Sphingosylphosphorylcholine induces apoptosis of endothelial cells through reactive oxygen species-mediated activation of ERK

Sphingosylphosphorylcholine (SPC) produces reactive oxygen species (ROS) in MS1 pancreatic islet endothelial cells. In the present study, we explored the physiological significance of the SPC-induced ROS generation in endothelial cells. SPC induced cell death of MS1 cells at higher than 10 microM concentration through a caspase-3-dependent pathway. SPC treatment induced sustained activation of an extracellular signal-regulated kinase (ERK), in contrast to transient activation of ERK in response to platelet-derived growth factor (PDGF)-BB, which stimulated proliferation of MS1 cells. Both the SPC-induced cell death and ERK activation were abolished by pretreatment of the cells with the MEK inhibitor U0126 or by overexpression of a dominant negative mutant of MEK1 (DN-MEK1). Pretreatment of the cells with N-acetylcysteine, an antioxidant, completely prevented the SPC-induced ROS generation, apoptosis, and ERK activation, whereas the ROS generation was not abrogated by treatment with U0126. Consistent with these results, SPC induced cell death of human umbilical vein endothelial cells (HUVECs) through ROS-mediated activation of ERK. These results suggest that the SPC-induced generation of ROS plays a crucial role in the cell death of endothelial cells through ERK-dependent pathway.     

Tumor necrosis factor-α-mediated suppression of dual-specificity phosphatase 4: crosstalk between NFκB and MAPK regulates endothelial cell survival

We investigated the effects of tumor necrosis factor-α (TNF-α) exposure on mitogen-activated protein kinase signaling in human microvascular endothelial cells. TNF-α caused a significant suppression of a dual specificity phosphatase, DUSP4, that regulates ERK1/2 activation. Thus, we hypothesized that suppression of DUSP4 enhances cell survival by increasing ERK1/2 signaling in response to growth factor stimulation. In support of this concept, TNF-α pre-exposure increased growth factor-mediated ERK1/2 activation, whereas overexpression of DUSP4 with an adenovirus decreased ERK1/2 compared to an empty adenovirus control. Overexpression of DUSP4 also significantly decreased cell viability, lessened recovery in an in vitro wound healing assay, and decreased DNA synthesis. Pharmacological inhibition of NFκB activation or a dominant negative construct of the inhibitor of κB significantly lessened TNF-α-mediated suppression of DUSP4 expression by 70–84 % and attenuated ERK activation, implicating NFκB-dependent pathways in the TNF-α-mediated suppression of DUSP4 that contributes to ERK1/2 signaling. Taken together, our findings show that DUSP4 attenuates ERK signaling and reduces cell viability, suggesting that the novel crosstalk between NFκB and MAPK pathways contributes to cell survival.     

Sphingosylphosphorylcholine stimulates expression of fibronectin through TGF-beta1-Smad-dependent mechanism in human mesenchymal stem cells

Sphingosylphosphorylcholine (SPC) has been reported to stimulate the expression of fibronectin (FN), which plays a key role in cell recruitment and adhesion during wound healing. In a previous study, we reported that SPC induces differentiation of human adipose tissue-derived mesenchymal stem cells (hATSCs) to smooth muscle-like cell types through ERK-dependent autocrine secretion of TGF-beta1 and delayed activation of the TGF-beta1-Smad pathway. In the present study, we demonstrated that SPC dose- and time-dependently increased the expression of FN in hATSCs. Pretreatment of the cells with U0126, an MEK inhibitor, markedly attenuated the SPC-induced expression of FN and delayed phosphorylation of Smad2, suggesting that ERK is involved in the SPC induction of FN expression through activation of Smad2. In addition, the SPC-induced expression of FN and delayed activation of Smad2 were abrogated by SB-431542, a TGF-beta type I receptor kinase inhibitor, or anti-TGF-beta1 neutralizing antibody. Furthermore, the SPC-induced expression of FN was abrogated by adenoviral expression of Smad7, an inhibitory Smad, or short interference RNA (siRNA)-mediated depletion of endogenous Smad2 expression, suggesting that SPC induces the expression of FN through ERK-dependent activation of the TGF-beta1-Smad2 crosstalk pathway. Adhesion of U937 monocytic cells to hATSCs was enhanced by pretreatment of hATSCs with SPC or TGF-beta1 for 4 days, and the peptide GRGDSP (an antagonist of fibronectin receptors) blocked the adhesion of U937 cells to the hATSCs. These results led us to suggest that SPC-induced FN expression plays a pivotal role in the wound healing by stimulating adhesion and recruitment of leukocytes.     

Increased levels of DUSP6 phosphatase stimulate tumourigenesis in a molecularly distinct melanoma subtype

The mitogen-activated protein kinase (MAPK) pathway is important in melanoma. In this pathway, DUSP6 phosphatase negatively controls the activation of extracellular signal-regulated (ERK) kinase. Through comparison of melanoma signalling pathways between immortal mouse melanocytes and their tumourigenic derivatives, retrieved from mouse xenografts, we identified a molecularly distinct subtype of melanoma, characterized by reduced ERK activity and increased DUSP6 expression. Overexpression of DUSP6 enhanced anchorage-independent growth and invasive ability of immortal mouse melanocytes, suggesting that increased DUSP6 expression contributes to melanoma formation in the mouse xenografts. In contrast, reduced tumourigenicity was observed after DUSP6 overexpression in human melanoma cells. A minority of thick human primary melanomas had high DUSP6 expression and the same poor melanoma-specific survival as the majority of thick primaries with low DUSP6 levels. We have demonstrated that DUSP6 is important in melanoma and that it plays a different role in our distinct subtype of mouse melanoma compared with that in classic human melanoma.     

Role of MEK-ERK pathway in sphingosylphosphorylcholine-induced cell death in human adipose tissue-derived mesenchymal stem cells

Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in a variety of cellular responses. In the present study, we demonstrated that treatment of human adipose tissue-derived mesenchymal stem cells (hATSCs) with D-erythro-SPC resulted in apoptosis-like cell death, as demonstrated by decreased cell viability, DNA strand breaks, the increase of sub-G1 fraction, cytochrome c release into cytosol, and activation of caspase-3. In contrast, the exposure of hATSCs to L-threo-SPC did not induce the cell death, suggesting that the SPC-induced cell death was selective for the D-erythro-stereoisomer of SPC. The D-erythro-SPC-induced cell death was prevented by DEVD-CHO, a caspase-3 specific inhibitor, and Z-VAD-FMK, a general caspase inhibitor, suggesting that the SPC-induced cell death of hATSCs occurs through the cytochrome c- and caspase-3-dependent pathways. In addition, D-erythro-SPC treatment stimulated the activation of mitogen-activated protein kinases, such as ERK and c-Jun NH2-terminal protein kinase (JNK), and the D-erythro-SPC-induced cell death was completely prevented by pretreatment with the MEK inhibitor, U0126, but not by pretreatment with the JNK inhibitor, SP600125, and the p38 MAPK inhibitor, SB202190, suggesting a specific involvement of ERK in the D-erythro-SPC-induced cell death. Pretreatment with U0126 attenuated the D-erythro-SPC-induced release of cytochrome c. From these results, we suggest that ERK is involved in the SPC-induced cell death of hATSC through stimulation of the cytochrome c/caspase-3-dependent pathway.     

Dual-specificity phosphatase 6 deletion protects the colonic epithelium against inflammation and promotes both proliferation and tumorigenesis

The Ras/mitogen-activated protein kinase (MAPK) pathway controls fundamental cellular processes such as proliferation, differentiation, and apoptosis. The dual-specificity phosphatase 6 (DUSP6) regulates cytoplasmic MAPK signaling by dephosphorylating and inactivating extracellular signal-regulated kinase (ERK1/2) MAPK. To determine the role of DUSP6 in the maintenance of intestinal homeostasis, we characterized the intestinal epithelial phenotype of Dusp6 knockout (KO) mice under normal, oncogenic, and proinflammatory conditions. Our results show that loss of Dusp6 increased crypt depth and epithelial cell proliferation without altering colonic architecture. Crypt regeneration capacity was also enhanced, as revealed by ex vivo Dusp6 KO organoid cultures. Additionally, loss of Dusp6 induced goblet cell expansion without affecting enteroendocrine and absorptive cell differentiation. Our data also demonstrate that Dusp6 KO mice were protected from acute dextran sulfate sodium-induced colitis, as opposed to wild-type mice. In addition, Dusp6 gene deletion markedly enhanced tumor load in Apc ^Min/+ mice. Decreased DUSP6 expression by RNA interference in HT29 colorectal cancer cells enhanced ERK1/2 activation levels and promoted both anchorage-independent growth in soft agar as well as invasion through Matrigel. Finally, DUSP6 mRNA expression in human colorectal tumors was decreased in advanced stage tumors compared with paired normal tissues. These results demonstrate that DUSP6 phosphatase, by controlling ERK1/2 activation, regulates colonic inflammatory responses, and protects the intestinal epithelium against oncogenic stress.     

Sphingosylphosphorylcholine induces proliferation of human adipose tissue-derived mesenchymal stem cells via activation of JNK

Sphingosylphosphorylcholine (SPC) has been implicated in a variety of cellular responses, including proliferation and differentiation. In this study, we demonstrate that d-erythro-SPC, but not l-threo-SPC, stereoselectively stimulated the proliferation of human adipose tissue-derived mesenchymal stem cells (hADSCs), with a maximal increase at 5 microM, and increased the intracellular concentration of Ca(2+) ([Ca(2+)](i)) in hADSCs, which do not express known SPC receptors (i.e., OGR1, GPR4, G2A, and GPR12). The SPC-induced proliferation and increase in [Ca(2+)](i) were sensitive to pertussis toxin (PTX) and the phospholipase C (PLC) inhibitor U73122, suggesting that PTX-sensitive G proteins, Gi or Go, and PLC are involved in SPC-induced proliferation. In addition, SPC treatment induced the phosphorylation of c-Jun and extracellular signal-regulated kinase, and SPC-induced proliferation was completely prevented by pretreatment with the c-Jun N-terminal kinase (JNK)-specific inhibitor SP600125 but not with the MEK-specific inhibitor U0126. Furthermore, the SPC-induced proliferation and JNK activation were completely attenuated by overexpression of a dominant negative mutant of JNK2, and the SPC-induced activation of JNK was inhibited by pretreatment with PTX or U73122. Treatment of hADSCs with lysophosphatidic acid (LPA) receptor antagonist, Ki16425, had no impact on the SPC-induced increase in [Ca(2+)](i). However, SPC-induced proliferation was partially, but significantly, attenuated by pretreatment of the cells with Ki16425.These results indicate that SPC stimulates the proliferation of hADSCs through the Gi/Go-PLC-JNK pathway and that LPA receptors may be responsible in part for the SPC-induced proliferation.     

Sphingosylphosphorylcholine generates reactive oxygen species through calcium-, protein kinase Cdelta- and phospholipase D-dependent pathways

Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in numerous biological processes. Treatment of MS1 pancreatic islet endothelial cells with SPC increased phospholipase D (PLD) activity in a time- and dose-dependent manner. In addition, treatment of the MS1 cells with 10 microM SPC induced stimulation of phospholipase C (PLC) activity and transient elevation of intracellular Ca2+. The SPC-induced PLD activation was prevented by pretreatment of the MS1 cells with a PLC inhibitor, U73122, and an intracellular Ca2+-chelating agent, BAPTA-AM. This suggests that PLC-dependent elevation of intracellular Ca2+ is involved in the SPC-induced activation of PLD. The SPC-dependent PLD activity was also almost completely prevented by pretreatment with pan-specific PKC inhibitors, GF109203X and RO-31-8220, and with a PKCdelta-specific inhibitor, rottlerin, but not by pretreatment with GO6976, a conventional PKC isozymes-specific inhibitor. Adenoviral overexpression of a kinase-deficient mutant of PKCdelta attenuated the SPC-induced PLD activity. These results suggest that PKCdelta plays a crucial role for the SPC-induced PLD activation. The SPC-induced PLD activation was preferentially potentiated in COS-7 cells transfected with PLD2 but not with PLD1, suggesting a specific implication of PLD2 in the SPC-induced PLD activation. SPC treatment induced phosphorylation of PLD2 in COS-7 cells, and overexpression of the kinase-deficient mutant of PKCdelta prevented the SPC-induced phosphorylation of PLD2. Furthermore, SPC treatment generated reactive oxygen species (ROS) in MS1 cells and the SPC induced production of ROS was inhibited by pretreatment with U73122, BAPTA-AM, and rottlerin. In addition, pretreatment with a PLD inhibitor 1-butanol and overexpression of a lipase-inactive mutant of PLD2 but not PLD1 attenuated the SPC-induced generation of ROS. These results suggest that PLC-, Ca2+-, PKCdelta-, and PLD2-dependent pathways are essentially required for the SPC induced ROS generation.     

Novel participation of transglutaminase-2 through c-Jun N-terminal kinase activation in sphingosylphosphorylcholine-induced keratin reorganization of PANC-1 cells

Sphingosylphosphorylcholine (SPC) is found at increased levels in the malignant ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments that contribute to the viscoelasticity of metastatic cancer cells. In this study, we investigated the role and molecular mechanisms of Tgase-2 in SPC-induced K8 phosphorylation and perinuclear reorganization in PANC-1 cells (PAN(WT)), and in PANC-1 cells that stably expressed shTgase-2 or Tgase-2 (PAN(shTg2) and PAN(Tg2)). SPC induces the expression of Tgase-2 in a time- and dose-dependent manner. Gene silencing of Tgase-2 or cystamine suppressed the SPC-induced phosphorylation and perinuclear reorganization of K8 and suppressed the SPC-induced migration of PANC-1 cells. An inhibitor of c-Jun N-terminal kinase (JNK), SP600125, suppressed the SPC-induced phosphorylation of serine 431 in K8 and keratin reorganization. Next, we examined the effect of Tgase-2 on JNK activation of serine 431 phosphorylation in K8. Tgase-2 gene silencing suppressed the expression of active form JNK (pJNK). Constitutive or tetracyclin-induced conditional expression of Tgase-2 increased the levels of pJNK. Tgase-2 was coimmunoprecipitated with K8 and JNK. In addition, K8 was coimmunoprecipitated with Tgase-2 and JNK. JNK was also coimmunoprecipitated with K8 and Tgase-2. Overall, these results suggest that Tgase-2 is involved in SPC-induced phosphorylation and perinuclear reorganization of K8 by activating JNK and forming a triple complex with K8 and JNK. Therefore, SPC-induced Tgase-2 might be a new target for modulating keratin reorganization, metastasis of cancer cells and JNK activation.     

Regulation of the inducible nuclear dual-specificity phosphatase DUSP5 by ERK MAPK

DUSP5 is an inducible, nuclear, dual-specificity phosphatase, which specifically interacts with and inactivates the ERK1/2 MAP kinases in mammalian cells. In addition, expression of DUSP5 causes nuclear translocation of ERK2 indicating that it may act as a nuclear anchor for the inactive kinase. Here we show that induction of DUSP5 mRNA and protein in response to growth factors is dependent on ERK1/2 activation and that the accumulation of DUSP5 protein is regulated by rapid proteasomal degradation. DUSP5 is phosphorylated by ERK1/2 both in vitro and in vivo on three sites (Thr321, Ser346 and Ser376) within its C-terminal domain. DUSP5 phosphorylation is absolutely dependent on the conserved kinase interaction motif (KIM) within the amino-terminal domain of DUSP5, indicating that the same protein–protein contacts are required for both the inactivation of ERK2 by DUSP5 and for DUSP5 to act as a substrate for this MAPK. Using a combination of pharmacological inhibitors and phospho-site mutants we can find no evidence that phosphorylation of DUSP5 by ERK2 significantly affects either the half-life of the DUSP5 protein or its ability to bind to, inactivate or anchor ERK2 in the nucleus. However, co-expression of ERK2 results in significant stabilisation of DUSP5, which is accompanied by reduced levels of DUSP5 ubiquitination. These changes are independent of ERK2 kinase activity but absolutely depend on the ability of ERK2 to bind to DUSP5. We conclude that DUSP5 is stabilised by complex formation with its physiological substrate and that this may reinforce its activity as both a phosphatase and nuclear anchor for ERK2.     

Ceramide inhibits cell proliferation through Akt/PKB inactivation and decreases melanin synthesis in Mel-Ab cells

Ceramide is a bioactive sphingolipid that mediates a variety of cell functions. However, the effects of ceramide on cell growth and the melanogenesis of melanocytes are not known. In the present study, we investigated the actions of cell-permeable ceramide and its possible role in the signaling pathway of a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Our results show that C2-ceramide inhibits DNA synthesis in Mel-Ab cells and G361 human melanoma cells in a dose-dependent manner. Cell cycle analysis confirmed the inhibition of DNA synthesis by a reduction in the S phase. To investigate the ceramide signaling pathway, we studied whether C2-ceramide is able to influence extracellular signal-regulated kinase (ERK) and/or Akt/protein kinase B (PKB) activation. We demonstrated that phosphorylated Akt/PKB is decreased by C2-ceramide, whereas phosphorylated ERK was only slightly affected. Therefore, the C2-ceramide-induced inactivation of Akt/PKB may be closely related to the reduced cell proliferation of Mel-Ab cells. Furthermore, we assessed the effects of C2-ceramide on the pigmentation of Mel-Ab cells. The results obtained showed that the melanin content of cells was significantly reduced by C2-ceramide at concentrations in the range of 1-10 microM, and that the pigmentation-inhibiting effect of C2-ceramide is much greater than that of kojic acid at 1-100 microM. In addition, we found that the activity of tyrosinase is reduced by C2-ceramide treatment. Our results demonstrate that C2-ceramide reduces the pigmentation of Mel-Ab cells by inhibiting tyrosinase activity.     

Protein phosphatase 2A activity associated with Golgi membranes during the G2/M phase may regulate phosphorylation of ERK2

The extracellular signal-regulated kinase (ERK) 1 and 2 proteins are mitogen-activated protein kinase (MAPK) members that regulate cell proliferation and differentiation. ERK proteins are activated exclusively by MAPK kinase 1 and 2 phosphorylation of threonine and tyrosine residues located within the conserved TXY MAPK activation motif. Although dual phosphorylation of Thr and Tyr residues confers full activation of ERK, in vitro studies suggest that a single phosphorylation on either Thr or Tyr may yield partial ERK activity. Previously, we have demonstrated that phosphorylation of the tyrosine residue (Tyr(P) ERK) may be involved in regulating the Golgi complex structure during the G2 and M phases of the cell cycle (Cha, H., and Shapiro, P. (2001) J. Cell Biol. 153, 1355-1368). In the present study, we examined mechanisms for generating Tyr(P) ERK by determining cell cycle-dependent changes in localized phosphatase activity. Using fractionated nuclei-free cell lysates, we find increased serine/threonine phosphatase activity associated with Golgi-enriched membranes in cells synchronized in the late G2/early M phase as compared with G1 phase cells. The addition of phosphatase inhibitors in combination with immunodepletion assays identified this activity to be related to protein phosphatase 2A (PP2A). The increased activity was accounted for by elevated PP2A association with mitotic Golgi membranes as well as increased catalytic activity after normalization of PP2A protein levels in the phosphatase assays. These data indicate that localized changes in PP2A activity may be involved in regulating proteins involved in Golgi disassembly as cells enter mitosis.     

Protein phosphatase 2A, a negative regulator of the ERK signaling pathway, is activated by tyrosine phosphorylation of putative HLA class II-associated protein I (PHAPI)/pp32 in response to the antiproliferative lectin, jacalin

Protein phosphatase 2A (PP2A) is a family of mammalian serine/threonine phosphatases that is involved in the control of many cellular functions including those mediated by extracellular signal-regulated kinase (ERK) signaling. While investigating the reversible antiproliferative effect of the dietary lectin, jacalin, which binds the Thomsen-Friedenreich antigen (galactose beta1-3 N-acetylgalactosamine alpha-), we have found that this lectin (30 microg/ml) induces rapid, transient, tyrosine phosphorylation of putative human HLA-DR-associated protein I (PHAPI, also known as the tumor suppressor pp32) in HT29 human colon cancer cells. This is accompanied by the release of PP2A from association with PHAPI, allowing increased phosphatase activity of PP2A (by 42 +/- 10% at 10 min) and consequent complete dephosphorylation of the ERK kinase, MEK1/2, by 10 min and of ERK1/2 by 60 min. PHAPI knockdown by RNA interference abolished the effects of jacalin on PP2A activation and MEK inhibition. Thus phosphorylation of PHAPI/pp32 is a critical regulatory step in PP2A activation and ERK signaling.