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Background In endothelial cells, phospholipase C (PLC) β1-activated Ca 2+ is a crucial second messenger for the signaling pathways governing angiogenesis. PLCβ1 is inactivated by complexing with an intracellular protein called translin-associated factor X (TRAX). This study demonstrates specific interactions between Globo H ceramide (GHCer) and TRAX, which highlight a new angiogenic control through PLCβ1 activation. Methods Globo-series glycosphingolipids (GSLs), including GHCer and stage-specific embryonic antigen-3 ceramide (SSEA3Cer), were analyzed using enzyme-linked immunosorbent assay (ELISA) and Biacore for their binding with TRAX. Angiogenic activities of GSLs in human umbilical vein endothelial cells (HUVECs) were evaluated. Molecular dynamics (MD) simulation was used to study conformations of GSLs and their molecular interactions with TRAX. Fluorescence resonance energy transfer (FRET) analysis of HUVECs by confocal microscopy was used to validate the release of PLCβ1 from TRAX. Furthermore, the in vivo angiogenic activity of extracellular vesicles (EVs) containing GHCer was confirmed using subcutaneous Matrigel plug assay in mice. Results The results of ELISA and Biacore analysis showed a stable complex between recombinant TRAX and synthetic GHCer with K d of 40.9 nM. In contrast, SSEA3Cer lacking a fucose residue of GHCer at the terminal showed ~ 1000-fold decrease in the binding affinity. These results were consistent with their angiogenic activities in HUVECs. The MD simulation indicated that TRAX interacted with the glycan moiety of GHCer at amino acid Q223, Q219, L142, S141, and E216. At equilibrium the stable complex maintained 4.6 ± 1.3 H-bonds. TRAX containing double mutations with Q223A and Q219A lost its ability to interact with GHCer in both MD simulation and Biacore assays. Removal of the terminal fucose from GHCer to become SSEA3Cer resulted in decreased H-bonding to 1.2 ± 1.0 by the MD simulation. Such specific H-bonding was due to the conformational alteration in the whole glycan which was affected by the presence or absence of the fucose moiety. In addition, ELISA, Biacore, and in-cell FRET assays confirmed the competition between GHCer and PLCβ1 for binding to TRAX. Furthermore, the Matrigel plug assay showed robust vessel formation in the plug containing tumor-secreted EVs or synthetic GHCer, but not in the plug with SSEA3Cer. The FRET analysis also indicated the disruption of colocalization of TRAX and PLCβ1 in cells by GHCer derived from EVs. Conclusions Overall, the fucose residue in GHCer dictated the glycan conformation for its complexing with TRAX to release TRAX-sequestered PLCβ1, leading to Ca 2+ mobilization in endothelial cells and enhancing angiogenesis in tumor microenvironments.

Lipid membrane curvature plays important roles in various physiological phenomena. Curvature-regulated dynamic membrane remodeling is achieved by the interaction between lipids and proteins. So far, several membrane sensing/sculpting proteins, such as Bin/amphiphysin/Rvs (BAR) proteins, are reported, but there remains the possibility of the existence of unidentified membrane-deforming proteins that have not been uncovered by sequence homology. To identify new lipid membrane deformation proteins, we applied liposome-based microscopic screening, using unbiased-darkfield microscopy. Using this method, we identified phospholipase Cβ1 (PLCβ1) as a new candidate. PLCβ1 is well characterized as an enzyme catalyzing the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). In addition to lipase activity, our results indicate that PLCβ1 possessed the ability of membrane tubulation. Lipase domains and inositol phospholipids binding the pleckstrin homology (PH) domain of PLCβ1 were not involved, but the C-terminal sequence was responsible for this tubulation activity. Computational modeling revealed that the C terminus displays the structural homology to the BAR domains, which is well known as a membrane sensing/sculpting domain. Overexpression of PLCβ1 caused plasma membrane tubulation, whereas knockdown of the protein reduced the number of caveolae and induced the evagination of caveolin-rich membrane domains. Taken together, our results suggest a new function of PLCβ1: plasma membrane remodeling, and in particular, caveolae formation.

Motherhood goes through preparation, onset and maintenance phases until the natural weaning. A variety of changes in hormonal/neurohormonal systems and brain circuits are involved in the maternal behavior. Hormones, neuropeptides, and neurotransmitters involved in maternal behavior act via G-protein-coupled receptors, many of which in turn activate plasma membrane enzymes including phospholipase C (PLC) β isoforms. In this study, we examined the effect of PLCβ1 knockout (KO) on maternal behavior. There was little difference between PLCβ1-KO and wild-type (WT) dams in the relative time spent in maternal behavior during the period between 24 h prepartum and 12 h postpartum (−24 h ∼ PPH 12). After PPH 18, however, PLCβ1-KO dams neglected their pups so that they all died in 2-3 days. In the pup retrieval test, latency was not different during the period within PPH 12, but after PPH 18, PLCβ1-KO dams could not finish pup retrieval in a given time. During both periods, FosB expression in the nucleus accumbens (NAcc) of PLCβ1-KO dams was significantly lower than WT, but not different in the medial preoptic area (mPOA). Given that mPOA activity is required for initiation of maternal behavior, and that NAcc is known to be involved in maternal motivation and maintenance of maternal behavior, our results suggest that PLCβ1 signaling is essential for transition from the onset to maintenance phase of maternal behavior.

The persistent administration of β2-adrenergic (β2AR) agonists has been demonstrated to increase the risk of severe asthma, partly due to the induction of tolerance to bronchoprotection via undefined mechanisms. The present study investigated the potential effect of the long-acting β2-adrenergic agonist, formoterol, on the expression of muscarinic M3 receptor (M3R) in rat airway smooth muscle cells (ASMCs). Primary rat ASMCs were isolated and characterized following immunostaining with anti-α-smooth muscle actin antibodies. The protein expression levels of M3R and phospholipase C-β1 (PLCβ1) were characterized by western blot analysis and the production of inositol 1,4,5-trisphosphate (IP3) was determined using an enzyme-linked immunosorbent assay. Formoterol increased the protein expression of M3R in rat ASMCs in a time- and dose-dependent manner, which was significantly inhibited by the β2AR antagonist, ICI118,551 and the cyclic adenosine monophosphate (cAMP) inhibitor, SQ22,536. The increased protein expression of M3R was positively correlated with increased production of PLCβ1 and IP3. Furthermore, treatment with the glucocorticoid, budesonide, and the PLC inhibitor, U73,122, significantly suppressed the formoterol-induced upregulated protein expression levels of M3R and PLCβ1 and production of IP3. The present study demonstrated that formoterol mediated the upregulation of M3R in the rat ASMCs by activating the β2AR-cAMP signaling pathway, resulting in increased expression levels of PLCβ1 and IP3, which are key to inducing bronchoprotection tolerance. Administration of glucocorticoids or a PLC antagonist prevented formoterol-induced bronchoprotection tolerance by suppressing the protein expression of M3R.

Myelodysplastic syndrome (MDS) is an adult hematological disease that evolves into acute myeloid leukemia (AML) in about 30% of the cases. The availability of a highly specific probe moved us to perform in patients affected with MDS/AML, associated with normal karyotype, painting and fluorescence in situ hybridization (FISH) analysis aimed to check the inositide-specific phospholipase C (PI-PLC) β1 gene, a player in the control of some checkpoints of the cell cycle. Here we present a preliminary observation in which FISH analysis disclosed in a small group of MDS/AML patients with normal karyotype the monoallelic deletion of the PI-PLCβ1 gene. On the contrary, PI-PLC β4, another gene coding for a signaling molecule, located on 20p12.3 at a distance as far as less than 1Mb from PI-PLCβ1, is unaffected in MDS patients with the deletion of PI-PLC β1 gene, hinting at an interstitial deletion. The MDS patients, bearing the deletion, rapidly evolved to AML. The data suggest the possible involvement of PI-PLCβ1 in the progression of the disease and pave the way for a larger investigation aimed at identifying a possible high-risk group among MDS patients with a normal karyotype.

By using a retrovirus‐derived system we generated derivatives of the human colon adenocarcinoma cell line LS174T (ATCC CL 188) that stably overexpress a full‐length cDNA encoding the β1 isoform of bovine phosphoinositides‐specific phospholipase C (PI‐PLC). This was confirmed by the elevated levels of catalytic activity to release phosphoinositides from phosphatidylinositol (PI‐PLC) or phosphatidylinositol‐bis‐phosphate (PIP2‐PLC), and the enhanced expressions of messenger RNA and protein. PI‐PLC β1 overexpresser clones grew to form cell clumps floating in liquid medium, whereas the pMV7‐introduced control clones displayed morphologic characteristics that were very similar to those of the parent LS174T cell line. Three individual PI‐PLC β1 overexpresser cell lines displayed increased doubling time (18.0 h, 21.5 h, and 23.8 h) when compared with 4 individual pMV7‐introduced control cell lines (13.1 h, 10.7 h, 12.9 h, and 9.3 h). Anchorage‐independent growth ability in soft agar medium was dramatically suppressed by overexpression of PLC β1, and the ability of PLC‐overproducer clones to form aggregates when cultured in liquid medium was dramatically enhanced when compared with that of pMV7‐introduced control clones. Tumorigenicity of PLC β1‐overproducers was much weaker than that of vector‐transduced control clones. The spontaneous release of carcinoembryonic antigen from PLC β1‐overproducer clones was much higher than that from pMV7 control clones. The ability of PLC β1‐overproducer clones to form aggregates during suspension culture was much stronger than that of the control clones. These results provide the first evidence that elevated levels of endogenous PI‐PLC β1 suppress tumor cell growth, but enhance the ability to form cell aggregates and to release carcinoembryonic antigen, an intercellular adhesion molecule.