Prolactin-Mediated Inhibition of 20α-Hydroxysteroid Dehydrogenase Gene Expression and the Tyrosine Kinase System

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 235, 587592 (1997)ARTICLE NO. RC976833

Prolactin-Mediated Inhibition of 20a-HydroxysteroidDehydrogenase Gene Expression and theTyrosine Kinase System

L. Zhong,* T. G. Parmer,* M. C. Robertson, and G. Gibori*,1,2

*Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612;and Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada

Received April 25, 1997

20aOH progesterone or 17OH progesterone as sub-The rat luteal 20a-hydroxysteroid dehydrogenase strate and the physiological importance of the 20a-

plays a key role at catabolizing progesterone and at HSD activity in these enzymes is not yet clear. In con-decreasing the level of this steroid secreted by the ova- trast the ovarian 20a-HSD utilizes primarily proges-ries. Throughout pregnancy and before parturition terone as substrate and plays an important role in inac-neither the mRNA nor the protein for this enzyme

tivating progesterone synthesized by the rat corpus lu-could be detected. In this investigation we set to exam-teum leading to a switch from the secretion ofine whether PRL and PRL-like hormone from placen-progesterone, a steroid essential for the maintenancetal origin silence the expression of this gene andof pregnancy, to that of 20aOH progesterone, which iswhether PRL action involves tyrosine kinase activityunable to sustain fetal survival (6-9). The pattern ofand/or de novo protein synthesis. The results revealedluteal 20a-HSD enzyme activity contributes to thethat PRL and PRL-like hormone from rat placentalchanges of progesterone levels in the circulation duringorigin (rPL-1 and rPL-2), but not rat growth hormone,gestation and ultimately controls the process of preg-caused a rapid and profound inhibition of 20a-HSD

mRNA expression in highly luteinized granulosa cells. nancy. Indeed it was known for years (9,10) that little,Immunoprecipition and western blot analysis indicate if any, 20a-HSD activity is found in the rat corpus lu-that PRL-R associates with JAK2 and Stat5, and this teum throughout pregnancy and that enzyme activityassociation is increased whithin 30 seconds with PRL becomes substantial just before parturition. The devel-treatment. Althrough both JAK2 and Stat5 were phos- opment of a highly specic antibody (11) and the suc-phorylated on tyrosine upon PRL treatment, the PRL cessful cloning of rat ovarian 20a-HSD by our and othermediated inhibition of 20a-HSD was not reversed by laboratories (4,5) have led us to demonstrate that theeither tyrosine kinase inhibitors, AG18 and genistein, absence of enzyme activity throughout pregnancy andbut was largely reversed by the protein synthesis in- its appearance just before parturition is not due to acti-hibitor cycloheximide. In summary, results of this in- vation/deactivation of already present enzyme butvestigation indicate that although PRL can activate

rather to lack of 20a-HSD gene expression throughoutthe JAK2/Stat5 system in the corpus luteum, the downgestation and to the rather abrupt and massive expres-regulation of 20a-HSD mRNA by PRL does not appearsion of both mRNA and protein of 20a-HSD just beforeto involve tyrosine kinase activity but depends on departurition.novo synthesis of protein(s). q 1997 Academic Press

Several investigators have examined the hormonalregulation of 20a-HSD activity and have establishedan important inhibitory role for PRL (12,13). Our re-

Several enzymes with intrinsic 20a-hydroxysteroid sults (14) have revealed that PRL inhibition of enzymedehydrogenase (20a-HSD) activity have been recently activity is due to an inhibitory effect on 20a-HSD genecloned. They are the 17bHSDs (1,2), the testicular al- expression. Indeed, sustained PRL treatment in vivodose reductase (3) and the ovarian 20a-HSD (4,5). The causes the virtual disappearance of 20a-HSD mRNA17bHSDs and the aldose reductase utilize either and protein from the corpus luteum. This led us to

suggest that PRL in the rst half of pregnancy andrat placental lactogen in the second half silence the1 Corresponding author. Fax: 312-996-1414; E-mail: GGibori@expression of this gene.uic.edu.

2 NIH Merit Awardee (HD 11119). PRL has been shown to stimulate the expression of

0006-291X/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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Vol. 235, No. 3, 1997 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 1. Inhibitory action of PRL and PRL-like hormones on 20a-HSD message in luteinized granulosa cells. A. hCG primed granulosacells were cultured in the presence of different doses of PRL (0.011 mg/ml) or absence of PRL for 12 h. Total RNA was extracted fromtriplicate dishes. 20 mg/lane RNA was fractionated in 1% agarose gel and transferred to nylon membrane and hybridized with 20a-HSDcDNA. 18 S ribosomal RNA is shown in the inset to assess the loading. B. Cells were treated with either rPL-1 (0.0110 mg/ml) or rPL-2(0.011 mg/ml) for 24 h. Total RNA was isolated as described in Materials and Methods. 20a-HSD mRNA was detected by Northern blotanalysis. C. Cells were treated with either PRL (1 mg/ml) or rat growth hormone (1 mg/ml) for 24h. 20a-HSD mRNA was detected usingNorthern blot analysis. D. RNA was reverse transcribed and amplied 20 cycles by PCR as described in Materials and Methods. Theamplied product of 20a-HSD is 440 bp long. L19 was used as an internal control.

through an afnity column. rPL-2 was puried from the conditionedseveral genes by activating JAK2/Sta5 system (15,16).medium of rat choriocarcinoma (RCHO) cells using immunoafnitySince PRL action on 20a-HSD is inhibitory rather thanchromatography as previously described (18).stimulatory and since PRL-mediated regulation of this

Granulosa cell culture. 27-28 days old immature female Sprague-gene is totally unknown, we set out to examine whetherDawley rats (Sasco, Madison, WI) were injected with 0.15 IU hCGPRL activates the JAK/Stat system in highly luteinized sc twice daily for two days, followed by 10 IU hCG i.v. on the third

granulosa cell and whether 20a-HSD inhibition in- day. Seven hours later ovaries were isolated and incubated sequen-volves tyrosine kinase mediated phosphorylation. tially in 6 mM EGTA in DMEM/F-12 and 0.5 M sucrose in DMEM/

F-12. Individual follicles were popped and granulosa cells were col-lected. Cells were cultured at 377C in a humidied atmosphere withMATERIALS AND METHODS 95% air and 5% CO2 in 60 mm petri dishes (Corning, NY) at about81105 cells/ml in the Dulbecco's modied Eagle's medium-Ham's F-

Materials. DMEM/F-12, trypan blue, 8-bromo-cAMP, AG18, so- 12 (DMEM/F-12, 1:1) with 15 mM HEPES, 3.15 g/L glucose, 1% fetaldium vanadate, cycloheximide and hCG were obtained from Sigma bovine serum, 100 IU penicillin G, 100 mg/ml streptomycin and 0.25Chemical Co. (St. Louis, MO). Genistein was obtained from ICN

mg/ml amphotericin B. After three days of culture medium wasBiomedicals (Aurora, Ohio). Antibiotic-antimycotic solution was ob- changed, cells were treated with different hormones or reagents, andtained from GIBCO BRL (Gaithersburg, MD), Taq DNA polymerase either RNA or proteins were extracted.from Perkin-Elmer Corporation (Foster City, CA), Klenow enzyme

Northern blot analysis. Total RNA was extracted and fraction-from Boehringer Mannheim Biochemicals (Indianapolis, IN), fetalated by electrophoresis in 1% agarose gels and blotted to nylon mem-bovine serum (FBS) from Hyclone (Logan, UT), GeneScreen Plusbranes. Ethidium bromide staining indicated whether ribosomalnylon membrane from New England Nuclear System (Boston, MA)RNAs were intact and whether equal amounts of RNA were loadedand [a-32P] deoxy-CTP and ECL detection kit from Amersham (Ar-in each lane. A full length rat 20a-HSD cDNA (4) was labeled usinglington Heights, IL). Antibodies to phosphotyrosine (PY20) and Stat5the random primer DNA-labeling method according to the manufac-were purchased from Transduction Laboratories (Lexington, KY).turer. Blots were prehybridized overnight at 427C in a solution con-JAK 2 antibody was purchased from Upstate Biotechnology Incorpo-taining 40% formamide, 61 SSC, 51 Denhardt's, 20 mM Na2HPO4,rated (Lake Placid, NY). Ovine prolactin (NIDDK, ovine PRL-18, 30pH 7.0, 0.2% Sodium Dodecyl Sulfate and 100 mg/ml heterologousIU/mg) and GH (NIDDK, rat GH-B-14-SIAFB, 1.8 IU/mg) were a giftDNA. Hybridization was completed in the same solution containingfrom NIDDK.32P-labeled cDNA probe (11106 CPM/ml) at 427C overnight. BlotsPurication of rPL-1 and rPL-2. The entire process for generat- were washed and then exposed to Kodak X-Omat lms (Eastmaning and purifying rat placental lactogen-I was reported before (17). Kodak, Rochester, NY) with intensifying screen at 0807C.Briey, rPL-1 cDNA was inserted into pMSXND expression vector

and stably transfected into CHO cells. Medium was collected from Immunoprecipitation and Western blot analysis. Cells weretreated with PRL for 0 to 60 minutes, washed twice with cold PBSthe transfected cells. Protein in the medium was concentrated. rPL-

1 was puried from the medium by passing the concentrated protein and lysed with 1X immunoprecipitation buffer (1% Triton X-100, 150

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mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mMsodium vanadate, 0.2 mM PMSF, 0.5% NP-40). 500 mg of total celllysates were incubated with 10 mg of monoclonal PRL-R antibody U6(kindly provided by Dr. Paul Kelly) overnight, and continued foranother 24 h after the addition of 50 ml of protein G sepharose 4 fastow. The pellet was washed three times with 11 immunoprecipita-tion buffer, resuspended in 20 ml of 21 concentrated electrophoresissample buffer and boiled for 5 minutes. The supernatant was loadedon a 7.5% SDS-PAGE gel and transferred to nitrocellulose mem-brane. Immunoblotting was performed by blocking non-specic bind-ing with 5% milk in TBS buffer containing 0.1% Tween 20. Blotswere then incubated 1 h with primary antibody, followed by a seriesof washes and incubated with a secondary antibody linked to horse-radish-peroxidase for 1 h. After extensive washing, blots were devel-oped using an enhanced chemiluminescence Western blotting detec-tion system (Amersham Corp., Arlington Heights, IL) and exposedfor 1-5 seconds to X-ray lms.

RT-PCR. 1 mg of total RNA was reverse transcribed at 427C in a20 ml reaction mix (11PCR buffer, 3.75 mM MgCl2, 0.2 mM dNTP,100 pmol Random hexamer primers, 0.01 M DTT, 200 units M-MLV

FIG. 2. Time course of PRL action of 20a-HSD mRNA expression.reverse transcriptase). A mix containing oligonucleotide primers (50Luteinized granulosa cells were treated with 1 mg/ml PRL for 0, 4,pmol each), [a-32P]d-CTP (2 mCi of 3000 Ci/mmol) and Taq DNA8 and 12h. Total RNA was isolated from triplicate dishes. 20a-HSDpolymerase (2.5 U) were added to each reaction. The total volumemRNA levels were detected by RT-PCR with ribosomal L19 used aswas made up to 90 ml with 11PCR buffer and the sample was overlaidan internal standard.with light mineral oil. Amplication was carried out for 20 cycles

using 957C for denaturing, 657C for annealing and 727C for extensionin a 480 Perkin-Elmer/Cetus thermal cycler. The RT-PCR reactionincluded L19 ribosomal protein mRNA primers to normalize the HSD gene expression in cultured luteinized cells, wedata. The conditions were such that amplication of the product was

examined whether PRL causes the association of JAK2in the exponential phase and the assay was linear with respect tothe amount of input RNA. and Stat5 with the PRL-R. Results shown in Fig. 3A

indicated that JAK2 co-precipitated with the PRL-Rdemonstrating an association of JAK2 with the PRL-RRESULTSin luteinized cells. This association increased within 30seconds of PRL treatment, remained elevated for 30Previous studies (11,14) have shown that adminis-

tration of sustained levels of PRL to hypophysecto- min and decreased thereafter. Similar results were ob-served with Stat5 (Fig. 3B). Since the association ofmized pregnant rats causes, within three days of treat-

ment, a drop in 20a-HSD expression. To examine the JAK2 and Stat5 with the PRL-R causes their phosphor-ylation on tyrosine, we examined the state of phosphor-time course and dose-related response of PRL action

and also to examine whether PRL-related hormones ylation of JAK2 and Stat5 by immunoblotting withphosphotyrosine antibody. Phosphorylation on tyrosinedown regulate the expression of this gene, we utilized

highly luteinized granulosa cells in culture. These cells of both JAK2 and Stat5 increased with PRL treatment(Fig. 3C).express both the short and the long form of the PRL-

R and also express the 20a-HSD gene (data not shown). Once we established that PRL causes the associationof JAK2 and Stat5 with the PRL-R and the phosphory-As shown in Fig. 1A within 12 h of culture relatively

low doses of PRL (0.01-1 mg/ml) caused a substantial lation of these proteins on tyrosine, we examinedwhether the inhibition of 20a-HSD by PRL is throughdecrease in 20a-HSD mRNA. Time course analysis

shown in Fig. 2 indicated that PRL inhibition of 20a- the tyrosine kinase system. For this purpose we usedtwo tyrosine kinase inhibitors (AG18 and genistein)HSD was rapid and occurred within 4 h of PRL treat-

ment. Since in pregnant rat PRL ceases to be secreted which have been used previously to prevent JAK medi-ated phosphorylation and PRL stimulation of gene ex-at mid pregnancy whereas the placenta produces se-

quentially PRL-like hormones termed placental lacto- pression (15,16). As shown in Fig. 4A PRL alonecaused, as expected, a marked inhibition of 20a-HSDgen I (rPL-1) and placental lactogen II (rPL-2) (19), we

examined the effect of both these hormones on 20a- mRNA expression (lane 2). However surprisingly AG18at both doses used (lanes 3 & 4), did not reverse theHSD expression. As shown in Fig. 1B, both rPL-1 and

rPL-2 caused a down regulation of 20a-HSD mRNA inhibitory effect of PRL. Furthermore, addition of 20mM AG18 alone caused a clear inhibition in 20a-HSDlevels. Treatment of these cells with rat growth hor-

mone, which is highly homologous to PRL but does not mRNA. Higher doses of AG18 (50, 80 and 100 mM) werealso unable to reverse PRL inhibition (data not shown).bind to PRL-R, had no inhibitory effect that could be

detected by either Northern blot analysis (Fig. 1C) or Similar results were obtained when cells were culturedin the presence of genistein (Fig. 4B). The inhibitoryRT-PCR (Fig. 1D).

Once we established that PRL down regulates 20a- effect of PRL on 20a-HSD mRNA was not reversed by

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genistein at any dose used (lanes 3-5). As a matter offact, genistein alone (lanes 6-8) caused an inhibition of20a-HSD expression. This inhibition appeared re-versely related to the dose used.

Since tyrosine kinase activation does not appear tomediate PRL-mediated inhibition of 20a-HSD gene ex-pression, we investigated whether protein synthesis isnecessary for such an effect by culturing luteinizedcells in the presence of PRL alone or PRL and cyclohex-amide. As shown in Fig. 5, the inhibitory action of PRLon 20a-HSD expression was largely reversed by cyclo-hexamide.

DISCUSSION

The results presented herein have revealed that theinhibition of 20a-HSD mRNA expression in the ratovary is PRL-specic, rapid and appear not to involve

FIG. 4. Effect of tyrosin kinase inhibitors, AG18 and Genistein,on PRL regulation of 20a-HSD message. A. Luteinized granulosacells were cultured with medium alone, 1 mg/ml PRL, PRL/AG18(10 mM), PRL/AG18 (20 mM), AG18 (10 mM) or AG18 (20 mM) for12 h. AG18 was added into the media 30 minutes before PRL treat-ment. 20a-HSD mRNA was detected using Northern blot analysis.B. Cells were treated with different doses of genistein (20, 50, 80 mg/ml) in the absence or presence of 1 mg/ml PRL. Genistein was addedinto the media 30 minutes before PRL treatment. 20a-HSD mRNAwas detected using RT-PCR, and the size of the amplied 20a-HSDmRNA was 440 bp. L19 was used as a internal control.

tyrosine kinase activity. Pituitary PRL and placentalPRL-related hormones were able to down regulate 20a-HSD expression whereas rat growth hormone which ishighly homologous to PRL but does not bind to PRL-Rhad no inhibitory action. This implies that the totalinhibition of 20a-HSD gene expression seen through-out pregnancy (14) is due to PRL and to PRL-like hor-mones secreted by the placenta. The luteotropic effectof prolactin-like molecule(s) secreted by the placenta iswell recognized. The rat placenta secretes many pro-

FIG. 3. Association and activation of JAK2 and Stat5 by PRL. teins structurally related to pituitary PRL referred toA: Cells were treated with 1 mg/ml PRL for 0 to 60 minutes. Cellular as rat placental lactogen (rPL-1, rPL-1 variant, rPL-2)protein were then immunoprecipitated with PRL-R monoclonal anti-

and PRL-like protein (PLPA, PLPB and PLPC) (19).body (U6) and immunoblotted with a polyclonal JAK2 antibody (Up-To date, only rPL-1 and rPL-2 have been shown to bindstate Biological Incorporation) followed by second antibody linked to

HRP. The detection was carried out by enhanced chemiluminescence to PRL receptor whereas the role of the other PRL-(ECL, Amersham). B: The blot was stripped and immunoblotted with related proteins remains undened. It is well estab-a monoclonal antibody against Stat5 (Transduction Laboratories). lished that PRL is secreted only until mid pregnancy,C: The same blot was stripped again and immunoblotted with a

followed by rPL-1 until day 13 and rPL-2 from thismonoclonal phosphotyrosine (P-TYR) antibody (Transduction Labo-ratories). stage until the end of pregnancy. Despite the apparent

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20a-HSD through the tyrosine kinase system is IL-6is not yet clear. However, IL-6 is known to decreaseprogesterone secretion by ovarian cells (25,26) and re-cent investigation from our laboratory has shown thatbefore parturition, the rise in 20a-HSD is accompaniedby an abrupt expression of IL-6 mRNA in the corpusluteum (27). In addition, the corpus luteum and lutein-ized granulosa cells express IL-6 receptor and respondto IL-6 with an increase in 20a-HSD mRNA (27). Ourrecent isolation of the promoter for 20a-HSD gene (28)indicates the presence of two GAS-like sites. Whetherthese GAS-like sites are response elements for IL-6stimulation remains to be investigated.

Whereas inhibition of tyrosine kinase did not reverseFIG. 5. CHX effect on PRL regulation of 20a-HSD message. Lu- the PRL-mediated down regulation of 20a-HSD, cyclo-

teinized granulosa cells were cultured with medium alone, 1 mg/ml hexamide, the protein synthesis inhibitor, did reversePRL, PRL and 10 mg/ml CHX for 12 h. CHX was added 30 minutes

in large part this PRL effect suggesting that the syn-before PRL treatment. 20a-HSD message was analyzed by Northernthesis of protein synthesis mightay be necessary forblot.PRL action on 20a-HSD gene expression. The identityof such putative protein is yet to be uncovered. WhetherPRAP, the novel protein we have recently identiedcapacity of both rPL-1 and rPL-2 to bind to PRL-R only

rPL-1 was considered to have PRL-like activity in the and cloned (29), is one such protein remains a possibil-ity. PRAP (for PRL Receptor Associated Protein) wascorpus luteum (20). This conclusion was based on the

fact that the luteotropic action was found in placental shown to bind to the short form of the PRL receptor.Although the short form of the PRL-R is expressed inextract and serum obtained only from rats between

days 11-13 of pregnancy. The cloning, expression and several tissues including the rat corpus luteum (30),its role is yet not known and the mechanism by whichpurication of rPL-1 and rPL-2 have led us to reexam-

ine the PRL-like activity of rPL-2. The results indicate PRL signaling is transduced through this receptor isyet to be established. It is possible that up regulationthat rPL-2 is also a potent inhibitor of 20a-HSD mRNA

expression. of gene expression by PRL in the ovaries such as a2-MG (16) may involve the long form of the receptor andIt is clear from this investigation that PRL triggers

in luteinized cells an increase in the association of the JAK/Stat system, whereas down regulation of the20a-HSD gene may involve the short form of the PRLJAK2 and Stat5 with the PRL receptor and their phos-

phorylation on tyrosine within 30 seconds. Recently, receptor and PRAP.two Stat5 genes designated Stat5a and Stat5b (21,22)as well as two splice variants of Stat5a (23) have been ACKNOWLEDGMENTScloned. The Stat5 antibody used in this investigation

We want to thank Dr. Paul Kelly for his PRL-R antibody andrecognizes all members of the Stat5 family and doesNIDDK for ovine PRL and rat growth hormone. We would like tonot allow for the identication of the type of Stat5 acti-express our sincere thanks to Jingsong Ou for his technical assis-vation by PRL. Nevertheless despite the activation of tance with the RT-PCR. We also thank Linda Alaniz for her photo-

JAK2/Stat5 by PRL, the inhibition of 20a-HSD expres- graphic work, Rosemary Clepper for animal care, Janice Gentry andVivian Rogala for assistance in the manuscript preparation.sion appears not to involve this system. The drop in

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Prolactin-Mediated Inhibition of 20α-Hydroxysteroid Dehydrogenase Gene Expression and the Tyrosine Kinase System