Doe Sun Na

Annexin-I inhibits phospholipase A2 by specific interaction, not by substrate depletion

Annexin‐I is a calcium dependent phospholipid binding and phospholipase A2 (PLA2) inhibitory protein. A ‘substrate depletion’ model has been proposed for the mechanism of PLA2 inhibition by annexin‐I in studies with 14 to 18 kDa PLA2s. Herein, we have studied the inhibition mechanism using 100 kDa cytosolic PLA2 from porcine spleen. The inhibition has been measured at various substrate and calcium ion concentrations. The pattern of PLA2 inhibition by annexin‐I was consistent with a ‘specific interaction’ mechanism rather than the ‘substrate depletion’ model. Apparent contradiction with previous studies can be explained by the calcium‐dependent binding of annexin‐I to the substrate.

Angiogenic activity of human CC chemokine CCL15 in vitro and in vivo

CCL15 is a novel human CC chemokine and exerts its biological activities on immune cells through CCR1 and CCR3. Because a number of chemokines induce angiogenesis and endothelial cells express CCR1 and CCR3, we investigated the angiogenic activity of CCL15. Both CCL15(1‐92) and N‐terminal truncated CCL15(25‐92) stimulate the chemotactic endothelial cell migration and differentiation, but CCL15(25‐92) is at least 100‐fold more potent than CCL15(1‐92). Treatment with pertussis toxin (PTX), with anti‐CCR1, or with anti‐CCR3 antibody inhibits the CCL15(25‐92)‐induced endothelial cell migration. CCL15(25‐92) also stimulates sprouting of vessels from aortic rings and mediates angiogenesis in the chick chorioallantoic membrane assay. Our findings demonstrate that CCL15(25‐92) has in vitro and in vivo angiogenic activity, and suggest roles of the chemokine in angiogenesis.

Differential effects of annexins I, II, III, and V on cytosolic phospholipase A2 activity: specific interaction model.

Annexins (ANXs) are a family of proteins with calcium‐dependent phospholipid binding properties. Although inhibition of phospholipase A2 (PLA2) by ANX‐I has been reported, the mechanism is still controversial. Previously we proposed a ‘specific interaction’ model for the mechanism of cytosolic PLA2 (cPLA2) inhibition by ANX‐I [Kim et al., FEBS Lett. 343 (1994) 251–255]. Here we have studied the cPLA2 inhibition mechanism using ANX‐I, N‐terminally deleted ANX‐I (ΔANX‐I), ANX‐II, ANX‐II2P112, ANX‐III, and ANX‐V. Under the conditions for the specific interaction model, ANX‐I, ΔANX‐I, and ANX‐II2P112 inhibited cPLA2, whereas inhibition by ANX‐II and ANX‐III was negligible. Inhibition by ANX‐V was much smaller than that by ANX‐I. The protein–protein interactions between cPLA2 and ANX‐I, ΔANX‐I, and ANX‐II2P112 were verified by immunoprecipitation. We can therefore conclude that inhibition of cPLA2 by specific interaction is not a general function of all ANXs, and is rather a specific function of ANX‐I. The results are consistent with the specific interaction model.

Human LZIP binds to CCR1 and differentially affects the chemotactic activities of CCR1-dependent chemokines

Signaling molecules that bind to chemokine receptors should play key roles in regulation of cell migration induced by chemokines. To characterize the CCR1‐mediated cellular signal transduction mechanism, we used the yeast two‐hybrid system to identify a cellular ligand for CCR1. LZIP, which has been known as a transcription factor in various cell types, was identified as a CCR1 binding protein. Although the ability of LZIP to bind DNA is possibly what allows it to function as a transcription factor, its detailed function and participation in chemotaxis have not been established. We found that LZIP binds to CCR1 based on results of a mammalian two‐ hybrid assay and immunoprecipitation experiments. The 21‐260 residues of LZIP were essential for interaction with CCR1. Results from a chemotaxis assay using LZIP transfected cells showed that LZIP enhanced Lkn‐1‐induced chemotaxis, whereas the chemotactic activities induced by other CC chemokines that bind to CCR1, including MIP‐1α, RANTES, or HCC‐4, were not affected by LZIP overexpression. These data indicate that LZIP binds to CCR1 and that the interaction between CCR1 and LZIP participates in regulation of Lkn‐1‐dependent cell migration without affecting the chemotactic activities of other CC chemokines that bind to CCR1.

Transgenic mouse model for breast cancer: Induction of breast cancer in novel oncogene HCCR-2 transgenic mice

Transgenic mice containing novel oncogene HCCR-2 were generated to analyse the phenotype and to characterize the role of HCCR-2 in cellular events. Mice transgenic for HCCR-2 developed breast cancers and metastasis. The level of p53 in HCCR-2 transgenic mice was elevated in most tissues including breast, brain, heart, lung, liver, stomach, kidney, spleen, and lymph node. We examined whether stabilized p53 is functional in HCCR-2 transgenic mice. Defective induction of p53 responsive genes including p21WAF1, MDM2, and bax indicates that stabilized p53 in HCCR-2 transgenic mice exists in an inactive form. These results suggest that HCCR-2 represents an oncoprotein that is related to breast cancer development and regulation of the p53 tumor suppressor.

Leukotactin-1/CCL15-induced chemotaxis signaling through CCR1 in HOS cells

Leukotactin‐1 (Lkn‐1)/CCL15 is a recently cloned CC‐chemokine that binds to the CCR1 and CCR3. Although Lkn‐1 has been known to function as a chemoattractant for neutrophils, monocytes and lymphocytes, its cellular mechanism remains unclear. To understand the mechanism of Lkn‐1‐induced chemotaxis signaling, we examined the chemotactic activities of human osteogenic sarcoma cells expressing CCR1 in response to Lkn‐1 using inhibitors of signaling molecules. Inhibitors of Gi/Go protein, phospholipase C (PLC) and protein kinase Cδ (PKCδ) inhibited the chemotactic activity of Lkn‐1 indicating that Lkn‐1‐induced chemotaxis signal is transduced through Gi/Go protein, PLC and PKCδ. The activities of PLC and PKCδ were also enhanced by Lkn‐1 stimulation. Chemotactic activity of Lkn‐1 was inhibited by the treatment of cycloheximide and actinomycin D suggesting that newly synthesized proteins are needed for chemotaxis. Nuclear factor‐κB (NF‐κB) inhibitor reduced chemotactic activity of Lkn‐1. DNA binding activity of NF‐κB was also enhanced by Lkn‐1 stimulation. These results suggest that Lkn‐1 transduces the signal through Gi/Go protein, PLC, PKCδ, NF‐κB and newly synthesized proteins for chemotaxis.

Annexin-I inhibits PMA-induced c-fos SRE activation by suppressing cytosolic phospholipase A2 signal

Annexin‐I (ANX‐I) is a 37‐kDa protein with a calcium‐dependent phospholipid‐binding property. Previously we have observed the inhibition of cytosolic phospholipase A2 (cPLA2) by ANX‐I in the studies using purified recombinant ANX‐I, and proposed a specific interaction model for the mechanism of cPLA2 inhibition by ANX‐I [Kim et al. (1994) FEBS Lett. 343, 251–255]. Here we have studied the role of ANX‐I in the cPLA2 signaling pathway by transient transfection assay. The stimulation of Rat2 fibroblast cells with phorbol 12‐myristate 13‐acetate (PMA) induced the c‐fos serum response element (SRE). The SRE stimulation by PMA was dramatically reduced by (1) pretreatment with a cPLA2‐specific inhibitor, arachidonyltrifluoromethyl ketone, or (2) co‐transfection with antisense cPLA2 oligonucleotide, indicating that the SRE activation was through cPLA2 activation. Co‐transfection with an ANX‐I expression vector also reduced the SRE stimulation by PMA, suggesting the inhibition of cPLA2 by ANX‐I. The active domain of ANX‐I was mapped using various deletion mutants. ANX‐I(1–113) and ANX‐I(34–346) were fully active, whereas ANX‐I(114–346) abolished the activity. Therefore the activity was in the amino acid 34 to 113 region, which corresponds to the conserved domain I of ANX‐I.

Leukotactin-1-induced ERK activation is mediated via Gi/Go protein/PLC/PKCδ/Ras cascades in HOS cells

Recently cloned leukotactin-1 (Lkn-1) that belongs to CC chemokine family has not been characterized. To understand the intracellular events following Lkn-1 binding to CCR1, we investigated the activities of signaling molecules in response to Lkn-1 in human osteogenic sarcoma cells expressing CCR1. Lkn-1-stimulated cells showed elevated phosphorylation of extracellular signal-related kinases (ERK1/2) with a distinct time course. ERK activation was peaked in 30 min and 12 h showing biphasic activation of ERK. Pertussis toxin, an inhibitor of G(i)/G(o) protein, and phospholipase C (PLC) inhibitor blocked Lkn-1-induced activation of ERK. Protein kinase C delta (PKC delta) specific inhibitor rottlerin inhibited ERK activation in Lkn-1-stimulated cells. The activities of PLC and PKC delta were also enhanced by Lkn-1 stimulation. Dominant negative Ras inhibited activation of ERK. Immediate early response genes such as c-fos and c-myc were induced by Lkn-1 stimulation. Lkn-1 affected the cell cycle progression by cyclin D(3) induction. These results suggest that Lkn-1 activates the ERK pathway by transducing the signal through G(i)/G(o) protein, PLC, PKC delta and Ras, and it may play a role for cell proliferation, differentiation, and regulation of gene expression for other cellular processes.

Calcium-dependent interaction of annexin I with annexin II and mapping of the interaction sites

Annexins are multifunctional intracellular proteins with Ca2+‐ and phospholipid‐binding properties. Their structures consist of four conserved repeat domains that form the core and a diverse N‐terminal tail, from which their functional differences may arise. We searched for cellular proteins that interact with the N‐terminal tail plus domain I of annexin I (ANX1) by using the yeast two‐hybrid method. Screening of a HeLa cell cDNA library yielded annexin II (ANX2) cDNA. The interaction between ANX1 and ANX2 also occurred in vitro in a Ca2+‐dependent manner. Mapping of the interaction sites revealed that interaction between domain I of ANX1 and domain IV of ANX2 was stronger than the other combinations.

Translocation of lipocortin (annexin) 1 to the membrane of U937 cells induced by phorbol ester, but not by dexamethasone

1 Induction of lipocortin 1 secretion by dexamethasone has been demonstrated, although the secretory mechanism is still unknown. We have studied the effects of 12‐tetradecanoyl phorbol 13‐acetate (TPA) and/or dexamethasone on the expression, translocation, and secretion of lipocortin 1 in U937 cells. 2 The expression of lipocortin 1 and its mRNA increased during TPA‐induced differentiation of U937 cells to a maximum of 1.9 fold and 8.2 fold, respectively, after 48 h. Both the protein and the mRNA levels decreased after 48 h. 3 TPA caused the translocation of lipocortin 1 from the cytosol to the membrane of U937 cells in a time‐dependent manner, as determined by Western blot analysis. The translocation was concurrent with the differentiation of the cells. After 48 h of TPA treatment, 82.6 ± 6.5% of lipocortin 1 was present in the membrane fraction compared to 41.6 ± 1.7% in untreated cells. 4 The amount of lipocortin 1 that was externally bound (associated) with the membrane increased to 3.2 fold as the cytosol to membrane translocation of lipocortin 1 increased. 5 Dexamethasone decreased the externally bound lipocortin 1, but had no effect on the cytosol to membrane translocation. 6 This offers a model system with which the function and the secretion mechanism of lipocortin 1 can be studied. Our data is consistent with the hypothesis that the secretory mechanism is through an unknown pathway, involving the translocation of lipocortin 1 from the cytosol to the internal membranes, and then, its secretion to the external membrane.