Human monocyte/neutrophil elastase inhibitor (MNEI) is regulated by PU.1/Spi-1, Sp1, and NF-kappaB.
Human monocyte/neutrophil elastase inhibitor (MNEI) is a specific inhibitor of the neutrophil azurophil granule proteases including elastase. To understand the physiological mechanisms that regulate expression of MNEI, we dissected a 1.0 kb region upstream of exon 1. On transient transfection, promoter activity of MNEI-luciferase constructs was highest in U937 myeloid cells, followed by K562 hematopoietic cells, followed by HeLa cervical carcinoma cells, indicating that the MNEI promoter is most active in myeloid cells and is also active in non-myeloid cells. Three transcription factor binding elements, which confer the majority of activity, are located within the first 180 base pairs of the promoter, one of which, located at -128, was active in U937 and K562 cells but inactive in non-myeloid HeLa cells. The three proximal elements were identified by transient transfection, mutation, gel shift and competition assays as Sp1 at -170, PU.1/Spi-1 at -128, and Sp1 at -66. The trans-acting factors that bind and control these elements were detected, and their identity confirmed by antibody supershift assays. Further upstream at -821, an additional regulatory element was identified controlled by NF-kappaB, which supports the highest levels of MNEI transcriptional activity. In U937 cells, reporter gene expression by the MNEI-luciferase construct that included the NF-kappaB element was two- to three-fold greater than the construct without the element. In addition, treatment of myeloid cells with lipopolysaccharide, a complex glycolipid of gram-negative bacteria, activated NF-kappaB to bind the -821 element, together suggesting that enhancement of expression of the anti-inflammatory MNEI gene is linked to innate immune responses to bacterial infection.
Zeng
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The Center for Blood Research and the Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Remold-O'Donnell
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D020413:3' Untranslated Regions; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002896:Chromosomes, Human, Pair 6; D004252:DNA Mutational Analysis; D004742:Enhancer Elements, Genetic; D005091:Exons; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D020014:K562 Cells; D008156:Luciferases / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism*; D015843:Serpins; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D020298:U937 Cells; D000099232:Proto-Oncogene Protein Spi-1
D020413:3' Untranslated Regions; D008156:Luciferases; D016328:NF-kappa B; D011506:Proteins; D011518:Proto-Oncogene Proteins; D015843:Serpins; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1; C075177:SERPINB1 protein, human