extfieldsofstudy: Chemistry Medicine

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Involvement of Sodium in Early Phosphatidylserine Exposure and Phospholipid Scrambling Induced by P2X7 Purinoceptor Activation in Thymocytes*

Extracellular ATP (ATPec), a possible effector in thymocyte selection, induces thymocyte death via purinoceptor activation. We show that ATPec induced cell death by apoptosis, rather than lysis, and early phosphatidylserine (PS) exposure and phospholipid scrambling in a limited thymocyte population (35–40%). PS externalization resulted from the activation of the cationic channel P2X7 (formerly P2Z) receptor and was triggered in all thymocyte subsets although to different proportions in each one. Phospholipid movement was dependent on ATPec-induced Ca2+ and/or Na+ influx. At physiological external Na+ concentration, without external Ca2+, PS was exposed in all ATPec-responsive cells. In contrast, without external Na+, physiological external Ca2+ concentration promoted a submaximal response. Altogether these data show that Na+ influx plays a major role in the rapid PS exposure induced by P2X7 receptor activation in thymocytes.

Extracellular ATP (ATP ec ), a possible effector in thymocyte selection, induces thymocyte death via purinoceptor activation. We show that ATP ec induced cell death by apoptosis, rather than lysis, and early phosphatidylserine (PS) exposure and phospholipid scrambling in a limited thymocyte population (35-40%). PS externalization resulted from the activation of the cationic channel P2X7 (formerly P2Z) receptor and was triggered in all thymocyte subsets although to different proportions in each one. Phospholipid movement was dependent on ATP ec -induced Ca 2؉ and/or Na ؉ influx. At physiological external Na ؉ concentration, without external Ca 2؉ , PS was exposed in all ATP ec -responsive cells. In contrast, without external Na ؉ , physiological external Ca 2؉ concentration promoted a submaximal response. Altogether these data show that Na ؉ influx plays a major role in the rapid PS exposure induced by P2X7 receptor activation in thymocytes.
Extracellular ATP (ATP ec ) 1 induces thymocyte apoptosis and may play a role in thymocyte maturation (1)(2)(3)(4). A major change of cell membrane during apoptosis, slightly preceding nuclear condensation (5), is the exposure on the exoplasmic cell surface of phosphatidylserine (PS) (6 -9), a phospholipid normally maintained on the inner membrane leaflet by the aminophospholipid translocase (APLT) (10). This process is associated with a general loss of the asymmetric distribution (scrambling) of all the other major phospholipids (6,7,9). The presence of specific PS receptors on macrophages potentially allows a rapid elimination of apoptotic cells by phagocytosis before their lysis (11). Rapid PS exposure and scrambling are generally observed after platelet activation or treatment of cells with a Ca 2ϩ iono-phore, both conditions promoting a drastic and rapid rise in cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) (7). An involvement of Ca 2ϩ has been also reported in PS exposure during apoptosis (6,7,12). Ca 2ϩ acts by inhibiting APLT and by targeting scramblase (6,7,13) or other molecular complexes, which could contain phosphoinositides (14,15). Recent studies show that the rapid Ca 2ϩ -induced PS exposure is correlated with scramblase expression (16), in contrast to the delayed PS exposure associated with apoptosis (17,18), in agreement with the involvement of other phospholipid transporters in apoptotic pathways.
ATP ec is able to evoke physiological responses in a wide variety of cells and tissues. ATP is concentrated in cell cytosol and in exocytic vesicles, reaching 2-4 and 100 mM, respectively (19), and can be merely released from damaged cells, transported through carriers or channels, or secreted (19 -21). In the extracellular medium, its lifetime is very short due to its rapid degradation by ectonucleotidases and ecto-ATPases (19). ATP ec binds to distinct types of purinergic receptors classified into two subfamilies: metabotropic P2Y receptors coupled to Gprotein (P2Y1-6 and P2Y11) and ligand-gated ion channel P2X receptors (P2X1-7) (22). P2X7 receptor, previously designated as P2Z receptor (23), utilizes extracellular ATP 4Ϫ to increase cationic permeability with consecutive plasma membrane depolarization. Another feature of the P2X7 receptor is its ability to form or activate a non-selective pore, permeable to molecules with molecular mass Ͻ400 Da in thymocytes, when the ATP ec application is prolonged or repeated (24,25). Activation of the P2X7 receptor triggers various physiological processes, including cell death by apoptosis or necrosis (26) or interleukin-1␤ release (22,27,28). PS is rapidly externalized upon P2X7 receptor activation during peripheral T cells apoptosis (29,30) and precedes interleukin-1␤ release from lipopolysaccharideprimed monocytes (28). PS exposure, Ca 2ϩ influx, and pore formation are dependent on the cytoplasmic domain of the P2X7 receptor, which harbors a putative tumor necrosis factor receptor-related death domain and a Src homology 3-binding domain (29).
In this work, we show that ATP ec induced early PS exposure, 1-oleoyl-2-[6-(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproylsn-glycero-3-phosphocholine (NBD-PC) scrambling, and apoptosis in 35-40% of the thymocytes. PS exposure and NBD-PC scrambling were dependent on an increase in Ca 2ϩ and/or Na ϩ influx resulting from P2X7 activation. At physiological external concentrations, Na ϩ was more potent than Ca 2ϩ to induce PS exposure. Portions of this work have appeared in a preliminary report (31). * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a grant from the Association de la Recherche contre le Cancer (ARC).

Flow Cytometry Analysis
Endogenous PS Externalization-When cells were incubated in Ca 2ϩ -containing medium, PS exposure was assayed from the binding of FITC-annexin-V in the presence of PI (50 g/ml), a marker of plasma membrane integrity used to evaluate the percentage of necrotic cells. PSϩ/PIϪ cell population represents non-necrotic cells with externalized PS. Alternatively, when cells were incubated in a medium devoid of Ca 2ϩ , factor Va, another specific ligand of PS whose binding is Ca 2ϩindependent, was used as described previously (32). Binding of factor Va was detected by indirect immunofluorescence. The fixation of factor Va on the cell surface altered the plasma membrane integrity as reflected by the uptake of PI in all PSϩ cells. Consequently the responsive cells were expressed as total PSϩ cells. However, the proportion of the necrotic cells, estimated in independent assays by measurement of PI uptake, was found to be less than 10% after ATP ec treatment, a proportion similar to that observed in control cells. The percentage of PSϩ cells determined by factor Va binding was always slightly higher than that obtained by FITC-annexin-V staining, suggesting that the affinity of factor Va for PS was higher than that of FITC-annexin-V.
NBD-PC Internalization-The transverse redistribution of the PC analog was assayed as described previously (6,33). Briefly, after incubation of thymocytes for 10 min with 1 mM ATP ec , the fluorescent analog NBD-PC (62.5 nM final concentration representing about 1.5% of cellular phospholipids) was loaded into the cells by 6-min incubation at room temperature. The percentage of cells with internalized PC was assayed after extraction by fatty acid-free bovine serum albumin (2 min at 4°C) of the fraction of the analog present in the external leaflet. Samples were sorted by flow cytometry after addition of PI to evaluate the proportion of necrotic cells.
Plasma Membrane Depolarization-Thymocytes (2⅐10 6 /ml) were stained with 300 nM DiBac4(3) for 20 min at 37°C. Fluorescence intensity of the DiBac4(3) probe increased when cells were depolarized. In these experiments, PI uptake was used to exclude necrotic cells and to determine plasma membrane integrity in non-necrotic cells.
Ethidium or Propidium Uptake-After various times of incubation at 37°C, an aliquot of cells (2⅐10 6 /ml) was stained with PI (50 g/ml) or ethidium bromide (10 g/ml) for 3 min at room temperature. The extent of the pore opening induced by ATP ec was evaluated from the fluorescence of propidium or ethidium bound to nuclear DNA and detected by flow cytometry.
Detection of Surface Markers CD4 and CD8 -The surface markers CD4 and CD8 were detected by flow cytometry after staining thymocytes with R-phycoerythrin-anti-CD4 and either FITC-anti-CD8 or biotin-anti-CD8 and peridin chlorophyll a protein-streptavidin.
Cytosolic Calcium Concentration-Thymocytes (2⅐10 6 /ml) were loaded with 2 M fluo4-AM (15 min at 37°C). PI uptake was used to exclude necrotic cells. The variations in [Ca 2ϩ ] i were determined in non-necrotic cells. [Ca 2ϩ ] i was calculated from the following equation: where K d is the dissociation constant of the Ca 2ϩ -fluo4 complex (345 nM) (34), F max is the maximum fluorescence (obtained after addition of 8 M A23187 and 1.4 mM Ca 2ϩ for 5 min), and F min is the minimum fluorescence (obtained after addition of 8 M A23187 and 5.4 mM EGTA for 5 min). In control cells one fluorescence peak was observed, whereas after ATP ec stimulation a second population with higher fluorescence was apparent (high Ca 2ϩ cells). F is the geometric mean fluorescence value of this high Ca 2ϩ cell population.
In all these experiments, cells were sorted by flow cytometry using a BD Biosciences FaxVantage cytometer. Fluorochrome excitation was performed by argon laser at 488 nm, and fluorescence emissions were analyzed with a 530 nm filter for FITC, NBD-PC, DiBac4(3), and fluo4; with a 575 nm filter for ethidium and R-phycoerythrin; with a 630 nm filter for PI; and with a 660 nm filter for peridin chlorophyll a protein.

Measurement of Calcium Influx
Cells (5⅐10 6 /ml) were loaded with 4 M fura2-AM (30 min at 37°C) and resuspended in a Ca 2ϩ -free RPMI 1640 medium supplemented with 150 M EGTA. Fura2 fluorescence was recorded continuously in a thermostatted and magnetically stirred cuvette of a spectrofluorometer. The fluorescence emission ratio of the probe excited at 340 and 380 nm reflected [Ca 2ϩ ] i .

Measurement of Nuclear Apoptotic Events
Nuclear condensation was quantified by fluorescence microscopy after DNA staining with Hoescht 33342 (10 g/ml) and PI (50 g/ml) for 10 min. Approximately 200 randomly selected cells were scored for each experimental condition. Cells exhibiting blue or red condensed or fragmented nuclei were considered apoptotic. Cells with red nuclei without signs of condensation or fragmentation were considered necrotic.
For detection of DNA fragmentation, cells (3⅐10 6 ) were washed and lysed in 10 mM Tris-HCl, pH 7.4, 0.2% Triton X-100, 1 mM EDTA, 100 g/ml proteinase K for 10 min on ice followed by 60 min at room temperature. After centrifugation (30 min, 13,000 ϫ g, 4°C), DNA in the supernatant was precipitated overnight in cold absolute ethanol containing 0.3 M sodium acetate at Ϫ20°C. After centrifugation (30 min, 13,000 ϫ g, 4°C), the pellet was resuspended in water and incubated for 30 min at 37°C with 50 g/ml DNase-free RNase. DNA concentration was quantified by UV spectrometry at 260 nm. About 5 g of DNA was mixed with gel loading solution and incubated for 5 min at 65°C before electrophoresis in a 1.5% agarose gel containing ethidium bromide. DNA was visualized by UV light.

Data Analysis
Data are means Ϯ S.E. of a minimum of three independent experiments. Differences between means were evaluated by paired t test with p Ͻ 0.05 being taken as the level of significance.

ATP ec Induces Apoptosis and PS Exposure in a Limited
Thymocyte Population-Cell death was measured after 5-h incubation of freshly isolated thymocytes in the presence of ATP ec . Nuclear condensation was significant at 0.5 mM ATP ec in 20 -25% of the cells and maximal at 1 mM affecting 35% of the total population (Fig. 1A). Thymocyte apoptosis was confirmed by visualization of DNA fragmentation on agarose gel electrophoresis. Oligonucleosome formation induced by 1 mM ATP ec was comparable to that promoted by 1 M dexamethasone, a well known inducer of thymocyte apoptosis (Fig. 1B). When compared with spontaneous thymocyte apoptosis, the effect of 1 mM ATP ec on nuclear condensation was significantly increased after 4-h treatment and reached a maximum (35-40%) after 6 h (Fig. 1C). Incubation for up to 9 h did not change significantly the percentage of cells with condensed nuclei and marginally increased the percentage of necrotic cells (11%), indicating that under these experimental conditions ATP ec induced apoptosis preferentially to necrosis in a limited population of thymocytes.
During the apoptotic process, PS exposure was reported to be concomitant or to precede slightly nuclear events such as nuclear condensation (5). ATP ec induced PS exposure and nuclear condensation within the same range of concentrations (0.5-3 mM) with a maximal effect at 1 mM (Figs. 1A and 2A). Surprisingly PS exposure was already significant in 15% of cells after 6-min treatment with 1 mM ATP ec and was maximal after 20 -30 min, affecting about 40% of the thymocytes (Fig. 2B). These results show that PS exposure was a very early event in thymocytes, preceding nuclear alterations. After 5-h incubation with 1 mM ATP ec , both PS exposure and nuclear condensation, observed by light microscopy after FITC-annexin-V binding and Hoescht staining, were detected in the same cell population (data not shown). . DNA fragmentation was determined by agarose gel electrophoresis. 100-base pair DNA ladder was used as molecular marker (M). C, thymocytes were incubated without (control) or with 1 mM ATP ec in 5% FCS/RPMI for various times. The percentage of condensed nuclei was evaluated as in A (means Ϯ S.E. of three to seven independent experiments).

FIG. 2. Effects of ATP ec on PS exposure.
A, thymocytes were incubated at 37°C for 30 min in 5% FCS/RPMI with different concentrations of ATP ec . The percentage of PS-exposing cells impermeable to PI (PSϩ/PIϪ) was evaluated from FITC-annexin-V binding and PI uptake by flow cytometry. B, thymocytes were incubated without (control) or with 1 mM ATP ec in 5% FCS/RPMI for various times, and the percentage of PSϩ/PIϪ was determined as above. Results are means Ϯ S.E. of three to seven independent experiments. C, after incubation for 30 min with 1 mM ATP ec , thymocyte subsets (DN, DP, SPCD4ϩ, and SPCD8ϩ) were characterized by CD4/CD8 staining. In each subset, PS exposure was detected simultaneously by FITC-annexin-V binding by flow cytometry. Results represent the percent distribution of thymocyte subsets and the percentage of PSϩ cells in each subset (mean Ϯ S.E. of three independent experiments).
Differentiation of immature thymocytes into mature T cells takes place in the thymus and proceeds via an ordered sequence of developmental steps characterized notably by variable expression of CD4 and CD8. Early precursor cells are initially double negative CD4ϪCD8Ϫ (DN) and go through a double positive CD4ϩCD8ϩ (DP) intermediate state before differentiating into single positive CD4ϩCD8Ϫ or CD4ϪCD8ϩ (SPCD4ϩ or SPCD8ϩ, respectively). The distribution of the thymocyte population and the percentage of PS-exposing cells in each subset (DN, DP, and SP, either CD4ϩ or CD8ϩ) were analyzed by flow cytometry. As previously reported (2), DP accounted for about 80% of the population (Fig. 2C). ATP ec induced PS externalization in each thymocyte subset. The most mature thymocytes (SPCD4ϩ or SPCD8ϩ) were also those in which the percentage of cells exposing PS was significantly the highest (68 and 73% versus 43 and 29% in DN and DP, respectively) (Fig. 2C). These data show that, as far as PS exposure is concerned, ATP ec responsiveness was dependent on the maturation state of thymocytes.
P2X7 Purinoceptor Is Involved in ATP ec -induced PS Exposure-Thymocytes express different classes of membrane receptors activated by purine ligands (1,3). To identify which purinoceptor(s) was mediating early PS exposure in thymocytes, the effect of various purine agonists and antagonists was investigated. Neither adenosine (a P1 receptor agonist) nor UTP (a P2Y1, P2Y4, and P2Y6 agonist) (22) was able to induce PS exposure. ␣␤mATP, a P2X1 or P2X3 receptor agonist (25), was unable to induce PS exposure (Fig. 3A). In contrast, 0.1 mM BzATP, a P2X7 agonist with a 10-fold higher affinity than ATP ec (22,25), promoted PS externalization to the same extent as did 1 mM ATP ec (Fig. 3A). Furthermore antagonists of P2X (suramin or PPADS) (22,25), at concentrations known to in-hibit P2X1 receptor (1), had no effect on PS exposure induced by 1 mM ATP ec (Fig. 3B), whereas oxidized ATP, a P2X7 antagonist (22,25), significantly inhibited ATP ec -induced PS externalization (Fig. 3B). As P2X7 is activated by ATP 4Ϫ but not by Mg-ATP (22,25), chelation of ATP ec by high Mg 2ϩ concentrations antagonizes P2X7 activation. Pretreatment of thymocytes with 15 mM Mg 2ϩ significantly inhibited PS exposure induced by ATP ec without affecting PS exposure in control cells (Fig.  3B). Furthermore ADP induced PS exposure in a significantly smaller percentage of cells than did ATP ec , whereas 1 mM AMP was without effect (Fig. 3A) as expected from their respective affinities for the P2X7 purinoceptor (2,23,35). PS exposure induced by 1 mM ADP was inhibited by oxidized ATP confirming that this effect was mediated through P2X7 activation (data not shown). PS exposure required ATP ec concentrations above 0.1 mM ( Fig. 2A) in agreement with the range of concentrations reported to activate P2X7, excluding a role for P2X1 purinoceptor, whose activation occurs at concentrations below 0.1 mM (25). Altogether these data provide evidence for the implication of P2X7 in ATP ec -induced PS exposure.
P2X7 Receptor Is Initially in Channel Conformation during PS Exposure-To determine whether P2X7 was in a channel or pore conformation under the conditions leading to early PS exposure, we measured the uptake of two impermeant nucleus markers of different molecular sizes, ethidium (314 Da) and propidium (414 Da), in response to 1 mM ATP ec . Fig. 4A shows that, during the first 20 min of incubation, there was no uptake of propidium, and the uptake of ethidium, observed in less than 15% of cells, was not significantly different with or without ATP ec . Furthermore the level of fluorescence intensity was the same in control or ATP ec -treated cells (data not shown). These data indicate that P2X7 was not in a pore conformation at least for the period during which PS became externalized in thymocytes upon treatment with 1 mM ATP ec . Nevertheless, after 40 min of ATP ec treatment, only ethidium and not propidium uptake was observed in a significant percentage of cells (Fig.  4A), showing that the pore formation was a time-dependent and size-limited process in thymocytes.
P2X7 is an ATP-gated nonspecific cation channel mediating fast permeability changes to Na ϩ , K ϩ , and Ca 2ϩ (22). When thymocytes were incubated in a Ca 2ϩ -free medium, there was no change in [Ca 2ϩ ] i upon addition of 1 mM ATP ec , showing that this treatment did not induce any Ca 2ϩ release from intracellular stores (Fig. 4B). Nevertheless subsequent addition of Ca 2ϩ to the medium triggered a significant increase in [Ca 2ϩ ] i , reflecting Ca 2ϩ influx in ATP ec -treated cells (Fig. 4B). Attempts to measure Na ϩ influx by using Sodium Green-AM or sodiumbinding benzofuran isophthalate-AM (Molecular Probes), two FIG. 4. Effects of ATP ec on PI and ethidium uptake, Ca 2؉ influx, and membrane depolarization. A, thymocytes were incubated without (control) or with 1 mM ATP ec for different times. After incubation, cells were stained with PI (Pr) or ethidium bromide (Et), and the percentage of cells with an increased fluorescence was measured by flow cytometry (means Ϯ S.E. of three independent experiments). B, after loading with fura2-AM, thymocytes were incubated in a Ca 2ϩ -free medium containing 150 M EGTA. The fura2 fluorescence ratio (340/ 380 nm) was used to estimate the variations in [Ca 2ϩ ] i . Gray line, control thymocytes before and after addition of 2 mM Ca 2ϩ ; black line, thymocytes incubated with 1 mM ATP ec before and after addition of 2 mM Ca 2ϩ . C, after loading with DiBac4(3), thymocytes were incubated with 1 mM ATP ec for various times. Histograms of DiBac fluorescence were obtained by flow cytometry. specific probes for Na ϩ detection, were unsuccessful, presumably as a result of inefficient probe loading in thymocytes. However, addition of 1 mM ATP ec induced a rapid depolarization of the plasma membrane detected from the increase in DiBac4(3) fluorescence (Fig. 4C), providing indirect evidence for the occurrence of ATP ec -induced Na ϩ influx (35). Altogether these data confirm that P2X7 in thymocytes is a rapidly gated cation channel.
ATP ec Induces Ca 2ϩ -dependent or Na ϩ -dependent PS Exposure-To determine a possible involvement of ATP ec -induced Ca 2ϩ influx in PS exposure, the percentage of cells with externalized PS (PSϩ cells) was determined after 20-min incubation with 1 mM ATP ec in Na ϩ -free choline media containing different Ca 2ϩ concentrations. Increasing external Ca 2ϩ concentration resulted in a progressive elevation in [Ca 2ϩ ] i (Fig. 5A), detectable as soon as 1 mM external Ca 2ϩ , in a maximum of about 40% of thymocytes (data not shown), named high Ca 2ϩ cells. Similarly the percentage of PSϩ cells induced by ATP ec increased with external Ca 2ϩ , reaching a maximum as soon as 2.5 mM Ca 2ϩ , not significantly different from the percentage detected in 5% FCS/RPMI (Fig. 5A, dotted line). Remarkably, at 0.5 mM Ca 2ϩ in choline medium, PS exposure was observed in only a significantly smaller percentage of cells than that detected in 5% FCS/RPMI, although the latter medium contained about 0.5 mM Ca 2ϩ (Fig. 5A, dotted line). This difference suggests that an additional mechanism, independent of Ca 2ϩ , was involved in PS exposure in 5% FCS/RPMI.
In addition to Ca 2ϩ uptake, P2X7 activation triggers K ϩ efflux and Na ϩ influx (22). Interestingly P2X7 channel can be activated in the absence of external Ca 2ϩ and Na ϩ (36). Thus, in the choline medium devoid of both Ca 2ϩ and Na ϩ , ATP ec could induce K ϩ efflux but was unable to promote significant PS exposure (Fig. 5, A and B), suggesting that K ϩ efflux alone could not be responsible for PS exposure. To study the possible involvement of Na ϩ in ATP ec -induced PS exposure, we determined the percentage of PSϩ cells in a Ca 2ϩ -free choline media containing different Na ϩ concentrations. The percentage of PSϩ cells, induced by 20-min treatment with 1 mM ATP ec , augmented at external Na ϩ concentrations above 70 mM, affecting 38% of cells at 139 mM Na ϩ , a percentage not significantly different from that observed in 5% FCS/RPMI (Fig. 5B, dotted line). Under these conditions, the effect of external Na ϩ on PS exposure can be attributed to ATP ec -mediated Na ϩ influx.
To provide further evidence for a Na ϩ -dependent mechanism of PS exposure, we tested the effect of gramicidin D and monensin, ionophores that induce Na ϩ influx in cells suspended in media containing exclusively Na ϩ . Fig. 5C shows that incubation of thymocytes for 15 min in NaCl medium, but not in choline medium, with 1.5 M gramicidin D induced PS exposure in about 25% of cells. Increasing the time of incubation or the gramicidin D concentration induced thymocyte necrosis as detected by PI uptake (data not shown). In the same way, PS exposure was induced by monensin in the presence of Na ϩ in the medium (Fig. 5C).
ATP ec Induces Ca 2ϩ -dependent or Na ϩ -dependent Phospholipid Scrambling-PS exposure is generally accompanied by the transmembrane redistribution of the other major phospholipids, a process known as scrambling. To investigate whether this scrambling process also occurs in thymocytes activated by ATP ec , PC redistribution was studied using a fluorescent analog of PC (NBD-PC). This method allows the determination of the percentage of cells exhibiting a transbilayer movement of PC at the time of measurement, whereas the factor Va binding assay provides the percentage of cells having exposed PS over the duration of the ATP ec treatment. In choline medium con-taining 5 mM Ca 2ϩ , 10 min after addition of 1 mM ATP ec NBD-PC was internalized in about 35-40% of thymocytes, a percentage similar to that of PSϩ cells under the same conditions (Fig. 6A). Addition of 15 mM MgCl 2 after 10-min incuba- tion with ATP ec to inactivate P2X7 did not affect significantly NBD-PC internalization and endogenous PS exposure (Fig.  6A), indicating that transient activation of P2X7 was sufficient for inducing and maintaining phospholipid scrambling. In fact, under these conditions, [Ca 2ϩ ] i remained elevated as detected by the maintenance of a high fluorescence level of fluo4 (data not shown).
Interestingly treatment of thymocytes for 10 min with 1 mM ATP ec in NaCl medium also induced NBD-PC redistribution (Fig. 6B), showing that Na ϩ influx, as well as Ca 2ϩ influx, was able to mediate ATP ec -induced phospholipid scrambling. When ATP ec after 10-min incubation was chelated by high Mg 2ϩ concentration, NBD-PC scrambling was stopped, and endogenous PS relocated on the inner membrane leaflet (Fig. 6B), showing that, in contrast to the effect of high external Ca 2ϩ oncentration, the Na ϩ effect on scrambling was rapidly reversible. However, under the same conditions, the plasma membrane remained depolarized as detected after DiBac4(3) staining (data not shown), suggesting that membrane depolarization was triggered at a much lower Na ϩ concentration than PS exposure. This hypothesis was supported by the observation that 25 mM external Na ϩ was sufficient to induce a significant depolarization of thymocytes (data not shown) but not PS exposure, which required about 105 mM external Na ϩ (Fig. 5B). DISCUSSION ATP ec has been reported to induce cell death by lysis or apoptosis in a variety of cells, including immune cells, such as dendritic cells, macrophages, and thymocytes (22). Our data show that ATP ec induced a rapid PS exposure in thymocytes, which was already significant after 10 -15 min. Following a longer time of incubation, PS exposure and nuclear condensation occurred in the same cell population, amounting to 35-40% of total thymocytes. Cells exhibiting PS exposure and nuclear condensation remained impermeable to PI, and apoptosis was confirmed by DNA fragmentation observed in cellular extracts. Consequently, under these experimental conditions, thymocyte death induced by ATP ec occurred by apoptosis rather than by lysis. ATP ec -induced apoptosis of thymocytes has been reported to be mediated through activation of P2X1 and P2X7 (1)(2)(3)(4). Our pharmacological studies provide evidence for the involvement of P2X7 in PS exposure. We confirm also that, in thymocytes, ATP ec triggers a significant and sustained Ca 2ϩ influx without releasing intracellular Ca 2ϩ (3,24,35,37), a rapid membrane depolarization (4,24,35), and the progressive formation of a limited size pore permeable to ethidium and not to PI (22,24,35). All these characteristics are hallmarks of P2X7 activation.
PS externalization is known to play a critical role in cell phagocytosis by macrophages (38 -40). Although PS exposure is considered to be a hallmark of apoptosis, it can be dissociated from other features of the apoptotic process, for instance caspase activation or DNA fragmentation, as PS-dependent phagocytic recognition of cells can occur earlier or independently of these latter events (41,42). We have observed that, after a short treatment (30 min) with ATP ec , thymocyte phagocytosis by thioglycollate-elicited peritoneal macrophages was significant and inhibited when external PS was masked by annexin-V binding. 2 Consequently early PS exposure, mediated by ATP ec via P2X7 activation and preceding further steps of apoptosis, could lead to recognition and phagocytosis of thymocytes. Such a possibility for a rapid thymocyte elimination is supported by the observations of Surh and Sprent (43) that apoptotic cells were mainly present within phagocytic cells in the thymus. Furthermore thymocyte elimination, studied in fetal thymic organ culture, suggests an implication of ATP ec and potentially of P2X7 in thymocyte death by neglect (3). In this context, the early P2X7-induced PS exposure could be involved in thymocyte elimination during thymic selection. From the various P2X7-mediated responses (Ca 2ϩ influx (35,37), plasma membrane depolarization (4), or ethidium uptake (35)) within the different subsets of thymocytes or in peripheric T lymphocytes, previous studies have attempted to implicate ATP ec in thymocyte maturation and/or in peripheral T cell regulation. DN subset was the most responsive to P2X7 activation according to Nagy et al. (4), whereas, in other studies, the most mature SP subsets, notably SPCD4ϩ, exhibited the strongest response (35,37). This latter observation was supported by the persistence of the P2X7 response in peripheral T cells (30,35). We show that P2X7 activation induced PS exposure in all subsets and predominantly in the most differentiated SP cells. This characteristic is likely due to a higher level of P2X7 expression in SP subsets in relation to the thymocyte maturation state (35). Furthermore P2X7 activation, induced by ATP ec in splenic T cells (29) or by ADP-ribosylation in lymph node T cells (30), leads to apoptosis with an early PS exposure, suggesting a role of P2X7 in T cell death, for instance to eliminate potentially autoreactive T cells during inflammatory reaction (30). Thus, early PS externalization induced by P2X7 activation could be essential in the rapid elimination of peripheral T cells by phagocytosis.
Our data show that ATP ec induced both early PS exposure and NBD-PC internalization, reflecting phospholipid scrambling, a process generally attributed to an increase in [Ca 2ϩ ] i in cell activation (7) and apoptosis (6,7,12). However, in many instances there was no correlation between scrambling and increased [Ca 2ϩ ] i . In Jurkat T cells, the increase in [Ca 2ϩ ] i induced by Ca 2ϩ ionophore was not sufficient by itself to trigger scrambling, leading to the hypothesis that the ionophore has an additional effect, possibly implicating mitochondria (44). In thymocytes, induction of apoptosis and PS exposure by dexamethasone, gliotoxin, and thapsigargin was not correlated with an increase in [Ca 2ϩ ] i (45). In the same way, during in vitro attack of hepatocytes by natural killer cells, there was no evidence for a straightforward relationship between elevation of [Ca 2ϩ ] i and PS externalization (46). Ca 2ϩ is not the only known inducer of phospholipid scrambling: a decrease in pH stimulated NBD-phospholipid scrambling in inside-out vesicles (13,47), and polyamines were implicated in PS exposure in apoptosis (48). In platelets stimulated with collagen, Na ؉ influx, mediated through activation of the Na ϩ /H ϩ exchanger, was involved in the generation of procoagulant activity (49), a process generally initiated by PS externalization.
We investigated the respective roles of Ca 2ϩ and Na ϩ in ATP ec -induced PS externalization by manipulating the cation concentration of the incubation media. First, we show that PS exposure was correlated with an increase in [Ca 2ϩ ] i , itself resulting from a stimulation of Ca 2ϩ influx, mediated by ATPgated channel P2X7. In our experimental conditions, we confirm that ATP ec did not induce any Ca 2ϩ release (Refs. 3, 24, 35, and 37 and the present data). Consequently the store-operated Ca 2ϩ influx, previously implicated in PS exposure in erythroleukemia cells treated with Ca 2ϩ ionophore (34), could not be involved in ATP ec -induced PS exposure. Second, in a medium devoid of Ca 2ϩ , PS externalization was correlated with the external Na ϩ concentration. This effect is attributable to Na ϩ influx mediated by ATP-gated channel P2X7 and expected to increase intracellular Na ϩ concentration. The role of Na ϩ influx in PS exposure was confirmed by using the Na ϩ ionophores gramicidin D or monensin. Third, at a 2 mM external Ca 2ϩ concentration, the physiological Ca 2ϩ level in biological fluids, and in the absence of Na ϩ , ATP ec induced PS exposure only in a fraction of the ATP ec -responsive cells as determined in the complete medium containing the physiological Na ϩ concentration. Altogether these data show that ATP ec was able to induce PS exposure by a process dependent solely on Na ؉ influx and that Ca 2ϩ , at physiological concentrations, was not sufficient, in the absence of Na ϩ , to elicit the full PS response in the entire responding cell population.
The mechanisms by which intracellular Ca 2ϩ and Na ϩ induced PS exposure and phospholipid scrambling remain hypothetical. As already described for Ca 2ϩ (6,7), both cations could act on the same target by directly activating a phospholipid transporter, such as scramblase, and inhibiting APLT. Alternatively both cations could modulate some regulators of phospholipid transport. For example, a depletion in intracellular ATP known to inhibit the activity of APLT (10, 50) has been observed and implicated in PS exposure during apoptosis (18,46,51). ATP depletion could result from its consumption by Na ϩ /K ϩ or Ca 2ϩ pumps as a result of their activation to remove Na ϩ and Ca 2ϩ from the cells (46). The hypothesis of APLT inhibition following ATP ec treatment could not be directly ver-ified by measuring NBD-PS uptake since this movement will include both rapid PS scrambling and APLT activity. However, even if it was not inhibited, APLT activity was insufficient to overcome the extremely fast PS randomization induced by AT-P ec . When, after a first application, ATP ec was chelated with Mg 2ϩ , NBD-PC scrambling and PS exposure persisted when they were induced by high Ca 2ϩ concentrations. Cytosolic Ca 2ϩ remained sufficiently elevated to maintain scrambling and APLT inhibition at least during the period of observation. On the contrary, in Na ϩ medium, ATP ec chelation stopped scrambling, allowing relocation of endogenous PS. Following ATP ec chelation, intracellular Na ϩ could be actively extruded through the Na ϩ /K ϩ pump, returning to a concentration below the threshold level required to maintain scrambling. Thus, APLT could be directly inhibited either by Na ϩ or by ATP consumption by the Na ϩ /K ϩ pump. Interestingly the presence of external PS in a subpopulation of sickle cells was correlated with APLT inhibition rather than with an elevated [Ca 2ϩ ] i (52). Furthermore this cell population had a low K ϩ and high Na ϩ content (52) in agreement with a potential role of Na ϩ in PS exposure and APLT inhibition. Although the possibility of regulation of PS externalization in apoptosis by modulating APLT activity is attractive (18,46,51,52), it cannot account for the attached scrambling of the other phospholipids. Consequently the most likely hypothesis could be that Na ϩ , as already described for Ca 2ϩ , acts by both inhibiting APLT and activating scramblase or other molecular complexes able to promote phospholipid transport. A role for intracellular Na ϩ has been proposed to explain other cellular processes such as reverse transport of dopamine (53), stress-activated protein kinase/c-Jun N-terminal kinase activation (54), P2X7-mediated membrane blebbing (36), and platelet procoagulant properties (49). In apoptosis, an early Na ϩ influx is involved in the control of cell shrinkage and activation of the apoptotic program (55). However, the mechanisms by which Na ϩ exerts its action remain largely unknown and would deserve to be investigated.
Here we present evidence that ATP ec induces early PS externalization and phospholipid scrambling, which precede other events of apoptosis, in a maximum of 35-40% of thymocytes. Phospholipid movements are dependent on an increase in intracellular Ca 2ϩ and/or Na ϩ concentrations resulting from activated P2X7 channel. We suggest that Na ϩ , rather than Ca 2ϩ , is the major mediator of the early PS externalization induced by ATP ec . The role of Na ϩ in PS externalization, and consequently cell elimination, would require to be investigated in other physiological or pathological conditions, particularly during apoptosis in which the role of Ca 2ϩ in mediating delayed PS exposure has been challenged.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
mRNA expression profiling reveals a role of Helicobacter pylori vacuolating toxin in escaping host defense.

AIM
To study the immune response of host to Helicobacter pylori VacA.

METHODS
The monocyte/macrophage-like U937 cells were infected with Helicobacter pylori vacA-positive strain NCTC 11638 or isogenic vacA-negative mutant. Differentially expressed genes were identified at 2, 6, 10, and 24 h post-infection by cDNA microarray. Differential expressions of some genes were confirmed by Northern blot.

RESULTS
More than 100 genes altered their mRNA expression at different time points respectively, many of which were identified to be related to immune evasion.

CONCLUSION
VacA is a crucial element for H pylori to escape from host immune defense by means of differentially regulating the expression of some related genes. These genes, previously known or unknown to be involved in the mechanism of immune evasion, deserve further investigation to unearth much more information complicated in the immune response.

INTRODUCTION
Helicobacter pylori infects about half of the world's population. Despite the induction of an immunological reaction, the infection of H pylori is commonly life-long, suggesting that this pathogen has evolved mechanisms to evade protective immune responses to achieve the state of host-microbial equilibrium [1] . Some products of H pylori have been determined to have immunosuppressive effects for prolonging the infection. A 100 ku H pylori protein inhibits proliferation of T-cell and macrophage [2] , and VacA, a cytotoxin that has been found to cause massive vacuolation in several mammalian cell lines and in the gastric epithelia of patients with active chronic gastritis associated with H pylori infection [3] , may perform targeted action to disable T cells. VacA interferes with proteolytic processing of tetanus toxin and toxoid and specifically inhibits the Ii-dependent pathway of antigen presentation mediated by newly synthesized major histocompatibility complex (MHC) class II, suggesting that VacA may contribute to the persistence of H pylori by interfering with protective immunity [4] . However, to the author's knowledge, the exact mechanism of such an immunosuppression effect has not been fully studied. Hence, in this study, we performed a large scale measurement of gene expression alteration in host cells using gene microarray technology, which provided the crucial information for interpreting the mechanisms of immunosuppression.

Bacterial strains and growth conditions
H pylori NCTC 11638 strain positive for vacA was given as a gift by Dr. Shi (Shanghai Institute of Gastroenterology). Isogenic vacA-negative mutant 11638-vacA was constructed by substitution of a kanamycin resistant gene for a short fragment of vacA through homologous recombination, as described previously [5] . H pylori strains were cultured routinely on brain heart infusion (BHI) agar plates with 5% sheep blood in mixed air containing 100 ml/L CO 2 , 50 mL/L O 2 , and 850 mL/L N 2 at 37 . Pharmacia, Sweden) and lyophilized. The probes were resuspended in 20 µL hybridization solution containing 8 µg of poly(dA), 2 µg of yeast tRNA, 10 µg of human Cot I DNA (Gibco-BRL, USA). After heated to 95 for 2 min and then cooled to room temperature, the mixture was applied to the slides and covered by a coverslip. The slides were incubated in a humid cabinet of an incubator for 16-18 h at 42 . Then the slides were washed at 60 for 10 min in solutions of 2×SSC with 2 g/L SDS, 0.1×SSC with 2 g/L SDS, and 0.1×SSC sequentially, and then dried at room temperature.

Array scanning and data processing
Each slide was scanned at 10 µm resolution on a GenePix 4000B scanner (Axon Instruments, Inc., Foster City, CA) at variable PMT voltage to obtain maximal signal intensities with no more than 1% probe saturation. The images were processed with GenePix Pro 3.0. Ratios were normalized by a linear regression between ln(Cy5) and ln(Cy3) of all the genes' background-corrected signal intensities on the microarray. Genes exhibited a 2-fold or greater change in expression level and exceeded 200 in signal intensity were considered true outliers.

Preparation of 32 P-labeled probes
The plasmids containing cDNA clone used for preparing probes were provided by Shanghai BioStar Genechip Inc. A total amount of 200 ng plasmids were used as templates for PCR amplification. PCR products were purified using QIAquick Gel Extraction Kit (Qiagen, Germany). The probes were labeled using random primed Strip-EZ DNA Kit (Ambion, Austin, TX). A total amount of 25 ng purified DNA diluted in 9 µL TE (10 mmol/L Tris-HCl, pH8, 1 mmol/L EDTA) was denatured at 95 for 5 min and then immediately frozen in liquid nitrogen, thawed, microfuged, and placed on ice. Afterwards, the following reaction was assembled as follows and mixed gently: A 9.0 µL of denatured DNA, 2.5 µL of 10×Decamer solution, 5.0 µL of 5×buffer-dATP/-dCTP, 2.5 µL of 10×dCTP, 5.0 µL of [α-32 P]dATP (3 000 Ci/mmol, 10 mCi/mL), 1.0 µL of exonuclease-free Klenow, and nucleasefree water to 25 µL. After 20 min incubation at 37 , 1 µL of 0.5 mol/L EDTA was added to stop the reaction. A  with ULTRArray hybridization solution (Ambion, Austin, TX) containing cDNA probes labeled with [α-32P] dCTP by random priming (Strip-EZ DNA labeling system, Ambion). The hybridized membranes were serially washed at 55 using 2×SSC with 1 g/L SDS solution, then exposed to a phosphoimager. Blots were scanned and quantified by a phosphoimager in combination with Optiquant software v. 2.50 (Cyclone Storage Phosphor System, Packard Instruments).

RESULTS
The mRNA expression in U937 cells was compared at 2, 6, 10, and 24 h after the infection with NCTC 11638 or 11638-vacA. More than 100 genes altered their mRNA expression at different time points respectively, among which, the genes related to immune evasion were selected (Table 1). To confirm the differential expression profiling of the cDNA microarrays, two genes: PSMD13 and ILK were chosen for northern blot analysis. As shown in Figure 1, the expression levels of these 2 genes were lower in the cells treated with H pylori NCTC11638 than those treated with 11638-vacA. In addition, in both cases, the fold ratio changes detected by the microarray were confirmed by Northern blot ( Table 2). The level of β-actin transcript was present at approximately the same level in both samples, providing an assessment of RNA content in each sample used for Northern blot analysis.

DISCUSSION
Helicobacter pylori has found its own way to thrive within host cells, despite the presence of a well-functioning immune system. In the present study, differential expression of macrophage upon stimulation of VacA gives clear evidence that this vacuolating cytotoxin has evolved with various tricks to escape from the innate and adaptive immunity of the host. Recognition of pathogen-infected host cells by effector T lymphocytes requires intracellular processing of microbial antigens and their presentation on the surface of the antigenpresenting cells (APCs) in association with MHC molecules. To avoid immune recognition, many kinds of microorganisms have evolved mechanisms which interfere with antigen presentation pathways. These mechanisms may be pivotal, especially for those pathogens causing persistent infections. The present differential expressions provide clear evidence that VacA downregulates the expression of MHC class Iand class II molecules in macrophage. Thus, specific T lymphocytes may not be activated, resulting in the evasion of H pylori from the host's immune response and colonization of the bacteria in gastric mucosa persistently, remaining "invisible" to both CD8 + and CD4 + T cells. Interference with the class II antigen presentation pathway by Listeria monocytogenes has been shown to result in a reduced proliferation of CD4 + T lymphocytes in response to heterologous antigens [6] . Interactions between MHC class I and human immunodeficiency virus (HIV) resulted in down-regulation of MHC-Isurface expression, contributing to pathogenesis by suppressing the host's immune response [7] . The role of MHC class I and class II-restricted functions in H pylori infection and immunity upon oral immunization has also been examined in vivo. It was found that experimental challenge with H pylori resulted in significantly greater colonization in MHC class I and class II mutant mice than in wild-type mice [8] . MHC class II-deficient mice were unable to respond to oral antigenic stimulation and remained persistently infected with H pylori [8] .
Nitric oxide (NO) is an important mediator of biological processes including inflammatory response. It is synthesized from L-arginine by a family of nitric oxide synthases (NOS), in which, endothelial NOS (eNOS) is a constitutively expressed isoform present in vascular endothelial cells, cardiac myocytes, and blood platelets [9] . eNOS interacting protein (NOSIP) is a 34 ku protein specifically binds to the carboxylterminal region of the human eNOS oxygenase domain, overexpression of which in eNOS-expressing cells has been demonstrated to inhibit NO sythase activity [10] . Thus, down-regulation of this protein upon stimulation of VacA may relieve such inhibition, leading to more NO production. The original concept that the small quantities of NO generated in a pulsatile fashion by constitutive eNOS mainly fulfil regulatory functions required for normal homeostatic function of the vasculature, while the high amount of NO produced by inducible NOS (iNOS) exerts antimicrobial and cytotoxic effects in the immune system, has recently been modified [11] . The expression of eNOS is not restricted to endothelial cells, as it has been found to be present in monocytes/macrophages and in B and T lymphocytes [12] . eNOS can also assume typical immunological functions previously assigned to iNOS, such as the induction of apoptotic cell death and the control of viruses [11] . NO endows macrophages with cytostatic or cytotoxic activity against microbes and tumor cells [13] . Nevertheless, more and more evidences have demonstrated a role for NO in the induction of immunosuppression by inhibiting T-cell proliferation during G1/S transition [14][15][16] . In mouse models of T-cell-mediated autoimmunity, such as myelin antigen-induced EAE, the disease was exacerbated by genetic deletion of iNOS, indicating that NO suppressed Tcell-mediated immunity in vivo [17] . In addition, NO induces apoptosis, which, the ubiquitin/proteasome and NF-κB pathway have been determined to be involved in [18] .
In eukaryotic cells, degradation of many proteins involves their initial modification by conjugation of ubiquitin (Ub). Ubiquinated proteins are rapidly degraded by the 26S proteasome [19] . NO can inhibit the activities of the 20S and 26S proteasomes, providing a likely mechanism for the accumulation of NO-induced pro-apoptotic proteins p53 and Bax, the substrates of the ubiquitin/proteasome system [18] . Apart from the indirect effect via NO functioning, the mRNA expression of proteasomes shows a significant downregulation as the result of being directly stimulated by VacA. Ub/proteasome pathway can catalyze the proteolytic processing of inactive 105 ku NF-κB precursor into 50 ku subunit. p50 is then maintained in the cytosol conjugated with the p65 subunit in an inactive complex bound to IκB. In addition, Ub/proteasome is involved in proteolytic digestion of IκB, which is required for NF-κB activation [19] . Therefore, decreased proteasome activity should inhibit the proteolysis of NF-κB precursor and inhibit IκB degradation, thus blocking NF-κB activation. Another involved protein, integrin-linked kinase (ILK), which has been determined to upregulate NF-κB activity [20] , also shows decreased expression in this study. It might be the result of VacA induction, and moreover, ILK mRNA expression was found to be downregulated by NO [21] , corresponding to the above speculation that more NO may be produced due to downregulation of NOSIP. NF-κB is known to be important to cell survival. Fibroblasts and macrophages from Rel A (p65 subunit of NF-κB) (-/-) mice were sensitive to TNF-α-induced cell death, and reintroduction of Rel A enhanced cell survival [22] . Activation of NF-κB is required for inflammatory cytokine release by macrophages during infection [23] . Consequently, inhibition of NF-κB activation may be responsible for decreased cytokine release from macrophages and the resulting immunosuppression. Additionally, because ILK is an apoptosis suppressor [24] , decreased production of this protein in macrophage may accelerate apoptosis of the cell that plays important roles in innate host defense and antigen presentation, leading to the evasion from host immunity against H pylori.
The capability of degrading proteins by the proteasome accounts for another important function to generate peptides presented on MHC-class I molecules to circulating lymphocytes. The presentation of these peptides enables the immune system to screen for and destroy cells expressing unusual polypeptides [19] . Selective preteasome inhibitors were determined to prevent MHC-class I presentation of the antigenic peptide [25] . Moreover, LMP2 and LMP7, two subunits of the proteasome, were found to be encoded in the major histocompatibility complex (MHC) [26][27][28] . The experiment using specific antibodies against LMP2 and LMP7 showed that they were co-expressed with MHC-class I molecules [29] . The levels of MHC-class I expression were shown to coincide perfectly with the LMP levels in different tissues, corresponding to the result in the present study, which shows simultaneous downregulation of MHCclass I and LMP7.
MSP, also known as HGF-like protein, is a serum protein belonging to the plasminogen-related growth factor family. It was originally discovered by Leonard and Skeel as a serum protein that stimulates shape change, movement, chemotaxis and phagocytosis of mouse peritoneal resident macrophages [30,31] . The other important effect of MSP on macrophages was to inhibit endotoxin-or cytokine-stimulated NO production [32] . Thereby, the decreased expression of MSP may contribute to deactivating macrophage and producing more NO, which, as described above, functions as an immunosuppressor.
The early growth response 1 (EGR1), a zinc-finger transcription factor that was shown to be significantly upregulated by 2 h postinfection in the present study, has been determined to induce downregulation of copper-zinc superoxide dismutase and manganese superoxide dismutase and stimulate the generation of reactive oxygen species (ROS) via the NADH/NADPH-oxidase system [33] . In addition to NO, ROS produced by NADPH oxidase also have the ability to inhibit the proliferation of lymphocytes by a mechanism that suppressor macrophages impair the proliferative response of T lymphocytes to antigens or mitogens [11] . Otsuji et al. [34] demonstrated that the oxidative stress from tumor-derived macrophages mediated the decrease of CD3ζ chain within T cells, which suppressed the antigen-specific T-cell responses. Pre-treatment of CTL or NK cells with nontoxic concentrations of H 2 O 2 severely reduced their cytotoxic activity, leading to the speculation that macrophage-derived reactive oxygen metabolites contribute directly to alterations in signal transducing molecules of T cells and NK cells and to the mechanism of immunosuppression [35] . Furthermore, the defective expression of CD3ζ on lymphocytes has been related to some kind of carcinoma, including gastric adenocarcinoma [36,37] .
From Table 1, we may find up-regulation of MRC OX-2 (the antigen identified by monoclonal antibody, MOX2), a broadly expressed membrane glycoprotein, which has been shown to be important for regulation of the macrophage lineage. In the OX-2-deficient spleens, the number of macrophages was nearly twice the number of those in normal spleens [38] , implying a role of OX-2 in suppressing the activation of macrophage. The immunosuppression effects of OX-2 could be further determined by many other studies. For example, several studies reported that increased expression of OX-2 in mice receiving renal allografts was associated with immunosuppression leading to increased graft survival, along with the polarization of cytokine production to type 2 cytokines in lymphocytes harvested from the transplanted animals [39][40][41] . Furthermore, infusion of a mAb to OX-2 blocks both the increased graft survival and the altered cytokine production [42] . All these data make clear that upregulation of OX-2 does favor to the immune evasion of H pylori. Table 1 also demonstrates significant downregulation of many cytoskeleton-related gene expression. For example, RhoG is a member of the Rho family of GTPases, which signals to actin assembly during phagocytosis [43] . Vav1 serves as a guanine nucleotide exchange factor (GEF) for Rho proteins, and establishes an essential and direct link between receptors with intrinsic or associated tyrosine kinase activity and the mitogenic and cytoskeletal pathways regulated by Rho proteins [44] . Cytoskeleton has been known to be an important structural basis for phagocytosis since cytochalasin B, a toxin that blocks actin polymerization, was shown to inhibit uptake of IgG-coated erythrocytes by mouse macrophages [45] . Phagocytosis is a process by which macrophages and leukocytes could ingest microbial pathogens to accomplish two essential immune functions, i.e., to initiate the microbial death pathway, and to direct antigens to both MHC I and MHC II compartments. That is to say, phagocytosis serves not only as an innate immune effector but as a bridge between the innate and acquired immune responses [1] . Thus, to destruct cytoskeleton may be another trick of VacA to evade host immune response.
In conclusion, we have shown that H pylori VacA induces the alteration of a series of genes related to immune evasion in macrophage, which ultimately establishes a state of hostmicrobial equilibrium. Some of these genes are for the first time made an association with VacA stimulation. Further investigations of the previously uncharacterized genes should be made to help us see through the underlying mechanisms utilized by H pylori to escape host immunity.


s2fieldsofstudy: Biology Chemistry

text:
cAMP Dose-dependently Prevents Palmitate-induced Apoptosis by Both Protein Kinase A- and cAMP-Guanine Nucleotide Exchange Factor-dependent Pathways in β-Cells*

Lipid accumulation in pancreatic β-cells is thought to cause its dysfunction and/or destruction via apoptosis. Our studies show that incubation of the β-cell line RINm5F with the saturated free fatty acids (FFA) palmitate caused apoptosis based on increases in caspase 3 activity, Annexin V staining, and cell death. Furthermore, exposure of RINm5F cells to cAMP-increasing agents, 3-isobutyl-1-methylxanthine (IBMX), and forskolin completely abolished palmitate-mediated caspase 3 activity and significantly inhibited Annexin V staining and cell death. The cyclic AMP analogs cpt-cAMP and dibutyryl-cAMP mimicked the protective effects of IBMX and forskolin, suggesting that cAMP is the mediator of the anti-apoptotic effects. The protective action of IBMX and forskolin was rapid and did not appear to require gene transcription or new protein synthesis. However, these protective effects were clearly independent of protein kinase A (PKA) because of the lack of inhibition by the PKA inhibitors H-89 and KT5720. In attempts to identify this PKA-independent mechanism, we found that the newly developed cAMP analog 8CPT-2Me-cAMP, which selectively activates the cAMP-dependent guanine nucleotide exchange factor (cAMP-GEF) pathway, mimicked the protective effects of IBMX and forskolin, suggesting that the cAMP-GEF pathway is involved. In addition, both glucagon-like peptide (GLP-1) and its receptor agonist, Exenatide, inhibited palmitate-mediated caspase 3 activation in a dose-dependent manner. Unexpectedly, H-89 partially reversed the protective effects of GLP-1 and Exenatide, suggesting that PKA may play a role in the protective effects of these incretins. To explain these seemingly conflicting results, we demonstrated that low concentrations of cAMP produced by GLP-1 and Exenatide preferentially activate the PKA pathway, whereas higher cAMP concentrations produced by IBMX and forskolin activate the more dominant cAMP-GEF pathway. Taken together, these results indicate that intracellular concentrations of cAMP may play a key role in determining divergent signaling pathways that lead to antiapoptotic responses.

Obesity is a leading factor associated with the development of type 2 diabetes. Elevated levels of FFA 1 in the circulation of obese subjects have been postulated to cause peripheral and hepatic insulin resistance and impairment of insulin secretion from pancreatic ␤-cells (1). Studies in Zucker diabetic fatty (ZDF) rats have indicated that chronically elevated levels of FFA cause lipid accumulation in ␤-cells and lead to ␤-cell dysfunction and destruction via apoptosis (2).
Elevated levels of intracellular cAMP have been reported to protect various cell types including neutrophils, hepatocytes, and neuronal cells from apoptosis stimulated by various agents (3)(4)(5)(6). The mechanisms responsible for the protective action of cAMP against apoptosis include the synthesis of antiapoptotic proteins, inactivation of pro-apoptotic proteins, and activation of phoshatidylinositol 3-kinase-dependent Akt. Interestingly, cAMP has been also shown to promote apoptosis in leukemia and glioma cells by abnormal cell cycle regulation (7,8). Therefore, it appears that cAMP can cause both apoptotic and antiapoptotic effects depending on the cell type and triggering stimulus. The antiapoptotic effects of cAMP on lipotoxicity, however, have not been previously reported.
The activation of protein kinase A (PKA) by cAMP is a classical cellular signaling pathway that regulates a variety of cellular responses. The specificity of this regulation by cAMP is provided by protein kinase A-anchoring proteins that target its receptor, PKA, to specific cellular locations (9). It has been shown that mitochondria-anchored PKA inactivates the proapoptotic factor BAD through phosphorylation in response to the survival cytokine interleukin-3. Interleukin-3-induced phosphorylation of BAD was blocked by the PKA inhibitors Rp-cAMP and H-89 consistent with anchored PKA as the principal kinase (9).
Recent studies indicate that cAMP causes an even broader range of cellular responses by activating signaling pathways that are independent of PKA. Cyclic AMP has been shown to regulate gene transcription, cellular proliferation, and cytokine signaling through PKA-independent pathways (10 -13). A novel PKA-independent pathway that is activated by cAMP involves the guanine nucleotide exchange factors (GEFs also designated as Epac) for the small GTPases Rap1 and Rap2. It has been demonstrated that cAMP-GEFs directly activate Rap1, which is clearly independent of PKA (14,15). However, the functional consequences of the activation of Rap1 by the cAMP-GEF pathway are not fully understood. The recent development of a novel cAMP analog, 8-(chloro-phenylthio)-2Ј-O-methyl-adenosine-3Ј,5Ј-cyclic monophosphate (8CPT-2Me-cAMP), which activates cAMP-GEF, but not PKA, has been used to identify the cAMP-GEF signaling pathway (16). Furthermore, two isoforms of GEF have been identified, GEF-1 and GEF-2, and pancreatic islets and ␤-cell lines express both isoforms (17). The cAMP-GEF pathway has been previously shown to regulate incretin-potentiated insulin secretion (18).
It is postulated that cAMP binds to GEF, and the cAMP-GEF complex activates Rap1, a ubiquitously expressed GTPase of the Ras superfamily. Both Ras and Rap1 can exist in an inactive GDP-bound form or an active GTP-bound form, and cAMP-GEF promotes the formation of the active GTP-bound form of GTPase. Although the precise function of Rap1 is not known, it is believed to exhibit both Ras-dependent and -independent cellular functions. Recent studies suggest that the Ras/mitogen-activated protein kinase proliferation signaling pathway may regulate GLP-1-mediated ␤-cell growth and proliferation (17). Recent evidence also indicates that Ca 2ϩ -induced Ca 2ϩ release is regulated by the cAMP-GEF pathway in the ␤-cell line, INS-1 (19), and the ability of incretins to potentiate glucose-stimulated insulin secretion from isolated mouse islets involves both a PKA-dependent pathway and a PKA-independent pathway. The PKA-independent pathway involves the interaction of GEF-2 and Rim2, an insulin granule-associated protein (18).
Glucagon-like peptide-1 (GLP-1) is a 300-amino acid peptide secreted from L-cells of the intestinal epithelium in response to food (20). GLP-1 is one of the most potent insulinotropic substances known with half-maximal effective concentrations around 10 pmol/liter (21) and a strict requirement for glucose concentrations above 4.5 mmol/liter (22). GLP-1 mediates its effects by binding to cell surface receptors coupled to heterotrimeric G proteins, which are expressed by pancreatic ␤-cells. The binding of GLP-1 to its receptor stimulates cAMP formation, activation of PKA-dependent and -independent pathways, and a rise in intracellular Ca 2ϩ concentrations (23,24). GLP-1 has been shown to be effective in normalizing fasting glucose levels of type 2 diabetic patients resistant to diet and sulfonylurea therapy (25). GLP-1 has also been shown to improve glucose control in ZDF rats by increasing islet cell proliferation and decreasing cellular apoptosis (20). The extensive and rapid degradation of GLP-1 after subcutaneous injection, however, limits its usage as a therapeutic agent. A number of strategies to prolong the half-life of GLP-1 are currently being explored. The GLP-1 receptor agonist, Exenatide, exhibits a longer halflife than GLP-1 because of its resistance to the ubiquitously expressed enzyme, dipeptidyl peptidase IV.
In this study, we have developed an in vitro model of palmitate-induced apoptosis of the ␤-cell line RINm5F and demonstrated that cAMP-increasing agents including IBMX and forskolin and the G protein-coupled receptor agonists GLP-1 and Exenatide provide protection in both a PKA-and cAMP-GEFdependent manner. Low concentrations of cAMP produced by GLP-1 and Exenatide preferentially activate the PKA pathway, whereas higher cAMP concentrations produced by IBMX and forskolin activate the more dominate cAMP-GEF pathway. These findings suggest that modulation of cAMP concentrations may provide a strategy to optimize antiapoptotic signaling pathways.

EXPERIMENTAL PROCEDURES
Materials-Tissue culture medium, CMRL-1066, was obtained from Invitrogen. Penicillin, streptomycin, Hanks' balanced salt solution, Lglutamine, and the insulinoma cell line RINm5F were obtained from the Washington University Tissue Culture Support Center. Fetal bovine serum was from Hyclone (Logan, UT). BSA (fraction V, fatty acid-free) was from ICN Biomedical (Irvine, CA). Palmitic acid, IBMX, forskolin, and GLP-1 were obtained from Sigma. DEVD-AMC, cpt-cAMP, dibutyryl-cAMP, and KT5720 were from Biomol (Plymouth Meeting, PA), and Annexin V-FITC was from BD Biosciences Phar-Mingen (San Diego, CA). Direct cAMP enzyme immunoassay kits were obtained from Assay Designs (Ann Arbor, MI). H-89 was obtained from Alexis (San Diego, CA), and 8CPT-2Me-cAMP was from the BioLog Life Sci. Institute (Bremen, Germany). Exenatide was a kind gift from Amylin Pharmaceuticals Inc. (San Diego, CA) and Eli Lilly (Indianapolis, IN).
BSA Complexed FFA Concentrations-Fatty acid supplemented medium was prepared as described previously (26). Briefly, a 20 mM solution of fatty acid in 0.01 N NaOH was incubated at 70°C for 30 min with mixing. The dropwise addition of 1 N NaOH facilitated solubilization of FFA. Fatty acid soaps were complexed with 5% fatty acid-free BSA in phosphate-buffered saline. The BSA complexed fatty acid was added to 10% serum-containing cell culture medium to achieve desired total fatty acid concentrations at a 6.6:1 fatty acid to BSA molar ratio. The pH of the medium did not differ significantly from the addition of BSA complexed fatty acids, and unbound free fatty acids remained solubilized under all experimental conditions. These conditions were chosen to model pathophysiologic states in which FFA:BSA molar ratios approach 7:1 and yield unbound FFA concentrations in the several hundred nanomolar range (26).
Cell Culture and Expression of Full-length FATP1 cDNA-RINm5F cells were grown in RPMI 1640 medium supplemented with 10% calf serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and a final glucose concentration of 16.8 mM. RINm5F cells (10 5 ) were transduced in a 35-mm well by two successive 7-h exposures to 1 ml of a virus containing hemagglutinin epitope-tagged murine FATP1 cDNA (⌬U3FATP1) prepared as described previously (27). FATP1-overexpressing RINm5F cells showed an approximate 4 -5-fold increase in fatty acid uptake, elevation in lipid accumulation, and higher apoptotic cell death as compared with control cells. The overexpression of FATP1 did not interfere with RINm5F cell growth in normal culture media. In addition, the parameters of nutrient-mediated activation of metabolism based on MTS assays and protein translation monitored by p70 s6k activation were similar in FATP1-overexpressing RINm5F cells in comparison with nontransfected control and LacZ cells. FATP1-overexpressing ␤-cells were used to optimize palmitate-induced apoptosis over shorter exposure periods in these in vitro studies compared with control RINm5F cells. Control and Lac Z RINm5F cells also showed stimulated caspase 3 activity in response to palmitate treatment, and the cAMPincreasing agents forskolin and IBMX completely inhibited palmitatemediated apoptosis as in FATP1-overexpressing cells. However, the magnitude of caspase 3 activation in response to palmitate treatment was smaller than FATP1-overexpressing RINm5F cells.
Apoptosis Assays-For fluorescent caspase 3 cleavage assays, the cells (3 ϫ 10 5 ) were plated in a 24-well plate. After treatment with palmitate, the cells were harvested and washed once with phosphatebuffered saline. The cells were lysed with 160 l of Buffer A (10 mM Hepes, pH 7.4, 42 mM KCl, 1 mM dithiothreitol, 5 mM MgCl 2 , 0.5% CHAPS, 1 mM phenylmethylsulfonyl fluoride, and 10 M leupeptin). Triplicate cell lysates (50 l) were transferred to a 96-well plate. Buffer B (150 l; 25 mM Hepes, pH 7.4, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, and 3 mM dithiothreitol) containing 10 M DEVD-AMC was added to the cell lysates and incubated at room temperature for 20 min in the dark. The plate was read at excitation and emission wavelengths of 380 and 460 nm, respectively. For linearity, the plate was read every 2 min for 90 min, and the slope was determined. A comparison of caspase 3 activities under different experimental conditions was made only within an individual experiment because the measurement of absolute caspase 3 activity was not determined.
Annexin V-FITC binding and propidium iodide staining were performed according to the manufacturer's protocol, and the cells were analyzed by flow cytometry. Apoptotic cells were defined as propidium iodide-negative (indicating an intact plasma membrane) and as Annexin V-FITC-positive in comparison with cells incubated in the absence of palmitate.
Cell Viability Assays-After experimental treatment, both floating and attached cells were harvested and transferred to microcentrifuge tubes, and cell viability assays were performed. The cells were counted using a hemacytometer (Hausser Scientific, Horsham, PA) after mixing with trypan blue in a 1:1 ratio.
Assay of Phosphorylated and Total cAMP Response Element-binding Protein (CREB)-The cells (3 ϫ 10 6 ) were plated and cultured overnight in 35-mm Petri dishes. The cells were then pretreated for 30 min with H-89 (10 M), followed by stimulation with forskolin (10 M) for 10 min. The cells were washed once in 1ϫ phosphate-buffered solution, solubilized with boiling SDS sample buffer, subjected to electrophoresis, and transferred to nitrocellulose. Anti-phosphorylated CREB and anti-CREB antibodies (Cell Signaling Technology, Beverly, MA) were used following the manufacturer's recommendations and detected by chemiluminescence using an horseradish peroxidase-conjugated donkey antirabbit antibody.
cAMP Assays-Cells (3 ϫ 10 5 ) were plated in a 24-well plate. After experimental treatment, incubation media were aspirated and placed in microcentrifuge tubes. The cells were lysed by the addition of 500 l of 0.1 N HCl containing 0.1% Triton X-100 and incubated for 10 min at room temperature. The lysed cells were scraped into microcentrifuge tubes. The samples were centrifuged at 1300 ϫ g for 5 min at room temperature. Cyclic AMP concentrations in these supernatants were determined with a direct enzyme immunoassay kit as described by the manufacturer. Cyclic AMP concentrations in the culture media were also determined. The total concentration of 5 cAMP produced by each condition was obtained by adding cellular and culture media cAMP contents.
Statistics-Statistical comparisons were made between groups using a one-way analysis of variance. Significant differences were evaluated using Tukey post hoc analysis.

Cyclic AMP Protects RINm5F Cells from Palmitate-mediated
Apoptosis-In this in vitro model, we have demonstrated that incubation of the ␤-cell line RINm5F with the saturated FFA palmitate causes an increase in apoptosis based on caspase 3 activity, Annexin V staining, and assays of cell death. The concentration dependence of this effect indicated that 300 M palmitate complexed with BSA in a ratio of 6.6:1 (FFA:BSA) for 12 h caused an optimal increase in the number of ␤-cells undergoing apoptosis (data not shown). Under these experimental conditions, the free or unbound FFA concentration is estimated to be in the several hundred nanomolar range (26).
As shown in Fig. 1A, incubation of RINm5F cells with palmitate and the cAMP-increasing agents IBMX (100 M) and forskolin (10 M) completely prevented palmitate-mediated increases in caspase 3 activity. In a similar manner, IBMX and forskolin also significantly prevented, albeit to a lesser degree, palmitate-mediated increases in Annexin V staining and cell death as shown in Fig. 1 (B and C). The cyclic AMP analogs, chlorophenylthio-cAMP (cpt-cAMP; 200 M) or dibutyryl-cAMP (1 mM) mimicked the effects of IBMX and forskolin, suggesting that the action of IBMX and forskolin is mediated by increases in intracellular cAMP (Fig. 2). Direct measurements of cAMP concentrations under these identical conditions have also been performed as shown in Table I. IBMX and forskolin stimulated cAMP production ϳ100-fold higher than control and palmitate conditions. The specific concentrations of IBMX, forskolin, and the cAMP analogs employed in these experiments were selected based on dose-response studies (data not shown).
The time required for cAMP-increasing agents to prevent palmitate-mediated increases in caspase 3 activity was next evaluated. As shown in Fig. 3, palmitate-mediated increases in caspase 3 activity were completely prevented by exposure of RINm5F cells to cAMP-increasing agents present for the duration of a 12-h incubation period. The addition of IBMX and forskolin to the culture media 1 h prior to the end of a 12-h incubation with palmitate was sufficient to cause a significant decrease in caspase 3 activity. These findings suggest that the effects of IBMX and forskolin are rapid and probably do not require gene transcription or new protein synthesis. These cAMP-increasing agents were also added after the 12-h incubation period with palmitate and processed for caspase 3 activity. This condition did not result in inhibition of palmitate- mediated apoptosis, suggesting that IBMX and forskolin did not interfere with the measurement of caspase 3 activity.
The Protective Effects of IBMX and Forskolin Are Dependent on cAMP-GEF and Independent of PKA-The involvement of PKA activation by cAMP was studied using two PKA inhibitors, H-89 and KT5720. As shown in Fig. 4 (A and B), various doses of H-89 (1-50 M) or KT5720 (0.1-10 M) did not reverse the inhibitory effects of IBMX and forskolin on palmitate-mediated caspase 3 activity. In the absence of either palmitate or IBMX ϩ forskolin, neither H-89 nor KT5720 affected basal levels of caspase 3 activity (Fig. 4, white columns). To show that H-89 was able to inhibit PKA in intact cells, its ability to block cAMP-stimulated phosphorylation of CREB was examined. As shown in Fig. 5, H-89 (10 M) was able to inhibit forskolinstimulated phosphorylation of CREB to basal levels. These studies suggest that the protective effects of these cAMP-increasing agents are independent of PKA activation.
Recent studies have shown that cAMP directly activates the guanine nucleotide exchange factor (cAMP-GEF) pathway in a PKA-independent manner (14). The novel cAMP agonist, 8CPT-2Me-cAMP, designed to selectively activate the cAMP-GEF pathway but not PKA, was used to determine whether this signaling pathway is involved in the protective effects of IBMX and forskolin on palmitate-mediated apoptosis. Fig. 6 shows that 8CPT-2Me-cAMP (10 and 100 M) completely prevented palmitate-mediated caspase 3 activity, suggesting that the cAMP-GEF pathway may be involved in the protective action of IBMX and forskolin on palmitate-mediated apoptosis.
GLP-1 and Its Receptor Agonist, Exenatide, Prevent Palmitate-mediated Apoptosis-GLP-1 has been shown to decrease ␤-cell apoptosis in ZDF rats (20). Based on our studies indicating that cAMP prevents palmitate-mediated apoptosis, we determined next whether the ability of GLP-1 to decrease apoptosis in ZDF islets may be due, in part, to increases in cAMP levels that directly regulate the cAMP-GEF pathway. RINm5F TABLE I cAMP production by RINm5F cells The cells were incubated in control medium, medium containing 300 M palmitate, or medium in the presence of palmitate with IBMX and forskolin for 12 h. After treatment, cAMP concentrations were measured by the protocol as described under "Experimental Procedures." The total concentrations of cAMP production by each condition were obtained by adding cellular content and cAMP present in the culture medium. The data are presented as the means Ϯ standard deviation.  To determine the time requirements for the protective effects of IBMX ϩ forskolin incubation on palmitate-mediated apoptosis, the cells were treated with medium containing 300 M palmitate for 12 h, but IBMX ϩ forskolin were present only during the final hour of the 12-h incubation (fourth column). As a control to verify that IBMX ϩ forskolin did not interfere with the caspase 3 activity assay, these agents were added after the 12-h incubation (fifth column), and the cells were assayed for caspase 3 activity. The results are representative of three individual experiments. The asterisk denotes a statistically significant difference (p Ͻ 0.0001) between the second and fourth columns. Pal, palmitate; For, forskolin.  2 and 3). Lane 1 contains untreated cells. The level of phosphorylation of CREB was estimated using antiphosphorylated CREB and anti-CREB antibodies. cells were treated with palmitate and increasing concentrations of GLP-1 or its receptor agonist, Exenatide. As shown in Fig. 7, both GLP-1 and Exenatide provided significant protection from palmitate-mediated caspase 3 activation. This protection by GLP-1 and Exenatide occurred in a concentrationdependent manner and maximally reduced the caspase 3 activity to ϳ65% of its activity in the presence of palmitate alone. GLP-1 or Exenatide alone did not alter basal caspase activity as indicated by the white columns.
Experiments were next performed to determine whether the ability of GLP-1 or Exenatide to provide protection against palmitate-induced apoptosis is due to activation of the cAMP-GEF pathway similar to the effects of IBMX and forskolin. Unexpectedly, an inhibitor of PKA, H-89 (1 and 10 M), almost completely reversed GLP-1-mediated (1 M) and Exenatidemediated (100 nM) protection against apoptosis as shown in Fig. 8. To understand these seemingly conflicting results, we hypothesized that cAMP may activate both PKA and cAMP-GEF pathways depending on its concentration. To test this hypothesis, RINm5F cells were incubated with two different concentrations of forskolin (1 and 10 M) or Exenatide, in the presence of palmitate, to determine whether these treatments could invoke different concentrations of intracellular cAMP. Fig. 9A indicates that 1 and 10 M forskolin (third and fourth columns) indeed resulted in dose-dependent increases in cAMP. Moreover, Exenatide (fifth column) and GLP-1 (data not shown) stimulated cAMP production to a level similar to 1 M forskolin. As shown in Fig. 9B (fourth and sixth columns), forskolin at 1 and 10 M also resulted in a concentration-dependent protection of RINm5F cells against palmitate-mediated apoptosis. In addition, 1 M forskolin-mediated protection was readily reversed by H-89 (Fig. 9B, fifth column), whereas 10 M forskolin-mediated protection was not (Fig. 9B, seventh  column), suggesting that cAMP activates these different signaling pathways in a concentration-dependent manner. Thus, lower concentrations of cAMP produced by 1 M forskolin stimulate the PKA pathway, which is reversed by H-89, whereas the higher concentrations of cAMP produced by 10 M forskolin stimulate the cAMP-GEF pathway, which is PKA-independent.
The dose-dependent activation by cAMP of two different cellular signaling pathways has been confirmed using Exenatide in the absence or presence of a phosphodiesterase inhibitor, IBMX. As shown in Fig. 9A, Exenatide (100 nM) stimulated cAMP production at a level similar to forskolin (1 M). The presence of IBMX in addition to Exenatide increases intracellular cAMP levels about 2-fold compared with Exenatide alone (data not shown). Although cAMP levels stimulated by Exenatide as shown in Fig. 9A are not as high as those stimulated by forskolin (10 M), these lower levels appear to be sufficient to further enhance the protective effects of Exenatide against apoptosis as shown in Fig. 10A (fifth column). In Fig.  10A, IBMX (sixth column) or Exenatide (fourth column) alone protected palmitate-mediated apoptosis ϳ25-30%. However, when these agents were combined (fifth column), the protection was further enhanced to ϳ75%.
To further support our findings that different cAMP levels activate alternative downstream signaling pathways, studies were performed with Exenatide as described in Fig. 10A but in   FIG. 6. The cAMP-GEF selective analog, 8CPT-2Me-cAMP, prevents palmitate-mediated apoptosis. RINm5F cells (3 ϫ 10 5 ) were incubated with control medium or medium containing 300 M palmitate, or palmitate ϩ IBMX (100 M) ϩ forskolin (10 M) for 12 h as previously described. To test the effects of the GEF-selective cAMP analog on palmitate-mediated caspase 3 activity, the cells were treated with various concentrations of 8CPT-2Me-cAMP (solid bars) for 12 h in the presence of 300 M palmitate, followed by determination of caspase 3 activity. The cells were also incubated with various concentrations of 8CPT-2Me-cAMP alone as indicated by the white bars. The results are representative of three individual experiments. The asterisk denotes statistically significant differences (p Ͻ 0.05) between palmitatetreated (second column) and palmitate ϩ 8CPT-2Me-cAMP-treated groups. Pal, palmitate; For, forskolin. the presence of the PKA inhibitor, H-89. As shown in Fig. 10B, H-89 (10 M) readily reversed Exenatide-mediated protection (fourth and fifth columns) against palmitate-induced apoptosis. In contrast, a combination of Exenatide and IBMX (sixth and seventh columns) that results in increased cAMP levels produced almost complete protection that was not reversed by H-89. DISCUSSION Our studies have demonstrated that palmitic acid induces apoptosis in the ␤-cell line RINm5F, and intracellular concentrations of cAMP exert a key role in modulating antiapoptotic responses. In this in vitro design, activation of caspase 3 activity provided greater sensitivity in assessing palmitate-mediated apoptosis than Annexin V or cell viability based on trypan blue staining.
In the course of these studies, we observed that the cAMPincreasing agents IBMX and forskolin completely prevented palmitate-mediated apoptosis of the ␤-cell line RINm5F. The protective action of IBMX and forskolin was rapid and did not require prolonged incubations, suggesting that gene transcrip-tion or new protein synthesis was not involved. Although increases in cAMP concentrations augment nutrient stimulation of insulin secretion from ␤-cells, alterations in insulin concentrations in the incubation media failed to affect palmitatemediated apoptosis (data not shown). Furthermore, different concentrations of glucose did not alter the protective effects of cAMP against palmitate-mediated apoptosis (data not shown). Separate experiments demonstrated that forskolin alone was almost as effective as the combination of IBMX and forskolin, and IBMX alone provided an intermediate level of protection based on this same comparison (data not shown). In this latter condition, it is believed that IBMX alone increases basal cAMP levels because of its ability to inhibit phosphodiesterase activity. Increases in intracellular levels of cAMP appeared to mediate the protective effects of IBMX and forskolin because the cAMP analogs cpt-cAMP and dibutyryl-cAMP also provided complete protection from palmitate-induced apoptosis. Furthermore, control studies indicated that these cAMP-increasing agents did not interfere with the caspase 3 activity measurements. Because PKA is a well established effector of cAMP that mediates prosurvival signaling, we examined the ability of the PKA inhibitors H-89 and KT5720 to reverse this protective action of cAMP against palmitate-mediated apoptosis. The in- were treated with various conditions as indicated for 12 h. After treatment, cAMP concentrations were measured as described under "Experimental Procedures." B, to determine whether the reversibility by H-89 depends on the concentrations of intracellular cAMP, RINm5F cells (3 ϫ 10 5 ) were incubated with medium as indicated for 12 h as previously described, followed by caspase 3 assays. The results are the averages of four individual experiments. The asterisk denotes a statistically significant difference (p Ͻ 0.001) between the indicated columns. Pal, palmitate; For, forskolin. ability of these two structurally unrelated PKA inhibitors to reverse palmitate-mediated apoptosis suggested that this protective action of cAMP occurred in a PKA-independent manner.
Interestingly, de Rooij et al. (14) reported that forskolin and cAMP activate Rap1, a small Ras-like GTPase, also in a PKAindependent manner. These investigators determined that cAMP directly activates a GEF (also designated as Epac) that in turn activates Rap1 in Chinese hamster ovary cells. More recently, Kang et al. (28) reported that cAMP triggers Ca 2ϩinduced Ca 2ϩ release in pancreatic ␤-cells through activation of cAMP-GEF in a PKA-independent manner. These findings were accomplished, in part, by the use of a newly developed cAMP analog, 8CPT-2Me-cAMP, that selectively activates the cAMP-GEF pathway (16). Our studies indicate that this selective cAMP analog for the cAMP-GEF pathway mimicked the ability of cAMP-increasing agents to provide complete protection from palmitate-induced apoptosis. This protection by 8CPT-2Me-cAMP also occurred over a similar concentration range in which 8CPT-2Me-cAMP did not activate PKA signaling mechanisms involving CREB in the ␤-cell line, INS-1, as reported by Kang et al. (28).
Recent studies have also indicated that the cAMP-GEF signaling pathway exerts a critical role in GLP-1-potentiated insulin secretion by ␤-cells (29). In addition to its insulinotropic effects, GLP-1 has also been reported to promote islet cell growth and to inhibit apoptosis in ZDF rats (20). Our studies have shown that GLP-1 and its receptor agonist, Exenatide, also provided a significant degree of protection in a concentration-dependent manner against palmitate-mediated apoptosis of the ␤-cell line, RINm5F (Fig. 6). The lack of complete protection in comparison with IBMX and forskolin (10 M) is believed to be due to the reduced concentrations of cAMP produced by these incretins (Fig. 8A). Paradoxically, the protection provided by GLP-1 or Exenatide against palmitate-mediated apoptosis was almost completely reversed by H-89, suggesting that a PKA-dependent pathway is involved in the protective actions of incretins unlike those of forskolin. A possible mechanism to explain these divergent findings is that the intracellular concentrations of cAMP generated by GLP-1 or its receptor agonist, Exenatide, in comparison with the cAMPincreasing agent, forskolin, may determine the signaling pathways that lead to antiapoptotic responses. In support of this mechanism, low concentrations of cAMP produced by 1 M forskolin or Exenatide (Fig. 9) appeared to activate the PKA pathway, which was reversed by H-89. In contrast, an approximately 3-fold higher concentration of cAMP produced by 10 M forskolin activated the more dominant cAMP-GEF pathway, which was not inhibited by H-89. This proposed mechanism is further supported by the differences in affinities of cAMP for PKA activation with a K d of ϳ100 nM and a K d of ϳ10 M for cAMP-GEF2 (18). A similar mechanism based on intracellular cAMP concentrations has been proposed to explain a PKAindependent pathway involving the cAMP-GEF⅐Rim2 complex in the potentiation of insulin secretion by incretins (18). Interestingly, our findings suggest that increasing intracellular concentrations of cAMP by using incretins in combination with specific phosphodiesterase inhibitors (Fig. 10A) may further enhance their antiapoptotic effects by activation of the cAMP-GEF pathway.
A postulated mechanism to explain the antiapoptotic actions of GLP-1, Exenatide, and forskolin as described in these studies is shown in Fig. 11. The interaction of GLP-1 or Exenatide To test whether IBMX enhances the protective effects of Exenatide, the cells were incubated with Exenatide (100 nM) alone or in the presence of IBMX (20 M). As a control, IBMX (20 M) alone condition was also included. After a 12-h incubation, caspase 3 activities were measured. B, to determine whether the reversibility by H-89 depends on the concentrations of intracellular cAMP, RINm5F cells (3 ϫ 10 5 ) were incubated with medium containing various agents as indicated for 12 h, followed by caspase 3 assays. The results are representative of three individual experiments. The asterisk denotes statistically significant differences (p Ͻ 0.001) between the indicated columns. Pal, palmitate; For, forskolin. with its receptor activates heterotrimeric G s proteins, leading to the dissociation of the G␣ s subunit that subsequently stimulates adenylyl cyclase to produce cAMP. Cyclic AMP produced by this G s -coupled receptor mechanism or by forskolin can activate either the PKA and/or cAMP-GEF pathway depending on its intracellular concentration. The specific downstream signaling mediators of the PKA or the cAMP-GEF pathway that are responsible for these antiapoptotic effects are currently unknown. An elucidation of this cellular mechanism and its relevance to FFA-induced apoptosis in primary ␤-cells is of importance to optimize conditions for ␤-cell growth and proliferation with minimal apoptosis.