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1.
Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the most abundant proteins in the cerebrospinal fluid. Nevertheless, its role in the central nervous system is far from clear. Here, we present evidence that L-PGDS induces glial cell migration and morphological changes in vitro and in vivo. We also identified myristoylated alanine-rich C-kinase substrate (MARCKS), heat shock proteins and actin as L-PGDS-binding proteins, demonstrating that MARCKS/Akt/Rho/Jnk pathways are involved in the L-PGDS actions in glia. We further show that the cell migration-promoting activity of L-PGDS is independent of PGD2 production. The results suggest a novel non-enzymatic function of L-PGDS protein in brain inflammation, and may have an impact on glial cell biology and brain pathology related with reactive gliosis. L-PGDS is a potential drug target that can be exploited for therapeutic intervention of glia-driven neuroinflammation and related diseases.  相似文献   

2.
Prostaglandin D synthase (PGDS) is responsible for the conversion of PGH(2) to PGD(2). Two distinct types of PGDS have been identified: hematopoietic-type PGDS (H-PGDS) and lipocalin-type PGDS (L-PGDS). L-PGDS acts as both a PGD(2)-synthesizing enzyme and as an extracellular transporter of various lipophilic small molecules. Although L-PGDS is one of the most abundant proteins in the cerebrospinal fluid, little is known about the function of L-PGDS in the central nervous system (CNS). To better understand the role of L-PGDS in the CNS, effects of L-PGDS on the migration and morphology of glial cells were investigated. The L-PGDS protein accelerated the migration of cultured glial cells. Expression of the L-pgds gene was detected in glial cells and neurons. L-PGDS protein also induced morphological changes in glia similar to the characteristic phenotypic changes in reactive gliosis. L-PGDS-induced cell migration was associated with augmented formation of actin filaments and focal adhesion, which was accompanied by activation of AKT, RhoA, and JNK pathways. L-PGDS protein injected into the mouse brain promoted migration and accumulation of astrocytes in vivo. Furthermore, the cell migration-promoting effect of L-PGDS on glial cells was independent of the PGD(2) products. The L-PGDS protein interacted with myristoylated alanine-rich protein kinase C substrate (MARCKS) to promote cell migration. These results demonstrate the critical role of L-PGDS as a secreted lipocalin in the regulation of glial cell migration and morphology. The results also indicate that L-PGDS may participate in reactive gliosis in an autocrine or paracrine manner, and may have pathological implications in neuroinflammatory diseases.  相似文献   

3.
Export of newly synthesized G protein–coupled receptors (GPCRs) remains poorly characterized. We show in this paper that lipocalin-type prostaglandin D2 (PGD2) synthase (L-PGDS) interacts intracellularly with the GPCR DP1 in an agonist-independent manner. L-PGDS promotes cell surface expression of DP1, but not of other GPCRs, in HEK293 and HeLa cells, independent of L-PGDS enzyme activity. In addition, formation of a DP1–Hsp90 complex necessary for DP1 export to the cell surface is dependent on the interaction between L-PGDS and the C-terminal MEEVD residues of Hsp90. Surprisingly, PGD2 synthesis by L-PGDS is promoted by coexpression of DP1, suggesting a possible intracrine/autocrine signaling mechanism. In this regard, L-PGDS increases the formation of a DP1–ERK1/2 complex and increases DP1-mediated ERK1/2 signaling. Our findings define a novel cooperative mechanism in which a GPCR (DP1) promotes the activity of the enzyme (L-PGDS) that produces its agonist (PGD2) and in which this enzyme in turn acts as a cofactor (of Hsp90) to promote export and agonist-dependent activity of the receptor.  相似文献   

4.
Translocation to the nucleus of diacylglycerol kinase (DGK)– ζ is dependent on a sequence homologous to the effector domain of Myristoylated Alanine Rich C-Kinase Substrate (MARCKS). These data would suggest that MARCKS could also localize to the nucleus. A single report demonstrated immunofluorescence staining of MARCKS in the nucleus; however, further experimental evidence confirming the specific domain responsible for this localization has not been reported. Here, we report that MARCKS is present in the nucleus in GBM cell lines. We then over-expressed wild-type MARCKS (WT) and MARCKS with the effector domain deleted (ΔED), both tagged with V5-epitope in a GBM cell line with low endogenous MARCKS expression (U87). We found that MARCKS-WT localized to the nucleus, while the MARCKS construct without the effector domain remained in the cytoplasm. We also found that over-expression of MARCKS-WT resulted in a significant increase in total cellular phosphatidyl-inositol (4,5) bisphosphate (PIP2) levels, consistent with prior evidence that MARCKS can regulate PIP2 levels. We also found increased staining for PIP2 in the nucleus with MARCKS-WT over-expression compared to MARCKS ΔED by immunofluorescence. Interestingly, we observed MARCKS and PIP2 co-localization in the nucleus. Lastly, we found changes in gene expression when MARCKS was not present in the nucleus (MARCKS ΔED). These data indicate that the MARCKS effector domain can function as a nuclear localization signal and that this sequence is critical for the ability of MARCKS to regulate PIP2 levels, nuclear localization, and gene expression. These data suggests a novel role for MARCKS in regulating nuclear functions such as gene expression.  相似文献   

5.
We have compared the properties of a rat aorta-derived protein kinase C substrate (p75) with those of 80 kDa kinase C substrates from rat brain (MARCKS) and rabbit aorta (p80). Rat aortic p75 appeared to be closely related to rat brain MARCKS on the basis of: solubility in perchloric acid and trichloroacetic acid, heat stability, isoelectric point (pI 4.2), overall V8 protease phosphopeptide map, and immunocrossreactivity with an antibody directed against the N-terminal domain of MARCKS. However, p75 could be distinguished from rat brain MARCKS and from the rabbit aorta-derived p80 on the basis of its consistently more rapid electrophoretic mobility in SDS-containing gels, and in terms of a unique proteolytic phosphopeptide found in MARCKS but not in aortic p75. We conclude that p75 probably belongs to the family of protein kinase C substrates represented by MARCKS, and that differences in post-translational processing (glycosylation) or mRNA processing may account for the unique properties of the p75 protein in rat aortic tissue.Abbreviations p75 75,000 Da protein - MARCKS Myristoylated Alanine-Rich C Kinase Substrate  相似文献   

6.
In the mammalian brain, nitric oxide (NO) has been implicated in neuronal signal transmissions. NO stimulates guanylate cyclase to increase intracellular cGMP, which in turn activates cGMP-dependent protein kinases (PKG), but the targets of PKG in the brain have not fully been understood. In this study, we examined cGMP-dependent phosphorylation of proteins in rat brain and found that one of the possible substrates was myristoylated alanine-rich C-kinase substrate (MARCKS), an actin-binding membrane-associated protein that regulates cell adhesion. In addition, possible degradation products of MARCKS were observed after transfection of PKG or stimulation with 8pCPT-cGMP. Western blot analysis showed that the MARCKS protein levels were decreased when the cells were stimulated with 8pCPT-cGMP. These results suggest that MARCKS is a target of PKG, and PKG-dependent phosphorylation of MARCKS results in its degradation to reduce its protein levels in the cells.  相似文献   

7.
Lipocalin-type Prostaglandin D synthase (L-PGDS) acts as the PGD2-synthesizing enzyme in the brain of various mammalian species. It belongs to the lipocalin superfamily and is the first member of this family to be recognized as an enzyme. Although the solution and crystal structure of L-PGDS has been determined to understand the molecular mechanism of catalytic reaction, the structural analysis of L-PGDS in complex with its substrate remains to be performed. Here, we present the nearly complete assignment of the backbone and side chain resonances of L-PGDS/substrate analog (U-46619) complex. This study lays the essential basis for further understanding the substrate recognition mechanism of L-PGDS.  相似文献   

8.
Abstract: Expression of the protein kinase C substrate MARCKS and other heat-stable myristoylated proteins have been studied in four cultured neural cell lines. Amounts of MARCKS protein, measured by [3H]myristate labeling and western blotting, were severalfold higher in rat C6 glioma and human HTB-11 (SK-N-SH) neuroblastoma cells than in HTB-10 (SK-N-MC) or mouse N1E-115 neuroblastoma cells. Higher levels of MARCKS mRNA were also detected in the former cell lines by S1 nuclease protection assay. At least two additional 3H-myristoylated proteins of 50 and 40–45 kDa were observed in cell extracts heated to >80°C or treated with perchloric acid. The 50-kDa protein, which bound to calmodulin in the presence of Ca2+, was more prominent in cells (N1E-115 and HTB-10) with less MARCKS, whereas neuromodulin (GAP-43) was detected in N1E-115 and HTB-11 cells only. Heating resulted in a fourfold increase in the detection of MARCKS by western blotting; this was not paralleled by a similar increase in [3H]myristate-labeled MARCKS and may be due to a conformational change affecting the C-terminal epitope or enhanced retention of the protein on nitrocellulose. Addition of β-12- O -tetradecanoylphorbol 13-acetate resulted in three- to fourfold increased phosphorylation of MARCKS in HTB-11 cells, with little increase noted in HTB-10 cells. These results indicate that MARCKS, neuromodulin, and other calmodulin-binding protein kinase C substrates exhibit distinct levels of expression in cultured neurotumor cell lines. Of these proteins, only MARCKS appears to be correlated with phorbol ester stimulation of phosphatidylcholine turnover in these cells.  相似文献   

9.
Mechanisms controlling the recycling of G protein-coupled receptors (GPCRs) remain largely unclear. We report that GGA3 (Golgi-associated, γ adaptin ear containing, ADP-ribosylation factor-binding protein 3) regulates the recycling and signaling of the PGD2 receptor DP1 through a new mechanism. An endogenous interaction between DP1 and GGA3 was detected by co-immunoprecipitation in HeLa cells. The interaction was promoted by DP1 agonist stimulation, which was supported by increased DP1-GGA3 colocalization in confocal microscopy. Pulldown assays showed that GGA3 interacts with the intracellular loop 2 and C-terminus of DP1, whereas the receptor interacts with the VHS domain of GGA3. The Arf-binding deficient GGA3 N194A mutant had the same effect as wild-type GGA3 on DP1 trafficking, suggesting a new mechanism for GGA3 in recycling. Depletion of Rab4 inhibited the GGA3 effect on DP1 recycling, revealing a Rab4-dependent mechanism. Interestingly, depletion of L-PGDS (L-type prostaglandin synthase, the enzyme that produces the agonist for DP1) impaired the ability of GGA3 to mediate DP1 recycling, while GGA3 knockdown prevented L-PGDS from promoting DP1 recycling, indicating that both proteins function interdependently. A novel interaction was observed between co-immunoprecipitated endogenous L-PGDS and GGA3 proteins in HeLa cells, and in vitro using purified recombinant proteins. Redistribution of L-PGDS towards GGA3- and Rab4-positive vesicles was induced by DP1 activation. Silencing of GGA3 inhibited ERK1/2 activation following DP1 stimulation. Altogether, our data reveal a novel function for GGA3, in a newly described association with L-PGDS, in the recycling and signaling of a GPCR, namely DP1.  相似文献   

10.
T Umekage  K Kato 《FEBS letters》1991,286(1-2):147-151
We have isolated a mouse brain cDNA clone encoding a protein of 200 amino acids (Mr 20,165) with partial homology with MARCKS (myristoylated alanine-rich C-kinase substrate). Two regions show similarity with MARCKS, one is the kinase C phosphorylation site domain which is supposed to bind calmodulin, and the other is the region near to the N-terminus, including the consensus sequence of myristoylation. It has a similar amino acid composition to MARCKS, but the content of alanine is not as high. It is distributed throughout the mouse brain, but the pattern is not identical with that of MARCKS. Both proteins may be members of a new protein family involved in coupling the protein kinase C and calmodulin signal transduction systems.  相似文献   

11.
The MARCKS protein (myristoylated alanine-rich C kinase substrate) is an actin- and calmodulin-binding protein that is expressed in many mammalian tissues. The role of MARCKS in endothelial signaling responses is incompletely understood. We found that siRNA-mediated knockdown of MARCKS in cultured endothelial cells abrogated directed cell movement in a wound healing assay. We used biochemical and cell imaging approaches to explore the role of MARCKS in endothelial signal transduction pathways activated by insulin. Insulin treatment of vascular endothelial cells promoted the dose- and time-dependent phosphorylation of MARCKS. Cell imaging and hydrodynamic approaches revealed that MARCKS is targeted to plasmalemmal caveolae and undergoes subcellular translocation in response to insulin. Insulin treatment promoted an increase in levels of the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) in plasmalemmal caveolae. The insulin-stimulated increase in caveolar PIP(2) was blocked by siRNA-mediated knockdown of MARCKS, as determined using both biochemical assays and imaging studies using FRET-based PIP(2) biosensors. The critical role of PIP(2) in MARCKS responses was explored by examining the PIP(2)- and actin-binding proteins Arp2/3 and N-WASP. Insulin promoted the rapid and robust phosphorylation of both N-WASP and Arp2/3, but these phosphorylation responses were markedly attenuated by siRNA-mediated MARCKS knockdown. Moreover, MARCKS knockdown effectively abrogated N-WASP activation in response to insulin, as determined using a FRET-based N-WASP activity biosensor. Taken together, these studies show that MARCKS plays a key role in insulin-dependent endothelial signaling to PIP(2) and is a critical determinant of actin assembly and directed cell movement in the vascular endothelium.  相似文献   

12.
We have examined the pattern of protein myristoylation in C3H10T1/2 fibroblasts during cell growth. During the growing phase of 10T1/2 cells, several proteins were radiolabelled with [3H]myristate, and among them proteins with molecular masses of 22, 35, a doublet of 42–45 and 67 kDa were labelled predominantly. The extent of myristoylation in each of these proteins changed with cell density. The amount of radioactivity incorporated into the 22 kDa protein in 10T1/2 cells decreased with increasing cell density and remained at a low level during the stationary phase. In contrast, the incorporation into the 67 kDa protein increased parallel to cell density. The density-dependent change of myristoylation was not observed in any of the transformants of 10T1/2 cells thus far examined. The 67 kDa protein was identified as MARCKS (myristoylated alanine-rich C kinase substrate) by immunoprecipitation with an anti-MARCKS antibody. By Western blot analysis, we found that the amount of MARCKS in 10T1/2 cells increased significantly analogous with cell density. Therefore, it is possible that MARCKS and the 22 kDa protein play a role in contact-mediated cell signalling in 10T1/2 cells, but the mechanism is lost in transformed cells. © 1997 John Wiley & Sons, Ltd.  相似文献   

13.
14.

Background

Overexpression of the myristolated alanine-rich C kinase substrate (MARCKS) occurs in vascular proliferative diseases such as restenosis after bypass surgery. MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation. We sought to identify the mechanism of differential regulation by MARCKS of VSMC and EC proliferation in vitro and in vivo.

Methods and Results

siRNA-mediated MARCKS knockdown in VSMCs inhibited proliferation and prevented progression from phase G0/G1 to S. Protein expression of the cyclin-dependent kinase inhibitor p27kip1, but not p21cip1 was increased by MARCKS knockdown. MARCKS knockdown did not affect proliferation in VSMCs derived from p27kip1-/- mice indicating that the effect of MARCKS is p27kip1-dependent. MARCKS knockdown resulted in decreased phosphorylation of p27kip1 at threonine 187 and serine 10 as well as, kinase interacting with stathmin (KIS), cyclin D1, and Skp2 expression. Phosphorylation of p27kip1 at serine 10 by KIS is required for nuclear export and degradation of p27kip1. MARCKS knockdown caused nuclear trapping of p27kip1. Both p27kip1 nuclear trapping and cell cycle arrest were released by overexpression of KIS, but not catalytically inactive KIS. In ECs, MARCKS knockdown paradoxically increased KIS expression and cell proliferation. MARCKS knockdown in a murine aortic injury model resulted in decreased VSMC proliferation determined by bromodeoxyuridine (BrdU) integration assay, and inhibition of vascular wall thickening. MARCKS knockdown increased the rate of re-endothelialization.

Conclusions

MARCKS knockdown arrested VSMC cell cycle by decreasing KIS expression. Decreased KIS expression resulted in nuclear trapping of p27kip1 in VSMCs. MARCKS knockdown paradoxically increased KIS expression in ECs resulting in increased EC proliferation. MARCKS knockdown significantly attenuated the VSMC proliferative response to vascular injury, but accelerated reestablishment of an intact endothelium. MARCKS is a novel translational target with beneficial cell type-specific effects on both ECs and VSMCs.  相似文献   

15.
Lipocalin-type prostaglandin D synthase (L-PGDS) is a member of the lipocalin superfamily, which is composed of secretory transporter proteins, and binds a wide variety of small hydrophobic molecules. Using this function, we have reported the feasibility of using L-PGDS as a novel drug delivery vehicle for poorly water-soluble drugs. In this study, we show the development of a drug delivery system using L-PGDS, one that enables the direct clinical use of 7-ethyl-10-hydroxy-camptothecin (SN-38), a poorly water-soluble anti-cancer drug. In the presence of 2 mM L-PGDS, the concentration of SN-38 in PBS increased 1,130-fold as compared with that in PBS. Calorimetric experiments revealed that L-PGDS bound SN-38 at a molecular ratio of 1:3 with a dissociation constant value of 60 μM. The results of an in vitro growth inhibition assay revealed that the SN-38/L-PGDS complexes showed high anti-tumor activity against 3 human cancer cell lines, i.e., Colo201, MDA-MB-231, and PC-3 with a potency similar to that of SN-38 used alone. The intravenous administration of SN-38/L-PGDS complexes to mice bearing Colo201 tumors showed a pronounced anti-tumor effect. Intestinal mucositis, which is one of the side effects of this drug, was not observed in mice administered SN-38/L-PGDS complexes. Taken together, L-PGDS enables the direct usage of SN-38 with reduced side effects.  相似文献   

16.
17.

Background and Purpose

Glucocorticoids pretreatment confers protection against neonatal hypoxic-ischemic (HI) brain injury. However, the molecular mechanism remains poorly elucidated. We tested the hypothesis that glucocorticoids protect against HI brain injury in neonatal rat by stimulation of lipocalin-type prostaglandin D synthase (L-PGDS)-induced prostaglandin D2 (PGD2)-DP1-pERK mediated signaling pathway.

Methods

Dexamethasone and inhibitors were administered via intracerebroventricular (i.c.v) injections into 10-day-old rat brains. Levels of L-PGD2, D prostanoid (DP1) receptor, pERK1/2 and PGD2 were determined by Western immunoblotting and ELISA, respectively. Brain injury was evaluated 48 hours after conduction of HI in 10-day-old rat pups.

Results

Dexamethasone pretreatment significantly upregulated L-PGDS expression and the biosynthesis of PGD2. Dexamethasone also selectively increased isoform pERK-44 level in the neonatal rat brains. Inhibitors of L-PGDS (SeCl4), DP1 (MK-0524) and MAPK (PD98059) abrogated dexamethasone-induced increases in pERK-44 level, respectively. Of importance, these inhibitors also blocked dexamethasone-mediated neuroprotective effects against HI brain injury in neonatal rat brains.

Conclusion

Interaction of glucocorticoids-GR signaling and L-PGDS-PGD2-DP1-pERK mediated pathway underlies the neuroprotective effects of dexamethasone pretreatment in neonatal HI brain injury.  相似文献   

18.
Lipocalin-type prostaglandin D synthase (L-PGDS) acts as both a PGD2 synthase and an extracellular transporter for small lipophilic molecules. From a series of biochemical studies, it has been found that L-PGDS has an ability to bind a variety of lipophilic ligands such as biliverdin, bilirubin and retinoids in vitro. Therefore, we considered that it is necessary to clarify the molecular structure of L-PGDS upon binding ligand in order to understand the physiological relevance of L-PGDS as a transporter protein. We investigated a molecular structure of L-PGDS/biliverdin complex by small-angle X-ray scattering (SAXS) and multi-dimensional NMR measurements, and characterized the binding mechanism in detail. SAXS measurements revealed that L-PGDS has a globular shape and becomes compact by 1.3 Å in radius of gyration on binding biliverdin. NMR experiments revealed that L-PGDS possessed an eight-stranded antiparallel β-barrel forming a central cavity. Upon the titration with biliverdin, some cross-peaks for residues surrounding the cavity and EF-loop and H2-helix above the β-barrel shifted, and the intensity of other cross-peaks decreased with signal broadenings in 1H–15N heteronuclear single quantum coherence spectra. These results demonstrate that L-PGDS holds biliverdin within the β-barrel, and the conformation of the loop regions above the β-barrel changes upon binding biliverdin. Through such a conformational change, the whole molecule of L-PGDS becomes compact.  相似文献   

19.
Recently wedemonstrated the induction of apoptosis by the addition ofrecombinant lipocalin-type prostaglandin D2 synthase (L-PGDS) to the culture medium of LLC-PK1 cells. Becauseprotein kinase C (PKC) has been shown to be involved in theapoptotic process of various cell types, we examined the potentialrole of L-PGDS in phorbol 12-myristate 13-acetate (PMA)-inducedapoptosis. We report here the enzymatic activation andphosphorylation of L-PGDS in response to phorbol ester in cellculture and the direct phosphorylation of recombinant L-PGDS by PKC invitro. Treatment of cells with PMA or L-PGDS decreasedphosphatidylinositol 3-kinase (PI3-K) activity and concomitantlyinhibited protein kinase B (PKB/Akt) phosphorylation, which led to thehypophosphorylation and activation of Bad. In addition,hypophosphorylation of retinoblastoma protein was also observed inresponse to L-PGDS-induced apoptosis. Cellular depletion ofL-PGDS levels by using an antisense RNA strategy prevented PI3-Kinactivation by phorbol ester and inhibited caspase-3 activation andapoptosis. We conclude that phorbol ester-induced apoptosis is mediated by L-PGDS phosphorylation and activation by PKC and is accompanied by inhibition of the PI3-K/PKBanti-apoptotic signaling pathways.

  相似文献   

20.
The regulation of vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis plays a clear role in the atherosclerotic process. Recently, we reported on the inhibition of the exaggerated growth phenotype of VSMCs isolated from hypertensive rats by lipocalin-type prostaglandin D2 synthase (L-PGDS). In the present study, we report the differential effects of L-PGDS on VSMC cell cycle progression, migration, and apoptosis in wild-type VSMCs vs. those from a type 2 diabetic model. In wild-type VSMCs, exogenously added L-PGDS delayed serum-induced cell cycle progression from the G1 to S phase, as determined by gene array analysis and the decreased protein expressions of cyclin-dependent kinase-2, p21Cip1, and cyclin D1. Cyclin D3 protein expression was unaffected by L-PGDS, although its gene expression was stimulated by L-PGDS in wild-type cells. In addition, platelet-derived growth factor-induced VSMC migration was inhibited by L-PGDS in wild-type cells. Type 2 diabetic VSMCs, however, were resistant to the L-PGDS effects on cell cycle progression and migration. L-PGDS did suppress the hyperproliferation of diabetic cells, albeit through a different mechanism, presumably involving the 2.5-fold increase in apoptosis and the concomitant 10-fold increase of L-PGDS uptake we observed in these cells. We propose that in wild-type VSMCs, L-PGDS retards cell cycle progression and migration, precluding hyperplasia of the tunica media, and that diabetic cells appear resistant to the inhibitory effects of L-PGDS, which consequently may help explain the increased atherosclerosis observed in diabetes. apoptosis; atherosclerosis; insulin resistance  相似文献   

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