Lauren Bryan

EGF regulates plasminogen activator inhibitor-1 (PAI-1) by a pathway involving c-Src, PKCδ, and sphingosine kinase 1 in 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 (PKCδ), and sphingosine kinase 1 (SphK1), the enzyme that produces sphingosine‐1‐phosphate. EGF induced rapid phosphorylation of c‐Src and PKCδ and concomitant translocation of PKCδ as well as SphK1 to the plasma membrane. Down‐regulation of PKCδ abolished EGF‐induced SphK1 translocation and up‐regulation of PAI‐1 by EGF;whereas, down‐regulation of PKCα 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 PKCδ 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, PKCδ, and SphK1 that have all been implicated in regulating motility and invasion of glioma cells.—Paugh, B. S., Paugh, S. W., Bryan, L., Kapitonov, D., Wilczynska, K. M., Gopalan, S. M., Rokita, H., Milstien, S., Spiegel, S., and Kordula, T. EGF regulates plasminogen activator inhibitor‐1 by a pathway involving c‐Src, PKCδ, and sphingosine kinase 1 in glioblastoma cells. FASEB J. 22, 4557–465 (2008)

Regulation and functions of sphingosine kinases in the brain

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.

Interleukin-1 regulates the expression of sphingosine kinase 1 in glioblastoma cells.

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.

Sphingosine-1-phosphate and interleukin-1 independently regulate plasminogen activator inhibitor-1 and urokinase-type plasminogen activator receptor expression in glioblastoma cells: implications for invasiveness.

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 S1P2 receptor antagonist JTE013 and by the down-regulation of S1P2 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 S1P2, 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. (Mol Cancer Res 2008;6(9):1469–77)

Nuclear Factor-1-X Regulates Astrocyte-specific Expression of the α1-Antichymotrypsin and Glial Fibrillary Acidic Protein Genes

Discrete tissue-specific changes in chromatin structure of the distal serpin subcluster on human chromosome 14q32.1 allow a single gene encoding α1-antichymotrypsin (ACT) to be expressed in astrocytes and glioma cells. This astrocyte-specific regulation involves activatory protein-1 (AP-1) because overexpression of dominant-negative 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 astrocyte-specific 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.

A Novel Mechanism of Tissue Inhibitor of Metalloproteinases-1 Activation by Interleukin-1 in Primary Human Astrocytes

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-κB 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-κB (IκBαSR), or by the knock-down of p65 subunit of NF-κB. Binding of activated NF-κB (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-κB 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.

Nuclear Factor I Isoforms Regulate Gene Expression During the Differentiation of Human Neural Progenitors to Astrocytes

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, bone morphogenic protein‐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 (NP) 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 NP 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 glial fibrillary acidic protein and secreted protein acidic and rich in cystein‐like protein 1, 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. Stem Cells 2009;27:1173–1181

Astrocyte-specific Expression of the α1-Antichymotrypsin and Glial Fibrillary Acidic Protein Genes Requires Activator Protein-1

An amyloid-associated serine proteinase inhibitor (serpin), α1-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 Unique Transcriptional Activation Domain of Nuclear Factor-I-X3 Is Critical to Specifically Induce Marker Gene Expression in Astrocytes

Transcription factors of the nuclear factor 1 (NFI) family regulate normal brain development in vertebrates. However, multiple splice variants of four NFI isoforms exist, and their biological functions have yet to be elucidated. Here, we cloned and analyzed human NFI-X3, a novel splice variant of the nfix gene, which contains a unique transcriptional activation (TA) domain completely conserved in primates. In contrast to previously cloned NFI-X1, overexpression of NFI-X3 potently activates NFI reporters, including glial fibrillary acidic protein (GFAP) reporter, in astrocytes and glioma cells. The GAL4 fusion protein containing the TA domain of NFI-X3 strongly activates the GAL4 reporter, whereas the TA domain of NFI-X1 is ineffective. The expression of NFI-X3 is dramatically up-regulated during the differentiation of neural progenitors to astrocytes and precedes the expression of astrocyte markers, such as GFAP and SPARCL1 (Secreted Protein, Acidic and Rich in Cysteines-like 1). Overexpression of NFI-X3 dramatically up-regulates GFAP and SPARCL1 expression in glioma cells, whereas the knockdown of NFI-X3 diminishes the expression of both GFAP and SPARCL1 in astrocytes. Although activation of astrocyte-specific genes involves DNA demethylation and subsequent increase of histone acetylation, NFI-X3 activates GFAP expression, in part, by inducing alterations in the nucleosome architecture that lead to the increased recruitment of RNA polymerase II.

Sphingosine-1-phosphate inhibits IL-1–induced expression of C-C motif ligand 5 via c-Fos–dependent suppression of IFN-β amplification loop

The neuroinflammation associated with multiple sclerosis involves activation of astrocytes that secrete and respond to inflammatory mediators such as IL‐1. IL‐1 stimulates expression of many chemokines, including C‐C motif ligand (CCL) 5, that recruit immune cells, but it also stimulates sphingosine kinase‐1, an enzyme that generates sphingosine‐1‐phosphate (S1P), a bioactive lipid mediator essential for inflammation. We found that whereas S1P promotes IL‐1‐induced expression of IL‐6, it inhibits IL‐1‐induced CCL5 expression in astrocytes. This inhibition is mediated by the S1P receptor (S1PR)‐2 via an inhibitory G—dependent mechanism. Consistent with this surprising finding, infiltration of macrophages into sites of inflammation increased significantly in S1PR2–/– animals. However, activation of NF‐κB, IFN regulatory f actor‐1, and MAPKs, all of which regulate CCL5 expression in response to IL‐1, was not diminished by the S1P in astrocytes. Instead, S1PR2 stimulated inositol 1,4,5‐trisphosphate‐dependent Ca++ release and Elk‐1 phosphorylation and enhanced c‐Fos expression. In our study, IL‐1 induced the IFNβ production that supports CCL5 expression. An intriguing finding was that S1P induced c‐Fos‐inhibited CCL5 directly and also indirectly through inhibition of the IFN‐β amplification loop. We propose that in addition to S1PR1, which promotes inflammation, S1PR2 mediates opposing inhibitory functions that limit CCL5 expression and diminish the recruitment of immune cells.—Yester, J. W., Bryan, L., Waters, M. R., Mierzenski, B., Biswas, D. D., Gupta, A. S., Bhardwaj, R., Surace, M. J., Eltit, J. M., Milstien, S., Spiegel, S., Kordula, T. Sphingosine‐1‐phosphate inhibits IL‐1‐induced expression of C‐C motif ligand 5 via c‐Fos‐dependent suppression of IFN‐β amplification loop. FASEB J. 29, 4853–4865 (2015). www.fasebj.org