Discrete tissue-specific changes in chromatin structure of the distal serpin subcluster on human chromosome 14q32.1 allow a single gene encoding ␣ 1 -antichymotrypsin (ACT) to be expressed in astrocytes and glioma cells. This astrocyte-specific regulation involves activatory protein-1 (AP-1) because overexpression of dominantnegative c-jun(TAM67) abolishes ACT expression in glioma cells. Here we identify a new regulatory element, located within the ؊13-kb enhancer of the ACT gene, that binds nuclear factor-1 (NFI) and is indispensable for the full basal transcriptional activity of the ACT gene. Furthermore, down-regulation of NFI expression by siRNA abolishes basal ACT expression in glioma cells. However, NFI does not mediate astrocyte-specific expression by itself, but likely cooperates with AP-1. A detailed analysis of the 14-kb long 5-flanking region of the ACT gene indicated the presence of adjacent NFI and AP-1 elements that colocalized with DNase I-hypersensitive sites found in astrocytes and glioma cells. Interestingly, knock-down of NFI expression also specifically abrogates the expression of glial acidic fibrillary protein (GFAP), which is an astrocyte-specific marker protein. Mutations introduced into putative NFI and AP-1 elements within the 5-flanking region of the GFAP gene also diminished basal expression of the reporter. In addition, we found, using isoform-specific siRNAs, that NFI-X regulates the astrocytespecific expression of ACT and GFAP. We propose that NFI-X cooperates with AP-1 by an unknown mechanism in astrocytes, which results in the expression of a subset of astrocyte-specific genes.2 is expressed at low levels by astrocytes in the brain under normal physiological conditions. However, elevated ACT levels have been observed in several neuropathological disorders of the central nervous system, including Alzheimer disease (1, 2). This drastic change in ACT expression is caused by proinflammatory cytokines, including IL-1, IL-6, oncostatin M (OSM), and tumor necrosis factor (TNF)␣, which are released at the site of tissue damage (3, 4). ACT secreted by reactive astrocytes subsequently associates with the ␤-amyloid peptide, which is the major component of pathological deposits found in the brains of Alzheimer disease patients (1).ACT belongs to the serine protease inhibitor (serpin) family of proteins and is also expressed in the liver and secreted into the plasma (5). The gene encoding ACT is clustered with 10 additional serpin genes on human chromosome 14q32.1 and resides within the distal serpin subcluster that also contains genes encoding kallistatin, protein C inhibitor, and the kallistatin-like protein (6, 7). The expression profile of the distal serpin subcluster is dramatically different between astrocytes and hepatocytes. All four genes are expressed in hepatocytes, whereas only the ACT gene is expressed in astrocytes (8). Investigations of the regulatory mechanisms controlling the selective expression of ACT in astrocytes and glioma cells demonstrated that the ACT gene is localize...

An amyloid-associated serine proteinase inhibitor (serpin), ␣ 1 -antichymotrypsin (ACT), is encoded by a gene located within the distal serpin subcluster on human chromosome 14q32.1. The expression of these distal serpin genes is determined by tissue-specific chromatin structures that allow their ubiquitous expression in hepatocytes; however, their expression is limited to a single ACT gene in astrocytes. In astrocytes and glioma cells, six specific DNase I-hypersensitive sites (DHSs) were found located exclusively in the 5-flanking region of the ACT gene. We identified two enhancers that mapped to the two DHSs at ؊13 kb and ؊11.5 kb which contain activator protein-1 (AP-1) binding sites, both of which are critical for basal astrocyte-specific expression of ACT reporters. In vivo, these elements are occupied by c-jun homodimers in unstimulated cells and c-jun/c-fos heterodimers in interleukin-1-treated cells. Moreover, functional c-jun is required for the expression of ACT in glioma cells because both transient and stable inducible overexpression of dominant-negative c-jun(TAM67) specifically abrogates basal and reduces cytokine-induced expression of ACT. Expression-associated methylation of lysine 4 of histone H3 was also lost in these cells, but the DHS distribution pattern and global histone acetylation were not changed upstream of the ACT locus. Interestingly, functional AP-1 is also indispensable for the expression of glial fibrillary acidic protein (GFAP), which is an astrocyte-specific marker. We propose that AP-1 is a key transcription factor that, in part, controls astrocyte-specific expression of genes including the ACT and GFAP genes.The 11 genes encoding the serine proteinase inhibitors (serpins) 2 are clustered on human chromosome 14q32.1 and occupy the ϳ370 kb region (1, 2). This serpin cluster can be divided into three subclusters that contain 4, 3, and 4 genes, respectively. The distal subcluster consists of the genes encoding the ␣ 1 -antichymotrypsin (ACT), kallistatin, protein C inhibitor, and the recently identified kallistatin-like protein (2). All of these genes are highly transcribed in hepatocytes and hepatoma cells, and their promoters are accessible to DNase I digestion (3). In contrast, only the ACT gene is expressed in brain astrocytes and glioma cells (3). Selective expression of ACT in these cells correlates with DNase I accessibility at the 5Ј-flanking region of the gene, whereas nonexpressed protein C inhibitor and kallistatin genes are localized in DNase I-inaccessible chromatin (3).In the liver, hepatocyte-specific gene expression is determined by transcription factors belonging to the hepatocyte nuclear factor (HNF) and CAAT enhancer-binding protein (C/EBP) families, with HNF-1 and HNF-4 being critical, but not sufficient, for the expression of the genes from the proximal serpin subcluster (4). The presence of binding sites for these transcription factors near the promoters of the distal serpin genes suggests that they play a critical role in their expression in hepatic cells. ...

Reactive astrogliosis is the gliotic response to brain injury with activated astrocytes and microglia being the major effector cells. These cells secrete inflammatory cytokines, proteinases, and proteinase inhibitors that influence extracellular matrix (ECM) remodeling. In astrocytes, the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) is up-regulated by interleukin-1 (IL-1), which is a major neuroinflammatory cytokine. We report that IL-1 activates TIMP-1 expression via both the IKK/NF-B and MEK3/6/p38/ATF-2 pathways in astrocytes. The activation of the TIMP-1 gene can be blocked by using pharmacological inhibitors, including BAY11-7082 and SB202190, overexpression of the dominant-negative inhibitor of NF-B (IB␣SR), or by the knock-down of p65 subunit of NF-B. Binding of activated NF-B (p50/p65 heterodimer) and ATF-2 (homodimer) to two novel regulatory elements located ؊2.7 and ؊2.2 kb upstream of the TIMP-1 transcription start site, respectively, is required for full IL-1-responsiveness. Mutational analysis of these regulatory elements and their weak activity when linked to the minimal tk promoter suggest that cooperative binding is required to activate transcription. In contrast to astrocytes, we observed that TIMP-1 is expressed at lower levels in gliomas and is not regulated by IL-1. We provide evidence that the lack of TIMP-1 activation in gliomas results from either dysfunctional IKK/NF-B or MEK3/6/p38/ATF-2 activation by IL-1. In summary, we propose a novel mechanism of TIMP-1 regulation, which ensures an increased supply of the inhibitor after brain injury, and limits ECM degradation. This mechanism does not function in gliomas, and may in part explain the increased invasiveness of glioma cells.The remodeling of the extracellular matrix (ECM), 2 including the degradation of the ECM by matrix metalloproteinases (MMPs) and its subsequent resynthesis, is critical during normal physiological processes, such as angiogenesis, embryonic development, organ morphogenesis, bone remodeling, and ovulation (1, 2). During these processes the proteolytic activity of MMPs is tightly controlled at the transcriptional level by growth factors, hormones, and cytokines, and at the protein level by proteolytic cleavage of inactive zymogens, and the inhibition by specific inhibitors, including tissue inhibitors of metalloproteinases (TIMPs) (2). The delicate balance between the activities of MMPs and TIMPs is critical to limit deleterious outcomes of uncontrolled degradation, which is manifested in pathological conditions such as periodontal disease, arthritis, tumor cell invasion, fibrosis, and neurodegenerative disorders (2-4). These pathological conditions often represent chronic inflammatory diseases suggesting that inflammatory mediators, including inflammatory cytokines, may disrupt the intricate balance between MMPs and TIMPs.In the central nervous system (CNS), infection and injury induce a histopathological response known as reactive astrogliosis, which is the primary cause of regenerative failure in ...

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