Down-regulated expression of Annexin A7 induces apoptosis in mouse hepatocarcinoma cell line by the intrinsic mitochondrial pathway.

Our previous studies have shown that decreased expression of Annexin A7 elevates apoptosis in Hca-P cells, a hepatocarcinoma cell line with lymphatic metastatic potential. In this study, RNA interference technique was used to down-regulate the expression of Annexin A7, and unmanipulated Hca-P cells and transfected nonspecific-sequence Hca-P cells as control. The down-regulation of Annexin A7 declined the cell viability after cisplatin exposure. And the reduced expression of Annexin A7 decreased the expression of Bcl2, increased the expression of Cytochrome-C in the cytoplasme, and then improved the expression of Caspase-3. However there was no significant effect on the expression of Bax, Caspase-12, Fas, FasL and Caspase-8. The results indicate that the decreased expression of Annexin A7 could inhibit the proliferation, and increase the apoptosis of Hca-P cells by affecting the expression of the apoptosis associated proteins by the mitochondrial pathway.

Annexin A7 and its binding protein galectin-3 influence mouse hepatocellular carcinoma cell line in vitro.

Lymph node metastasis is recognized as an important mode of liver cancer metastasis. Our previous study has built two hepatocarcinoma cell lines, Hca-F with high (75%) and Hca-P with low (25%) incidences of lymph node metastasis, and has indicated that annexin A7 is an important factor in the lymphatic metastasis of tumors. There is evidence that galectin-3 is the binding protein of annexin A7 and works in protein complexes. Our current study shows that both annexin A7 and galectin-3 express higher in Hca-F than Hca-P. Annexin A7 was successfully down-regulated in Hca-P by RNA interference, and this resulted in concomitant reduction of galactin 3 expression in annexin A7 down regulated compared to the control and N-control cells. Using CCK-8 assay, the expression level of annexin A7 and galectin-3 were found to have correlation with the proliferation ability; Transwell assay showed annexin A7 and galectin-3 are involved in cell migration and invasion regulation in mouse hepatocellular carcinoma cell lines, immunofluorescence assay indicate annexin A7 and galectin-3 were co-located annexin A7 and galectin-3 played roles in DNA damage and cell proliferation cycle checkpoint arrest pathway. Those phenomena indicated that annexin A7 influences lymphatic metastasis of tumors by interacting with galectin-3 through the regulation of tumor cell proliferation, attachment, migration and invasion.

Ech1 is a potent suppressor of lymphatic metastasis in hepatocarcinoma.

We have previously demonstrated that Ech1 is involved in the lymphatic metastasis of tumors in vitro. Here, we gain an insight into the role that Ech1 is playing in Hca-F cell. The expression of Annexin A7, Gelsolin and Clic1 genes, which were also relevant to tumor lymphatic metastasis, had been inhibited due to downregulation Ech1 gene by Western blot analysis. And downregulated of Ech1 inhibits the metastasic capability of Hca-F cells to peripheral lymph nodes in vivo. Our work indicates although the involvement of Ech1 in tumor metastasis development and progression, but the subcellular location of Ech1 has not much contribution to that.

ANXA7, PPP3CB, DNAJC9, and ZMYND17 genes at chromosome 10q22 associated with the subgroup of schizophrenia with deficits in attention and executive function.

BACKGROUND: A genome scan of Taiwanese schizophrenia families suggested linkage to chromosome 10q22.3. We aimed to find the candidate genes in this region. METHODS: A total of 476 schizophrenia families were included. Hierarchical clustering method was used for clustering families to homogeneous subgroups according to their performances of sustained attention and executive function. Association analysis was performed using family-based association testing and TRANSMIT. Candidate associated regions were identified using the longest significance run method. The relative messenger RNA expression level was determined using real-time reverse transcriptase polymerase chain reaction. RESULTS: First, we genotyped 18 microsatellite markers between D10S1432 and D10S1239. The maximum nonparametric linkage score was 2.79 on D10S195. Through family clustering, we found the maximum nonparametric linkage score was 3.70 on D10S195 in the family cluster with deficits in attention and executive function. Second, we genotyped 79 single nucleotide polymorphisms between D10S1432 and D10S580 in 90 attention deficit and execution deficit families. Association analysis indicated significant transmission distortion for nine single nucleotide polymorphisms. Using the longest significance run method, we identified a 427-kilobase region as a significant candidate region, which encompasses nine genes. Third, we studied messenger RNA expression of these nine genes in Epstein-Barr virus-transformed lymphoblastic cells. In schizophrenic patients, there was significantly lower expression of ANXA7, PPP3CB, and DNAJC9 and significantly higher expression of ZMYND17. CONCLUSIONS: ANXA7, PPP3CB, DNAJC9, and ZMYND17 genes are potential candidate genes for schizophrenia, especially in patients with deficits in sustained attention and executive function. The responsible functional variants remained to be clarified.

Increased expression of annexin A7 in temporal lobe tissue of patients with refractory epilepsy.

Annexin A7 is a member of the family of annexins, which are thought to function in the regulation of calcium homeostasis and the fusion of vesicles. Refractory epilepsy may be related to the imbalance of calcium homeostasis. Our aims are to investigate the expression of Annexin A7 in epileptic brains in comparison with human controls and to explore Annexin A7's possible role in refractory epilepsy. We examined the expression of Annexin A7 via immunohistochemistry, double-label immunofluorescence and western blot. The expression of Annexin A7 was shown to be significantly increased in patients with refractory epilepsy. Double-label immunofluorescence and confocal microscopy disclosed Annexin A7 immunoreactivity in the neurons, which were recognized by the antibody of neuron specific enolase (NSE). The result showed that Annexin A7 may be involved in the pathophysiology of refractory epilepsy and may play a role in developing and maintaining the epilepsy.

Proteomics analysis of two mice hepatocarcinoma ascites syngeneic cell lines with high and low lymph node metastasis rates provide potential protein markers for tumor malignancy attributes to lymphatic metastasis.

Lymph node metastasis (LNM) is recognized as an important factor involved in the tumor malignancy progression. Our previous study has indicated that the hepatocarcinoma cell line with 75% of LNM (Hca-F)-cell-induced neoplasia and the hepatocarcinoma cell line with 25% of LNM-induced neoplasia are accompanied with high (75%) and low (25%) incidences of LNM. In the current study, 62 and 54 protein spots were observed up-regulated and down-regulated in Hca-F cell relative to the hepatocarcinoma cell line with 25% of LNM by 2-D DIGE. Totally, 113 unique proteins were identified by HPLC-nano ESI-MS/MS analysis. The expression levels of Annexin A7, Ulch3, and ER protein 29 were validated by Western blotting analyses. The abnormally regulated proteins were categorized and annotated by protein analysis through evolutionary relationships analysis with the aid of the database for annotation, visualization and integrated discovery tool. Seventeen gene candidates concordantly expressed both at mRNA and protein levels. By making a challenge, we detected expression levels of Annexin A7 in primary gastric cancer (GC) and primary GC cancer tissues with LNMs by immunohistochemisty. Higher ratio of positive and strong expressions Annexin A7 in GC might correlate with the tumor progression. The repression of Annexin A7 inhibits the mobility and invasion abilities of Hca-F cell, increases the apoptosis rate of Hca-F cell. Current study narrows and provides certain specific protein candidates potentially playing important roles in LNM-associated cancers.

The significance of ANXA7 expression and its correlation with poor cellular differentiation and enhanced metastatic potential of gastric cancer.

BACKGROUND: Annexin-A7 (ANXA7) exhibits biological and genetic properties expected of a tumor suppressor gene and may play a role in cancer progression. However, the ANXA7 expression in different histological subtypes of gastric adenocarcinomas and its correlation with invasive potentials has not been elucidated. METHODS: Immunohistochemical staining of ANXA7 for 84 primary gastric adenocarcinomas was performed, and data was correlated with clinicopathological parameters of patients. RESULTS: The ANXA7 expression was well correlated with the grade of differentiation of primary tumors. Its expression was detected in 100% (8/8), 64.9% (24/37), 66.7% (2/3), 31.9% (13/31), 0% (0/3), and 0% (0/2) of well-differentiated tubular, moderately-differentiated tubular, papillary, poorly differentiated, signet-ring cell, and mucinous adenocarcinoma, respectively. According to the Lauren's classification, the ANXA7 expression was higher in intestinal type than in diffuse type tumor (71.9% vs. 6.1%, P = 0.003). The loss of expression of ANXA7 expression was significantly related to distant metastasis (P = 0.04). However, there were no significant associations between the ANXA7 expression and survival of cancer patients (P = 0.159). CONCLUSIONS: A striking correlation between ANXA7 expression and cell differentiation of gastric cancer was observed. The loss of expression of ANXA7 is associated with distant metastasis.

Nuclear localization of Annexin A7 during murine brain development.

BACKGROUND: Annexin A7 is a member of the annexin protein family, which is characterized by its ability to interact with phospholipids in the presence of Ca2+-ions and which is thought to function in Ca2+-homeostasis. Results from mutant mice showed altered Ca2+-wave propagation in astrocytes. As the appearance and distribution of Annexin A7 during brain development has not been investigated so far, we focused on the distribution of Annexin A7 protein during mouse embryogenesis in the developing central nervous system and in the adult mouse brain. RESULTS: Annexin A7 is expressed in cells of the developing brain where a change in its subcellular localization from cytoplasm to nucleus was observed. In the adult CNS, the subcellular distribution of Annexin A7 depends on the cell type. By immunohistochemistry analysis Annexin A7 was detected in the cytosol of undifferentiated cells at embryonic days E5-E8. At E11-E15 the protein is still present in the cytosol of cells predominantly located in the ventricular germinative zone surrounding the lateral ventricle. Later on, at embryonic day E16, Annexin A7 in cells of the intermediate and marginal zone of the neopallium translocates to the nucleus. Neuronal cells of all areas in the adult brain present Annexin A7 in the nucleus, whereas glial fibrillary acidic protein (GFAP)-positive astrocytes exhibit both, a cytoplasmic and nuclear staining. The presence of nuclear Annexin A7 was confirmed by extraction of the nucleoplasm from isolated nuclei obtained from neuronal and astroglial cell lines. CONCLUSION: We have demonstrated a translocation of Annexin A7 to nuclei of cells in early murine brain development and the presence of Annexin A7 in nuclei of neuronal cells in the adult animal. The role of Annexin A7 in nuclei of differentiating and mature neuronal cells remains elusive.

Prognostic impact of ANX7-GTPase in metastatic and HER2-negative breast cancer patients.

PURPOSE: ANX7-GTPase located on chromosome 10q21 is significantly altered and associated with hormone-refractory metastatic prostate cancers. Therefore, we investigated whether levels of ANX7 correlate with breast cancer progression and survival EXPERIMENTAL DESIGN: A diagnostic tumor tissue microarray containing 525 human breast tissue specimens at different stages of the disease was assayed for ANX7 using immunocytochemical methods with ANX7 monoclonal antibody. A separate prognostic tumor tissue microarray containing 553 human breast tissue specimens annotated with clinicopathological parameters was assayed for ANX7, HER2, estrogen receptor, progesterone receptor, and p53 protein. RESULTS: We report here for the first time that the expression of ANX7-GTPase is significantly enhanced and associated with the presence of metastatic disease (P < 0.0001) in the 525 human breast tissue specimens analyzed. Furthermore, using a separate 553 case retrospective prognostic tumor tissue microarray, we found that increased ANX7 expression is also significantly associated with poor overall patient survival (P < 0.014). This is particularly true when restricted to patients in whom the BRE clinical grade is 2 (P < 0.001) or for whom there is a lack of HER2 expression (P < 0.002). Finally, Cox regression analysis shows that as the expression of ANX7 rises, the probability of survival decreases by more than 10-fold for those patients with HER2-negative tumors. These latter patients represented 66% of the population affected with breast cancer in this study. CONCLUSIONS: High levels of ANX7 in tumor correlate strongly with poor survival of HER2-negative patients and the most aggressive forms of breast cancer. This is the first study to demonstrate that ANX7 antibody has the potential for development into an in vivo diagnostic and therapeutic tool. This simple and reliable immunohistochemical assay may therefore become an important biomarker for metastatic breast cancer diagnosis and management of HER2-negative breast tumor patients.

Expression of annexin I, II and VII proteins in androgen stimulated and recurrent prostate cancer.

PURPOSE: We performed a comprehensive survey of annexin I, II and VII protein expression in patient matched benign prostatic epithelium (BPE), androgen stimulated prostate cancer (AS-CaP) and recurrent prostate cancer (R-CaP). MATERIALS AND METHODS: Annexin I, II and VII was immunostained in 23 matched pairs of BPE and AS-CaP specimens, and in 25 R-CaP specimens. Protein expression was assessed visually and using color digital video image analysis. RESULTS: The expression levels of annexins I and II was decreased from BPE to AS-CaP in all patients examined using visual assessment (22 of 22) or digital image analysis (18 of 18 for annexin I and 22 of 22 for annexin II). Annexin I mean optical density (MOD) decreased by 69% (0.718 to 0.222, p <0.0001) and annexin II MOD decreased by 71% (0.820 to 0.238, p <0.0001). Annexin I MOD further decreased almost 50% in R-CaP compared with AS-CaP (0.117 vs 0.222, p <0.0065) and annexin II MOD further declined by 37% (AS-CaP 0.238 vs R-CaP 0.150, p <0.0001). Annexin VII MOD showed no significant change between BPE and AS-CaP (0.229 and 0.214, respectively) but it decreased 14% in R-CaP vs AS-CaP (0.184 vs 0.214, p <0.0051). CONCLUSIONS: Annexins I and II expression are decreased in AS-CaP compared with BPE and a further reduction is observed with progression from AS-CaP to R-CaP. Annexin VII protein expression is decreased in R-CaP, although AS-CaP and BPE are similar. This study suggests that the dysregulations of annexin proteins I, II and VII are important events in prostate carcinogenesis and progression.

Mapping protein expression in mouse pancreatic islets by immunolabeling and electron energy loss spectrum-imaging.

A combination of immuno-electron microscopy and electron energy-loss spectrum-imaging was used to map the distributions of endocrine polypeptide hormones and proteins in mouse pancreatic islet of Langerhans. Tissue was analyzed from control animals and from mice that were heterozygous for the Anx7 gene, which defines a Ca2+/GTP-dependent membrane fusion and ion channel protein. The heterozygous Anx7 (+/-) mouse displays defects in IP3 receptor mediated Ca2+ signaling and insulin secretion. Therefore, information was obtained about the distributions of the hormones insulin and glucagon, as well as the proteins ANX7 and the IP3 receptor. Insulin secretion appears to be defective in the mutants. It was found from immunolabeling experiments that expression of the IP3 receptor is reduced in mutant islets compared to control islets. Subcellular distributions of sulfur and nitrogen obtained by electron energy-loss spectrum-imaging showed that the insulin concentrations of beta granules were essentially the same in control and mutant islets. By contrast, immunogold labeling of mutant islets shows more insulin immunoreactivity in the beta granules. It follows that insulin may be packaged differently in mutant islets, making antigenic determinants more available to the labeling antibody. The increased rate of insulin secretion in the hyperplastic mutant islets can be explained by compensatory increases in islet size, rather than by an increased insulin concentration in the beta cells. The results indicate that reduced ANX7 expression leads to defects in the IP3 receptor expression in the endocrine cells of the mutant mouse. Increased size of the islet or of adrenal medulla may be a compensatory mechanism for secretion defect by individual endocrine cells. Defects in IP3 receptor expression, and documented consequences of a Ca2+ signaling defect, lead to other changes in organelles such as the mitochondrial number in islet beta-cells. The effects and consequences of reduced ANX7 expression on mitochondria are evident in ultrastructural observations.

Annexin VII: an astroglial protein exhibiting a Ca2+-dependent subcellular distribution.

A fundamental issue in neuronal and glial cells is how intracellular rises in Ca2+ are coupled to signaling cascades and changes in subcellular morphology. We studied the expression and localization of annexin VII (synexin), a Ca(2+)-/GTP-dependent membrane fusion protein, in the human CNS. Here, we demonstrate the presence of two annexin VII isoforms (47 and 51 kDa) in human brain tissue as well as its exclusive expression in astroglial cells. An in vitro study of astrocyte-derived C6 rat glioblastoma cells expressing a GFP tagged annexin VII fusion protein demonstrates a sequential redistribution of the fusion protein in response to rising intracellular Ca2+ concentrations. Our findings indicate a role of annexin VII in the regulation of intracellular Ca(2+)-dependent processes in astroglial cells.

Annexin VII as a novel marker for invasive phenotype of malignant melanoma.

Both F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. While examining the genetic difference between the two sublines, we found a marked reduction of annexin VII expression in BL6 cells. In addition, fusion cell clones of both sublines were as poorly metastatic as F10 cells after subcutaneous injection, and contained the annexin VII message as abundantly as F10 cells. Hence, we examined whether the annexin VII expression was correlated with the less malignant phenotype of clinical cases by immunohistochemistry. Immunoreactivities to anti-annexin VII antibody in melanoma cells were evaluated quantitatively by using skin mast cells as an internal positive control. Eighteen patients with malignant melanoma were divided into two groups: lymph node metastasis-negative and positive groups. The ratio of numbers of patients positive versus negative to the antibody was significantly larger in the former than in the latter group. These results not only indicated that annexin VII serves as a marker for less invasive phenotype of malignant melanoma, but also suggested a possible role of annexin VII in tumor suppression.

Expression and localisation of annexin VII (synexin) isoforms in differentiating myoblasts.

Annexin VII exists in a 47 kDa and a 51 kDa isoform with the 51 kDa protein being the only isoform present in skeletal muscle. Expression of the 51 kDa isoform during myogenesis and localization was studied in cells after conversion into myogenic cells by transduction with MyoD and in mouse and human myogenic cell lines. MyoD expression in NIH3T3 and C3H10T1/2 fibroblasts led to disappearance of the mRNA specific for the 47 kDa isoform and appearance of the 51 kDa isoform-specific mRNA. The overall amount of annexin VII protein was reduced in myogenic converted cells. Both in undifferentiated and differentiated cells annexin VII was localized by immunofluorescence microscopy to punctate structures which were distributed all over the cell. A GFP annexin VII fusion protein showed a similar distribution. Cell fractionation studies indicated that annexin VII is equally distributed between cytosol and membrane fractions in undifferentiated cells, while in differentiated cells it is exclusively present in the membrane fraction. By sucrose gradient centrifugation of postnuclear supernatants we identified two distinct annexin VII-containing membrane populations that cofractionated with caveolin 3- and sorcin-containing membranes.

Antisense-mediated regulation of Annexin VII gene expression during the transition from growth to differentiation in Dictyostelium discoideum.

Annexin VII is believed to be required for proper Ca(2+)-homeostasis in Dictyostelium discoideum cells. As was previously reported, the expression of Annexin VII gene increased during the transition of D. discoideum Ax-2 cells from growth to differentiation. We have casually cloned an interesting gene, Quit3, by the differential plaque hybridization. Quit3 had no coding region, and was expressed more predominantly in the growth phase than in the differentiation phase. Unexpectedly, this gene was found to encode the complementary sequence of Annexin VII. Therefore, it is most likely that the Quit3 mRNA may regulate the Annexin VII synthesis by the natural antisense transcript via an antisense RNA-RNA interaction, thus resulting in striking increase of Annexin VII production in the phase-shift of cells from growth to differentiation. Since Annexin VII is known to be coded for by a single gene in Dictyostelium, the antisense RNA seemed to be encoded in the same genetic locus as the Annexin VII mRNA.

Detection and localization of synexin (Annexin VII) in Xenopus adult and embryonic tissues using an antibody to the Xenopus N-terminal PGQM repeat domain.

Synexin (Annexin VII) is a widely distributed member of the annexin gene family which forms calcium channels and drives calcium-dependent membrane fusion. In Xenopus laevis, different synexins contain two to six tandem repeats of the tetra amino acid sequence PGQM in the unique N-terminal, with a distribution specific to adult tissues and embryonic stages. Immunogold studies using the PGQM-specific polyclonal antibody showed that synexin is localized in adult muscle to myosin-rich A-bands, Z-bands, and T-tubules, and in other adult tissues to nuclei and mitochondria and other formed elements. In oocytes, synexin was also found associated with yolk granules. The PGQM tandem repeats could represent interaction sites for other proteins, and we therefore synthesized a synthetic peptide containing the maximum six tandem repeats [NH2-(PGQM)6-Y-COOH] to test this hypothesis. We found that the peptide alone could specifically bind and crosslink to different proteins in a tissue-specific manner. In liver, it bound to a single 35-kDa protein. In muscle, it bound to four proteins (35, 45, 48, and 116 kDa). Therefore, we conclude that the PGQM domain is accessible to specific antibodies and that the PGQM repeat is sufficiently ordered to unambiguously identify specific binding proteins in different Xenopus tissues.

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.

Calcium-dependent binding of the plasma protein apolipoprotein A-I to two members of the annexin family.

Affinity chromatography with purified annexins coupled to CNBr-activated Sepharose 4B was used to determine the capacity of proteins found in cytosolic fractions of the bovine adrenal medulla to bind to an immobilized annexin in a Ca2+-dependent manner. Several proteins were eluted from a recombinant annexin I column in the presence of 2 mM EGTA, including protein kinase C (PKC), members of the annexin family, and a 26 kDa protein that appeared as the most prominent band on SDS-PAGE. The identities of PKC, annexin I, annexin IV, annexin VI, and annexin VII were confirmed by Western blotting. The 26 kDa protein was purified by anion exchange chromatography on a Poros Q column and determined to be apolipoprotein A-I (apoA-I) by peptide sequencing. Comigration of apoA-I and chromobindin 2 on two-dimensional gels identified apoA-I as chromobindin 2. Overlay assays were performed to verify the apoA-I-annexin I interaction using apoA-I immobilized on nitrocellulose and annexin I in solution with binding detected using anti-annexin I antiserum. Additionally, the ability of biotin-labeled apoA-I in solution to bind to several purified annexins immobilized on nitrocellulose was determined by detection with horseradish peroxidase-conjugated avidin. Using these methods, it was shown that both annexin I and annexin VII bind to bovine apoA-I in a Ca2+-dependent manner. Other annexins, such as annexin IV and annexin VI, do not exhibit this binding. The results suggest that certain annexins may function as extracellular binding sites for plasma proteins.

Effect of protein synthesis inhibitors on synexin levels and secretory response in bovine adrenal medullary chromaffin cells.

The effects of the protein synthesis inhibitors actinomycin D and cycloheximide on the cellular content of the calcium binding protein synexin, and on the secretory response of cultured bovine adrenal medullary chromaffin cells were determined. Both protein synthesis inhibitors produced a slow decrease in the cellular synexin content. The synexin level was reduced by 50% after 133 h of incubation in the presence of 2 micrograms/ml actinomycin D or 5 micrograms/ml cycloheximide. However, this was partly due to an artefactual stabilization of synexin, since metabolic labelling of synexin with [35S]methionine showed that the half-time of degradation was only 40 h. The secretory response of chromaffin cells was quickly diminished in the presence of protein synthesis inhibitors. Catecholamine secretion induced by membrane depolarization or barium stimulation of intact cells, or by calcium stimulation of digitonin-permeabilized cells was decreased by 77-82% after 24 h of incubation in the presence of 5 micrograms/ml cycloheximide. These results suggest that, in addition to synexin, at least one or more proteins with a shorter half-time of degradation than synexin are involved in the secretory response of adrenal chromaffin cells.

Expression and localization of annexin VII (synexin) in muscle cells.

Annexin VII (synexin) is a member of the annexin family of proteins, which are characterized by Ca(2+)-dependent binding to phospholipids. We used PCR to isolate from a lambda gt11-mouse fibroblast library annexin VII cDNA fragments corresponding to the two isoforms found in both humans and Dictyostelium discoideum. The two isoforms of 47 kDa and 51 kDa differed by 22 amino acids inserted into the proximal third of the hydrophobic N terminus. Annexin VII-specific polypeptides expressed in Escherichia coli were used to generate isoform-specific monoclonal antibodies. Expression of the two isoforms during myogenesis was followed in the myogenic cell lines BC3H1 and L6. Only the 47 kDa isoform was present in undifferentiated L6 or BC3H1 myoblasts. The 51 kDa isoform appeared after myogenesis had been induced and in striated muscle only the 51 kDa isoform was observed. Immunofluorescence showed that annexin VII was located in the cytosol of mononucleated and fused polynucleated cultured cells, whereas in striated muscle, annexin VII was located preferentially at the plasma membrane and the transverse tubules. However, there was also some residual cytosolic staining, which was more abundant in type II (fast twitch) than in type I (slow twitch) fibers. Permeabilization of L6 cells with digitonin in the presence of 5 mM EGTA led to a release of annexin VII from the cells, which paralleled the loss of cytosolic lactate dehydrogenase (LDH) at low detergent concentrations (50 microM). In the presence of 100 microM extracellular Ca2+, annexin VII remained bound to the plasma membrane even in the presence of high digitonin concentrations. Incubation with the Ca(2+)-specific ionophore A23187 and 100 microM extracellular Ca2+ led to a redistribution of annexin VII from the cytosol to the plasma membrane after 30 minutes of incubation. The results obtained indicate a developmentally and Ca(2+)-regulated localization and expression of annexin VII and raise the possibility that annexin VII may play a role in excitation-contraction coupling in skeletal muscle.