Annexin-A7 protects normal prostate cells and induces distinct patterns of RB-associated cytotoxicity in androgen-sensitive and -resistant prostate cancer cells.

The tumor suppressor role of annexin-A7 (ANXA7) was previously demonstrated by cancer susceptibility in Anxa7(+/-)-mice and by ANXA7 loss in human cancers, especially in hormone-resistant prostate tumors. To gain mechanistic insights into ANXA7 tumor suppression, we undertook an in vitro study in which we compared wild-type (WT)-ANXA7 and dominant-negative (DN)-ANXA7 effects to a conventional tumor suppressor p53 in prostate cancer cells with different androgen sensitivity. Unlike p53 (which caused cell growth arrest and apoptosis to a noticeable extent in benign PrEC), WT-ANXA7 demonstrated profound cytotoxicityin androgen-sensitive LNCaP as well as in the androgen-resistant DU145 and PC3 prostate cancer cells, but not in PrEC. In androgen-sensitive LNCaP, WT-ANXA7 decreased low-molecular-weight (LMW) AR protein forms and maintained higher retinoblastoma 1 (RB1)/phospho-RB1 ratio. In contrast, DN-ANXA7 (which lacks phosphatidylserine liposome aggregation properties) increased LMW-AR forms and hyperphosphorylated RB1 that was consistent with the lack of DN-ANXA7 cytotoxicity. According to the microarray-based Ingenuity Pathways Analysis, a major WT-ANXA7 effect in androgen-sensitive LNCaP constituted of upregulation of the RB1-binding transcription factor E2F1 along with its downstream proapoptotic targets such as ASK1 and ASPP2. These results suggested a reversal of the RBdependent repression of the proapoptotic E2F-mediated transcription. However, DN-ANXA7 increased RB1/2 (but not E2F1) expression and induced the proliferation-promoting ERK5, thereby maintaining the RB-dependent repression of E2F-mediated apoptosis in LNcaP. On the other hand, in androgen-resistant cells, WT-ANXA7 tumor suppressor effects involved PTEN and NFkB pathways. Thus, ANXA7 revived the RB-associated cell survival control and overcame androgen resistance and dysfunctional status of major tumor suppressors commonly mutated in prostate cancer. Published 2009 UICC.

Synexin and GTP increase surfactant secretion in permeabilized alveolar type II cells.

We have previously suggested that synexin (annexin VII), a Ca(2+)-dependent phospholipid binding protein, may have a role in surfactant secretion, since it promotes membrane fusion between isolated lamellar bodies (the surfactant-containing organelles) and plasma membranes. In this study, we investigated whether exogenous synexin can augment surfactant phosphatidylcholine (PC) secretion in synexin-deficient lung epithelial type II cells. Isolated rat type II cells were cultured for 20-22 h with [(3)H]choline to label cellular PC. The cells were then treated with beta-escin, which forms pores in the cell membrane and releases cytoplasmic proteins including synexin. These cells, however, retained lamellar bodies. The permeabilized type II cells were evaluated for PC secretion during a 30-min incubation. Compared with PC secretion under basal conditions, the presence of Ca(2+) (up to 10 microM) did not increase PC secretion. In the presence of 1 microM Ca(2+), synexin increased PC secretion in a concentration-dependent manner, which reached a maximum at approximately 5 microg/ml synexin. The secretagogue effect of synexin was abolished when synexin was inactivated by heat treatment (30 min at 65 degrees C) or by treatment with synexin antibodies. GTP or its nonhydrolyzable analog beta:gamma-imidoguanosine-5'-triphosphate also increased PC secretion in permeabilized type II cells. The PC secretion was further increased in an additive manner when a maximally effective concentration of synexin was added in the presence of 1 mM GTP, suggesting that GTP acts by a synexin-independent mechanism to increase membrane fusion. Thus our results support a direct role for synexin in surfactant secretion. Our study also suggests that membrane fusion during surfactant secretion may be mediated by two independent mechanisms.

Calcium-dependence of synexin binding may determine aggregation and fusion of lamellar bodies.

Synexin (annexin VII) is a member of the annexin family of calcium and phospholipid binding proteins that promote calcium-dependent aggregation and fusion of lipid vesicles or secretory granules. We have previously suggested that synexin may be involved in membrane fusion processes during exocytosis of lung surfactant since it promotes fusion in vitro of lamellar bodies with plasma membranes. In this study, we characterized calcium-dependency of synexin binding to lamellar bodies and plasma membranes, since such binding is the initial, and, therefore, may be the rate-limiting step in membrane aggregation and fusion. The binding of biotinylated synexin to lamellar bodies and plasma membranes increased in a calcium-dependent manner reaching a maximum at approx. 200 microM Ca2+. Binding to lamellar bodies was completely inhibited by unlabelled synexin. Gel-overlay analysis showed that synexin bound to an approx. 76 kDa protein in the lamellar body and plasma membrane fractions. The calcium kinetics were noticeably similar for synexin binding to lamellar bodies and plasma membranes, aggregation of lamellar bodies, and fusion of lamellar bodies with lipid vesicles. At low calcium concentrations, aggregation of lamellar bodies could be increased with increasing synexin concentration, and arachidonic acid increased all three parameters (binding, aggregation, and fusion) in a similar manner. The effects of calcium and arachidonic acid on these three parameters suggest that synexin binding to lamellar bodies may be a rate-determining step for fusion during surfactant secretion. Furthermore, at near physiological calcium levels, the membrane fusion may be enhanced by elevated concentrations of synexin and polyunsaturated fatty acids.

Novel isoforms of synexin in Xenopus laevis: multiple tandem PGQM repeats distinguish mRNAs in specific adult tissues and embryonic stages.

Synexin (annexin VII) is a calcium-dependent, phospholipid-binding and membrane fusion protein in the annexin gene family, which forms calcium channels and may play a role in exocytotic secretion. We report here the cloning and characterization of five novel isoforms of cDNAs encoding Xenopus synexin from brain, oocyte and stage 24 cDNA libraries. The most prevalent Xenopus synexin has 1976 bp of cDNA sequence, which contains a 1539 bp open reading frame of 512 amino acids encoding a 54 kDa protein. This Xenopus protein is 6 kDa larger than the previously reported human and mouse synexins with which it shares approx. 73% identity in the C-terminal region and approx. 44% identity in the N-terminal region. Further studies with PCR revealed the molecular basis of the substantial divergence in the Xenopus synexin's N-terminal domain. The domain equivalent to the mammalian tissue-specific cassette exon occurs at a different position and is variable in size and sequence. The most interesting observation relates to the occurrence of different forms of synexin due to the varying numbers of tandem PGQM repeats that are expressed differently in different adult tissues and embryonic stages. For these reasons we have labelled this set of unique isoforms annexin VIIb, referring to mammalian forms, which lack the PGQM tandem repeats, as annexin VIIa. In spite of these differences from annexin VIIa, the form of recombinant annexin VIIb with three PGQM repeats was found to be catalytically active. We interpret these results to indicate that the actual calcium and phospholipid binding sites are conserved in Xenopus, and that the variations observed between members of the synexin gene family in the regulatory domain clearly point towards the tissue- and stage-specific roles of individual members, possibly involving the exocytotic process.