Calcium-independent phospholipase A2 through arachidonic acid mobilization is involved in Caco-2 cell growth

Several studies indicate that phospholipase A(2) (PLA(2)) expression and/or activation account for the high levels of arachidonic acid (AA) detected in cancer and, together with the elevated expression of cyclooxygenase-2, lead to cell proliferation and tumor formation. Using Caco-2 cells, a human colorectal carcinoma cell, we studied the role of high-molecular-weight PLA(2)s, cytosolic PLA(2) (cPLA(2)), and calcium-independent PLA(2) (iPLA(2)) in the AA cascade and in cell growth. Treatment with an antisense oligonucleotide against cPLA(2)alpha decreased [(3)H]AA release induced by ionophore A23187 or by a phorbol ester but did not affect the release of [(3)H]AA, [(3)H]thymidine incorporation, or Caco-2 growth induced by fetal calf serum (FCS). However, these parameters were significantly modified by iPLA(2) inhibitors and by an antisense oligonucleotide against iPLA(2)beta. Our results show that iPLA(2) was involved in AA release and the subsequent prostaglandin production induced by serum. Moreover, these data indicate that iPLA(2) may be involved in the signaling pathways involved in the control of Caco-2 proliferation.     

Kidney injury molecule-1 is involved in the chemotactic migration of mesenchymal stem cells

A better understanding of the organ specific factors that regulate the migration of mesenchymal stem cells (MSCs) into the target organ is essential for optimization of strategies to improve the repair after injury. In the present study, we showed that the kidney injury molecule-1 (KIM-1), a well-known kidney-specific biomarker, enhanced the in vitro migration capacity of MSCs as a potent kidney-specific chemo-attractant or an inducer. The in vitro roles were verified by migration assay using KIM1-PK1 cell lines, the mouse proximal tubular epithelial cells (mPTEs) and recombinant human KIM-1 proteins (rhKIM-1). Immunofluorescence staining displayed specific ectodomain binding of KIM-1 on the surface of MSCs. Upregulation of chemokine receptor type 4 (CXCR4) protein when treated with tumor necrosis factor alpha (TNF-α) was shown. The effect of KIM-1 on migration of MSCs was augmented by TNF-α pretreatment in a dose-dependent manner, and reduced by AMD3100, an antagonist of CXCR4. These results suggest that KIM-1 is a potential chemo-ligand of CXCR4 and may play an important role in kidney-specific migration of MSCs via interaction between KIM-1 and CXCR4.     

Phosphoinositide-specific phospholipase C9 is involved in the thermotolerance of Arabidopsis

Intracellular calcium (Ca(2+)) increases rapidly after heat shock (HS) in the Ca(2+)/calmodulin (Ca(2+)/CaM) HS signal transduction pathway: a hypothesis proposed based on our previous findings. However, evidence for the increase in Ca(2+) after HS was obtained only through physiological and pharmacological experiments; thus, direct molecular genetic evidence is needed. The role of phosphoinositide-specific phospholipase C (PI-PLC) is poorly understood in the plant response to HS. In this work, atplc9 mutant plants displayed a serious thermosensitive phenotype compared with wild-type (WT) plants after HS. Complementation of atplc9 with AtPLC9 rescued both the basal and acquired thermotolerance phenotype of the WT plants. In addition, thermotolerance was even improved in overexpressed lines. The GUS staining of AtPLC9 promoter:GUS transgenic seedlings showed that AtPLC9 expression was ubiquitous. The fluorescence distribution of the fusion protein AtPLC9 promoter:AtPLC9:GFP revealed that the subcellular localization of AtPLC9 was restricted to the plasma membrane. The results of a PLC activity assay showed a reduction in the accumulation of inositol-1,4,5-trisphosphate (IP(3)) in atplc9 during HS and improved IP(3) generation in the overexpressed lines. Furthermore, the heat-induced increase in intracellular Ca(2+) was decreased in atplc9. Accumulation of the small HS proteins HSP18.2 and HSP25.3 was downregulated in atplc9 and upregulated in the overexpressed lines after HS. Together, these results provide molecular genetic evidence showing that AtPLC9 plays a role in thermotolerance in Arabidopsis.     

Localization of Tie2 and phospholipase D in endothelial caveolae is involved in angiopoietin-1-induced MEK/ERK phosphorylation and migration in endothelial cells

Angiopoietin-1 (Ang1) and its receptor, Tie2, play critical roles in blood vessel formation. Ang1 triggers a variety of signaling events in endothelial cells leading to vasculogenic and angiogenic processes. However, the underlying mechanism for Ang1/Tie2 signaling is not fully understood. Here, we show that Tie2 and phospholipase D (PLD) are localized in the caveolae, specialized subdomains of the endothelial cell plasma membrane enriched with signaling molecules. Interestingly, Ang1 increased PLD activities in a dose- and time-dependent manner. Ang1-induced MEK/ERK activation was abrogated when PLD was inhibited, suggesting that PLD mediates Ang1-induced MEK/ERK activation. Moreover, PLD inhibitor, 1-butanol, inhibited Ang1-induced endothelial cell migration. Our results indicate that: (1) caveolae may be the platform for Tie2/PLD association in endothelial cells; (2) PLD is a new mediator of Ang1/Tie2-induced signaling pathway, and it participates in MAPK activation and endothelial cell migration.     

Migration through host cells activates Plasmodium sporozoites for infection

Plasmodium sporozoites, the infective stage of the malaria parasite transmitted by mosquitoes, migrate through several hepatocytes before infecting a final one. Migration through hepatocytes occurs by breaching their plasma membranes, and final infection takes place with the formation of a vacuole around the sporozoite. Once in the liver, sporozoites have already reached their target cells, making migration through hepatocytes prior to infection seem unnecessary. Here we show that this migration is required for infection of hepatocytes. Migration through host cells, but not passive contact with hepatocytes, induces the exocytosis of sporozoite apical organelles, a prerequisite for infection with formation of a vacuole. Sporozoite activation induced by migration through host cells is an essential step of Plasmodium life cycle.     

A putative phospholipase C is involved in Pichia fermentans dimorphic transition

Background

Pichia fermentans DiSAABA 726 is a dimorphic yeast that reversibly shifts from yeast-like to pseudohyphal morphology. This yeast behaves as a promising antagonist of Monilia spp. in the yeast-like form, but becomes a destructive plant pathogen in the pseudohyphal form thus raising the problem of the biological risk associated with the use of dimorphic yeasts as microbial antagonists in the biocontrol of phytopathogenic fungi.

Methods

Pichia fermentans DiSAABA 726 was grown in urea- and methionine-containing media in order to induce and separate yeast-like and pseudohyphal morphologies. Total RNA was extracted from yeast-like cells and pseudohyphae and retro-transcribed into cDNA. A rapid subtraction hybridization approach was utilized to obtain the cDNA sequences putatively over-expressed during growth on methionine-containing medium and involved in pseudohyphal transition.

Results

Five genes that are over-expressed during yeast-like/pseudohyphal dimorphic transition were isolated. One of these, encoding a putative phospholipase C, is involved in P. fermentans filamentation. In fact, while the inhibition of phospholipase C, by means of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (Et-18), is accompanied by a significant reduction of pseudohyphae formation in P. fermentans, the addition of exogenous cAMP fully restores pseudohyphal growth also in the presence of Et-18.

Conclusion

Phospholipase C is part of a putative “methionine sensing machinery” that activates cAMP-PKA signal transduction pathway and controls P. fermentans yeast-like/pseudohyphal dimorphic transition.

General significance

Phospholipase C is a promising molecular target for further investigations into the link between pseudohyphae formation and pathogenicity in P. fermentans.     

Cell-membrane phospholipase C is involved in inducing the antiviral effect of interferon

A monospecific inhibitory antibody directed to phospholipase C (phosphoinositidase C) blocked the antiviral effect of human interferons alpha and beta when tested on human quiescent fibroblasts challenged with the vesicular stomatitis virus. This action was due to specific inhibition of polyphosphoinositide hydrolysis because (a) the F(ab)₂ fragment of the antibody molecule was also inhibitory; (b) excess antibodies directed to phospholipase A₂ and to a phosphatidylcholine-preferring phospholipase C did not have any inhibitory effect, and (c) the combination of 12-O-tetradecanoylphorbol-acetate and calcium ionophore A23187 had an interferon-like antiviral effect which was not influenced by the inhibitory anti-phospholipase C antibodies. To avoid an interferon-like effect due to induction of interferon by second messengers, Vero cells, which lack interferon biosynthesis, were also used. Liposomes containing inositol 1,4,5-triphosphate and 1-oleoyl-2-acetyl-rac-glycerol protected Vero cells against the infection with the vesicular stomatitis virus. These results taken together show that phosphoinositide-derived second messengers are involved in triggering the antiviral effect of interferons alpha and beta.     

Merozoite surface antigen-I of plasmodium

The merozoite is the invasive form of the asexual stage of Plasmodium species. At least two polymorphic glycoproteins have been found on its surface in the human malaria parasite Plasmodium falciparum. The best-characterized of these is known as merozoite surface antigen-1 (MSA1) (185-200 kDa) (Ref. 1). Similar molecules are found in other malaria species. The other merozoite surface antigen, MSA2 (35-48 kDa) (Ref. 2), is distinct from MSA1 but is equally polymorphic. In this review, Juan Cooper condenses the body of structural information on MSA1 known to date. A database compiled from MSA1 sequences from several species used together with sequence comparisons and predicted secondary structure reveals interesting features of this molecule.     

Annexin A5 is involved in migration and invasion of oral carcinoma

Annexin A5 is a Ca2+-binding protein which is involved in membrane organization and dynamics. As recent data suggest a role of annexin A5 in cancer we aimed to gain more insight into the biological function of endogenous annexin A5 and assessed its possible influence on proliferation and invasion capacity. We down-regulated annexin A5 by RNA interference in HaCaT keratinocytes, squamous carcinoma cell line A431 as well as in a primary cell culture of a human oral carcinoma. Hereby, we detected reduced migration and invasion capacity of HaCaT cells which was even stronger in the oral carcinoma. To determine target genes of annexin A5 we used a metastasis specific microarray. Thereby, genes implicated in cell motility including S100A4, TIMP-3, and RHOC were observed to be regulated. These deregulations were confirmed by RT-PCR or western blots, respectively. These observations suggest that the invasion capacity, a main characteristic of tumors, is at least partially regulated by annexin A5 in oral carcinoma.     

MUC1 is involved in trophoblast transendothelial migration

The factors that regulate trophoblast invasion of the uterine vasculature are incompletely understood. In this paper we show that macaque trophoblasts express the mucin, MUC1, and that it is involved in trophoblast-endothelial interaction. Immunocytochemistry, Western blotting and RT-PCR analyses confirmed that MUC1 was expressed by isolated early gestation macaque trophoblasts. MUC1 was also detected in endovascular trophoblasts in sections of placental-decidual tissue during early gestation. A blocking antibody against MUC1 reduced trophoblast adhesion to uterine endothelial cells and also blocked trophoblast transendothelial migration. MUC1 is known to bind to Intercellular Adhesion Molecule-1 (ICAM-1) in other systems. Incubation in the presence of a blocking antibody against Intercellular Adhesion Molecule-1 (ICAM-1) or recombinant ICAM-1 modestly, but significantly, reduced transendothelial trophoblast migration. These results are consistent with the idea that MUC1 is involved in trophoblast adhesion to uterine endothelial cells and in trophoblast transendothelial migration.     

Petunia phospholipase c1 is involved in pollen tube growth

Although pollen tube growth is essential for plant fertilization and reproductive success, the regulators of the actin-related growth machinery and the cytosolic Ca2+ gradient thought to determine how these cells elongate remain poorly defined. Phospholipases, their substrates, and their phospholipid turnover products have been proposed as such regulators; however, the relevant phospholipase(s) have not been characterized. Therefore, we cloned cDNA for a pollen-expressed phosphatidylinositol 4,5-bisphosphate (PtdInsP2)-cleaving phospholipase C (PLC) from Petunia inflata, named Pet PLC1. Expressing a catalytically inactive form of Pet PLC1 in pollen tubes caused expansion of the apical Ca2+ gradient, disruption of the organization of the actin cytoskeleton, and delocalization of growth at the tube tip. These phenotypes were suppressed by depolymerizing actin with low concentrations of latrunculin B, suggesting that a critical site of action of Pet PLC1 is in regulating actin structure at the growing tip. A green fluorescent protein (GFP) fusion to Pet PLC1 caused enrichment in regions of the apical plasma membrane not undergoing rapid expansion, whereas a GFP fusion to the PtdInsP2 binding domain of mammalian PLC delta1 caused enrichment in apical regions depleted in PLC. Thus, Pet PLC1 appears to be involved in the machinery that restricts growth to the very apex of the elongating pollen tube, likely through its regulatory action on PtdInsP2 distribution within the cell.     

Epimorphin is involved in differentiation of rat hepatic stem-like cells through cell-cell contact

Epimorphin, a mesenchymal cell surface-associated molecule, is detected on hepatic stellate cells (HSCs) in the liver. Here, we show the involvement of epimorphin in differentiation of rat hepatic stem-like cells (HSLCs) through contact with HSCs. HSLCs, isolated from adult rats, cultured in stellate cell-conditioned medium had no phenotypic and morphological changes, whereas HSLCs co-cultured with HSCs expressed albumin, transferrin, and tyrosine aminotransferase. An anti-epimorphin antibody inhibited hepatocytic differentiation of HSLCs in co-culture. Furthermore, epimorphin induced mRNA expression of albumin, transferrin, tyrosine aminotransferase, and gamma-glutamyl transpeptidase with decrease of c-kit and musashi-1. Morphologically, HSLCs piled up when co-cultured with HSCs, which was dramatically inhibited by an anti-epimorphin antibody. HSLCs contact with epimorphin started piling up, changed their shape from flat to cuboidal, and subsequently developed bile-canaliculi-like structures. In conclusion, epimorphin is a factor that induces differentiation of hepatic stem-like cells through epithelial-mesenchymal cell contact.     

Nonmuscle myosin IIb is involved in the guidance of fibroblast migration

Although myosin II is known to play an important role in cell migration, little is known about its specific functions. We have addressed the function of one of the isoforms of myosin II, myosin IIB, by analyzing the movement and mechanical characteristics of fibroblasts where this protein has been ablated by gene disruption. Myosin IIB null cells displayed multiple unstable and disorganized protrusions, although they were still able to generate a large fraction of traction forces when cultured on flexible polyacrylamide substrates. However, the traction forces were highly disorganized relative to the direction of cell migration. Analysis of cell migration patterns indicated an increase in speed and decrease in persistence, which were likely responsible for the defects in directional movements as demonstrated with Boyden chambers. In addition, unlike control cells, mutant cells failed to respond to mechanical signals such as compressing forces and changes in substrate rigidity. Immunofluorescence staining indicated that myosin IIB was localized preferentially along stress fibers in the interior region of the cell. Our results suggest that myosin IIB is involved not in propelling but in directing the cell movement, by coordinating protrusive activities and stabilizing the cell polarity.     

MiR-129-5p is down-regulated and involved in the growth,apoptosis and migration of medullary thyroid carcinoma cells through targeting RET

Dysregulation of the REarranged during Transfection proto-oncogene (RET) pathway and microRNA (miRNAs) are crucial for the development of medullary thyroid carcinomas (MTC). Here we demonstrate that miR-129-5p is down-regulated in MTC tissues and cell lines and inhibits RET expression by directly binding its 3′ untranslated regions. Ectopic expression of miR-129-5p significantly decreases cell growth, induces apoptosis and suppresses migration ability in MTC cells through decreasing the phosphorylated AKT, thus functioning as a tumor suppressor. These findings give new clues for understanding MTC carcinogenesis and may help in developing a therapeutic approach for the treatment of RET-activated MTC.     

Migration through host cells: the first steps of Plasmodium sporozoites in the mammalian host

Malaria starts with the infection of the liver by Plasmodium sporozoites. This form of the parasite migrates through several host cells breaching their plasma membranes before infecting a final hepatocyte which they enter forming a parasitophorous vacuole. It is still controversial why Plasmodium sporozoites migrate through host cells. By reviewing the most recent literature, we hope to give an insight on the different steps of host invasion in which migration through cells is involved and on the possible role for this mechanism in infection.     

A rat spermatocyte surface protein is involved in adhesion of pachytene spermatocytes to Sertoli cells in vitro.

Immunochemical approaches were used in trying to identify rat spermatocyte molecules involved in the adhesion to Sertoli cells in coculture. The results show that a surface protein of 80,000 apparent molecular weight strongly inhibits spermatocyte adhesion, suggesting it to be the germ cell surface component involved in the recognition of Sertoli cells.     

Fas-mediated activation of phospholipase D is coupled to the stimulation of phosphatidylcholine-specific phospholipase C in A20 cells.

The activation of phospholipase D in murine B cell lymphoma A20 cells treated with anti-Fas monoclonal antibody has been investigated. Fas cross-linking resulted in a both dose- and time-dependent increases in phospholipase D activity. There was a nearly maximum saturated rise in phospholipase D activity at the dose of 200 ng/ml anti-Fas monoclonal antibody showing a fourfold increase within 3 h. Fas activation also caused an approximately twofold increase of phosphatidylcholine-specific phospholipase C activity and 1,2-diacylglycerol release, which could be blocked by 30 min pretreatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 (50 microgram/ml). Pretreatment of D609 also effectively inhibited the translocation of protein kinase C betaI and betaII from the cytosol to the membrane and the activation of phospholipase D induced by Fas cross-linking, suggesting that 1, 2-diacylglycerol released from the cellular phosphatidylcholine pool through phosphatidylcholine-specific phospholipase C plays a major role in protein kinase C/phospholipase D activation. Anti-Fas monoclonal antibody failed to elicit phosphoinositide-specific phospholipase C activation and any changes in the intracellular Ca2+ level in A20 cells, indicating that the phosphoinositide-mediated pathway is not involved in this Fas signaling. Therefore, these results suggest that Fas-mediated phospholipase D activation may be a consequence of primary stimulation of phosphatidylcholine-specific phospholipase C and that phospholipase D may play a role in Fas cross-linking signaling downstream from phosphatidylcholine-specific phospholipase C.     

Induction of cytosolic phospholipase A2 by oncogenic Ras is mediated through the JNK and ERK pathways in rat epithelial cells

Mutations in ras genes have been detected with high frequency in nonsmall cell lung cancer cells (NSCLC) and contribute to transformed growth of these cells. It has previously been shown that expression of oncogenic forms of Ras in these cells is associated with elevated expression of cytosolic phospholipase A(2) (cPLA(2)) and cyclooxygenase-2 (COX-2), resulting in high constitutive levels of prostaglandin production. To determine whether expression of constitutively active Ras is sufficient to induce expression of these enzymes in nontransformed cells, normal lung epithelial cells were transfected with H-Ras. Stable expression of H-Ras increased expression of cPLA(2) and COX-2 protein. Transient transfection with H-Ras increased promoter activity for both enzymes. H-Ras expression also activated all three families of MAP kinase: ERKs, JNKs, and p38 MAP kinase. Expression of constitutively active Raf did not increase either cPLA(2) or COX-2 promoter activity, but inhibition of the ERK pathway with pharmacological agents or expression of dominant negative ERK partially blocked the H-Ras-mediated induction of cPLA(2) promoter activity. Expression of dominant negative JNK kinases decreased cPLA(2) promoter activity in NSCLC cell lines and inhibited H-Ras-mediated induction in normal epithelial cells, whereas expression of constructs encoding constitutively active JNKs increased promoter activity. Inhibition of p38 MAP kinase or NF-kappaB had no effect on cPLA(2) expression. Truncational analysis revealed that the region of the cPLA(2) promoter from -58 to +12 contained sufficient elements to mediate H-Ras induction. We conclude that expression of oncogenic forms of Ras directly increases cPLA(2) expression in normal epithelial cells through activation of the JNK and ERK pathways.