Vol. 275, Issue 4, E553-E557, October 1998
Transduction pathways involved in rapid hormone receptor
regulation in the mammary epithelium
Franklyn F.
Bolander Jr.
Department of Biological Sciences, University of South Carolina,
Columbia, South Carolina 29208
 |
ABSTRACT |
Previous studies
have shown that the envelope protein of the mouse mammary tumor virus
(MMTV) rapidly upregulates prolactin (PRL) receptors by shifting them
from internal pools to the cell surface and downregulates epidermal
growth factor (EGF) receptors by inducing their internalization and
degradation. This study shows that the effect on PRL receptors is
mediated by the nitric oxide (NO)/cGMP pathway, since it can be
mimicked by an NO donor or 8-bromo-cGMP and can be blocked by an NO
synthase inhibitor. In contrast, the effect on EGF receptors is
mediated by tyrosine phosphorylation and phosphatidylinositol 3-kinase
(PI3K), since it can be blocked by either a tyrosine
kinase inhibitor or by a PI3K inhibitor. Both of these pathways can be
activated by a calcium ionophore and inhibited by calcium chelation.
Therefore, it appears that the mouse mammary tumor virus envelope
protein, like other retroviral envelope proteins, initially elevates
cytoplasmic calcium, which can then stimulate both the NO/cGMP and the
tyrosine phosphorylation/PI3K pathways, leading to PRL receptor
upregulation and EGF receptor downregulation, respectively.
nitric oxide; cGMP; phosphatidylinositol 3-kinase; epidermal growth
factor receptor; prolactin receptor
| |
INTRODUCTION |
THIS LABORATORY has previously shown that, in mammary
epithelium, the envelope protein (gp52) of the mouse mammary tumor
virus (MMTV) can rapidly elevate prolactin (PRL) receptors by
recruiting them from internal pools (7) and lower epidermal growth
factor (EGF) receptors by inducing their internalization and
destruction (5). These actions are presumed to facilitate viral
propagation, since the pups are infected by MMTV in the milk, a product
of the differentiated mammary gland. By upregulation of receptors for
PRL, a differentiative hormone, and downregulation of those for EGF, a
growth/anti-differentiative hormone, the MMTV can enhance mammary gland
differentiation, milk production, and its own propagation. This work
attempts to determine which transduction pathways are being used to
effect these changes in receptor levels.
Several molecular mechanisms have been associated with receptor
regulation. For example, the most intensive investigations in the field
of receptor cycling have been done on the receptor tyrosine kinases, in
which it has been shown that the EGF, insulin, insulin-like growth
factor II (IGF-II), platelet-derived growth factor, and stem cell
factor receptors all require tyrosine phosphorylation for receptor
processing (15, 17, 24, 32, 33, 35). Although the PRL receptor is not a
tyrosine kinase, it is closely associated with soluble tyrosine kinases
(11) and therefore may be subject to a similar control. As a result,
this pathway is a potential mediator of the actions of MMTV. The role
of this phosphorylation can be examined by the use of genistein, a
tyrosine kinase inhibitor, and pervanadate, a phosphotyrosine
phosphatase inhibitor that would allow tyrosine phosphorylation to
accumulate.
Another second messenger that has been implicated in receptor
processing is phosphatidylinositol 3-kinase (PI3K), which is believed
to be involved in vesicular trafficking. PI3K is required for the
redistribution of the IGF-II, platelet-derived growth factor, and
possibly the stem cell factor receptor (12, 15, 17, 35). However, it is
not involved in the EGF-induced downregulation of its own receptor
(32). The participation of PI3K in the MMTV-induced effects can be
tested with wortmannin, a PI3K inhibitor.
Although the nitric oxide (NO)/cGMP pathway has not yet been associated
with receptor relocation, previous studies have demonstrated that it
can be activated by the envelope protein of MMTV (4) as well as by
several other retroviral envelope proteins (8, 27). Therefore, this
transduction system was also investigated. NO synthase (NOS), a
calcium-dependent enzyme, can be inhibited by calcium depletion or by
substrate antagonists; the pathway can be activated by calcium
ionophores or NO donors, for example sodium nitroprusside (SNP).
Finally, one of the major effectors for NO is cGMP, the synthesis of
which is stimulated by NO; the role of this nucleotide can be examined
employing any of several cGMP agonists.
With the use of these pharmacological tools, the contribution of these
various transducing pathways to the effects of MMTV on PRL and EGF
receptors can be evaluated.
| |
EXPERIMENTAL PROCEDURES |
Materials.
Mouse EGF (lot 908374) was purchased from Collaborative Biomedical
Products (Bedford, MA), and ovine PRL (oPRL-19) was kindly provided by
the Hormone Distribution Program (National Institute of Diabetes and
Digestive and Kidney Diseases, Bethesda, MD). HEPES, genistein,
wortmannin, 8-bromo-cGMP, A-23187, EGTA, sodium orthovanadate, hydrogen
peroxide, catalase, lactoperoxidase, and BSA were obtained from Sigma
Chemical (St. Louis, MO).
NG-monomethyl-L-arginine
(L-NMMA) and SNP were purchased
from Alexis (San Diego, CA). Medium 199 with Earle's salts was from
GIBCO (Grand Island, NY), and collagenase type I (179 U/mg) was
obtained from Worthington Biochemicals (Freehold, NJ).
Na125I (carrier free) was
purchased from NEN (Boston, MA).
The MMTV envelope protein was prepared in the author's laboratory by
the method of Marcus et al. (20); milk from C3H/HeN MMTV+ mice was used
as the source for the virus. Pervanadate was prepared from
orthovanadate and hydrogen peroxide according to the procedure of
Pumiglia et al. (23) and used immediately.
Organ culture.
Virgin mice (C3H/HeN MMTV+ and MMTV
) were obtained from the
Frederick Cancer Research Facility (Frederick, MD). The mice (MMTV) were killed by cervical dislocation, and explants were prepared from the fourth pair of mammary glands under sterile conditions, as previously described (16). Explants were cultured on
siliconized lens paper in medium 199 containing 20 mM HEPES (pH 7.6)
and combinations of the following reagents, as required by the
individual experiment: SNP (10 µM), 8-bromo-cGMP (10 µM), L-NMMA (100 µM), genistein (50 µM), pervanadate (100 µM), wortmannin (100 nM), A-23187 (32.5 nM),
EGTA (3 mM), and/or the MMTV envelope protein (1 µg/ml). The
tissue was incubated under air at 37°C.
The concentrations of the above reagents were determined empirically
using mammary epithelium cultured for 30 min, as described above. Under
these conditions, 32.5 nM A-23187 increased
45Ca2+
fluxes 8.5-fold (3). During the short incubation period without hormones, neither pervanadate nor genistein affected
32P incorporation into proteins
immunoprecipitated with anti-phosphotyrosine antibody, but both
significantly affected the actions of PRL. PRL increased tyrosine
phosphorylation 440%, as determined by the method of Said and Medina
(25); 100 µM pervanadate amplified this effect 2.5-fold, whereas 50 µM genistein reduced PRL stimulation by 77%. PRL also stimulated
PI3K 370%, as measured by the method of Whitman et al. (34). Again,
100 nM wortmannin had no effect under basal conditions, but it did
completely suppress the stimulation by PRL. Finally, the effect of
L-NMMA on NOS was
ascertained indirectly by determination of cGMP levels with the use of
an RIA, as previously described (4). The MMTV envelope protein
stimulated cGMP levels 220%, whereas
L-NMMA totally blocked this
elevation.
Cell isolation and receptor assay.
EGF and insulin receptors were measured on an epithelial cell-enriched
fraction isolated from mammary explants, as previously described (31).
Briefly, the tissue was finely minced and digested with collagenase
[1.5 mg/ml of medium 199 containing 20 mM HEPES (pH 7.6) and 4%
(wt/vol) BSA] at 37°C. During this incubation, the tissue
fragments were pipetted through successively smaller bore pipettes.
After 30 min, the cells were centrifuged and washed three times in
medium 199 containing 20 mM HEPES (pH 7.6) and 2% BSA.
EGF and insulin were iodinated by a modification (6) of the
lactoperoxidase method of Miyachi et al. (22). The resulting 125I-labeled hormone was used in
binding studies, as previously described (2). Receptor assays were
performed after a 30-min incubation; this time period was chosen
because it is the shortest interval during which one can easily see
receptor redistribution within the mammary epithelial cell.
Protein was determined by the method of Lowry et al. (19), and binding
data were analyzed by the method of Scatchard (26). Because of the low
epithelial content of mouse mammary glands, five to six animals were
required to generate enough cells to construct a single Scatchard plot;
all experiments were replicated six times, and resulting data were
subjected to ANOVA.
| |
RESULTS |
The envelope protein of MMTV has previously been demonstrated to induce
RNA synthesis in mammary epithelium via the NO/cGMP pathway (4), and
Fig. 1 shows that these same transducers
can recruit PRL receptors to the cell surface during the 30-min
incubation period. SNP, an NO donor, and 8-bromo-cGMP both mimic the
envelope protein, whereas
L-NMMA, an NOS inhibitor, blocks
the effects of MMTV. Tyrosine phosphorylation does not appear to be
involved, because pervanadate, a phosphotyrosine phosphatase inhibitor, does not shift PRL receptors, and genistein, a tyrosine kinase inhibitor, does not block the effects of MMTV. Finally, the inhibition of PI3K by wortmannin also has no effect on MMTV induction of PRL
receptors.
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Fig. 1.
Effect of modulators of nitric oxide (NO)/cGMP, tyrosine kinase, and
phosphatidylinositol 3-kinase (PI3K) pathways on plasma membrane
prolactin (PRL) receptors. Receptors were measured 30 min after
addition of envelope protein (mouse mammary tumor virus, MMTV); other
culture conditions and compound concentrations are given in
EXPERIMENTAL PROCEDURES. Data are
means ± SE of 6 separate experiments. SNP, sodium nitroprusside;
L-NMMA,
NG-monomethyl-L-arginine.
|
|
In contrast to the stimulation of PRL receptors, EGF receptors are
downregulated by the MMTV envelope protein (5). Surprisingly, the
NO/cGMP pathway does not appear to play any role in this process, because neither SNP nor 8-bromo-cGMP can induce internalization, and
L-NMMA cannot block the effect
of MMTV (Fig. 2). There is evidence that
tyrosine phosphorylation is involved, since genistein can block the
MMTV-triggered downregulation. However, pervanadate cannot mimic the
effects of the MMTV envelope protein. Although pervanadate can augment
PRL-induced tyrosine phosphorylation (see EXPERIMENTAL
PROCEDURES), it was unable to affect tyrosine
phosphorylation during a 30-min incubation. Similar results have been
reported in rabbit mammary epithelium, in which no effects of
pervanadate were seen on gene expression before a 24-h exposure (1),
and in mouse mammary epithelium, in which no effects of this compound were observed on mitosis before 5 days (21). Apparently, the basal
activity of tyrosine kinases in the mammary gland is so low that the
inhibition of phosphotyrosine phosphatases is not sufficient to elevate
net tyrosine phosphorylation during short incubation periods. Like
tyrosine phosphorylation, PI3K also appears to be important, as
wortmannin blocks the downregulation induced by the MMTV envelope
protein.
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Fig. 2.
Effect of modulators of NO/cGMP, tyrosine kinase, and PI3K pathways on
plasma membrane epidermal growth factor (EGF) receptors. Receptors were
measured 30 min after addition of envelope protein (MMTV); other
culture conditions and compound concentrations are given in
EXPERIMENTAL PROCEDURES. Data are
means ± SE of 6 separate experiments.
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|
Calcium can integrate all of these transduction pathways. First,
calcium can activate NOS, the product of which, NO, can stimulate the
soluble guanylate cyclase. In addition, calcium can activate several
soluble tyrosine kinases, the substrates of which include docking
proteins for PI3K. Therefore, it was of interest to examine the role of
calcium in receptor regulation. Cytosolic calcium levels were elevated
by the calcium ionophore A-23187, which allows extracellular calcium to
leak into the cell. Calcium stores were depleted by using A-23187 in
combination with the calcium chelator EGTA. With EGTA in the medium,
the calcium concentration is reversed, and A-23187 now transports
calcium out of the cell. Cells were preincubated with A-23187 and EGTA
for 30 min before the addition of the MMTV envelope protein. The
elevation of cytoplasmic calcium was indeed able both to increase the
plasma membrane receptors for PRL and decrease those for EGF (Fig.
3). Furthermore, calcium-depleted cells
were unable to respond to MMTV with respect to receptor redistribution.
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Fig. 3.
Effect of modulators of cellular calcium on plasma membrane PRL
(left) and EGF receptors
(right). Receptors were measured 30 min after addition of envelope protein (MMTV); other culture conditions
and compound concentrations are given in EXPERIMENTAL
PROCEDURES. Data are means ± SE of 6 separate
experiments.
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Finally, all agonists at maximally effective concentrations were
equipotent to the MMTV envelope protein (Figs. 1-3) and were not
additive with the envelope protein (Fig. 3 and unpublished observations), suggesting that MMTV and these effectors are utilizing the same pathway.
| |
DISCUSSION |
Most hormones downregulate their own receptors, especially those
binding receptor tyrosine kinases or G protein-coupled receptors. In
the former case, it has been shown that tyrosine receptor
phosphorylation is essential for internalization (12, 15, 24, 32, 33, 35); in addition, PI3K activation is often required (12, 15). These two
processes are frequently linked, since PI3K contains an
src-homology 2 domain
that binds phosphotyrosines; this interaction not only allosterically
stimulates the enzyme but also brings it to the plasma membrane near
its substrates. The exact role of PI3K in receptor regulation is not
clear, but PI3K appears to be involved with membrane trafficking,
probably through protein kinase B, protein kinase C
, or other
transducers that its product is known to affect (29). Therefore, it is
not surprising that the MMTV envelope protein also utilizes this
pathway to induce EGF receptor internalization. It is interesting that
EGF itself requires receptor tyrosine phosphorylation but not PI3K
stimulation to downregulate its own receptor (32), suggesting that
different stimuli can have similar effects on receptor regulation via
different mechanisms.
Fewer investigations have been done on receptor upregulation via
recruitment from internal pools, probably because most receptors are
already at the cell surface, so that few microsomal receptors are
available to recruit. However, PRL does have a large reservoir of
internal receptors (7). In contrast to the situation with the EGF
receptor, the relocalization of the PRL receptor appears to be mediated
by the NO/cGMP pathway (Fig. 4). It is not
clear why two different pathways are employed by the envelope protein to move hormone receptors within a cell. The use of two different pathways may be required to ensure directional specificity. However, IGF-II recruits its own receptors to the cell surface in a
PI3K-dependent manner (17), suggesting that the PI3K pathway can be
used to shuttle receptors in either direction. Alternatively, the
mechanisms may be receptor specific: EGF and IGF-II bind receptor
tyrosine kinases, whereas PRL interacts with a cytokine receptor.
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Fig. 4.
A hypothetical pathway integrating data in this study. NOS, NO
synthase; PKB, protein kinase B; PKC, protein kinase C;
Y-PO4, a phosphotyrosine docking
site.
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|
Although studies of receptor exocytosis are limited, there are several
examples of nonreceptor membrane proteins being induced to migrate to
the cell surface in response to cyclic nucleotides: the renal water
channel, aquaporin-2 (10), the epithelial chloride channel (28), and
the cystic fibrosis transmembrane conductance regulator (18) are all
translocated to the plasmalemma in response to the elevation of cAMP.
The effect on aquaporin-2 is mediated by direct phosphorylation by the
cAMP-dependent protein kinase (protein kinase A, PKA), but
the effect on the chloride channel is PKA independent. The mechanism
for the effect on cystic fibrosis transmembrane conductance regulator
is unknown. It is interesting to note that cGMP, which induces PRL
receptor redistribution, also has the potential to activate PKA at high
physiological concentrations (9, 14) in addition to stimulating its own
kinase, the cGMP-dependent protein kinase.
Finally, there are also a few examples of calcium-regulated membrane
protein redistribution. Acetylcholine activation of the muscarinic
receptor in the parotid gland recruits the aquaporin-5 channel to the
cell surface by a calcium-dependent, but protein kinase C-independent,
mechanism (13). In addition, Ret, a member of the receptor complex for
the glial cell line-derived neurotropic factor, requires calcium for
posttranslational processing and migration to the cell surface (30).
Neither study examined the role of the NO/cGMP pathway in their
respective system.
Receptor number is an important factor in determining the sensitivity
of a cell toward hormones. Although the concentration can be regulated
by altering receptor synthesis, this usually requires several hours.
Receptor redistribution is a mechanism that can dramatically change
receptor number at the cell surface within minutes. For many receptor
tyrosine kinases, this process appears to be mediated by components of
the tyrosine phosphorylation/PI3K pathway. However, this study has
shown that, for at least one cytokine receptor, another transduction
system predominates: the NO/cGMP pathway.
| |
ACKNOWLEDGEMENTS |
The technical assistance of William McAmis is gratefully
appreciated.
| |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: F. F. Bolander, Jr., Dept. of Biological
Sciences, Univ. of South Carolina, Columbia, SC 29208.
Received 15 April 1998; accepted in final form 23 June 1998.
| |
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Am J Physiol Endocrinol Metab 275(4):E553-E557
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Abstract
Introduction
