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Department of Animal Sciences, Rutgers, State University of New Jersey (A.D., N.B., D.K.S.), New Brunswick, New Jersey 08901; and Departments of Veterinary and Comparative Anatomy (S.H., M.P.), and Pharmacology and Physiology, Washington State University, Pullman, Washington 99164-6520
Address all correspondence and requests for reprints to: Dr. D. K. Sarkar, Department of Animal Sciences, Rutgers, State University of New Jersey, New Brunswick, New Jersey 08901.
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Abstract |
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Introduction |
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We have previously shown that TGFß1 is produced in the pituitary of normal rats (6). Immunohistochemical methods revealed the presence of TGFß1 in the lactotropes of the anterior pituitary gland. Pituitary levels of TGFß1 messenger RNA (mRNA) and protein decrease during estradiol-induced cell proliferation in the pituitary gland (6, 7, 8). Determination of the effects of TGFß1 on estradiol-induced lactotropic cell proliferation and PRL secretion from pituitary cells in culture indicated that TGFß1 inhibits the growth of lactotropes and decreases PRL secretion (7, 8, 9, 10). The action of this growth factor on the secretion of other pituitary hormones was not evident.
Recently, the inhibitory action of TGFß1 on lactotropic proliferation has been attributed to actions through the type II TGFß receptors (9, 11). Additionally, altered expression of these receptor subtypes on lactotropes may in part be responsible for the loss of the ability of TGFß1 to suppress cell proliferation in transformed lactotropes that result from estradiol exposure (7, 11). The levels of both immunoreactive TGFß receptor type II protein and in situ TGFß receptor type II mRNA hybrids in the pituitary were significantly decreased during estradiol-induced tumorigenesis (7, 9). Determination of [125I]TGFß1-binding sites in lactotropes by double immunohistochemistry and receptor autoradiography revealed specific binding sites of TGFß1 in lactotropes of the anterior pituitary (9). [125I]TGFß1 binding in the anterior pituitary was reduced after estradiol treatment. Hence, it appears that lactotropes are not only the site of TGFß1 production, but may also be a site of TGFß1 action. The expression and actions of the other two TGFß isoforms in lactotropes have not previously been described.
Given the similarity of the TGFß proteins to one another and the fact that they are thought to act via similar complexes of TGFß receptor subtypes, it is not surprising that in many tissues TGFß isoforms elicit similar responses. However, the differential expression (reviewed in Ref. 12) and receptor affinities (13) of the TGFß isoforms indicate that there may be isoform-specific functions for the TGFß proteins. Hence, we studied the production and actions of the various TGFß isoforms in lactotropes in the presence and absence of estradiol.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunohistochemical localization of TGFß2, TGFß3, and
PRL
Anterior pituitary tissues were obtained from cyclic female rats
on the day of estrus or from ovariectomized rats with or without
estradiol treatment after perfusion with 4% formalin in 10
mM PBS (pH 7.4). The pituitaries were postfixed with 4%
buffered-formaldehyde, paraffin embedded, and sectioned into 2-µm
sections. The sections were deparaffinized and rehydrated through a
descending series of alcohol solutions to water. The tissues were
processed for immunostaining using TGFß2 or TGFß3 antibody (gifts
from Dr. K. C. Flanders) and PRL antibody (PRL-S9, NIDDK) and
double immunohistochemical procedures as previously described (6, 8).
Sections were first treated with hydrogen peroxide in absolute methanol
for 30 min at room temperature to block the endogenous peroxidase
activity. This was followed by incubation with 10% normal serum for 30
min to further block nonspecific binding. Sections were incubated with
primary antibodies overnight at 4 C. The antiserum specificity was
characterized (14) and verified by no staining in tissues when reacted
without primary antibody or with primary antibodies preabsorbed with a
100-fold excess of antigen. The antibodies were detected using
immunocytochemistry kits (Vector Laboratories, Inc.,
Gilroy, CA; or BioGenex Laboratories, Inc., San Ramone,
CA). The sections were counterstained with Gill’s hematoxylin to
detect nuclei. Two investigators independently performed cell counts,
which involved counting five separate areas in each tissue and 500
cells/area.
Primary cultures of anterior pituitary cells
Anterior pituitaries from ovariectomized rats implanted with
estradiol-containing capsules for 7–10 days were dissociated
enzymatically with Hanks’ Balanced Salt Solution containing
collagenase, deoxyribonuclease, and BSA and were grown on
poly-L-lysine-coated coverslips as described previously
(7). Cells were maintained in DMEM (1:1; Sigma, St. Louis,
MO; containing 100 U/ml penicillin and 100 µg/ml streptomycin) with
high serum (10% FCS) for 1 day and then in medium containing 2.5% FCS
and 10% horse serum for another 2 days. Cultures were maintained in
serum-free DMEM containing human transferrin (100 µM),
insulin (5 µM), putrescine (1 µM), and
sodium selenite (30 nM) at 37 C in 7.5%
CO2. The lactotropic cell population, as
determined by identifying PRL-immunoreactive (PRL-IR) cells, was
69 ± 2% in these cultures (7).
Treatment of primary pituitary cell cultures
Cells were plated on poly-L-lysine-coated 24-well
plates. For immunostaining studies the cells were plated onto
poly-L-lysine-coated coverslips that were placed in the
24-well plates. Cells were plated at 250,000 cells/well. Primary
cultures were maintained for 4 days before the onset of
experimentation. During experimentation, cells were maintained in
DMEM-Ham’s F-12 containing serum supplement (described above).
Cultures were treated as indicated in the figures and in
Results. Treatments were changed at 48-h intervals for total
treatments of 96 h. In studies involving bromodeoxyuridine
(BrdUrd) incorporation assays, BrdUrd was added for 4 h in fresh
medium containing serum supplement and treatment after the initial 96-h
treatment.
Lactotropic cell proliferation
Lactotropic cell proliferation was determined by identifying the
cells that had both BrdUrd and PRL immunoreactivities, as described
previously (8). In brief, cultures were treated with 0.1 mM
BrdUrd 4 h before harvesting. Cells were then fixed with 99%
ethanol and treated with hydrogen peroxide to block endogenous
peroxidase activity. Cells were incubated at 4 C overnight with BrdUrd
monoclonal mouse IgG (1:200; Becton Dickinson and Co., San
Jose, CA) and stained using the Vectastain ABC kit
(Vector Laboratories, Inc., Burlingame, CA) with
diaminobenzidine as the chromagen. The cells were then incubated
with PRL antibody (1:100,000; PRL-S9, NIDDK) at 4 C overnight and
stained using the Vectastain ABC-AP kit (Vector Laboratories, Inc.) with alkaline phosphatase as the chromogen.
As BrdUrd is incorporated into cells during S phase, cells with
immunoreactivity for BrdUrd were considered to be dividing. Cells
immunoreactive for both BrdUrd and PRL were considered to be dividing
lactotropes. Two investigators independently performed cell counts,
which involved counting 5 separate areas in each coverslip and 500
cells/area.
Western blot analysis of TGFß3 protein
Immunoblotting was performed on pituitary tissue homogenates.
One hundred micrograms of total protein from each pituitary, as
determined using the DC protein quantification reagents
(Bio-Rad Laboratories, Inc., Hercules, CA), were run out
on 12% denaturing polyacrylamide minigels and transferred to
nitrocellulose in a semidry transfer chamber (Bio-Rad Laboratories, Inc.). The nitrocellulose membranes were stained
with Ponceau S solution (Sigma) to check the efficiency of
transfer and to verify equal protein levels in each lane. The membranes
were then placed in 5% milk block for 5 h, followed by incubation
in primary antibody (monoclonal TGFß3 antibody, R&D Systems,
Minneapolis, MN; 1 µg/ml) in blocking buffer at 4 C overnight.
Horseradish perioxidase-conjugated antimouse IgG (1:1500; 30 min at
room temperature) was used for detection of the antibody by enhanced
chemiluminescence (Amersham Pharmacia Biotech, Arlington
Heights, IL; film was exposed to blotted membranes for 10 min).
Assays of TGFß1 and TGFß3 mRNA levels
Total cellular RNA was isolated from anterior pituitary tissue
by guanidium isothiocyanate-phenol extraction (15) and used for
Northern blot analysis of TGFß1 or TGFß3. RNA samples were resolved
on denaturing 1% agarose gels containing formaldehyde and transferred
to MSI nylon filters (MSI, Westboro, MA) by blotting. The RNA was UV
cross-linked to the filter and then hybridized sequentially with DNA
probes. Prehybridizations were performed for 2 h, and
hybridizations were carried out for 24 h in a solution containing
50% formamide, 1 M NaCl, 10% dextran sulfate, 1 x
Denhardt’s solution, 2% SDS, and 0.1 mg/ml denatured salmon sperm DNA
at 42 C. The filters were then exposed to Kodak XAR-5 film
(Eastman Kodak Co., Rochester, NY) at -70 C. Probes were
purified gene fragments labeled by random priming with
[32P]deoxy-CTP to a specific activity of more
than 2 x 109 cpm/µg. The 985-bp TGFß1
probe contained the entire rat complementary DNA (cDNA) and was the
XbaI to HindIII fragment from prTGFß1
provided by Drs. S. Quian and A. B. Roberts. The TGFß3 probe
included sequences from position 831-1449 of the mouse cDNA and was
provided by Dr. H. Moses. The cyclophilin probe was the 117-bp
PstI to XmnI fragment including the 5'-portion
from the rat cDNA. The 18S probe was the 1.5-kb EcoRI
fragment from the human 18S gene cloned into HHCSA 65 and obtained from
American Type Culture Collection (Manassas, VA).
Statistics
The data shown in the figures and text are the mean ±
SE. Data were analyzed using one-way ANOVA. The
post-hoc test used was Student-Newmann-Keuls test;
P < 0.05 was considered significant. Comparisons
between points on two different dose-response curves were performed
using the Bonferroni multiple comparison test; P <
0.01 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Together, these observations suggest
that both TGFß1 and TGFß3 were produced in the lactotropes.
TGFß2-IR cells were identified in the anterior pituitary, but
TGFß2-positive cells were not lactotropes because they did not show
colocalization with PRL-IR (data not shown).
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) and the number of
TGFß3-IR cells (Fig. 2B). As TGFß2 was not localized to the
lactotropes, the effects of estradiol on TGFß2-IR and mRNA levels
were not determined.
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). Conversely,
ovariectomy increased TGFß1 and decreased TGFß3 mRNA levels (Fig. 3).
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). The
Western blot data shown in Fig. 4 are representative of six blots, all
of which show this same pattern. The TGFß3 band was specific and was
the only band that appeared. Additionally, the specificity of this
antibody was checked by preabsorption with excess TGFß3 peptide as
described for immunohistochemistry. This was not repeated in the
Western blot analysis. Figure 4 indicated a more diffuse band for the
TGFß3 standard than for the treated samples. The reason for this
difference in migration is not fully understood, but possibly results
from some degradation of this peptide. It may also be that as the
TGFß3-probed test samples were prepared from tissue, the peptide was
modified slightly in some way that was not present in the purified
peptide. Also, the samples had to be acid activated because we were
probing for intracellular peptide, and it may be possible that this
treatment caused slight conformational change, resulting in slower
migrating bands. We have no positive explanation for the slight
migration difference, but we believe that this does not negate the
observed changes in TGFß3 protein.
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Effects of TGFß1, TGFß2, and TGFß3 on lactotropic cell
proliferation
To determine the biological actions of these growth factors on the
lactotropes, we used primary cultures of pituitary cells to determine
the proliferating response of lactotropes to TGFß isoforms in
vitro. TGFß3 increased the number of lactotropes undergoing DNA
replication in a dose-dependent manner (Fig. 5F). The TGFß3 concentration required
for half-maximal stimulation was approximately 0.01 ng/ml. TGFß1
alone produced no significant effect on the growth of low proliferating
lactotropes in primary culture (Fig. 5F). TGFß2 also had no
significant effect on lactotrope growth in primary cultures (data not
shown).
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). The cell-proliferating effects of TGFß3 appear to be
specific, as the related factor, inhibin (10 ng/ml), did not affect
lactotropic proliferation (85± 18% of the control value; n = 4).
In contrast to the action of TGFß3, TGFß1 concentration-dependently
inhibited estradiol-induced lactotropic cell proliferation (Fig. 5F).
TGFß2 had no significant effect on estradiol-induced lactotropic cell
proliferation. Thus, it appears that although TGFß1 inhibits
estradiol-induced lactotropic cell proliferation, TGFß3 stimulates
lactotropic proliferation.
Effects of TGFß1- and TGFß3-neutralizing antibodies on
lactotropic cell proliferation
Determinations of the blocking effects of the TGFß1 and TGFß3
antibodies indicated that the actions of TGFß1 and TGFß3 on
lactotropes are specific. Ten micrograms of the TGFß3 antibody were
able to block the effect of 10 ng TGFß3 on estradiol-induced
lactotropic cell proliferation (Fig. 4, G and F). The action of TGFß1
on lactotropes was completely blocked by a neutralizing antibody (10
ng/ml TGFß1, 23.2 ± 7.9% of control; 10 ng/ml TGFß1 and 10
µg/ml anti-TGFß1, 105 ± 7.9% of control; n = 6;
P < 0.001).
Studies involving immunoneutralization of endogenous TGFß3 using
neutralizing antibodies during estradiol-activated cell proliferation
indicated that TGFß3 antibody completely prevented the
cell-proliferating action of estrogen on lactotropes (Fig. 5G). Hence,
TGFß3 was required for estradiol to stimulate lactotropic cell
proliferation.
Immunoneutralization of TGFß1 did not affect the ability of estradiol
to induce lactotropic proliferation (Fig. 5G). Hence, the TGFß1
inhibitory influence is absent during estradiol-activated cell
proliferation when the production of TGFß1 in the cells is lowered.
These data together with the data showing estradiol inhibition of
lactotropic production of TGFß1 suggest that lactotrope growth is
kept under control in basal conditions by TGFß1.
In the absence of estrogen, when TGFß1 activity was immunoneutralized by TGFß1 antibody in cultured cells, and the cells were subsequently subjected to exogenous TGFß3, lactotropic growth in response to TGFß3 was markedly increased. The TGFß3 growth response in the presence of TGFß1 antibody was similar to that observed in the presence of estradiol. Hence, the reduced TGFß1 production observed after estradiol exposure is important for lactotropes to respond maximally to TGFß3.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the anterior pituitary, estradiol appears to be a key regulator of TGFß1 and TGFß3 expression and, possibly, action. Our finding that estradiol increases TGFß3 production in the pituitary is in agreement with the findings of other investigators that TGFß3 mRNA levels are up-regulated by estradiol treatment in bone. Yang et al. demonstrated that in bone, TGFß3 expression (but not TGFß1 or TGFß2) is increased with estradiol treatment and is dependent on estrogen receptor mediation (16). The TGFß3 gene does not contain a typical palindromic estrogen response element. Yang et al. suggest that estrogen bound to its receptor recognizes an alternative responsive element on the TGFß3 gene. Further studies are needed to determine the nature of the estrogen-induced TGFß3 increase that we observed in the anterior pituitary.
Estradiol stimulated lactotrope proliferation and decreased TGFß1, but increased TGFß3 levels in anterior pituitary tissue. Therefore, it may be that estradiol has a dual action involving down-regulation of TGFß1 and up-regulation of TGFß3. The combined effect of releasing an inhibitory control and increasing a stimulatory factor could result in the potent mitogenic effect that estradiol has only on lactotropes in the pituitary. Estrogen potentiation of the TGFß3 response can be explained by the fact that the steroid also inhibits TGFß1 growth inhibitory influences. Indeed, the suppression of TGFß1 levels appears to be necessary for TGFß3 to exert the maximal proliferative effect; TGFß3 alone elicits only moderate stimulation of lactotropic proliferation, whereas neutralizing TGFß1 during the addition of TGFß3 results in a robust proliferative response. Additionally, neutralization of TGFß3 diminished the ability of estradiol to stimulate lactotropic proliferation in primary cultures of anterior pituitary cells. Although estradiol acts to increase TGFß3 production, it also functions to increase TGFß3 action, as demonstrated by the enhanced response observed with TGFß3 in the presence of estradiol. Thus, it appears that the lactotrope is a unique cell type where, under estrogenic conditions, TGFß isoforms act as both positive and negative growth regulators.
The mechanisms by which TGFßs regulate cell proliferation have not been well established. Our data indicate the need to investigate the mechanisms of TGFß action in an isoform-specific manner. Primary cultures of anterior pituitary cells from estradiol-treated Fischer-344 rats contained approximately 69% lactotropic cells. It is feasible that TGFß3 could have a paracrine action on neighboring cells to induce the release of other growth-stimulating factors. In aortic smooth muscle cells, TGFß stimulates cell proliferation by increasing platelet-derived growth factor (PDGF) synthesis (3). For endothelial cells, factors other than PDGF would be expected to be indirect mitogenic factors, because these cells usually lack PDGF receptors (3). In fibroblasts, the stimulation of cell growth by TGFß involves the ability of TGFß to transiently increase cellular responses to a variety of exogenously added growth-promoting factors, such as epidermal growth factor, bombesin, and vasopressin (17). TGFß may also participate in the stimulation of fibroblast proliferation by increasing collagen production (18). These data suggest that mitogenesis by TGFß may be mediated indirectly through other growth stimulatory peptide factors; we have studied this possibility (18A ). Whether the action of TGFß3 on lactotropes is mediated by other growth factors as well as the receptor system used by TGFß3 will need to be addressed in future studies.
Estradiol enhances cell proliferation in tissues such as bone, kidney, uterus, and mammary gland (19, 20). These estradiol-responsive tissues have also been identified as TGFß target tissues (21). The estrogen agonist/antagonist drug raloxifene acts as an antagonist to estrogen’s actions in some tissues, such as breast and uterus, while acting to mimic estrogen’s protective actions in bone. The selective action of raloxifene has been correlated to an isoform-specific increase in TGFß expression, where TGFß3 is selectively increased; estradiol has a similar action, but at higher concentrations than those needed with raloxifene (16). The pituitary of F-344 rats appears to be another tissue where estrogen may exert its actions by increasing TGFß3 expression.
The results presented here suggest that under basal conditions lactotropic growth is halted or maintained at a low rate by TGFß1 produced locally in lactotropes and that estradiol, by reducing the inhibitory influence of TGFß1 and enhancing the production of stimulatory TGFß3, activates DNA replication and lactotropic proliferation. Whether this bifunctional regulation of growth by two closely related TGFß proteins is unique to lactotropes or exists in other estradiol-responsive cells will be an interesting question to further the understanding of the mitogenic actions of estradiol.
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Acknowledgments |
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Footnotes |
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2 Present address: Vollum Institute, Oregon Health Science University,
L-474, Portland, Oregon 97201.
Received April 7, 1999.
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-globulin (Calbiochem, La Jolla, CA). n =
5–6/group.