre-exposed to ambient hypoxia. Such results indicate that HIF-1-dependent down-regulation of PPAR-alpha may provide an adaptive response to proinflammatory stimuli during cellular hypoxia. These studies provide unique insight into the regulation of PPAR-alpha expression and, importantly, provide an example of a down-regulatory pathway mediated by HIF-1.

Hypoxia-inducible factor 1-dependent induction of intestinal trefoil factor protects barrier function during hypoxia.

Mucosal organs such as the intestine are supported by a rich and complex underlying vasculature. For this reason, the intestine, and particularly barrier-protective epithelial cells, are susceptible to damage related to diminished blood flow and concomitant tissue hypoxia. We sought to identify compensatory mechanisms that protect epithelial barrier during episodes of intestinal hypoxia. Initial studies examining T84 colonic epithelial cells revealed that barrier function is uniquely resistant to changes elicited by hypoxia. A search for intestinal-specific, barrier-protective factors revealed that the human intestinal trefoil factor (ITF) gene promoter bears a previously unappreciated binding site for hypoxia-inducible factor (HIF)-1. Hypoxia resulted in parallel induction of ITF mRNA and protein. Electrophoretic mobility shift assay analysis using ITF-specific, HIF-1 consensus motifs resulted in a hypoxia-inducible DNA binding activity, and loading cells with antisense oligonucleotides directed against the alpha chain of HIF-1 resulted in a loss of ITF hypoxia inducibility. Moreover, addition of anti-ITF antibody resulted in a loss of barrier function in epithelial cells exposed to hypoxia, and the addition of recombinant human ITF to vascular endothelial cells partially protected endothelial cells from hypoxia-elicited barrier disruption. Extensions of these studies in vivo revealed prominent hypoxia-elicited increases in intestinal permeability in ITF null mice. HIF-1-dependent induction of ITF may provide an adaptive link for maintenance of barrier function during hypoxia.

Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia.

The transcription factor C/EBPalpha (for CCAAT/enhancer binding protein-alpha; encoded by the gene CEBPA) is crucial for the differentiation of granulocytes. Conditional expression of C/EBPalpha triggers neutrophilic differentiation, and no mature granulocytes are observed in Cebpa-mutant mice. Here we identify heterozygous mutations in CEBPA in ten patients with acute myeloid leukemia (AML). We found that five mutations in the amino terminus truncate the full-length protein, but did not affect a 30-kD protein initiated further downstream. The mutant proteins block wild-type C/EBPalpha DNA binding and transactivation of granulocyte target genes in a dominant-negative manner, and fails to induce granulocytic differentiation. Ours is the first report of CEBPA mutations in human neoplasia, and such mutations are likely to induce the differentiation block found in AML.

Regulation of endothelial CD73 by adenosine: paracrine pathway for enhanced endothelial barrier function.

During episodes of inflammation, multiple cell types release adenine nucleotides in the form of ATP, ADP, 5'-AMP, and adenosine. In particular, following activation, polymorphonuclear leukocytes release larger quantities of 5'-AMP. Extracellular 5'-AMP is metabolized to adenosine by surface-expressed 5'-ectonucleotidase (CD73). Adenosine liberated by this process activates surface adenosine A(2B) receptors, results in endothelial junctional reorganization, and promotes barrier function. We hypothesized that adenosine signaling to endothelia provides a paracrine loop for regulated expression of CD73 and enhanced endothelial barrier function. Using an in vitro microvascular endothelial model, we investigated the influence of 5'-AMP; adenosine; and adenosine analogues on CD73 transcription, surface expression, and function. Initial experiments revealed that adenosine and adenosine analogues induce CD73 mRNA (RT-PCR), surface expression (immunoprecipitation of surface biotinylated CD73), and function (HPLC analysis of etheno-AMP conversion to ethenoadenosine) in a time- and concentration-dependent fashion. Subsequent studies revealed that similar exposure conditions increase surface protein through transcriptional induction of CD73. Analysis of DNA-binding activity by EMSA identified a functional role for CD73 cAMP response element and, moreover, indicated that multiple cAMP agonists induce transcriptional activation of functional CD73. Induced CD73 functioned to enhance 5'-AMP-mediated promotion of endothelial barrier (measured as a paracellular flux of 70-kDa FITC-labeled tracer). These results provide an example of transcriptional induction of enzyme (CD73) by enzymatic product (adenosine) and define a paracrine pathway for the regulated expression of vascular endothelial CD73 and barrier function.

PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding.

The lineage-specific transcription factors GATA-1 and PU.1 can physically interact to inhibit each other's function, but the mechanism of repression of GATA-1 function by PU.1 has not been elucidated. Both the N terminus and the C terminus of PU.1 can physically interact with the C-terminal zinc finger of GATA-1. It is demonstrated that the PU.1 N terminus, but not the C terminus, is required for inhibiting GATA-1 function. Induced overexpression of PU.1 in K562 erythroleukemia cells blocks hemin-induced erythroid differentiation. In this system, PU.1 does not affect the expression of GATA-1 messenger RNA, protein, or nuclear localization. However, GATA-1 DNA binding decreases dramatically. By means of electrophoretic mobility shift assays with purified proteins, it is demonstrated that the N-terminal 70 amino acids of PU.1 can specifically block GATA-1 DNA binding. In addition, PU.1 had a similar effect in the G1ER cell line, in which the GATA-1 null erythroid cell line G1E has been transduced with a GATA-1-estrogen receptor fusion gene, which is directly dependent on induction of the GATA-1 fusion protein to effect erythroid maturation. Consistent with in vitro binding assays, overexpression of PU.1 blocked DNA binding of the GATA-1 fusion protein as well as GATA-1-mediated erythroid differentiation of these G1ER cells. These results demonstrate a novel mechanism by which function of a lineage-specific transcription factor is inhibited by another lineage-restricted factor through direct protein-protein interactions. These findings contribute to understanding how protein-protein interactions participate in hematopoietic differentiation and leukemogenesis. (Blood. 2000;96:2641-2648)

A nuclear factor Y (NFY) site positively regulates the human CD34 stem cell gene.

Proper regulation of the human CD34 gene requires a combinatorial action of multiple proximal and long-range, cis elements. This report shows that, like the murine CD34 5' untranslated region (UTR), the corresponding region of the human CD34 gene is necessary for optimal promoter activity. We localized the most critical element of this region to base pairs +48/+75. Through oligonucleotide competition and antibody supershift experiments in electrophoretic mobility shift assays, we found that this sequence contains a binding site (CCAAT box) for the transcription factor NFY (nuclear factor Y), a factor mediating cell type-specific and cell-cycle regulated expression of genes. Mutating this site led to a 5-fold decrease in CD34 promoter activity in transient transfection experiments. Interestingly, NFY binds adjacently to the earlier identified c-myb binding site. Here we show that both binding sites are important for CD34 promoter function: mutating either site alone decreased CD34 promoter-driven reporter gene activity 4-fold. We also show that the integrity of the c-myb binding site is necessary for stabilization of NFY binding to its site. Such cooperation between c-myb, which is expressed in early hematopoietic cells, and NFY, which is expressed in many cell types, might contribute to specific activation of CD34 in stem cells. The CCAAT box motif was also noted in the 5' UTR of the murine CD34 gene, however, NFY did not bind to this region. Thus, our results indicate that the functional similarities between the human and murine CD34 5' UTRs are achieved through different molecular mechanism(s).