Gonadotropin-releasing Hormone Receptor mRNAExpression by Human Pituitary Tumors In VitroJoseph M. Alexander and Anne KlibanskiNeuroendocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Abstract

An important question in the pathogenesis and regulation ofhumangonadotroph adenomas is whether heterogeneous gonad-otropin responses to gonadotropin-releasing hormone (GnRH)are due to dysregulation of GnRHreceptor biosynthesis and/or cell-signaling pathways. Weinvestigated gonadotropin re-sponsiveness to pulsatile GnRHin 13 gonadotroph adenomas.All tumors had evidence of follicle-stimulating hormone (FSH)ft and a subunit biosynthesis using reverse transcriptase/poly-merase chain reaction (RTPCR) techniques. Four tumors sig-nificantly increased gonadotropin and/or free subunit secretionduring pulsatile 10-8 MGnRHadministration. The GnRHan-tagonist Antide (10-' to 108 M) blocked secretory increasesin all GnRH-responsive tumors. Gonadotropin and/or free sub-unit secretion increased after 60 mMKCl, confirming thatGnRHnonresponsiveness was not due to intracellular gonado-tropin depletion. Wehypothesized that GnRHnonresponsive-ness in these tumors may be due to GnRHreceptor (GnRH-Rc) biosynthetic defects. RTPCRanalyses detected GnRH-Rctranscripts only in responsive tumors and normal human pitu-itary. This is the first demonstration of a cell-surface receptorbiosynthetic defect in human pituitary tumors. Weconclude (a)one third of gonadotroph tumors respond to pulsatile GnRHinvitro, (b) GnRH-Rc mRNAis detected in human gonadotrophadenomas and predicts GnRHresponsiveness, and (c) GnRH-Rc biosynthetic defects may underlie GnRHnonresponsivenessin gonadotroph tumors. (J. Clin. Invest. 1994.93:2332-2339.)Key words: gonadotropin * adenoma * pathogenesis * pulsatile.perifusion

Introduction

The majority of clinically nonfunctioning pituitary adenomassynthesize gonadotropin subunit mRNAsand secrete intactgonadotropins and/or their free subunits ( 1, 2) and are there-fore considered to be of a gonadotroph cell origin. There areseveral lines of evidence suggesting dysregulation of gonadotro-pin and free subunit secretion by gonadotropin-releasing hor-mone (GnRH)I in such tumors. GnRHconsistently stimu-

Address correspondence to Dr. Anne Klibanski, Neuroendocrine Unit,Jackson 1021, Massachusetts General Hospital, Boston, MA02114.

Receivedfor publication 30 June 1993 and in revisedform 9 No-vember 1993.

1. Abbreviations used in this paper: AMV, avian myeloblastosis virus;GapDH, glyceraldehyde phosphate dehydrogenase; GH, growth hor-mone; GnRH, gonadotropin-releasing hormone; GnRH-Rc, gonado-tropin-releasing hormone receptor; GPH, glycoprotein hormone;PRL, prolactin; RTPCR, reverse transcriptase/polymerase chain reac-tion; TSH, thyroid-stimulating hormone.

lates gonadotropin secretion in normal individuals (3). In con-trast, < 50% of patients with gonadotropin-secreting tumorsexhibit in vivo increases in serum levels of intact gonadotro-pins and free subunits after an intravenous bolus of GnRH(4,5). Second, desensitization rarely occurs in neoplastic gonado-trophs. Although gonadotropin secretion decreases during con-tinuous GnRH administration in normals, patients withGnRH-responsive pituitary adenomas demonstrate a persis-tent agonist effect with sustained increases in gonadotropin se-cretion (5). GnRH-induced secretory responses have beendemonstrated in static cell cultures of pituitary tumors (6-8).However, the physiological regulation of neoplastic gonado-trophs by pulsatile GnRHhas not been examined in vitro, andlittle is known about the mechanism underlying GnRHrespon-siveness in neoplastic gonadotrophs.

Although human pituitary adenomas are clonal in origin, itis unknown how hypothalamic or other factors may potentiatetumor development or lead to specific tumor phenotypes (9,10). An important question in the pathogenesis and regulationof human neoplastic gonadotrophs is whether the lack ofGnRHresponsiveness in a subset of tumors is due to dysregula-tion of GnRHreceptor (GnRH-Rc) biosynthesis and/or cell-signaling pathways. The recent cloning of human receptors forhypothalamic releasing peptides has offered the potential fornew insights into receptor expression and function in neoplas-tic pituitary cells( 1 1-13). Data indicate that GnRH-Rcbiosyn-thesis can be regulated by physiological factors such as gonadalsteroids as well as homologous regulation by GnRHitself( 14).Therefore, the investigation of GnRH-Rc expression is criticalfor determining both GnRHregulation and potential cell-sig-naling defects in human tumors of gonadotroph origin. Se-quence analysis of the human GnRH-Rc has revealed that it isa member of the Gprotein-coupled receptor family ( 13 ). Theconserved structure ofthe cloned GnRH-Rcconfirms biochem-ical data that its effects on phospholipases and subsequentphosphatidylinositol hydrolysis is likely mediated by interac-tions with the Gqa family of GTP-binding proteins ( 15, 16).Constitutive activation of this cell-signaling pathway has mito-genic and transforming effects in NIH3T3 mouse fibroblasts( 17 ). Therefore, GnRH-Rc could potentially modulate tumorgrowth as well as gonadotropin biosynthesis and secretion.

Because of the potential importance of GnRHin pituitarytumor pathogenesis, we investigated (a) intact gonadotropinand free subunit responses to pulsatile physiological GnRHinhuman gonadotropin-secreting adenomas, (b) blockade with aGnRHantagonist, and (c) whether the presence of GnRH-RcmRNApredicts GnRHresponsiveness in neoplastic gonado-trophs.

Methods

Clinical data. Adenoma tissue was obtained from 13 patients with go-nadotropin hormone-producing pituitary tumors who underwenttranssphenoidal surgery. Patients ranged in age from 48 to 80 yr (me-dian age 58 yr, see Table I). All patients presented with visual field

2332 J. M. Alexander and A. Klibanski

J. Clin. Invest.© The American Society for Clinical Investigation, Inc.0021-9738/94/05/2332/08 $2.00Volume 93, June 1994, 2332-2339

Table I. Patient Data and Preoperative Serum Levels

No. Age Sex a Subunit LH FSH FSHjS Prolactin

yr usg/liter lU/liter IU/liter aig/liter jug/liter

1 58 Male 0.6 n/d n/d 0.3

14%. Cross-reactivities of the assay with each of the other intact glyco-protein hormones and free # subunits are < 0.2%. Intact LH and FSHwere measured by RIA using polyclonal antibodies specific for the (#subunit, and quantitated using World Health Organization IU Stan-dards IRP 78/ 549 (FSH) and IRP 68/40 (LH). The detection limit ofboth RIAs is 0.8 IU/liter. Cross reactivities of the LH assay are 3.9% forboth FSHand TSH, and 1.2% and 1.3% for FSH# and TSHI3, respec-tively. The cross-reactivities of the FSHassay are < 0.03% for all glyco-protein hormones and free subunits.

RIAs of other pituitary hormones. TSH and prolactin (PRL) hor-mone concentrations in perifusion media were determined using com-mercially available double-antibody chemiluminescent assays (Ni-chols Institute, San Juan Capistrano, CA). Cross-reactivities of eachassay with all other pituitary hormones are < 0.1%. Growth hormone(GH) concentrations in perifusion media were determined using acommercially available double-antibody immunoradiometric assay(Nichols Institute). Cross-reactivities of each assay with all other pitu-itary hormones are < 0. 1%.

Statistical analysis. Secretory baselines were determined as themean hormone level released per column fraction during the 20 minpreceding each GnRHpulse. Secretory peaks were calculated as maxi-mal hormone released after the GnRHpulse minus the preceding se-cretory baseline. Secretory responses that were > 2 SDs above preced-ing baseline were considered significant.

Results

Basal hormone secretion and immunocytochemistry. All 13 tu-mors secreted one or more intact gonadotropins and/or freesubunits during perifusion experiments. Detectable levels ofFSH were found in 10 tumors with mean secretory rates rang-ing from 1.1 to 10.0 IU/liter-min-' (median 2.6 IU/li-ter min- '). Detectable levels of LH were found in 1 tumorswith mean secretory rates ranging from 1.7 to 7.9 IU/li-ter min-' (median 2.6 IU/liter min'-). Free a and FSH,3subunit levels were detected in eight and two tumors, respec-tively. The mean a subunit secretory rate ranged from 0.2 to0.6 ,gg/liter min-' (median 0.4 ,gg/liter - min-'). The meanFSH# subunit secretory rates were 3.3 and 1.7 ,gg/liter - min-'in tumor 9 and 10, respectively. Immunocytochemical stainingwas performed on pituitary tumor tissue from each patientusing specific antibodies for LH(B, TSHj3, FSH(B, a subunit,GH, PRL, and ACTH(26). Nine of 13 tumor specimens im-munostained positively for one or more gonadotropin sub-units. Immunocytochemical staining for GH, PRL, andACTHwas negative in all tumors, and 2 of 13 tumors had rareTSHj3 immunostaining.

To exclude the possibility that normal pituitary tissue con-tamination was responsible for the observed tumor secretoryphenotype in perifusion, medias were assayed for the nongona-dotroph hormones, GH, PRL, and TSH (Table II). Secretionof these nongonadotroph pituitary hormones from perifusedgonadotroph tumors was negligible for both GnRH-responsiveand -unresponsive tumors. GHwas undetectable in all tumors(< 1.0 ,ug/liter). TSHlevels were near the detection limit in alltumors, and ranged from 0.06 to 0. 13 mIU/liter (median, 0.07mIU/liter), whereas PRL levels were also low, ranging from0.1 to 1.5 ag/liter (median, 0.2 ,ug/liter).

Gonadotropin subunit biosynthesis in perifused tumors. Toconfirm the gonadotroph origin ofthese tumors, RTPCRanaly-sis was performed for a gonadotropin-specific marker, FSH3,as well as GPHa subunit and GapDH. As shown in Fig. 1, all12 perifused tumors showed positive RTPCRamplification forFSHI3 mRNA,as well as GPHa subunit and GapDH. RTPCR

Table II. Basal Secreetion of GH,by Perifused Pituitary Tumors

PRL, and TSH

Tumor No. GH PRL TSH

ug/liter gg/liter mlUlfiter

1 < 1.0 0.2 0.072 < 1.0 0.3 0.063

Non-respondingtumors

Responding Normaltumors pituitary

Tumor # 1 2 4 6 7 8 11 12 13 3 5 9

221 bp-w-

535 bp-p-

340 bp-mm- rnww- rn.1.

ministered to four of the nine GnRH-nonresponsive tumors.In each case, a significant transient secretory burst of gonado-tropins and/or free subunits was observed. The response intumor 2 is also shown in Fig. 3. Secretion of LH as well as a-and FSH,3-free subunit were undetectable in this pituitary tu-mor throughout the perifusion experiment.

Gonadotropin responses to GnRHstimulation during An-tide blockade. In all four GnRH-responsive tumors, gonadotro-pin secretory pulses were blocked by I0-6- 10-8 MAntide. TheLH responses to GnRHin tumor 3 during coadministration ofincreasing doses of Antide are shown in Fig. 4. LH responsesafter each GnRHpulse in column A were significantly in-creased over basal levels. LH stimulation after GnRHwasblocked by simultaneous administration of o0-8 MGnRHwith either l0-7 or I0-1 MAntide. However, lower concentra-tions of Antide ( 10-9 and 10-10 M) failed to block GnRH-stim-ulated LH secretion. Mean basal FSH secretion rates in col-umns A and B were 1.2 and 1.1 IU/liter * min-', respectively.After GnRHadministration, mean FSHsecretion rates in col-umns Aand B were significantly increased to 2.5 and 2.0 IU/li-ter * min-', respectively. However, FSH secretory baselines in

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Figure 2. Coordinate secretion of intact FSH and free FSH,3 subunitby tumor 9 in response to pulsatile GnRH. 10-min GnRHpulseswere administered hourly. Arrows indicate the timing and durationof each GnRHpulse. Fractions were collected every 5 min.

GPHa-subunit

Figure 1. RTPCRanalysis of gonadotropin subunitmRNAsfrom perifused tumors. Analysis of gonado-

FSHf- tropin subunit mRNAswas examined by RTPCRsubunit amplification of tumor total RNA, followed by

Southern blot hybridization with human GPHa sub-unit, FSHf3, or GapDHcDNAprobes. Normal pitu-itary total RNAwas used as a control. RT was omit-

GapDH ted from one normal pituitary PCRreaction to assurethat the observed amplified products were RNAspe-cific. All RTPCRproducts were of the expected size.

both columns, as well as responses to GnRH, were attenuatedafter the first two GnRHpulses, probably because of depletionof intracellular FSH. After 60 mMKCl, there was a significantincrease in both LH and FSH secretion. LH increased overbaseline 4.2- and 5.0-fold in columns A and B, respectively.Release of intracellular FSHafter KCl administration was lesspronounced, but secretion increased over baseline 0.7- and 2.4-fold in columns A and B, respectively.

Recovery of gonadotropin responses after Antide blockade.GnRHresponses of tumor 9 after administration of Antide areshown in Fig. 5. LH secretory responses to GnRH(column A)ranged from 7.7 to 28.1 IU/liter * min ', with a mean responseof 260% over the preceding secretory baseline. LH secretoryresponses to GnRHand 10-6 MAntide (column B) rangedfrom 5.2 to 9.1 IU/liter * min-', with a mean response of 45%.FSH secretory responses to GnRHranged from 5.4 to 7.3 IU/liter * min'-, with a mean response of 85% over the preceding

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Figure 3. Secretion of FSH in perifusion by GnRH-nonresponsive pi-tuitary tumor 2. 5-min fractions of perifusion media were assayed forintact FSH secretion by tumor 2 during the 5-h experiment. 10-minGnRHpulses were given every 45 min, followed by a single 10-minpulse of 60 mMKCl at the conclusion of the experiment. Arrows in-dicate the timing and duration of each GnRHpulse.

GnRHReceptor mRNAExpression by HumanPituitary Tumors In Vitro 2335

+ - R.T.

I

f Fa f f 10 to 10 MAKCI _.r frrr "r r 1O MGnRH

IK I

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Figure 4. Dose-dependent Antide inhibition of secretory responses bytumor 3. 10-min GnRHpulses were given every 45 min. Antide[10-7-l0-bo M] was coadministered. A single 10-min pulse of 60 mMKCI was given at the conclusion of the experiment. Arrows indicatethe timing and duration of each GnRH, Antide, or KCI pulse. (-----)Control perifusion column A profile of GnRHalone. ( ) Perifu-sion column B profile of GnRH+ simultaneous Antide.

secretory baseline. FSHsecretory responses to GnRHand 10-6MAntide ranged from 3.5 to 4.1 IU/liter * min', with a meanresponse of 8%. Pretreatment with 10-6 MAntide significantlydecreased the secretory responses of both LH and FSH toGnRH.

RTPCRof human GnRHreceptor from perifused gonado-troph tumors and normal human pituitary. The results ofRTPCRanalyses of GnRH-Rc mRNAfrom perifused tumorsand normal pituitary controls are shown in Fig. 6. GnRH-Rcwas identified in all GnRH-responsive tumors, and GnRH-Rcamplification was similar to that seen in two normal human

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Figure S. Recovery of secretory responses by tumor 9 after antideblockade. 10-min GnRHpulses were coadministered with Antide orat 10 or 20 min after Antide treatments. Arrows indicate the timingand duration of each GnRHand Antide pulse. (-----) Perifusion col-umn profile of GnRHalone. ( ) Perifusion column profile ofGnRHpreceded by Antide.

a. Responsive tumors and normal pituitaryA B C 1) A\ B C1 1) A 1B C 1) A BS C' 1) A\ B (: D

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Figure 6. RTPCRanalysis of human GnRHreceptor mRNAfromperifused pituitary tumors and normal pituitary. Lane A, GnRHre-ceptor RTPCRreactions. The demarcated band represents the ex-pected 293-bp PCRproduct. Lane B, control GnRHreceptor RTPCRwithout RT. Lane C, GapDHRTPCRreactions. The demarcatedband represents the expected 340-bp PCRproduct. Lane D, controlGapDHRTPCRreactions without RT.

pituitary samples. GnRH-unresponsive tumors failed to showamplification of the GnRH-Rc mRNAcomparable to levelsdetected for either responsive tumors or normal pituitary. Alltumor mRNApreparations had detectable levels of controlGapDHPCRproduct, and GapDHmRNAfrom unresponsivetumors amplified with comparable signal intensity as that seenfor GnRH-responsive tumors and normal pituitary.

Discussion

Wehave shown that approximately one third of gonadotrophtumors are responsive to pulsatile GnRHand that GnRH-in-duced secretory increases can be blocked in a dose-responsiveand time-dependent manner by administration of a GnRHantagonist. Wereport the first identification of human GnRH-Rc mRNA in neoplastic pituitary tissue using sensitiveRTPCRtechniques. All GnRH-responsive tumors studied haddetectable levels of GnRH-Rc mRNAcomparable to thosefound in normal human pituitary tissue. In contrast, the major-ity of gonadotroph tumors were unresponsive to pulsatileGnRHin perifusion and did not synthesize detectable levels ofGnRH-Rc mRNA. Therefore, the presence of GnRH-RcmRNApredicts tumor responsiveness to GnRHstimulation invitro. The lack of detectable GnRH-Rc mRNAin GnRH-unresponsive tumors suggests that tumor receptor biosynthesisis a critical determinant of gonadotroph responses to GnRHstimulation. These data are consistent with the hypothesis thatcell-surface receptor biosynthetic defects are present in humanpituitary tumors.

What are the differential effects of GnRHon normal versusneoplastic gonadotrophs? This question has been addressed invivo by studies investigating gonadotroph desensitization inpatients with gonadotropin-secreting pituitary tumors. In nor-mal patients, administration of a GnRHagonist leads to pitu-itary desensitization and subsequent decreases in gonadotropinsecretion. In contrast, patients with gonadotropin-secreting pi-

2336 J. M. Alexander and A. Klibanski

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tuitary tumors receiving chronic GnRH agonists typicallyshow a persistent agonist effect (5). These data suggest thatgonadotroph tumors may be associated with abnormalities inreceptor structure and / or function. It is unknown whether con-tinuous GnRHadministration in vitro will lead to desensitiza-tion of gonadotropin secretion in human pituitary tumors. Ex-periments using normal perifused rat pituitary cells have dem-onstrated that gonadotropin secretion decreases duringcontinuous GnRHadministration for 30-120 min (27). Re-covery of secretory responses is not immediate, and completerecovery can require up to 30 min. Studies investigating gonad-otropin responses to continuous GnRH in human gonado-troph tumors in vitro will be necessary to investigate this ques-tion.

Administration of a GnRHantagonist to normal subjects(28) and normal rat pituitary cells in vitro ( 19) causes a consis-tent decrease in intact gonadotropin and a subunit secretionwithout a stimulatory agonist phase. In one study, a 3-12-mocourse of the Nal-Glu GnRHantagonist to five menwith FSH-secreting pituitary tumors significantly decreased serum FSH,LH, and testosterone levels, without affecting adenoma size(29). In vivo studies are limited by several factors. First, itcannot be determined whether changes in serum hormone lev-els in treated patients represent tumor versus normal gonado-tropin responses. Second, how pulsatile GnRHstimulationcontrols neoplastic gonadotrophs has not been investigated.Therefore, the physiological regulation of human pituitary tu-mors by GnRH is unknown. Our present data demonstrateresponsiveness to pulsatile GnRHin approximately one thirdof gonadotroph tumors. Wefound that all tumors included inthis series had evidence of FSHJ3 biosynthesis using RTPCRtechniques. Gonadotropin secretion was found in GnRHnonresponsive, as well as GnRH-responsive tumors. The pres-ence of FSH,3 biosynthesis in tumors nonresponsive to GnRHand lacking GnRH-RcmRNAsuggests that FSH3biosynthesisin these tumors maybe regulated by GnRH-independent mech-anisms. In all GnRH-responsive tumors, administration of theGnRHantagonist Antide blocked gonadotroph secretory re-sponses, consistent with effects seen in normal pituitary tissue.Recovery times of up to 20 min were tested, however, GnRHresponsiveness after Antide blockade could not be demon-strated in the tumors studied. These experiments suggest thatlonger recovery periods are required to reestablish GnRHresponsiveness of neoplastic gonadotrophs after Antideblockade.

Altered GnRHpulse characteristics are one mechanism ofgenerating differential gonadotropin subunit gene expressionand secretion in normal gonadotrophs. In vitro pituitary peri-fusion systems have facilitated the precise study of hormoneand subunit regulation in response to physiological GnRHinexperimental animals (27, 30-32). Recent studies using peri-fused normal rat pituitary cells have shown that variations inGnRHpulse amplitude have preferential effects on FSH(3 sub-unit mRNAsteady state levels (33). Therefore, gonadotropinsubunit secretory phenotype in normal pituitary tissue mayreflect underlying GnRHpulse patterns. Imbalanced gonado-tropin subunit biosynthesis and secretion in gonadotroph tu-mors have been confirmed in vivo and in vitro. Elevated serumFSH13-free subunit levels have been shown in patients withgonadotroph adenomas. Wehave recently demonstrated ex-cess FSH3 subunit mRNAexpression and FSHB-free subunit

secretion relative to a subunit (34). This finding is in contrastto normal pituitary tissue where a subunit is synthesized inexcess of B subunit. Two of four of the GnRH-responsive tu-mors secreted FSH3, whereas none of the GnRH-nonrespon-sive tumors had detectable levels of FSHB-free subunit secre-tion during perifusion. It is unknown whether different fre-quencies of GnRHadministration will alter gonadotropin-freesubunit biosynthetic and/or secretion patterns in human go-nadotroph tumors. The responses of normal and neoplasticgonadotrophs to variable intervals of GnRHadministrationmay differ. Therefore, alterations in pulsatile GnRHmay favorFSHB secretion and play a role in the FSHI3 phenotype ob-served in a large subset of tumors. However, we have not inves-tigated whether GnRH-responsive tumors have higher levels ofFSHOmRNAlevels than nonresponsive tumors. RTPCRdataon FSHOmRNAin perifused tumors in Fig. 1 are designed tobe a qualitative marker of the gonadotroph origin of these tu-mors and do not reflect FSHf3 mRNAsteady state levels. Fu-ture perifusion studies using quantitative RTPCRtechniqueswill be necessary to determine if GnRH pulsatility affectsFSHBmRNAlevels in neoplastic gonadotrophs, as has beenshown for normal individuals.

Studies of clinically nonfunctioning human pituitary tu-mor responses to hypothalamic signaling peptides, such asGnRHand TRH, may provide significant insights into thepathogenesis of neoplastic gonadotrophs. Studies using molecu-lar genetic techniques have demonstrated the monoclonal na-ture of clinically nonfunctioning pituitary tumors and indicatethat a somatic mutation is a required event for tumor initiation(9). The single-cell origin of such adenomas makes it unlikelythat circulating factors (either hypothalamic peptides or otherhormones) initiate tumor pathogenesis. However, studies oftumor responses to such peptides may elucidate underlyingmutations that are responsible for biosynthetic and secretorydefects. In addition, genomic alterations may also give pitu-itary cells a distinct growth advantage, as has been shown insomatotroph tumors (35).

The lack of detectable GnRH-Rc mRNAin GnRHunre-sponsive tumors suggests that receptor biosynthesis is deficientin a large subset of human gonadotroph adenomas. Alterationsin GnRH-Rc biosynthesis may be the result of cell-signalingdefects in neoplastic gonadotrophs. In one model of constitu-tive activation of GnRHpathways, continuous in vitro admin-istration of GnRHto normal rat pituitary cells downregulatedpituitary GnRH-Rc mRNAlevels (14). This finding suggeststhat one mechanism of long-term desensitization of GnRHresponses by GnRHagonists is to downregulate constitutiveGnRH-Rc gene expression and mRNAsteady state levels.Therefore, mutations that activate gonadotroph-specificGnRHsecond messenger pathways could chronically downreg-ulate GnRH-Rcbiosynthesis and desensitize neoplastic gonad-otrophs to GnRHstimulation. The conserved structure of thecloned GnRH-Rc confirms biochemical data that its effects onphospholipases and subsequent phosphoinositol turnover islikely mediated by interactions with the Gqa family of GTP-binding proteins ( 15, 16). Constitutive activation of Gqa hasmitogenic and transforming effects in NIH3T3 mouse fibro-blasts ( 17). It is unknown whether activating mutations in sec-ond messenger systems could potentially have cell proliferativeeffects as well as desensitizing GnRH-signaling pathways thatmodulate gonadotropin biosynthesis and secretion.

GnRHReceptor mRNAExpression by HumanPituitary Tumors In Vitro 2337

The gonadotroph origin and phenotype of clinically non-functioning pituitary tumors have been described by severalgroups. We have confirmed the gonadotroph phenotype ofthese tumors both at the biosynthetic and secretory levels. All13 tumors secreted LH, FSH, and/or FSHI3 in perifusion. Wehave previously shown underlying biosynthetic and secretoryabnormalities in clinically nonfunctioning pituitary tumors. Incontrast to the secretion of both LH and FSHby normal pitu-itary tissue, preferential secretion of FSHand FSH3-free sub-unit is seen in a subset of tumors (34). Wehave confirmedthese findings in this group of tumors by performing RTPCRfor FSH,3 mRNA.All 13 tumors synthesized FSH/3 mRNAinaddition to GPHa subunit and GapDH, consistent with theirgonadotroph phenotype in perifusion. Several lines of evidencesuggest that normal tissue contamination is not a complicatingfactor in this study. First, the 13 perifused gonadotroph tumorsin this study do not exhibit significant in vitro secretion of GH,PRL, and TSH, an expected result if perifused gonadotrophtumors were composed of large numbers of normal pituitarycells. Second, we might also expect a histological description ofnormal tissue in resected tumor samples, however, all tissuereceived was histologically diagnosed as adenoma tissue. Third,human pituitary tumors have been demonstrated to be geneti-cally monoclonal in origin (9, 36-40). Because contaminationof normal pituitary tissue would give a polyclonal result, theconsistent finding of monoclonality in both macro- and micro-adenomas argue strongly against normal tissue contaminationin our samples. Fourth, our finding that one third of humangonadotroph adenomas are responsive to GnRHin vitro is inagreement with previous studies documenting both secretory(6, 7) and intracellular (8) responses to GnRHin static cul-ture. These complementary approaches strongly suggest thatnormal pituitary tissue contamination of resected human go-nadotroph tumors is not a factor in our analysis.

In contrast to the uniform LH and FSHresponses to physio-logical GnRH in normal pituitary cells, only one third ofpituitary tumors are GnRHresponsive in vitro. Wehave dem-onstrated the presence of GnRH-Rcin human pituitary gonad-otropin-secreting adenomas. GnRH-Rc transcripts were de-tected only in responsive tumors, and the levels were compara-ble to those seen in normal human pituitaries. Therefore,GnRH-Rc mRNAsteady state levels in such tumors predictGnRH responsiveness in vitro. These studies suggest thatGnRHnonresponsiveness in gonadotroph tumors may be dueto GnRH-Rcbiosynthetic defects. In addition, the documenta-tion of FSH3biosynthesis in tumors lacking GnRH-RcmRNAand responsiveness to exogenous GnRHsuggests the impor-tance of GnRH-independent mechanisms in the regulation ofgonadotropin biosynthesis in a subset of tumors. Future inves-tigations will need to characterize such defects and determinetheir role in tumor pathogenesis.

Acknowledgments

Wethank Dr. S. Raiti of the National Pituitary Agency for supplyingassay reagents for human gonadotropins and subunit RIAs, Dr. A.Bauer-Dantoin and Dr. A.N. Hollenberg for providing a partial se-quence of the human GnRH-Rc, and H.A. Bikkal for technical assis-tance.

This work was supported in part by National Institutes of Healthgrant DK-40947, P30HD28 138, and an American Cancer Society IRG

Award. Dr. Alexander is a recipient of the SmithKline Beecham Medi-cal Discovery Fellowship Award.

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