It has long been known that sphingolipids, especially sphingomyelin, a principal component of myelin, are highly enriched in the central nervous system and are structural components of all eukaryotic cell membranes. In the last few years, substantial evidence has accumulated from studies of many types of cells demonstrating that in addition to their structural roles, their breakdown products form a new class of signaling molecules with potent and myriad regulatory effects on essentially every cell in the body. While the sphingolipid metabolites sphingosine and its precursor ceramide have been associated with cell growth arrest and apoptosis, sphingosine-1-phosphate (S1P) enhances proliferation, differentiation, and cell survival as well as regulates many physiological and pathological processes. The relative levels of these three interconvertible sphingolipid metabolites, and thus cell fate, are strongly influenced by the activity of sphingosine kinases, of which there are two isoforms, designated SphK1 and SphK2, the enzymes that phosphorylate sphingosine to produce S1P. Not much is yet known of the importance of S1P in the central nervous system. Therefore, this review is focused on current knowledge of regulation of SphK1 and SphK2 on both transcriptional and post-translational levels and the functions of these isozymes and their product S1P and its receptors in the central nervous system.

Chronic inflammation and inflammatory cytokines have recently been implicated in the development and progression of various types of cancer. In the brain, neuroinflammatory cytokines affect the growth and differentiation of both normal and malignant glial cells, with interleukin 1 (IL-1) shown to be secreted by the majority of glioblastoma cells. Recently, elevated levels of sphingosine kinase 1 (SphK1), but not SphK2, were correlated with a shorter survival prognosis for patients with glioblastoma multiforme. SphK1 is a lipid kinase that produces the pro-growth, anti-apoptotic sphingosine 1-phosphate, which can induce invasion of glioblastoma cells. Here, we show that the expression of IL-1 correlates with the expression of SphK1 in glioblastoma cells, and neutralizing anti-IL-1 antibodies inhibit both the growth and invasion of glioblastoma cells. Furthermore, IL-1 up-regulates SphK1 mRNA levels, protein expression, and activity in both primary human astrocytes and various glioblastoma cell lines; however, it does not affect SphK2 expression. The IL-1-induced SphK1 up-regulation can be blocked by the inhibition of JNK, the overexpression of the dominant-negative c-Jun(TAM67), and the down-regulation of c-Jun expression by small interference RNA. Activation of SphK1 expression by IL-1 occurs on the level of transcription and is mediated via a novel AP-1 element located within the first intron of the sphk1 gene. In summary, our results suggest that SphK1 expression is transcriptionally regulated by IL-1 in glioblastoma cells, and this pathway may be important in regulating survival and invasiveness of glioblastoma cells.

Patients with gliomas expressing high levels of epidermal growth factor receptor (EGFR) and plasminogen activator inhibitor-1 (PAI-1) have a shorter overall survival prognosis. Moreover, EGF enhances PAI-1 expression in glioma cells. Although multiple known signaling cascades are activated by EGF in glioma cells, we show for the first time that EGF enhances expression of PAI-1 via sequential activation of c-Src, protein kinase C delta (PKCdelta), and sphingosine kinase 1 (SphK1), the enzyme that produces sphingosine-1-phosphate. EGF induced rapid phosphorylation of c-Src and PKCdelta and concomitant translocation of PKCdelta as well as SphK1 to the plasma membrane. Down-regulation of PKCdelta abolished EGF-induced SphK1 translocation and up-regulation of PAI-1 by EGF; whereas, down-regulation of PKCalpha had no effect on the EGF-induced PAI-1 activation but enhanced its basal expression. Similarly, inhibition of c-Src activity by PP2 blocked both EGF-induced translocation of SphK1 and PKCdelta to the plasma membrane and up-regulation of PAI-1 expression. Furthermore, SphK1 was indispensable for both EGF-induced c-Jun phosphorylation and PAI-1 expression. Collectively, our results provide a functional link between three critical downstream targets of EGF, c-Src, PKCdelta, and SphK1 that have all been implicated in regulating motility and invasion of glioma cells.

Even though astrocytes are critical for both normal brain functions and the development and progression of neuropathological states, including neuroinflammation associated with neurodegenerative diseases, the mechanisms controlling gene expression during astrocyte differentiation are poorly understood. Thus far, several signaling pathways were shown to regulate astrocyte differentiation, including JAK-STAT, BMP-2/Smads, and Notch. More recently, a family of Nuclear Factor-1 (NFI-A, -B, -C, and -X) was implicated in the regulation of vertebral neocortex development, with NFI-A and -B controlling the onset of gliogenesis. Here, we developed an in vitro model of differentiation of stem cells towards neural progenitors and subsequently astrocytes. The transition from stem cells to progenitors was accompanied by an expected change in the expression profile of markers, including Sox-2, Musashi-1, and Oct4. Subsequently, generated astrocytes were characterized by proper morphology, increased glutamate uptake, and marker gene expression. We used this in vitro differentiation model to study the expression and functions of NFIs. Interestingly, stem cells expressed only background levels of NFIs, while differentiation to neural progenitors activated the expression of NFI-A. More importantly, NFI-X expression was induced during the later stages of differentiation towards astrocytes. In addition, NFI-X and -C were required for the expression of GFAP and SPARCL1, which are the markers of astrocytes at the later stages of differentiation. We conclude that an expression program of NFIs is executed during the differentiation of astrocytes, with NFI-X and -C controlling the expression of astrocytic markers at late stages of differentiation.

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...

Glioblastoma multiforme is an invasive primary brain tumor, which evades the current standard treatments. The invasion of glioblastoma cells into healthy brain tissue partly depends on the proteolytic and nonproteolytic activities of the plasminogen activator system proteins, including the urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), and a receptor for uPA (uPAR). Here we show that sphingosine-1-phosphate (S1P) and the inflammatory mediator interleukin-1 (IL-1) increase the mRNA and protein expression of PAI-1 and uPAR and enhance the invasion of U373 glioblastoma cells. Although IL-1 enhanced the expression of sphingosine kinase 1 (SphK1), the enzyme that produces S1P, down-regulation of SphK1 had no effect on the IL-1 -induced uPAR or PAI-1 mRNA expression, suggesting that these actions of IL-1 are independent of S1P production. Indeed, the S1P-induced mRNA expression of uPAR and PAI-1 was blocked by the S1P 2 receptor antagonist JTE013 and by the down-regulation of S1P 2 using siRNA. Accordingly, the inhibition of mitogen-activated protein kinase/extracellular signal -regulated kinase kinase 1/2 and Rho-kinase, two downstream signaling cascades activated by S1P 2 , blocked the activation of PAI-1 and uPAR mRNA expression by S1P. More importantly, the attachment of glioblastoma cells was inhibited by the addition of exogenous PAI-1 or siRNA to uPAR, whereas the invasion of glioblastoma cells induced by S1P or IL-1 correlated with their ability to enhance the expression of PAI-1 and uPAR. Collectively, these results indicate that S1P and IL-1 activate distinct pathways leading to the mRNA and protein expression of PAI-1 and uPAR, which are important for glioblastoma invasiveness.

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 ...