From the
Parathyroid hormone-related protein (PTHrP) is
endoproteolytically processed to yield a family of mature secretory
forms. These include an amino-terminal, a mid-region, and a
carboxyl-terminal form. Prior studies suggested that the mid-region
form is secreted via the regulated secretory pathway, whereas the
amino- and carboxyl-terminal forms are secreted via the constitutive
pathway. Further, PTHrP is unusual in that it is produced under normal
circumstances by neuroendocrine cell types as well as by prototypical
constitutively secreting cell types. The potential for cell-specific
secretory pathway use by PTHrP has not been explored. Using
immunohistochemical and perifusion techniques, we demonstrate that all
three PTHrP daughter peptides are secreted via the regulated pathway in
neuroendocrine cells. In contrast, all three daughter peptides are
secreted in a constitutive fashion by non-neuroendocrine cells. Thus,
the secretion of PTHrP is unique in that it appears to be
cell-specific. When it is expressed in neuroendocrine cells that
contain the regulated pathway, it is secreted in a regulated fashion;
when it is expressed in non-neuroendocrine cells, it defaults to the
constitutive pathway. This phenomenon has not previously been described
for a polypeptide hormone in naturally occurring cells.
Parathyroid hormone-related protein (PTHrP)
Finally, PTHrP is produced by
a broad range of cell types (see ). Many of these cell
types are known to secrete neuroendocrine peptides such as PTHrP in a
regulated fashion. Others are cell types that most would view as
belonging to the constitutively secreting family. Although the
processing of PTHrP is known to proceed in a cell-specific
fashion
(6) , little is known regarding the cell-specific
mechanisms that govern its secretion.
With these considerations in
mind, we sought to answer two questions in the current study. First,
are the amino-terminal, mid-region, and carboxyl-terminal forms of
PTHrP all secreted via the regulated or the constitutive pathway, or
can the PTHrP daughter peptides generated in a single cell type employ
different secretory pathways? Second, because PTHrP is produced in both
constitutive and regulated secretory cell types, can the PTHrP
secretory pathway vary in a cell-specific manner? The results indicate
that the three PTHrP daughter peptides can be secreted by either the
regulated or the constitutive secretory pathway and that the secretory
pathway employed is cell-specific. Whereas this type of cell-specific
secretory pathway use may well exist for other peptides, it has not
previously been described.
Insulin was measured in the perifusate using an insulin RIA
described previously
(18) . Briefly, this assay uses a polyclonal
guinea pig insulin antiserum (ICN Biomedicals, Costa Mesa, CA) and a
rat insulin standard (Eli Lilly, Indianapolis, IN). The detection limit
of the assay is 195 pg/ml.
Neuroendocrine cells typically
secrete approximately 3-5% of their stored hormone with each
secretory burst. We were curious to determine if the secretion of PTHrP
follows this same general rule. A perifusion was performed with
RIN-(1-139) cells stimulated with 40 mM KCl.
shows the values of these parameters for different
secretory forms of PTHrP and for insulin. As can be seen,
2.2-5.0% of each of these proteins is secreted with each
secretory burst, numbers compatible with neuroendocrine secretion via
the regulated pathway.
PTHrP is a prohormone that is post-translationally,
endoproteolytically processed to yield a family of mature secretory
forms of the
peptide
(2, 3, 4, 5, 6, 12) .
Previous work has shown that post-translational processing of PTHrP is
tissue-specific. That is, different PTHrP-producing cell types appear
to secrete different forms of the peptide
(2, 6) . At
present, at least three major secretory forms of the peptide have been
shown to exist (Fig. 1): an amino-terminal
species
(2, 4, 5, 6) , a mid-region
species
(5, 6, 9) , and a carboxyl-terminal
species
(6, 10, 17) . As suggested by the
dotted boxes in Fig. 1C, other mature forms of
the protein are likely to be identified with further investigation. As
outlined in the Introduction, little information was previously
available directly defining the secretory pathway(s) employed by the
various PTHrP secretory species.
Using a combination of
immunohistochemistry and a perifusion system, the findings in the
current study clearly indicate that secretion of all three of the major
mature PTHrP species by RIN cells occurs via the regulated pathway
described initially by Kelly
(13) . This is in contrast to our
own prior immunohistochemical findings
(6) suggesting that,
although the mid-region species was indeed present in secretory
vesicles in RIN and other cells, the amino- and carboxyl-terminal forms
of the peptide were present in the Golgi apparatus but not in secretory
granules. If this were true, it would suggest that the prohormone
cleavages of PTHrP that yield the three mature secretory forms must
occur in the Golgi, prior to sorting and targeting to different classes
of secretory/transport vesicles. The observations in
Fig. 4
displaying the manner of secretion of the three species
from potassium-stimulated perifused RIN-(1-139) cells indicate
that these secretory forms are most likely packaged into secretory
granules prior to secretion. The immunohistochemical co-localization
with insulin in Fig. 3suggests that the mature PTHrP secretory
forms, at least the mid-region species, are packaged into the same
granules as insulin in RIN cells. The observation that all three
species are contained within secretory vesicles is compatible with
prohormone cleavages occurring either in the Golgi system (as seen in
many propeptides cleaved by the furin/PACE-4 family of processing
proteases
(7, 8) ) or within the secretory granules
themselves after sorting into immature secretory vesicles (as occurs
with pro-opiomelanocortin (POMC) and insulin by the prohormone
convertases PC-1/3 and PC-2
(7, 8) ). The monobasic
cleavage at Arg
PTHrP is unusual in that it is produced by such a
broad array of different cell types, some of which are outlined in
. As indicated in the table, some of these are bona fide
members of the neuroendocrine family and might be expected to secrete a
peptide such as PTHrP via the regulated pathway. Others are typical
examples of cells that cause exocytosis of peptides via the
constitutive pathway. Given these observations and given prior
observations indicating that PTHrP can be post-translationally
processed in a cell-specific manner (6), it was of some interest to
determine whether PTHrP would be secreted constitutively by these types
of cells. We chose two prototypical constitutive cell types: a
fibroblast and a squamous epithelial cell. Both secreted PTHrP in a
constitutive fashion. At one level, these findings are intuitive; how
could one expect cells such as these, which do not contain secretory
granules, to secrete via a pathway other than the constitutive pathway?
What is surprising here is the documentation that a single
neuroendocrine peptide may be secreted via either the regulated or the
constitutive pathway in a cell-specific fashion.
To our knowledge,
other examples of this phenomenon have not been described. To be sure,
there are numerous examples in which a regulated, neuroendocrine
peptide has been diverted to the constitutive pathway by transfection
into a constitutive cell type
(13) . Conversely, there are many
examples in which a constitutively secreted peptide is transfected into
a regulated secretory cell type and is nonetheless secreted by the
constitutive pathway
(13) . There are also examples in which
deletion of key targeting sequences diverts a neuroendocrine peptide
such a POMC into the constitutive pathway
(22) . What is
different in the PTHrP situation is that all of the above examples of
``secretory pathway overlap'' have been observed not in
naturally occurring cells but in cellular models of gene transfer. In
contrast, this appears to be the normal course of events for PTHrP.
Although this phenomenon is unusual, we suspect that PTHrP is not the
only peptide for which it occurs. Analogous situations may occur for
POMC products secreted by lymphocytes or keratinocytes, both of which
express the POMC gene
(23, 24) , or for products of the
atrial natriuretic peptide gene that are secreted by
macrophages
(25) . Further studies will need to be performed to
clarify these issues.
In summary, PTHrP is post-translationally
cleaved into a family of mature secretory forms. These mature secretory
forms appear to be secreted via the regulated secretory pathway in
neuroendocrine cell types such as RIN cells, and by extrapolation we
presume that they are also secreted by the other neuroendocrine cells
that produce PTHrP: pancreatic beta cells, pituitary somatotropes,
adrenal medullary cells, parathyroid cells, central nervous system
neurons, perhaps atrial cardiocytes, and others. In contrast, but as
expected, the broad range of other cell types that express the PTHrP
gene most likely secrete the peptide(s) via the constitutive pathway.
The basis for this difference in secretory pathway targeting lies most
likely not in the targeting/sorting mechanisms present in these two
cell types but more proximally in the promoters and transcription
factors that allow expression of PTHrP peptides in such a broad
spectrum of tissues. We presume that proPTHrP contains a peptide
sequence that targets it to the regulated secretory pathway in cell
types that possess this pathway, as occurs, for example, in the
targeting of POMC
(22) . On the other hand, when the PTHrP gene
is expressed in constitutively secreting cell types such as the
osteoblast, the chondrocyte, the keratinocyte, the smooth muscle cell,
the hepatocyte, and the renal proximal tubular cell, we presume that it
is secreted via the constitutive pathway by default. The existence of
putatively specific peptide sequences that target PTHrP to the
regulated secretory pathway and the cellular mechanisms responsible for
that targeting will require further study.
The first column indicates the number of picomoles
of peptide secreted during stimulation with 40 mM KCl. The
second column indicates the picomoles of peptide extracted from the
cells after the secretory burst. The values in the third column are
obtained by adding the values in columns one and two to give the total
number of picomoles of peptide in the cells. The percent secreted is
then obtained by dividing the peptide secreted by the total peptide.
We thank Drs. K. Insogna and M. Bellantoni for
providing us with the RIN-(1-139) cells. We also thank Drs. W.
Zawalich and K. Zawalich for performing the insulin immunoassays. We
also thank Dr. Neil Soifer for advice and guidance in the performance
of the immunohistochemical studies. Finally, we thank Dr. P. Dannies
for unfailing support in designing, performing, and interpreting these
studies.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
undergoes extensive endoproteolytic post-translational
processing prior to secretion (1-6). The endoproteases that are
responsible for this processing are currently unknown but, given the
abundance of basic residues within the PTHrP sequence (see
Fig. 1
), are likely to include members of the subtilisin family
of endoproteases such as furin, PC-1/3, PC-2, and
PACE-4
(7, 8) . This endoproteolytic processing leads to
the secretion of at least three secretory forms of PTHrP (see
Fig. 1
)
(2, 3, 4, 5, 6) .
One is an amino-terminal species,
PTHrP-(1-36)
(4, 5) . The second is a mid-region
species that begins at amino acid 38, the carboxyl terminus of which is
as yet undetermined. It is recognized by an antibody directed at an
epitope in the 49-59 region
(5, 9) . The third
secretory peptide is a carboxyl-terminal species recognized by an
antibody directed against the 109-138
region
(9, 10) . Neither the amino nor the carboxyl
terminus of this peptide have been precisely defined. This peptide
corresponds closely with the bioactive PTHrP-(107-139)
synthesized by Fenton et al.(11) , which they have
named ``osteostatin.'' Other secretory forms of PTHrP are
likely to exist. For example, Wu et al.(12) have
demonstrated that keratinocytes produce a glycosylated form of the
peptide. A peptide bounded by amino acids 141-173 is also likely
to be generated (see Fig. 1).
Figure 1:
Post-translational processing of PTHrP.
A shows the three initial PTHrP translation products, each
comprised of a -36 to -1 prepro region, followed by 139
identical amino acids. The middle isoform has a two-amino-acid
extension to amino acid 141, and the bottom isoform extends an
additional 34 amino acids to amino acid 173. These three isoforms are
results of alternative splicing. K indicates lysine, and R indicates arginine. The multibasic (arginine- and lysine-rich)
regions are potential endoproteolytic proc-essing sites. B shows the antisera used in the RIAs and IRMAs described under
``Materials and Methods.'' C shows the secretory
forms of PTHrP that have been identified to date (solid boxes)
as well as those that are likely to exist (dotted
lines).
Kelly
(13) has described
the existence of two secretory pathways in eukaryotic cells: the
``regulated'' and the ``constitutive'' secretory
pathways. Little is known regarding the pathways employed in the
secretion of the various PTHrP secretory forms. Work from Deftos'
group
(1) has shown that the phorbol ester, phorbol 12-myristate
13-acetate, and ionomycin led to the appearance in conditioned medium
of PTHrP from carcinoid cell lines over a period of 5-10 min.
Whether this represented regulated or constitutive secretion was not
clarified in formal terms. Rizzoli et al.(14) have
suggested that secretion of PTHrP in response to cyclic AMP by a
squamous carcinoma cell line requires as long as 40 min. Again, whether
this secretion was of the regulated or the constitutive variety is
uncertain. In addition, these studies focused only on the
amino-terminal secretory form of PTHrP. Work from our laboratory, using
immunohistochemistry, has suggested that, although the mid-region PTHrP
secretory peptide is packaged into secretory granules in rat insulinoma
cells
(5, 6) , the amino- and carboxyl-terminal species
are not
(6) . If true, this would have two implications. First,
it would indicate that the endoproteolytic processing enzymes that
process PTHrP are based in the Golgi apparatus or the trans-Golgi
network. This is compatible with processing by the Golgi-based enzymes,
furin or PACE-4, but would be less compatible with processing by PC-1/3
or PC-2, which are believed to function within the secretory
granule
(7, 8) . Second, it would suggest that the
mid-region PTHrP species is secreted via the regulated secretory
pathway, whereas the amino- and carboxyl-terminal species would be
secreted via the constitutive secretory pathway. Although precedent
exists for the sorting of the daughter peptides derived from a single
precursor into different classes of secretory
vesicles
(15, 16) , this type of differential sorting of
daughter peptides appears to be uncommon. Further, immunohistochemical
studies reported by Deftos' group
(17) have shown that a
carboxyl-terminal form of PTHrP can clearly be visualized in secretory
granules in atrial cardiocytes. Thus, an alternate explanation for our
immunohistochemical findings could be that all three secretory forms of
PTHrP are present within secretory granules but that they are packaged,
processed, or folded in such a way that they cannot be detected by our
amino- and carboxyl-terminal antisera.
Cell Lines
The rat insulinoma (RIN)
1036-48 cells were a generous gift of Dr. Michael Appel of the
University of Massachusetts, Worcester, MA. In this manuscript, these
untransfected RIN cells are referred to as RIN-0 cells to contrast them
with RIN 1036-48 cells stably transfected with the
PTHrP-(1-139) isoform. These latter cells are referred to as
RIN-(1-139) cells. RIN-(1-139) cells were prepared using
the pLJ vector as described previously in detail
(5, 6) .
Dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cells
were stably transfected with a PTHrP-(1-139) construct in the
vector pFR-SR as described previously in detail
(6) and are
referred to as CHO(1-139) cells. RWG-T2 is a squamous carcinoma
line that naturally expresses PTHrP and was a generous gift from Dr.
Theresa Guise of the University of Texas, San Antonio, TX. The RIN
lines were grown in RPMI 1640 medium, and the RWG-T2 cells were grown
in Dulbecco's modified Eagle's medium; both media contained
10% fetal bovine serum, 1% glutamine, and 200 IU/ml
penicillin/streptomycin. The CHO cells were grown in nucleotide-free
-minimum essential medium supplemented with nucleotide-free 5%
fetal bovine serum, 200 IU/ml penicillin/streptomycin, and 200
µM methotrexate.
Perifusion
Preliminary experiments comparing
secretion rates from cells in culture flasks with rates from cells
perifused in the system described below revealed that PTHrP
concentrations were approximately 10-fold higher in perifusate than in
conditioned medium harvested from cells. This reflects the greater
surface area (and therefore the cell number to medium volume) of the
perifusion bead system as compared with standard planar culture flasks.
Accordingly, for these experiments, cells were grown to confluence in
plastic flasks, trypsinized, and then seeded to 10-cm Petri dishes
containing Cytodex-3 collagen-coated microcarrier beads (Pharmacia
Biotech Inc.). On day 2 after seeding, 1.0 ml of the cell-laden beads,
containing approximately 10 cells, were loaded onto a 0.5
3.0-cm plastic column. The column was connected via
polypropylene tubing to a peristaltic pump. The column was submerged in
a water bath at 37 °C and perifused with medium appropriate for the
cell type as described in the previous section at a rate of 0.33
ml/min. After a 20-30-min equilibration period, 1-ml fractions
were collected at 3-min intervals. Secretogogues were added to the
perifusate at the time points and in the concentrations shown in the
figures and the figure legends. The fractions collected were frozen
immediately at -70 °C for subsequent immunoassay.
PTHrP and Insulin Immunoassays
These assays have
been described in detail previously. The regions of PTHrP that they
detect are shown schematically in Fig. 1. The amino-terminal
PTHrP-(1-36) RIA employs the sheep antiserum Sheep-2 and uses
TyrPTHrP-(1-36)NH
as both radiolabel and
standard
(6, 10) . The mid-region PTHrP RIA employs
antiserum Sheep-1 and uses PTHrP-(37-74) as both radioligand and
standard
(6, 9) . The carboxyl-terminal PTHrP RIA uses
Tyr
PTHrP-(109-138) as radioligand and
standard
(6, 10) . The specificities, sensitivities, and
inter/intra-assay variation have been summarized previously
(6) .
For the RIN-0 and RWG-T2 cells, which produce less PTHrP than the other
lines under basal conditions, we developed a two-site immunoradiometric
assay (IRMA) for PTHrP-(1-36). This PTHrP-(1-36) IRMA is
identical in all respects to a PTHrP-(1-74) IRMA reported earlier
(10) except that it employs affinity-purified rabbit-6 antiserum
directed against PTHrP-(1-36) instead of Sheep-1
anti-PTHrP-(37-74) as the ``capture'' antibody. The
sensitivity of this PTHrP-(1-36) IRMA is 5 pM.
Immunohistochemistry
Immunohistochemistry was
performed using methods we have described
previously
(5, 6) . The PTHrP antiserum employed was
affinity-purified Sheep-1
(5) , and the insulin antiserum was a
guinea pig anti-insulin antiserum purchased from ICN Biomedicals. The
second antibodies were a rhodamine-labeled goat anti-sheep antiserum
and a fluorescein-labeled sheep anti-guinea pig antiserum, both from
Tago Inc. (Burlingame, CA). Insulin and PTHrP immunofluorescence could
be completely extinguished by preincubating the appropriate antiserum
with excess insulin or PTHrP, respectively. Co-localization studies
were performed with the appropriate filters using a Zeiss Axiophot
microscope. When cells were single-stained for only PTHrP or insulin,
fluorescence could be visualized only through the appropriate color
filter.
Protein Extraction
PTHrP or insulin secreted with
each episode of stimulation by KCl was expressed as a percentage of the
corresponding total peptide as shown in . ``Total
peptide'' is defined as the sum of the cellular and the secreted
peptides (insulin or PTHrP) as described below; a perifusion experiment
as described above was performed with RIN-(1-139) cells. At 30
min, the cells were stimulated with 40 mM KCl, and the PTHrP
and insulin secreted were quantitated by RIA on perifusion fractions.
After the peak of secretion was collected (15 min following exposure to
KCl, see Fig. 6), the cells and beads from the perifusion column
were immediately transferred to a Petri dish and extracted using
guanidine thiocyanate as described previously
(5) . The extract
was then analyzed by RIA for insulin and PTHrP immunoactivity.
Figure 6:
Secretion of the three PTHrP species in
response to leucine (10 mM) and KCl (40 mM). Note
that all three species of PTHrP are released in response to leucine
(dark bar labeled leucine), although in smaller
amounts than are observed following KCl (dark bar labeled
KCl). This figure is representative of three similar
experiments. Similar results were observed with arginine. ,
PTHrP-(1-36);
, PTHrP-(37-74);
,
PTHrP-(109-138).
PTHrP Is Secreted by the Regulated Secretory Pathway in
RIN Cells
Previous work
(19, 20) has shown that
PTHrP is normally produced by the insulin-secreting beta cells of the
pancreatic islet. The beta cell is a classical example of a
neuroendocrine cell that employs the regulated secretory pathway
(). RIN cells, beta cell models, were therefore chosen to
determine whether PTHrP was secreted by the regulated or constitutive
pathway in these typical neuroendocrine cells. In initial studies,
untransfected RIN cells, which we previously showed to contain small
quantities of PTHrP (5), were selected for perifusion. Potassium
chloride (40 mM) was selected as a secretagogue, because it is
well known to depolarize RIN cells and lead to the secretion of insulin
(21). The results of a perifusion study using RIN-0 cells is shown in
Fig. 2
. As can be seen in the figure, amino-terminal PTHrP is
secreted by these cells in response to two separate episodes of
stimulation by KCl. Although these results suggest that amino-terminal
PTHrP is secreted by RIN cells via the regulated pathway, the magnitude
of the response was small, and neither basal nor stimulated secretion
of the mid-region nor carboxyl-terminal forms of PTHrP could be
detected.
Figure 2:
Perifusion of RIN-0 cells. Immunoreactive
PTHrP-(1-36) was measured in the perifusate by IRMA following two
episodes of stimulation with 40 mM KCl shown as the dark
bars at the top of the figure. Both applications of KCl
led to secretory bursts of PTHrP-(1-36), but these were
quantitatively small and were near the detection limit of the assay.
Neither PTHrP-(37-74) nor PTHrP-(109-136) could be
detected.
Demonstration of co-packaging of insulin and PTHrP within
the same granules in RIN cells would provide additional evidence that
PTHrP is secreted via the regulated secretory pathway. In an effort to
determine whether such co-packaging occurs, RIN-0 cells were
simultaneously immunostained for insulin and mid-region PTHrP. As can
be seen in Fig. 3, the pattern for insulin staining is
characteristic of staining within secretory granules. Interestingly,
PTHrP appears to stain the very same secretory vesicles that are
stained by the insulin antiserum; these findings suggest that insulin
and mid-region PTHrP are contained within the same granules or
vesicles. Formal proof that PTHrP is localized within dense core
insulin-containing granules will require immunohistochemistry at the
electron microscopic level.
Figure 3:
Immunofluorescent co-localization of PTHrP
and insulin in RIN-0 cells using rhodamine for PTHrP-(37-74)
(left panel) and fluorescein for insulin (right
panel). The white arrowheads indicate clusters of
secretory granules that stain for both insulin and PTHrP. The open
arrows indicate Golgi staining. There is an identical staining
pattern for PTHrP and insulin.
Because the quantities of mid-region and
carboxyl-terminal PTHrP produced by RIN-0 cells (Fig. 2) are
undetectable by the PTHrP immunoassays and because amino-terminal PTHrP
is close to the detection limit of the assay, RIN-(1-139) cells
that overexpress PTHrP were selected for further study. The
PTHrP-(1-139) (Fig. 1) isoform was chosen over
the(1-141) or(1-173) isoforms, because RIN and CHO cells
transfected with this isoform produce amino-terminal, mid-region, and
carboxyl-terminal PTHrP secretory species, whereas RIN-(1-141)
and RIN-(1-173) cells produce only amino-terminal and mid-region
secretory forms of PTHrP
(6) . As can be seen in Fig. 4,
secretion of all three PTHrP species is easily detected from these
cells in response to 40 mM KCl. Four sequential episodes of
KCl administration led to four distinct episodes of secretion,
indicating that 1) only a portion of PTHrP-containing secretory
granules are released during each episode of secretion, as is typical
of secretion from neuroendocrine cells, and 2) the phenomenon being
observed is true secretion and not simply death or lysis of cells with
the administration of a lethal, hyperpolarizing dose of KCl. The
observation that secretion occurs within 3 min of administration of
KCl, the earliest timepoint sampled following exposure to KCI,
indicates that secretion is occurring from RIN cells via the regulated
and not the constitutive pathway. As can be seen in Fig. 4,
immediate secretion of all three PTHrP species occurred. This indicates
that, despite immunohistochemical data indicating that amino- and
carboxyl-terminal PTHrP species cannot be detected within secretory
vesicles in RIN cells
(5, 6) , they are there nonetheless
and are presumably folded, processed, or packaged in a way that makes
them ``invisible'' to the same antisera that can easily
detect them following secretion.
Figure 4:
Perifusion of RIN-(1-139) cells.
Following stimulation with 40 mM KCl (dark bars at
top of figure), each of the three PTHrP species was secreted
in a burst that begins within 3 min of stimulation. Note that the basal
secretion of PTHrP between episodes of KCl stimulation is also easily
detectable for all three secretory forms. , PTHrP-(1-36);
, PTHrP-(37-74);
,
PTHrP-(109-138).
The immunohistochemical data in
Fig. 3
and the secretory data in Fig. 4suggest that insulin
and PTHrP are packaged in the same granules. If this were the case,
they should be released from RIN cells following stimulation with KCl
with the same kinetics. This is shown to be true in Fig. 5. This
figure makes the additional point that PTHrP and insulin are secreted
from these cells in roughly equimolar amounts. This is significant, for
it suggests that RIN-(1-139) cells do not sort, process, package,
and secrete PTHrP in an anomalous fashion due to overexuberant
expression or saturation of sorting and processing mechanisms.
Figure 5:
Co-secretion of insulin and PTHrP by
RIN-(1-139) cells following 40 mM KCl (dark bar at top of figure) stimulation. See the text for
details. , PTHrP-(1-36);
;
insulin.
We
remained concerned that stimulation with KCl was nonphysiologic and
might lead to artifactual ``secretion'' through cell lysis
and death. We therefore employed leucine, a physiologic islet
secretagogue that stimulates insulin secretion via its action on the
glycolytic pathway (21). Although glucose might seem the natural
secretagogue, RIN cells respond poorly to glucose, for they express
glucose transporters and glucokinase at levels significantly below
normal. As can be seen in Fig. 6, RIN-(1-139) cells display
prompt secretory bursts of all three peptides in response to 10
mM leucine, the first peak in the figure. The secretory
response to KCl, the second peak, is more pronounced. Of note, the
leucine also caused secretion of insulin during the same time course as
PTHrP secretion, as was described above with KCl (data not shown). A
similar response was seen in response to arginine, another beta cell
secretagogue
(21) (not shown).
PTHrP Is Secreted in a Constitutive Manner by Fibroblasts
and by Squamous Carcinoma Cells
In order to study the mechanism
of secretion of PTHrP by classical constitutively secreting cell types,
we employed Chinese hamster ovary fibroblasts. As these cells do not
normally express PTHrP, they were stably transfected with the same
PTHrP-(1-139) isoform used in the RIN-(1-139) cells.
Fig. 7
shows the results of an experiment in which
CHO(1-139) cells were perifused with 40 mM KCl. As can
be seen in Fig. 7, in contrast to the results observed with
RIN-(1-139) cells, no secretory response is observed from
CHO(1-139) cells. That this is not a reflection of low levels of
expression of the three PTHrP species is evident from the figure; the
concentrations of the three peptides in the perifusate ranged from 100
to almost 400 pM and were higher on average than basal
secretion from RIN-(1-139) cells. Similar results were observed
following perifusion of these cells with the calcium ionophore A23187
(0.5 µM) (not shown). Thus, CHO(1-139) cells secrete
all three PTHrP species at a steady, basal constitutive rate but do not
appear to have the capacity to secrete in a regulated fashion.
Figure 7:
Perifusion of CHO(1-139)
fibroblasts. In contrast to the events shown in Fig. 6 in RIN cells, no
secretion is observed from CHO(1-139) fibroblasts following
stimulation with 40 mM KCl (dark bars at
top). Similar results were observed following exposure to 0.5
µM of the calcium ionophore A23187 (not shown). Basal
secretion, however, is easily measurable in all three assays, which
have detection limits ranging from 5-50 pM. ,
PTHrP-(1-36);
, PTHrP-(37-74);
,
PTHrP-(109-138).
Concerned that cells engineered to secrete PTHrP might not be
reflective of events occurring under normal conditions, we chose a
second typical constitutively secreting cell type for study, a cell
type that naturally secretes PTHrP. The human squamous carcinoma cell
line RWG-T2 was selected as it fits these criteria, as well as causing
hypercalcemia when passaged in mice, and is presumed to be
representative of events occurring in human keratinocytes, which also
normally produce PTHrP. The results of perifusion of these cells are
shown in Fig. 8. The concentrations of PTHrP in the perifusate
are lower than those seen in the CHO(1-139) cells, and
carboxyl-terminal PTHrP was unmeasurable by our assays. But
importantly, as clearly seen in the figure, these squamous carcinoma
cells secrete amino-terminal and mid-region PTHrP in a constitutive
fashion.
Figure 8:
Perifusion of RWG-T2 squamous carcinoma
cells. As with CHO fibroblasts, no secretion is observed following KCl
stimulation (dark bars at top). Basal levels of
PTHrP-(1-36) () and PTHrP-(37-74) (
) secretion
are readily detectable. Carboxyl-terminal PTHrP secretion was
undetectable.
may occur in either the Golgi system or
the secretory granules. One cautionary note should be added to these
conclusions; the current study contains no direct evidence documenting
that the PTHrP species secreted into the perifusate are discrete,
separate secretory species and not, for example, unprocessed
prohormone. Attempts to chromatographically separate the various
species of PTHrP in the perifusate were unsuccessful due to the small
amounts of these peptides in the perifusate. We believe that processing
is complete, however, by the time secretion occurs, for prior studies
by ourselves and others have shown that the secretory forms of PTHrP
identified in the perifusate are present in protease-protected RIN and
squamous cell extracts and are therefore generated prior to secretion
(2, 4-6).
Table:
Examples of tissues and cell types that produce
PTHrP
Table:
Cellular
protein extraction from RIN-(1-139) after perifusion with 40
mM KCl
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.