-
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
Go
6W.S
cq
t
9.00
8.00-
7.00
6.00-
5.00
4.00
3.00-
2.00-
1.00'
f f ui8N
I I I I I I0 5 10 15 20 25 30 35
Fraction # (5 minutes each)
4 GnRH -4
10.00
s9.00 -
_I1 x8.00 =
7.000
u
6.00 x
5.00 3
4.00 - $L4
3.00
2.00 w
1.00 t0.00
40
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
18.00-
16.00-
14.00-
12.00-
10.00-
8.00-
4.00-
2.00-
-810 MGnRH KCaf f r f f r r
, I I I I I I I
0 10 20 30 40 50 60 70Fraction # (5 minutes each)
80
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
II I I I I I I
10 20 30 40 50 60 70Fraction # (5 minutes each)
Wnide
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
F10 MAntide
10 MGnRH30.00- ' , , ,
25.00-
20.00-
15.00 k
10.00 1 /
5.004
0.005 10 15 20 25 30
Fraction # (5 minutes each)35 40
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
_-S -~ _ ~ -.-0^*Tumor 3 Tumor 5 rumor 9 Normal Normal
pituitary I pituitary 2
b. Non-responsive tumors
A B C 1) A it C 1) A 1; C D A1* C 1) B C 1)
I'1on r 1 Tumor 2 'I'limor 4
A B C 1) A B 1 1) Xi C 1)
* - a
*1 le'91 111l0 1 II
a
I2llilr
1uuin1o1r 6 'I Lainor 7
a, 1B 1)
Tumoller I.;
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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0
8
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Of 10, 20'
C ffm f"
-
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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