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INFLAMMATION/IMMUNITY/MEDIATORS

PGE₂ inhibits apoptosis in human adenocarcinoma Caco-2 cell line through Ras-PI3K association and cAMP-dependent kinase A activation

¹Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano, Università degli Studi di Napoli Federico II; and ²Centro di Microcitemia A. Mastrobuoni, Azienda Ospedaliera Cardarelli, Naples, Italy

Submitted 22 December 2006 ; accepted in final form 13 July 2007

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION AND CONCLUSIONS GRANTS REFERENCES

 
PGE₂ plays a critical role in colorectal carcinogenesis. We have previously shown that COX-2 expression and PGE₂ synthesis are mediated by IGF-II/IGF-I receptor signaling in the Caco-2 cell line and that the pathway of phosphatidylinositol 3-kinase (PI3K)/Akt protects the cell from apoptosis. In the present study, we demonstrate that PGE₂ has the ability to increase Ras and PI3K association and decrease the level of apoptosis in the same experimental system. The effect of PGE₂ on PI3K/Ras association is dependent on the activation of EP4 receptor, the increase of cAMP levels, and the activation of PKA. In fact, treatment of cells with the PKA inhibitor H89 decreases the association of Ras and PI3K and Ras-associated PI3K activity. PKA inhibitor H89 is able to decrease threonine phosphorylation of Akt and to increase serine phosphorylation of Akt by p38 MAPK and counteracts the cytoprotective effect induced by PGE₂. In addition, PGE₂ is able to activate p38 MAPK and the inhibition of p38 MAPK, with SB203580 specific inhibitor or with dominant negative MKK6 kinase, is able to revert the apoptotic effect of H89 and serine phosphorylation of Akt. The effect of PGE₂ on Caco-2 cell survival through PKA activation is mediated and regulated by the balance of threonine/serine phosphorylation of Akt by p38 kinase and PI3K. In conclusion, our data elucidate a novel mechanism for regulation of colon cancer cell survival and provide evidences for new combinatory treatments of colon cancer.

colon cancer; prostaglandin E₂; Akt; protein kinase A; phosphatidylinositol 3-kinase

In conclusion, PKA is able to stabilize the Ras/PI3K complex and to balance serine/threonine phosphorylation of Akt by modulating the PI3K p38 MAPK activity.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION AND CONCLUSIONS GRANTS REFERENCES

 
Cell growth and culture. Caco-2 cells were routinely grown in 100-mm plastic dishes at 37°C in a humidified atmosphere of 5% CO₂ in air in DMEM supplemented with 10% fetal calf serum, glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml) and buffered with HEPES (20 mM). Caco-2 cells were seeded at 5 x 10⁴ cells/ml and were routinely subcultured when 80% confluent. The culture medium was changed every other day. Cells were serum withdrawn 3 days after plating at the beginning of the proliferative phase of the growth. Drugs were added for 24 h after 48 h of the serum withdrawal. H89 (Calbiochem, Darmstadt, Germany) was dissolved in DMSO and prepared as an 8 mM stock and added at the final concentration of 10 µM. PGE₂ (Sigma) was dissolved in ethanol and prepared as a 1 mM stock solution and added at the final concentration of 2.5 µM. LY294002 (Calbiochem, Darmstadt, Germany) was dissolved in DMSO and prepared as a 16.2 mM stock solution and added at the final concentration of 50 µM. PD98059 (Calbiochem, Darmstadt, Germany) was dissolved in DMSO and prepared as an 8 mM stock solution and added at the final concentration of 40 µM. SB203580 p38 inhibitor (Calbiochem, Darmstadt, Germany) was dissolved in DMSO and added at the final concentration of 5 µM. ONO-AE1-329 (an EP4 agonist) was gift kindly provided by Ono Pharmaceutical (Osaka, Japan) and was dissolved in DMSO and prepared as a 10 mM stock solution and added at the final concentration of 1 µM.

Transfection. Cells were seeded at 5 x 10⁴ cells/ml and after 24 h transfected by the Lipofectamine Plus procedure as indicated by the manufacturer (GIBCO-BRL, Life Technologies, Milan, Italy). Stable transfectants were isolated as single clones in the presence of 0.4 mg/ml G418 during the course of all the experiments. The plasmid with dominant negative of MKK6K was kindly provided by Dr. Mario Chiariello, Istituto di Endocrinologia ed Oncologia Sperimentale G. Salvatore, Naples, Italy. The efficiency of transfection was tested by Western Blot with anti-GST antibody.

Apoptosis detection. To define the level of apoptosis, Caco-2 cells were trypsinized, pelleted, fixed, and propidium iodide (PI) stained. PI staining fluorescence of individual cells was analyzed by using a FACSCalibur flow cytometer apparatus (Becton-Dickinson, Mountain View, CA) and the MODFIT analysis software. For each sample, at least 20,000 events were stored. Each experiment was repeated at least three times.

Western blot analysis. Cells were washed in cold PBS and lysed for 10 min at 4°C with 1 ml of lysis buffer (50 mM Tris, pH 7.4, 0.5% NP40, 0.01% SDS) containing complete protease inhibitor cocktail (Roche, Mannheim, Germany). Lysates from adherent cells were obtained by scraping and centrifuging cells at 12,000 g for 15 min at 4°C. The supernatants were collected and protein concentration in cell lysates was determined by Bio-Rad Protein Assay (Bio-Rad, Richmond, CA) and 70 µg of total protein from each sample was analyzed. Proteins were separated by a 12% SDS-PAGE and transferred on nitrocellulose membrane (Hybond-ECL Nitrocellulose, Amersham, Rainham, UK). The membrane was blocked with TBS-Tween 0.1% containing 5% nonfat dry milk for 1 h at room temperature. After the blocking, the membranes were incubated with the relative primary antibody overnight at 4°C. Mouse monoclonal anti-p-ERK1/2 and ERK2 and anti-p38 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were diluted 1:1,000 in PBS-Tween 0.1%, BSA 5%; anti-phosphoserine 473 Akt (Cell Signaling), anti-phosphothreonine 308 (Upstate, Lake Placid, NY), and anti-Akt (Cell Signaling) rabbit polyclonal antibodies were diluted 1:1,000 in 1 x PBS in the presence of 0.1% Tween and 5% BSA. After the incubation, the membranes were washed six times with PBS-Tween 0.1% and were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Bio-Rad, Richmond, CA) diluted 1:2,000 in PBS, 0.2% Tween, the membranes were washed, and protein bands were detected by an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Uppsala, Sweden). For quantization of immunoblots, relative intensities of bands were quantified by densitometry with a desk scanner (Pharmacia Discovery System) and RFLPrint software (PDI).

Immunoprecipitation. Fresh cells lysates (300 µg) were incubated overnight with 10 µl of pan-Ras antibody (Oncogene, Boston, MA) or with 5 µl of p38 antibody (Santa Cruz Biotechnology) at 4°C. After the incubation, protein A-G Sepharose was added for 1 h. Pellets were washed in cold lysis buffer 5–6 times and resuspended in Laemmli 2x for Western blot analysis.

PI3K assay. Fresh cells lysates (300 µg) were incubated overnight with 10 µl of pan-Ras antibody (Oncogene) or 3 µl of PI3K p85 antibody (Upstate Biotechnology) at 4°C. After the incubation, protein A-G Sepharose was added for 1 h. Pellets were washed in cold lysis buffer, then in 100 mM Tris·HCl (pH 7.4) supplemented with 500 mM LiCl, 1 mM EDTA, and 0.2 mM NaVO₄. Pellets were further resuspended in 30 mM HEPES (pH 7.5) and 6.25 mM MgCl₂ and 125 µM cold ATP; the kinase reaction was initiated by addiction of 2 µg/µl phosphatidylinositol (Sigma, Milan, Italy) and 10 µCi ³²P-ATP (3,000 Ci/mmol) and performed for 15 min at 37°; the reaction was stopped by the addition of 5 M HCl and 0.5 M EDTA and methanol-chloroform (1:1). After mixing vigorously and centrifuging to separate the phases, the organic layer was collected and separated by thin-layer chromatography. [³²P]phosphoinositides were visualized by autoradiography. The autoradiographs were quantified by a Phosphorimager using the program ImageQuant (Amersham Pharmacia Biotech).

Statistical analysis. Statistical comparisons were performed by the Mann-Whitney U-test. A P value < 0.05 was considered a significant difference.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION AND CONCLUSIONS GRANTS REFERENCES

 
PGE₂ through CAMP/PKA inhibits apoptosis in Caco-2 cells and stabilizes the association of Ras with PI3K. We have previously demonstrated that IGF-II peptides were secreted at high levels in the medium collected from actively proliferating Caco-2 cells after 48 h of serum deprivation and that IGF-II through the IGF-I receptor pathway upregulated COX-2 mRNA expression and PGE₂ synthesis (5, 37). Our data, also, demonstrated that IGF-II/IGF-Ir reduced apoptosis through the activation of PI3K pathway and the inhibition of this pathway induced apoptosis and decreased PGE₂ levels (5, 22).

To understand how PGE₂ can cooperate to induce proliferation and inhibit apoptosis in Caco-2 cells, we analyzed the level of PGE₂ produced in Caco-2 cells after 72 or 168 h of serum deprivation. The intracellular PGE₂ concentrations were 326 and 125 pg/mg after 72 and 168 h of serum deprivation, respectively, whereas no detectable amounts of PGE₂ were found in the medium at the same time points (data not shown). Also, the addition of exogenous PGE₂ was able to induce by twofold the proliferation of Caco-2 cells that have been serum deprived for 72 and 168 h (data not shown). To demonstrate that exogenous PGE₂ was able to inhibit apoptosis induced by serum deprivation and that the effect was mediated by the PGE₂ cooperation with the autocrine loop of IGF-II on the activation of PI3K and MAPK, we analyzed the effect of exogenous PGE₂ addition on apoptosis and MAPK and PI3K kinase pathways activation in Caco-2 cells after 72 h of serum deprivation. As shown in Fig. 1A, the addition of exogenous PGE₂ at 2.5 µM decreased by 50% the levels of apoptosis, whereas the treatment with PI3K inhibitor (LY) or with competitive inhibitor of EP4 PG receptor (ONO-AEI-329) was able to revert the cytoprotective effect of PGE₂. The treatment with MAPK inhibitor was not able to revert the effect of PGE₂. In these experimental conditions, PGE₂ increased twofold the levels of Ser-p-Akt, whereas this increase was reverted by PI3K inhibitor (LY) or EP4 inhibitor (ONO-AEI-329) (Fig. 1B). To demonstrate that the effect of PGE₂ was mediated by receptors coupling G(s) adenylate cyclase, we analyzed the effects on serine Akt phosphorylation following treatment with 10 µM forskolin or forskolin plus H89. We obtained the same effect of PGE₂, thus suggesting that the PGE₂ effect might be mediated by cAMP-dependent PKA activation (data not shown). Concordantly, exogenously added PGE₂ increased 2.5-fold the levels of pERK-1/2, whereas the effect was abolished by the addition of EP4 inhibitor and inhibited by 50% in the presence of PI3K or MAPK inhibitors (Fig. 1B). Since it is known that the effects of PGE₂ through EP4 receptor were mediated by cAMP-dependent PKA, we decided to analyze the effect of PKA on PI3K and Akt activation. To analyze the total PI3K activity and Ras-associated PI3K activity, we immunoprecipitated the lysates of cells treated with PGE₂ or PGE₂ plus PKA inhibitor (H89) with p85 and pan-Ras antibody. As shown in Fig. 2A, exogenous PGE₂ slightly increased the association of p85 with Ras, whereas basal and PGE₂-induced levels of p85 regulatory subunit of PI3K associated with Ras decreased by fourfold by treatment with PKA inhibitor H89 (Fig. 2A). On the other hand, the total PI3K activity increased following treatment with PGE₂ alone or in combination with H89 PKA inhibitor, whereas PI3K activity associated with Ras increased following PGE₂ treatment and decreased in the presence of H89 (Fig. 2B). Moreover, the ratio of Ras-associated PI3K activity vs. total PI3K activity Ras/PI3K increased by twofold in the cells treated with PGE₂ while decreased by twofold in cells treated with H89 and with PGE₂ plus H89 (Fig. 2B, bottom histogram).

View larger version (26K): [in this window] [in a new window]   Fig. 1. Effect of PGE2 treatment on serum-deprived-induced apoptosis and Akt and ERK1/2 phosphorylation in Caco-2 cells. A: Caco-2 cells after 48 h of serum deprivation were treated for 24 h with 2.5 µM PGE2 (PGE), alone or in combination with 40 µM PD98059 (PD), 50 µM LY294002 (LY), or 1 µM EP4 PG receptor inhibitor AE1–329 (ONO). Ctrl, control. Apoptosis was calculated as the percentage of cells showing a subdiploid DNA peak, as described in MATERIALS AND METHODS. Data are expressed as means ± SD. B: Western immunoblot analysis using anti-(Ser 473) phosphorylated Akt and phosphorylated ERK1/2 antibodies was performed on protein lysates from cells cultured in the absence of serum for 48 h and incubated for 24 h with the above-indicated drugs. The immunoblots were stripped and reblotted with antibodies against total Akt or ERK-2 protein. Representative autoradiographs are shown. Histograms represent the mean densitometric analysis of 3 separate experiments ± SD.

View larger version (33K): [in this window] [in a new window]   Fig. 2. Effect of PGE2 treatment on Ras-associated phosphatidylinositol 3-kinase (PI3K) activity. A: Caco-2 cells were cultured in the absence of serum for 48 h and then treated with 2.5 µM PGE2, alone or in combination with H89 at 10 µM, for an additional 24 h. The cells extracts (200 µg) were immunoprecipitated (IP) with a pan-Ras monoclonal antibody and analyzed through Western blot (WB) with anti-p85 and pan-Ras antibodies. Histograms represent the mean densitometric analysis of 3 experiments ± SD. B: effect of 2.5 µM PGE2 treatment, alone or in combination with H89 at 10 µM, on PI3K activity associated with pan-Ras immunoprecipitate in Caco-2 cells. The cell extracts (300 µg) were immunoprecipitated with p85 or pan-Ras antibody and the precipitate was assayed for the presence of a PI3K activity by using phosphatidylinositol as a substrate as described in MATERIALS AND METHODS. PtdInsP, phosphatidylinositol 3 phosphate; ORI, origin. Histograms represent the mean densitometric analysis of 32P PtdIns autoradiographs of 3 separate experiments.

View larger version (37K): [in this window] [in a new window]   Fig. 3. Effect of PGE2 treatments, alone and with H89, on Akt and ERK1/2 phosphorylation in Caco-2 cells. Caco-2 cells were cultured in the absence of serum for 48 h and then treated with 2.5 µM PGE2, alone or in combination with H89 at 10 µM for 24 h. Western immunoblot analysis using anti-(Ser 473) phosphorylated Akt and pERK1/2 antibody was performed on protein lysates from cells cultured in the absence of serum for 48 h and incubated for 24 h with the above-indicated drugs. The immunoblots were stripped and reblotted with antibodies against total Akt and ERK2 protein. Representative autoradiographs are shown. Histograms represent the mean densitometric analysis of 3 separate experiments ± SD.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Effect of PGE2 treatments, alone and with H89 on apoptosis of Caco-2 cells. Apoptosis analysis was performed on cells cultured in the absence of serum for 48 h, following treatment with PGE2, alone and with H89. Apoptosis was calculated as the percentage of cells showing a subdiploid DNA peak, as described in MATERIALS AND METHODS. Data are expressed as means ± SD.

View larger version (36K): [in this window] [in a new window]   Fig. 5. Effect of PGE2 treatments, alone and with H89 on glycogen synthase kinase (GSK) phosphorylation. Caco-2 cells were cultured in the absence of serum for 48 h and then treated with 2.5 µM PGE2, alone or in combination with H89 at 10 µM for 24 h. Western immunoblot analysis using anti-phosphorylated GSK antibody was performed on protein lysates from cells cultured in the absence of serum for 48 h and incubated for 24 h with the above-indicated drugs. The immunoblots were stripped and reblotted with antibodies against total GSK protein. Representative autoradiographs are shown. Histograms represent the densitometric analysis of the representative autoradiographs.

View larger version (40K): [in this window] [in a new window]   Fig. 6. Effect of PGE2 treatments, alone and with H89, on threonine and serine phosphorylation of Akt. Caco-2 cells were cultured in the absence of serum for 48 h and then treated with 2.5 µM PGE2, alone or in combination with H89, at 10 µM for 24 h. Western immunoblot analysis using anti-threonine and serine Akt-phosphorylated antibody was performed on protein lysates from cells cultured in the absence of serum for 48 h and incubated for 24 h with the above-indicated drugs. The immunoblots were stripped and reblotted with antibodies against total Akt protein. Representative autoradiographs are shown. Histograms represent the densitometric analysis of the representative autoradiographs.

View larger version (26K): [in this window] [in a new window]   Fig. 7. Effect of PGE2 treatments, alone and with H89 or p38 MAPK inhibitor (SB203580), on serine phosphorylation of Akt and apoptosis. Caco-2 cells were cultured in the absence of serum for 48 h and then treated with 2.5 µM PGE2, alone or in combination with H89 at 10 µM or p38 MAPK inhibitor at 5 µM for 24 h. A: Western immunoblot analysis using anti-serine Akt phosphorylated antibody was performed on protein lysates from cells cultured in the absence of serum for 48 h and incubated for 24 h with the above-indicated drugs. The immunoblots were stripped and reblotted with antibodies against total Akt protein. Representative autoradiographs are shown. Histograms represent the mean densitometric analysis of 3 separate experiments ± SD. B: level of apoptosis by FACS analysis.

View larger version (33K): [in this window] [in a new window]   Fig. 8. Effect of 30-min and 1-h PGE2 treatment, alone and with H89 or p38 MAPK inhibitor, on Akt serine phosphorylation. Caco-2 cells were cultured in the absence of serum for 48 h and then treated with 2.5 µM PGE2, alone or in combination with H89 at 10 µM or p38 MAPK inhibitor at 5 µM for 30 min and 1 h. Western immunoblot analysis using anti-serine Akt-phosphorylated antibody was performed on protein lysates from cells cultured in the absence of serum for 48 h and incubated for 30 min and 1 h with the above-indicated drugs. The immunoblots were stripped and reblotted with antibodies against total Akt protein. Representative autoradiographs are shown. Histograms represent the densitometric analysis of the autoradiographs.

View larger version (31K): [in this window] [in a new window]   Fig. 9. Effect of PGE2 treatment, alone and with H89, on p38 activation (A) and serine Akt phosphorylation (B). A: Caco-2 cells were cultured in the absence of serum for 48 h and then treated with 2.5 µM PGE2 for 24 h. Western immunoblot analysis using anti-phosphorylated p38 (p-p38) antibody was performed on protein lysates from cells cultured in the absence of serum for 48 h and treated with PGE2 for 24 h. The immunoblots were stripped and reblotted with antibodies against total p38 protein. B: Caco-2 cells were transfected with purified recombinant full-length human MKK6 kinase mutated in S207/T211. Stable clones were cultured in the absence of serum for 48 h and then treated with 2.5 µM PGE2, alone or in combination with H89 at 10 µM for 24 h. Western immunoblot analysis using anti-serine Akt-phosphorylated antibody was performed on protein lysates from cells cultured in the absence of serum for 48 h and incubated for 24 h with the above-indicated drugs. The immunoblots were stripped and reblotted with antibodies against total Akt protein. Representative autoradiographs are shown in both panels. Histograms represent the densitometric analysis of the autoradiographs.

View larger version (35K): [in this window] [in a new window]   Fig. 10. Effect of PGE2 treatments, alone and with H89, on serine phosphorylation of Akt associated to p38 MAPK. Effect of 2.5 µM PGE2 treatment, alone or in combination with H89 at 10 µM, on serine phosphorylation of Akt associated with p38 MAPK in Caco-2 cells. The cell extracts (300 µg) were immunoprecipitated with p38 MAPK antibody. Western immunoblot analysis using anti-serine Akt-phosphorylated antibody was performed on protein lysates immunoprecipitated from cells cultured in the absence of serum for 48 h and incubated for 24 h with the above-indicated drugs. The immunoblots were stripped and reblotted with antibodies against total Akt protein. Representative autoradiographs are shown. Histograms represent the mean densitometric analysis of 3 separate experiments ± SD.

View larger version (14K): [in this window] [in a new window]   Fig. 11. PGE2 regulation of PI3K pathway in Caco-2 cells. Through cAMP and PKA activation, PGE2 stabilizes the complex of PI3K/Ras activated by the receptor tyrosine kinase signaling. PI3K/Ras and/or PI3K alone may modulate the serine and threonine phosphorylation of Akt and, thus, the survival of the cells. The inhibition of PKA, with H89, decreases PI3K/Ras association and increases PI3K activity not associated to Ras. Consequently, PI3K pathway activates p38 MAPK, increases serine phosphorylation of Akt, and induces apoptosis.

	DISCUSSION AND CONCLUSIONS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION AND CONCLUSIONS GRANTS REFERENCES

Our previous data demonstrated that IGF-II/IGF-Ir is able to upregulate COX-2 expression and PGE₂ synthesis through the PI3K activation pathway (5). It has been demonstrated that PGE₂ transactivates EGFR and triggers mitogenic signaling in gastric epithelial and colon cancer cells as well as in rat gastric mucosa in vivo (38). In addition, the COX-2/PGE₂ signaling pathway and the receptor tyrosine kinase-dependent signaling system promote the growth of colon cancer in a synergistic fashion (22). In this study we demonstrate that PGE₂ is able to inhibit apoptosis induced by serum deprivation through PI3K activation and thus the inhibition, at different levels, of PI3K pathway is able to revert this protective mechanism. Cumulative evidence suggests that COX-2 is activated through a Ras-dependent pathway (30, 35, 40). In addition, the inhibition of COX-2 activity suppresses the growth of xenograft of Ras-transformed rat intestinal epithelial cells in nude mice (28). Also, PGE₂ synergistically enhance Ras-induced transcription of the prooncogenic gene amphiregulin (22), whose overexpression in colorectal tumor has been well investigated (3, 16). These findings suggest that COX-2-generated PGs may contribute to Ras-mediated transformation of intestinal epithelial cells through different mechanisms. It has clearly been demonstrated that EP2 and EP4, the major PGE₂ receptor on intestinal epithelial cells, mediate their activity through increase in cAMP production, and it has been demonstrated that increased cellular cAMP stimulates the expression of COX-2 and vascular endothelial growth factor in the stroma (3). In addition, one possible mechanism for COX-2 inhibition of apoptosis is the induction of cAMP-dependent cellular inhibitor of apoptosis (IAP2) and the induction of amphiregulin transcription via the cAMP/PKA pathway mediated by PGE₂ signaling (15). The observation that cAMP suppresses apoptosis in transformed colon epithelial cells suggests that this second messenger might play a role in the formation of colorectal adenoma and cancer. Whether the antiapoptotic function of cAMP agonists, including PGE₂, can interfere with activation of proliferative signals has not been elucidated yet. We demonstrate that exogenous PGE₂ is able to induce proliferation in colon carcinoma Caco-2 cell line and protect them by serum deprivation-induced apoptosis, increasing Ras/PI3K association and the activation of p-Akt and MAPK. Also, the effect of PGE₂ is mediated by PKA. In fact, H89, a PKA-selective inhibitor, is able to decrease the association of Ras with PI3K and to revert the cytoprotective effect. Recent studies suggest that cAMP and PKA participate in the regulation of growth factor signaling by interfering with activation of the Raf1 Ras-mediated pathway or by regulating several phosphatases that act to dephosphorylate various components of the Ras-ERK pathway (20). In our experimental system, the inhibition of PKA in the presence of PGE₂ decreases cell proliferation and MAPK activation but strongly increases activation of Akt. However, the activation of Akt does not correlate with cell survival and with serine 9 phosphorylation of GSK, the substrate of Akt. We demonstrate that inhibition of PKA is able to reduce the Akt-threonine phosphorylation and, conversely, to increase the Akt-serine phosphorylation and this mechanism may modulate the Akt activity. We show that PGE₂ plus H89 treatment activates serine Akt phosphorylation and induce apoptosis. The proapoptotic effect of H89 might be dependent on the unbalance between the presences of higher serine than threonine phosphorylation. It has been demonstrated that PGE₂ inhibits leukotriene synthesis through inhibition of translocation of 5-lipoxygenase. The PKA inhibitor allows p38 phosphorylation and 5-lipoxygenase translocation and activity in thapsigargin-stimulated neutrophils (6). Also, it has been demonstrated that cAMP, through ERK1/2 and p38 MAPK, promotes CREB-dependent induction of cellular inhibitor apoptosis protein 2 (14). The data presented herein demonstrate that treatment of Caco-2 cells with p38 kinase inhibitor or transfection with MKK6 kinase mutated in S270/TR211 decreases the serine phosphorylation of Akt in the presence of H89. The effect of H89 is decreased only by 50% in the presence of dominant negative MKK6K, and this may be related to p38 activation by MKK3 kinase. On the basis of the above findings, it is possible to postulate that PKA inhibitor-dependent apoptosis may be mediated through p38 kinase activation. In support of this hypothesis, it has been recently shown that p38 activation is able to increase the levels of phosphoserine Akt-2 and that modulation of PI3K pathways in myogenesis regulate p38 activity (8). We also demonstrate that the H89-dependent increase of Akt serine phosphorylation is associated to p38 MAPK. Additional experiments will be necessary to confirm the hypothesis that Ras/PI3K association may modulate the activation of p38 kinase in colon cancer cells. In conclusion, our study suggests that, through PKA, PGE₂ can enhance or stabilize Ras/PI3K association and modulate the Akt phosphorylation and, thus, the cell survival, and this may be one mechanism responsible for the involvement of COX-2 in Ras-mediated transformation (4, 30, 40) and PGE₂ in Ras-mediated activation of peroxisome proliferator-activated receptor- (27).

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION AND CONCLUSIONS GRANTS REFERENCES

 
This work was supported in part by a grant from Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Italy.

	ACKNOWLEDGMENTS

 
We thank Maria Grazia Catenacci for the artwork and Rita Cerillo for technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. Acquaviva, Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano," Università degli Studi di Napoli "Federico II", Via Pansini, 5, 80131, Napoli, Italy (e-mail: angacqua{at}unina.it)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^* V. Leone and A. di Palma contributed equally to this work.

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