Thymosin β4 promotes the migration of endothelial cells without intracellular Ca2+ elevation

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318 (2012) 1659–1666

Available online at www.sciencedirect.com

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Research Article

Thymosin b4 promotes the migration of endothelial cells without intracellular Ca2þ elevation Anna Selmia,1, Mariusz Malinowskib,1, Wojciech Brutkowskic, Radoslaw Bednareka, Czeslaw S. Cierniewskia,b,n a

Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz, Poland Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland c Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warsaw, Poland b

article information

abstract

Article Chronology:

Numerous studies have demonstrated the effects of Tb4 on cell migration, proliferation,

Received 9 January 2012

apoptosis and inflammation after exogenous treatment, but the mechanism by which Tb4

Received in revised form

functions is still unclear. Previously, we demonstrated that incubation of endothelial cells with

23 March 2012

Tb4 induced synthesis and secretion of various proteins, including plasminogen activator

Accepted 10 April 2012

inhibitor type 1 and matrix metaloproteinases. We also showed that Tb4 interacts with Ku80,

Available online 29 May 2012

which may operate as a novel receptor for Tb4 and mediates its intracellular activity. In this

Keywords:

paper, we provide evidence that Tb4 induces cellular processes without changes in the

Calcium signaling

intracellular Ca2þ concentration. External treatment of HUVECs with Tb4 and its mutants

Thymosin b4

deprived of the N-terminal tetrapeptide AcSDKP (Tb4AcSDKPT/4A) or the actin-binding sequence

HUVECs

KLKKTET (Tb4KLKKTET/7A) resulted in enhanced cell migration and formation of tubular structures

Adhesion and migration

in Matrigel. Surprisingly, the increased cell motility caused by Tb4 was not associated with the

Angiogenesis

intracellular Ca2þ elevation monitored with Fluo-4 NW or Fura-2 AM. Therefore, it is unlikely that externally added Tb4 induces HUVEC migration via the surface membrane receptors known to generate Ca2þ influx. Our data confirm the concept that externally added Tb4 must be internalized to induce intracellular mechanisms supporting endothelial cell migration. & 2012 Elsevier Inc. All rights reserved.

Introduction Intracellular Ca2þ elevation plays a key role in the regulation of endothelial cell migration [1–5]. It activates the transcription of a number of immediate early genes which are required for the induction of cell proliferation and motility [6]. Moreover, an increase in [Ca2þ]i frequently results in nitric oxide production, which in turn promotes the proliferation of endothelial cells during n

wound healing [7]. Growing evidence suggests that altered Ca2þsignaling contributes to carcinogenesis, increased migration and proliferation, decreased apoptosis, dedifferentiation, metastasis and therapy-resistance [8]. Interestingly, all of these cellular processes can be stimulated by thymosin b4 (Tb4), but the mechanism explaining how this happens is not yet fully understood. Tb4 is a multifunctional protein that has pleiotropic activities, which are important in cell survival and repair. In contrast to numerous

Corresponding author at: Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz, 6/8 Mazowiecka St., Poland. Fax: þ48 42 678 94 33. E-mail address: [email protected] (C.S. Cierniewski). 1 These authors contributed equally to this work. 0014-4827/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2012.04.009

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studies demonstrating the effects of Tb4 on various cellular processes, little is known about the downstream molecules or signaling mechanisms that modulate Tb4-induced activation of endothelial cells and, therefore, affect their migration. Recently, a novel mechanism explaining the numerous extracellular activities of Tb4 has been proposed [9]. Tb4 was identified to interact in endothelial cells with ecto-ATP synthase and to increase cell surface ATP levels. Consequently, the extracellular signaling pathway involving the ATP-responsive P2  4 receptor leading to enhanced HUVEC migration was proposed [9]. P2  4 belongs to the 7 mammalian P2X receptor family members which are ATP-gated ion channels [10,11], and all these receptors, with the exception of P2  5, can facilitate entry of Ca2þ in response to stimulation by extracellular ATP [11–13]. Therefore, in this study we attempted to explain whether Tb4 generates Ca2þ signals in migrating endothelial cells.

Material and methods Reagents and antibodies Synthetic thymosin Tb4 was from Bachem. The MMPs and their inhibitors used in this study included MMP-2 and MMP-9 (Chemicon, (Temecula, CA), TIMP-1, TIMP-2 and TIMP-3 (R&D Systems), MMP-2 Inhibitor II and MMP-9 Inhibitor I (Calbiochem), and GM6001 Chemicon, (Temecula, CA). Recombinant Tb4 and its mutants, modified by substitution of functional motifs with alanine using scanning mutagenesis, were obtained as previously described [14]. Thus, mutants were produced that were deprived of the N-terminal tetrapeptide AcSDKP (Tb4(AcSDKPT/4A)) and the actin-binding sequence KLKKTET (Tb4(KLKKTET/7A)).

Cell cultures HUVECs cultured in Medium 200, supplemented with Low Serum Growth Supplement containing 2% FBS and antibiotics (streptomycin and penicillin), were grown in monolayer in flasks or dishes at 37 1C in 5% CO2. For the experiments, HUVECs were harvested at confluence with trypsin/EDTA and suspended in Medium 200 containing 1% FBS and antibiotics. Human endothelial cell line EA.hy926, derived by fusion of human umbilical vein endothelial cells with continuous human lung carcinoma cell line A549, was obtained from ATCC (Manassas, VA). The EA.hy926 cells were cultured in growth medium DMEM with high glucose and Lglutamine, supplemented with 10% fetal bovine serum (FBS) and antibiotics. The culture flasks were maintained in a 90–95% humidified atmosphere of 5% CO2 at 37 1C. Cells were harvested at confluence with trypsin/EDTA. Cell viability was determined microscopically by Trypan blue exclusion. Growth media and supplements for cell culture were from Sigma-Aldrich (St. Louis, CA).

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the cells with a 200 ml pipette tip. Afterwards, the cells were rinsed twice with M199 medium to remove any wound-derived, loose and dislodged cells, and further cultured in a replaced M199 medium containing 1% BSA for 2 h to 24 h, with or without 200 nM Tb4 or its mutants Tb4(SDKP/4A) or Tb4(KLKKTET/7A, used at a concentration of 200 nM. In some experiments, the cells were additionally treated with 25 mM GM6001. Images were recorded immediately (time zero), 4 h and 24 h later and stored for analysis. Migration of cells into the denuded area was evaluated with an inverted Nikon phase-contrast microscope (Tokyo, Japan) at a magnification of 400X and photographed with a digital camera. The migration of cells was quantified using image analysis of a minimum of seven randomly selected fields of view of the denuded area. The mean wound area is expressed as the percent of recovery (% R) from three identically treated plates using the equation: % R¼ [1(Tt/T0)]  100, where T0 is the wounded area at 0 h, and Tt is the wounded area 4–24 h post-injury. All experiments were performed in quadruplicate, and each experiment was repeated at least three times. Migration assay was also conducted in Transwells chambers (pores 8 mm, Costars). The filters of the chambers were precoated with collagen type I or fibronectin (15 mg/ml in PBS, pH 7.5) for 3 h at 37 1C. Cells were treated with 200 nM Tb4 or its mutants Tb4(SDKP/4A) or Tb4(KLKKTET/7A) for 24 h, harvested with trypsin/EDTA and suspended in Medium 200 with 0.1% BSA to a final concentration of 1  106/ml. Before placement in the Transwells, 100 ml aliquots of cell suspensions were preincubated for 30 min with 25 mM GM6001. The chambers were incubated for 6 h at 37 1C in 5% CO2. Then the cells on the upper surface of the filters were removed using a cotton swab. The filters were fixed in methanol and stained with Mayer’s hematoxylin (AQUAMEDICA) and eosin (PPH Poch. S.A.). Cells present on the lower surface of the filter were counted. Nonspecific migration was assessed by subtraction of that produced by the cells migrated in 0.5% BSA precoated filters. Each experiment was performed in triplicate and repeated at least 3 times.

Capillary tube formation in MatrigelTM The wells of a 48-well plate were coated with MatrigelTM according to the manufacturer’s instructions (Becton Dickinson, Bedford, MA) and were incubated at 37 1C for 30 min. HUVECs were grown in 6-well dishes in the presence of 200 nM Tb4 or its mutants Tb4(SDKP/4A) or Tb4(KLKKTET/7A) or MMP inhibitors for 24 h. The following inhibitors were used: TIMP 1, 2, or 3 (1 mg/ml), MMP-2 inhibitor II (3 mM), MMP-9 inhibitor (1 mM), GM6001 (Ilomastat, 25 mM), e-amino capronic acid (1 mg/ml). Then cells were detached with 1 mM EDTA, sedimented by centrifugation for 5 min and resuspended in cell culture medium. The HUVECs were added to MatrigelTM-coated wells and incubated for 24 h. Photomicrographs at 100X magnification were taken using the digital camera Photo CL50 AGFA attached to a Nikon TMS-F microscope. The average cell length was measured for at least 100 cells.

Endothelial cell migration and proliferation assays Measurement of intracellular calcium HUVECs were grown to confluence in M199 medium supplemented with 15% FCS, 150 mg/ml endothelial growth factor (Clonetics, San Diego, CA) and 90 mg/ml heparin (Sigma) using 24-well plates. Cell culture was maintained in 1% FCS for 20 h before experiments. Then the cells were starved for 4 h in FBS-free medium and wounded across the cell monolayer by scraping away a swathe of

Intracellular Ca2þ was measured by two approaches using the Fluo-4 NW Calcium Assay Kit (Molecular Probes) and the Fura-2 AM Calcium Assay Kit (Molecular Probes) according to the manufacturer’s protocols. Cells were plated in 96-well plates covered with poly-D-lysine (Sigma) and incubated overnight at

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37 1C/5% CO2 in culture medium to allow the cells to adhere. The following day the medium was removed and the cells were gently washed once with PBS and were loaded with Fluo-4 NW (100 ml). Incubation was continued for 45 min at 37 1C/5% CO2. The plates were then placed in room temperature for 15 min until the time of the assay. Fluorescence intensity was measured by the EnVision 2103 spectrofluorometer (Perkin Elmer). To measure Ca2þ using Fura-2 AM, the cells were cultured in 10 cm2 dishes at 37 1C/5% CO2 in culture medium. After a few days, the medium was removed and the cells were gently washed once with PBS and loaded with Fura-2 AM (1 mg/ml) in 1 ml culture medium. Incubation was continued for 30 min at 37 1C/5% CO2. After incubation the dye was removed and the cells were washed once with calcium buffer. The fluorescence signal was monitored using the Leica AF7000 Live Imaging System.

Confocal microscopy The HT29 and EA.hy926 cells were mixed with biotinylated Tb4 for 20–30 min on ice. The ice-cold medium was then replaced by warm medium and incubated at 37 1C for different periods of time. The cells were later washed with ice-cold stripping buffer (50 mM sodium citrate and 280 mM sucrose, pH 4.6 to remove unbound biotinylated Tb4). Then the cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Biotinylated Tb4 was detected with avidin conjugated with fluorescein. The cells were then visualized using a helium/neon ion laser (543 nm excitation) and analyzed with MultiScan version 8.08 software. For intracellular probe visualization, the confocal laser microscope Nicon system was used.

Statistical analysis We analyzed the statistical significance of our data by using a paired Student t-test or ANOVA. Statistically significant data bearing P value o0.05 or o0.001 are annotated by the  and  symbols, respectively. Data are expressed as a mean7SD.

Results Effects of Tb4 on endothelial cell migration and branching morphogenesis Given the role of Tb4 in modulating endothelial cells during angiogenesis or wound healing, we attempted to characterize the role of Ca2þ signaling in a Tb4-induced transition of endothelial cells from the quiescent into the proliferating-migrative phenotype. To address this issue, the phenotypic properties of HUVECs induced by Tb4 or its mutants were characterized in the preliminary experiments. Tb4 was modified by substitution of functional motifs with alanine using scanning mutagenesis. Thus, mutants were produced that were deprived of the N-terminal tetrapeptide AcSDKP (Tb4AcSDKPT/4A) and the actin-binding sequence KLKKTET (Tb4KLKKTET/7A). The cells were treated with Tb4 or its mutants (200 nM) for 0–24 h and subjected to several functional assays. Migration of Tb4-treated HUVECs was tested by a wound healing-like test and the transwell migration assay. In agreement with previous studies, the incubation of HUVECs

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with Tb4 resulted in their enhanced migration (Fig. 1A, B) and increased MMP-2 secretion (Fig. 1C). Interestingly, mutation of the N-terminal tetrapeptide in Tb4 only slightly decreased activity of the resultant Tb4AcSDKPT/4A mutant when compared to the wild-type Tb4. This increase in cell migration was not mediated by altered actin dynamics since the Tb4 mutant with a damaged G-actin-binding motif (Tb4KLKKTET/7A) showed almost the same activity as the wild-type Tb4. This indicates that in addition to both biological active regions, namely the G-actinbinding motif and the N-terminal tetrapeptide, there are some additional mechanisms by which Tb4 stimulates endothelial cell migration. Consistent with the role MMPs play in cell motility, Tb4-induced cell migration was abolished by GM6001 – the broad-spectrum inhibitor of MMPs (Fig. 1D). Similarly, when tested using Transwell filters coated on the lower side with collagen type I, cells treated with Tb4 or its mutants showed increased migration when compared to the control cells. Migration was efficiently blocked by GM6001 (Fig. 1E). HUVECs seeded onto MatrigelTM spontaneously acquired an elongated morphology and formed a capillary network within the gel (Fig. 2A). Repositioning of the endothelial cells, invasion into the gel and the beginning of cell elongation were all evident 6 h after plating the cells onto the gel, and capillary tube formation was clearly visible by 24 h. In the presence of Tb4 and all its mutants, both the onset of tubular structures and their growth into complex networks were much faster. Quantitative analysis of the activating effect included determining the length of 100 cells selected randomly 24 h after placing the HUVECs on Matrigel. The data shown in Fig. 2B reveal that the substitution of both biologically active motifs in the Tb4 molecule did not significantly influence its activity in inducing the endothelial cells to form capillary-like tubes. When the HUVECs were seeded on Matrigel and subsequently stimulated with Tb4 in the presence of proteolytic inhibitors, we observed that MMP activity predominantly contributed to invasion and tube formation by endothelial cells (Fig. 2C). Tube formation was partly inhibited by epsilon-aminocapronic acid, and almost abolished by the synthetic inhibitors of metalloproteinases (GM6001, MMP-2 inhibitor II, MMP-9 inhibitor I). Similarly, natural TIMPs, particularly TIMP-1 and TIMP-2, significantly inhibited the process stimulated by Tb4. The endothelial cells remained quiescent in their presence.

Tb4 does not affect the intracellular Ca2þ level Having so defined Tb4, next we tested whether all of the abovementioned cellular processes were mediated by intercellular Ca2þ elevation. An increase in [Ca2þ]i has been widely implicated in the migration and proliferation of cells. Hence, the aim of our next experiments was to explore whether Tb4 caused a change in [Ca2þ]i in the endothelial cells, and if so, whether Ca2þ signaling mediated the Tb4 activation of these cellular processes. In the course of our studies we used two fluorescent probes, Fluo-4 NW and Fura-2 AM, to monitor the Ca2þ signals in endothelial cells. Two potential activation pathways were considered: the release of Ca2þ by Tb4-generated secondary messengers or due to the direct action of Tb4 on the membrane [Ca2þ]i store. When the HUVECs were treated with trypsin, they showed an initial peak and a subsequent sustained phase in [Ca2þ]i. The peak was caused by the Ca2þ release from the intracellular Ca2þ stores, and the sustained phase was due to the influx of extracellular

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Fig. 1 – Migration of human endothelial cells induced by Tb4. Confluent HUVEC culture was starved for 4 h. After wounding, the cells were maintained in M199 containing 1% bovine serum albumin for 24 h, with or without 200 nM Tb4 or its mutants Tb4SDKP/4A or Tb4KLKKTET/7A, used at a concentration of 200 nM. Cell culture images were recorded immediately (0 h), and after 24 h (A). Migration of wounded cells was estimated by quantification of % of recovery as described in Materials and Methods (B). Panel (C) shows increased secretion of MMP-2 by endothelial cells activated by Tb4 and its mutant Tb4KLKKTET/7A and analyzed by gelatin zymography. Panel (D) shows that the migration of HUVECs is inhibited by GM6001, the inhibitor of MMPs. Alternatively, HUVECs were grown on polycarbonate filters precoated with collagen type I, and migration into the lower part of the filter was evaluated. Cells on the lower side of the filter were counted, and their number was expressed in relation to the control cells. Panel (E) shows that treatment of cells with Tb4 or its mutants significantly increased the number of migrating cells. The promoting effect of Tb4 was abolished by GM6001. Data are shown as a mean of at least three determinations 7SD. P values o0.05 and o0.001 are annotated by n and nn, respectively. Ca2þ across the cell membrane (Fig. 3A and B). A similar calcium mobilization response was observed when the HUVECs were treated with thrombin reacting with a Gq protein-coupled receptor (Fig. 3B). In contrast, HUVECs treated with 200 nM Tb4 or its mutants (Tb4KLKKTET/7A, Tb4AcSDKPT/4A) did not show any intracellular Ca2þ elevation, thus indicating that the Ca2þ influx was not induced by these peptides (Fig. 3A and B). There was no visible effect even when higher concentrations of Tb4, up to 1 mM, were used (not shown). The same phenomenon was also observed when Fluo-4 NW was replaced by Fura-2 AM (Fig. 3C), or when the experiments were repeated using other types of cells, e.g. EA.hy926 or HT29 cells (Fig. 4A and D). Interestingly, Tb4 appears to be rapidly internalized in these cells. Fig. 5 shows the uptake of Tb4 by HT29 and EA.hy926 cells incubated with biotinylated Tb4 for 0 to 45 min and detected by staining with avidin-conjugated fluorescein. A Tb4 influx was observed already after 5 min of incubation. This is clearly seen when fluorescence intensity was quantitated using ImageJ program (Fig. 5C and D). Its distribution matched that of the endogenous Tb4, i.e. there were heavy accumulations of Tb4 close to the membranes, particularly at the cell–cell contact sites, as we previously described (14). These findings indicate that the

stimulation of HUVECs with Tb4, which leads to increased migration and their organization into a capillary tube network, is not mediated by Ca2þ influx, but results from the intracellular activity of Tb4.

Discussion Tb4 is now recognized as a potent regenerative peptide in a variety of tissues [15]. However, it is still uncertain which function of Tb4, i.e. intracellular and/or extracellular, has greater importance for its biological activity. As a major G-actin-sequestering protein in cells, Tb4 maintains a dynamic equilibrium between G-actin and F-actin, which is critical for the rapid reorganization of the cytoskeleton. Hence, the mechanism by which Tb4 influences cell proliferation, migration and differentiation is generally believed to be linked with its role in controlling the polymerization of actin. On the other hand, Tb4 can be released from cells and found in large amounts in extracellular fluids, thus showing a broad range of biological activities [16]. Furthermore, Tb4, when externally added to endothelial cells: (a) induces expression and releases

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Fig. 2 – Effect of Tb4 and its mutants on capillary tube formation by HUVECs in Matrigel. HUVECs were placed on Matrigel in the presence or absence of Tb4 and incubated for 24 h. The tubes were observed under a microscope at 100X magnification. Representative images are shown in panel (A). Panel (B) shows the quantitative analysis of mean tube lengths from three independent experiments in which Tb4 or its mutants, Tb4SDKP/4A or Tb4KLKKTET/7A, were used. nn indicates po0.001. All p values were calculated using a paired Student t test. Panel (C) shows the inhibition of tube formation by protease inhibitors, such as TIMP-1, TIMP-2 or TIMP-3 used at 1 lg/ml, MMP-2 inhibitor II (3 lM), MMP-1 inhibitor I (1 lM), GM6001 (25 lM) and EACA (1 mg/ml). Data are expressed as mean 7S.D. and were collected during three separate experiments. n po0.05, and nn po0.001 were determined using the t test for independent samples.

plasminogen activator inhibitor type 1 (PAI-1) by a mechanism involving the activation of the mitogen-activated protein kinase cascade, thus leading to enhanced c-Fos/c-Jun binding to the AP-1-like element present in the PAI-1 promoter [17,18]; (b) activates the survival kinase Akt, and thus promotes myocardial and endothelial cell migration in the embryonic heart [19], (c) promotes skin and corneal wound healing through its effects on cell migration, angiogenesis and possibly cell survival [20,21], and (d) shows anti-inflammatory properties by directly targeting the NF-kB subunit RelA/p65 and inhibiting the sensitizing effects of its intracellular binding partners, PINCH-1 and ILK, in an actin-independent manner [22]. All these observations suggest that Tb4 can express its activity toward different cells via receptor-mediated mechanisms. Indeed, several studies focused on searching for its putative cellular receptor resulted in identifying several proteins that, upon interaction with Tb4, start to modulate important cell functions. More than a few proteins were identified as intracellular binding partners for Tb4, including focal adhesion proteins such as PINCH-1 and ILK, hMLH1, Ku80, and stabilin-2 [14,19,23]. Since all these proteins are located inside cells, externally added Tb4 must first be internalized to form a complex with the proteins and thus to modulate important cell functions. The present studies came to the surprising conclusion that activation of endothelial cells with Tb4 and the resulting enhanced cell motility are not mediated by Ca2þ signaling. Similarly, immortalized endothelial cells EA.hy926 and colon cancer cell line HT29 did not show any Ca2þ influx when activated with Tb4. Our data are contradictory to those published

by Huang et al. [24], who showed an extracellular Ca2þ influx after treatment of HL-60 cells with Tb4. However, the calcium signal which they described appears to be very weak; it started from about 40 nmoles (in resting cells) and after activation with Tb4 reached 116 nmoles (in calcium-free medium). It seems that it could be caused by mechanosensitive Ca2þ-permeable channels, activated by shear stress, induced during the mixing and adding of reagents to the cell culture. In endothelial cells, Ca2þ signals can be evoked by a number of mechanisms. Many agonists, such as nucleotides, acetylcholine and growth factors, are able to trigger phospholipase C activation, inositol 1,4,5-trisphosphate production and Ca2þ release from the endoplasmic reticulum. In the absence of Ca2þ influx from the extracellular medium, a short-duration [Ca2þ]i increase occurs [25]. This mode of Ca2þ inflow, which depends on the physical coupling between the STIM protein on the Ca2þ store and the plasma membrane Ca2þ channels [26,27], is the predominant pathway of Ca2þ entry in non-excitable cells, including endothelial cells [28–31]. Additional routes for Ca2þ influx may be provided by: (a) receptor-activated cation channels, activated by intracellular second messengers, such as diacylglycerol [32]; (b) mechanosensitive Ca2þ-permeable channels, activated by stretch, pressure and shear stress [32]; and (c) L- and T-like voltage-dependent Ca2þ channels, whose expression in endothelial cells is rather limited [33]. None of these mechanisms is initiated by external Tb4, which suggests that the extracellular actions of Tb4 result rather from its interaction with intracellular receptors, and takes place after its internalization. Our data are in contradiction with a recently proposed mechanism connecting

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Fig. 3 – Tb4 has no effect on the intracellular Ca2þ concentration in HUVECs. Cells grown in 96-well plates loaded with Fluo-4 NW were treated with 200 nM Tb4 and its mutants Tb4SDKP/4A, Tb4KLKKTET/7A, or trypsin or thrombin. Panel (A) shows cells treated either with trypsin or Tb4. Panel (B) shows the kinetic data of Ca2þ response to trypsin, thrombin, Tb4 and its mutants monitored using Fluo-4 NW by the EnVision 2103 spectrofluorometer. Panel (C) and (D) show the effect of trypsin and Tb4 on [Ca2þ]i in Fura-2-loaded HUVECs. The experiments were performed in a Ca2þ-containing medium. The time-response plots of Ca2þ signals in HUVECs induced by trypsin or Tb4 and its mutant Tb4KLKKTET/7A were monitored using the Leica AF7000 Live Imaging System (panel D). Data shown are representative of three to five independent experiments.

Fig. 4 – Tb4 has no effect on the intracellular Ca2þ concentration in EA.hy926 and HT29, but is rapidly internalized. EA.hy926 (panel A) or HT29 cells (panel B) grown in 96-well plates loaded with Fluo-4 NW were treated with 200 nM Tb4 and its mutant Tb4KLKKTET/7A or trypsin, and fluorescence was measured by the EnVision 2103 spectrofluorometer. In panel (C and D), EA.hy926 and HT29, loaded in Fura-2, were tested using the Leica AF7000 Live Imaging System. Data shown are representative of 3–5 independent experiments.

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Fig. 5 – Tb4 is rapidly internalized in cells. Panels (A and B) and (C and D) show the internalization of Tb4 by HT29 cells and EA.hy926 cells, respectively, incubated with biotinylated Tb4 for 0 to 45 min, followed by avidin conjugated with fluorescein. These cells are representative of a number of cells analyzed during three separate experiments. The image brightness (panels B and D) of the samples taken from sections close to the cell edges (n ¼30) from different samples was measured using ImageJ program (panel B). It reflects the concentration of the biotinylated Tb4 uptaken by cells.

Tb4 to purinergic receptors [9]. According to this mechanism, Tb4, upon binding to F1-F0 ATP synthase, causes increased extracellular levels of ATP which, in turn, activates the P2  4, i.e. the ATP-gated ion channel, to promote cell migration. However, if this mechanism is correct, one would expect that upon treatment of endothelial cells with Tb4, intracellular Ca2þ elevation should be observed. There are two bioactive fragments of Tb4, namely the Nterminal tetrapeptides AcSDKP and LKKTET, which are derived from its central actin-binding domain [15]. The present observations, in which purified mutants of Tb4 were used together with our previous studies utilizing vectors expressing the same mutants, demonstrated that increased endothelial cell motility did not require the presence of these two active motifs of Tb4. Therefore, our data indicate that the release of Ca2þ from intracellular stores, following stimulation of HUVECs with Tb4, does not occur, and the data further confirm that Tb4 may exert its effects in multiple ways: (a) directly on the actin cytoskeleton as a monomer-sequestering protein to alter actin dynamics; (b) indirectly via activation and transcription of signaling molecules which alter the actin cytoskeleton; and (c) completely independent of actin but via specific intracellular receptors. Since treatment with wild-type Tb4 and with the actin-sequestering mutant variant Tb4KLKKTET/7A induces similar migration behavior of HUVECs, our data support the concept that the pro-migration activity of Tb4 does not involve its effect on actin dynamics.

Acknowledgments This work was supported by the National Science Center project N301 4392 38.

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