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Cell Growth & Differentiation Vol. 10, 447-456, June 1999
© 1999 American Association for Cancer Research

Retinoic Acid Induces Selective Expression of Phosphoinositide 3-Kinase {gamma} in Myelomonocytic U937 Cells

Reinhard Baier, Tzvetanka Bondeva, Reinhard Klinger, Andrey Bondev and Reinhard Wetzker1

Research Unit "Molecular Cell Biology" [R. B., T. B., A. B., R. W.] and Institute for Biochemistry II [R. B., R. K.], Medical Faculty, Friedrich-Schiller-University, D-07747 Jena, Germany


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Retinoic acid provokes growth inhibition and differentiation of the human leukemic cell line U937 to macrophage-like cells. We report that treatment of U937 cells with all-trans retinoic acid (ATRA), but not the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, results in an increased gene expression of the phosphoinositide 3-kinase (PI3K) species PI3K{gamma}. PI3K{gamma} expression was transcriptionally elevated, indicating that the PI3K{gamma} gene may be a direct target for ATRA. In contrast to its effect on PI3K{gamma} expression, ATRA did neither affect the levels of the PI3K species ß and {delta} nor the adapter proteins p85 and p101. Enhanced expression by ATRA of PI3K{gamma} correlated with an increase of PI3K lipid kinase activity. Additionally, ATRA induced significant and lasting stimulation of mitogen-activated protein kinase/Erk2 activity. This effect was sensitive to the PI3K inhibitors wortmannin or LY294002 and, therefore, attributed to the up-regulation of PI3K{gamma} expression. Our findings suggest that sustained MAPK activation via PI3K{gamma} precedes ATRA-dependent differentiation or growth inhibition.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Retinoids are known to induce growth retardation and differentiation in numerous tumors and cellular systems, including human myeloid cell lines (1, 2, 3, 4) . RA2 -dependent differentiation is associated to a changed pattern of gene expression and the appearance of a more mature phenotype.

The effects of ATRA and its derivatives are thought to be mediated by cognate RARs belonging to the superfamily of nuclear steroid/nonsteroid hormone receptors (5, 6, 7, 8) . Although the differentiation processes induced by ATRA are very complex, only few direct target genes of ATRA have been described (9) . By consequence, the molecular mechanisms and proteins mediating the RA effects are only poorly understood thus far. These direct target genes may play important roles in the complex response to RA (e.g., the maintenance of a functional balance between proliferation of premature cells and the appearance of a characteristic differentiated phenotype with reduced proliferation activity).

The human myelomonocytic cell line U937 is a well established cell system to study induced differentiation of leukemic cells. ATRA directly or indirectly causes up-regulated expression of several genes, related to a more monocyte-/macrophage-like phenotype of these cells. Thus, an ATRA-dependent synthesis of type II transforming growth factor ß1 receptor (10) , of IFN-regulating factors 1 and 2 and 2'-5' oligoadenylatesynthetase (11) , CD11b, CD23, and cyclooxygenase (12 , 13) , {gamma}-glutamyltransferase (14) , AML-1 (10) , protein tyrosine phosphatase-U2 (15) , type II transglutaminase (16 , 17) , and of thrombomodulin (18 , 19) has been observed. Only the latter two genes may be considered as direct targets for RA/RAR.

PI3Ks represent a group of intracellular signaling proteins that express in vitro dual intrinsic functions as lipid kinases and protein kinases (20, 21, 22, 23) . An increasing body of experimental evidence characterize PI3Ks as regulators of a variety of cellular functions including proliferation, differentiation, and cell survival. In some cellular systems, PI3K seems to direct more specialized functions like cell migration, membrane ruffling, production of reactive oxygen species and secretory processes (reviewed in Refs. 23 and 24 ).

Several species of PI3Ks have been cloned and characterized. The PI3K species {alpha}, ß, and {delta} occur as heterodimeric proteins consisting of a p110 catalytic subunit and a p85 regulatory adapter subunit, and are regulated by receptors with intrinsic or associated tyrosine kinase activity (23) . Another species, PI3K{gamma}, has been cloned in our laboratory and found to be activated by {alpha} and ß{gamma} subunits of heterotrimeric G proteins proteins (25, 26, 27) . In contrast to the other members of the PI3K family, PI3K{gamma} is not activated by receptor tyrosine kinases and does not bind to the p85 family of adapter proteins. PI3K{gamma} was recently found to form a heterodimer with a protein named p101 (28) and seems to be preferentially expressed in cells of the hematopoietic lineage (29) .

We recently reported that overexpression of PI3K{gamma} in COS-7 cells stimulates the MAPK Erk2 in a Gß{gamma}-dependent fashion (30 , 31) . Activation of Erk2 induced by stimulation of the muscarinic (m2) receptor with carbachol, was abolished by expression of a catalytically inactive mutant of PI3K{gamma}. These data suggest that PI3K{gamma} might constitute an important element of the signal transduction pathway from G-protein-coupled receptors to the MAPK cascade.

The MAPK pathway has been shown to play a pivotal role in the regulation of proliferation, differentiation, and numerous responses of eucaryotic cells (32, 33, 34, 35, 36) . However, little is known about the function of these cascades in hematopoietic systems (37) . Recent studies demonstrate that the kinetics of MAPK activation provides a rationale by which PC-12 cells discriminate between the proliferation response to epidermal growth factor or differentiation induced by nerve growth factor (reviewed in Ref. 38 ). In addition, erythroleukemic K562 cells and monoblastoid U937 cells have been shown to undergo growth inhibition or differentiation processes dependent on sustained activation of the MAPK/Erk pathway by phorbol esters or by overexpression of constitutively active members of the MAPK cascade (39, 40, 41) .

In this study, we addressed the question whether gene expression of a specific PI3K is controlled by inducers of hematopoietic cell differentiation as ATRA or TPA. We demonstrate that a unique PI3K isoform, PI3K{gamma}, represents a direct target gene for ATRA and its cognate RAR. Gene expression of PI3K{gamma} is transcriptionally up-regulated on treatment with ATRA of U937 cells, whereas PI3Kß or {delta} isoforms and the adapter proteins p85 or p101 did not respond. The up-regulation of PI3K{gamma} is associated with a sustained increase of endogenous PI3K activity and elevation of the activity of the MAP kinase Erk2. The data suggest that increased PI3K{gamma} expression mediates the prolonged Erk2 activation and might play an important role in the control of growth and differentiation of U937 cells induced by ATRA.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
ATRA Selectively Induces Expression of PI3K{gamma} in U937 Cells.
Exposure of U937 cells to ATRA or TPA leads to up-regulation of several genes and development of a more mature monocyte-like phenotype of the cells (10 , 11 , 13 , 17 , 19 , 42) . To investigate the role of PI3Ks during differentiation, U937 cells were treated with increasing concentrations of both agents. After 24 h, cells were harvested for parallel investigation of endogenous protein expression or cytoplasmic mRNA levels. Fig. 1ACitation , shows that only PI3K{gamma} responded in a dose-dependent manner to ATRA treatment, whereas the expression of other PI3K isoforms (ß,{delta}) and PKC{alpha},ß were unaffected. Significant induction of PI3K{gamma} protein was already observed at 0.01 µM ATRA (370% of control) and reached a maximum of about 650% of control at 5 µM ATRA. In contrast to ATRA, TPA failed to induce any of the proteins tested, but slightly reduced expression of PI3K{gamma} at higher concentrations. Investigation of PI3K{gamma} cytoplasmic mRNA level induced after a 7-h exposition of U937 cells to ATRA confirmed a clear dose-dependent induction of PI3K{gamma} gene expression (Fig. 1B)Citation . Again, TPA failed to show any effect on PI3K{gamma} mRNA levels.



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Fig. 1. ATRA, but not TPA, induces PI3K{gamma} gene expression. U937 cells were treated with the indicated concentrations of ATRA or TPA. After 24 h, the cells were harvested, and aliquots were used for analysis of expression of protein and RNA. A, Western assay. Blots were probed with antibodies against PI3K{gamma}, PI3Kß, PI3K{delta}, PKC{alpha}, and PKCß. Equivalent loading of protein was assessed by use of an antibody against vinculin. B, Northern analysis. Total RNA was size-fractionated on a denaturing agarose gel, transferred onto nylon membranes, and hybridized with a NH2-terminal 1.2-kb cDNA probe specific for PI3K{gamma}. Equivalence in loading was confirmed by hybridization to a GAPDH (1.4 kb) cDNA probe. Films and autoradiographs are representative for two independent experiments with similar results.

 
To investigate the kinetics of growth inhibition and PI3K{gamma} gene expression, U937 cells were treated with 1 µM ATRA for different time intervals. Chronic exposure for 48 h to ATRA of U937 cells decreased cell proliferation to about 65% of vehicle-treated control (data not shown). Protein and RNA from cell lysates were analyzed by Western and Northern blotting for endogenous levels of PI3K{gamma} protein and mRNA. Western blot analysis revealed a strong up-regulation of PI3K{gamma} protein expression within 6–8 h. After 48 h, PI3K{gamma} reached about a 20-fold level versus initial expression (Fig. 2A)Citation . PI3K{gamma} mRNA investigated in parallel started to increase within 2–3 h after the addition of 1 µM ATRA and reached a maximum after a 48-h incubation (Fig. 2B)Citation . The early response of PI3K{gamma} mRNA suggests that the PI3K{gamma} gene might be a direct target for ATRA-activated RARs. In a similar experiment, TPA did not exhibit any effects on PI3K{gamma} mRNA levels (data not shown).



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Fig. 2. Time-dependent induction of PI3K{gamma} gene expression by ATRA. U937 cells were exposed to ATRA for the time periods indicated. Each experimental group was treated, as described in Fig. 1Citation , for analysis of PI3K{gamma} protein expression (A) and for PI3K{gamma} steady-state mRNA levels (B). The values given in the graphs (top, A and B) represent the means of two independent experiments with similar results. Films and autoradiographs (bottom, A and B) show one representative experiment.

 
Direct Transcriptional Induction by ATRA of PI3K{gamma} Gene Expression.
Regulation of RA-responsive genes may occur by direct binding of activated RARs to the cognate cis-regulating DNA binding sites of target genes or, alternatively, by induction of de novo synthesized transcription factors, which in turn regulate respective target genes. To investigate the molecular mechanism underlying the up-regulation by ATRA of PI3K{gamma} gene expression, U937 cells were preincubated for 30 min with the inhibitor of protein synthesis Chx, followed by treatment with ATRA for different time intervals. Although blocking PI3K{gamma} protein synthesis completely (data not shown), Chx did not abolish PI3K{gamma} mRNA induction by ATRA after 3, 6, and 9 h (Fig. 3A)Citation , but led to a slight accumulation of cytoplasmic PI3K{gamma} mRNA itself. This explains the additive effects of ATRA and Chx within the first 6 h of treatment. These results indicate a direct up-regulation by ATRA/RAR of PI3K{gamma} mRNA expression without essential de novo protein synthesis. Instead, de novo synthesis of PI3K{gamma} mRNA seemed to be required for the observed ATRA-dependent increase of PI3K{gamma} gene expression because Act D, an inhibitor of de novo RNA synthesis, completely silenced induction of PI3K{gamma} mRNA and protein (Fig. 3A)Citation .



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Fig. 3. Induction by ATRA of PI3K{gamma} mRNA is independent of intermediate protein biosynthesis, but requires de novo mRNA synthesis. A, de novo synthesis. U937 cells were pretreated for 30 min with Act D or Chx. Thereafter, the cells were exposed to ATRA or vehicle for 3, 6, and 9 h. The cells were harvested and subsequently analyzed for mRNA levels ({blacksquare}) and protein expression ({square}), as described in Fig. 1Citation . The values represent mean mRNA and protein levels of three independent experiments. B, turn-over of PI3K{gamma} mRNA. U937 cells were pretreated for 24 h with ATRA (1 µM) or vehicle. Thereafter, Act D was added at a given time point (t = 0). Cells were then harvested after the time periods indicated, and remaining fractions (N/No) of PI3K{gamma} mRNA were assessed by Northern analysis. Autoradiographs are representative for two independent experiments.

 
Inhibition of the decay of cytoplasmic mRNA is a common mechanism to induce a net increase of mRNA species. To address this point, we pretreated U937 cells with ATRA for 24 h and added Act D (at t0) thereafter. RNA from cells was prepared after the time intervals indicated, and remaining fractions of PI3K{gamma} mRNA were estimated by Northern analysis. Fig. 3BCitation demonstrates relative high stability of the PI3K{gamma} transcript, showing a half-life time of about 6 h. No posttranscriptional stabilization by ATRA of PI3K{gamma} mRNA could be observed compared with vehicle-treated cell populations. In an attempt to assess PI3K{gamma} protein stability, we used a similar experimental design, but exposed U937 cells to Chx instead of Act D. PI3K{gamma} protein remained stable for at least 9 h after the addition of Chx to ATRA- or vehicle-treated U937 cell populations (data not shown). Time intervals thereafter have not been evaluated because the cells started to suffer from Chx treatment. As for the PI3K{gamma} transcript, no posttranslational effect of ATRA on PI3K{gamma} protein could be detected, again suggesting a direct effect of ATRA on PI3K{gamma} gene expression. Taken together, these results exclude any posttranscriptional or nongenomic effects, but favor a direct and transcriptional mechanism of ATRA-dependent PI3K{gamma} gene expression.

ATRA Increases Cytoplasmic PI3K Lipid Kinase Activity.
Next, we were interested to investigate whether ATRA could influence basal PI3K activity in nonstimulated U937 cells. As shown in Fig. 4, A and BCitation , after 24 h exposure of U937 cells to ATRA PI3K activity in total cell lysates was enhanced by 2.5–3.5-fold, using phosphatidylinositide (4 , 5) bisphosphate [PI (4 , 5) P2] as substrate. Wortmannin (50 nM), a specific PI3K inhibitor, completely abolished production of three phosphorylated lipids, indicating specificity of the assay. In addition, a specific inhibitor for PKC, bisindolylmaleimide 1 (5 µM), did not significantly reduce ATRA-enhanced production of PI3K products, excluding any trans-activation effects by PKC under these experimental conditions. To exclude direct effects of ATRA on PI3K activity, the compound was added directly to vehicle-treated U937 cell lysates. No effect on PI3K activity has been observed. In a parallel experiment TPA did not change PI3K activity and PI3K{gamma} gene expression after a 5-h incubation of U937 cells (Fig. 4, B and D)Citation . Reprobing the membranes with antibodies against PI3Kß, PI3K{delta} (data not shown), and the adapter protein p85 demonstrated unaltered gene expression of these proteins. To investigate a possible effect of ATRA on p101 expression, an antibody was raised against amino acids 398–411 of pig p101 (28) . The specificity of this antibody was verified by Western blotting of purified GST-p101 fusion protein [cDNA of pig p101 was a gift from Dr. Len Stephens (Brabham Institute, Cambridge, United Kingdom)] and the same amount of His-tagged PI3K{gamma} or a GST-PI3K{gamma} fusion protein. The fusion proteins have been expressed and purified from sf9 insect cells after infection with recombinant baculoviruses. The antibody proved to be sensitive to p101 and showed no cross-reactivity to PI3K{gamma} fusion proteins (Fig. 4C)Citation . We used this antibody to investigate whole cell lysates from pretreated U937 cells (with or without ATRA or TPA). A specific and dominant band of about 100 kDa was clearly visible applying 100 µg of protein (Fig. 4D)Citation , whereas p101 was hardly detectable using 50 µg of cell lysates. Although it is possible that the antibody against pig p101 shows reduced sensitivity to human p101, it seems to be more likely that p101 is expressed in U937 cells on a significant lower level as compared with PI3K{gamma} expression. Thus, the enhanced total basal PI3K activity by ATRA seems to be due to the selective expression of the PI3K{gamma} gene and seems to be independent on the adapter protein p101 recently identified.



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Fig. 4. ATRA-induced PI3K{gamma} gene expression increases PI3K lipid kinase activity. U937 cells were treated with ATRA for 24 h or TPA for 5 h. Thereafter, the cells were harvested and lysed by ultrasonication in lysis buffer, as described in "Materials and Methods." A, ATRA enhances total PI3K activity after 24 h in an in vitro kinase assay. Whole cell lysate/sample (10 µg) was applied for in vitro lipid kinase assays, and production of three phosphorylated lipids was investigated by TLC and demonstrated by exposure of the TLC plate to a phosphoimager. B, quantification of lipid production was performed by densitometric scanning of TLC signals. Values represent mean ± SD of three independent in vitro lipid kinase assays. C, immunoblot detection of GST-p101 fusion protein from baculovirus-infected sf-9 insect cells. Purified fusion proteins (as indicated) were subjected to Western analysis using a polyclonal antibody against p101 (amino acids 398–411). Specificity of the antibody was demonstrated by parallel application of His-PI3K{gamma} and GST-PI3K{gamma}. D, expression of PI3K{gamma} and adapter proteins p101 and p85 after ATRA or TPA treatment. Cell lysate/lane (100 µg) was applied to Western assay, as described in Fig. 1Citation .

 
PI3K{gamma} Enhances MAPK Activity in U937 Cells.
We recently showed that PI3K{gamma} activates the MAPKs Erk and JNK in a Gß{gamma}-dependent fashion in the COS-7 cell system for transient expression (30 , 31) . We concluded that PI3K{gamma} could constitute an important signaling element linking G protein-coupled receptors to MAPK pathways. MAPKs are known to be involved in the regulation of numerous complex cellular functions, such as proliferation and differentiation (32, 33, 34, 35, 36) . Therefore, we were interested to investigate whether ATRA influences MAPK activity in U937 cells. Using an in vitro MAPK assay based on Erk2 immunoprecipitates from cell lysates, we looked for a possible relation of ATRA-dependent induction of PI3K{gamma} expression and increase of MAPK activity. Fig. 5ACitation demonstrates that treatment of U937 cells with ATRA for 24 h induced a sustained increase of Erk2 activity in a dose-dependent manner (maximal 5.4-fold). TPA, a known effector of MAPK that acts via a PKC-dependent pathway, induced an about 2.5-fold increase of Erk2 activity. To look for a possible role of PI3K(s) in the control of Erk2 activity, ATRA-pretreated U937 cells were incubated with the PI3K inhibitors Wortmannin and LY294002 for 6 h (Fig. 5)Citation . Wortmannin (in nanomolar concentrations) and LY294002 (in low micromolar concentrations), which are considered to specifically inhibit PI3Ks, abolished the ATRA-induced Erk2 activity in a concentration-dependent manner.



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Fig. 5. ATRA specifically induces dose-dependent increase of MAPK activity in U937 cells. A, dose response. U937 cells were treated with ATRA in the concentrations indicated or vehicle alone for 24 h. A parallel set of cell populations received TPA in the indicated concentrations. Top, MAPK activity in Erk2 immunoprecipitates was assessed by incorporation of [32P] into MBP as substrate. MAPK activity from vehicle-treated populations was set to 100%. Values represent the mean of two independent experiments. Bottom, MBP phosphorylation and Western blot analysis of Erk2 immunoprecipitates are shown. B, PI3K inhibition. U937 cells were pretreated with ATRA (1 µM) for 24 h. Then, Wortmannin or LY294002 (LY) was added, and the cells were further incubated for 6 h. Endogenous MAPK activity was assessed as described above. Top, values represent the means of two MAPK assays from typical experiments. Bottom, MBP phosphorylation and Western blot analysis of Erk2 immunoprecipitates are shown.

 
In addition, we asked whether ATRA induces any autocrine or paracrine stimulation of U937 cells, which could be responsible for the observed induction of Erk2 activity. Treatment of U937 cells with conditioned growth medium derived from U937 cells pretreated with ATRA did not induce Erk2 stimulation above the level observed for U937 cells incubated with conditioned medium derived from vehicle-treated cells (data not shown). This suggests that autocrine feed-back loops, potentially initiated by ATRA treatment of U937 cells, do not feed into the observed increased Erk activity after ATRA treatment.


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The preferred expression of PI3K{gamma} in leukocytes (29) seems indicative for the prominent regulatory function of this PI3K species in these cells. Here, we present evidence for the engagement of PI3K{gamma} in ATRA-mediated early responses of the promyeloic cell line U937, which precede the growth-inhibitory effects of this drug. The rapid increase of PI3K{gamma} gene expression points to the involvement of this signaling protein in the regulation of ATRA-dependent differentiation or growth inhibition. Up-regulation of PI3K{gamma} expression during differentiation occurs on the transcriptional level, independent from de novo biosynthesis, thus suggesting that the PI3K{gamma} gene might be a direct target for ATRA and its RAR.

Remarkably, the ATRA effect on PI3K{gamma} level expresses strong selectivity. TPA, another known inducer of U937 cell differentiation, did not affect PI3K{gamma} gene expression neither on the level of mRNA nor protein, indicating distinct signaling pathways. Furthermore, in our hands, ATRA did not induce any significant alteration in the expression levels of PKC {alpha} and ß species or other PI3K species. Neither the catalytic subunits p110ß and {delta} nor their p85 adapter proteins exposed any remarkable change of their expression levels. Thus, up-regulation of PI3K{gamma} gene expression seems to be involved in the phenotypic alterations produced by ATRA in U937 cells.

Recently, Stephens et al. (28) discovered an adapter protein, p101, which specifically binds to the PI3K{gamma} species of PI3K. This adapter protein has been proposed to regulate the Gß{gamma}-sensitivity of PI3K{gamma}, in analogy to the function of p85 as regulatory subunit of the tyrosine kinase-dependent PI3Ks {alpha},ß, and {delta}. We could not detect any effect of ATRA on the protein level of p101. This observation suggests a regulatory role of PI3K{gamma} during differentiation of U937 cells, which is independent on p101. Consequently, the functional interdependence of PI3K{gamma} and p101 seems to be more relaxed than previously assumed. Recent results on the regulation of PI3K{gamma} lipid kinase activity by either Gß{gamma} (26) or the G protein-coupled fMLP receptor (25) support this view. Both the stimulation of the lipid kinase by Gß{gamma} in vitro and the activatory effect of fMLP on PI3K{gamma} in permeabilized neutrophils take place in the absence of p101.

Several signaling events can be induced by PI3K{gamma}. Thus, the enzyme expresses lipid kinase activity that is sensitive to agonists of G protein-coupled receptors. We show that PI3K activity can be induced by ATRA during differentiation of U937 cells. The synthesis of three phosphorylated phosphoinositides parallels strictly the increase of PI3K{gamma} expression induced by ATRA. These data point to PI3K{gamma} as the main source of elevated basal PI3K activity and suggest a regulatory function of the three phosphorylated lipids during the development of the differentiated phenotype of U937 cells.

Recently, we reported that the catalytic PI3K{gamma} subunit mediates {gamma}-dependent activation of the MAPK Erk2 and concluded that PI3K{gamma} might participate in the signaling from G protein-coupled receptors to the MAPK pathway (30) . This prompted us to investigate effects of ATRA-dependent up-regulation of PI3K{gamma} on Erk2 protein kinase activity. The present data reveal a significant and sustained increase of Erk2 activity after treatment with ATRA. This effect was not mediated by an up-regulation of the Erk2 protein expression level, but rather indicated an activation of the enzyme. The ATRA-mediated response on Erk2 activity seemed to be specific because it was dependent from the ATRA concentrations applied. In addition, ATRA-induced stimulation of Erk2 seemed to be dependent on PI3K activity because it was sensitive to the PI3K inhibitors Wortmannin and LY294002. Indirect stimulation of Erk activity caused by autocrine or paracrine loops, which might be induced by ATRA, could be excluded. We did not detect any additional stimulatory effect on Erk2 activity in U937 cells after incubation with conditioned medium derived from cells pretreated with ATRA. Taken together, our data suggest an essential cooperation of PI3K{gamma} and enzymes of the MAPK cascade during the differentiation process or inhibition of growth induced by ATRA.

Up-regulation by ATRA of PI3K{gamma} gene expression is sufficient to increase MAPK activity for prolonged periods of at least up to 40 h. The significance of this observation is corroborated by several recent studies, demonstrating a direct relationship of a maintained activation of the MAPK pathway and the induction of differentiation and growth inhibition in PC-12 cells (38) and, more specifically, in myeloid K562 and U937 cells or in CTLL-20 T-cells (39, 40, 41 , 43) . Thus, in leukocytes, expression of PI3K{gamma} and subsequent prolonged stimulation of Erk2 activity might be generally involved in the control of differentiation or growth inhibition induced by ATRA.

Another feature of PI3K-dependent signaling is the recently described activation of an antiapoptotic pathway via PI3K-dependent kinase-1-mediated activation of AKT/PKB (Ref. 44 and references therein). Apoptosis frequently occurs at late stages after terminal differentiation of WBCs (45 , 46) . We recently demonstrated that PI3K{gamma} is able to activate AKT kinase in COS-7 cells, transiently overexpressing PI3K{gamma} and AKT (20) . Therefore, it is possible that transcriptionally induced endogenous PI3K{gamma} provides a signal to activate a differentiation stage-dependent antiapoptotic program in U937 cells that enables the cells to promote through the differentiation process initiated by ATRA. Interestingly, ATRA has recently been shown to induce resistance to idarubicin-induced apoptosis in U937, HL-60, and KG-1 cells (47) . Such an antiapoptotic program, mediated at least in part via induced PI3K{gamma}, could allow the cells to execute their specialized functions as differentiated cells before undergoing apoptosis. This hypothesis for an antiapoptotic effect and a putative dual physiological role of PI3K{gamma} in U937 cells is currently under detailed investigation in our laboratory.

The data presented in this study support the idea of a pleiotropic role of PI3K{gamma} in the regulation of cell functions. Together, with our previous investigations on an involvement of PI3K{gamma} in the control of fMLP receptor-dependent events in mature neutrophils (25) , this study suggests an additional, rather G protein-coupled receptor-independent potential of PI3K{gamma} in directing the maturation of hematopoietic cells.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture.
U937 cells were grown and subcultured every 2 days in RPMI 1640 supplemented with 10% FCS at 37°C, 5% CO2 in humidified atmosphere. After seeding the cells in fresh growth medium at an initial density of 2–5 x 105 cells/ml, ATRA (Sigma Chemical Co.) or TPA (Sigma Chemical Co.) were added as stock solutions in ethanol or DMSO, respectively, to achieve the indicated final concentrations of ATRA and TPA and a constant vehicle concentration of 0.01%. Control cells always received an equal amount of ethanol or DMSO, respectively. The stock solution of Chx (10 mg/ml; Sigma Chemical Co.) was prepared in ethanol to achieve a final concentration of 10 µg/ml in the cell culture medium. Act D (Sigma Chemical Co.) was dissolved in DMSO to obtain a 10 mg/ml stock solution (final concentration, 4–8 µg/ml medium). Wortmannin (Sigma Chemical Co.) was dissolved in DMSO as a 1 mM stock solution, and LY294002 (Calbiochem) stock solution (20 mM) was prepared in ethanol. Conditioned medium was prepared by treatment of U937 cells with ATRA (1 µM) or vehicle (0.01% ethanol) for 24 h. Then, cells were washed twice and incubated with fresh medium without ATRA for 16 h. In some experiments, conditioned media were saved and subsequently applied to parental, untreated U937 cells. Then, the cells were further incubated for 7 h and harvested thereafter.

Cell Proliferation Assay.
Cell numbers from aliquots of time course experiments were estimated after 48 h of continuous treatment with ATRA (1 µM) or vehicle (0.01% ethanol) alone by use of a cell counter (Casy1; Schärfe System, Reutlingen, Germany).

Western Blot and Antibodies.
Aliquots of cells from all experiments were saved and stored at -80°C until further analysis. Whole cell lysates were prepared by suspending thawed cells in lysis buffer (20 mM Hepes, 150 mM NaCl, 3.0 mM MgCl2, 2 mM EGTA, 0.2 mM EDTA, and 1% (v/v) Triton-X100) supplemented with aprotinin (2.8 mg/ml), leupeptin (20 µg/ml), PMSF (0.1 mM), pepstatin A (15 µg/ml), ß-glycerophosphate (5 mM), and Na-orthovanadate (1 mM), followed by incubation for 10 min on ice. Cell lysates were cleared by centrifugation for 15 min (17000 rpm at 4°C), and supernatants were saved for protein analysis. Concentration of whole cell protein was assessed by modified Bradford method (Sigma Chemical Co.). For Western blot analysis, 50 µg of protein/sample were denatured in 6 x SDS-PAGE buffer (pH6.8; final concentration, 62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM DTT, and 0.1% bromphenol blue), boiled for 5 min at 94°C, and size-fractionated by SDS-PAGE on a 7.5% gel. Transfer of proteins onto PVDF membranes (Schleicher and Schuell) was accomplished by semi-dry blotting (semi-dry transfer cell; Bio-Rad). The following antibodies have been used in this study: a mouse monoclonal antibody against PI3K{gamma} (NH2-terminal and mab PI3K{gamma}), a rabbit polyclonal antibody to PI3Kß (pab PI3Kß and NH2- and COOH-terminal; Santa Cruz Biotechnology, Santa Cruz, CA), a rabbit polyclonal antibody to PI3K{delta} (pab PI3K{delta}; kindly provided by Bart Vanhaesebroeck; Ref. 48 ), monoclonal antibodies to p85 adapter subunit of PI3K{alpha},ß,{delta} (mab p85; Santa Cruz Biotechnology), monoclonal antibodies to PKC{alpha} and ß (Santa Cruz Biotechnology), and a monoclonal antibody against pan-Erk/MAPKs (Transduction Laboratories). A rabbit polyclonal antibody was raised by Eurogenetec (Brussels, Belgium) against amino acids 398–411 of pig p101 (YERPRRPGGHERRG; Ref. 28 ), according to their proposal derived from sequence analysis. A monoclonal antivinculin antibody (Serotec) served as loading control. Visualization of immune complexes was performed using secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology) and ECL detection kit (Amersham). Membranes were exposed to Kodak BIOMAX-MR5 imaging films, and resulting signals were quantified as ratio of target protein:vinculin using OfotoD1–2.0 and NIH-Image 1.57 software.

Construction and Expression of PI3K{gamma} and p101 Fusion Proteins.
Construction, expression, and purification of recombinant baculoviruses for expression of human GST-PI3K{gamma} and pig GST-p101 have been described previously (22 , 26, 27, 28) . cDNA encoding pig p101 was kindly provided by Len Stephens (Brabham Institute, Cambridge, United Kingdom). His-tagged PI3K{gamma} was prepared by cloning wild-type PI3K{gamma} cDNA in the open reading frame into BaculoGold/pAcHLT-A baculovirus expression vector (PharMingen Europe, Germany). Purification of epitope-tagged His-PI3K{gamma} was performed on nickel-NTA agarose column (Ni-NTA; Qiagen, Hilden, Germany). Bound His-PI3K{gamma} was eluted with 100 mM imidazol, and eluted protein was further purified by Q-Sepharose (Pharmacia, Uppsala, Sweden). His-PI3K{gamma} was finally eluted using a gradient of 0–200 mM NaCl. Purified proteins were verified on 7.5% SDS-PAGE after Coomassie staining or Western blotting, respectively.

RNA Purification and Northern Analysis.
Total cellular RNA was isolated according to the single-step procedure originally described by Chomczynski and Sacchi (49) . In brief, before harvesting, cells were rinsed with PBS, and up to 106 cells were lysed in 100 µl of 4 M guanidine isothiocanate, 25 mM sodium citrate (pH 7.0), 0.5% sodium lauryl sarcosine, and 0.15 M 2-mercaptoethanol. Genomic DNA was sheared by 15 passages through a 20-G needle, and total cytoplasmic RNA was prepared from cell lysates by the addition of 2 M sodium acetate (pH 4.0; final concentration, 0.2 M), 1 volume of water-saturated acidic phenol. and one-fifth volume of chloroform/isoamylalcohol (24:1). Samples were vortexed for 1 min and incubated on ice for 20 min to separate organic and aqueous phases. After spinning for 15 min at 12,000 rpm, the RNA-containing aqueous upper phase was carefully removed. RNA was precipitated by the addition of 0.7 volumes of 2-propanol for several hours at 4°C, centrifuged as above, and pellets were washed once with 70% ethanol. After the last centrifugation, RNA was air-dried and dissolved in DEPC-treated H2O. Northern blot hybridization was performed by size-fractionation of 25 µg of total cytoplasmic RNA on denaturing 0.8% agarose/formaldehyde gels, followed by capillary transfer of RNA onto nylon membranes (Hybond N; Amersham) with 10 x SSC [1.5 M NaCl, 0.15 M Na-citrate (pH 7.0)]. Immobilized RNA was fixed by UV cross-linking. Hybridization to cDNA probes was performed under stringent conditions in the presence of 50% formamide at 42°C for 18 h. Thereafter, membranes were washed at 62°C for 30 min: once with 2 x SSPE/0.1% SDS to remove unbound radioactivity, once with 0.5 x SSPE/0.1 x SDS, and a final washing step with 0.1 x SSPE/0.1% SDS [20 x SSPE adjusted to pH 7.4 contains 3.6 M NaCl, 200 mM NaH2PO4·H20, and 20 mM EDTA (pH 8.0)]. cDNA probes were labeled using a random-prime labeling kit (prime-it II; Stratagene) and [{alpha}-32P]dCTP (3000 Ci/mmol; Amersham). The following cDNA probes were used in Northern blot hybridizations: a NcoI/StuI, 1228-bp NH2-terminal fragment of human PI3K{gamma} and a 1400-bp PstI fragment of mouse GAPDH, as housekeeping gene used for normalizing equivalence of loaded RNA amounts. Membranes were exposed to Kodak BIOMAX-MR5 imaging films with two intensifying screens (Cronex Plus) at -80°C. The mRNA signals were quantified as a ratio of PI3K{gamma}/GAPDH after densitometric scanning of the resulting autoradiographs using OfotoD1–2.0 and NIH-Image 1.57 software. Scanning of the images after different exposure times (1–4 days) was performed to ensure linearity in film sensitivity.

In Vitro PI3K Lipid Kinase Assay.
Total PI3K activity from whole cell lysates was assayed, as described previously (25) . In brief, cell lysates were prepared in lysis buffer (without Tritron X-100) by two freeze-thaw cycles, followed by 10 strokes of ultrasonication (10 s, duty cycle 75%, output control 6; Branson Sonifier 250) on ice. Lysates were cleared from cell debris by centrifugation, and total protein concentration was assessed from the supernatant. For in vitro kinase assays, 10 µg of soluble whole cell protein was mixed with 125 µM sonicated lipid substrates [phosphatidylinositol 4-phosphate, PI(4)P, or phosphatidylinositol 4,5-bisphosphate, PI (4 , 5) P2; Sigma Chemical Co.] dissolved in H20 and 3 x PI3K buffer [20 mM Tris-HCl (pH 7.4), 4 mM MgCl2, and 100 mM NaCl]. Samples were preincubated for 10 min on ice and subsequently exposed to 40 µM ATP/0.2 µCi [{gamma}-32P]ATP (3000 Ci/mmol; Amersham) for 15 min at 37°C. After stopping the reaction with 2.5 volumes of 1 M HCl, lipids were extracted with chloroform:methanol (1:1, vol/vol), followed by two washes with HCl (1 M). The yield of PI3K product PI (3, 4, 5)P3 was quantified after TLC on preactivated (1.2% K2-oxalat in 40% methanol) silica gel 60 plates (Merck), using 1-propanol/2 M acetic acid (13:7, vol./vol.) as eluent. TLC plates were exposed to a phosphoimager. Quantification of signals was performed by the aid of Molecular Analyst software for phosphoimager.

MAPK Assay.
ATRA-treated U937 cells were lysed, as described above, in the presence of 1 mM DTT and 1% NP40. Endogenous MAPK/Erk2 was precipitated using Erk2-coupled agarose beads (Santa Cruz Biotechnology). Immuncomplexes were washed three times with PBS, 1% NP40, and 2 mM orthovanadate; once with 100 mM Tris-HCl (pH 7.5) and 0.5 M LiCl; and once with kinase reaction buffer [12.5 mM MOPS (pH 7.5), 12.5 mM ß-glycerophosphate, 7.5 mM MgCl2, 0.5 mM EGTA, 0.5 mM NaF, and 0.5 mM orthovanadate]. In vitro MAPK assay was performed in the presence of 3.3 µM DTT, 1 µCi [{gamma}-32P]ATP, and 20 µM cold ATP using MBP as substrate (final concentration, 1.5 µg/µl). After 20 min at 30°C, the reaction was terminated by the addition of 6 x SDS-PAGE sample buffer. Samples were boiled at 95°C for 5 min and analyzed by SDS-PAGE (12%). The gel was cut into two parts, and the upper part was transferred onto polyvinylidene difluoride membranes and subjected to immunoblotting using anti-pan-Erk mab (Transduction Laboratories) to control for Erk2-precipitation. The lower part was dried, and phosphorylation of MBP was quantitated by exposure to a phosphoimager, as described above.


    Footnotes
 
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.

1 To whom requests for reprints should be addressed, at Research Unit "Molecular Cell Biology," Medical Faculty, Friedrich-Schiller-University Drackendorfer Str. 1, D-07747 Jena, Germany; Phone: 49-3641-304-460; Fax: 49-3641-304-462; E-mail: i5rewe{at}rz.uni-jena.de Back

2 The abbreviations used are: RA, retinoic acid; ATRA, all-trans RA; RAR, RA receptor; PI3K, phosphoinositide 3-kinase; MAPK, mitogen-activated protein kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate; Chx, cycloheximide; PKC, protein kinase C; Act D, actinomycin D; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MBP, myelin basic protein. Back

Received for publication 11/20/98. Revision received 2/ 1/99. Accepted for publication 4/14/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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