(Received for publication, August 14, 1995; and in revised form, November 10, 1995)
From the
The rat pituitary cell line GHC
secretes
granins (chromogranin B and secretogranin II) and prolactin by the
regulated secretory pathway. The intracellular storage of prolactin is
preferentially induced by hormone treatment with estradiol, insulin,
and epidermal growth factor. The goal of this study was to determine
the effect of hormone treatment on storage of granins and constitutive
secretory markers. The granins were efficiently stored in both
hormone-treated and -untreated cells (17% of total secreted in 4 h).
Secreted alkaline phosphatase (SEAP), a truncated membrane protein that
would not be expected to enter secretory granules, and
glycosaminoglycan, a marker for the constitutive secretory pathway,
exhibited 70-80% secretion under both conditions. In comparison,
the relative prolactin secretion was 31 and 68% from hormone-treated
and -untreated cells, respectively. Phorbol ester and KCl stimulated
prolactin secretion 2.3-fold from untreated cells and 5.5-fold from
hormone-treated cells. In contrast, SEAP secretion was stimulated
1.5-fold from both treated and untreated cells, consistent with
secretion by the constitutive secretory pathway. Stimulated secretion
of granins was detected from both hormone-treated and -untreated cells.
These results suggest that granin and prolactin storage are
differentially regulated and that the constitutive secretory pathway is
not affected by hormone treatment.
Endocrine cells exhibit a constitutive secretory pathway, common to all eukaryotic cells, as well as a regulated secretory pathway (Kelly, 1985). The latter is characterized by intracellular storage of secretory proteins, slow basal release, and stimulated secretion in response to secretogogues. Granins (chromogranin A, chromogranin B, secretogranin II) are a family of sulfated, calcium-binding proteins that are co-stored with peptide and amine hormones in secretory granules of endocrine cells. Thus, granins can serve as general markers for the regulated secretory pathway in endocrine cells (Scammell, 1993). It has been suggested that granins play a direct role in the sorting and packaging of peptide hormones in secretory granules (Rosa et al., 1985; Gorr et al., 1987a; Huttner et al., 1991; Scammell, 1993). In support of this hypothesis, granins exhibit calcium-induced aggregation at low pH, i.e. the conditions found in the trans-Golgi network and secretory granules of endocrine cells (Gorr et al., 1987b, 1988, 1989; Gerdes et al., 1989; Chanat and Huttner, 1991; Thompson et al., 1992). These aggregates can include other regulated secretory proteins but exclude constitutive secretory proteins (Gorr et al., 1989; Huttner et al., 1991). Specific sorting receptors have also been proposed to act in the segregation of regulated secretory proteins (Kelly, 1985). However, such sorting receptors have not been conclusively identified (Gorr et al., 1992).
The rat
pituitary cell line GHC
, a subclone of the GH3
cell line, stores prolactin, chromogranin B, and secretogranin II in
secretory granules (e.g. Scammell et al. (1990a)) and
secretes all three proteins in response to extracellular stimulation
(Hinkle et al., 1992). The expression of prolactin and granins
is induced by treating the cells with a combination of estradiol,
insulin, and epidermal growth factor (EGF) (
)(Scammell et al., 1986, 1990a; Thompson et al., 1992). In
addition, this hormone treatment preferentially induces prolactin
granulogenesis and the intracellular storage of prolactin (Scammell et al., 1986; Reaves et al., 1990). However, the
expression or storage of growth hormone, which is present in small
amounts in these cells, is not induced by hormone treatment (Scammell et al., 1986). Also, when insulin is expressed in transfected
GH
C
cells, the peptide is sorted to the
regulated secretory pathway, but insulin does not exhibit a
preferential increase in storage in hormone-treated cells (Reaves et al., 1990). To determine if induction of storage and
granulogenesis is specific for prolactin, the storage of the
endogenous-regulated secretory proteins secretogranin II and
chromogranin B, as well as constitutive secretory markers were
compared. Hormone treatment induced granin synthesis, but it did not
preferentially stimulate granin storage. The secretion and storage of
secreted placental alkaline phosphatase (SEAP) and glycosaminoglycans
was not affected by hormone-treatment.
In
some experiments, 4-methylumbelliferyl -D-xyloside was
added from a 100 mM stock solution in Me
SO
to a final concentration of 1 mM. The xyloside was added
to the preincubation and labeling buffers. Control incubations
contained 1% Me
SO
.
Fluorographs and dot-blots were quantitated by densitometric scanning using a Bio Image Visage 60 image scanner (Bio Image, Ann Arbor, MI). The film exposure times were adjusted to ensure that the bands were in the linear range of the film. In general, the samples from chase incubations were exposed longer than the cell extracts and labeling medium samples and are, therefore, not directly comparable. Due to some interexperimental variation, the data on stimulated secretion of prolactin and SEAP were normalized by dividing all values in an experimental group (± stimulation) by the lowest value from the unstimulated samples. The data were analyzed by two-tailed Student's t test or analysis of variance with Student-Newman-Keuls multiple comparison post test. p < 0.05 was considered statistically significant.
Rat pituitary GHC
cells were cultured
for 4 days with or without hormone treatment (EIE: 1 nM estradiol, 300 nM insulin, 10 nM EGF).
Immunofluorescence microscopy of untreated cells indicated that
prolactin was predominantly found in a perinuclear region consistent
with localization in the Golgi complex. Only few cytoplasmic granules
were detected in these cells. Hormone-treated cells, on the other hand,
exhibited a bright punctuate pattern throughout the cytoplasm,
consistent with storage of prolactin in secretory granules (not shown).
Prolactin was quantitated by dot-immunoblotting of cell extracts and
media samples. Densitometric scanning of the immunoblots indicated that
hormone treatment induced the cellular and extracellular prolactin
levels by 20- and 3-fold, respectively, in agreement with earlier
reports (Scammell et al., 1986; Reaves et al., 1990;
Hinkle et al., 1992).
To determine if the storage of secretogranin II was similar to that of prolactin, the relative secretion of both proteins in 4 h was quantitated (Fig. 1). The relative prolactin secretion was 68% from untreated cells and 36% from hormone-treated cells, consistent with increased prolactin storage in hormone-treated cells. In contrast, secretogranin II exhibited 15-20% secretion under both conditions, suggesting that the protein is predominantly stored in both untreated and hormone-treated cells.
Figure 1:
Relative secretion of prolactin (PRL) and secretogranin II (SgII) from
GHC
cells. The secreted and cellular amounts of
PRL and SgII from untreated (-EIE) and hormone-treated cells
(+EIE) were quantitated by dot-immunoblotting after 4 h of
incubation. % Secreted = secreted amount/total
(secreted + cellular) amount
100%. Results from seven (PRL) and four (SgII; n = 14)
independent experiments were analyzed and presented as mean ±
S.E. Asterisk, different from untreated PRL samples (p < 0.0001; n =
21-23).
Since an endogenous marker protein for the constitutive
secretory pathway has not been identified in GHC
cells, a truncated form of human placental alkaline phosphatase
(SEAP) was expressed in these cells and evaluated as a potential marker
for constitutive secretion. SEAP was released linearly from transfected
cells, and about 80% of total SEAP activity was found in the medium of
untreated cells after 4 h (Fig. 2). Hormone treatment had no
significant effect on the relative secretion of SEAP. When the cells
were incubated at 16 °C, to block protein export, only about 30% of
total SEAP activity was secreted in 4 h (Fig. 2). Incubation at
16 °C did not lead to a build-up in intracellular SEAP, suggesting
that synthesis or degradation of SEAP was affected in addition to
secretion.
Figure 2:
Relative secretion of SEAP. The secreted
and cellular amounts of SEAP from untreated (-EIE) and
hormone-treated cells (+EIE) were quantitated enzymatically after
4 h of incubation at 37 or 16 °C. % Secreted =
secreted amount/total (secreted + cellular) amount 100%.
Results from two independent experiments were analyzed and presented as
mean ± S.E. asterisk, different from 37 °C samples (p < 0.001; n =
6-11).
The rapid basal secretion of SEAP suggested that the
enzyme was secreted constitutively from both untreated and
hormone-treated GHC
cells. To further test
this, secretion was stimulated with phorbol ester and KCl and compared
with unstimulated secretion. SEAP secretion was stimulated 1.5-fold
from both untreated and hormone-treated cells (Fig. 3A), consistent with secretion by the
constitutive secretory pathway. Basal SEAP secretion resumed when cells
that had been incubated at 16 °C were warmed to 37 °C, but
stimulated secretion did not increase (not shown). Phorbol ester and
KCl stimulated prolactin secretion 2.3-fold from untreated cells and
5.5-fold from hormone-treated cell, respectively (Fig. 3B). The stronger stimulation of hormone-treated
cells was in agreement with an earlier report (Reaves et al.,
1990).
Figure 3:
Stimulated secretion of SEAP (A)
and prolactin (B) from untreated (-EIE) and
hormone-treated cells. The cells were incubated with 50 mM NaCl + 100 nM 4-phorbol 12,13-didecanoate (CTRL) or 50 mM KCl + 100 nM phorbol
12-myristate 13-acetate (STIM). Data from two to six
independent experiments, each performed in triplicate, were normalized
as described under ``Experimental Procedures,'' and expressed
as mean ± S.E. Asterisk, different from control (SEAP,
-EIE, p < 0.03, n = 6; SEAP,
+EIE, p < 0.02, n = 6; prolactin,
-EIE, p < 0.02, n = 9; prolactin,
+EIE: p < 0.0001, n =
17-18).
Sulfate-labeled GHC
cells were used
to further analyze the secretion of granins from untreated and
hormone-treated cells. Since tyrosine-sulfation of secretogranin II and
chromogranin B occurs in the trans-Golgi network, this modification is
a specific marker for the late stages of the secretory pathways
(Huttner, 1988). GH
C
cells were radiolabeled
with [
S]sulfate for 4 h to allow sufficient time
for the sulfated granins to reach the storage compartment. Medium and
extracts of hormone-treated cells contained a high molecular weight
sulfated molecule, presumably representing a heparansulfate
proteoglycan (Hinkle et al., 1992), chromogranin B, and
secretogranin II (Fig. 4, lanes 1 and 3).
Similar results have been reported for untreated cells (Hinkle et
al., 1992). The cellular and secreted amounts of secretogranin II
each increased about 6-fold upon hormone treatment, while chromogranin
B synthesis was increased about 2-fold (Table 1). The higher
stimulation of secretogranin II synthesis compared with chromogranin B
synthesis reflects similar differences in mRNA levels after
hormone-treatment (Thompson et al., 1992). Hormone treatment
did not appear to selectively increase the cellular amounts of sulfated
granins (Table 1). To further determine if hormone-treatment
affected granin storage, the relative secretion of sulfated granins was
calculated for hormone-treated and untreated cells. Approximately 33%
of each granin was secreted from both hormone-treated and untreated
cells, confirming that hormone-treatment did not increase the fraction
of stored granins (Fig. 5).
Figure 4:
Metabolic labeling of
GHC
cells with
Na
SO
. Hormone-treated cells were
labeled with [
S]sulfate in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of methylumbelliferyl xyloside. Aliquots of labeling medium (MED) and cell extracts (CELL) were analyzed by
SDS-PAGE and fluorography. The positions of sulfated proteoglycan (p), chromogranin B (c), secretogranin II (s), and glycosaminoglycans (g) are indicated. Lane 5 is a shorter exposure of lane 3 to better
visualize the chromogranin B band.
Figure 5:
Relative secretion of granins and
glycosaminoglycan from [S]sulfate-labeled
GH
C
cells. Hormone-treated (EIE) or
untreated (blank) cells were radiolabeled in the presence (MUX) or absence (blank) of methylumbelliferyl xyloside.
Aliquots of labeling medium and cell extracts were analyzed by SDS-PAGE
and fluorography. Radioactive chromogranin B (CgB),
secretogranin II (SgII), and glycosaminoglycans (GAG)
were quantitated by densitometric scanning of the radiographic film. % Secreted = secreted amount/total (secreted +
cellular) amount
100%. Results from three independent
experiments were analyzed and presented as mean ± S.E. Asterisk, different from granin samples (p <
0.001, n = 3-11).
To test if the 30% secretion rate of sulfated granins represented the regulated (slow) or constitutive (fast) secretory pathway, the cells were labeled in the presence of methylumbelliferyl xyloside to stimulate synthesis of sulfated glycosaminoglycans, a marker for the constitutive secretory pathway (Fig. 4; lanes 2 and 4). The xyloside did not affect the proportion of sulfated granins detected in the secretion medium (Fig. 5). Glycosaminoglycan synthesis was induced approximately 11-fold by xyloside treatment. In contrast to the granins, the glycosaminoglycans were predominantly (67% of total) found in the secretion medium ( Fig. 4and Fig. 5). These results are consistent with sorting and storage of granins in the regulated secretory pathway in both hormone-treated and untreated cells, while glycosaminoglycans are rapidly secreted by the constitutive secretory pathway. Stimulated secretion of sulfated granins was tested by incubating the cells for 15 min in the presence of 50 mM KCl. In two experiments, secretion of chromogranin B was stimulated about 2.3-fold from both hormone-treated and untreated cells (Fig. 6A), while secretion of secretogranin II was stimulated 2-fold from hormone-treated cells and 1.6-fold from untreated cells (Fig. 6B). Due to the small amounts of the diffuse sulfated glycosaminoglycan band present in the chase media, the constitutive marker could not be reliably quantitated in the stimulation experiments. However, in a control experiment, SEAP secretion was stimulated 1.2-fold (p < 0.06) in 1 h by 50 mM KCl (not shown).
Figure 6:
Stimulated secretion of granins from
hormone-treated (+EIE) and untreated
(-EIE) cells. [S]sulfate-labeled
GH
C
cells were chase incubated for 15 min in
the presence of 50 mM NaCl (control) or 50 mM KCl
(stimulated). Secreted chromogranin B (top) and secretogranin
II (bottom) were quantitated by SDS-PAGE and fluorography and
presented as mean ± S.D. Asterisk, different from
control; CgB, -EIE, p < 0.0002, n =
2-3; CgB, +EIE, p < 0.002; n =
3; SgII, -EIE, p < 0.004, n =
2-3; SgII, +EIE, p < 0.005; n =
3). IOD, integrated optical
density.
GHC
cells
express a cell-associated heparansulfate proteoglycan (Hinkle et
al., 1992). Methylumbelliferyl xyloside reduced the amount of
sulfated proteoglycan in the cells about 30% (Fig. 4), but the
xyloside did not prevent stimulated secretion of sulfated granins (Fig. 7). Thus, neither the reduction of proteoglycan synthesis
nor the increased constitutive secretion of glycosaminoglycans
prevented sorting of granins to the regulated secretory pathway.
Figure 7:
Stimulated secretion of granins in the
presence of methylumbelliferyl xyloside. GHC
cells were labeled with [
S]sulfate in the
presence of 1 mM methylumbelliferyl xyloside and then chase
incubated for 15 min in the presence of 50 mM NaCl (control)
or 50 mM KCl (stimulated). Secreted granins were quantitated
by SDS-PAGE and fluorography and presented as mean ± S.D. Asterisk, different from control (CgB, p < 0.0007; SgII, p < 0.007; n = 3). IOD, integrated optical density.
Chromogranin B and secretogranin II, which serve as markers
for the regulated secretory pathway in most endocrine cell types
(Scammell, 1993), are expressed in GHC
cells,
and their expression is induced by hormone treatment with estradiol,
insulin, and EGF (Scammell et al., 1990a; Thompson et
al., 1992). In contrast to prolactin (Scammell et al.,
1986; Reaves et al., 1990), the storage of granins is not
preferentially induced by hormone treatment, although the relative
secretion remained constant, indicating that granin storage was
increased in parallel with granin expression in hormone-treated cells.
Thus, both secretogranin II and chromogranin B are efficiently stored
in untreated cells, while prolactin is rapidly secreted from these
cells, as measured by the relative secretion in the absence of
stimulation. In hormone-treated cells, on the other hand, both granins
and prolactin are efficiently stored, i.e. they exhibit low
relative secretion (Table 2). The relative secretion of prolactin
in 4 h reported here is similar to that previously reported (Scammell et al., 1986; Reaves et al., 1990; Hinkle et
al., 1992) (Table 2), assuming constant cellular amounts and
linear secretion in 24 h (see Reaves et al. (1990)). These
results indicate that prolactin and granin storage are differentially
regulated in GH
C
cells.
Granins have been
suggested to play a role in the sorting and packaging of peptide
hormones in the regulated secretory pathway (e.g. Gorr et
al. (1987a) and Huttner et al.(1991)). This is supported
by the co-aggregation in vitro of parathyroid hormone and
chromogranin A and B under the conditions of low pH and millimolar
concentrations of calcium that are found in the trans-Golgi network and
secretory granules (Gorr et al., 1989). Similarly, prolactin
exhibits low pH-induced aggregation (Thompson et al., 1992).
Recent evidence, however, suggests that calcium-induced aggregation at
low pH is neither necessary (Schmidt and Moore, 1994) nor sufficient
(Chanat et al., 1994) for sorting of regulated secretory
proteins. The present findings that granins, but not prolactin, are
efficiently stored in untreated GHC
cells
indicates that granin storage is not sufficient for the efficient
storage of prolactin. This finding suggests that the granins do not
directly interact with prolactin in the formation of a storage complex.
Indeed, it appears that the prolactin storage complex is highly
specific since it is disrupted by the expression of small amounts of
human prolactin in the rat cell line (Arrandale and Dannies, 1994).
One possible explanation for the differential storage of prolactin
and granins is that the proteins are stored in separate granule
populations. This is directly supported by the finding that prolactin
and secretogranin II are stored in distinct secretory granule
populations in bovine pituitary somatomammotrophs (Hashimoto et
al., 1987). Similarly, peptide hormones derived from a common
prohormone are sorted to, and stored in, separate granule populations
in Aplysia neuronal cells (Jung and Scheller, 1991). Thus,
individual cell types can contain separate regulated secretory pathways
including separate granule populations. Differential storage in
separate granule populations may be mediated by distinct sorting
signals (Gorr and Darling, 1995), presumably including Asn and Ser
in human prolactin (Arrandale and Dannies,
1994) or differential retention during granule maturation (Kuliawat and
Arvan, 1994). A second, but not mutually exclusive, explanation for
differential storage is that prolactin and granin storage involves
different subpopulations of GH
C
cells. Thus,
this cell line is heterogeneous and prolactin and granin expression are
not uniformly induced in hormone-treated cells, although most prolactin
expressing cells also express at least one of the two granins (Scammell et al., 1990a). Scammell and co-workers (Scammell et
al., 1990a; Thompson et al., 1992) also noted that granin
expression is less strongly induced than that of prolactin upon hormone
treatment, although granins exhibited a redistribution to secretory
granules, similar to that of prolactin, as judged by immunofluorescence
(Scammell et al., 1990a). It is possible that the preferential
storage of prolactin correlated with the large increase in expression
levels in hormone-treated cells. However, prolactin storage is not
directly induced by high prolactin levels, since cells treated with EGF
alone exhibit increased prolactin expression with no preferential
increase in prolactin storage (Reaves et al., 1990).
A
truncated, soluble form of vesicular stomatitis virus G-protein has
been used as a marker for constitutive secretion from endocrine cells (e.g. Moore and Kelly(1986)). Here it is shown that a
truncated form of human placental alkaline phosphatase (SEAP) is
secreted constitutively from GHC
cells. Since
alkaline phosphatase activity is readily quantitated, SEAP is a
convenient marker for the constitutive secretory pathway. SEAP and the
constitutive secretory marker glycosaminoglycan (Burgess and Kelly,
1984) were rapidly secreted from both untreated and hormone-treated
cells. Thus, it appears that the constitutive secretory pathway is not
affected by hormone treatment. Furthermore, the rapid secretion of SEAP
and glycosaminoglycans from hormone-treated cells, demonstrates that
the slow release of prolactin and granins is not due to a general block
of the secretory pathways due to hormone-treatment. The relative
secretion of SEAP was blocked at 16 °C, but without an increase in
cellular storage of SEAP, suggesting that SEAP is not shifted to a
storage compartment at the lower temperature. Similarly, the absence of
increased stimulated SEAP secretion after return to 37 °C suggests
that reducing its rate of secretion is not sufficient to target SEAP to
the regulated secretory pathway.
Stimulated secretion of prolactin has been detected in both untreated and hormone-treated cells (this study and Reaves et al. (1990)). The stronger stimulation of prolactin secretion from hormone-treated cells is presumably accounted for by the increased intracellular storage of the hormone. In untreated cells, the stimulation of prolactin secretion is somewhat stronger than that of SEAP. However, taken together with the Golgi localization of prolactin and the relative secretion data, it appears that prolactin is mainly secreted by the constitutive secretory pathway in untreated cells. Since untreated and hormone-treated cells exhibit both regulated and constitutive secretory pathways, as evidenced by granin and SEAP secretion, respectively, these results suggest that a regulated pathway for prolactin secretion is up-regulated by hormone treatment.
Chromogranin B and secretogranin II are tyrosine-sulfated in the
trans-Golgi network (Rosa et al., 1985; Huttner, 1988); thus,
analysis of granins in untreated and hormone-treated cells allowed the
use of sulfation as a marker for the late stages of the secretory
pathway, thereby eliminating differences due to transit through the
endoplasmic reticulum and Golgi apparatus. Stimulation of sulfated
chromogranin B secretion was similar in both untreated and
hormone-treated cells, while a consistent but small difference was
detected for secretogranin II secretion. These results suggest that the
two granins are sorted to separate granule populations that are
exocytosed at different rates upon stimulation. These granules may
exist in the same cells or in different subpopulations of
GHC
cells, in agreement with fluorescent
localization of granins in different cells (Scammell et al.,
1990a), as discussed above. If chromogranin B and secretogranin II are
differentially sorted in individual cells, this would imply the
presence of functionally distinct sorting signals on chromogranin B and
secretogranin II. Recent reports indicate that this is indeed the case.
Thus, chromogranin B, but not secretogranin II, is rerouted to the
constitutive pathway in dithiothreitol-treated PC12 cells (Chanat et al., 1993). In addition, our recent proposal that an
amino-terminal hydrophobic peak serves as the sorting signal for
chromogranin B, but not secretogranin II (Gorr and Darling, 1995), is
consistent with this possibility.
Methylumbelliferyl xyloside
inhibits proteoglycan synthesis but stimulates synthesis of free
glycosaminoglycan chains (Schwartz, 1977; Burgess and Kelly, 1984).
GHC
cells express a heparan sulfate
proteoglycan that is cellularly located (Hinkle et al., 1992).
Xyloside treatment reduced sulfate incorporation into this proteoglycan
about 30% and reduced sulfation of granins while strongly stimulating
glycosaminoglycan synthesis. The decrease in granin sulfation does not
prevent their sorting to the regulated secretory pathway in
hormone-treated cells. Similarly, inhibition of sulfation with sodium
chlorate does not prevent sorting of granins in untreated cells (Hinkle et al., 1992). Based on these and earlier data (Burgess and
Kelly, 1984; Gorr and Cohn, 1990; Gorr et al., 1991), it is
concluded that tyrosine-sulfation, oligosaccharide-sulfation, or
proteoglycan synthesis are not required for sorting or storage of
secretory proteins in the regulated secretory pathway in endocrine
cells.