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Activin-A up-regulates type I activin receptor mRNA levels in human immortalized extravillous trophoblast… Chen, Victor T; Peng, Chun; Leung, Peter C Mar 24, 2003

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ralReproductive Biology and ssBioMed CentEndocrinologyOpen AcceResearchActivin-A up-regulates type I activin receptor mRNA levels in human immortalized extravillous trophoblast cells.Victor TS Chen1, Chun Peng2 and Peter CK Leung*1Address: 1Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, BC, Canada and 2Department of Biology, York University, Toronto, Ontario, CanadaEmail: Victor TS Chen - victorchen@shaw.ca; Chun Peng - cpeng@yorku.ca; Peter CK Leung* - peleung@interchange.ubc.ca* Corresponding author    ActivinFollistatinActivin ReceptorImmortalized Extravillous Trophoblast CellsCompetitive PCRAbstractActivin is known to play an important regulatory role in reproduction, including pregnancy. Tofurther examine the role and signaling mechanism of activin in regulating placental function, thesteady-state level of activin type I receptor (ActRI) mRNA in immortalized extravilloustrophoblasts (IEVT) cells was measured using competitive PCR (cPCR). An internal standard ofActRI cDNA for cPCR was constructed for the quantification of ActRI mRNA levels in IEVT cells.ActRI mRNA levels were increased in a dose-dependent manner by activin-A with the maximaleffect observed at the dose of 10 ng/ml. Time course studies revealed that activin-A had maximaleffects on ActRI mRNA levels at 6 hours after treatment. The effects of activin-A on ActRI mRNAlevels was blocked by follistatin, an activin binding protein, in a dose-dependent manner. In addition,inhibin-A inhibited basal, as well as activin-A-induced ActRI mRNA levels. These findings provideevidence, for the first time, that activin-A modulates ActRI mRNA levels in human trophoblast cells.IntroductionAlthough activins and inhibins were originally isolatedfrom follicular fluids and identified as stimulators and in-hibitors, respectively, of pituitary follicle-stimulating hor-mone (FSH), the identification of inhibins and activins ina wide variety of tissues suggest that these factors playmuch greater roles than the control of FSH secretion [1–5]. Also, it has become evident that these factors exerttheir effects mostly in autocrine/paracrine manners.Similar to other members of the transforming growth fac-tor-β (TGF-β) family, activins exert their actions by inter-acting with both type I and type II membrane serine/been shown to interact with activins [5] and their mRNAshave been detected in human placental trophoblast cells[8–10], as well as in choriocarcinoma cells [11]. Activins,particularly activin-A, has been shown to be produced byhuman placenta [5,11–13]. Many studies have demon-strated that activin-A plays important regulatory roles inhuman placenta, including stimulation of cytotrophob-last differentiation into invasive extravillous cytotrophob-last [14]; stimulation of progesterone [11,14–16], humanchorionic gonadotropin (hCG)[15,17,18], estradiol [11],gonadotropin-releasing hormone (GnRH) [15] and oxy-tocin [19] secretion. However, regulation of activin sign-aling at the receptor level has not been examined.Published: 24 March 2003Reproductive Biology and Endocrinology 2003, 1:29Received: 7 March 2003Accepted: 24 March 2003This article is available from: http://www.RBEj.com/content/1/1/29© 2003 Chen et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.Page 1 of 9(page number not for citation purposes)threonine kinase receptors [6,7]. Two type I (ActRI and Ac-tRIB) and two type II (ActRII and ActRIIB) receptors haveReproductive Biology and Endocrinology 2003, 1 http://www.RBEj.com/content/1/1/29The human placenta provides specialized functions dur-ing gestation that is critical for the development of the em-bryo and fetus. Among these important functions are theproduction of hormones, cytokines and growth factorsthat contribute to the gestational coordination of mater-nal, extraembryonic, and embryonic tissues. Develop-ment of the human placenta depends on proliferationand differentiation of certain trophoblast cells as well asinvasion to the endometrium and its vasculature by ahighly proliferative, migratory and invasive subpopula-tion of extravillous trophoblast (EVT) cells [20].To further study activin signaling in human placenta, wehave developed a competitive quantitative PCR to meas-ure ActRI mRNA levels in an immortalized EVT cell line,HTR8/SVneo. We report here the first evidence that ac-tivin-A regulates its own receptor mRNA levels in a dose-and time-dependent manner and this effect can beblocked by its binding protein, follistatin, or its antago-nist, inhibin-A in human placenta.Materials and MethodsCell lineThe use of placental tissue samples and cell lines was ap-proved by the Clinical Screening Committee for Researchand Other Studies Involving Human Subjects, Universityof British Columbia. The HTR-8/SVneo trophoblast cellline was generously provided by Dr. PK Lala (University ofWestern Ontario). This cell line was obtained from hu-man first trimester placenta explant cultures and immor-talized using SV40 large T antigen [21]. Cells werecultured in RPMI 1640 medium containing 10% FBS andantibiotics (Invitrogen Canada Inc., Burlinton, ON) aspreviously described [21].PCR PrimersOligonucleotide primers were synthesized based on thepublished sequences of the human ActRI [22], and β-actin[23]. The upstream primer (ActRI-1), 5'-GATGAGAAGT-CATGGTTCAGG-3', and downstream primer (ActRI-2),5'-TATGTTTGGCCTTTGTTGATC-3' were designed suchthat the predicted sizes of PCR products are 700 bp for na-tive ActRI cDNA. Primers are chosen to flank introns sothat the amplified ActRI cDNA is readily distinguishedfrom a possible contaminating genomic DNA. Anotherpair of primers, ActRI-3 (5'-GATGAGAAGTCATGGTTCAGG-3') and ActRI-4 (5'-TATGTTTGGCCTTTGTTGATC-3")were synthesized and used as nested primers to validatethe internal standard and measuring the regulation of Ac-tRI mRNA levels in HTR8/SVneo cells. The human β-actinprimers, used as another internal control to normalizecDNA amount in different samples, are AC1 upstreamand AC2 downstream primers of which sequence are 5'-Hormonal treatmentHuman recombinant activin-A, inhibin-A and follistatin-288 were kindly provided by Dr. Parlow at the NationalHormone and Pituitary Program. The HTR8/SVneo cellswere plated in 24-well culture plates in FBS supplementedRPMI medium. Two days after plating when cells reachabout 70% confluence, the culture medium was replacedwith serum-free RPMI 1640. For dose-response studies,cells were treated with various doses (0.1 to 30 ng/ml) ofactivin-A for 12 hours. For time-course experiments, cellswere cultured in the presence or absence of 10 ng/ml ac-tivin-A for 1, 3, 6, 12, 24 hours. To determine the interac-tion between activin and follistatin, cells were treated withactivin-A (10 ng/ml) alone, or activin-A with differentconcentrations of follistatin-288 (10, 20, 50, or 100 ng/ml) for 6 h. Similarly, to examine the effect of inhibin onbasal and activin-A-stimulated ActRI mRNA levels, cellswere treated with Activin-A, inhibin-A, either alone or incombination for 6 h. There are four wells in each treat-ment group and each experiment was conducted threetimes.Construction of an internal standard for comparative PCRCloning of native ActRI PCR product using primers ActRI-1 and ActRI-2 was performed by ligating the PCR productinto a pDirect (BD Biosciences Clontech, Palo Alto, CA),followed by sequencing analysis to confirm the identity.To construct an internal standard for comparative PCR, a156 base pair segment was removed from the cloned na-tive ActRI PCR product by restriction digestion using StyIand Eco47III (Fig. 1). With the same pair of primers isused in PCR, the subcloned mutant ActRI template yield-ed a PCR product 156 base pairs smaller than that fromthe native cDNA template.Total RNA extraction, Reverse transcription and Compet-itive PCRTotal RNA from HTR-8/SVneo cells was extracted usingthe RNaid Kit (Bio/Can Scientific Inc., Mississauga, ON)following manufacture's suggested procedures. Two µg oftotal RNA was used to synthesized cDNA using FirstStrand cDNA Synthesis Kit (Amersham Pharmacia Bio-tech Inc., Oakville, ON) as previously described [9]. cPCRis similar to PCR except that it includes an internal stand-ard to monitor the variation between different tubes aswell as among different experiments. In the cPCR reac-tion, target sequences of native cDNA or sample cDNAcombined with 0.5 pg/µl of the internal standard of ActRIcDNA (internal standard, IS) are co-amplified in a tubewith a volume of 50 µl containing 5 µl of 10 × PCR buffer[200 mM Tris-HCl (pH 8.4), 500 mM KCl], 1.5 µl MgCl2(50 mM), 1 µl dNTPs (10 mM), 0.5 µl Taq DNA polymer-ase (5 units/µl) (Gibco BRL), 38 µl sterile Milli-Q water, 1Page 2 of 9(page number not for citation purposes)GGACCTGACTGACTACCTCATGAA-3' and 5'-GGT-GGAAGGTGGTCAACACCTAG-3', respectively.µl each (5 µM) of specific primers (ActRI-3 and ActRI-4)and equal volume (1 µl) of IS and cDNA sample or nativeReproductive Biology and Endocrinology 2003, 1 http://www.RBEj.com/content/1/1/29cDNA. PCR amplifications were performed for 35 cyclesin a Perkin-Elmer/Citus DNA thermal cycler with denatur-ation at 94°C for 30 seconds, annealing at 53°C for 30seconds and extension at 72°C for 90 seconds. The finalcycle was followed by a 15 minutes extension step at72°C. PCR for β-actin was conducted for 25 cycles usingsimilar conditions as described for ActRI expect that 2 µlof cDNA samples were used and annealing temperaturewas 55°C.Assessment and quantification of ActRI mRNA levelsAfter cPCR, 10 µl from each PCR reaction was subjected togel electrophoresis and recorded with a negative film. ThemRNA levels were used to normalize the samples for var-iation in cDNA concentrations. Statistical significance ofthe data was determined by one-way analysis of variancefollowed by Scheffe's test. p < 0.05 was consideredsignificant.ResultsValidation of cPCRTo validate the cPCR for measurement of ActRI mRNA lev-els, PCR was conducted using a constant amount of IS(0.5 pg) and an increasing amount of native ActRI cDNA(0.01, 0.1, 1, 10 pg). As shown in Fig. 2A, the ratio of bandintensity (native ActRI cDNA/IS), measured by densitom-Figure 1Schematic structure of the ActRI cDNA and primers used in PCR or cPCR. An 156 bp internal deletion has been done between the restriction sites Sty I and Eco47 III. Co-amplification of the ActRI native cDNA and mutant cDNA template using primer ActRI-1 and primer ActRI-2 resulted in 700 bp and 544 bp PCR product, respectively. Co-amplification of the ActRI native cDNA and mutant cDNA template using primer ActRI-3 and primer ActRI-4 resulted in 650 bp and 494 bp PCR prod-uct, respectively.5' 3'5' 3'Native ActRI cDNA1478Mutant ActRI cDNA (Internal Standard)ActRI-1ActRI-1ActRI-2ActRI-2Sty I Eco47III1318 1478132213221730173010311031ActRI-3 ActRI-4ActRI-3 ActRI-4Page 3 of 9(page number not for citation purposes)intensity of PCR products was determined using a densit-ometer and expressed as the ratio of ActRI and IS. β-actinetry scanning, was nearly linear when plotted as a func-tion of the amount of native ActRI cDNA in the cPCRReproductive Biology and Endocrinology 2003, 1 http://www.RBEj.com/content/1/1/29reaction. To control for variation in sample cDNA concen-trations, β-actin mRNA levels in each sample was alsomeasured and used to normalize the ActRI mRNA levels.To validate a semi-quantitative PCR for β-actin, PCR wasfirst conducted for 20 to 40 cycles. There is a linear rela-tionship between the amount of PCR product generatedand the number of PCR cycle when PCR was conductedbetween 20 to 30 cycles (data not shown) and therefore,subsequent β-actin PCR was conducted for 25 cycles. Inaddition, when different amount of cDNA (from 0.5 to 4µl) was used in PCR, there is a linear relationship betweenthe amount of initial cDNA and PCR product (Fig. 2B).Activin-A stimulated ActRI mRNA expressionThe ActRI mRNA level, measured at 12 hours after treat-ment, was increased in a dose-dependent manner by ac-tivin-A (Fig. 3). A significant stimulation (P < 0.05) ofActRI mRNA levels was observed at the dose of 3, 10 and30 ng/ml. When IEVT cells were treated with activin-A (10ng/ml) for different period of time, a significant increasein ActRI mRNA level was found at 3 hours (P < 0.05) andthe maximal response observed at 6 hours (P < 0.01) aftertreatment. By 24 h after treatment, ActRI mRNA levels inactivin-A treated cells were not significantly different fromthose in the control cells (Fig. 4).Figure 2Validation of PCR. A) Competitive PCR for ActRI was performed using a constant amount of internal standard (0.5 pg) and increasing amount of native ActRI cDNA. A linear relationship was found when the ratio of native ActRI cDNA versus internal standard was plotted as a function of the amount of native ActRI cDNA. B) β-actin PCR was conducted using different amount of template cDNA. There is a linear relationship between the amount of cDNA used and the amount of PCR product generated.Molecular  Negative     0.01         0.1             1          10  pg cDNAMarker        control        0.5          0.5 0.5 0.5 pg IS1018bp506bp652bp492bpA.0.01          0.1                   1                 10Native ActRI cDNA (pg)Log (ActRI/Internal Standard)-1                          0                          1B.0.5             1             2             4    µl cDNA524bp246810121416cDNA (µl)Spot density(X105 )4210.5Page 4 of 9(page number not for citation purposes)Follistatin blocked the effect of activin-A on ActRI mRNA expressionThe activin-binding protein, follistatin, has been shown toneutralize the effects of activin in several biologicalReproductive Biology and Endocrinology 2003, 1 http://www.RBEj.com/content/1/1/29systems [24–27]. To determine the specificity of activin-Aaction, we examined the effects of increasing concentra-tions of recombinant human follistatin-288 on activin-A-induced ActRI mRNA levels. Treatment with activin-Aalone resulted in a significant increase in ActRI mRNA lev-els (P < 0.05); however, when activin-A and follistatinwere added to the culture together, the activin-A-stimulat-ed ActRI mRNA levels were decreased. At the doses of 50and 100 ng/ml, follistatin-288 completely blocked the ef-fects of activin-A on the ActRI mRNA levels (Fig. 5).Inhibin-A antagonized the effect of activin-A on ActRI mRNA expressionof inhibin-A on ActRI mRNA levels. Inhibin-A (10 ng/ml)induced a 60% reduction in ActRI mRNA levels (p < 0.01)within six hours of treatment. When inhibin-A was ap-plied together with activin-A (10 ng/ml), significantreductions in activin-A-stimulated ActRI mRNA levelswere observed (Fig. 6).DiscussionFor many years the placental cytotrophoblasts obtainedby disaggregation of placental tissue have served as themost widely investigated model for the study of placentalfunction in vitro. However, the purity of cytotrophoblastswas not consistent. The IEVT cells used in the study exhibitFigure 3Dose-dependent stimulation of ActRI mRNA levels by activin-A. Cells were incubated for 12 h in the absence or presence of activin-A (0.1–30 ng /ml). cPCR was performed to measure ActRI mRNA levels. The data were pooled from 3 separate exper-iments and expressed as a percentage of control value (mean ± SEM). Data were normalized for β-actin mRNA levels meas-ured by PCR. *(p < 0.05) or **(p < 0.01) versus control.0100200300400ActRImRNAlevels(%ofControl)0.1 1 3 10 30[Activin-A] (ng/ml)Control* ***Page 5 of 9(page number not for citation purposes)Inhibin-A has opposing effects as activin-A in many bio-logical systems. In this study, we also examined the effectsimilar cellular function to normal EVT cells [21] and theyReproductive Biology and Endocrinology 2003, 1 http://www.RBEj.com/content/1/1/29provide a promising model to study the function of EVTcells. Using these cells, we have demonstrated that activin-A stimulates, while inhibin-A inhibits, ActRI mRNA levels.Our results showed that ActRI mRNA accumulation at 12hours after treatment was stimulated in a dose-dependentmanner by activin-A. The time course of the activin-A ef-fect on ActRI mRNA showed responses at 3 to 12 hours af-ter treatment, with a maximal increase occurring at 6hours. This response pattern of activin-A on ActRI mRNAsuggests that activin-A exerts a positive feedback effect onits own receptor and that the action of activin-A on ActRImRNA level is transient. The transient effect of activin-A islikely due to the termination of activin signaling. Activinby activin, and in turn, Smad7 inhibits further activin sig-naling by blocking the phosphorylation of Smad2/3 byactivin type I receptors [28,29]. In addition, activin signal-ing can also be terminated by the degradation of Smadproteins through the ubiquitination pathway [30].The changes in ActRI mRNA levels as determined by cPCRin this study could have resulted from changes in the tran-scriptional rate of ActRI gene and/or the stability of the Ac-tRI mRNA. Activin is known to regulate gene expression atthe level of transcription via the Smad signaling pathway[5–7] and expression of Smads in trophoblast cells includ-ing the HTR8/SVsno have been demonstrated [31,32], it istherefore possible that activin-A may regulate its receptorFigure 4Time course effects of activin-A on ActRI mRNA levels. IEVT cells were incubated with or without activin-A (10 ng/ml) for 1, 3, 6, 12, 24 hours. cPCR was performed to measure ActRI mRNA levels and data were also normalized for β-actin value. *,(p < 0.05) versus control. **,(p < 0.01) vs control. Data present mean ± SEM of three experiments.0100200300400500control 1 h 3 h 6 h 12 h 24 hDuration of activin-A treatment****ActRImRNA levels (% of Control)Page 6 of 9(page number not for citation purposes)signaling has been shown to be terminated by negativefeedback mechanisms involving the activation of Smad7expression at the level of gene transcription. FutureReproductive Biology and Endocrinology 2003, 1 http://www.RBEj.com/content/1/1/29studies are also needed to confirm that there is a correlat-ed change at the protein level.Although this is the first study demonstrating that activin-A regulates its own receptor expression in human placen-ta, several studies in other tissues have also observed mod-ulation of activin signaling by activin itself. In the ratpituitary, activin-A stimulated ActRI and ActRIIB mRNAlevels without altering ActRII mRNA expression [33]. Onthe other hand, activin-A downregulated ActRIB, ActRIIand ActRIIB mRNA levels in a cell line derived from ap53(-/-)inhibin-α (-/-) mouse testicular tumor [34].anatomically and temporally limit the local activities ofactivin. Follistatin mRNA transcripts and immunoreactiv-ities have been detected in placental trophoblast cells [16]Similar to many other systems [24,25], follistatin alsoneutralizes activin actions in human placenta [16]. In thepresent study, we also found that the stimulatory effect ofactivin-A on ActRI mRNA levels could be completelyblocked by follistatin. Since it is known that follistatin in-hibits activin actions by binding to activin and thus pre-venting the interaction between activin and its type IIreceptor [35] the neutralization of activin-A action on Ac-tRI mRNA levels by follistatin suggests that the action ofactivin is a receptor-mediated event.Figure 5Interaction between activin-A and follistatin-288 on ActRI mRNA expression. Cells were incubated for 6 hours in the presence or absence of activin-A (10 ng/ml) alone, or activin-A(10 ng/ml) incombination with increasing concentrations of follistatin (10–100 ng/ml). cPCR was performed to obtain ActRI mRNA levels which were then normalized for β-actin mRNA levels meas-ured by PCR. Data present mean ± SEM of three experiments. Different letters indicate statistically significant differences (p < 0.05).0100200300400Activin-A (ng/ml)        0  10 10 10 10 10Follistatin (ng/ml)       0 0 10 20 50   100TreatmentabbbaaActRImRNA levels (% of Control)Page 7 of 9(page number not for citation purposes)Follistatin is widely distributed and produced by many ac-tivin-responsive tissues [1,25] and may, therefore, serve toReproductive Biology and Endocrinology 2003, 1 http://www.RBEj.com/content/1/1/29Inhibin and activin possess opposing activities in severalbiological systems including pituitary FSH secretion,erythroid differentiation, and gonadal sex-steroidproduction [4]. In the present study, we found that inhib-in-A caused a 60 % reduction in basal ActRI mRNA levelsand completely inhibited activin-A-induced ActRI mRNAexpression. This finding is in agreement with the observa-tion that inhibin counteracts the effects of activin on cul-tured first trimester trophoblast cells [17].In summary, we have demonstrated that in activin-A up-regulates mRNA levels of it own receptor ActRI in humanextravillous trophoblast cells. Since activin-A has beenA suggests a positive feedback regulatory mechanism bywhich activin-A modulates its own signaling in these cells.AcknowledgementsThis research was supported by grants from the Canadian Institutes of Health Research to PCKL and CP. PCKL is a Distinguished Scholar of the Michael Smith Foundation for Health Research. We thank NHPP and Dr. Parlow for providing recombinant human activin-A, inhibin-A and follistatin-288.References1. DePaolo LV, Bicsak A, Erickson GF, Shimasaki S and Ling N Follista-tin and activin: a potential intrinsic regulatory system withindiverse tissues Proc Soc Exp Biol Med 1991, 18:500-5122. Bilezikjian LM and Vale WW Local extragonadal roles of activinsFigure 6Effects of inhibin-A on basal and activin-A-induced ActRI mRNA levels in IEVT cells. Cells were treated with inhibin-A (10 ng/ml), activin-A (10 ng/ml) or the combination of both for 6 h. ActRI mRNA levels were measured by cPCR and then normalized for β-actin mRNA levels. Data present mean ± SEM of three experiments. Different letters indicate statistically significant dif-ferences (p < 0.05).Activin-A (ng/ml)        0  10               0 10 10Inhibin-A (ng/ml)        0 0               10 10 50   050100150200250300acbActRImRNA levels (% of Control)TreatmentdaPage 8 of 9(page number not for citation purposes)shown to stimulate the differentiation of extravillous tro-phoblast cells [14], the up-regulation of ActRI by activin-Trends Endocrinol Metab 1992, 3:218-223Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central Reproductive Biology and Endocrinology 2003, 1 http://www.RBEj.com/content/1/1/293. 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