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The Plant Hormone Abscisic Acid Is a Prosurvival Factor inHuman and Murine Megakaryocytes*

Received for publication, August 4, 2016, and in revised form, December 23, 2016 Published, JBC Papers in Press, January 3, 2017, DOI 10.1074/jbc.M116.751693

Alessandro Malara‡, Chiara Fresia§, Christian Andrea Di Buduo‡, Paolo Maria Soprano‡, Francesco Moccia¶,Cesare Balduini¶, Elena Zocchi§, Antonio De Flora§, and Alessandra Balduini‡�1

From the Departments of ‡Molecular Medicine, Laboratories of Biotechnology, IRCCS San Matteo Foundation, and ¶Biology andBiotechnology, University of Pavia, Pavia 27100, Italy, the §Department of Experimental Medicine, Section of Biochemistry,University of Genova, Genova 16132, Italy, and the �Department of Biomedical Engineering, Tufts University,Medford, Massachusetts 02155

Edited by Roger J. Colbran

Abscisic acid (ABA) is a phytohormone involved in pivotalphysiological functions in higher plants. Recently, ABA hasbeen proven to be also secreted and active in mammals, where itstimulates the activity of innate immune cells, mesenchymaland hematopoietic stem cells, and insulin-releasing pancreatic� cells through a signaling pathway involving the second mes-senger cyclic ADP-ribose (cADPR). In addition to behaving likean animal hormone, ABA also holds promise as a nutraceuticalplant-derived compound in humans. Many biological functionsof ABA in mammals are mediated by its binding to the LANCL-2receptor protein. A putative binding of ABA to GRP78, a keyregulator of endoplasmic reticulum stress, has also been pro-posed. Here we investigated the role of exogenous ABA in mod-ulating thrombopoiesis, the process of platelet generation. Ourresults demonstrate that expression of both LANCL-2 andGRP78 is up-regulated during hematopoietic stem cell differen-tiation into mature megakaryocytes (Mks). Functional ABAreceptors exist in mature Mks because ABA induces an intracel-lular Ca2� increase ([Ca2�]i) through PKA activation and sub-sequent cADPR generation. In vitro exposure of human ormurine hematopoietic progenitor cells to 10 �M ABA does notincrease recombinant thrombopoietin (rTpo)-dependent Mkdifferentiation or platelet release. However, under conditions ofcell stress induced by rTpo and serum deprivation, ABA stimu-lates, in a PKA- and cADPR-dependent fashion, the mitogen-activated kinase ERK 1/2, resulting in the modulation of lym-phoma 2 (Bcl-2) family members, increased Mk survival, andhigher rates of platelet production. In conclusion, we demon-strate that ABA is a prosurvival factor for Mks in a Tpo-indepen-dent manner.

Abscisic acid (ABA)2 is a hormone involved in many physio-logical and developmental processes throughout the life cycle

of plants. In the early phase of the life of a plant, ABA regulatesseed maturation and the maintenance of embryo dormancy (1,2). Later, at the onset of ontogenesis, it mediates several adapt-ive responses toward environmental cues such as desiccation,cold, or salt stress and acts as a negative growth regulator.Recently, ABA has been shown to be also present and active inmammals, where it stimulates the functional activity of innateimmune cells (3, 4), insulin biosynthesis, release in pancreatic �cells, and glucose uptake by adipocytes and myoblasts (5, 6). Inthe bone marrow (BM), high levels of ABA were detected inmesenchymal stem cells, which play an essential role in the BMmicroenvironment by providing hematopoietic progenitorswith soluble factors essential to their proliferation and differ-entiation and to prevent detrimental lymphocyte activation (7).Consistently, micromolar amounts of ABA were demonstratedto expand uncommitted human hematopoietic progenitors (8).

Because of these potentially beneficial effects and its pres-ence in fruits and vegetables, ABA is currently investigated as apossible nutraceutical compound (9). In this regard, recent evi-dence has been provided regarding the effects of low-dose ABAintake, in the form of fruit extracts, on the improvement ofglucose tolerance and decrease of insulinemia in rats andhumans (10).

ABA effects on mammalian cells are dependent on the Gprotein-coupled and peripheral membrane protein lanthioninesynthetase C-like protein 2 (LANCL-2) (11–13). Binding ofABA to LANCL-2 triggers activation of a PKA-mediated signal-ing pathway involving the ADP-ribosyl cyclase-catalyzed con-version of NAD� to the second messenger and potent Ca2�

mobilizer cyclic ADP-ribose (cADPR) (14 –17). Recently, mem-bers of the heat shock protein 70 family of chaperones (includ-ing GRP78 and HSP70-2) have been identified as putative ABAbinding proteins (18). The functional and mechanistic signifi-cance of an interaction between ABA and HSP-70 proteins inmammalian cells remains unknown (18). However, the increas-ingly recognized role of GRP78 as a key marker and mechanisticplayer of the unfolded protein response triggered by severalendoplasmic reticulum (ER) stress conditions (19 –22) cer-

* This work was supported by Italian Ministry of University and Research FIRBGrant RBFR1299KO (to A. M. and C. F). The authors declare that they haveno conflicts of interest with the contents of this article.

1 To whom correspondence should be addressed: Dept. of Molecular Medi-cine, Laboratories of Biotechnology, IRCCS San Matteo Foundation, Uni-versity of Pavia, Via Forlanini 6, Pavia 27100, Italy. Tel.: 39-382502968; Fax:39-382502990; E-mail: [email protected].

2 The abbreviations used are: ABA, abscisic acid; BM, bone marrow; cADPR,cyclic ADP-ribose; ER, endoplasmic reticulum; Mk, megakaryocyte; Tpo,

thrombopoietin; rTpo, recombinant thrombopoietin; rhTpo, recombinanthuman thrombopoietin; rmTpo, recombinant mouse thrombopoietin;RyR, ryanodine receptor; 7-AAD, 7-amino actinomycin D; HSC, hematopoi-etic stem cell; RT-PCR, real-time PCR.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 8, pp. 3239 –3251, February 24, 2017

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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tainly demands further investigations of possible ABA-GRP78interactions.

Megakaryocytes (Mks) differentiate from stem cells and areresponsible for platelet release into the bloodstream (23). Dur-ing differentiation, Mks become giant and polyploid cells underthe effects of thrombopoietin (Tpo), the main thrombopoieticfactor in the BM. Tpo acts by binding to a specific cell surfacereceptor, the cellular homolog of the myeloproliferative leuke-mia virus oncogene (Mpl), leading to receptor dimerization,activation of intracellular signal transduction pathways, andresponses of target cells. Many of the effects of Tpo on cellsurvival and proliferation have been ascribed to the activationof the Jak/STAT and Ras/Raf/MAPK pathways (24). The use ofrecombinant Tpo (rTpo) has facilitated the development of invitro Mk culture systems, improving the study of the mecha-nisms of platelet formation. However, its role in this process isstill debated (25).

At the end of their maturation process, Mks extend longbranching processes, designated proplatelets, into sinusoidalblood vessels where platelets are released (26, 27). Althoughplatelet release shares several features with the apoptotic pro-cess, recent findings have demonstrated that platelet produc-tion proceeds independently of both the intrinsic and extrinsicapoptotic pathways (28 –32).

Here we demonstrate that in vitro differentiated Mks expressfunctional LANCL-2. Moreover, ABA promotes Mk survival ina Tpo-independent manner through ERK 1/2-dependent mod-ulation of Bcl-2 family members implicated in the regulation ofcell apoptosis.

Results

Expression of ABA-binding Proteins during Thrombopoiesis—After the cloning and characterization of Tpo, several in vitroculture systems have been developed to obtain highly enrichedpopulations of Mks. Under our well characterized culture con-ditions, human Mks are differentiated with recombinanthuman Tpo (rhTpo), IL-11, and IL-6 in serum-free medium,whereas mouse Mks are differentiated in serum-containingmedium in the presence of recombinant mouse Tpo (rmTpo)only (33, 34).

Specifically, in this study, enriched CD34� hematopoieticstem cells were purified from cord blood and differentiated for13 days in the presence of rhTpo, IL-11, and IL-6 to generatemature CD34�, CD41�, CD42b� Mks (Fig. 1, A and B). Underthese culture conditions, expression of the ABA main receptorLANCL-2 and of the ABA-binding protein GRP78 was mea-sured in freshly isolated CD34� cells and cells on days 7, 10, and13 of Mk differentiation. Increased expression of GRP78 andLANCL-2 mRNAs was detected during Mk maturation, with apeak on day 10 of differentiation (p � 0.001 and 0.05 for the twotranscripts, respectively, compared with time 0) and a subse-quent, comparable fall on day 13 (Fig. 1C). The time-dependentpattern of GRP78 changes confirms the earlier results of Lopezet al. (35), who suggested that transient up-regulation ofGRP78, peaking on day 11 of Mk maturation, marks the shift ofthe first step of differentiation to the stage of proplatelet forma-tion. In addition, they demonstrated that these patterns ofGRP78 expression indicate a parallel transient activation of ER

stress, which seems to be strictly required for Mk maturation(35).

The increased synthesis of GRP78 and LANCL-2 was con-firmed by Western blotting analysis in CD61 (�3 integrin)-pos-itive Mks (Fig. 1D). As shown in Fig. 1E, Mks cultured in thepresence of 10 �M ABA showed higher levels of LANCL-2 andGRP78 proteins at the end of culture (day 13). In addition, short(16-h) stimulation of mature Mks with ABA only was sufficientto induce a significant increase in LANCL-2 and GRP78 mRNAs(p � 0.05) (Fig. 1F) and a slight increase in protein expression(Fig. 1G). Overall, these results demonstrated that differenti-ated Mks express both ABA-binding proteins whose levels canbe further and steadily up-regulated in an autocrine manner bythe addition of ABA to the culture medium.

ABA Promotes a Cytosolic Ca2� Increase in Mature Mks—We demonstrated previously that ABA evokes, in mammaliancells, a LANCL-2-initiated signaling cascade characterized bythe two-step activation of PKA and the PKA substrate and theADP-ribosyl cyclase enzyme CD38, with the sequential gener-ation of the 2 s messengers cAMP and cADPR and resulting ina rise in intracellular Ca2� concentration ([Ca2�]i) (3–5). Totest the functionality of LANCL-2 in human Mks, cells on day13 of culture were seeded in fresh medium only and stimulatedwith 10 �M ABA to analyze changes in the phosphorylationlevels of PKA substrates. As shown in Fig. 2A, increased phos-phorylation of PKA substrates was detected after 15, 30, and 60min of ABA stimulation. The specificity of ABA effects on cellactivation was further confirmed by the reduced phosphoryla-tion of PKA substrates in Mks pretreated with a well knownPKA inhibitor (H89) prior to ABA stimulation (Fig. 2A). Next,the effects of 10 �M ABA on cytosolic Ca2� concentration wereevaluated. Addition of ABA to mature human Mks induced asignificant increase in [Ca2�]i compared with controls (water;Fig. 2, B and C). The [Ca2�]i increase was almost completelyinhibited by preincubation of the cells with the cADPR antag-onist 8-Br-cADPR or the PKA inhibitor H89 (Fig. 2, D and E),confirming LANCL-2 functionality and involvement of cAMPand cADPR in eliciting the ABA-induced and Ca2�-dependenteffects on Mks (see below).

ABA Does Not Synergize with Cytokines in Sustaining Mk Dif-ferentiation and Platelet Release—To define possible effects ofABA on Mk differentiation, 10 �M ABA was added to the stan-dard culture medium used to differentiate human and mouseMks from their progenitors. As shown in Fig. 3, the addition ofABA did not enhance the percentage of Mks differentiatingfrom both human and mouse hematopoietic progenitor cells(Fig. 3, A and B). The maturation profile of human Mks wassimilar in control and ABA-treated cultures, as comparable lev-els of low and high ploidy Mks were detected by flow cytometryat the end of culture (Fig. 3C). Consistently, when humanmature Mks were seeded in fresh medium containing rhTpo foran additional 24 h in the presence or absence of ABA, no differ-ences were observed in the percentage of proplatelet formation(Fig. 3, D and E) or in the rate of platelet release, as quantifiedwith cell counting beads by flow cytometry (Fig. 3, F and G).Altogether, these data demonstrate that ABA does not increase/amplify Mk differentiation, maturation, and platelet produc-tion under standard culture conditions in vitro. However, the

The Role of Abscisic Acid in Thrombopoiesis

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finding that ABA significantly increases the [Ca2�]i in matureMks when seeded in fresh medium without Tpo and other dif-ferentiating factors (Fig. 2) prompted us to focus on the mech-anisms of ABA signaling on fully differentiated cells in theabsence of culture supplements (e.g. cytokines and serum).

ABA Increases Survival of Mature Mk in a Tpo-independentManner by Up-regulating ERK 1/2 and the Expression of Anti-apoptotic Bcl-2 Family Members—Tpo deprivation in vitro hasbeen demonstrated to reduce Mk viability and increase apopto-sis (36). In plants, ABA regulates cell survival during stress con-ditions and induces the phosphorylation of several componentsof the MAPK cascade (37). To determine whether ABA caninduce activation of MAPKs in human Mks independently ofTpo, we investigated the phosphorylation levels of the ERK 1/2and p38 MAP kinases upon ABA stimulation under conditionsof cytokine deprivation.

As shown in Fig. 4A, differentiated human Mks displayedrapid and sustained phosphorylation of ERK 1/2 in the presenceof 10 �M ABA with respect to untreated Mks. These effectswere dose-dependent, as only micromolar concentrations ofABA were able to induce ERK 1/2 phosphorylation (Fig. 4B).On the contrary, the phosphorylation level of p38 MAPK was

not affected by ABA stimulation (Fig. 4C). To test whether ERK1/2 activation was dependent on the upstream PKA/CD38pathway, the phosphorylation levels of ERK 1/2 and p38 MAPKwere evaluated in Mks treated with specific inhibitors. Consis-tently with results on [Ca2�]i increase (Fig. 2, D and E), phos-phorylation of ERK 1/2 was significantly reduced by antagoniz-ing both PKA activity (with H89) and cADPR function (with8-Br-cADPR) in Mks stimulated with ABA for 30 min (Fig. 4D).On the other hand, p38 MAPK was neither activated by ABAnor inhibited by antagonizing the PKA/CD38 pathway (Fig.4E). Together, these findings raise the possibility that the PKA/CD38 pathway targets the MEK/ERK 1/2 pathway to sustainABA-induced and cADPR-mediated modulation of gene tran-scription. In mammals, ERK 1/2 is implicated in the promotionof cell survival through the regulation of antiapoptotic proteinexpression (38), and, recently, ABA has been demonstrated toinduce apoptosis in glioblastoma cell lines (39). Thus, we stud-ied the ability of ABA to induce the transcription of ERK 1/2target genes to address a potential prosurvival role of ABA instressed Mks. Therefore, differentiated human Mks weredeprived of rhTpo and cultured in fresh medium in the pres-ence or absence of 10 �M ABA for 24 h. Members of the Bcl-2

FIGURE 1. Expression of ABA-binding proteins during human thrombopoiesis. A, flow cytometry analysis of CD34 and CD41 cell marker expression infreshly isolated CD34� hematopoietic stem cells (HSCs) and after 13 days of differentiation toward the Mk lineage. B, percentages of CD34� or CD41� cells infreshly isolated CD34� HSCs and after 13 days of differentiation toward the Mk lineage. Results from three independent experiments are shown. C, RT-PCR ofLANCL-2 and GRP78 expression in CD34� HSCs and Mks on days 3, 7, 10, and 13 of differentiation. At least three independent experiments were performed.Data are presented as mean � S.D. *, p � 0.05; ***, p � 0.001. D, representative Western blot of LANCL-2 and GRP78 expression on days 7, 10, and 13 of Mkdifferentiation. Calreticulin was revealed to ensure equal protein levels, whereas �3 integrin was revealed to monitor Mk differentiation. OD, opticaldensity. E, analysis of LANCL-2 and GRP78 protein levels in Mks differentiated for 13 days in the presence or absence (Ctrl, water) of 10 �M ABA. ABA wasadded at the beginning of the culture and with every medium change on days 3, 7, and 10 of differentiation. F, RT-PCR analysis of LANCL-2 and GRP78mRNA levels in mature Mks stimulated or not (Ctrl, water) for 16 h with 10 �M ABA. Three independent experiments were performed. Data are presentedas mean � S.D. *, p � 0.05. G, representative Western blot analysis of LANCL-2 and GRP78 expression in mature human Mks stimulated or not (Ctrl, water)for 16 h with 10 �M ABA.




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family with antiapoptotic activity (Bcl-2 and Bcl-XL), but notwith proapoptotic activity (Bax), were significantly up-regu-lated at both the mRNA and protein levels after 24 h of ABAtreatment (Fig. 4, F and H). Further, inhibition of PKA andcADPR function by H89 (10 �m) and 8-Br-cADPR (50 �M),respectively, significantly reduced the ABA-dependent effectson expression of the antiapoptotic proteins Bcl-2 and Bcl-XL inhuman Mks (Fig. 4, I and J, p � 0.001). Overall, these datademonstrated that, independent of Tpo, ABA may function as asignal to promote Mk survival through ERK 1/2 activation andmodulation of the pro/antiapoptotic protein ratio.

The cADPR-dependent [Ca2�]i Increase in ABA-treated MksIs Mediated by Ryanodine Receptors and Not by TRPM-2Activation—cADPR has been recognized as a universal Ca2�

mobilizer by activating ryanodine receptors (RyRs) in manytypes of cells (16, 17). In addition, cADPR has been reported tomediate Ca2� entry by activating transient receptor potentialcation channel melastatin 2 (TRPM-2) (16, 17, 40). To investi-gate whether RyRs or TRPM-2 channels are involved in thecADPR-induced increase in [Ca2�]i, we first evaluated theexpression of these receptors in human Mks by RT-PCR. Ourresults demonstrated that RyR1 and RyR3 were expressed indifferentiated human Mks on day 13 of culture, whereas RyR2was not detected under the same experimental conditions (Fig.5A). TRPM-2 was also expressed by terminally differentiatedhuman Mks (Fig. 5A). Expression of RyRs was further con-firmed by RT-PCR in mouse BM-derived Mks (data notshown). However, RyRs were hardly detectable by Westernblotting analysis in human and mouse Mk extracts, probablybecause of the low abundance with which the RyRs areexpressed within these cells (data not shown).

These data prompted us to investigate, at a functional level,whether the ABA effects on mature Mks were dependent onCa2� mobilization from intracellular stores or on extracellularCa2� entry. For this purpose, we first measured the [Ca2�]i inMks loaded with the Ca2�-sensitive fluorochrome Fura-2 in theabsence of extracellular Ca2� (0 Ca2�) prior to stimulation with10 �M ABA (Fig. 5B). Interestingly, when ABA was applied in 0Ca2�, the initial increase in [Ca2�]i still occurred (peak inten-sity, see the legends for Figs. 2 and 5 for details), whereas thefollowing plateau phase (peak duration, see the legend forFig. 5) was significantly decreased (Fig. 5, B, D, and E; p �0.001). Thus, intracellular Ca2� release plays a major role ineliciting the Ca2� peak induced by ABA, whereas extracel-lular Ca2�influx might be involved in sustaining a late phaseof Ca2� signaling.

In the second approach, we performed fluorescence measure-ments of Ca2� in mature Mks pretreated with the RyRs inhib-itor tetracaine or with the specific TRPM-2 inhibitor econazoleprior to ABA stimulation (Fig. 5C). The results confirmed aprominent role of RyRs in mediating cADPR-dependent effectsin Mks stimulated with ABA, as tetracaine completely abro-gated both the peak intensity and duration of the intracellularCa2� increase in treated Mks (Fig. 5, D and E; p � 0.001). On thecontrary, inhibition of TRPM-2 by econazole in ABA-treatedcells did not affect the early peak of calcium increase but signif-icantly decreased the plateau phase to a value comparable withthat observed in Mks stimulated with ABA in 0 Ca2� (Fig. 5, Dand E, p � 0.001). Overall, these data support the view thatCa2� increase after ABA stimulation is mediated by an initialCa2� mobilization from ER stores through RyRs activation,whereas TRPM-2 sustains Ca2� entry from the extracellular

FIGURE 2. ABA elicits an intracellular calcium increase in mature human Mks through protein kinase A- and cADPR-dependent mechanisms. A,representative Western blot analysis of PKA substrate phosphorylation levels in differentiated human Mks treated or not with 10 �M ABA for 15, 30, and 60 minor treated with 10 �M PKA inhibitor H89 for 15, 30, and 60 min at 37 °C prior to ABA stimulation. Actin was revealed to ensure equal protein levels. Left panel,band intensities from eight selected bands per lane were analyzed for quantitative comparison using Quantity One software (Bio-Rad). *, p value � 0.05; ***,p � 0.001. Ctrl, control. B, cytosolic Ca2� concentration was monitored in Fura-2-loaded Mks in the presence (arrow) or absence (water) of 10 �M ABA. C,statistical analysis of the cytosolic Ca2� peak in the presence or absence of 10 �M ABA. The peak was calculated as the difference between the maximumfluorescence intensity reached upon stimulation and the baseline fluorescence intensity. Results are presented as the mean � S.D. of four independentexperiments (total number of cells analyzed, n � 200). ***, p � 0.001. D, the cytosolic Ca2� concentration was monitored in Fura-2-loaded human mature Mkspreincubated for 30 min with 50 �M 8-Br-cADPR (trace 1) or 10 �M PKA-specific inhibitor H89 (trace 2) prior to ABA stimulation. E, statistical analysis of cellpercentage with cytosolic Ca2� increase after stimulation with 10 �M ABA in the presence or absence of 50 �M 8-Br-cADPR or 10 �M PKA inhibitor. Results arepresented as mean � S.D. of three independent experiments (total number of cells analyzed, n � 150). ***, p � 0.001.




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space secondary to, and dependent on, initial RyRs-mediatedCa2� release. To analyze the functional contribution of thesetwo separate phases of Ca2� mobilization/entry during ABA-dependent MAPK activation, the phosphorylated levels of ERK1/2 and p38 MAPK after ABA stimulation were evaluated inMks pretreated with tetracaine or econazole. As shown in Fig. 5,F and G, inhibition of Ca2� release from ER stores by tetracainesignificantly abolished ERK 1/2 activation, whereas it had neg-ligible effects on p38 MAPK phosphorylation (Fig. 5, H and I).On the contrary, inhibition of Ca2� entry with econazole didnot affect the phosphorylation level of ERK 1/2 after ABA stim-ulation (Fig. 5, J and K; p � 0.001). Similarly, the increasedexpression of anti-apoptotic proteins was abrogated by tetra-caine but not prevented by TRPM-2 inhibition with econazolein ABA-stimulated Mks (Fig. 5, L–O; p � 0.001). Thus, theobserved effects of ABA on the modulation of ERK 1/2 activityand gene transcription in human Mks are dependent on themobilization of Ca2� stores from the ER via activation of RyRsby the second messenger cADPR. Whether cADPR by itselfsynergizes with Ca2� released from the ER in enhancing ERK1/2 phosphorylation is still undetermined.

ABA Increases Mk Survival under Stress Conditions andSustains Prolonged Platelet Release in Vitro—To determinewhether ABA has a measurable effect on Mk survival, weexplored the ability of ABA to increase the survival of differen-tiated Mks under conditions of cell stress. For this purpose,human Mks at the end of culture were reseeded under Tpo-deprived conditions, whereas mouse Mks differentiated fromfetal liver progenitor cells were purified with a BSA gradientand cultured under Tpo- and serum-deprived conditions.Under both experimental conditions, Mk properties were mon-itored over time for 2 additional days in the absence or presenceof 10 �M ABA. Cell viability was evaluated by staining the cellswith a specific Mk marker (CD41 or CD45) and measuring theexclusion of the DNA dye 7-amino actinomycin D (7-AAD) byflow cytometry. As shown in Fig. 6, A and B, ABA increased therate of Mk survival, with a peak after 48 h in human Mks andafter 24 h in mouse Mks. Moreover, the increased survival rateunder conditions of cell stress in ABA-treated Mks was paral-leled by a significant increase at 48 h in the percentage of mouseMks forming proplatelets with respect to the untreated control(Fig. 6C). Consistently, the absolute number of released plate-

FIGURE 3. ABA does not modulate Tpo-dependent differentiation, maturation, and platelet release in human and mouse Mks. A, Flow cytometryanalysis of percentages of CD41� Mks differentiated from human cord blood-derived CD34� cells in standard cultures (Ctrl) or with addition of 10 �M ABA for13 days. ns, not significant. B, flow cytometry analysis of percentages of CD41�/CD45� double-positive Mks differentiated from mouse BM hematopoieticprogenitor cells. Cells were cultured for 3 days in the presence of 10 ng/ml rhTpo alone (control) or Tpo plus 10 �M ABA. C, analysis of cell ploidy in human Mksdifferentiated in standard cultures (Ctrl) or with addition of 10 �M ABA. ABA was added at the beginning of the culture and with every medium change on days3, 7, and 10 of differentiation. N, nuclei. D, representative phase-contrast images of human Mk-forming proplatelets seeded in fresh medium for 16 h in thepresence of 10 ng/ml rhTpo alone or rhTpo plus exogenous 10 �M ABA. Scale bar � 50 �m. Arrows indicate proplatelets. E, on day 13 of maturation, human cordblood-derived Mks were seeded in fresh medium for 16 h in the presence of 10 ng/ml rhTpo alone or rhTpo plus exogenous 10 �M ABA, and proplateletformation was quantified (mean � S.D., n � 3 separate experiments). F, on day 13 of maturation, human Mks were seeded in fresh medium for 16 h in thepresence of rhTpo alone or rhTpo plus 10 �M ABA. Samples were mixed with counting beads to quantify the absolute number of released platelets (PLT) by flowcytometry. Platelets were identified as CD41� events in the FL1 green channel with the same physical parameters of peripheral blood platelets from a controlsubject, whereas beads were visualized in the FL2 detector (red channel). SSC, side scatter; FSC, forward scatter; B, beads, P, platelets. G, absolute numbers ofCD41�-released platelets analyzed by flow cytometry. On day 13 of maturation, 2 � 105 human Mks were seeded in fresh medium for 16 h in the presence ofrhTpo alone or rhTpo plus 10 �M ABA. Results are presented as mean � S.D. n � 3 separate experiments.




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lets was increased in mouse Mk cultures treated with ABA withrespect to the untreated control after 48 h (Fig. 6, D and E, p �0.05). Finally, to elucidate whether the prosurvival effects ofABA on Mks was dependent on activation of the mitogen-acti-vated kinase ERK 1/2, human mature Mks were pretreated witha specific ERK 1/2 inhibitor, PD98059 (10 �M), prior to treat-ment with 10 �M ABA for 24 h. Increased expression of antiapo-ptotic proteins (Bcl-2 and Bcl-XL) induced by ABA was signif-icantly abrogated by ERK 1/2 inhibition (Fig. 6, F and G, p �0.001). Further, as shown in Fig. 6, H and I, ERK 1/2 inhibitionsignificantly reduced the beneficial effects of ABA in terms ofcell survival, as measured by exclusion of the dye 7-AAD by flowcytometry. These results clearly demonstrate that ABA can actas an Mk prosurvival agonist in response to cytokine and serumdeprivation through an ERK 1/2-dependent mechanism.

Discussion

ABA is a phytohormone involved in the control of severalphysiological and developmental processes in higher plants

(1–2). We have recently demonstrated that ABA is producedand released by several human cell types, which also respond tothis hormone with functional activities triggered by cytosolicCa2� increases. The pathways of ABA biosynthesis have beenwidely explored and elucidated in plants, but less is known inanimals. Concerning the mechanisms of ABA release in mam-malian cells, it has been reported recently that ABA transport ismediated by members of the anion exchanger (AE) family ofanion exchangers and is bidirectional (41, 42). This featureaccounts for ABA uptake and release from mammalian andhuman cells and, more generally, for the mechanisms of ABA-related autocrine and paracrine functions in the animal king-dom (43). Established sources of plasmatic ABA, in addition todietary intake of fruits and vegetables (10), are pancreatic �cells, adipocytes, and, to a lesser extent, granulocytes, mono-cytes, and mesenchymal stem cells (43).

Despite the completely different ABA receptors in plants andhumans, the remarkable conservation of cADPR-Ca2� signal-ing pathways from vegetal (44) to animal kingdoms demon-

FIGURE 4. ABA induces ERK phosphorylation and modulation of anti-apoptotic Bcl-2 family proteins in a Tpo-independent manner. A, representativetime course analysis of ERK 1/2 phosphorylation levels in Tpo-deprived human Mks in the presence (ABA) or absence of 10 �M ABA. Vinculin was revealed toensure equal protein loading. Bottom panel, a graph quantifying the ratio of phosphorylated ERK 1/2 and total ERK 1/2. Results from three independentexperiments are presented. ***, p � 0.001. B, dose-dependent analysis of ERK 1/2 phosphorylation after 30 min in control Mks or Mks stimulated with ABA at1, 10, and 100 ng/ml or 2 and 10 �g/ml concentrations. Bottom panel, a graph quantifying the ratio of phosphorylated ERK 1/2 and total ERK 1/2. Results fromthree independent experiments are presented. ***, p � 0.001. C, representative time course analysis of p38 phosphorylation levels in Tpo-deprived human Mksstimulated with 10 �M ABA or not. Vinculin was revealed to ensure equal protein loading. Bottom panel, a graph quantifying the ratio of phosphorylated p38and total p38. Results from three independent experiments are presented. D, representative Western blotting analysis of ERK 1/2 phosphorylation in controlTpo-deprived human Mks (water) and Mks stimulated with 10 �M ABA for 30 min and Mks pretreated with 8-Br-cADPR (50 �M) or H89 (10 �M) prior to ABAstimulation. Bottom panel, a graph quantifying the ratio of phosphorylated ERK 1/2 and total ERK 1/2 of at least three independent experiments. *, p �0.05; **,p � 0.01; ***, p � 0.001. E, representative Western blotting analysis of p38 phosphorylation in control Tpo-deprived human Mks (water), Mks stimulated with10 �M ABA for 30 min, and Mks pretreated with 8-Br-cADPR (50 �M) or H89 (10 �M) prior to ABA stimulation. Bottom panel, a graph quantifying the ratio ofphosphorylated (pp38) and total p38 levels of at least three independent experiments. F, RT-PCR analysis of Bcl-2, Bcl-XL, and Bax mRNA levels in Tpo-deprivedhuman Mks treated (ABA) or not (Ctrl, water) with 10 �M ABA for 24 h (mean � S.D., n � 3 separate experiments). *, p � 0.05; **, p � 0.01. G, cord blood-derivedhuman Mks were cultured overnight in the absence of rhTPO and stimulated with exogenous 10 �M ABA or not (Ctrl) for 24 h. The protein levels of Bcl-2, Bcl-XL,and Bax were analyzed by Western blotting. A representative experiment is shown. H, quantification of Bcl-2, Bcl-XL, and Bax by relative densitometric analysisof the protein/vinculin ratio in a Western blot of control or ABA-treated Mks after 24 h of culture in the absence of rhTPO. Results are expressed as mean � S.D.of three independent experiments. *, p � 0.05; **, p � 0.01. I, Western blotting analysis of Bcl-2, Bcl-XL, and Bax in cord blood-derived human Mks culturedovernight in the absence of Tpo and presence of 10 �M ABA or absence (Ctrl) for 24 h or treated with 8-Br-cADPR (50 �M) or H89 (10 �M) prior to ABA stimulation.J, quantification of Bcl-2, Bcl-XL, and Bax protein levels in Mks stimulated with ABA in the absence of Tpo or pretreated with 8-Br-cADPR (50 �M) or H89 (10 �M)prior to ABA stimulation. At least three independent experiments were performed. ***, p � 0.001.




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strates that ABA can be considered a universal signaling mole-cule (3– 8). In in vivo models, administration of ABA wasdemonstrated to have beneficial effects on several conditionsand diseases, including obesity-related inflammation, diabetes,atherosclerosis, and inflammatory bowel disease (10, 45– 48).Here we demonstrate, for the first time, that exogenous ABAcan act as a prosurvival factor for Mks, leading to increasedplatelet generation and release in a Tpo-independent manner.Our results demonstrate that the expression of the main ABAreceptor in mammalian cells, LANCL-2, increases during Mkdevelopment and that addition of exogenous ABA to matureMks elicits a signaling pathway involving PKA/cAMP andcADPR-mediated cytosolic Ca2� increase, as described previ-

ously in other human cell types (3–5). Next, we show that, dif-ferent from previous studies in rats (49), human and mouseMks express functional RyRs that are responsible for the cyto-solic Ca2� increase mediated by ABA.

The failure to detect RyRs proteins by Western blotting,despite identification of the two corresponding transcripts byRT-PCR, can therefore be ascribed to their low abundance inMks, which is still sufficient, however, to elicit a measurableCa2� release following ABA stimulation. Indeed, the Ca2�

response to ABA consists of an initial [Ca2�]i peak that is due toRyRs-dependent Ca2� release, followed by influx of Ca2�

through TRPM-2 channels. Interestingly, pharmacologicalblockade of RyRs with tetracaine fully abrogated ABA-induced

FIGURE 5. Inhibition of intracellular calcium increase abolishes ABA-dependent effects on the expression of proteins of the Bcl-2 family. A, RT-PCRanalysis of RyRs and TRPM-2 expression in human Mks differentiated from cord blood-derived CD34� cells for 13 days in the presence of Tpo, IL-11, and IL-6.Relative expression (�Ct) was calculated on GAPDH expression. At least three independent cell cultures were analyzed. B, cytosolic Ca2� concentration wasmonitored in Fura-2-loaded Mks in the presence of 10 �M ABA or absence of extracellular Ca2� prior to ABA stimulation (arrow). C, cytosolic Ca2� concentrationwas monitored in the presence of 10 �M ABA (black trace) or in Mks pretreated with 10 �M econazole (blue trace 1) or 100 �M tetracaine (blue trace 2) prior to ABAstimulation (arrow). D, statistical analysis of the cytosolic Ca2� peak in Fura-2-loaded human mature Mks stimulated with 10 �M ABA, stimulated with 10 �M ABAin the absence of extracellular Ca2�, or preincubated for 30 min with 10 �M econazole or 100 �M tetracaine prior to ABA stimulation. Peak intensity wascalculated as the difference between the maximum fluorescence level reached upon stimulation and the baseline fluorescence intensity. Results are presentedas mean � S.D. (total number of cells analyzed, n � 310). ***, p � 0.001. E, statistical analysis of cytosolic Ca2� peak duration in human Mks stimulated with 10�M ABA, stimulated with 10 �M ABA in the absence of extracellular Ca2�, or preincubated for 30 min with 100 �M tetracaine or 10 �M econazole prior to ABAstimulation. Peak duration was calculated as time elapsed between the achievement of maximum fluorescence intensity and return to the ground fluores-cence intensity. Results are presented as mean � S.D. of four independent experiments (total number of cells analyzed, n � 310). ***, p � 0.001. F, represen-tative Western blot of ERK 1/2 phosphorylation in control human Mks stimulated with 10 �M ABA for 30 min or pretreated with 100 �M tetracaine prior to ABAstimulation. G, quantification of the pERK/ERK ratio in control human Mks (water), stimulated with 10 �M ABA for 30 min, or pretreated with 100 �M tetracaineprior to ABA stimulation. At least three independent experiments were performed. ***, p � 0.001. H, representative Western blot of p38 phosphorylation incontrol human Mks stimulated with 10 �M ABA for 30 min or pretreated with 100 �M tetracaine prior to ABA stimulation. I, quantification of the pp38/p38 ratioin control human Mks stimulated with 10 �M ABA for 30 min or pretreated with 100 �M tetracaine prior to ABA stimulation. At least three independentexperiments were performed. ns, not significant. J, representative Western blot of ERK 1/2 phosphorylation in control human Mks (water) stimulated with 10�M ABA for 30 min or pretreated with 10 �M econazole prior to ABA stimulation. K, quantification of the pERK/ERK ratio in control human Mks stimulated with10 �M ABA for 30 min or pretreated with 10 �M econazole prior to ABA stimulation. At least three independent experiments were performed. ***, p � 0.001. L,representative Western blotting analysis of Bcl-2, Bcl-XL, and Bax in cord blood-derived human Mks cultured in the presence of 10 �M ABA or water (control) for24 h or treated with tetracaine (100 �M) prior to ABA stimulation. M, quantification of Bcl-2, Bcl-XL, and Bax protein levels in human Mks stimulated with water(Ctrl) or 10 �M ABA or pretreated with tetracaine was performed from three independent experiments. ***, p � 0.001. N, representative Western blottinganalysis of Bcl-2, Bcl-XL, and Bax in cord blood-derived human Mks cultured overnight in the presence of 10 �M ABA or water (control) for 24 h or treated witheconazole (10 �M) prior to ABA stimulation. O, quantification of Bcl-2, Bcl-XL, and Bax protein levels in human Mks stimulated with water (Ctrl) or 10 �M ABA orpretreated with econazole (10 �M). Three independent experiments were performed.




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Ca2� signaling. This observation is coherent with the notionthat intracellular Ca2� acts synergistically with cADPR (orADPR) to gate TRPM-2 channels (50). This feature stronglysuggests that ABA is unlikely to gate TRPM-2 without the priorstimulation of RyRs-dependent Ca2� mobilization. Alterna-tively, TRPM-2-mediated Ca2� entry could be locally amplifiedby RyRs through the Ca2�-induced Ca2� release process, asobserved for TRP vanilloid 4 channels (51). In this case, uponRyR blockade with tetracaine, TRPM-2-dependent Ca2� sig-nals could remain confined beneath the plasma membrane andfall below the resolution of our Ca2� imaging system.

Further, we show that, in the unique and complex biologicalprocess of Mk development, ABA does not synergize with Tpoin supporting Mk differentiation, maturation, or platelet release.However, in differentiated Mks and under conditions of cellularstress, stimulation with ABA induces, through the cAMP/cADPR signaling cascade, the peculiar activation of the mito-gen activated kinase ERK 1/2, resulting in the up-regulation ofantiapoptotic Bcl-2 family members, thus increasing Mk sur-vival and increasing platelet release in vitro in the absence ofrecombinant Tpo and serum.

The involvement of members of the Bcl-2 protein family inapoptotic mechanisms triggered by micromolar ABA is notunprecedented. An earlier study has demonstrated a significantup-regulation of Bcl-2 in human uncommitted hemopoieticprogenitors (CD34 cells) following incubation with 2 �M ABAfor 24 h (8). More recently, ABA has been reported to induceapoptosis in glioblastoma cells via up-regulation of Bax andconcomitant down-regulation of Bcl-2 compared with ABA-untreated cells (39). These ABA-induced effects, opposite tothe pro-survival outcome observed in our study with stressedMks, are mediated by other players of apoptosis/differentiationand especially by the involvement of the retinoic acid signalingpathway (39). Conversely, the role of ERK 1/2 in ABA-respon-sive glioblastoma cells was not investigated. Finally, a recentstudy demonstrated that, in human mesenchymal stem cells,cADPR stimulates cell proliferation by inducing repetitiveCa2� oscillations that, in turn, lead to phosphorylation of ERK1/2 (52). Therefore, it appears that, in different types of humancells, cADPR, either autocrinally generated (52) or intracellu-larly produced as a second messenger of the ABA signalingcascade (this study), expands the stem cell precursors through

FIGURE 6. ABA sustains Mk survival and platelet release during cell stress. A, human cord blood-derived Mks at the end of culture were seeded for 24 or 48 hin fresh medium deprived of Tpo and supplemented or not (control, water) with 10 �M ABA. Viability was assessed after CD41 and 7-AAD staining by flowcytometry. Results are expressed as normalized mean � S.D. over control cells of at least three independent experiments. *, p � 0.05. B, mouse fetalliver-derived Mks were purified with a BSA gradient and seeded for 24 or 48 h in fresh medium deprived of Tpo and serum and supplemented or not with 10�M ABA. Viability was then assessed after CD41 and 7-AAD staining by flow cytometry. Results are expressed as normalized mean � S.D. over control conditionsof at least three independent experiments. *, p � 0.05; **, p � 0.01. C, percentages of mouse fetal liver-derived Mks extending proplatelets after 24 or 48 h ofculture in the presence or absence of 10 �M ABA following Tpo and serum withdrawal. Results are expressed as mean � S.D. of four independent experiments.**, p � 0.01. Ctrl, control. D, representative flow cytometry analysis of released platelets from 2 � 105 mouse fetal liver-derived Mks that were seeded inserum-free medium, deprived of Tpo, and stimulated or not (Ctrl, water) with 10 �M ABA for 48 h. Total cell cultures were mixed with counting beads, andplatelets were enumerated as CD41� events with the same physical parameters of mouse peripheral blood platelets. Beads were visualized in the FL2 channel.B, beads; PLT, platelets. E, quantification of released platelets after 24 or 48 h in fetal liver-derived Mks cultured in serum-free medium, deprived of Tpo, andstimulated or not (Ctrl, water) with 10 �M ABA. *, p � 0.05. F, representative Western blotting analysis of Bcl-2, Bcl-XL, and Bax in cord blood-derived human Mkscultured overnight in the presence of 10 �M ABA or water (Ctrl) for 24 h or treated with PD98059 (10 �M) for 30 min prior to ABA stimulation. G, quantificationof Bcl-2, Bcl-XL, and Bax protein levels in human Mks stimulated with water (Ctrl) or 10 �M ABA or pretreated with PD98059 (10 �M) was performed from threeindependent experiments. ***, p � 0.001. H, representative flow cytometry histograms of CD41/7-AAD double-positive mouse Mks cultured for 24 h inserum-free medium, deprived of Tpo, and stimulated with 10 �M ABA (ABA), 10 �M ABA and 10 �M PD98059 (ABA�PD98059), or water (Ctrl). I, quantification ofmouse Mk viability in cells cultured for 24 h in serum-free medium, deprived of Tpo, and stimulated with 10 �M ABA, 10 �M ABA and 10 �M PD98059, or water.Results are expressed as mean � S.D. of three independent experiments. *, p � 0.05; **, p � 0.01.




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multiple, still ill-defined mechanisms downstream of ERK 1/2activation. Indeed, the ERK 1/2 pathway is known to be associ-ated with increased cell proliferation (52) but also with decreasedapoptosis (53, 54), depending on different cell types and context-specific conditions (55).

Tpo, also known as c-Mpl ligand, is the primary physiologicalgrowth factor for the Mk lineage that also plays a central role inthe survival and proliferation of hematopoietic stem cells (56,57). However, although Tpo is one key driver of Mk differenti-ation, mice lacking either c-Mpl or Tpo are able to successfullyproduce platelets, indicating a role for other regulators in theend stage of Mk maturation (58, 59). ABA did not synergizewith Tpo to sustain or increase Mk differentiation, but its func-tional consequences on Mk signaling and function become evi-dent only after deprivation of Tpo and serum from the media ofhuman and mouse Mks cultures. These effects might beexplained by the ability of ABA to activate in Mks the samebiochemical pathways normally regulated by Tpo, such as arapid and sustained ERK 1/2 phosphorylation (60) as well asCa2� signaling (61). Therefore, as in plants, the beneficialeffects of ABA on Mks and platelet production are prominentwhen cells experience a transient stress condition, such as thewithdrawal of their main physiological regulator.

Data on the signals that drive terminal Mk maturation arestill insufficient to elucidate the exact mechanisms of plateletproduction. It is known that Ca2� fluxes play a crucial role inthe regulation of mature Mk functions and platelet formation(62), whereas the balance between cell survival, apoptosis, andplatelet biogenesis in Mks is still debated and far from beingcompletely deciphered (28 –32). An example of such complex-ity is a different time schedule between Mk survival and pro-platelet formation in ABA-treated human and murine Mks,respectively (Fig. 6, A–C). In particular, it is not understoodhow Mks can undergo apoptosis and yet produce viable plate-lets that circulate in the bloodstream for days. Related to this,Lopez et al. (35) recently suggested that ER stress-induced apo-ptosis signaling is involved and strictly required in the processof thrombopoiesis and might be the missing link responsible forcontrol of both induction and down-regulation of the apoptosissignal. This view is supported by the parallel behavior of expres-sion of the ABA receptors LANCL-2 and GRP78 during throm-bopoiesis (Fig. 1, C and D). The up-regulation of LANCL2 andGRP78 expression levels by ABA in mature Mks, althoughmechanistically unexplained, seems to represent an interestingexample of positive feedback regulation.

In mammals, proteins of the Bcl-2 family display a range ofbioactivities and are critical regulators of the “intrinsic” apopto-sis pathway. This family contains both prosurvival and proapo-ptotic members, and the balance between these competing sys-tems regulates the apoptotic switch. The prosurvival familyconsists of Bcl-2, Bcl-XL, Mcl-1, A1, and Bcl-w and maintainscellular viability by restraining the proapoptotic effectors Bakand Bax. When activated, Bak and Bax damage mitochondriaand trigger a cascade of events that ultimately leads to activa-tion of caspase-9 (63). As a whole, Mks seem to have evolved aunique apoptotic pathway to regulate their survival and plateletgeneration that seems independent of the canonical intrinsicand extrinsic apoptosis pathways (30). In this work, we demon-

strate for the first time the potential action of a plant hormone/nutraceutical agent in sustaining thrombopoiesis. Further, weextend the previously described mechanisms of ABA action onmammalian cells by delineating ERK 1/2 as a linker betweenPKA activation and cADPR increase with the modulation of theratio between anti- and proapoptotic proteins in stimulatedcells. Fig. 7 shows the cADPR-mediated and Ca2�-dependentmechanisms involved in the enhanced survival of ABA-stimu-lated Mks.

The identification of ABA as a novel Mk agonist opens newavenues of investigation and raises interesting questions. A sofar unanswered one concerns whether ABA-Mk interactionsoccur in vivo. With respect to this, we have demonstrated pre-viously that a significant source of BM-derived ABA is repre-sented by mesenchymal stem cells and other hematopoietic lin-eages as well as by granulocytes and macrophages (3, 7), whichare closely associated with Mks in the BM, thereby ensuringoverall BM homeostasis (64).

Interestingly, ABA might be autocrinally released by Mks, asdemonstrated by the detection of ABA in a range of 0.5–1.5pmol/106 cells in human Mk extracts on day 13 of culture (datanot shown). This suggest that in vivo Mk-ABA interactionswithin the BM are likely to occur.

The use of micromolar concentrations of ABA in this study,remarkably higher than the nanomolar basal levels of ABAdetectable in human plasma (10), does not exclude that func-tionally active concentrations may be reached in vivo underspecific conditions and at selected sites of the organism (e.g. inthe BM hematopoietic niche). Moreover, our recent study onadministration in rats and humans of low-dose dietary ABA(0.5–1.0 �g/kg of body weight) demonstrates that nanomolar

FIGURE 7. Schematic of ABA signaling in megakaryocytes. Following influxof extracellular ABA in Mks across any of the transporters of the AE family (39,40), it interacts with the G protein-coupled, N-terminal glycine myristoylatedperipheral membrane protein LANCL-2 (11) This interaction triggers G pro-tein-mediated activation of adenylate cyclase (AC), overproduction of cAMP,PKA-mediated stimulation of CD38, and increase in [cADPR]i. Downstream ofcADPR, two mechanisms might be involved to induce the observed increasein [Ca2�]i: extracellular Ca2� influx through activation of TRPM-2 by cADPR(50) or mobilization of intracellular Ca2� from cytoplasmic stores throughryanodine receptor activation. The latter event is responsible for the in-creased ERK 1/2 phosphorylation in Mks with changes in gene transcription inthe nucleus. Among the potential ERK gene targets, members of the Bcl-2family of proteins are specifically up-regulated by ABA treatment, resulting inincreased cell survival under stress conditions.




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plasma concentrations were functionally effective to the extentof improving glucose tolerance and decreasing insulinemia,whereas much higher doses of ingested ABA (50 mg/kg of bodyweight) resulted in enhanced insulin release (10). This discrep-ancy provides evidence that in vitro experiments may not nec-essarily enable the prediction of the in vivo relevance of mech-anisms of ABA effects in specific target cells, including Mks. Inthis regard, there is a plethora of in vivo insults, including che-motherapeutic agents, autoantibodies, and viruses, that causeMk stress, resulting in thrombocytopenia, reduced Mk survival,or increased apoptotic death (65– 67). Conditions of reducedTpo production have also been reported during liver diseases inwhich thrombocytopenia is caused not only by peripheral bloodplatelet destruction but also by decreased platelet productionbecause of a reduction of mRNA for Tpo in the liver (65). Onthese grounds, it would be interesting to analyze the effects ofABA as a nutritional intervention during these circumstancesof thrombopoietic stress (9, 10).

Finally, the positive effect of ABA on thrombopoiesis may beexploitable to improve the production of platelets in appropri-ate bioreactors of industrial interest, such as the recentlyobtained silk protein-based systems (68). Indeed, the silk modelhas already been proven to identify endothelium-specific mol-ecules such as VEGF and VCAM-1, useful for ex vivo produc-tion of functional platelets (69).

Experimental Procedures

Materials—The LANCL-2 antibody was described previ-ously (42). Anti-GRP78 (3183), anti-phospho-ERK 1/2 (Thr-202/Tyr-204) (4377), anti-ERK 1/2 (9102), Ser(P)/Thr(P) PKAsubstrate (9621), Bax (2772), and Bcl-XL (2762) antibodies werepurchased from Cell Signaling Technologies (Euroclone,Milan, Italy). Rabbit anti-RyR1 and anti-RyR3 polyclonal anti-bodies (70) were kindly provided by Prof. Vincenzo Sorrentino(University of Siena). Anti-phospho-p38 MAPK (Thr-180/Tyr-182) (AB32557), anti-p38 MAPK (AB89454), and anti-Calreti-culin (ab31290) were from Abcam (Cambridge, UK). Anti-�3integrin (clone C-20, SC6626) and anti-Bcl-2 (clone C2, SC8372)were from Santa Cruz Biotechnology (Santa Cruz, CA). ABA,8-Br-cADPR, H89 dihydrochloride hydrate, tetracaine hydro-chloride, PD98059, and econazole nitrate salt were all fromSigma-Aldrich (Milan, Italy).

Animals and Megakaryocyte Cell Cultures—Human umbili-cal cord blood was collected following normal pregnancies anddeliveries upon informed parental consent in accordance withthe Ethical Committee of the IRCCS San Matteo Foundationand the principles of the Declaration of Helsinki. CD34� cellswere separated and cultured as described previously (33).Briefly, CD34� cells were cultured for 13 days in the presence of10 ng/ml of rhTpo, IL-6, and IL-11 with medium change ondays 3, 7, and 10. ABA content in extracts from cells was mea-sured as reported previously (5). C57BL/6 and CD1 wild-typemice were from Charles River Laboratories. 6- to 8-week-oldmice were used in all experiments. Mice were housed at theanimal facility of the Department of Physiology, Section of Gen-eral Physiology, University of Pavia (approval nos. 1/2010,24/06/2010, and 3/2013, November 19, 2013). All animals weresacrificed according to the current European legal animal prac-

tice requirements. Mouse Mks were obtained from BM andfetal liver cells as described previously (34). Briefly, BM cellswere flushed from femora, and lineage-negative cells were puri-fied with a lineage cell depletion kit (Miltenyi Biotech). Cellswere cultured for 4 days in DMEM (Gibco) supplemented with1% penicillin/streptomycin, 1% L-glutamine, 10% FBS, and 10ng/ml rmTpo (Peprotech, London, UK). For fetal liver cells,fetal livers were obtained from embryonic days 12–15 timedpregnant CD1 mice. A single-cell suspension was made bydrawing the cells into a syringe through a 25-gauge needle andthen expelling the cells into a tube through a nylon screen. Cellswere differentiated in vitro for 4 days in DMEM supplementedwith 1% penicillin/streptomycin, 1% L-glutamine, 10% FBS, and10 ng/ml rmTpo (Peprotech). At the end of the culture, Mkswere purified using a 1.5/3% gradient of bovine serum albumin(Sigma-Aldrich).

Western Blotting—Human Mks were lysed in Hepes-glycerollysis buffer (50 mM Hepes, 150 mM NaCl, 10% glycerol, 1% Tri-ton X-100, 1.5 mM MgCl2, and 1 mM EGTA) containing leupep-tin (1 �g/ml) and aprotinin (1 �g/ml) for 30 min at 4 °C. Sam-ples were clarified by centrifugation at 15,700 � g at 4 °C for 15min. Laemmli sample buffer was then added to supernatants.Samples were heated at 95°C for 3 min, separated by electro-phoresis on 8% or 12% sodium dodecyl sulfate-polyacrylamidegel and then transferred to polyvinylidene fluoride membranes.Membrane were probed with primary antibodies, washed threetimes with PBS and Tween 0.1%, and incubated with an appro-priate peroxidase-conjugated secondary antibody. Membraneswere visualized using Immobilon Western chemiluminescentHRP substrate (Millipore, Milan, Italy) and the ChemiDoc XRSimaging system (Bio-Rad). Densitometry analysis was per-formed using Quantity One software (Bio-Rad). The relativeratio of optical density units was calculated regarding the gelband corresponding to the internal control for each lane andeach type of protein after subtracting the background noise.

Real-time-PCR—Total cellular RNA was extracted andtreated with DNase as described previously (34). Retrotrans-cription was performed using the iScript cDNA synthesis kitaccording to the instructions of the manufacturer (Bio-Rad).For quantitative real-time PCR, RT samples were diluted up to60 �l with double-distilled H2O, and 3 �l of the resulting cDNAwas amplified in duplicate in 20-�l reaction mixtures with eachspecific primer (200 nM) and SsoFastTM Evagreen� Supermix(Bio-Rad) at 1� final concentration. The amplification reactionwas performed in a CFX real-time system (Bio-Rad). Primersfor human genes used in the study were as follows: �2-micro-globulin forward 5-CCCCCACTGAAAAAGATGAGT-3and reverse 5-TGATGCTGCTTACATGTCTCG-3, GAPDHforward 5-ACAGTTGCCATGTAGACC-3 and reverse5-TTTTTGGTTGAGCACAGG-3, Bcl-2 forward 5-GGA-GGCTGGGATGCCTTT-3 and reverse 5-ACCCATGG-CGGTGACCATGC-3, Bax forward 5-GAGAGGTCTTT-TTCCGAGTGG-3 and reverse 5-CCTTGAGCACCAGT-TTGCTG-3, Bcl-XL forward 5-ATGAACTCTTCCGGGA-TGG-3 and reverse 5-TGGATCCAAGGCTCTAGGTG-3,LANCL-2 forward 5-ACAAGGTCTTTAAGGAGGAG-3and reverse 5-TAAAGGGACAGGAAGGAATAG-3, GRP78forward 5-TCTATGAAGGTGAAAGACCC-3 and reverse




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5-TCTCAAAGGTGACTTCAATC-3, and TRPM2 forward5-TCGGACCCAACCACACGCTGTA-3 and reverse 5-CGTCATTCTGGTCCTGGAAGTG-3. All were from Sigma-Aldrich. Predesigned primers for RyR1 (catalog no. HS00166991_m1), RyR2 (catalog no. HS00181461_m1), and RyR3 (catalogno. HS00168821_m1) were from Applied Biosystems (ThermoFisher Scientific). CFX Manager� software 3.0 was used fornormalization of the samples (Bio-Rad). �-2 microglobulin andGAPDH gene expression was used for the comparative conc-entration analysis.

Flow Cytometry—Cell viability was assessed by incubatingMks with 5 �l of 7-AAD viability staining solution (BioLegend,Milan, Italy). Analysis of human Mks differentiation was per-formed on day 13 of culture. Mks were identified as the percent-age of CD41� events in the fractions of CD45� cells. Formurine BM cultures, Mks on day 4 of culture were analyzedusing allophycocyanin-conjugated anti-mouse CD45 (clone30F11, Miltenyi Biotech) and FITC-conjugated anti-mouseCD41 (clone MWReg30, BioLegend) antibodies. Mk outputwas calculated as the percentage of side scatterhigh/forwardscatterhigh/CD45�/CD41� cells and normalized to the totalnumber of CD45� cells. For Mk ploidy, cells were fixed over-night in ice-cold 70% ethanol at �20 °C. Samples were incu-bated in PBS with 100 �g/ml of RNase and propidium iodidesolution and stained with FITC-conjugated anti-human CD41antibody (clone HIP8, eBioscience, Milan, Italy). All sampleswere acquired with a Beckman Coulter Navios flow cytometer.Non-stained samples and isotype control antibodies wereused to set the correct analytical gating. Offline data analysiswas performed using the Beckman Coulter Navios softwarepackage.

Released Platelet Quantification—Platelets released in cul-ture were quantified as described previously (69). Platelets pro-duced in vitro were analyzed using the same forward and sidescatter pattern as human and mouse peripheral blood platelets.Platelets were identified as CD41�, and their number was cal-culated using a TruCount bead standard by flow cytometry.

Measurement of Cytosolic Ca2� Concentration—12-mm glasscoverslips were coated overnight at 4 °C with 100 �g/mlfibrinogen. On day 14 of culture, 1 � 105 human Mks wereharvested and allowed to adhere at 37 °C and 5% CO2 for 16 h.Then Mks were loaded with 4 �M Fura-2/AM (MolecularProbes Europe BV, Leiden, The Netherlands) in physiologicalsalt solution (150 mM NaCl, 6 mM KCl, 1.5 mM CaCl2, 1 mM

MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4)) for anadditional 30 min at 37 °C and 5% CO2. After being washed inphysiological salt solution, the coverslip was fixed to the bottomof a Petri dish, and the cells were observed using an uprightepifluorescence Axiolab microscope (Carl Zeiss, Arese, Italy)equipped with a Zeiss X63 Achroplan objective (water immer-sion, 2.0 mm working distance, 0.9 numerical aperture). Mkswere stimulated or not (control) with 10 �M ABA and excitedalternately at 340 and 380 nm, and the emitted light wasdetected at 510 nm. A first neutral density filter (1 or 0.3 opticaldensity) reduced the overall intensity of the excitation light, anda second neutral density filter (0.3 optical density) was coupledto the 380-nm filter to approach the intensity of the 340-nmlight. The excitation filters were mounted on a filter wheel

(Lambda 10, Sutter Instruments, Novato, CA). Custom soft-ware, working in the LINUX environment, was used to drivethe camera (Extended ISIS camera, Photonic Science, Roberts-bridge, UK) and the filter wheel to measure and plot the fluo-rescence from rectangular regions of interest, each enclosingevery Mk present within the analyzed field. In some experi-ments, Mks were incubated with PKA (10 �M, 30 min at 37 °C),cADPR (50 �M, overnight at 37 °C), econazole (10 �M, 1 h at37 °C) and tetracaine (100 �M, 1 h at 37 °C) inhibitors prior toABA stimulation. Analysis of Ca2� signals were performedaccording to methods published previously (62).

Statistics—For all experiments, values are expressed asmean � S.D. Student’s t test was performed for paired observa-tions. ANOVA followed by post hoc Bonferroni t test, was per-formed for grouped observations. p � 0.001, p � 0.01, or p �0.05 were considered statistically significant. All experimentswere independently replicated at least three times unless oth-erwise specified.

Author Contributions—A. M., C. F., C. A. D. B., and P. M. S. con-ducted the experiments and analyzed the results. F. M. analyzed theresults. C. B., E. Z., A. D. F., and A. B. conceived the idea for the pro-ject. A. M. and A. B. wrote the paper.

Acknowledgments—We are indebted to Prof. Vincenzo Sorrentino(University of Siena) for kindly providing the rabbit anti-RyR1 andanti-RyR3 antibodies and for helpful discussions and advice.

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BalduiniFrancesco Moccia, Cesare Balduini, Elena Zocchi, Antonio De Flora and Alessandra Alessandro Malara, Chiara Fresia, Christian Andrea Di Buduo, Paolo Maria Soprano,

MegakaryocytesThe Plant Hormone Abscisic Acid Is a Prosurvival Factor in Human and Murine

doi: 10.1074/jbc.M116.751693 originally published online January 3, 20172017, 292:3239-3251.J. Biol. Chem.

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