The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin.

Clostridium perfringens ε-toxin (ETX) is a potent pore-forming toxin responsible for a central nervous system (CNS) disease in ruminant animals with characteristics of blood-brain barrier (BBB) dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS), a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL). While native Chinese Hamster Ovary (CHO) cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/-) mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity.

High expression of ncRAN, a novel non-coding RNA mapped to chromosome 17q25.1, is associated with poor prognosis in neuroblastoma.

Neuroblastoma shows complex patterns of genetic aberrations including MYCN amplification, deletion of chromosome 1p or 11q, and gain of chromosome 17q. The 17q gain is frequently observed in high-risk neuroblastomas, however, the candidate genes still remain elusive. In the present study, we integrated the data of comparative genomic hybridization of 236 tumors by BAC array and expression profiling of 136 tumors by using the in-house cDNA microarray carrying 5,340 genes derived from primary neuroblastomas. A novel candidate gene mapped to chromosome 17q25.1 with two splicing variants, Nbla10727 and Nbla12061, was identified. The transcript size appeared to be 2.3 kb by Northern blot, however, the cDNA sequences had no obvious open reading frame. The protein product was undetectable by both in vivo and in vitro translation assays, suggesting that the transcript might not encode any protein product. Therefore, we named it as ncRAN (non-coding RNA expressed in aggressive neuroblastoma). In analysis of 70 patients with sporadic neuroblastoma, the high levels of ncRAN mRNA expression were significantly associated with poor outcome of the patients (p<0.001). The multivariate analysis showed that expression of ncRAN mRNA was an independent prognostic factor among age, stage, origin and MYCN expression. Ectopic expression of ncRAN induced transformation of NIH3T3 cells in soft agar, while knockdown of endogenous ncRAN with RNA interference significantly inhibited cell growth in SH-SY5Y cells. Collectively, our results suggest that ncRAN may be a novel non-coding RNA mapped to the region of 17q gain and act like an oncogene in aggressive neuroblastomas.

Intracellular role for sphingosine kinase 1 in intestinal adenoma cell proliferation.

Sphingosine kinase (Sphk) enzymes are important in intracellular sphingolipid metabolism as well as in the biosynthesis of sphingosine 1-phosphate (S1P), an extracellular lipid mediator. Here, we show that Sphk1 is expressed and is required for small intestinal tumor cell proliferation in Apc Min/+ mice. Adenoma size but not incidence was dramatically reduced in Apc Min/+ Sphk(-/-) mice. Concomitantly, epithelial cell proliferation in the polyps was significantly attenuated, suggesting that Sphk1 regulates adenoma progression. Although the S1P receptors (S1P1R, S1P2R, and S1P3R) are expressed, polyp incidence or size was unaltered in Apc Min/+ S1p2r(-/-), Apc Min/+ S1p3r(-/-), and Apc Min/+ S1p1r(+/-) bigenic mice. These data suggest that extracellular S1P signaling via its receptors is not involved in adenoma cell proliferation. Interestingly, tissue sphingosine content was elevated in the adenomas of Apc Min/+ Sphk1(-/-) mice, whereas S1P levels were not significantly altered. Concomitantly, epithelial cell proliferation and the expression of the G1/S cell cycle regulator CDK4 and c-myc were diminished in the polyps of Apc Min/+ Sphk1(-/-) mice. In rat intestinal epithelial (RIE) cells in vitro, Sphk1 overexpression enhanced cell cycle traverse at the G1/S boundary. In addition, RIE cells treated with sphingosine but not C6-ceramide exhibited reduced cell proliferation, reduced retinoblastoma protein phosphorylation, and cyclin-dependent kinase 4 (Cdk4) expression. Our findings suggest that Sphk1 plays a critical role in intestinal tumor cell proliferation and that inhibitors of Sphk1 may be useful in the control of intestinal cancer.

The cysteine-cluster motif of c-Yes, Lyn and FAK as a suppressive module for the kinases.

The Src family of non-receptor protein tyrosine kinases plays a critical role in the progression of human cancers so that the development of its specific inhibitors is important as a therapeutic tool. We previously reported that cysteine residues in the cysteine-cluster (CC) motif of v-Src were critical for the kinase inactivation by the SH-alkylating agents such as N-(9-acridinyl) maleimide (NAM), whereas other cysteine residues were dispensable. We found similar CC-motifs in other Src-family kinases and a non-Src-family kinase, FAK. In this study, we explored the function of the CC-motif in Yes, Lyn and FAK. While Src has four cysteines in the CC-motif, c-Yes and Lyn have three and two of the four cysteines, respectively. Two conserved cysteines of the Src family kinases, corresponding to Cys487 and Cys498 of Src, were essential for the resistance to the inactivation of the kinase activity by NAM, whereas the first cysteine of c-Yes, which is absent in Lyn, was less important. FAK has similar CC-motifs with two cysteines and both cysteines were again essential for the resistance to the inactivation of the kinase activity by NAM. Taken together, modification of cysteine residues of the CC-motif causes a repressor effect on the catalytic activity of the Src family kinases and FAK.

Extracellular export of sphingosine kinase-1a contributes to the vascular S1P gradient.

Sphingosine 1-phosphate (S1P), produced by Sphks (sphingosine kinases), is a multifunctional lipid mediator that regulates immune cell trafficking and vascular development. Mammals maintain a large concentration gradient of S1P between vascular and extravascular compartments. Mechanisms by which S1P is released from cells and concentrated in the plasma are poorly understood. We recently demonstrated [Ancellin, Colmont, Su, Li, Mittereder, Chae, Stefansson, Liau and Hla (2002) J. Biol. Chem. 277, 6667-6675] that Sphk1 activity is constitutively secreted by vascular endothelial cells. In the present study, we show that among the five Sphk isoforms expressed in endothelial cells, the Sphk-1a isoform is selectively secreted in HEK-293 cells (human embryonic kidney cells) and human umbilical-vein endothelial cells. In sharp contrast, Sphk2 is not secreted. The exported Sphk-1a isoform is enzymatically active and produced sufficient S1P to induce S1P receptor internalization. Wild-type mouse plasma contains significant Sphk activity (179 pmol x min(-1) x g(-1)). In contrast, Sphk1-/- mouse plasma has undetectable Sphk activity and approx. 65% reduction in S1P levels. Moreover, human plasma contains enzymatically active Sphk1 (46 pmol x min(-1) x g(-1)). These results suggest that export of Sphk-1a occurs under physiological conditions and may contribute to the establishment of the vascular S1P gradient.

SH2 domain containing protein tyrosine phosphatase 2 regulates concanavalin A-dependent secretion and activation of matrix metalloproteinase 2 via the extracellular signal-regulated kinase and p38 pathways.

We investigated the role of SH2 domain containing protein tyrosine phosphatase (SHP) 2 in Concanavalin A (Con A) -dependent signaling that leads to the augmented secretion and activation of matrix metalloproteinase (MMP) 2. In cells expressing mutant SHP-2 in which 65 amino acids in the SH2-N domain were deleted, we found that production, secretion, and proteolytic activation of MMP-2 in response to Con A treatment was severely impaired. Under Con A stimulation, complex formation of SHP-2 with SOS-1 and Grb-2 together with the activation of Ras signaling was clearly observed in wild-type cells, but not in SHP-2 mutant cells. In wild-type cells, Con A-treatment activated dual signaling pathways, extracellular signal-regulated kinase (Erk) and p38, in a Ras-dependent manner, whereas Con A-dependent activation of these signaling pathways was absent in SHP-2 mutant cells. In addition, pretreatment of wild-type cells with U0126, a potent inhibitor for mitogen-activated protein/ERK kinase 1, or with SB203580, a specific inhibitor for p38, significantly inhibited the Con A-dependent secretion and activation of MMP-2. However, overexpression of active mitogen-activated protein/ERK kinase 1 in SHP-2 mutant cells could not induce clear activation of MMP-2 secretion, although these cells responded well to the Con A treatment in a p38-dependent manner. Finally, reintroduction of wild-type SHP-2 into SHP-2 mutant cells rescued Erk and p38 activation, and also MMP-2 secretion, whereas dominant-negative SHP-2 could block the Con A-dependent activation of Erk and p38. Taken together, our results strongly suggest that SHP-2 plays a critical role as a positive mediator for Con A-dependent activation of MMP-2 secretion via Ras-Erk and Ras-p38 signalings.

Cysteine residues in the C-terminal lobe of Src: their role in the suppression of the Src kinase.

To evaluate the function of cysteine residues of the Src kinase, we constructed a series of Src mutants in which some of cysteines were replaced to alanines. With these mutants, we studied the effect of SH-alkylating agents, N-[p-(2-benzimidazolyl)phenyl] maleimide (BIPM) and N-(9-acridinyl) maleimide (NAM), on their kinase activity. Of 10 cysteine residues scattered over v-Src, either a single mutation at Cys520 or multiple mutations at the four clustered cyteines, Cys483, Cys487, Cys496 and Cys498, yielded clear resistance to the treatment with 10 microM BIPM or 1 microM NAM. In contrast, other cysteines including those in the SH2 domain and those in the catalytic cleft of the kinase domain were dispensable for the inactivation by BIPM and NAM. Similarly, deletion of SH2 and SH3 did not confer the resistance to v-Src, suggesting the inactivation by the SH-alkylating agents is SH2/SH3-independent. Although Cys520-mutated v-Src was resistant to 1 microM NAM, it was inactivated by 5 microM NAM. However, combined mutation including all of Cys483, Cys487, Cys496, Cys498 and Cys520 yielded clear resistance to 5 microM NAM. Among these mutants, those with double mutations in the four clustered cysteines yielded a temperature sensitive phenotype in the transfected cells, whereas Cys520 did not, suggesting that Cys520 has, at least in part, a discrete function. In contrast to v-Src, c-Src, which lacks cysteine at position 520, was resistant to 1 microM NAM but sensitive to 5 microM NAM. While replacement of Phe520 of c-Src to cysteine made it sensitive to 1 microM NAM, double mutation in clustered cysteines again yielded resistance to 5 microM NAM. Taken together, our results strongly suggest that the multiple cysteine residues clustered at the end of the C-terminal lobe are critical for the inhibition by the SH-alkylating agents and, thereby, have an allosteric repressor effect on the catalytic activity of Src in a SH2-phosphoTyr527 independent manner.

A role for SHPS-1/SIRPalpha1 in IL-1beta- and TNFalpha-dependent signaling.

We investigated the role of SHPS-1/SIRPalpha1 in IL-1beta- and TNFalpha-dependent signaling that leads to the activation of Erk 1/2 and Akt. Treatment of Balb3T3 cells with IL-1beta or TNFalpha activated tyrosine phosphorylation of SHPS-1, its association with SHP-2 and the phosphorylation of Erk 1/2 and Akt. PP1, a specific inhibitor for the Src family protein tyrosine kinases, strongly inhibited tyrosine phosphorylation of SHPS-1 and complex formation of SHPS-1 with SHP-2 by IL-1beta. In addition, PP1 substantially inhibited the IL-2beta- and TNFalpha-dependent activation of Erk 1/2 and Akt. Exogenous expression of either SHPS-1 mutants that lack SHP-2 binding function or a dominant negative mutant of SHP-2 markedly inhibited the activation of Erk 1/2 and Akt by IL-1beta, whereas wild type SHPS-1 did not. Moreover, IL-1beta-stimulation induced association of SHPS-1 with IL-1RAcP, a second subunit of IL-1 receptor, whereas expression of SHPS-1 mutant that lack SHP-2 binding function clearly blocked the association and tyrosine phosphorylation of endogenous SHPS-1. Taken together, our results strongly suggest that activation of Erk 1/2 and Akt by proinflammatory cytokines requires tyrosine phosphorylation of SHPS-1 and subsequent association of SHPS-1 with SHP-2.

The PLC-PKC cascade is required for IL-1beta-dependent Erk and Akt activation: their role in proliferation.

We investigated the signaling mechanisms that lead to IL-1beta-induced cell proliferation. Treatment of Balb 3T3 cells with IL-1beta activated two signaling pathways, Erk and Akt. IL-1beta also increased tyrosine phosphorylation of PLC-gamma in Src kinase-dependent manner. Pharmacological inhibition of the PLC-PKC cascade by using specific inhibitor for PLC-gamma (U73122) and PKC (GFX) strongly inhibited IL-1beta-induced Erk and Akt activation. Inhibition of MEK1 by its specific inhibitor, PD98059 substantially inhibited Erk activation. Similarly, inhibition of PI3K activation by its specific inhibitor LY294002 suppressed Akt phosphorylation. Moreover, IL-1beta-induced association of PLC-gamma with SHPS-1. SHPS-1 mutants lacking the tyrosine phosphorylation sites failed to associate with PLC-gamma. Finally, IL-1beta-induced proliferation of Balb 3T3 cells and inhibition of Erk and Akt signalings or their upstream signaling molecules, Src kinase and PKC by their inhibitors strongly inhibited IL-1beta-dependent cell proliferation. Taken together, our results suggest that a SHPS-1-PLC-gamma complex activate the PLC-PKC cascade, which is required for the activation of IL-1beta-dependent Erk and Akt signalings and cell proliferation.

Tyrosine phosphorylation of NOS3 in a breast cancer cell line and Src-transformed cells.

We investigated the tyrosine phosphorylation of NOS3 by active Src. In a cell line derived from human breast cancer, BT474, we found activation of c-Src and tyrosine phosphorylation of NOS3. Phosphorylation of NOS3 was suppressed by treatment of BT474 with PP1, an Src kinase inhibitor, in a dose-dependent manner, suggesting that phosphorylation of NOS3 is catalyzed by active c-Src. Phosphorylation of NOS3 was further examined by a series of Src mutants. In cells expressing v-Src, substantial phosphorylation of NOS3 was observed, whereas NOS3 phosphorylation was not evident in cells expressing c-Src. Similarly, NOS1 was also phosphorylated in cells expressing v-Src. Consistently, in cells expressing a temperature-sensitive mutant of v-Src, NOS3 phosphorylation was temperature-dependent. Moreover, transforming mutant of c-Src, Y527Fc-Src, could activate NOS3 phosphorylation. In contrast, non-myristoylated form of v-Src, G2Av-Src and a kinase-inactive mutant of v-Src, K295Mv-Src, could not activate NOS3 phosphorylation. Taken together, our results suggest that active, membrane-bound form of Src can induce constitutive phosphorylation of NOS3.

Engagement of S1P1-degradative mechanisms leads to vascular leak in mice.

GPCR inhibitors are highly prevalent in modern therapeutics. However, interference with complex GPCR regulatory mechanisms leads to both therapeutic efficacy and adverse effects. Recently, the sphingosine-1-phosphate (S1P) receptor inhibitor FTY720 (also known as Fingolimod), which induces lymphopenia and prevents neuroinflammation, was adopted as a disease-modifying therapeutic in multiple sclerosis. Although highly efficacious, dose-dependent increases in adverse events have tempered its utility. We show here that FTY720P induces phosphorylation of the C-terminal domain of S1P receptor 1 (S1P1) at multiple sites, resulting in GPCR internalization, polyubiquitinylation, and degradation. We also identified the ubiquitin E3 ligase WWP2 in the GPCR complex and demonstrated its requirement in FTY720-induced receptor degradation. GPCR degradation was not essential for the induction of lymphopenia, but was critical for pulmonary vascular leak in vivo. Prevention of receptor phosphorylation, internalization, and degradation inhibited vascular leak, which suggests that discrete mechanisms of S1P receptor regulation are responsible for the efficacy and adverse events associated with this class of therapeutics.

Ramping up RANTES in the acute response to arterial injury.

Arterial injury results in the formation of neointimal lesions. Lack of resolution of the pathologic neointima leads to stenosis, tissue ischemia, and organ dysfunction. In this issue of the JCI, Kovacic et al. show that, in response to arterial injury in mice, the cytokine TNF-alpha triggers a novel signaling pathway involving the combinatorial action of two transcription factors, STAT3 and NF-kappaB (p65 subunit), in VSMCs (see the related article beginning on page 303). Upon activation, these factors turn on transcription of a potent T cell chemokine, RANTES, which selectively recruits T cells into the vessel wall as part of the vascular wound-healing response.

Cell-surface residence of sphingosine 1-phosphate receptor 1 on lymphocytes determines lymphocyte egress kinetics.

The sphingosine 1-phosphate receptor 1 (S1P(1)) promotes lymphocyte egress from lymphoid organs. Previous work showed that agonist-induced internalization of this G protein-coupled receptor correlates with inhibition of lymphocyte egress and results in lymphopenia. However, it is unclear if S1P(1) internalization is necessary for this effect. We characterize a knockin mouse (S1p1r(S5A/S5A)) in which the C-terminal serine-rich S1P(1) motif, which is important for S1P(1) internalization but dispensable for S1P(1) signaling, is mutated. T cells expressing the mutant S1P(1) showed delayed S1P(1) internalization and defective desensitization after agonist stimulation. Mutant mice exhibited significantly delayed lymphopenia after S1P(1) agonist administration or disruption of the vascular S1P gradient. Adoptive transfer experiments demonstrated that mutant S1P(1) expression in lymphocytes, rather than endothelial cells, facilitated this delay in lymphopenia. Thus, cell-surface residency of S1P(1) on T cells is a primary determinant of lymphocyte egress kinetics in vivo.

S1P/S1P1 signaling stimulates cell migration and invasion in Wilms tumor.

Sphingosine-1-phosphate (S1P) is an important regulator of cellular functions via interaction with its receptors S1P(1-5). To date, nothing is known about the S1P receptor expression and the effects of S1P signaling in Wilms tumor. In this study, we found ubiquitous expression of S1P receptors in Wilms tumor specimens and cell lines. We demonstrated that S1P(1) acted as a promigratory modulator by employing S1P(1) antagonist VPC44116, S1P(1) siRNA and adenoviral transduction in Wilms tumor cells. Further, we clarified that S1P(1)-mediated migration occurred via Gi coupling and activation of PI3K and Rac1. In addition, S1P stimulated WiT49 cell invasion through S1P(1)/Gi signaling pathway. We consider that targeting S1P(1) may be a point of therapeutic intervention in Wilms tumor.

Immunosuppressive and anti-angiogenic sphingosine 1-phosphate receptor-1 agonists induce ubiquitinylation and proteasomal degradation of the receptor.

Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the S1P1 receptor. This receptor is activated by FTY720-P, a phosphorylated derivative of the immunosuppressant and vasoactive compound FTY720. However, in contrast to the natural ligand S1P, FTY720-P appears to act as a functional antagonist, even though the mechanisms involved are poorly understood. In this study, we investigated the fate of endogenously expressed S1P1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S1P1. We show that FTY720-P is more potent than S1P at inducing receptor degradation. Pretreatment with an antagonist of S1P1, VPC 44116, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient S1P1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and beta-arrestins abolished FTY720-P-induced S1P1 receptor degradation. These data suggest that agonist-induced phosphorylation of S1P1 and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S1P1 degradation is blocked by MG132, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of S1P1 receptor, whereas S1P at concentrations that induced complete internalization was not as efficient, suggesting that receptor internalization is required but not sufficient for ubiquitinylation and degradation. We propose that the ability of FTY720-P to target the S1P1 receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties.

Secretion of matrix metalloproteinase-9 by the proinflammatory cytokine, IL-1beta: a role for the dual signalling pathways, Akt and Erk.

BACKGROUND: Matrix metalloproteinases including MMP-9 mediate matrix destruction during chronic inflammatory diseases such as arthritis and atherosclerosis. MMP-9 up-regulation by inflammatory cytokines involve interactions between several transcription factors including activator protein-1 and NFkappaB. The upstream regulatory pathways are less well understood. RESULTS: To search for the mechanism of tissue destruction in the process of inflammatory disorders, we investigated the signalling pathway critical for the activation of MMP-9 expression and secretion by IL-1beta. Treatment of Balb 3T3 cells with IL-1beta activated MMP-9 transcription and subsequent secretion in a time- and dose-dependent manner. Concomitantly, IL-1beta treatment of cells activated phosphorylation of Akt, Erk and p38. Treatment of cells with either LY294002, a PI3K inhibitor, or expression of a dominant negative form of Akt drastically suppressed the IL-1beta-dependent secretion of MMP-9. Pretreatment of cells with a MEK1 inhibitor, U0126, also strongly inhibited IL-1beta-dependent secretion of MMP-9. In contrast, pre-treatment with a specific p38 kinase inhibitor, SB203580, had no effect on IL-1beta-dependent secretion of MMP-9. In addition, cells expressing constitutively active form of Akt or MEK1 showed no clear activation of MMP-9 secretion, whereas these cells responded well to IL-1beta treatment. However, co-transfection of cells with both active Akt and MEK1 was sufficient to induce MMP-9 secretion without stimulation with IL-1beta. CONCLUSION: Taken together, our results suggest that IL-1beta stimulation of cells activates MMP-9 secretion by the activation of the dual signalling pathways, the PI3K-Akt and MEK1-Erk and constitutive activation of these pathways were sufficient to induce MMP-9 secretion.