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The gep proto-oncogene Gα12 mediates LPA-stimulated activation ofCREB in ovarian cancer cells
Ji Hee Ha a,b,c, Jeremy D. Ward a,c, Lakshmi Varadarajalu a,c, Sang Geon Kim b, Danny N. Dhanasekaran a,b,c,⁎a Peggy and Charles Stephenson Cancer Center, The University of Oklahoma Health Sciences Center, 975 NE 10th Street, BRC West 1468, Oklahoma City, OK 73104, United Statesb College of Pharmacy, Seoul National University, Seoul, South Koreac Department of Cell Biology, The University of Oklahoma Health Sciences Center, 975 NE 10th Street, BRC West 1468, Oklahoma City, OK 73104, United States
Abbreviations: LPA, Lysophosphatidic Acid; LPAR, LPResponse Element Binding Protein; ERK, Extracellular-sMitogen and Stress-activated protein Kinase-1; p38Protein Kinase.⁎ Corresponding author at: Peggy andCharles Stephenso
of Oklahoma Health Sciences Center, 975 NE 10th Street, BOK 73104, United States. Tel.: +1 405 271 6850; fax: +1
Article history:Received 20 July 2013Accepted 24 August 2013Available online 19 September 2013
Keywords:LPAGα12
Ovarian cancerOncogeneCREB
Lysophosphatidic acid (LPA) plays a critical role in the pathophysiology of ovarian cancers. Previous studies haveshown that LPA stimulates the proliferation of ovarian cancer cells via Gα12. The present study utilizing Protein/DNA array analyses of LPA-stimulated HeyA8 cells in which the expression of Gα12 was silenced, demonstratesfor the first time that Gα12-dependent mitogenic signaling by LPA involves the atypical activation cAMP-response element binding protein (CREB). Results indicate that the robust activation of CREB by LPA is an earlyevent that can be monitored by the phosphorylation of SER133 of CREB as early as 3 min. The findings that theexpression of the constitutively activatedmutant of Gα12 stimulates CREB even in the absence of LPA inmultipleovarian cancer cell lines confirm the direct role of Gα12 in the activation of CREB. This is further substantiated bythe observation that the silencing of Gα12 drastically attenuates LPA-stimulated phosphorylation of CREB. Our re-sults also establish that LPA–Gα12-dependent activation of CREB is through a cAMP-independent, but Ras–ERK-dependent mechanism. More significantly, our findings indicate that the expression of the dominant negativeS133A mutant of CREB leads to a reduction in LPA-stimulated proliferation of HeyA8 ovarian cancer cells. Thus,results presented here demonstrate for the first time that CREB is a critical signaling node in LPA–LPAR andGα12/gep proto-oncogene stimulated oncogenic signaling in ovarian cancer cells.
Ovarian cancer remains as the most fatal gynecological cancers inthe world with a five-year survival rate of only approximately 45% [1].This is primarily due to our poor understanding of the disease in addi-tion to the asymptomatic nature of this cancer in the early stages. Inthis context, the identification of the lysophosphatidic acid (LPA) as anovel “ovarian cancer activating factor” present in ascetic fluid samplesfrom ovarian cancer patients is a highly significant finding [2,3] that ledto the characterization of LPA as a potential biomarker for ovarian can-cers [4–7] in addition to being considered as a possible therapeutic tar-get for ovarian cancers [8–11].
LPA is known to elicit its diverse cellular responses by stimulatingspecific set of heterotrimeric G proteins via a distinct family of G proteincoupled receptors [12–15]. Studies from several laboratories, includingours, have shown that LPA plays a crucial role in the progression, if
n Cancer Center, TheUniversityRC West 1468, Oklahoma City,405 271 2507.. Dhanasekaran).
ghts reserved.
not in the genesis, of ovarian cancer [8,13,16–20]. We have previouslyshown that LPA stimulates the proliferation of ovarian cancer cellsthrough GNA12, the gep proto-oncogene, Gα12 [19]. Our previousstudy, using a model system that utilizes a panel of ovarian cancercells in which the expression of Gα12 was stably silenced hasestablished the critical role of Gα12 in LPA-mediated oncogenic prolifer-ation of ovarian cancer cells [19]. Therefore, our present study is focusedon defining whether the mitogenic pathways stimulated by LPA viaGα12 involve any novel, thus far uncharacterized, signaling pathway(s).Towards this goal, we carried out a protein/DNA array analysis usingLPA-stimulated, but Gα12-silenced, HeyA8 (shGα12-Hey8A) cells. Ourresults presented here demonstrate that LPA stimulates the potent acti-vation of CREB via the proto-oncogene Gα12 by stimulating the phos-phorylation of Ser133 of CREB, leading to activation of CREB, whichhas been implicated in ovarian cancer cell proliferation [21]. We alsoshow that the activation of CREB by LPA is very rapid that it can be ob-served at least as early as 3 min following LPA-treatment. Furthermore,we demonstrate that the expression of the constitutively activated mu-tant of Gα12 stimulates the phosphorylation of CREB even in the ab-sence of LPA, whereas silencing of Gα12 abrogates LPA-stimulatedactivation of CREB. Our results further establish that LPA-mediated acti-vation of CREB via Gα12 is through a cAMP-independentmechanism in-volving a Ras–ERK-dependent signalingpathway.More importantly, wealso show that the expression of the dominant negative S133A mutant
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CREB leads to an attenuation of LPA-stimulated proliferation of ovariancancer cells. Taken together with the previous finding that the LPA–Gα12 signaling axis is critically involved in ovarian cancer cell prolifera-tion, our present study unravels a unique Gα12-dependent mechanismthroughwhich LPA signaling converges on CREB to stimulate the prolif-eration of ovarian cancer cells.
2. Materials and methods
2.1. Cells, plasmids, and transfections
The ovarian cancer cell lines SKOV3, and HeyA8 and OVCAR-3 weremaintained in Dulbecco's modified Eagle's medium (Cellgro, Manassas,VA) containing 10% fetal bovine serum (Gemini Bio-Products, WestSacramento, CA), 50 U/mL penicillin, and 50 μg/mL streptomycin at37 °C in a 5% CO2 incubator as previously described [20]. LPA wasobtained from (Avanti Polar Lipids, Alabaster, AL). It was dissolvedinto 20 mM stock solutions in sterile water, and stored at −20 °Cuntil use. shRNA-mediated silencing of Gα12 was carried out accordingto previously published methods [19]. Briefly, pLKO.1 vectors encodinga set of human shRNA targeting Gα12 (RHS4533—NM_007353) and thecontrol shRNA-vectorwere obtained fromOpen Biosystems (Huntsville,AL). SKOV3, HeyA8, and OVCAR3 cells were transfected with pLKO.1-shRNA/Gα12 or pLKO.1 vector control, respectively using AmaxaNuclearfector II system. To select for stably transfected shRNA-Gα12
cell colonies, puromycin (2 μg/ml; MP Biomedicals, Solon, Ohio) wasadded 24 h post-transfection. Single clones were scored and the silenc-ing of Gα12 expression was determined by immunoblot analysis. pCMVVectors encoding wild-type CREB and CREB-S133A mutant constructs(631925) were obtained from Clontech Laboratories, Mountain View,CA. The transfection studies presented here were carried out using anAmaxa Nucleofector II system (Lonza, Walkersville, MD) using themanufacturer's protocol for the respective cell types.
2.2. Protein/DNA array
HeyA8 cells thatwere stably expressing either shRNA directed againstGα12 or vector backbone alone (pcDNA3) were plated at a density of1.5 × 106 cells on 100 mm plates. The cells were allowed to adhere forapproximately 8 h, then washed thrice with PBS, and then placed inserum-free media. The cells were left in serum-free media overnight.The following day, the cells were either treated with 20 μM of LPA (onegroup of vector control cells and the stably silenced Gα12 cells) or left inserum-starvation conditions. The cells were incubated for 16 h with LPAor left in serum free media for this time. After 16 h, the cells were lysedand thenuclear extractwas obtainedusing theAffymetrixNuclear Extrac-tion Kit (Santa Clara, CA) according to the manufacturer's instructions.The nuclear lysate was then analyzed for transcription factor activationusing an Affymetrix Combo Protein/DNA Array (MA1215; Santa Clara,CA) according to themanufacturer's instructions. Briefly, nuclear extractsfrom unstimulated, LPA-stimulated or LPA-stimulated, but Gα12-silencedHeyA8 cells were incubated with biotin-labeled DNA probe encoding thebinding motifs of specific transcription factors. The transcription factors/proteins bound to the probes were isolated and the bound probes werereleased and hybridized to an array membrane containing 345 distinctspots for different transcription factor consensus sites. The hybridizedbiotin-labeled probe, indicative of the activated transcription factors inthe lysates, were identified by streptavidin–HRP based chemilumines-cence detection method. The intensities of the spots in each of the arrayswere quantified using CarestreamMolecular Imaging Software version 5(Rochester, NY).
2.3. qRT-PCR analysis
HeyA8 stably-silenced cell clones 1 through 3 and the vector controlcells were plated at 700,000 cells on 100 mm plates. The cells were
allowed to adhere and grow overnight at 37 °C with 5% CO2. The cellswere then lysed using the Qiagen RNeasy Mini Kit (Valencia, CA)according to the manufacturer's instructions. The quantity of RNA wasquantified using an Implen NanoPhotometer (Westlake Village, CA).The integrity of isolated RNAwas analyzed using Bio-Rad's Experion au-tomated electrophoresis system (Hercules, CA) according to themanufacturer's instructions. Two step qRT-PCR was performed by firststrand synthesis with SuperScript First-Strand Synthesis, primed withOligo(dT) from Invitrogen (Carlsbad, CA) following the manufacturer'sprotocol. All samples were processed at the same time to avoid batch-batch variation. A minus reverse transcriptase (RT) control was alsogenerated for each sample. The cDNA constructs were then stored at−80 °C until qRT-PCR was performed. qRT-PCR was performed usingBioRad's iQ SYBR green supermix kit (Hercules, CA). qRT-PCR mixturesconsisted of 1× SYBR Green, 300 nM of each primer, 1 μl of templatecDNA and water to 10 μl. The thermal profile for all genes was as fol-lows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and58 °C for 30 s. Ct was determined by single threshold for each well.Each sample was done in triplicate on the plate. Negative RT controlsfor each stably silenced Gα12 clone and the vector control were alsodone at the same time. A non-template control was also included to en-sure there was no primer, dimer or template contamination. Six endog-enous reference genes: actB, alas1, gusB, hprt, PP1A, and tbp were alsoamplified during the reaction. PCR was carried out using the followingprimers:
The reference genes with the most stable expression were chosenfor normalization using the geNorm method [22] by obtaining the Mvalue for each reference gene and using theMvalue to stepwise excludethosewith the highest M value until the stepwise inclusion did not con-tribute to the calculated normalization factor. Quality control for the
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qRT-PCR reaction involved checking the negative RT samples for a Ctless than 40, the NTC had a Ct less than 38, the positive control had aCt greater than 30, there was not efficiency greater than 110 or lessthan 90 and each replicate group Ct had a standard deviation less than0.20. A melting curve analysis was conducted from 55 °C to 90 °C with0.5 °C increases per cycle insure therewas nomis-annealing or contam-inated cDNA in the sample.
2.4. cAMP chemiluminescent immunoassay
A 96-well plate was used to plate out 1 × 105 HeyA8, SKOV3, andstably-silenced Gα12 HeyA8 cells per well into three treatment groups.The three treatment groups were serum-starved (untreated), LPA(20 μM), and Forskolin (20 μM). Each treatment group had six replicatesplated out per cell line. The cells were counted and allowed to adhereovernight. The following day the cells were washed 2 times in serum-free media and then left in serum-free media overnight. The followingday, the serum-free media was aspirated off the LPA and Forskolin treat-ment groups. The cells were then incubated in serum free media thatcontained either LPA or Forskolin for 10 min. The untreated group ofeach cell line was left in the serum free media. After 10 min, all themedia was aspirated from the wells and the cells were lysed using thelysis buffer provided by the manufacturer. After lysis, cAMP levels weredetermined using Invitrogen's cAMP Chemiluminescent ImmunoassayKit (Carlsbad, CA) following the manufacturer's instructions.
2.5. Cell proliferation assays
Quantification of cell proliferation was carried out using crystal vio-let staining according to previously published protocols [19]. Equalnumber of cells (2.5 × 104)were seeded in 12-well culture dishes over-night, serum-deprived for 24 h, then stimulated as described above. Atthe indicated time-point, cells were fixed using 10% formalin (FisherScientific, Pittsburgh, PA) dissolved in PBS for 10 min. Triplicate sampleswere fixed in this manner immediately before stimulation (0 h) and at24, 48, and 72-h. After fixation, all of the samples were stored in sterilePBS at 4 °C. At the conclusion of the experiment, the fixed samples werestained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO) for 6 h.The samples were then washed extensively to remove excess dye, anddried overnight. The cell-associated dye was then extracted by incuba-tion with 1 mL acetic acid (Fisher Scientific) for 60 s.
2.6. Immunoblot analysis
Immunoblot analyses with specific antibodies were carried out fol-lowing the previously published procedures [19,20]. The antibodiesthat were used for immunoblot analyses of Ser133-phospho-CREB(9198), CREB (9197), MSK1 (3489), phospho-MSK1 (9595), AKT(9272), phospho-AKT (9271), phospho-ERK (9106), phospho-p38MAPK(9211), HA-tag (2367), and GAPDH (2118) antibodies were obtainedfrom Cell Signaling Technology (Boston, MA). Antibodies to ERK (sc-93),p38MAPK (sc-535), and Gα12 antibodies were purchased from SantaCruz Biotechnology (Santa Cruz, CA) were from Ambion, Austin, TX(GAPDH antibody #4300) and Santa Cruz Biotechnology, Santa Cruz, CA(Gα12 # sc-409 and HA-epitope # sc-805). Peroxidase-conjugated anti-rabbit IgG (W401B) and anti-mouse IgG (NA93IV) were purchasedfrom Promega (Madison, WI) and GE Healthcare (Buckinghamshire,UK), respectively.
2.7. Statistical analysis
All statistical analysiswas performed usingGraphPad Prism (La Jolla,CA) by two-tailed Student's t-test with Welch's correction.
3. Results
3.1. LPA stimulates multiple transcription factors through Gα12
Since our previous studies have shown that LPA stimulates cell pro-liferation via Gα12 [19], we investigated whether there is any novel,thus far unidentified, Gα12-specific unique transcriptionally-regulatedevent regulated by LPA. To identify transcription factors potentially ac-tivated by Gα12, we stably silenced Gα12 in HeyA8 ovarian cancer cells[19]. First,we verified the silencing of Gα12 in these cells by immunoblotanalysis and by quantitative RT-PCR (Fig. 1A & B). Clone number 2showed the highest level of Gα12-silencing and was selected for allthe studies presented hereafter. The stably silenced Gα12 HeyA8 cellswere subjected to a Protein/DNA interaction array analysis. Of the 345transcription factors analyzed in this array, 34 transcription factorsshowed a 5-fold or greater increase in their activation profile, as inferredby the increase in their DNA-binding activity, upon LPA-treatment in aGα12-dependent manner (Fig. 1C; Table 1). It is significant to notehere that the DNA-binding activity of these transcription factors weregreatly reduced or absent in lysates derived from the cells in whichthe Gα12 was silenced (Fig. 1Cmiddle panel versus bottom panel), indi-cating that they are activated by LPA via a Gα12-dependentmechanism.Analyzing the previously published physiological roles of these tran-scription factors [23–59], it can be seen that the aberrant expressionand activation of many of the transcription factors identified in ourstudy have previously been associatedwith the genesis and/or progres-sion of many cancers, including ovarian cancer (Table 2), potentially in-dicating their importance in LPA-mediated oncogenic signaling.
3.2. Activation of CREB by LPA is Gα12-dependent early signaling event
Our finding that CREB is one of the transcription factors stimulatedby LPA via a Gα12-dependent mechanism is quite intriguing andnovel. Indeed, Gα12 has never been associated with the stimulation ofcAMP or CREB-dependent mitogenic pathways [60,61]. Taking into ac-count of the recent findings that CREB is overexpressed in ovarian can-cer and its suppression leads to a decrease in ovarian cancer cellproliferation [21] and that CREB has been linked to inducing oncogene-sis in several different cancer types [62], we focused on investigating therole of CREB in LPA–Gα12-mediated oncogenic proliferation of ovariancancer cells. Since the protein/DNA array analysis presented in Fig. 1Cwas carried out with cells that were exposed to LPA for 16 h, we rea-soned that it is of critical importance to clarify whether the activationof CREB by LPA is an early event or an event occurring much later. Ourreasoning for clarifying this was if CREB activation occurs early in LPA-mediated signaling, this would indicate that CREB activation is morethan likely due to immediate downstream signaling of Gα12. However,if CREB activation occursmuch later, e.g. after 1 h, thiswould potentiallyindicate that CREB-activation via Gα12 is due to a sequential relay oftemporal signaling events and thus indirectly activated by Gα12. There-fore, we carried out a time course analysis of LPA-stimulated CREB-activation in HeyA8 cells using Ser133 phosphorylation of CREB as anindex of CREB-activation [63]. As shown in Fig. 2, we found the phos-phorylation of Ser133 on CREB could be observed from 3 min onwardsfollowing LPA-stimulation and persisted beyond 6 h.
3.3. LPA-stimulated phosphorylation of CREB requires Gα12
To further validate that the CREB activation is a Gα12-mediatedevent, an immunoblot analysis was carried out to test whether the si-lencing of Gα12 abrogates LPA-stimulated Ser133-phosphorylation ofCREB. Our results indicated that the stimulation of the control HeyA8cells with LPA resulted in the phosphorylation of Ser133 by 3 min(Fig. 3A, Lane 1 versus Lane 3), whereas silencing of Gα12 led to a drasticreduction in LPA-mediated activation of CREB (Fig. 3A Lane 3 versusLane 4). Quantification of these results indicated that the silencing of
Fig. 1. LPA stimulates the activation of diverse transcription factors via Gα12. A. Silencing of Gα12 in HeyA8 cells using Gα12-specific shRNAwas monitored by immunoblot analysis usinglysates of 25 μg protein derived from three distinct clones ofGα12-silenced cells alongwith cells fromvector control clone. B. Gα12-shRNA-HeyA8 cloneswere analyzedbyquantitative RT-PCR for Gα12 expression. The expression levels of Gα12 for each clone in relation to vector control cells are presented in the bar graph. C. Hey cells stably expressing shRNA against Gα12 orthe vector alone (non-specific scrambled shRNA vector) were serum-starved overnight. The stably silenced Gα12 cells were treated with 20 μM of LPA for 16 h along with one group ofHeyA8 cells stably-expressing the vector alone. Additionally, one group of the vector control cells was left in serum-free media for the 16-hour treatment period. After the 16-hour treat-ment, nuclear lysate was obtained from each cell group and analyzed by a Protein/DNA array according to manufacturer's protocol. Representative array data from two independent ex-periments are presented. Each spot on the array that corresponds to a specific transcription factor was identified according tomanufacturer's protocol. Transcription factors stimulated byLPA but absent or down-regulated in Gα12-silenced cells are scored. The arrows indicate the spots corresponding to CREB. The profiles of activated transcription factors as indicated by thebinding of the respective transcription factors to theDNA-elements printed in the arraywere analyzed in serum-starvedHeyA8 cells (Upper Panel), HeyA8 cells stimulatedwith LPA (Mid-dle Panel), and LPA-stimulated HeyA8 cells in which the expression of Gα12 was silenced (Lower Panel).
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Gα12 attenuated LPA-stimulated phosphorylation of CREB bymore than50% (Fig. 3B). Similar attenuation of LPA-stimulated phosphorylation ofCREB-Ser133was observed, albeit to a varying extent, in Gα12-silenced-SKOV-3 cells (Fig. 3C), indicating that the observed Gα12-dependent ac-tivation of CREB by LPA is not unique to HeyA8 cells alone.
To rule out the possibility that the results obtained were due to acompensatory mechanism induced by the persistent silencing of Gα12
in these cells, we analyzed LPA-stimulated CREB phosphorylation inHeyA8 cells in which the expression of Gα12 was transiently silencedfor only 24 h via Gα12-targeting shRNA. As shown in Fig. 3D, transientsilencing of Gα12 led to the abrogation of LPA-stimulated phosphoryla-tion of CREB-Ser133. These results further confirm that the activation ofGα12 by LPA initiates a signaling cascade(s) that leads to the down-stream phosphorylation of CREB.
3.4. Constitutively activatedmutant of Gα12 stimulates the phosphorylationof Ser-133 of CREB
In order to directly demonstrate that the activation of Gα12 is suffi-cient for the observed Ser133-phosphorylation of CREB, we utilized an-other approach involving the expression of the constitutively active,GTPase-deficient mutant of Gα12, Gα12Q229L (Gα12QL) in ovarian can-cer cell lines. Since the GTPase deficient mutant of Gα12 would be anal-ogous to LPA-activated GTP-bound configuration of Gα12, if Gα12 is
truly involved in the activation of CREB, the expression of such a consti-tutively active mutant should lead to the Ser133-phosphorylation ofCREB. Following this rationale, HeyA8, OVCAR3, or SKOV3 cells weretransiently transfected with a vector encoding Gα12QL for 48 h. The ly-sates from the transfected cells were resolved by SDS-PAGE andsubjected to immunoblot analysis using antibodies specific to Ser133-phosphorylated CREB. Our results indicated that the expression ofGα12QL stimulated a significant increase (N60%) in the phosphorylationof CREB in all of the tested cell lines (Fig. 4). These results, together withthe data obtainedwith the use of Gα12-silenced ovarian cancer, confirmthat the activation of Gα12 –whether it is throughmutational activationor via LPA–LPAR-mediated activation – plays a determinant role in theactivation of CREB.
3.5. Stimulation of CREB by LPA involves is via cyclic AMP-independent burERK-dependent pathway
Canonical signaling involved in the activation of CREB involves thegeneration of cyclic AMP (cAMP) and subsequent activation of PKA isone of the major pathways known to activate CREB. On the other hand,past studies have shown that Gα12 activation does not lead to the produc-tion of cAMP [60,64]. This led us to hypothesize that LPA activation ofCREB was independent of cAMP signaling. To test this hypothesis, weperformed a chemiluminescent immunoassay to determine if LPA
Table 1LPA-stimulated and Gα12-dependent transcription factors in HeyA8 cells. Control HeyA8cell expressing non-specific sh-vector or HeyA8 cells in which Gα12 were stimulatedwith 20 μM LPA for 16 h. Nuclear extracts from these cells along with unstimulated con-trols were analyzed for the activation of different transcription factors using “AffymetrixCombo Protein/DNA Array” as described under Materials and methods section. Represen-tative array data from two independent experiments are presented here. Each spot on thearray, which corresponds to a specific transcription factor, was identified using the tem-plate from the user manual. The intensities of the spots were quantified using CarestreamMolecular Imaging Software version 5. Transcription factors stimulated by LPA but absentor down-regulated in Gα12-silenced cells were scored, quantified, and tabulated.
26 HINF Histone nuclear factor 7.05 10027 c-Myb Transforming oncogene 6.93 10028 E4F/ATF Activating transcription factor 6.62 10029 TR Thyroid hormone receptor 6.52 10030 Stat-1/3 Signal transducers and activators of
transcription 1/36.27 100
31 IRF-1/2 Interferon Regulatory Factor 6.25 10032 XBP-1 X-box binding protein-1 6.05 10033 Stat-4 Signal transducers and activators of
transcription 45.91 100
34 SP-1/ASP Sp 1 transcription factor 5.77 100
Table 2LPA-responsive and Gα12-dependent transcription factors associated with cancer. Basedon previously published data, at least seventeen of the transcription factors tabulatedhere have been shown to be either overexpressed or activated in different cancers.
Fig. 2. LPA-stimulated activation of CREB is an early event. A. HeyA8 cells (1 × 106) werestimulated with 20 μM LPA for varying lengths of time as indicated. Lysates from thesecells were analyzed by immunoblot for the phosphorylation of CREB on Ser133. The blotwas stripped and analyzed for total CREB and GAPDH, as loading controls. B. The phos-phorylated levels of CREB were quantified from three independent experiments usingHeyA8 cells as shown in the time course in part A.
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signaling and/or LPA–Gα12-dependent signaling activated cAMP inHeyA8 and SKOV3 cells. Briefly, Gα12-silenced HeyA8 or SKOV3 cellswere plated and allowed to adhere overnight, then serum-starved over-night and then stimulated with LPA (20 μM) for 10 min along with anuntreated control group and a positive control of Forskolin (20 μM) treat-ed cells, which activates adenylyl cyclase independent of receptor/G pro-tein coupling,was used as a positive control. Lysates from these cellswereanalyzed for cAMP levels using a chemiluminescent cAMP-immunoassay.As expected, Forskolin stimulated an increase in cAMP levels in the cells,however LPA-stimulation of these cells failed to elicit any significantchanges in cAMP levels (Fig. 5A). Our observation that cAMP-levels
were not affected by LPA stimulation pointed to a signaling mechanismindependent of cAMP in LPA–Gα12-mediated activation of CREB inthese cells.
Although CREB can be activated by the phosphorylation of Ser133 byvarious kinases including the canonical signaling pathways involvingcAMP and protein kinase A [65,66], previous studies have also shownLPA can stimulate the phosphorylation of CREB via cAMP-dependent[67] as well as -independent mechanisms [68]. It has been shown thatcAMP-independent Ser133 phosphorylation of CREB can be mediatedvia AKT or via ERK/p38MAPK-dependent pathways that result in the
Fig. 3. LPA-stimulated phosphorylation of CREB is Gα12-dependent. A. HeyA8 cells (1 × 106) in which Gα12 was silenced by stable expression of shRNA to Gα12 were stimulated with20 μMof LPA for 10 min or left unstimulated. Vector control HeyA8 cells (1 × 106) were also treatedwith 20 μMof LPA for 10 min as a positive control or left unstimulated, as a negativecontrol. Immunoblots were performed for phosphorylation of CREB on Ser133 The blots were then sequentially stripped and probed for the expressions of CREB, Gα12 and GAPDH. B. Thephosphorylated levels of CREB in relation to total levels of CREBwere quantified for each group and presented as bar graph inwhich the bars representmean ± SEM (n = 3). Anunpairedtwo-tail t-testwithWelch's correctionwas performed to determine statistical significance. *p b 0.05. C. Similar analysiswas carried out in SKOV3 cells inwhich the expression of Gα12wassilenced by the stable expression of shRNA directed against Gα12. The experiment was repeated 3 times and a representative immunoblot is shown. D. Transient silencing of Gα12 wascarried out by transfecting HeyA8 cells with shRNA specific for Gα12 or scrambled shRNA control for 48 h. These transfectants were stimulated with 20 μM of LPA for 10 min or leftunstimulated. Immunoblot analysis was carried out to monitor the phosphorylation of Ser133 of CREB. The blot was then sequentially stripped and probed for the expressions of CREB,Gα12 and GAPDH. The experiment was repeated three times and a representative immunoblot is shown.
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activation of the kinase MSK1 [63]. To identify the candidate pathwaythat was activating CREB in our system, we first monitored which ofthese aforementioned kinases were activated by LPA in a Gα12-dependent manner. The activation profiles of these kinases in response
Fig. 4.Activatedmutant ofGα12 stimulates the phosphorylation of CREB. A.) 1 × 106HeyA8, SKOLysates from these transfectants were resolved by SDS-PAGE and subjected to immunoblot anawere stripped and re-probed for total CREB and GAPDH to ensure equal loading. Levels of Gα1
phorylated levels of CREB in relation to total levels of CREBwere quantified and presented as barWelch's correction was performed to determine statistical significance. *p b 0.05.
to LPA were analyzed by immunoblot analysis using antibodies specificto the activated, phosphorylated forms of these kinases. Vector controlor Gα12-silenced HeyA8 cells (2 × 106) were serum starved and stimu-lated with 20 μM LPA for 10 min. Lysates from these cells were analyzed
V3, andOVCAR3 cellswere transiently transfectedwith vectors encodingGα12QL for 48 h.lysis using antibodies specific to Ser133-phosphorylated CREB. The same respective blots
2 were also probed to ensure expression of the constitutively active mutant. B.) The phos-graph inwhich the bars representmean ± SEM (n = 3). Anunpaired two-tail t-test with
Fig. 5. LPA stimulates the phosphorylation of CREB via cyclic AMP-independent but ERK-dependent pathway.A. LPA–Gα12-mediated activation of CREB is independent of cAMP-signaling.A 96-well plate was used to plate out 1 × 105 HeyA8 cells per well into three treatment groups. The three treatment groups were [1] serum-starved (untreated), [2] LPA (20 μM), and [3]Forskolin (20 μM)with each treatment group having a six replicates. The cells were counted and allowed to adhere overnight. The following day cells were washed 2-times in serum-freemedia and left to incubate overnight. The following day, the serum-free media was aspirated off and the cells were treated with either LPA or Forskolin prepared in serum-free media for10 min or cells were treatedwith serum-freemedia alone. After 10 min of treatment, themediawas aspirated and the cellswere lysed using the lysis buffer providedby themanufacturer.After lysis, cAMP levels were determined using a cAMP Chemiluminescent Immunoassay Kit following themanufacturer's instructions. Quantification of the data was done via GraphPadby interpolating the unknowns from a standard curve using the log(inhibitor) vs. response function. ***p = 0.0007. Control cells expressing non-specific scrambled sh-vector or HeyA8cells stably expressing shRNA targeting Gα12 cells were serum starved for 16 h followed by stimulationwith 20 μMof LPA for 10 min. The cells were lysedwith RIPA buffer and blotted forp-AKT (B), p-MSK-1 (C), p-P38MAPK (D), and p-ERK (E), respectively. The same blot was stripped and re-probed for total protein for AKT (B), MSK-1 (C), P38MAPK (D), ERK (E) andGAPDH. F. To validate whether ERK is activating CREB, a pharmacological inhibitor of the ERK pathway (PD98059) was used. HeyA8 cells (1 × 106) were pre-treated with 10 μMPD98059 for one hour followed by stimulation with 20 μM of LPA for 10 min. These cells were lysed and analyzed by immunoblot for phosphorylation of CREB on Ser133, then strippedand re-probed for total CREB and GAPDH to ensure equal loading of protein.
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for the activation of ERK, p38MAPK, or AKT. As shown in Fig. 5, LPA-stimulation of HeyA8 cells resulted in the activation of AKT (Fig. 5B),p38MAPK (Fig. 5C), MSK1 (Fig. 5D) and ERK (Fig. 5E) to a varying extent.However, the silencing of Gα12 in these cells abrogated only the activa-tion of ERK (Fig. 5E), suggesting the possible role for ERK in LPA–Gα12-mediated CREB activation. To test whether ERK is truly involved in LPA-mediated activation of CREB, we analyzed Ser133 phosphorylation ofCREB in response to LPA in cells treatedwith PD98059, a potent inhibitorof MEK, the upstream activator of ERK. Immunoblot analyses revealedtreatment of cells with PD98059 resulted in significant attenuation of
CREB phosphorylation (Fig. 5F), indicating the activation of CREB byLPA in these cells is ERK-dependent.
3.6. LPA–Gα12-stimulated phosphorylation of Ser133-CREB involves Ras
Thus far our study has demonstrated LPA-mediated activation ofCREB involves Gα12 and ERK and we therefore wanted to determine ifGα12-mediated activation of ERK followed the stereotypical activationof the MAP kinase pathway via Ras. Previous studies have establishedRas plays a determinant role in the activation of ERK [69–71].
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Furthermore, our laboratory aswell as others, has shown Gα12 can acti-vate Ras as well as the Ras–ERK signaling nexus [72–74]. Therefore, dueto thewell-characterized involvement of Ras in activating ERK and ERK-mediated downstream signaling involved in the direct activation ofCREB we tested whether inhibition of Ras led to attenuation of CREBphosphorylation. In addition, we investigated whether any of theother Gα12-responsive GTPases such as CDC42, Rac-1, and RhoA wereinvolved in the activation of CREB. To characterize the involvement of
Fig. 6. LPA and Gα12 stimulated phosphorylation of CREB requires Ras. A.) 1 X 106 SKOV3 cellsRasN17. The cells were serum starved for 16 h and were then stimulated with 20 μM of LPA forsame blot was stripped and re-probed for total CREB and GAPDH to ensure equal loading. Thnegative Ras constructs. The phosphorylated levels of CREB in relation to total levels of CREB(n = 3). An unpaired two-tail t-test with Welch's correction was performed to determine stwith vector alone orwith dominant-negativemutantHA-CDC42N17 (Upper Panel), HA-RacN17for 16 h and were then stimulated with 20 μMof LPA for 10 min. These cells were lysed and imfor total CREB and GAPDH to ensure equal loading. The stripped blots were also probed for thetransfected with Gα12QL, alongwith HA-RasN17, or vector alone. These cells were lysed and imfor total CREBandGAPDH to ensure equal loading. The strippedblotswere also assayed for totalthe dominant-negative Ras construct.
these small GTPases in LPA–Gα12 mediated activation of CREB, HeyA8cells were transiently transfected with dominant negative mutants ofH-Ras- (N17-H-Ras), Rac-1- (N17-Rac-1), CDC42- (N17-CDC42), andRhoA (N19-RhoA)-GTPases with subsequent immunoblot analysesbeing carried out to analyze the effect of expressing these dominantnegative mutants on LPA-stimulated phosphorylation of Ser133-CREB.Our results indicated that the stimulation of CREB by LPAwas drasticallyinhibited by the expression of the dominant negative mutant of Ras
were transfected for 48 h either with vector alone or with dominant-negative mutant HA-10 min. These cells were lysed and immunoblotted for phosphorylated CREB (S133). The
e stripped blots were also probed for the HA-tag to confirm expression of the dominant-were quantified and presented as bar graph in which the bars represent mean ± SEM
atistical significance. * p b 0.05. B.) 1 × 106 SKOV3 cells were transfected for 48 h either(Middle Panel), andHA-RhoN19 (Lower Panel), respectively. The cellswere serumstarvedmunoblotted for phosphorylated CREB (S133). The same blot was stripped and re-probedHA-tag to confirm expression of the dominant-negative constructs. C.) SKOV3 cells weremunoblotted for phosphorylated CREB (S133). The same blot was stripped and re-probedGα12, to showexpression theGα12QL construct and for theHA-tag to confirmexpression of
130 J.H. Ha et al. / Cellular Signalling 26 (2014) 122–132
(Fig. 6A). In contrast, LPA-stimulated phosphorylation of CREB was notattenuated by the expression of the dominant negative mutants ofCDC42, Rac, or RhoA (Fig. 6B). To confirm the specific role of Gα12 intransmitting signals through Ras to stimulate CREB, we analyzed the ef-fect of the dominant negativemutant of Ras on phosphorylation of CREBstimulated by the expression of the constitutively activatedGα12QLmu-tant on the activation of CREB. Our results indicated that the expressionof dominant negative Ras attenuated Gα12QL-stimulated phosphoryla-tion of CREB in two different ovarian cancer cell lines (Fig. 6C),establishing Gα12-mediated activation of CREB is via the Ras–ERK sig-naling pathway.
3.7. CREB is involved in LPA-mediated ovarian cancer cell proliferation
It is significant to note here that our previous studies have shownthat LPA stimulates the proliferation of ovarian cancer cells via Gα12.Similarly, CREB has been shown to be involved in the regulation of ovar-ian cancer cell proliferation [21]. In conjunction with our resultspresented here that LPA–Gα12 signaling axis stimulates the phosphory-lation of CREB, it can be hypothesized that the activation of CREB plays acrucial role in LPA-mediated proliferation of ovarian cancer cells. Tocharacterize the involvement of CREB in LPA-mediated proliferationwe utilized a dominant negative mutant of CREB. The CREB dominantnegative mutant was created bymutating serine 133 of CREB to alanine(S133A), which has been well characterized as a dominant negative in-hibitor of CREB that exerts its inhibitory effect by sequestering the up-stream kinase involved in Ser133-phosphorylation of CREB [75–78].
To test the hypothesis that CREB plays a role in LPA-mediated prolif-eration, HeyA8 cells were transfected with vectors encoding S133A-mutant CREB or a control vector. After 24 h, the transfected cells wereplated at 2 × 104, allowed to adhere overnight then serum-starvedthe following day. After serum-starvation the transfected cells werestimulated with 20 μM LPA for the indicated times and proliferation of
Fig. 7. LPA-induced proliferation of ovarian cancer cells requires CREB.A. HeyA8 cells (1 × 106)w(S133A). 2.5 × 104 of the transfected cells were seeded in triplicate into 12-well culture dishesthe indicated time-point, cells were fixed using 10% formalin dissolved in PBS for 10 min. Triplicand 72-h, respectively. The fixed samples were stained with 0.1% crystal violet for 6 h. The samassociated dye was then extracted by incubation with 1 mL acetic acid for 60 s and absorbancnegative inhibitory effect of the S133A-CREB mutant, lysates from S133A-CREB transfectants (to immunoblot analysis using antibodies to CREB and SER133-phospho-CREB respectively. The bPanel). B. The cellswere lysed and immunoblotted for Cyclin A (Upper Panel) followingwhich itof Cyclin A in relation to total levels of GAPDHwere quantified and presented as bar graph (Lowith Welch's correction was performed to determine statistical significance. ** p = 0.0018.
these cells were quantified following previously published methods[19]. The lysates from these transfected cells were also analyzed forthe expression levels of Cyclin A, which is known to be regulated byCREB and as an additional marker of proliferation [79]. The dominantnegative inhibitory effect of CREB-S133Awas verified by its ability to in-hibit Ser-133 phosphorylation of endogenous CREB in these cells. Anal-ysis of the proliferation of these cells indicated that the expression of thedominant negative CREB-mutant reduced the proliferation of ovariancancer cells by 40% compared to both vector control cells (Fig. 7A)with the concomitant analogous reduction in Cyclin A levels (Fig. 7B),thus establishing a role for CREB in LPA-stimulated proliferation ofthese cells.
4. Discussion
Although the potential oncogenic role of LPA in ovarian cancer path-ophysiology is increasingly being realized, its role and the underlyingmechanism in promoting ovarian cancer cell proliferation is not fullyclarified. In this context, a recent report from our laboratory was oneof the first to clearly demonstrate that the LPA-LPAR-Gα12 signalingaxis specifically induces proliferation in ovarian cancer cells, but not innormal epithelial ovarian cells [19]. Our current study expands our pre-vious finding further by establishing here for the first time that Gα12-dependent proliferative signaling stimulated by LPA involves the activa-tion of CREB. In this study, we were able to show that stimulation withLPA activates the transcription factor CREB in a Gα12-dependent man-ner by the DNA/protein array based functional assay – which is basedon the ability of activated CREB binding to the CRE-containing DNA-fragment printed in the array – as well as Ser133-phosphorylation ofCREB (Figs. 1 & 2). Furthermore, by using three different strategies,namely, 1) shRNA-mediated silencing of Gα12-expression (Figs. 1 & 3,2) dominant negative mutant-mediated inhibition of receptor-mediated activation of Gα12 (data not shown) and 3) expression of
ere transfected for 24 h eitherwith vector alone orwith dominant-negative CREBmutantand the cells were serum-starved for 16 h and were then stimulatedwith 20 μMof LPA. Atate sampleswere fixed in thismanner immediately before stimulation (0 h) and at 24, 48,ples were then washed extensively to remove excess dye, and dried overnight. The cell-e at 590 nm was quantified (upper panel). To confirm the expression and the dominant-CREB-S133A) along with those obtained from vector control cells (pCMV) were subjectedlot was stripped and reprobedwith antibodies to GAPDH tomonitor equal loading (Lowerwas stripped and re-probed for total GAPDH to ensure equal loading. The expression levelswer Panel) in which the bars represent mean ± SEM (n = 3). An unpaired two-tail t-test
131J.H. Ha et al. / Cellular Signalling 26 (2014) 122–132
constitutively activatedmutant of Gα12 (Fig. 4), we firmly establish thatthe phosphorylation of Ser133 is indeed mediated by Gα12. Our studiesusing the dominant negative inhibitory mutant of Gα12 that exerts itsinhibitory effect by competing with the endogenous Gα12 [19,80,81]in its interaction with the receptor further confirms that the LPA–LPAR–Gα12 signaling axis is involved in Ser133-phosphorylation andsubsequent activation of CREB. In addition, our results presented heresuggest that this phosphorylations is dependent on Ras, which in turninvolves ERK-mediated activation of CREB. Although our present studydoes not focus on the mechanism by which ERK stimulates thephosphorylation of Ser133 of CREB, it has been previously shown thatERK-mediated phosphorylation of Ser133 of CREB involves eitherMAPKAP-K1/RSK2 [82] or MSK1 [83]. Relatedly, Fig. 5D indicated thatthe silencing of Gα12 does not modulate MSK1 activation; therefore, itis more likely that the phosphorylation of CREB–Ser-133 involvesMAPKAP-K1. In fact, our preliminary data indicate that LPA-mediatedSER-133 phosphorylation of CREB is also sensitive to Ro-318220,which is known to inhibit MAPKAP-K1/RSK2 (Ha and Dhanasekaran,unpublished observation). However, since Ro-318220 can also inhibitother protein kinases such as MSK1, PKC, GSK3, and S6K1 [84], furtherstudies should identify the kinase downstream of ERK in LPA–Gα12-mediated activation of CREB. Finally, we show that inhibition of CREBin ovarian cancer cells leads to a marked decrease in proliferation com-pared to LPA-stimulated cells.We show that LPA-mediatedproliferationinvolves CREB.
It is worth noting that this is the first study, to our knowledge, toshow CREB is activated by LPA signaling in a Gα12-dependent mannervia the Ras–ERK signaling conduit (Fig. 8). The signaling paradigmpresented here is consistent with a recent observation that CREB isboth overexpressed and active in ovarian cancer patient samples and
Fig. 8. Schematic model for Gα12-dependent activation of CREB. LPA binds to one or moreLPA receptors that activate Gα12 and Gα12 thenmediates the activation of ERK1/2 via Ras.Activation of ERK1/2 leads to Ser133-phosphorylation of CREB. CREB, thus activated in anLPA–Gα12 dependent manner, stimulates the proliferation of ovarian cancer cells throughthe transcriptional activation of proliferation-specific genes. However, the kinase(s) thattransmits signals from ERK1/2 to CREB remains to be clarified.
RNAi-mediated silencing of CREB significantly reduced proliferation ofovarian cancer cells while having no effect on cell death [21]. In addi-tion, our data presented here identifies the potentialmechanism under-lying the hyperactive CREB levels seen in ovarian cancer patients. Due tothe fact that CREB can activate conservatively over 1000 different genes[63], it is a definite possibility that CREB can induce other phenotypic ef-fects of LPA besides proliferation, includingmigration, invasion and sur-vival, that warrants future investigation. Although our present studydoes not focus on the other identified transcription factors stimulatedby LPA via Gα12 (Tables 1 & 2), contextual characterization of thesetranscription may provide further insight to the mechanisms by whichGα12 and LPA signaling contribute to the oncogenesis and progressionof ovarian and other cancers. Since Gα12 stimulates the generationand coordination of multiple, often oncogenic, signaling inputs, it ismore likely that the transcription factors identified in our array analysesare activated by different branches of the signalingnetwork coordinatedby Gα12.
Since the identification of Gα12 as a potential oncogene that can in-duce neoplastic transformation [85], several studies including ours haveshown the ability of Gα12 in regulating multiple growth promoting ac-tivities [86]. In this regard, Gα12 acts as a typical oncogene in coordinat-ing the regulation multiple growth-promoting signaling nodes. Thefindings presented here provide another set of evidence that the aber-rant or asynchronous activation of Gα12 by increased levels of LPAcould lead to the activation of multiple oncogenic pathways (Table 1and 2). Although an activating mutation in Gα12 has not been reportedin any cancers including ovarian cancer, overexpression as well ashyper-activation of Gα12 has been observed in ovarian cancer celllines [19]. Therefore it appears that the increased receptor-activationof Gα12 is likely to play an oncogenic role in ovarian cancer pathophys-iology. Interestingly, this is in agreement with the earlier findings thatled to the characterization of Gα12 as an oncogene in Ewing's sarcomacells in which increased expression rather than mutational activationof Gα12 was shown to be the causative factor for oncogenic transforma-tion [85]. At present, we are pursuing studies to test whether anoverexpression or activation of Gα12 can be seen in ovarian cancer sam-ples in addition to defining the LPAR(s) that transmitsmitogenic signal-ing via Gα12 and the signaling network coordinated by this systeminvolving themultiple transcription factors identified here in promotingovarian cancer progression.
Overall, this study conclusively demonstrates silencing Gα12 can at-tenuate the signaling inputs involved in ovarian cancer cell proliferationand that Gα12–Ras–ERK-dependent signaling leads to activation of thetranscription factor CREB, in a cAMP-independentmanner. Additionally,our studies also suggest the interesting possibility that the delivery of aminigene or a small molecular inhibitor that could inhibit the LPAR–Gα12 interaction, or delivery of siRNA against Gα12 to ovarian cancercells could represent a novel way to block the pathogenic proliferationof ovarian and potentially other types of cancers that involve patholog-ical LPA-meditated signaling.
Acknowledgments
This work was supported by grants from the National Institutes ofHealth (CA116984, CA123233) and The World Class University projectfunded byMinistry of Education, Science and Technology Development,S. Korea [No. R32-2008-000-10098-0].
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