©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cell Type-specific Secretion of Parathyroid Hormone-related Protein via the Regulated versus the Constitutive Secretory Pathway(*)

Lauren L. Plawner , William M. Philbrick , William J. Burtis , Arthur E. Broadus , Andrew F. Stewart (§)

From the (1) Divisions of Endocrinology and Metabolism, West Haven Veterans' Affairs Medical Center, West Haven, Connecticut 06516 and Yale University School of Medicine, New Haven, Connecticut 06520

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Parathyroid hormone-related protein (PTHrP)() 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.

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.


MATERIALS AND METHODS

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 TyrPTHrP-(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.

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.

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).




RESULTS

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).

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 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.




DISCUSSION

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 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).

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.

  
Table: Examples of tissues and cell types that produce PTHrP


  
Table: Cellular protein extraction from RIN-(1-139) after perifusion with 40 mM KCl

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.



FOOTNOTES

*
This study was supported by the Department of Veterans Affairs (West Haven, CT), by the Connecticut Affiliate of the American Heart Association, and by NIH Grants DK47168 and AR 30102. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Research/151C, West Haven VA Medical Center, 950 Campbell Ave., West Haven, CT 06516. Tel.: 203-932-5711 (ext. 3385); Fax: 203-937-3829.

The abbreviations used are: PTHrP, parathyroid hormone-related protein; RIN, rat insulinoma; CHO, Chinese hamster ovary; RIA, radioimmunoassay; IRMA, immunoradiometric assay; POMC, pro-opiomelanocortin.


ACKNOWLEDGEMENTS

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.


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