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Cell Biology International (2008) 32, 121127 (Printed in Great Britain)
Translocation of annexin B1 in response to the stimulation of PMA and ionomycin in cervical cancer cells
Jing‑Jing Huang1, Hong‑Li Yan1, Yuan‑jian Gao, Shu‑Han Sun*, Yan He, Fei‑Xiang Ding, Qian Mei and Geng Xue
Institute of Molecular Genetics and Engineering, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, PR China
Annexin B1 is a novel member of the annexin superfamily which was isolated from a Cysticercus cellulosae cDNA library. To investigate the physiological roles of annexin B1, we firstly performed immunohistochemical analysis on frozen Cysticercus cellulosae sections and found that annexin B1 was present not only in the tegument of the bladder wall, but also in the host-derived inflammatory layer; In addition, ELISA analysis revealed that annexin B1 could be detected in the cystic fluid of Cysticercus cellulosae and the sera of pigs with cysticercosis. These findings indicated that annexin B1 might be a secretary protein. We further constructed a pEGFP–annexin B1 plasmid and transfected it into SiHa cells. We found that GFP–annexin B1 was stimulated to translocate to the plasma membrane by phorbol 12-myristate 13-acetate (PMA). By contrast, it was induced to distribute at the plasma and nuclear membranes by treatment with calcium ionophore ionomycin. PMA increased annexin B1 membrane binding, which might facilitate exocytosis. Moreover, translocation of the protein to the plasma and nuclear membranes after stimulated by ionomycin, was predicted to be related to an additional function.
Keywords: Annexin B1, Calcium, Immunohistochemistry, Protein kinase C.
1These authors contributed equally to this work.
*Corresponding author. Present address: Department of Medical Genetics, The Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, PR China. Tel./fax: +86 21 2507 0331.
Annexins are Ca2+-dependent phospholipid-binding proteins that belong to an evolutionarily-conserved multigene family, the members of which are expressed throughout the animal and plant kingdoms. These mainly intracellular proteins are localized either free in the cytosol or associated with cellular membranes or cytoskeletal proteins (Gerke and Moss, 2002). Despite the fact that these proteins lack secretary signals, many previous reports have documented their presence in the extracellular milieu. Annexin A1 was detected by immunoblotting analysis in the supernatants of colonic biopsies incubated in culture media, and in the luminal colonic perfusates of ulcerative colitis (UC) patients (Vergnolle et al., 2004). Annexin A6 was shown to be translocated from an intracellular pool to the external basolateral surface of the mouse mammary-duct epithelium upon the onset of lactation (Rocha et al., 1990). Moreover, annexin A2 showed a similar pattern of expression on endothelial cell surfaces (Hajjar et al., 1994).
Nevertheless, annexins are not secreted through the classical exocytic pathway, and the lack of a hydrophobic leader peptide prevents them from being targeted to the endoplasmic reticulum. Experiments in many cell lines have shown a direct correlation between the protein kinase C (PKC) phosphorylation and annexins' calcium-dependent membrane binding or exocytosis (Creutz et al., 1978; Liu et al., 1996; Gerke and Moss, 1997; Chander et al., 2001).
Taenia solium metacestode infection is a zoonotic disease with a wide distribution throughout the world. It presents a serious threat to human health by causing neurocysticercosis, and is responsible for important economic losses in pig breeding, especially in developing countries (Garcia and Brutto, 2000). In order to obtain the protective antigens required to develop nucleic-acid vaccines against Cysticercus cellulosae, an expression library was screened using sera from infected humans and/or pigs (Sambrook et al., 1989). One clone, designated as cC1, was recognized by sera from both humans and pigs. Sequence analysis revealed that it had typical carboxy (C)-terminus and distinct amino (N)-terminus structures. The cDNA encoded a novel member annexins family, which was designated as annexin B1 (Yan et al., 2002).
Annexin B1 was isolated from a Cysticercus cellulosae library by immunological screening, implying that it might be able to be secreted into the extracellular milieu. It was in this context that we investigated whether annexin B1 was in fact secreted, and if this is true, what is the mechanism underlying the secretion. We firstly isolated Cysticercus cellulosae and peripheral muscles from infected pigs and examined the histological distribution of annexin B1 by using immunohistochemical analysis. Then we constructed a pEGFP–annexin B1 plasmid and stimulated the pEGFP–annexin B1 transfected SiHa cells with both phorbol 12-myristate 13-acetate (PMA) and ionomycin.
2 Material and methods
We obtained permission for performing the research protocols and all experiments, following the guidelines of the ethics committee of the Second Military Medical University (SMMU). Muscle samples from five condemned pigs naturally infected with the larvae of T. solium were obtained from Experimental Animal Centre of Hebei province. These pigs were diagnosed according to the national standard and confirmed at slaughter. Samples, including one or more larvae and surrounding muscle tissues, were selected from heavily infected skeletal muscles. A total of 32 lesions were divided in two groups randomly: some were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 5
2.2 Detection of annexin B1 by double antibodies sandwich ELISA (Ag-ELISA)
We have previously obtained a monoclonal antibody against annexin B1 (2B10H5) with high sensitivity and specificity (Gao et al., 2007). The serum samples were tested in duplicate for the detection of annexin B1 using a sandwich ELISA (Ag-ELISA) as described before (Dorny et al., 2000, Pouedet et al., 2002). Ninety-six-well plates (Sino-American Biotechnology Company) were coated with 1
Plates were washed again followed by the addition of sheep anti-pig IgG coupled to horseradish peroxidase (HRP, Jingmei Biotech), which was developed with 3,3′-diaminobenzidine (DAB) substrate. Plates were washed three times after each reaction step. Optical density readings at 450
The small pieces of tissue were fixed in acetone at 4
2.4 Plasmid construction
The full-length cDNA of annexin B1 and human annexin A5 was originally cloned into the pJLA503 vector (Zhang et al., 2004). The resultant constructions, designated as pJLA503–annexin B1 and pJLA503–annexin A5, were used as template DNA for PCR. The oligonucleotide primers designed to amplify the coding region of annexin B1 were forward primer: 5′-CGCCTACTGTCGCTCCCTGGTTC-3′ and reverse primer: 5′-GCTATTATGCAGGGCCGATGAGTTTCAAG-3′. Full-length human annexin A5 cDNA was amplified by PCR using forward primer 5′-GGCCCAGCTAGCTTCAAAATGTCTACTG-3′ and reverse primer 5′-ACCATTTGTCGACGCTCAGGCCGTGT-3′ based on the cDNA sequence of annexin A5. The eukaryotic expression recombinant plasmids, pEGFP–annexin B1 and pEGFP–annexin A5, were constructed by inserting the amplified fragment into the EcoRI and SalI sites of pEGFP–N1 (Clontech, USA) and transformed into Escherichia coli DH5α. The transformants were confirmed by DNA sequencing.
2.5 Cell culture, transfection and treatment
SiHa cells were grown to 90% confluence at 37
2.6 Western blotting
After being transfected for 48
2.7 Measurement of intracellular calcium in cells after treatment with PMA and ionophore
The transfected SiHa cells were grown overnight in glass bottom dishes (Willco Wells, USA). After being starved for 24
2.8 Statistical analysis
Data are expressed as mean±S.E. and the number of experiments is shown as n. Statistical comparisons were made with the use of Student's t-test. A value of P
3.1 Histological distribution of annexin B1
We first determined whether annexin B1 could be found in the cystic fluid of T. solium cysticerci and the sera of infected humans/pigs. Double-antibody sandwich ELISA demonstrated the following scores: 19 out of 27 (70%) in the cystic fluid of T. solium cysticerci; 4 out of 5 (80%) in the sera of pigs with cysticercosis; and 2 out of 3 (67%) in human sera with cysticercosis. No antigens were detected in the sera of patients with the helminth infections mentioned above or in cysticercosis-free pigs.
We further detected the histological distribution of annexin B1 in the T. solium cysticerci. Strong brown signals were mainly detected in the bladder wall of the T. solium cysticerci and the layer derived from the host. Intriguingly, signals were only present in the inflammatory layer consisted of macrophages that transformed into epithelioid cells (Fig. 1), but not in the fibrous layer mainly consists of mesenchymal cells. These data suggested that annexin B1 was able to be present out of the parasite and might be related to inflammatory reaction.
Histological distribution of annexin B1. The procedure of immunohistochemical staining is described in Section
3.2 Identification of annexins in transfected SiHa cells
Western blot analysis was used to determine whether these GFP chimeras were expressed as stable and intact proteins. Fig. 2 shows that GFP–annexin B1 and GFP–annexin A5 were indeed present in these cells. The molecular masses of the antigens corresponded to those reported in the literature (those being GFP–annexin B1, 65
SDS–PAGE of total lysates of transfected SiHa cells and Western blotting of GFP–annexin B1 and GFP–annexin A5. (A) Protein markers were loaded in lane 1; total cellular lysates from pEGFP–annexin B1 transfected cells was loaded in lane 2; the lysates from pEGFP–annexin A5 transfected cells were loaded in lane 3. (B) Immunoblot analysis of GFP–annexin B1 (lane 4) and GFP–annexin A5 (lane 5). The Western blotting used monoclonal antibody against annexin B1 or human annexin A5 followed by HRP-conjugated secondary antibody.
3.3 Translocation of GFP–annexin B1 after treatment with PMA
To examine the subcellular localization of GFP–annexin B1 in response to PMA, transfected SiHa cells were incubated in the presence or absence of 20
Subcellular localization of GFP, GFP–annexin B1 and GFP–annexinA5 in SiHa cells after treatment with PMA in the presence of calcium. SiHa cells were transfected with expression constructs encoding GFP (A) or GFP–annexin B1 (B), or GFP–annexin A5 (C) and processed for confocal laser scanning microscopy as described. The cells were fixed with 3.7% formaldehyde in the absence of PMA (1), after 15
3.4 Translocation of GFP–annexin B1 after stimulation by ionomycin
To investigate the subcellular localization of GFP–annexin B1 after increasing the intracellular calcium level, transfected SiHa cells were stimulated with 10
Subcellular localization of GFP, GFP–annexin B1 and GFP–annexinA5 in SiHa cells after stimulation by ionomycin in the presence of calcium. SiHa cells were transfected with expression constructs encoding GFP (A), GFP–annexin B1 (B) or GFP–annexin A5 (C), and processed for confocal laser scanning microscopy. SiHa cells were fixed with 3.7% formaldehyde in the absence of ionomycin (1), after 10
3.5 Changes in cytosolic Ca2+ concentration induced by PMA or ionomycin
Calcium-imaging studies were performed to determine the extent to which the calcium levels rose in response to be treated with PMA or ionomycin. The results showed the relative changes of intracellular fluorescence. For the pEGFP–annexin B1-transfected cells, the fluorescence intensity increased sharply to the maximal value (200
Intracellular free calcium transients induced by ionomycin and PMA in SiHa cells transfected with pEGFP–annexin B1 and pEGFP–annexin A5. The cells were grown on glass-bottom dishes and loaded with rhod-2/AM. Changes in calcium levels were induced by perfusing the cell layer with ionomycin (10
We have detected strong brown signals in the host-derived layer with granulomatous infiltration by using immunohistochemical staining. In addition, annexin B1was present both in cystic fluid of T. solium cysticerci and sera of infected humans/pigs. These findings indicate that annexin B1 can be detected outside the parasite, and may belong to the few members of annexins which can be secreted, as has been reported previously in studies of other members of this protein family (Christmas et al., 1991; Yeatman et al., 1993; Solito et al., 1994; Coméra and Russo-Marie, 1995; Thorin et al., 1995; Vergnolle et al., 1995; Siever and Erickson, 1997; Perretti, 1998).
Our previous study demonstrated an anti-PLA
In a previous study, we also found that apoptotic signals were detected from the metacestodes isolated from pigs that were inoculated with pcDNA3–annexin B1, but not in those injected with pcDNA3 (Wang et al., 2003). In cardiomyocytes, annexin A5 was externalized at an early stage of apoptosis and might have had a proapoptotic effect (Monceaua et al., 2004). In human neutrophils, a novel calcium-dependent proapoptotic effect of annexin A1 has been confirmed (Solito et al., 2003). It can be assumed that that annexin B1 might be secreted from T. solium cysticerci and then engulfed into the bladder wall cells to protect the parasite by its proapoptotic effect.
Further studies are needed to confirm these assumptions and to explore the real physiological roles of annexin B1. Annexins have been shown to bind to negatively charged phospholipids, such as those enriched in the inner leaflet of the plasma membrane and the cytoplasmic aspect cellular organelles such as secretary vesicles, in a calcium-dependent manner (Raynal and Pollard, 1994). In the present study, we generated chimeric constructs containing GFP tag, and monitored the intracellular localization of GFP–annexin B1 in living cells. Our previous study (Yan et al., 2002) has shown that annexin A5 is more homologous to annexin B1 than other human annexins, and therefore we selected it for parallel studies. The results show that GFP–annexin A5 are mainly located in the nucleus in unstimulated cells; after raising intracellular calcium by ionomycin, GFP–annexin A5 relocated from its homogeneous distribution in nucleus to the periphery of the nuclear and plasma membrane. These findings are consistent with those of previous studies (Sun et al., 1992; Koster et al., 1993), indicating that the GFP tag on the N-terminus had little influence on the function of annexins.
Our results indicated that GFP–annexin B1 and GFP–annexin A5 were both translocated to the plasma and nuclear membrane when intracellular Ca2+ was raised by ionomycin. However, GFP–annexin B1 and GFP–annexin A5 showed a different response to the stimulation of PMA. PMA stimulation not only elevated the intracellular Ca2+ but also increased PKC activity. PKCs are serine–threonine kinases that are activated by diverse stimuli, including mitogens, and participate in a variety of cellular processes (such as proliferation, differentiation and apoptosis) (Battaini, 2001; Nishizuka, 2001). Annexin B1 has a short motif “Thr-Ile-Thr” in its N-terminus, which is similar to “Thr-Val-Thr” in the N-terminus of annexin A5. Previous studies have shown that human annexins A1, A2, A3 and A4, Drosophila annexin B10 and Hydra annexin B12 possess a “Thr/Ser-Val/Ile-Lys/Arg” motif in their N-termini. This conserved motif has been proposed to be a PKC phosphorylation site (Raynal and Pollard, 1994; Rothhut, 1997). In contrast, in the N-termini of annexin A5 and A6, the third basic residue in the short motif has been substituted by an uncharged Thr residue; as a consequence, annexins A5 and A6 inhibit the protein kinase C activity via a mechanism of phospholipid sequestration (Dubois et al., 1998). Thus, we assumed that intracellular PKC activity increased upon PMA stimulation, and annexin B1 might inhibit the PKC activity by binding plasma membrane. However, the mechanism needs further investigation.
In conclusion, by using immunochemical analysis and ELISA assay, we first identified that annexin B1 could indeed be secreted from the cell. When the intracellular Ca2+ concentrations were raised by treatment with PMA or ionomycin, we found that GFP–annexin B1 was able to translocate from the cytoplasm to the plasma membrane or nuclear membrane. These findings shed light on further exploration of the secretion mechanism and real physiological roles of annexin B1.
We wish to acknowledge the financial support the grants from the National Natural Science Foundation of China (No. 30271167), and Key Fundamental Research Programs of Shanghai (No. 05JC14043) and National key fundamental research programs of “973” project (No. 2006CB504105).
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Received 24 March 2007/28 June 2007; accepted 27 August 2007doi:10.1016/j.cellbi.2007.08.022