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Collagen-induced STAT family members activation in Entamoeba histolytica trophozoites

Jorge Cruz-Vera , L. Clara , R. Hernández-Kelly , J. Alfredo Méndez , Eduardo Pérez-Salazar , Arturo Ortega
DOI: http://dx.doi.org/10.1016/S0378-1097(03)00822-X 203-209 First published online: 1 December 2003

Abstract

The interaction of Entamoeba histolytica trophozoites with collagen type I and calcium induces a membrane to nuclei signaling. The transduction pathways involved in such phenomena are still poorly understood. Using a combination of immunoprecipitation assays, Western immunoblot analysis, electrophoretic mobility shift assays and immunocytochemistry we demonstrate here the expression, tyrosine phosphorylation, nuclear translocation and DNA binding of two members of the signal transducers and activators of transcription family of inducible transcription factors in the protozoan parasite E. histolytica. These results support the notion that the interaction of the extracellular matrix components with the parasite turns on a genetic program that facilitates the invasion of the host.

Keywords
  • Amoeba
  • Signal transduction
  • Collagen
  • Transcriptional regulation
  • Extracellular matrix

1 Introduction

The trophozoites of the human invasive parasite Entamoeba histolytica, bind extracellular matrix (ECM) components, such as collagen, fibronectin and laminin. This interaction results in adherence of the parasite with the formation and release of electron-dense granules that contain a collagenase activity involved in the pathogenic capacity of the parasite [1]. ECM components have a dynamic interaction through specific recognition sites, such as the RGD sequence [2,3]. This tripeptide binds with high affinity to the integrin receptor superfamily. These receptors are transmembranal glycoproteins formed by two non-identical subunits involved in the signaling between the ECM and the cytoskeletal network, although they participate in the regulation of gene expression [4].

The interaction between ECM components and integrins leads to the appearance of focal adhesion plaques that regulate cell attachment and actin polymerization through a tyrosine kinase cascade. In this context the non-receptor tyrosine kinase, pp125FAK plays a major role in cell spreading since overexpression of mutant version of this protein disrupts focal contact formation [5]. This non-receptor tyrosine kinase is activated through tyrosine phosphorylation during integrin-mediated cell adhesion [6,7]. Interestingly enough, an amoeba 140-kDa protein with the features of a β-1 integrin receptor that associates with tyrosine kinases and a collagen-binding protein has been reported [8,9].

Several signal transduction mechanisms are triggered in E. histolytica trophozoites after its interaction with ECM components [10]. Specifically, type-I collagen and calcium activate a kinase cascade that includes the focal adhesion kinase (pp1255FAK), the extracellular regulated kinases 1 and 2 (ERK1-2) and results in an increase in Fos expression that leads to a significant augmentation of activator protein 1 (AP-1) DNA binding [11]. This enhanced transcriptional activity might be involved in the synthesis and secretion of protease activities important for the parasite invasiveness [12]. AP-1 DNA binding has been reported to be induced through distinct second messenger systems by means of the concerted activation of multiple control elements present in the promoter region of early responsive genes like c-fos [13,14]. One of these cis-acting elements, known to be determinant for Fos expression is the sis-inducible element (SIE) [15].

The signal transducers and activators of transcription (STAT) family of inducible transcription factors are cytoplasmic proteins that after tyrosine-phosphorylation form homo- or heterodimers that are translocated to the nucleus where they bind DNA within a well defined consensus sequence such as the SIE [16]. Multiple STAT isoforms exist and a number has been added to the term STAT in order to differentiate the isoforms [17]. Tyrosine phosphorylation is essential for their function, although recently, it has been demonstrated that serine phosphorylation is also important for STAT function [18]. In the present work, we demonstrate the expression and activation of the E. histolytica homologs of STAT1 and STAT3 as a result of the activation of trophozoites with collagen type I and calcium. These results further support the hypothesis that ECM components trigger the expression of genes are relevant for the pathogenicity of this parasite.

2 Materials and methods

2.1 Cells

E. histolytica strain HM1: IMSS trophozoites were cultured in TYI S-33 medium [19]. All the experiments were carried out with trophozoites harvested after 48 h of culture.

2.2 Preparation of nuclear extracts from activated trophozoites

Approximately, 107 million trophozoites grown in culture bottles were rinsed with 10 mM K2HPO4/KH2PO4, 150 mM NaCl, pH 7.4 (PBS). Cells were incubated for 5 h in serum-free medium, subsequently were treated with or without (control) 3 mg ml−1 of human placenta collagen type I and 1 mM CaCl2 in serum-free medium for the indicated time periods. The cells were harvested and washed several times with PBS, pelleted and immediately incubated in serum-free medium containing 10 µg ml−1 of cytochalasin B (Sigma, St. Louis, MO, USA) for 10 min at 37°C. After another cycle of washing, the cells were resuspended in 400µl of ice-cold buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, pH 7.9). The cells were allowed to swell on ice for 15 min after which 25 µl of 10% NP-40 was added and the tube vigorously vortexed for 10 s. The homogenate was centrifuged for 50 s at top speed. The supernatant containing the cytosolic fraction was transferred to a fresh tube, whereas the nuclear pellet was resuspended in 50 µl of ice-cold buffer C (20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, pH 7.9) and the tube was vigorously rocked for 15 min and centrifuged for 5 min. The supernatant containing the nuclear proteins was stored at −70°C until use.

2.3 Gel electrophoresis and DNA binding assay

Nuclear extracts were prepared as outlined above from treated or untreated cells and approximately 15 µg were incubated on ice with 1 µg of poly (dI-dC) as non-specific competitor and 1 ng of 32P end-labeled double-stranded oligonucleotides: SIE: 5′-GTCGACATTTCCCGTAAATCGTC-3′. The reaction mixtures were incubated for 10 min at 4°C and electrophoresed in 5% polyacrylamide gels using a low ionic strength 0.5× Tris–borate–EDTA buffer [20]. The gels were dried and exposed to autoradiographic film or scanned using a Typhoon optical scanner (Molecular Dynamics, Piscataway, NJ, USA). For competitive studies, the reaction mixtures were pre-incubated with the indicated amount of unlabeled competitor oligonucleotide before the addition of labeled DNA. For gel supershift experiments, the reactions with the DNA–protein complexes were incubated at 4°C with anti-STAT1, anti-STAT3 or p53 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h prior to electrophoresis.

2.4 Immunoprecipitation and Western immunoblots

Cell lysates of control and treated cells were prepared by boiling for 5 min in 150 mM Tris containing 1 mM PHMB and solubilized in 200 µl of 100 mM Tris–HCl, 150 mm NaCl, 2 mM EDTA, 20 mM sodium molybdate, and 50 mM sodium fluoride, 0.1% sodium deoxycholate, and 2 mM PMSF, pH 7.4 (RIPA buffer). The cell debris was removed by centrifugation (14 000×g for 5 min). The lysate was preabsorbed with 15 µl of protein A or G coupled to Sepharose 4B (Invitrogen) for 15 min at 4°C. The primary antibody (0.125 ng) was then incubated (10 h, 4°C) with samples of the supernatant. The immune complexes were collected after incubation with Protein A or G-conjugated Sepharose 4B beads for 2 h at 4°C. The beads were pelleted, washed several times with NET buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA, 20 mM sodium molybdate, and 50 mM sodium fluoride, 0.1% bovine serum albumin (BSA), 0.1% SDS, 0.1% sodium deoxycholate, 0.5% NP-40, pH 7.0), and boiled for 5 min in Laemmli's sample buffer. Equal volumes of solubilized material were analyzed through 7.5% SDS–polyacrylamide gels, and the proteins were blotted to nitrocellulose membranes. Filters were stained with Ponceau S to ensure that equal levels of protein were present in all lanes. The membranes were washed with PBS and incubated 1 h to block excess protein binding sites using 5% non-fat dried milk in PBS, pH 7.2. Filters were then incubated for 1 h with the appropriate primary antibody, followed by either anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase. Finally, the proteins were detected using an ECL chemiluminescence kit (Amersham).

2.5 Staining procedures

Cell staining with anti-phospho-STAT1 or anti-phospho-STAT3 monoclonal antibodies was performed. Cells were grown in ethanol-washed and poly-l-lysine-treated (0.01 mg ml−1) glass coverslips for 48 h. Cells were incubated for 5 h in serum-free medium, subsequently were treated with or without (control) 3 mg ml−1 of human placenta collagen type I and 1 mM CaCl2 in serum-free medium for 60 min, then trophozoites were fixed for 15 min with 1.85% formaldehyde and 0.125% glutaraldehyde. They were then incubated with 1% BSA in PBS (BSA/PBS) for 60 min at room temperature. Cells were then exposed for 60 min to anti-phospho-STAT1 or anti-phospho-STAT3 monoclonal antibodies. The cells were washed several times with BSA/PBS and finally exposed for 60 min to rhodamine-labeled anti-mouse IgGs, washed three times in BSA/PBS. The preparation was mounted in 90% glycerol/PBS. Control immunolabeling was performed with the same staining procedures described above, without the primary antibodies.

3 Results

3.1 Collagen type I and calcium induce tyrosine phosphorylation of STAT1 and STAT3 in E. histolytica trophozoites

We have previously shown that human collagen type I and calcium induce a membrane to nuclei signaling that up-regulates Fos expression [21]. In order to gain insight into the molecular mechanisms involved in this phenomenon, we decided to explore the participation of STAT proteins in collagen signaling in amoeba. Control and collagen-treated trophozoites were subjected to immunoprecipitation assays with the anti-phosphotyrosine antibodies followed by Western immunoblot analysis with anti-STAT1 antibodies. The results are presented in Fig. 1. Collagen induces a time dependent increase in tyrosine phosphorylation of both STAT1α and STAT1β. These proteins become tyrosine-phosphorylated as early as 15 min of stimulation with collagen, reaching a maximal stimulation after 120 min of collagen treatment. Note that even in serum-free medium a basal STAT1 phosphorylation is present in both isoforms. When the phosphorylation status of STAT3 was explored, again, both isoforms (α and β) increase their phosphorylation content after exposure to collagen and calcium (Fig. 2). In this case a transient effect was detected, reaching a maximum after 45 min of treatment but returning to basal levels after 1 h of collagen exposure. These results suggest that STAT might be translocated to the nuclei after the interaction of E. histolytica trophozoites with collagen type 1 and calcium.

Figure 1

Tyrosine phosphorylation of STAT1 in E. histolytica trophozoites following collagen type I and calcium stimulation. Trophozoites were stimulated with 3 mg ml−1 collagen for the indicated time periods and cytoplasmic extracts were immunoprecipitated with anti-phosphotyrosine antisera, and analyzed via Western blots with anti-STAT1 monoclonal antibodies, that recognize the α- and the β-isoforms of the protein. Immunopositive polypeptides were detected with the ECL kit (Amersham). A representative autoradiography is presented whereas the graph represents the average of at least three independent experiments. *P<0.05 as compared to control values (Student's t-test).

Figure 2

Tyrosine phosphorylation of STAT3 in E. histolytica trophozoites following collagen stimulation type I and calcium stimulation. Trophozoites were stimulated with 3 mg ml−1 collagen for the indicated time periods and cytoplasmic extracts were immunoprecipitated with anti-phosphotyrosine antisera, and analyzed via Western blots with anti-STAT3 monoclonal antibodies, that recognize the α and the β isoforms of the protein. Immunopositive polypeptides were detected with the ECL kit (Amersham). A representative autoradiography is presented whereas the graph represents the average of at least three independent experiments. *P<0.05 as compared to control values (Student's t-test).

3.2 Collagen type I and calcium induce STAT1/STAT3 heterodimer formation and nuclear translocation in E. histolytica trophozoites

As stated, above, it has been well documented in a number of systems that once tyrosine phosphorylated, STAT proteins are translocated into the nucleus where the dimmer binds to the SIE in a number of genes, including c-fos. To explore the identity of STAT homo- or heterodimers formed as result of collagen and calcium induction in E. histolytica, we performed immunoprecipitation assays coupled with Western immunoblots assay anti-phospho-STAT antibodies. As depicted in Fig. 3, panel A, we were able to detect phospho-STAT3 in phospho-STAT1 immunoprecipitates in nuclear extracts prepared from collagen-treated trophozoites. Interestingly, this effect is time-dependent with a maximal association after 45 min of treatment. As expected when we immunoprecipitate with anti-phospho-STAT3 antibodies we can detect phospho-STAT1 in the immunoprecipitate (not shown). These results strongly suggest that collagen and calcium induce an association between phospho-STAT1/phospho-STAT3 that is targeted to the nuclei. Immunocytochemical evidence of the latter interpretation is provided in panel B of Fig. 3, collagen treatment results in a dramatic targeting of both phospho-STAT1 and phospho-STAT3 to the nucleus.

Figure 3

STAT1–STAT3 association and nuclear translocation. Panel A: E. histolytica trophozoites were stimulated as in Figs. 1 and 2. Nuclear extracts were prepared as detailed under Materials and methods and immunoprecipitated with anti-phospho-STAT1 monoclonal antibodies. The immunoprecipitates were analyzed via Western blot with anti-STAT3 monoclonal antibodies. Immunopositive polypeptides were revealed with the ECL kit (Amersham). Panel B: Immunostaining of control and collagen treated trophozoites with anti-phospho-STAT1 and anti-phospho-STAT3 antibodies. Trophozoites were treated with collagen type I and calcium for 60 min. Lower panels, phase contrast of the immunostained areas in the upper panels. Staining with anti-phospho-STAT1 or anti-phospho-STAT3 antibodies followed by rhodamine-labeled goat anti-mouse IgGs. Trophozoites were examined under 40× objective. Bars=10 µm.

3.3 Involvement of ERK1/2 in STAT signaling in E. histolytica trophozoites

It has recently been documented that although STAT tyrosine phosphorylation is a prerequisite for dimmer formation, nuclear translocation, binding to its cognate DNA sequences, and regulation of the target gene transcription, serine phosphorylation plays an additional role in the regulation of transcription [22]. Among the enzymes known to phosphorylate STAT3 in serine residues are ERK1 and ERK2. These proteins are an important point of convergence of different signaling pathways that regulate gene expression [16,17]. With this in mind, we hypothesized that an association between ERK and STAT proteins would be taking place as a consequence of collagen treatment. As shown in Fig. 4, this is indeed the case, in STAT1 immunoprecipitates, we were able to detect ERK1 and to a lesser extent, ERK2. To clarify if this association depends upon ERK activation, we exposed collagen-treated trophozoites to 7 µM of the MEK inhibitor PD98059. Under such circumstances, a significant reduction in the ERK levels in the phospho-STAT1 immunoprecipitates was found. It should be noted, however that a basal phospho-STAT1–ERK interaction is present. The origin of this basal association is not known at this moment.

Figure 4

Collagen type I-induced STAT1/ERK1/2 association in E. histolytica trophozoites. Trophozoites were stimulated with 3 mg ml−1 collagen with or without 7 µM PD98059 for the indicated time periods and cytoplasmic extracts were immunoprecipitated with anti-phospho-STAT1 antibodies, and analyzed via Western blot with anti-ERK1/2 polyclonal antibodies. Immunopositive polypeptides were detected with the ECL kit (Amersham). A representative autoradiography is presented whereas the graph represents the average of at least three independent experiments. *P<0.05 as compared to control values (Student's t-test).

3.4 Collagen type I and calcium induces STAT DNA binding activity in E. histolytica trophozoites

As already stated, STAT homo- or heterodimers once in the nuclei bind DNA in consensus sequences such as the SIE. Taking into consideration that collagen and calcium up regulate Fos expression, we decided to explore if we could detect the interaction of STAT heterodimers with a double-stranded oligonucleotide probe that contains the SIE of the human fos promoter [23]. As shown in panel A of Fig. 5, collagen treatment leads to the appearance of two retarded bands that are efficiently competed with a 100 excess of unlabeled probe. Supershift experiments with either anti-phospho-STAT1 or anti-phospho-STAT3 antibodies, clearly demonstrate that STAT1 and STAT3 participate in the DNA–protein complexes (panels B and C). These results are in line with the immunoprecipitation assays (Figs. 1 and 2).

Figure 5

Collagen-induced STAT DNA binding activity. Panel A: Nuclear extracts were prepared from control or collagen-treated trophozoites and incubated with a [32P]SIE probe. Specificity of the DNA binding activity was confirmed with a 100-fold excess of the unlabeled probe. Panel B: Supershift analysis of components of STAT complexes. Incubation of protein–DNA complexes with anti-phospho-STAT1 antibodies shifts the mobility of the lower complex (lower arrow). As a control anti-pp125FAK antibodies were used. Panel C: Phospho-STAT3 is present in the upper DNA complex. Supershift experiments were performed as in panel B. Results are representative of at least three independent experiments.

4 Discussion

We provide here evidence, not only for the expression, but most important, for the involvement of the STAT signaling cascade in the signaling transaction triggered by collagen type I and calcium in E. histolytica. One could argue that in the ∼70% of the amoeba genome one cannot identify the presence of any STAT member (data available on the TIGRdb via the World Wide Web-address: http://www.tigr.org/tdb/e2k1/eha1). A clear possibility is that these genes are located in the 30% of the genome no sequenced to date. In favor of this interpretation is the fact that at least in two E. histolytica promoters, bona fide STAT cognate sequences are present [24,25].

We have previously reported that collagen type I and calcium treatment upregulates Fos expression and that this is well correlated with an acute augmentation of AP-1 DNA binding [21]. Unable at this moment to characterize the E. histolytica c-fos promoter, we decided to focus on cis regulatory elements known to be critical for Fos expression in other systems. The SIE element has been demonstrated to play a pivotal role in c-fos transcription [15]. Therefore, we hypothesized that the STAT signaling cascade could be activated in response to collagen treatment in E. histolytica trophozoites.

Using a combination of immunoprecipitation assays coupled to Western immunoblots, first we were able to show that among the proteins that undergo tyrosine phosphorylation after collagen treatment two members of the STAT family are present, namely STAT1 and STAT3 (Figs. 1 and 2). These results were confirmed by the use of anti-phospho-STAT antibodies (Fig. 3). The identity of the tyrosine kinase responsible of STAT phosphorylation in response to collagen, is not known at this moment, but it may well be that pp125FAK plays this role, as demonstrated for 293T and A431 cells [5]. One has to keep in mind that this non-receptor tyrosine kinase is activated in response to collagen and calcium in E. histolytica trophozoites [11].

Phosphorylated STAT1 and STAT3 form complexes that co-immunoprecipitate. Interestingly, not only tyrosine phosphorylation of these proteins is needed for their interaction, but apparently also serine phosphorylation is necessary, since is STAT1–STAT3 interaction is sensitive to the MEK inhibitor PD90187 (Fig. 4). In this regard, it has been shown that ERKs phosphorylates STAT3 in Ser 727 [22]. This phosphorylation is crucial for DNA binding and transcriptional enhancement [18].

At this stage, the results presented thus far, suggested that STAT amoeba homologs were involved in membrane to nuclei signaling, so it was imperative to evaluate whether collagen and calcium could induce STAT1 and STAT3 DNA binding to the SIE of the fos promoter. The data in Fig. 5 detected two DNA–protein complexes which are supershifted by anti-phospho-STAT antibodies after exposure to collagen. Note that anti-STAT1 antibodies shift the lower complex, whereas anti-STAT3 shift the upper band. A summary of our current knowledge of collagen signaling in E. histolytica trophozoites is depicted in Fig. 6.

Figure 6

Current model of the signaling cascades activated by collagen type I and calcium. The stimulation of a putative β1-integrin like collagen receptor leads to a membrane to nuclei signaling that up-regulates the expression of genes most possibly involved in the pathogenic capacity of the parasite. Continuous arrows refer to established cascades whereas broken arrows describe plausible pathways.

In summary, we provide here evidence for the involvement of the STAT signaling pathway in the biochemical response of E. histolytica trophozoites to collagen type I and calcium. The present findings should pave the way to a better understanding of the molecular mechanisms involved in the pathogenesis of amoebiasis.

Acknowledgments

This work was supported by a Conacyt-México Grant (33058-N) to A.O. J.C.-V. and J.A.M. are supported by Conacyt doctoral training fellowships.

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